role of spleen tyrosine kinase in tumor necrosis factor
TRANSCRIPT
Graduate Theses and Dissertations Iowa State University Capstones, Theses andDissertations
2017
Role of spleen tyrosine kinase in tumor necrosisfactor like weak inhibitor of apoptosis induceddopaminergic neuronal cell death and microglialactivation in invitro and in vivo models ofParkinson diseaseSri Harsha KanuriIowa State University
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Recommended CitationKanuri, Sri Harsha, "Role of spleen tyrosine kinase in tumor necrosis factor like weak inhibitor of apoptosis induced dopaminergicneuronal cell death and microglial activation in invitro and in vivo models of Parkinson disease" (2017). Graduate Theses andDissertations. 16389.https://lib.dr.iastate.edu/etd/16389
Role of spleen tyrosine kinase in tumor necrosis factor like weak inhibitor of apoptosis
induced dopaminergic neuronal cell death and microglial activation in invitro and in vivo
models of Parkinson disease
by
Sri Harsha Kanuri
A dissertation submitted to the graduate faculty
in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY
Major: Biomedical Sciences (Physiology)
Program of Study Committee:
Arthi Kanthasamy, Major Professor
Anumantha Kanthasamy
Manju Reddy
Wilson K Rumbeiha
Bryan H Belliare
Jesse Paul Goff
The student author, whose presentation of the scholarship herein was approved by the
program of study committee, is solely responsible for the content of this dissertation.
The Graduate College will ensure this dissertation is globally accessible and will not
permit alterations after a degree is conferred
Iowa State University
Ames, Iowa
2018
Copyright © Sri Harsha Kanuri, 2018. All rights reserved.
ii
TABLE OF CONTENTS
Page
NOMENCLATURE .................................................................................................. iv
ABSTRACT………………………………. .............................................................. Vi
CHAPTER I: LITERATURE REVIEW .................................................................. 1
General Introduction .......................................................................................... 1
Dopamine Neurochemistry ............................................................................... 4
Basal Ganglia Neuroanatomy .......................................................................... 5
Lewy body pathology ....................................................................................... 6
Braak Hypothesis of Parkinson Disease .......................................................... 10
Cell Death in PD .............................................................................................. 12
Genetics and PD ............................................................................................... 17
Pathogenic Mechanisms in PD ......................................................................... 19
Inflammasome ................................................................................................. 31
Microglia and Neuro-inflammation in PD ....................................................... 40
Astrocytes ........................................................................................................ 45
Animal Models of PD ...................................................................................... 46
Spleen Tyrosine Kinase ...................................................................................... 57
Tweak Neuro-inflammation ................................................................................ 64
Bibliography ...................................................................................................... 76
CHAPTER II: INVOLVEMENT OF MITOCHONDRIA MEDIATED
OXIDATIVE STRESS DEPENDENT CELL SIGNALING EVENTS AND SYK
TYROSINE KINASE ACTIVATION IN TUMOR NECROSIS LIKE WEAK
STIMULATOR OF APOPTOSIS (TWEAK) INDUCED DOPAMINERGIC CELL
DEATH
......................................................................................................... 139
Abstract ......................................................................................................... 139
Introduction ......................................................................................................... 140
Methods ......................................................................................................... 145
Results ........................................................................................................ 151
Discussion ......................................................................................................... 162
Bibliography ........................................................................................................ 171
iii
CHAPTER III: ROLE OF SPLEEN TYROSINE KINASE (SYK) IN
MEDIATINGTUMOR NECROSIS FACTOR LIKE WEAK
INHIBITOROFAPOPTOSIS (TWEAK) INDUCED INFLAMMASOME
ACTIVATION IN BV2 MICROGIAL CELLS IN CELL CULTURE MODELS OF
PD
......................................................................................................... 197
Abstract ......................................................................................................... 197
Introduction ......................................................................................................... 198
Methods …..................................................................................................... 202
Results Section ..................................................................................................... 207
Discussion ......................................................................................................... 220
Bibliography ........................................................................................................ 228
CHAPTER IV: SPLEEN TYROSINE KINASE MEDIATES
NLRP3 INFLAMMASOME ACTIVATION IN LPS STIMULATED
MICROGLIA VIA NF-KB ACTIVATION, MAP KINASE ................................... 263
AND ROS GENERATION
Abstract ......................................................................................................... 263
Introduction ......................................................................................................... 264
Methods ......................................................................................................... 267
Results Section ..................................................................................................... 270
Discussion ......................................................................................................... 280
Bibliography ........................................................................................................ 282
CHAPTER V: CONCLUSIONS................................................................................ 291
Conclusions .......................................................................................................... 291
Bibliography ........................................................................................................ 297
CHAPTER VI : FUTURE AREAS OF INVESTIGATION ...................................... 299
Bibliography ........................................................................................................ 303
ACKNOWLEDGMENTS ......................................................................................... 306
iv
NOMENCLATURE
PD Parkinson Disease
AD Alzheimer Disease
TWEAK Tumor Necrosis Factor like Weak stimulator of Apoptosis
TNF, Tumor Necrosis Factor; ALS
Autophagy Lysosomal system
UPS Ubiquitin Proteasome system
SLE Systemic Lupus Erythematosis
BBB Blood brain barrier
MMP Mitochondrial membrane potential
LC3 Light Chain 3
Fn14 Fibroblast Growth Factor Inducible 14
TH Tyrosine Hydroxylase
MCP-1 Monocyte chemoattractant protein
EAE, Experimental Autoimmune Encephalitis
MPTP Methyl Phenyl Tetrahydroperidine
MAPK Mitogen activated Protein Kinase
ERK Extracellular signal regulated kinase
NF-kB Nuclear factor kappa-light-chain-enhancer of activated B cells
FPN Fipronil
GABA Gamma amino butyric acid
PKC-δ Protein Kinase C Delta
ER Endoplasmic Reticulum
MMP Mitochondrial Membrane Potential
SHSY Bone marrow biopsy-derived line SK-N-SH cell line
v
GSK-3B Glycogen Synthase Kinase – 3 Beta
N27 Rat dopaminergic neuronal cell line
BV2 Mouse microglial cell line
ROS Reactive oxygen species
RNS Reactive Nitrite species
GSH Glutathione
DA Dopaminergic neurons
PERK PKR-like ER kinase
CHOP CCAAT-enhancer-binding protein homologous protein
IRE1 Inositol Requiring 1
ATF-4 Activating Transcription Factor 4
Elf-2 Eukaryotic Initiation Factor 2
SYK Spleen Tyrosine Kinase
ZAP-70 Zeta-chain-associated protein kinase 70
BLINK B-cell Linker protein
PAMP Pathogen associated molecular patterns
DAMP Damage associated molecular patterns
LPS Lipo-polysaccaride
HBSS Hank Balanced Salt solution
G3BP1 G3BP Stress Granule Assembly Factor 1
NLRP3 nucleotide-binding domain, leucine-rich-containing family, pyrin domain-3
IL-1 B Interleukin-1 Beta
IL-18 Interleukin 18
R788 Spleen Tyrosine Kinase inhibitor
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ABSTRACT
Tweak and TNF family members are novel regulators of acute and chronic
inflammation. Tweak / Fn14 interaction appears to be involved in angiogenesis, inflammation,
proliferation, migration, cytokine production, cytotoxicity and apoptosis. Thus, TWEAK can
induce both cell death and proliferation. Recently, TWEAK has been shown to be associated with
neurodegenerative effects in an MPTP mouse model of PD. Despite numerous studies
demonstrating TWEAK’s ability to cause cell death in diverse cell types the mechanism(s) by
which TWEAK-induce dopaminergic cell death remain poorly defined. Therefore in an attempt to
better understand the molecular basis of TWEAK-induced dopaminergic neurotoxicity we
evaluated the apoptotic effect of TWEAK and associated molecular mechanisms using N27
dopaminergic neuronal cells. TWEAK-induced a time dependent increase in ROS generation,
mitochondrial dysfunction, caspase activation, and NF-kB activation. Additionally, a concurrent
activation of SYK and proteolytic cleavage of PKC delta was evidenced in TWEAK treated cells.
In contrast a marked down regulation of p-GSK 3b (Ser 9) and Akt activity was evidenced in
TWEAK treated cells. Intriguingly, inhibition of SYK activity via R708 attenuated TWEAK-
induced loss of dopaminergic cell viability. Likewise, SN50, NF-kB inhibitor and Quercetin, a
ubiquitous bioactive plant flavonoid attenuated TWEAK-induced apoptotic cell death further
highlighting the pivotal role of NF-kB and mitochondria dependent oxidative stress signaling
events in the mechanism of dopaminergic neurodegeneration. Taken together, our studies
demonstrate the involvement SYK/NF-kB signaling axis and mitochondrial dysfunction in
TWEAK-induced apoptotic cell death. In the next series of studies we determined the effects of
TWEAK on microglia. A growing body of evidence suggests that persistent microglial activation
and accompanying oxidative stress may act as co-conspirators in mediating dopaminergic
vii
neurodegeneration in the substantia nigra in PD pathogenesis. Previously we demonstrated that
PKC delta a redox sensitive kinase is a critical determinant of microglial activation response in
response to diverse inflammogens. More recently, SYK, has been implicated in the activation of
inflammatory cells in response to infection. Therefore, in the current study we hypothesized that
SYK may act as an upstream regulator of NLRP3 inflammasome thereby leading to a heightened
microglial activation response in TWEAK stimulated cells. In the present study we systematically
elucidated the signaling network underlying TWEAK-induced microglial activation response. A
concentration dependent increase in SYK activation and accompanying increase in kinase activity
was evidenced in TWEAK treated cells. Our results with pharmacological inhibitors and siRNA
mediated gene silencing revealed the regulatory role of SYK in ER stress response (ERS), NOX2
upregulation, GSK 3β activation as well as autophagolysosomal system (ALS) and mitochondrial
dysfunction. Taken together our findings demonstrate a role for SYK signaling network in
mediating TWEAK triggered inflammatory response by positively regulating NLRP3
inflammasome activation and ERS in an autophagy dependent manner. We have discovered the
pivotal role of SYK in mediating dopaminergic neurodegeneration as well as heightened
microglial activation response upon TWEAK treatment. By inhibiting SYK activation we can
limit dopaminergic neuronal degeneration as well as microglial activation response in response to
diverse inflammagens including TWEAK. Our studies further highlight the therapeutic advantage
of targeting SYK for the treatment of inflammation related disorders including PD.
1
CHAPTER 1. LITERATURE REVIEW
General Introduction
Parkinson’s disease (PD) is the second most common chronic neurodegenerative disease
only next to Alzheimer's disease. The economic burden of PD was 14.4 billion dollars in United
States in year 2010, and this burden would increase over coming years as size of the elderly
population also, grows substantially (Kowal et al., 2013). The annual incidence rate of Parkinson
Disease varies from 1.5- 14.8 cases per 100,000 populations worldwide (de Lau & Breteler, 2006;
Elbaz et al., 2016; Kasten et al., 2007; Tanner & Goldman, 1996; Wright Willis et al., 2010).
Caucasians have higher prevalence and incidence of Parkinson’s Disease as compared to African
Americans and Asians (Wright Willis et al., 2010). United States, mid-west and northeastern
regions have a relatively higher incidence and prevalence of Parkinson’s disease as compared to
other regions (Wright Willis et al., 2010). Parkinson’s disease is slightly more common in men
than women (de Lau et al., 2006; Elbaz et al., 2016; Kasten et al., 2007). The incidence and
prevalence of Parkinson’s disease were more common after 50 years of age and increases steadily
until 9th decade (de Lau et al., 2006; Elbaz et al., 2016; Kasten et al., 2007; Tanner et al., 1996).
Approximately, 630,000 people have been diagnosed with PD in 2010 and it is estimated that, by
the year 2050 the disease prevalence would be doubled (Kowal et al., 2013). The prevalence of
Parkinson Disease is 1-2 per 1000 population (Tysnes & Storstein, 2017). The prevalence of
Parkinson Disease increases gradually after 60 years age and becomes highest after 80 years of
age (1663 per 100,000 population) (C. L. Ma et al., 2014) . The mean age of disease onset is 48
years while average disease duration is 21 years (S. Rocha et al., 2014). The annual incidence rate
for males and females with PD includes 207-396 and 127-259 per 100, 000 respectively (C. L.
Ma et al., 2014). The estimated worldwide population with PD by the year 2030 would be around
2
8.67 million (Allyson Jones et al., 2012). The national annual heath care costs per person with PD
for the year 2017 is around $25,000 with additional out of pocket expenses $15,137 per person
(Bohingamu Mudiyanselage et al., 2017). The annual health care health cost per person with
moderate disease is 4 fold very higher than person with mild disease ($63,579 Vs $17,537). The
patients with PD will have severe disability and decreased quality of life attributed to varied
disease symptomatology. These figures highlight the importance of developing appropriate
clinical interventions which arrest the disease progression, reduce disability, improve the quality
of life and alleviate the clinical symptoms in PD patients.
It is hypothesized that, PD pathogenesis originates from gastrointestinal tract and gradually
spreads through brain stem, mid brain and subsequently other regions of the brain according to the
the recently described BRAAK staging (Braak et al., 2003; Robert E. Burke et al., 2008; D. W.
Dickson et al., 2010). The pathophysiological cellular hallmarks of PD includes apoptosis, protein
misfolding, neuroinflammation, mitochondrial dysfunction, calcium dyshomeostasis, lysosomal
dysfunction and proteosomal dysfunction (Antony et al., 2013) among others. A well characterized
histological hallmark of Parkinson’s disease is Lewy bodies, are mainly composed of alpha
synuclein proteins in substantia nigra of midbrain which has been implicated in the prion like
disease progression in the affected brain regions (Baba et al., 1998; DelleDonne et al., 2008; J. L.
Eriksen et al., 2005a; Wakabayashi et al., 2007).
Although mechanisms like oxidative stress, proteosomal dysfunction, mitochondrial
dysfunction and alteration of calcium homeostasis are proposed to be instigating factors (J. L.
Eriksen et al., 2005a). Recent studies implicate a combination of environmental and genetic factors
in the mechanism of dopaminergic neurodegeneration (Riess & Kruger, 1999). Most of the PD
cases are idiopathic but upto 10% of them are caused due to genetic mutations of PD genes like
3
LRRK2, Parkin, PINK, DJ-1 and SNCA (Hardy et al., 2003; Christine Klein & Westenberger,
2012). Genetic PD cases usually have early age of onset with most cases having the disease onset
around 40 years (Hardy et al., 2003; Christine Klein et al., 2012).
The cardinal features of parkinsonism includes resting tremor, rigidity, bradykinesia and
loss of postural reflexes (Jankovic, 2008). Sometimes the diagnosis of PD patients gets delayed
because they do not present with primary motor symptoms always and they present with non-
specific motor symptoms. Few non-specific motor symptoms of PD patients include dysarthria,
dysphagia, sialorrhoea, micrographia, shuffling gait, festination, freezing and dystonia (Jankovic,
2008). Additionally, non-motor symptoms in PD patients includes autonomic dysfunction,
cognitive / neurobehavioral abnormalities, sleep disturbances and sensory abnormalities
(Jankovic, 2008). The most common sleep disturbances in PD patients includes nocturnal sleep
disturbance and excessive day time sleepiness (Davie, 2008). The differential diagnosis of patients
presenting with motor symptoms includes progressive Supra nuclear palsy, Dementia with Lewy
bodies, multiple system atrophy and drug induced Dementia (Frank et al., 2006). Depression and
psychosis can be associated with Parkinson disease (Dooneief et al., 1992; Naimark et al., 1996).
Cognition impairment and depression are very common in PD developing in 40% of the patients
and can develop as late as 10 years after symptoms have emerged (Davie, 2008). The most
commonly used drugs for Parkinson’s disease include levodopa, dopamine agonists
(Bromocriptine, Roprinirole and Pramipexole), MAO inhibitors (Selegiline and Rasagiline),
COMT inhibitors (Entacapone and Tolcapone), neuroleptics and cholinesterase inhibitors (Davie,
2008; Korczyn, 2004). Apart from medications, surgical options are also available for providing
symptomatic relief in PD patients. Bilateral subthalamic stimulation is one of the viable surgical
therapies which provides substantial symptomatic relief from bradykinesia, tremor and rigidity in
4
PD patients (Davie, 2008). Understanding the disease pathogenesis is very important for
developing pharmacological interventions. In this context, animal models of PD have been
beneficial in studying the mechanistic basis of dopaminergic neurodegeneration.
Dopamine Neurochemistry
Parkinson Disease is characterized by dopaminergic cell loss in the substantia nigra and
depletion of striatal dopamine content with associating motor abnormalities. Therefore, it is
important to understand the role of dopamine metabolism and its metabolites. Tyrosine is
converted into L-DOPA by tyrosine hydroxylase by a rate limiting enzyme of dopamine
biosynthesis (Bjorklund & Dunnett, 2007; Johannes Meiser et al., 2013b). Next, L-DOPA is
converted to dopamine by DOPA decarboxylase by removing the carboxyl group from L-DOPA
(Bjorklund et al., 2007; Johannes Meiser et al., 2013b). Additionally, Dopamine is converted to
norepinephrine by dopamine beta hydroxylase. Furthermore, norepinephrine is converted into
epinephrine by phenylethanolamine – S-methyl transferase (Bjorklund et al., 2007; Johannes
Meiser et al., 2013b). Dopamine is released from synaptic vesicles upon stimulation of
dopaminergic neurons which tends to act on the post synaptic receptors or presynaptic receptors
(Werkman et al., 2006; Hui Zhang & Sulzer, 2012). The dopamine which is present in the synaptic
cleft can be taken up by dopaminergic neurons by dopamine transporters (DAT) or taken up by
surrounding glial cells (J. Eriksen et al., 2010). The dopamine that is up taken by the dopaminergic
neurons can present in the cytosol or taken back into synaptic vesicles by vesicular monoamine
transporters (VMAT) (J. Eriksen et al., 2010; Werkman et al., 2006; H. Zhang et al., 2012). The
dopamine which is up taken by glial cells can be metabolized by various enzymes like MAO
(monamine oxidase) or COMT (Catecholamine o-methyl transferase) into various metabolites
(Johannes Meiser et al., 2013a). The main conjugation reactions of dopamine include sulfation and
5
glucuronidation (Eisenhofer et al., 1999; Uutela et al., 2009). The main end products of dopamine
metabolism that are excreted in the urine includes HVA (Homo-vanilic acid), DOPAC (3,4-
dihydroxyphenylacetic acid) and their conjugation metabolites (Swahn & Wiesel, 1976). It is
widely believed that, dopamine metabolites like catecholamine oxidation products can lead to
production of superoxide radicals, deplete glutathione levels and promote the formation of alpha
synuclein formation (Johannes Meiser et al., 2013a; Perez et al., 2002). Neuro-melanin is another
byproduct of metabolism of cytoplasmic dopamine in neurons that can activate microglia mediated
neuro-inflammation and ROS production (Johannes Meiser et al., 2013b; Wilms et al., 2003;
Zecca et al., 2006). These findings indicate the dopamine and its metabolites can accentuate
dopaminergic neurodegeneration in PD.
Basal Ganglia Neuroanatomy
The basal ganglia consist of striatum, pallidum, subthalamic nucleus and substantia nigra
(Groenewegen, 2003). There are two pathways of basal ganglia-thalamocortical circuit.
The direct pathway projects directly from striatum to globus pallidus internus / Substantia nigra
reticulata and it expresses mainly dopamine D1 receptor (Groenewegen, 2003). The indirect
pathway projects from striatum to globus pallidus externus and then to globus pallidus internus /
Substantia nigra reticulata and it expresses mainly dopamine D2 receptor (Groenewegen, 2003).
Generally, the direct pathway is inhibitory whereas the indirect pathway is stimulatory
(Groenewegen, 2003). In fact, direct and indirect pathways act in opposing manner to control the
neurak output from globus pallidus and substantia nigra. The synchronous integration of both the
pathways will help initiation and stoppage of movements (Calabresi et al., 2014). Asynchronous
interaction of direct and indirect pathways such as that occurs in PD results in impairment of start
and stop actions, including bradykinesia, rigidity and gait abnormalities (Calabresi et al., 2014;
6
Dennis W. Dickson, 2012; Ferrer, 2011; G. M. Halliday & McCann, 2010). There are other
functional characteristics of direct and indirect pathways. Mounting evidence suggests that, direct
pathway is associated with reward learning and indirect pathway is involved with aversive
behavior (Hikida et al., 2010). Additionally, activation of direct pathway causes positive
reinforcement whereas activation of indirect pathway leads to punishment (Kravitz et al., 2012).
It is important to know that abnormalities of direct and indirect pathways are seen in other
psyhiatric diseases where dopamine metabolism is altered such as obsessive control disorders and
depression (Wichmann & DeLong, 2011). Dysregulation of direct and indirect pathways due to
dopamine metabolism, can lead to impulse control disorders in PD like pathological gambling,
hypersexuality, shopping and compulsive eating (Voon et al., 2009; G. Zhang et al., 2014). Apart
from PD, other neurodegenerative disorders that are associated with imbalance of direct and
indirect pathways includes Huntington disease (Galvan et al., 2012; Vonsattel et al., 1985). Deep
brain stimulation affords symptomatic relief in PD due to modulation of imbalance of direct /
indirect pathways in PD (DeLong & Wichmann, 2015; Tronnier et al., 2002; Wichmann et al.,
2011). Further studies to characterize the signaling pathways underlying dopamine interactions
with direct and indirect pathways are needed to facilitate the discovery of therapeutic options for
PD.
Lewy body pathology
The pathological hallmark and diagnostic marker of PD includes Lewy bodies which are
intra-neuronal inclusions in the substantia nigra of mid brain (Braak & Braak, 1998; Del Tredici
et al., 2002; Gibb & Lees, 1988; Wakabayashi & Takahashi, 1997). An earlier study, revealed that
Lewy bodies are most prevalent in PD and the brain regions encompass trans-entorhinal, entorhinal
regions, the limbic thalamic nuclei, the anterior cingulated areas, agranular insular cortex, and
7
components of the amygdala (Braak et al., 1998). The other specific brain regions where Lewy
bodies can be located in PD includes hypothalamus, thalamus, cerebral cortex and autonomic
ganglia (Gibb et al., 1988). In PD, the formation of Lewy bodies occurs first in areas like dorsal
vagus-glossophayrngeal complex, locus ceruleus, reticular nucleus, olfactory bulb, olfactory tract
and anterior olfactory nucleus before its spread to the substantia nigra of the mid brain (Del Tredici
et al., 2002). In PD, the systemic sites where Lewy bodies can also be found include sympathetic
nervous system, parasympathetic system, alimentary tract, cardiac plexus, adrenal plexus and
adrenal medulla (Wakabayashi et al., 1997). Lewy body related disorders include Lewy body
dementia, idiopathic Lewy body disease, Parkinson disease, subacute sclerosing pan encephalitis,
ataxia telangiectasia, motor neuron disease and juvenile Parkinson syndrome (Gibb et al., 1988).
The significance of Lewy bodies is difficult to predict as they can be neuroprotective in some
patients whereas in other patients they might represent pre-symptomatic cases that can progress to
full blown PD cases in the near future (Wakabayashi et al., 2007). Pathologically, they are
characterized by intracytoplasmic eosinophilic dark inclusions surrounded by paler halo
structurally composed of abnormally processed neurofilaments (Gibb et al., 1988; Wakabayashi
et al., 2007).
The specific age related prevalence of Lewy body in the general population is as follows
10% in age group 60 -69 years, 12% in age group 70 -79 years, 18% in age group 80 - 89 years
and 16% in age group 90 -99 years (Gibb et al., 1988). The prevalence of finding Lewy bodies
increases gradually from 6th to 9th decade. Mounting evidence suggests that, neural expression
of alpha synuclein leads to Lewy body like inclusions in neocortex, hippocampus and substantia
nigra and is associated with loss of dopamine in basal ganglia and motor abnormalities (Masliah
et al., 2000). It is interesting to know that, mutations of autosomal recessive gene like parkin can
8
cause early onset Parkinson Disease with Lewy bodies (Farrer et al., 2001). The major component
of Lewy body bodies in sporadic PD and Lewy body dementia is mainly alpha Synuclein (Duda
et al., 2000). Accordingly, it is widely believed that, accumulation of Lewy bodies occurs due to
aggregation of poorly degraded alpha-synuclein due to pertubations in autophago-lysosomal
system (ALS), ubiquitin-protesomal system (UPS) and induction of ER stress pathways (Y. Chu
et al., 2009; Mark R. Cookson, 2009; Cuervo et al., 2004; Fujiwara et al., 2002; Smith et al., 2005;
Stefanis et al., 2001).
The ability of alpha-synuclein to form beta pleated structures has been identified as a
critical factor that promotes aggregation and formation of Lewy bodies and Lewy neurites (Vilar
et al., 2008). Phosphorylation, truncation and nitration are the important predisposing factors that
accentuate beta pleated oligomerization and Lewy body formation (Mark R. Cookson, 2009).
Formation of dopamine-alpha-synuclein adducts results in Alpha-Synuclein profibril stabilization,
aggregation and accentuation of dopaminergic neurotoxicity (Conway et al., 2001). Aggregation
of Alpha-Synuclein is the most important factor that leads to formation of inclusion bodies,
accumulation of high molecular weight Alpha-Synuclein and enhanced neurotoxicity (Periquet et
al., 2007). It is interesting to know that serine 129 phosphorylation of Alpha-Synuclein is
neurotoxic and promotes neurodegeneration whereas tyrosine 125 phosphorylation is
neuroprotective and protects against neurodegeneration (L. Chen et al., 2009). Alpha-Synuclein
induced dysfunction of ubiquitin-proteosome system and chaperone mediated autophagy creates a
vicious loop that enhances its own aggregation and subsequent neurodegeneration (Alvarez-Erviti,
Rodriguez-Oroz, Cooper, Caballero, et al., 2010; Snyder et al., 2003). Furthermore, Alpha-
Synuclein causes mitochondrial dysfunction, golgi fragmentation, ER stress and impairs ER-golgi
transport in dopaminergic neuronal cells (Cooper et al., 2006; Devi et al., 2008; Gosavi et al., 2002;
9
Smith et al., 2005). In animal models of PD, it was shown that alpha Synuclein causes activation
of caspase-9 and caspase-3 in substantia nigra of midbrain ultimately leading to neurodegeneration
(L. J. Martin et al., 2006; Yamada et al., 2004). Alpha synuclein also tend to have neuro-
inflammatory effects on the microglia which is associated with the production of pro-inflammatory
mediators, regulation of of NADPH oxidase which ultimately leads to dopaminergic
neurodegeneration (Béraud et al., 2011; Marques & Outeiro, 2012; W. Zhang, Wang, et al., 2005).
Alpha synuclein can be transmitted from cell to cell via prion like mechanism leading to
pathological consequences characterized by cell death and associated neuronal injury (Desplats et
al., 2009; K. Luk et al., 2012; Recasens & Dehay, 2014). In this regard, direct intracerebral
injection of recombinant alpha synuclein into striatum of wild type mice resulted in its gradual
spreading to the neighboring substantia nigra ultimately leading to neuronal loss and behavioral
abnormalities (Desplats et al., 2009; K. Luk et al., 2012; Recasens et al., 2014). Furthermore, alpha
synuclein which is located within the neuronal cells can be transferred to neighboring microglia
and astrocytes and can lead to neuro-inflammation (H. J. Lee et al., 2010; W. Zhang, Wang, et al.,
2005). Alpha synuclein also tends to accumulate in the presynaptic terminals and thus might lead
to defects in neurotransmitter release (Spinelli et al., 2014). Taken together accumulation of alpha
synuclein into proteinaceous deposits known as Lewy body in mid brain region is deleterious and
can lead to progressive neurodegeneration and pathological consequences in PD patients. Studying
the mechanisms that underlie accumulation of these intra cellular inclusions in brain tissues and
developing pharmacological interventions against their formation would improve the survival of
dopaminergic neurons and survival of PD patients.
10
Braak Hypothesis of Parkinson Disease
Alpha Synuclein aggregation and Lewy body inclusions are pathological features of
Parkinson Disease (PD) (Spillantini et al., 1998). Dr Heiko Braak has proposed a hypothesis that
describes mode of spread; with pathology that starts peripherally and sequentially entering central
nervous system (Braak et al., 2003). 51-73% of the PD patients tend to follow the staging system
of Braak regarding initiation, propagation and spread of Lewy body and alpha-synuclein
aggregates into the central nervous system system (Rietdijk et al., 2017). According to Braak
hypothesis, pathological process starts systemically within the gut, spreads through vagus nerve
and olfactory bulb; sequentially entering the substantia nigra (Mid brain) in PD (Braak et al., 2003;
Brooks, 2010; R. E. Burke et al., 2008). In Braak staging, Stages I & II represent early Stage of
disease whereas as stages III, IV, V & VI represent late stages of disease with the presence of
Lewy neuritis and Lewy bodies within the tissues (Braak et al., 2003; Kurt A. Jellinger, 2009). In
Stage I, Lewy body pathology is present within olfactory bulb, vagus nerve and enteric nervous
system (Braak et al., 2003; Brooks, 2010; Kurt A. Jellinger, 2009). Non-motor symptoms like
constipation and loss of smell signifying the involvement of gastrointestinal and olfactory systems
with presence of Lewy bodies is also evidenced (Y. Saito & Murayama, 2012). According to the
dual hit hypothesis, neutrophic pathogen can enter the central nervous system through nasal or
gasterointestinal route thereby leading to pathological changes in the brain (Mulak & Bonaz, 2015;
Visanji et al., 2013). In the nasal route, there is early involvement of anterior olfactory nucleus or
olfactory bulb ultimately spreading to the central nervous system (Chandra et al., 2017; Mulak et
al., 2015; Visanji et al., 2013). In gastric route, the pathogen enters the gastrointestinal system and
localizes in the enteric nervous system (ENS) (Chandra et al., 2017; Mulak et al., 2015; Visanji et
al., 2013). The visceral motor projections of the dorsal motor nucleus of vagus nerve (DMV)
11
synapse with ENS (Chandra et al., 2017; Visanji et al., 2013). So the pathogen spreads retrograde
from ENS through dorsal motor nucleus of vagus nerve into central nervous system (Chandra et
al., 2017; Mulak et al., 2015; Visanji et al., 2013). In central nervous system, the pathology initially
localizes to medulla oblongata (Stage II) and gradually progresses to substantia nigra (Stage III).
(Braak et al., 2003; W. J. Burke, 2003; K. A. Jellinger, 2010). Stages IV, V and VI represent late
Stage of PD with the presence of Lewy body pathology in dorsal motor nucleus of mesocortex /
thalamus, high order neocortex and primary neocortex respectively (Braak et al., 2003; W. J.
Burke, 2003; K. A. Jellinger, 2010). There are few pitfalls that were associated with Braak
hypothesis. It is very highly unlikely that all the patients will have pattern of spread as described
according to Braak staging (Kalaitzakis & Pearce, 2009). It is possible that Lewy body pathology
is absent in dorsal motor nucleus of vagus nerve but still the patient might be presenting with wide
distribution of pathology within the brain (Kalaitzakis et al., 2009). There might be no correlation
between the pathology between the ENS and olfactory bulb which contradicts the dual hit
hypothesis (Lebouvier et al., 2011). Braak hypothesis is based on patterns of neurodegeneration in
different post mortem PD patients at various stages of diagnosis and spread of pathology was not
based in a single case of PD (Koprich et al., 2017). Furthermore, there is no conclusive evidence
that alpha-synuclein pathology undergoes spread during physiological conditions in humans and
is not always associated with onset of symptoms (Walsh & Selkoe, 2016). Additionally, there is
no specific in-vivo marker that can track the progression of disease spread sequentially from
gastrointestinal tract to brain stem (G. Halliday et al., 2012). Only Lewy body pathology is
considered for Braak staging while other earlier events like neuronal loss and microglial activation
were not considered thus creating a selection bias for staging of disease pathology (Milber et al.,
2012; Rietdijk et al., 2017).
12
Cell Death in PD
Broadly, the programmed cell death of dopaminergic neurons can be of three types namely,
apoptotic cell death, autophagic cell death and necrotic cell death (Kostrzewa, 2000; Venderova
& Park, 2012).
Apoptotic Cell Death
Evidence of apoptotic cell death like DNA cleavage, chromatin condensation positive
TUNEL staining, caspase-3 activation caspase-8 activation, TNF-alpha upregulation were
demonstrated in substantia nigra of human PD brains and MPTP treated mice brains (Hartmann,
Troadec, et al., 2001; K. A. Jellinger, 2000; Mochizuki et al., 1996; Mogi et al., 2000; N. A. Tatton
& Kish, 1997; Venderova et al., 2012). Moreover, sometimes apoptotic cell death can be
accompanied with autophagic degeneration in substantia nigra of PD patients (Anglade et al.,
1997; W. G. Tatton et al., 2003).
MPP+ induced apoptotic cell death is the most common form of cell death and is
characterized by positive TUNEL staining, DNA fragmentation, chromatin condensation, caspase
3,8 & 9 activation, endoplasmic reticulum (ER) dysfunction and mitochondrial fragmentation (R.
E. Burke, 2008; Hartmann et al., 2000; Suen et al., 2008; N. A. Tatton, 2000; N. A. Tatton et al.,
1998; Yasuda et al., 2013). Apoptotic cell death is also characteristic of 6-OHDA and alpha-
synulcein induced dopaminergic degeneration in animal models of PD (Saha et al., 2000; Zuch et
al., 2000). In MPTP treated mice, activation of caspase-8, 9& 3 precedes the apoptotic death of
dopaminergic neurons (Mark R. Cookson, 2009; Hartmann, Michel, et al., 2001; Turmel et al.,
2001). In N27 dopaminergic neuronal cells, alpha-synuclein treatment resulted in oxidative stress,
caspase-3 & 9 activation and apoptotic cell death (Chong et al., 2017). In MPTP treated animal
models, excessive BAX (pro-apoptotic protein) activation leads to mitochondrial membrane
13
permeability, excessive ROS production, release of cytochrome C into cytosol leading to apoptotic
cell death of dopaminergic neurons (Hassouna et al., 1996; Venderova et al., 2012; Vila et al.,
2001). MPTP treated mice have decreased expression of Bcl2 (Anti-apoptotic protein) and it’s
over expression affords protection against apoptosis of dopaminergic neurons (Offen et al., 2000;
Vila et al., 2001).
Previous studies support a role for ER stress in PD (Michel et al., 2016). MPP+, rotenone
and 6-OHDA cause ER stress associated apoptosis of dopaminergic neurons (E. J. Ryu et al.,
2002). MPP+ treatment causes ER stress via disruption of calcium metabolism and leads to
apoptotic cell death in human neuroblastoma cell lines (Selvaraj et al., 2012). Disruption of
calcium metabolism leads to apoptosis through pro-apoptotic proteins BAX / BAK and caspase-
12 activation (Nakagawa & Yuan, 2000; R. Sano & J. C. Reed, 2013). Some of the mediators of
ER stress induced apoptosis includes ATF-4, CHOP, GADD34, IRE1 alpha and caspase-12
(Szegezdi et al., 2006). Another study mentions that, ER stress plays a pivotal role in the alpha-
synuclein induced apoptosis and progressive degeneration of dopaminergic neurons in substantia
nigra (Smith et al., 2005). Interestingly, administration of ER stress antagonist protected against
dopaminergic neurodegeneration in alpha-synulcein over expressed mice (Colla et al., 2012b).
Perturbations of the protein degradation machinery namely autophago-lysosomal system
(ALS) and ubiquitin – proteosomal system (UPS) can lead to accumulation of Lewy bodies and
Lewy neurites containing aggregated a-synuclein, a distinctive hallmark of PD which can lead to
oxidative stress and cell death of dopaminergic neurons (Moore et al., 2005; Pan et al., 2008;
Przedborski, 2005; Tan et al., 2009). Formation of inclusion bodies have been believed to have a
deleterious effect on the survival of dopaminergic neurons. It has been shown that, matrix
metalloproteinase-3 (MMP-3) induced cleavage of alpha-synuclein leads to generation of several
14
truncated peptide fragments (1-78, 1-91and 1-93) which accentuates proto-fibril like aggregation
of alpha-synuclein, accumulation of Lewy Bodies and dopaminergic neurotoxicity (D. H. Choi et
al., 2011). It is hypothesized that, alpha synuclein causes downregulation of 14-3-3 chaperones
and upregulation of proapoptotic signals like BAD ultimately leading to apoptotic cell death of
dopaminergic neurons (Yasuda & Mochizuki, 2010).
Necrotic Cell Death
Necrotic cell death is associated with activation of PARP-1 [poly (ADP ribose)
polymersase-1] activation and translocation of AIF (Apoptosis Inducing Factor) from the outer
mitochondrial membrane to the nucleus (Venderova et al., 2012; H. Wang et al., 2003). Migration
of AIF to nucleus is associated with selective degeneration of dopaminergic neurons in substantia
nigra (Burguillos et al., 2011). Nuclear translocation of AIF was previously demonstrated in
ventral mesencephalon of human PD brains (Burguillos et al., 2011; Venderova et al., 2012). Poly
(ADP-ribose) polymerase-1 activation and nuclear AIF translocation were primarily implicated in
the LPS induced dopaminergic cell loss in rat ventral mesencephalon (Burguillos et al., 2011).
PARP activation is responsible for dopaminergic neurodegeneration in MPTP treated rats (Mandir
et al., 1999). Following PARP activation, there is activation of nuclear cell death signaling with
depletion of NAD+ and ATP followed by release of PAR polymers into cytosol and mitochondria
resulting in migration of AIF into the nucleus (S. J. Hong et al., 2004; Y. Wang et al., 2009). PARG
is an important metabolic enzyme that degrades PAR polymers from the cytosol and regulates
PARP induced neuronal cell death (Y. Wang et al., 2009). Previous studies overexpressing PARG
metabolic enzyme resulted in decreased AIF translocation and prevented necrotic cell death (Y.
Wang et al., 2009; Yu et al., 2006). Treatment of MN9D cells (dopaminergic neuronal cell line)
with MPP+ resulted in nuclear translocation of AIF and wide spread DNA fragmentation (C. T.
15
Chu et al., 2005). The predisposing factors that favor nuclear translocation of AIF include
decreased Bcl2 and increased BAX (E. C. Cheung et al., 2005; Cregan et al., 2002).
The mechanistic aspects of programmed cell necrosis includes PARP activation of calpain
followed by BAX upregulation, mitochondrial outer membrane permeabilization (MOMP)
resulting in AIF translocation to the nucleus leading to DNA breaks and chromatin condensation
(S. J. Hong et al., 2004; Moubarak et al., 2007). Previous studies show that, PARP causes
mitochondrial calpain activation through calcium dysregulation and JNK signaling pathway
(Douglas & Baines, 2014; Vosler et al., 2009; Xu et al., 2006). It is important to know that calpain
activation results in mitochondrial release of AIF into nucleus resulting in necrotic cell death
(Polster et al., 2005; Y. Wang et al., 2009). Accumulating evidence suggests that, heat shock
protein (Hsc-70) and Bcl2 are protective factors that delay and impede the migration of AIF into
nucleus preventing necrotic cell death (S. J. Hong et al., 2004; Y. Wang et al., 2009). The proposed
mechanism through which nuclear translocation of AIF induces chromatin condensation and DNA
fragmentation is through recruitment of nucleases and proteases (S. J. Hong et al., 2004). Inhibition
of PARP, calpain, BAX and AIF resulted in neuroprotection against dopaminergic cell loss and
behavioral abnormalities in MPTP mouse models of PD (Burguillos et al., 2011; Crocker et al.,
2003; S. J. Hong et al., 2004; C. Ma et al., 2016; Yokoyama et al., 2010). PARP and AIF are also
implicated in excitotoxic neuronal death induced by excess glutamate levels in ischemia and acute
neurotrauma (E. C. Cheung et al., 2005).
Autophagic Cell Death
Marked reduction of lysosomal markers like LAMP2A and Hsc-70 along with
upregulation of LC3-II levels and accumulation of autophagy vacuoles were demonstrated in
dopaminergic neurons of substantia nigra of PD brains highlighting the involvement of autophagic
16
process in dopaminergic neurodegeneration in PD (Alvarez-Erviti, Rodriguez-Oroz, Cooper,
Caballero, et al., 2010; B. Wang et al., 2016). Autophagic type of cell death in PD is second most
common form of death and is characterized by formation of large cytoplasmic vacuoles and
expression of molecular markers of autophagy (R. A. Nixon & D.-S. Yang, 2012; Venderova et
al., 2012). Mounting evidence suggests that, autophagic cell death is associated with commonly
associated neurotoxins like MPP+, rotenone and paraquat (Dadakhujaev et al., 2010; Niso-Santano
et al., 2011; J. H. Zhu et al., 2007). Alpha-synuclein accumulation or deletion of PD genes like
DJ1, LRRK2, PINK and Parkin can lead to dysfunctional autophagy and dopaminergic cell death
(Gomez-Suaga et al., 2012; McCoy & Cookson, 2011; Scarffe et al., 2014; Winslow &
Rubinsztein, 2011). Alpha-synuclein induced downregulation of chaperone mediated autophagy
leads to excess activation of autophagy resulting in accumulation of autophagosomes and
autophagy-induced cell death (Melinda A. Lynch-Day et al., 2012). Sometimes, autophagy
alterations can also be accompanied with apoptotic cell death in the substantia nigra of the PD
brains (Anglade et al., 1997). Accordingly, in a recent study, MPP+ induced apoptotic cell death
in dopaminergic neuronal cells is associated with activation of autophagy process by decreasing
the interaction of beclin-bcl2 complex (Nopparat et al., 2014). Inhibition of key autophagic
proteins like Atg5, Atg7 & Atg8 has offered neuroprotection against MPP+ induced dopaminergic
neuronal cell death (C. T. Chu et al., 2007). Furthermore, deletion of beclin and Atg16L1 resulted
in decreased accumulation of autophagosomes and offered neuroprotection against protein
aggregate induced neurotoxicity in neurodegenerative disorders like PD (Button et al., 2017).
Alternatively, overexpression of autophagy proteins like LAMP2 resulted in neuroprotection
against alpha-synuclein toxicity (B. Wang et al., 2016; Xilouri et al., 2013).
17
Genetics and PD
PD is a multifactorial disease caused by environmental factors, susceptible genetic factors
and ageing brain (Christine Klein et al., 2012). Although considered rare, these genetic cases
account for few documented cases of reported PD. In sporadic cases of PD, genetic mutations
account for 5-10% of the cases (Pirkevi et al., 2009). In contrast, the incidence of PD in the family
members of documented cases of PD is usually little higher between 10%-30% (von Coelln et al.,
2004). The autosomal dominant genes that was most commonly associated with PD includes alpha
synuclein (SNCA) and Leucine rich repeat kinase (LRRK2) (Christine Klein et al., 2012; Pirkevi
et al., 2009). The autosomal recessive genes that was most commonly associated with PD includes
Parkin, DJ1, PINK1 (PTEN induced kinase-1) and ATPase type 13A (Christine Klein et al., 2012;
Pirkevi et al., 2009). The exact mechanism due to which these genetic mutations lead to occurrence
of PD is not very clear but might be related to induction of specific cell signaling mechanisms.
Parkin
Parkin is cytosolic protein that is mainly located near mitochondrial membrane and it helps
in processing and degrading damaged proteins through ubiquitin proteasome system (von Coelln
et al., 2004). More specifically, it acts as an E3 ubiquitin ligase which attaches ubiquitin to
damaged proteins so that they will be recognized by proteasome complex and then degraded later
(Shimura et al., 2000). Parkin helps in processing alpha synuclein through UPS (ubiquitin-
proteasome system) and prevents the formation of Lewy bodies in PD (Lo Bianco et al., 2004).
Accordingly, parkin associated PD is associated with absence of Lewy bodies (Mori et al., 1998;
Yamamura et al., 1993). Parkin associated PD closely resembles idiopathic Parkinson Disease in
clinical findings although the age of onset relatively earlier with some cases occurring as early as
38-40 years (C. Klein et al., 2000; Lucking et al., 2000). In animal studies, parkin knock out mice
18
show some clinical and behavioral abnormalities which might due to neurodegeneration seen in
PD (Goldberg et al., 2003; Itier et al., 2003).
PINK
PINK is normally a cytosolic protein and gets localized to dysfunctional and depolarized
mitochondria to facilitate their removal through mitophagy (Lazarou et al., 2012). It is important
to know that, PINK migration to depolarized mitochondria leads to activation and recruitment of
parkin to mitochondria for their removal through autophagy (A. W. Greene et al., 2012; Matsuda
et al., 2010; Narendra et al., 2008). Accordingly, loss of PINK physiological function will result
in accumulation of dysfunctional mitochondria thereby leading to cell death. In addition, loss of
PINK1 function leads to abnormalities of mitochondrial morphology and loss of mitochondrial
membrane potential (Exner et al., 2007). Genetic mutations which lead to loss of PINK1 function
leads to clinical phenotype which is very similar to early onset parkin associated PD (Ibanez et al.,
2006). PINK1 knock out studies done in drosophila were associated mitochondrial abnormalities
implying their physiological role in mitochondrial dynamics (Ibanez et al., 2006).
DJ1
Mounting evidence suggests that, in autosomal recessive early onset PD, mutations of DJ1
are least common and are of less clinical significance in neurological practice (Healy et al., 2004).
DJ1 is widely expressed in neurons and glial cells in the brain (Ariga et al., 2013; Bonifati et al.,
2003). It is neuroprotective because, it is involved in physiological functions like mitochondrial
regulation and antioxidative stress reaction (Hao et al., 2010; Hayashi et al., 2009; Junn et al.,
2009; Taira et al., 2004; Yanagida et al., 2009; T. Yokota et al., 2003). Accordingly, genetic
mutations of DJ1 lead to loss of its neuroprotective functions thus making the cells vulnerable to
oxidative stress and cell death (Canet-Avilés et al., 2004). Loss of neuroprotective DJ1 function
19
might render the dopaminergic neurons more vulnerable to environmental toxins like Paraquat and
rotenone in DJ1 drosophila mutants (Meulener et al., 2005). Additionally, DJ1 deficient mice show
increased striatal denervation and dopaminergic neuronal loss emphasizing its neuroprotective
function (R. H. Kim et al., 2005). Finally, another study done on DJ1 null mice showed significant
behavioral abnormalities and alteration of dopaminergic neuronal function (J.-Q. Li et al., 2014).
LRRK2
The physiological functions associated with LRRK2 gene includes neuronal overgrowth,
cytoskeletal maintenance, vesicular trafficking and autophagic protein degradation (Rideout &
Stefanis, 2014). The various mechanisms involved with LRRK2 gene associated PD includes
oxidative stress, mitochondrial dysfunction, synaptic dysfunction, tau phosphorylation and alpha
synuclein accumulation (J.-Q. Li et al., 2014). Most important histopathological features of
LRRK2 gene associated PD includes hyperphosphorylated tau, ubiquitin inclusions and lewy body
neurities (Santpere & Ferrer, 2009). Over expression of LRRK2 gene in drosophila resulted in
behavioral abnormalities and dopaminergic neuronal cell death (Z. Liu et al., 2008). Additionally,
LRRK2 transgenic mice showed substantial behavioral abnormalities with minimal dopaminergic
neurodegeneration (Y. Li et al., 2009).
Pathogenic Mechanisms in PD
Oxidative stress
Parkinson Disease (PD) is a multifactorial disease with wide variety of molecular
mechanisms converging together leading to dopaminergic neurodegeneration. Oxidative stress is
proposed to be one of the most important molecular mechanisms that can potentially attenuate
dopaminergic degeneration in PD. Examination of post mortem brains of PD revealed lipid
peroxidation and GSH (Glutathione) depletion; both signifying the role of oxidative stress in
20
dopaminergic cell death (Hwang, 2013; P. Jenner et al., 1992; Sian et al., 1994). In normal
circumstances, ROS (Reactive Oxygen Species) production within the cell is dissipated by cellular
antioxidant defense mechanisms. Oxidative stress occurs when excess ROS generation is
associated with diminished function of antioxidant enzymes; allowing ROS to accumulate and
ultimately leading to deleterious consequences (Hwang, 2013; John Betteridge, 2000; Sies, 2015).
ROS and RNS can cause DNA fragmentation, lipid peroxidation and protein oxidation ultimately
leading to cell death (Hirsch & Hirsch, 1993; Peter Jenner, 2003).
The external factors that can cause oxidative stress includes parkinsonian neurotoxins like
MPTP, rotenone and paraquat (Arif & Khan, 2010). The internal causative factors that mediate
oxidative stress include alpha-synuclein and genetic PD mutations (PINK1, Parkin, DJ-1&
LRRK2) (Arduíno et al., 2009; Hauser & Hastings, 2013). Notably, the most common molecular
mechanisms that can lead to oxidative stress in PD include, dopamine metabolism, mitochondrial
dysfunction, neuro-inflammation, iron metabolism, calcium metabolism, ubiquitin-proteosome
dysfunction and GSH depletion (Carvalho et al., 2012; Ermak & J.A Davies, 2002; Hwang, 2013;
P. Jenner et al., 1992; J. Meiser et al., 2013)
Dopamine metabolism can yield intermediate products such as DA quinone which can be
detrimental to neuronal survival (Hwang, 2013; C. Zhou et al., 2008). DA quinone can induce
production of superoxide and hydrogen peroxide radicals thereby contributing to oxidative stress.
Mitochondrial dysfunction is the second most important factor contributing to oxidative stress in
PD. Neurotoxins can potentially cause disruption of complex I of ETC (Electron Transport Chain)
thereby leading to ROS accumulation and oxidative stress (Hwang, 2013; Marella et al., 2009;
Perier & Vila, 2012; Winklhofer & Haass, 2010). Mutations of mitochondrial genes (PINK, Parkin
and DJ-1) can cause mitochondrial damage and dysfunctioning of the ETC; both of which can
21
possibly mediate oxidative stress (M. R. Cookson, 2012; Hwang, 2013). Alpha-synuclein,
neuromelanin and MMP-3 are important mediators which are released from degenerating
dopaminergic neurons can stimulate the neighboring microglia to produce ROS and NOS (Nitric
oxide species) ultimately leading to oxidative stress (Hwang, 2013; C. Zhou et al., 2008).
Abnormal iron metabolism is associated with neurodegenerative diseases like PD and there is
excessive iron accumulation in post mortem brain samples of PD patients (Peter Jenner & Olanow,
1998; Perier et al., 2012). The various mechanisms through which iron accumulation can mediate
oxidative stress include dysregulation of mitochondrial function, attenuation of antioxidant
defense mechanism, GSH depletion and lipid peroxidation (Bresgen & Eckl, 2015; Marcio S.
Medeiros et al., 2016; Puntarulo, 2005).
Calcium metabolism is related to mitochondrial function and can be regarded as an
important contributor factor for oxidative stress (Ermak & Davies, 2002; Peng & Jou, 2010). L-
type calcium channels, which are preferentially concentrated in dopaminergic neurons of
substantia nigra can be activated by alpha-synuclein thereby leading to oxidative stress and death
of neurons (Venda et al., 2010).
Pathological aggregation of misfolded and toxic proteins is the one of the important
triggering factor for progression of neurodegenerative diseases like PD (J. Blesa et al., 2015; V.
Dias et al., 2013; Jha et al., 2014). Ubiquitin proteasome system (UPS) mainly functions to remove
unwanted accumulation of oxidized proteins (K.-L. Lim & Tan, 2007). Malfunctioning of
ubiquitin proteasome system can cause accumulation of misfolded, aggregated and oxidized
proteins ultimately leading to excessive ROS production and cell death (Di Domenico et al., 2014).
Recent reports suggest that, UPS dysfunction is followed by mitochondrial dysfunction,
accumulation of ROS and neuronal death (Sunita Maharjan et al., 2014). It is reported that, Lewy
22
body accumulation, impairment of mitochondrial complex I and Parkin gene mutations associated
with PD pathogenesis trigger impairment of proteosome system ultimately resulting in
accumulation of misfolded proteins and oxidative stress (McNaught et al., 2001; Olanow &
McNaught, 2006)
GSH (Glutathione) is generally regarded an antioxidant and mitochondrial protectant in
physiological conditions (Lushchak, 2012). The ratio of reduced glutathione (GSH) to oxidized
glutathione (GSSH) is regarded as indicator of oxidative stress (Zitka et al., 2012). GSH depletion
leads to oxidative stress due to compromise of antioxidant and cellular defense mechanisms and
is commonly associated with Parkinson Disease (Mytilineou et al., 2002). Examination of
postmortem brains revealed depleted GSH levels in substantia nigra of PD patients (Peter Jenner,
2003; Sian et al., 1994). GSH deficiency has been shown be associated with excessive
mitochondrial ROS production, oxidative stress and death of dopaminergic neurons (Peter Jenner
et al., 1998).
Autophagy is a physiological dynamic process that helps in degradation and recycling of
cellular proteins and organelles for maintaining homeostasis within the cell (Hansen & Johansen,
2011; Levine et al., 2011). Autophagy induction is generally used a compensatory defense
mechanism against accumulation of oxidized proteins and dysfunctional mitochondria (Dasuri et
al., 2013). Alpha-synuclein (characteristic feature of PD) has been shown to be associated with
downregulation of autophagy process (Dasuri et al., 2013). Dysregulation of autophagy is usually
associated with the accumulation ROS generated from damaged mitochondria thereby
accentuating oxidative stress and contributing to the demise of dopaminergic neurons in PD
(Dasuri et al., 2013). Caplains are cysteine proteases that are mainly involved in degradation of
cytoskeleton and signaling proteins in the presence of calcium (Dasuri et al., 2013). Caplain
23
activation along with calcium accumulation can lead to impairment of mitochondrial respiratory
function ultimately leading excess ROS production and oxidative stress (Dasuri et al., 2013; V.
Dias et al., 2013).
The ends products or biochemical sequelae of oxidative stress in substantia nigra of PD
patients can include lipid hyper-peroxides, malondialdehyde and 4-hydroxy nonenal (4-HNE)
(Peter Jenner, 2003; P. Jenner et al., 1992; C. Zhou et al., 2008). Various mechanisms by which
these end products potentiate dopaminergic neurodegeneration can include caspase activation,
PARP cleavage, DNA fragmentation, NF-kB inhibition, JNK activation, depletion of
peroxiredoxin-2 (Prx-2), BAX activation and disruption of electron transport chain (Peter Jenner,
2003; C. Zhou et al., 2008)
Therapeutic interventions aimed at oxidative stress might be beneficial in rescuing
dopaminergic neurons from neurodegeneration in PD. Doxycycline has been shown to be effective
in reducing RNS and MMP3 production; thus, offering protection in MPTP model of PD (Hwang,
2013). NQO1 (Quinone reductase) which converts pathogenic quinone into more stable metabolite
has shown therapeutic benefit in MPTP model of PD (Hwang, 2013). Coenzyme Q (CoQ) was
beneficial against oxidative stress in MPTP induced PD model and in double blinded randomized
clinical trials (Henchcliffe & Beal, 2008). The other therapeutic modalities that are effective
against oxidative stress in animal models of PD include dopamine antagonists and creatinine
through improvement of mitochondrial function and restoration of GSH levels (V. Dias et al.,
2013; Henchcliffe et al., 2008).
24
ER stress
The Endoplasmic reticulum (ER) is the largest organelle composed of nuclear envelope
and peripheral ER which are connected with each other through cisternae (Schwarz & Blower,
2016). The major significant functions of ER include protein synthesis, protein folding, post
translational modification, include lipid metabolism, calcium storage and carbohydrate
metabolism (Breckenridge et al., 2003; Lindholm et al., 2006; Paschen & Frandsen, 2001; Rao et
al., 2004; Schwarz et al., 2016). Rough ER (RER) with high concentration of ribosomes works in
conjunction with Golgi apparatus in synthesis of proteins and shuttling proteins to their appropriate
destinations (Chaudhari et al., 2014). Ribosome free smooth ER (RER) ribosomes is mainly
involved in carbohydrate metabolism, lipid metabolism and drug metabolism (Bravo et al., 2013).
Sarcoplasmic reticulum is specialized type of smooth ER that is specifically present in the muscle
cells that is associated with excitation contraction coupling and muscle contraction (Rossi & T.
Dirksen, 2006).
Aberrant ER functions can result in accumulation of misfolded proteins and mounting an
unfolded protein response (UPR) in the ER ultimately leading to pathological consequences
(Breckenridge et al., 2003; Paschen et al., 2001; Rao et al., 2004). Unfolded protein response
(UPR) in the ER includes simultaneous activation of three proteins in the ER membrane namely,
IRE1, PERK and ATF-6 in a three-pronged approach (Cullinan & Diehl, 2004; Oyadomari &
Mori, 2004; Ron & Hubbard, 2008; R. Sano et al., 2013; Yamaguchi & Wang, 2004). Primarily,
IRE1 mediated XBP1 mRNA splicing leads to stimulation JNK pathway and activation of
autophagy (Urano et al., 2000). Secondarily, PERK mediated Elf-2 alpha phosphorylation leads to
increased transcription of antioxidant enzymes for protection against oxidative stress (Cullinan et
al., 2004; DuRose et al., 2006). Lastly, ATF-6 activation by proteolytic activation in Golgi
25
apparatus leads to increased production of ER chaperones which prevent misfolding of proteins in
ER (Renata Sano & John C. Reed, 2013; L. Wu et al., 2013). Short term UPR response helps to
restore normal physiological function of ER whereas long term and sustained UPR response leads
deleterious consequences in the cell (Lindholm et al., 2006). It is interesting to note that, ER stress
stimulates autophagy to promote the clearance of misfolded proteins as a compensatory
mechanism thereby preventing their aggregation and further deleterious consequences (Ogata et
al., 2006). It is hypothesized that, excessive and prolonged UPR response leads to increased
production of ROS, mitochondrial depolarization and subsequent release of pro-apoptotic factors
into cytosol causing deleterious consequences namely cell death (Cao & Kaufman, 2014).
ER stress is implicated in the pathogenesis of neurodegenerative diseases like Parkinson
Disease, Alzheimer Disease and Prion Diseases (Mercado et al., 2015). Accumulating evidence
suggests that, pathological sequelae of ER stress were commonly witnessed in postmortem tissue
analysis of sporadic cases and animal models of PD (Mercado et al., 2015). Activation of UPR
(unfolded response) pathway was shown to be associated with human and cellular models of PD.
Studies had shown that there is activation of PERK-Elf-2 alpha pathway in the substantia nigra of
human PD brains (Hoozemans et al., 2007). Activation of PERK-Elf-2 pathway leads to CHOP
activation, ATF-4 upregulation and apoptosis of dopaminergic neurons in PD (Hoozemans et al.,
2007). Accumulation of ER stress markers including CHOP, IRE and PERK were observed in the
neuronal cultures treated with neurotoxins like MPP+, 6-OHDA and Rotenone (Holtz & O'Malley,
2003; Elizabeth J. Ryu et al., 2002). MPP+ induced dopaminergic neurotoxicity involves
transcriptional activation of ATF-6 and upregulation of ER chaperones via MAPK P38
phosphorylation (Egawa et al., 2010). It is interesting to note that, apoptosis in tunicamycin
induced ER stress in PC12 cells is regulated by combination of signaling molecules including
26
CHOP, AKT, P38 MAPK, TRB3 (Novel target gene of CHOP), FOXO 3a (Important transcription
factor) and PUMA (BH3 only subgroup of Bcl2 family) (C. G. Zou et al., 2009). It should be noted
that, ER stress and disturbance of calcium homeostasis are involved in pathogenesis of Parkinson
Disease (Arduíno et al., 2009). Studies show interaction of genes and ER stress in pathogenesis of
Parkinson Disease. It is proposed that, downregulation of DJ-1 leads to chronic ER stress,
oxidative stress and UPS dysfunction there leading to early onset juvenile Parkinson Disease
(Takanori Yokota et al., 2004). Decreased expression of Parkin leads to accumulation of Pear R
receptor in ER ultimately leading to sustained ER stress and dopaminergic cell death in autosomal
recessive early onset PD (Imai et al., 2001).
Previous reports indicate that, Alpha-Synuclein accumulation leads to the impairment in
processing of ATF-6 in COP-II vesicles through ER-golgi transit ultimately resulting in decreased
associated degradation and pro-apoptotic signaling (Credle, Forcelli, et al., 2015). Aggregation of
Alpha-Synuclein in ER results in sustained and prolonged ER stress induced UPR response thereby
leading to dopaminergic neurodegeneration and neuronal cell death in Alpha- Synuclein induced
PD cases (Colla et al., 2012a; Hugo J. R. Fernandes et al.; Jiang et al., 2010). Furthermore, ER
stress and autophagy dysfunction are together responsible for Alpha-Synuclein accumulation in
independent pleuri-potent stem cell derived dopaminergic neurons from PD patients heterozygous
for GBA-N370S mutation (Hugo J. Fernandes et al., 2016). Additionally, previously published
reports indicate that ER stress and mitochondrial dysfunction can potentiate accumulation of
Alpha-Synuclein in PC12 cell lines (W Smith et al., 2006). ER stress along with impaired clearance
of toxic protein aggregates by ubiquitin-proteosome system contributes to dopaminergic
neurodegeneration (Calì et al., 2011). In paraquat induced PD, there is marked upregulation of
CHOP and GRP-78 along with stimulation of IRE1/ASK/JNK pathway thereby leading to
27
neuronal cell death (W. Yang et al., 2009). Another report reveals that, altered calcium signaling
and inhibition of AKT / mTOR signaling leads to elevated ER stress in neurotoxin induced PD
cases (S et al., 2012). In the rat model of PD, overexpression of ATF-4 resulted in severe
dopaminergic cell loss and neurodegeneration via apoptotic cell death (Gully et al., 2016). Reports
suggest that, over expression of XBP-1 (X-box binding protein) results in dopaminergic neuronal
survival whereas deficiency of XBP-1 leads to sustained ER stress and dopaminergic
neurodegeneration (Valdés et al., 2014). Additionally, over expression of ER chaperone GRP-78
resulted in diminished Alpha-Synuclein toxicity, decreased apoptosis and increased survival of
dopaminergic neurons in rat model of PD (Gorbatyuk et al., 2012).
In 6-OHDA induced PD, basic fibroblast growth factor offered neuroprotection against ER
stress through activation of AKT and ERK ½ pathways (P. Cai et al., 2016). ER stress protective
agents like salubrinol were shown to provide neuroprotection in Alpha-synuclein over expressing
experimental models of PD (Colla et al., 2012a). Methoxyflavones are anti-inflammatory agents
which are present in citrus peel were shown offer protection against ER stress in MPTP induced
mice (Takano et al., 2007). Lastly, Insulin like growth factor also offered protection ER stress
against tunicamycin induced ER stress in PC12 cells through activation of AKT and P38 pathways
(C. G. Zou et al., 2009). Zonasimide (Antiepileptic drug) was proven to afford therapeutic benefit
in human PD by protection against ER stress (Tsujii et al., 2015). In rat model of PD, Telmisartan
and Candesartan celexitil offered protective benefits by protecting dopaminergic neurons against
ER stress (Tong et al., 2016; J. Wu et al., 2007). Taken together, it is likely that neuroprotective
strategies against ER stress would be beneficial in preventing dopaminergic neurodegeneration in
PD.
28
Autophagy
The hallmark of Parkinson Disease is the accumulation of protein aggregates in
dopaminergic neuronal cells which promote cell death through release of toxic intermediates.
Autophagy is a protective mechanism that helps in the clearance of the protein aggregates and
prevents their accumulation which can lead to detrimental outcomes (Eskelinen & Saftig, 2009;
Hansen et al., 2011; Nedelsky et al., 2008). They are three types of autophagy namely
Microautophagy, Macroautophagy and Chaperone mediated Autophagy (Boya et al., 2013).
Macroautophagy involves formation of isolation membrane which engulfs cytoplasmic material
which proceeds through autophagosome, auto-phagolysososome and degradation (A Lynch-Day
et al., 2012). Chaperone mediated autophagy involves receptor mediated uptake of cytoplasmic
contents by lysosome and their subsequent degradation (Kaushik et al., 2011). Microautophagy
involves nonreceptor mediated uptake of cytoplasmic aggregates into lysosomal lumen (W. W. Li
et al., 2012). Autophagic reflux describes the whole process of autophagy which begins with
formation of phagopore in cytoplasm and proceeds through the following stages; Autophagosome
and Autolysosomesome and finally ending with complete degradation of cargo material (Hansen
et al., 2011; Loos et al., 2014; X. J. Zhang et al., 2013).
The mechanism of autophagy is a complex process which includes three steps namely
autophagosome biogenesis, nucleation step and elongation step (Eskelinen et al., 2009; J. Kaur &
Debnath, 2015; Levine et al., 2011). In autophagosome biogenesis step, the ULK complex helps
to initiate the formation of autophagosome (J. Kaur et al., 2015). In the nucleation step, ULK1
complex migrates to the ER membrane and associates with PI-3K complex (Phosphotidylinositol-
3OH Kinase) which eventually get phosphorylated (PtdIns3-P) (Eskelinen et al., 2009; Levine et
al., 2011). Next, in the elongation step, ATG5-12 and LC3 present on the isolation membrane will
29
help in elongation and maturation of autophagosome (Eskelinen et al., 2009; Levine et al., 2011).
Finally, autophagosome gradually fuses with lysosome resulting in formation of auto-
phagolysosome thereby resulting in degradation and clearance of cargo (Eskelinen et al., 2009;
Hansen et al., 2011; J. Kaur et al., 2015; Levine et al., 2011; Loos et al., 2014; X. J. Zhang et al.,
2013). The most important regulatory factors for autophagy includes availability of aminoacids
and cytosolic calcium through TOR protein kinases (Eskelinen et al., 2009). The major
physiological functions of autophagy includes stress response, housekeeping function, innate
immunity, cell death and ageing (Eskelinen et al., 2009). Dysfunction of autophagy is associated
with accumulation of protein aggregation as seen in neuro-degenerative diseases like Parkinson
Disease, Alzheimer Disease and Prion diseases (Eskelinen et al., 2009; Hansen et al., 2011; Loos
et al., 2014; Nah et al., 2015; X. J. Zhang et al., 2013). Neurodegenerative diseases like Huntington
Disease and Alzheimer disease is associated with increased accumulation of autophagic vacuoles
implying activation of autophagic process (Nixon, 2013). It is interesting to note that, short lived
proteins are usually degraded by Ubiquitin Proteasome System (UPS) whereas long lived proteins
are degraded by autophagy (Fuertes et al., 2003; Kraft et al., 2010; D. Li, 2006; Lilienbaum, 2013).
It is believed that, autophagy process becomes stimulated when UPS is overwhelmed and there is
accumulation of misfolded proteins in the cytoplasm (Iwata et al., 2005; Lilienbaum, 2013; Taylor
et al., 2003; Yamamoto et al., 2006). Additionally, it is hypothesized that, impairment of UPS
leads to stimulation of autophagic process to clear the excess aggregation of protein aggregates in
the cytoplasm (Lilienbaum, 2013; Rideout et al., 2014; Yamamoto et al., 2006).
P62 is an important cytosolic protein that is frequently associated with ubiquitin positive
aggregates so that they will be degraded by autophagic process (Bjorkoy et al., 2009; Pankiv et al.,
2007). During oxidative stress, parkin induced ubiquitination of damaged mitochondria causes
30
migration of P62 which ultimately leads to their degradation through autophagic process (M. A.
Lynch-Day et al., 2012; Narendra et al., 2010). It has been reported that, P62 plays an important
role in autophagic degradation of ubiquition tagged cytoplasmic inclusions and Lewy bodies in
Parkinson Disease (Kuusisto et al., 2003; Watanabe et al., 2013; Zatloukal et al., 2002). Recently,
it was reported that, parkin degrades P62 associated with ubiquitinated positive inclusions and
deficiency of parkin leads to accumulation of P62 in dopaminergic neuronal cells and increases
their vulnerability to dopaminergic neurodegeneration (P. Song et al., 2016). Another study
mentions that, depletion of P62 resulted in decreased mitophagy and increased vulnerability to cell
death (Huang et al., 2011).
In paraquat induced PD models, production of ROS and loss of mitochondrial membrane
potential leads to parkin relocation to depolarized mitochondria and subsequent removal
autophagic removal of damaged mitochondria (Mitophagy) (A Lynch-Day et al., 2012). Previous
reports indicate that, autophagic rflux helps in removal of misfolded proteins and relieving ER
stress in neurodegenerative disorders like PD (Y. Cai et al., 2016). Autophagy cannot be
considered always neuroprotective. Chronic and excessive activation of autophagic process could
be detrimental and can be associated with neuronal loss in PD (Bredesen et al., 2006; Chung et al.,
2009).
Autophagy is usually protective mechanism that promotes cell survival through clearance
of aggregate proteins. But excessive activation of autophagy can be detrimental by
causing autophagic cell death (Kroemer & Levine, 2008). Excessive activation of autophagy can
engulf massive amount of cytosol and organelles leading cellular demise (Maiuri et al., 2007). In
other circumstances, excessive autophagy activation can degrade key antioxidant enzymes like
catalase which ultimately results in cell death (Maiuri et al., 2007). Previous reports have shown
31
that, over expression of ATG can lead to excessive activation of autophagy and cell death (R. C.
Scott et al., 2007). Autophagic cell death is defined as cell death accompanied by excessive
accumulation of autophagic vacuoles within the cell in the absence of chromatin condensation
(Kroemer et al., 2008). Reports indicate that, there is excess accumulation of autophagosomes with
co-localization of of LC3-II with Lewy bodies and alpha-synuclein inclusions in brain of PD
patients as compared to normal population (Alvarez-Erviti, Rodriguez-Oroz, Cooper, & et al.,
2010; Z. H. Cheung & Ip, 2009). Anticancer treatments often cause excessive activation of
autophagy ultimately leading to autophagic cell death of tumor cells (Shen et al., 2011).
Inflammasome
NLRP3 inflammasome is an intracellular multi-meric protein complex composed of
three proteins namely, NLR containing N-terminal pyrin domain, apoptosis associated speck like
protein containing CARD (ASC) and pro-caspase-1 (Haitao Guo et al., 2015; Schroder & Tschopp,
2010). Upon NLRP3 complex formation, it leads to its activation via caspase-1 mediated cleavage
of pro-inflammatory cytokines into active IL-1 beta and IL-18. Secreted pro-inflammatory
cytokines mount an innate immune response as a defense mechanism (Haitao Guo et al., 2015;
Kate Schroder et al., 2010). Their assembly usually occurs upon stimulation of external stimuli
by wide variety of pathogens, environmental irritants, toxins and PAMPs (Pathogen associated
molecular patterns) (Haitao Guo et al., 2015; Kate Schroder et al., 2010). The most common
microorganisms that are hypothesized to induce NLRP3 inflammasome activation include candida
albicans, saccharomyces cerevisiae, Listeria monocytogenes, Staphylococcus aureus, adenovirus
and Influenze virus (Latz et al., 2013). Inflammasomes are implicated in initiation and progression
of neurodegerative disorders like Alzheimer Disease and Parkinson Disease (Freeman & P.-Y.
32
Ting, 2015). Using experimental models of PD, previous studies reported NLRP3 inflammasome
activation as a potential mechanism in PD associated neuro-inflammatory response (Lamkanfi et
al., 2010; Mao et al., 2017; Mustafa et al., 2016a).
There are two types of inflammasome namely, canonical inflammasome which leads to
activation of procaspase-1 whereas non-canonical leads to activation of procaspase-11 (Lamkanfi
et al., 2010). Both types of inflammasome leads to production of pro-inflammatory cytokines like
IL-1 beta and IL-18. Canonical type of inflammasome is activated in a 2 step mechanism. The
two steps for NLRP3 inflammasome activation include, priming and activation. The priming step
involves NF-kB activation which results in production of pro-IL-1 beta which is a pre-requisite for
the activation step (E. I. Elliott & F. S. Sutterwala, 2015; Haitao Guo et al., 2015; Lamkanfi et al.,
2010; Kate Schroder et al., 2010). The underlying mechanisms that are responsible for priming
step includes mainly, IRAK1 activation, ERK activation and spleen tyrosine kinase dependent
ASC phosphorylation (E. I. Elliott et al., 2015). Next, activation step involves the assembly of
NLRP3 scaffold, ASC adaptor and caspase-1 together to form a multimeric protein complex that
is capable is activating pro-IL-1 beta and Peo-IL-18 to active IL-1 beta and active IL-18
respectively (E. I. Elliott et al., 2015; Haitao Guo et al., 2015; Lamkanfi et al., 2010; Kate Schroder
et al., 2010).
Non canonical-inflammasome is usually activated by gram negative bacteria due to
the presence of Lipopolysaccharide (LPS) in their plasma membrane (P. Broz & Monack, 2013).
Gram negative bacteria engulfed by macrophages can trigger immune response via caspase-11
dependent pyroptosis (P. Broz et al., 2013; Ding & Shao, 2017; Pellegrini et al., 2017). Some of
the important functions of non-canonical inflammasome includes clearing of bacterial infections,
spread of infection, sepsis and activation of canonical inflammasome (P. Broz et al., 2013; Ding
33
et al., 2017; Pellegrini et al., 2017). Non-canonical inflammasome is activated via three step
process (Ding et al., 2017; Latz et al., 2013). In the first step, LPS and IFN-gamma induced
expression of pro-caspase-11 through NF-kB pathway (Petr Broz & Dixit, 2016; Ding et al., 2017;
Latz et al., 2013; V. A. Rathinam & Fitzgerald, 2016). In the second step, lipid A component of
the LPS binds to pro-caspase-11 and promotes oligomerization and activation of caspase-11 (Ding
et al., 2017). In the third step, activated caspase-11 leads to cleavage of gasdermin D (GSDMD)
resulting its migration to the plasma membrane and formation of pores resulting in osmotic lysis
(Petr Broz et al., 2016; Ding et al., 2017; Kayagaki et al., 2015; Latz et al., 2013; V. A. Rathinam
et al., 2016; Shi et al., 2015). Non-canonical inflammasome might be specifically associated with
production of pro-inflammatory cytokines like IL-1 alpha an HMGB1 (Lamkanfi et al., 2010).
Some of the important functional roles of non-canonical inflammasome include cell death,
cytokine maturation, actin remodeling, enterocolitis, renal fibrosis and clinical sepsis (Diamond et
al., 2015; Lorenz et al., 2014).
Both types of inflammasome activation ultimately results in pyroptosis. Pyroptosis is
caspase-1 mediated cell death and is regarded as an important hallmark of inflammasome
activation in tissue damage (W. Chang et al., 2013; Lamkanfi et al., 2010). The most important
distinguishing features include loss of cell membrane, DNA fragmentation, water influx, cellular
swelling and release of pro-inflammatory content into surrounding areas to mount an inflammatory
response (W. Chang et al., 2013; Lamkanfi et al., 2010).
Different models proposed to inflammasome activation
A wide variety of host derived factors can activate the NLRP3 inflammasome including
ATP, Asbestos, Silica, uric acid and amyloid (Halle et al., 2008; L Cassel et al., 2008; Sanjeev
34
Mariathasan et al., 2006; Martinon et al., 2006). Phosphorylation of ASC adaptor protein is the
most important factor leading to inflammasome activation (H. Hara, K. Tsuchiya, I. Kawamura,
R. Fang, E. Hernandez-Cuellar, Y. Shen, J. Mizuguchi, E. Schweighoffer, V. Tybulewicz, et al.,
2013). The most common models that are proposed for NLRP3 activation step includes the
following, K+ efflux, Lysosomal damage, ROS production, ER stress, calcium signaling and
mitochondrila damage (Cristiane M. Cruz et al., 2007a; Catherine Dostert, Virginie Pétrilli, Robin
Van Bruggen, Chad Steele, Brooke Mossman, et al., 2008; Halle et al., 2008; Hornung et al., 2009;
T.-D. Kanneganti et al., 2006; L Cassel et al., 2008).
K+ efflux
Extracellular ATP, pore forming toxins, Nigericin and MSU crystals are the most important
triggering factors for K+ efflux model of NLRP3 inflammasome activation (E. I. Elliott et al.,
2015; Haitao Guo et al., 2015; Lamkanfi et al., 2010; Kate Schroder et al., 2010). Activation of
P2X7 purinergic receptor by ATP results in increase in potassium efflux and decrease in
intracellular K+ (Abais-Battad et al., 2014). Additionally, ATP can also induce pore formation
through Pannexin-1 which mediates inflammasome activation and release of proinflammatory
cytokines (Pelegrin & Surprenant, 2006).
Lysosomal Damage
Particulate structures like silica, alum, amyloid and monosodium urate are engulfed by
lysosomes which is followed by lysosomal rupture and release of cathepsin-B into cytosol thereby
leading to inflammasome activation (E. Elliott & F. Sutterwala, 2015; Haitao Guo et al., 2015;
Lamkanfi et al., 2010; Kate Schroder et al., 2010). Accordingly, use of acidificaton inhibitors
which neutralize the acidification of lysosomal PH decrease the release of lysosomal cathepsin-B
and inflammasome activation (Hornung et al., 2009). Lysosomal calcium metabolism is also
35
involved in stabilization of pro-IL-1 beta mRNA synthesis; thus, emphasizing its role in regulation
of assembly and activation of inflammasome (Kassandra Weber & Joel D. Schilling, 2014).
Lysosomal membrane permeabilization (LMP) together with release of cathepsin-B and NADPH
oxidases (NOXs) can cause mitochondrial destabilization, thereby leading to mitochondrial ROS
production and NLRP3 inflammasome activation (Heid et al., 2013). In PD disease models, alpha-
synuclein aggregates induced production of cathepsin-B is responsible for triggering NLRP3
inflammasome activation (Codolo et al., 2013; Haitao Guo et al., 2015).
Reactive Oxygen Species
In the third model, ROS production is implicated in NLRP3 inflammasome activation.
Mitochondrial and non-mitochondrial ROS generation has been implicated in NLRP3
inflammasome (Catherine Dostert, Virginie Pétrilli, Robin Van Bruggen, Chad Steele, Brooke T.
Mossman, et al., 2008; K. Schroder et al., 2010; R. Zhou et al., 2011). The source of major ROS
production is usually mitochondria with minor contribution from NADP oxidases (Y. He et al.,
2016; Rodgers et al., 2014; Kate Schroder et al., 2010). Co-localization studies demonstrate that,
there is increased migration of NLRP3 near to damaged ROS generating mitochondria upon
treatment with inflammasome stimulants thus highlighting the importance of mitochondrial ROS
in NLRP3 inflammasome activation (Rodgers et al., 2014; R. Zhou et al., 2011). Complex I
inhibitors like rotenone leads to mitochondrial membrane depolarization, ROS production, NLRP3
inflammasome activation and pro-inflammatory cytokine production (Won et al., 2015; R. Zhou
et al., 2011). Accordingly, ROS inhibitors efficiently block the NLRP3 inflammasome activation
in rotenone treated cells (R. Zhou et al., 2011). Alternatively blockage of mitophagy leads to
accumulation of damaged ROS producing mitochondria and sustained NLRP3 inflammasome
activation (C. S. Shi et al., 2012). Voltage dependent calcium channels (VDCC), which are the
36
important channels of communication between ER and mitochondria for exchange of metabolites
and ions play an important role in mitochondrial ROS production and NLRP3 inflammsome
activation (R. Zhou et al., 2011). Inhibition of voltage dependent calcium channels resulted in
decreased caspase-1 activation and reduced IL-1 beta secretion (Rodgers et al., 2014; R. Zhou et
al., 2011). Additionally, phagocytosis of asbestosis and silica particles can lead to activation of
NADPH oxidase complex in the plasma membrane which triggers ROS production and ultimately
NLRP3 inflammasome activation (Catherine Dostert, Virginie Pétrilli, Robin Van Bruggen, Chad
Steele, Brooke T. Mossman, et al., 2008). Moreover, in ATP treated macrophages elevated
intracellular calcium can lead to enhanced ROS production and NLRP3 inflammasome activation
(Cristiane M. Cruz et al., 2007b). ROS can lead to loss of mitochondrial membrane potential and
opening of mitochondrial permeability transition pore causing the release of mitochondrial DNA;
a potent inducer of NLRP3 inflammsome activation (E. Elliott et al., 2015). Mitochondrial ROS
can also lead to lysosomal damage resulting in increasing in lysosomal membrane permeabilizaton
(LMP), release of lysosomal proteases and inflammasome activation (Heid et al., 2013). In PD
disease models, alpha- Synucluein aggregates induced production ofROS is responsible for
triggering NLRP3 inflammasome activation (Codolo et al., 2013; Haitao Guo et al., 2015).
Autophagy Dysfunction
Autophagy and inflammasome regulate each other thus playing a mutually important role
in disease progression and pathogenesis of many inflammatory diseases (Martins et al., 2015; S.
Qian et al., 2017). Autophagy induction is one of the main cellular defense mechanism that helps
in limiting and destruction of NLRP3 inflammasome assembly and activation (Harris et al., 2017;
S. Qian et al., 2017; Tatsuya Saitoh & Shizuo Akira, 2016; C.-S. Shi et al., 2012; Zhenyu Zhong
et al., 2016). The various mechanisms through which autophagy suppresses NLRP3
37
inflammasome activation includes removing mitochondrial DAMPs, degrading inflammasome
complexes and negatively regulating IL-1 beta synthesis, release and assembly (S. Qian et al.,
2017). Accumulation of damaged mitochondria during NLRP3 inflammasome activation leads to
compensatory upregulation of mitophagy and increased synthesis of P62 (M. J. Kim et al., 2016).
Migration of P62 to parkin associated depolarized mitochondria will ultimately facilitate their
removal by autophagy process (M. J. Kim et al., 2016; S. Qian et al., 2017; Tatsuya Saitoh et al.,
2016; Zhenyu Zhong et al., 2016). Similarly, P62 molecule migrates to ubiquitinated ASC proteins
thereby resulting in their destruction through autophagy process (S. Qian et al., 2017; Tatsuya
Saitoh et al., 2016). Interestingly, ubiquitinated NLRP3 proteins are made inactive until they are
deubiquitinated by BRCC3 enzyme but they are not targeted by autophagosomes for destruction
(Py et al., 2013; Rodgers et al., 2014). NLR proteins like NOD2 and NLRX1 interact with
autophagy proteins like Atg5-12 and Atg16 thus favoring autophagosome formation and clearance
of inflammatory proteins (Homer et al., 2012; Lei et al., 2012; Rodgers et al., 2014). In a similar
fashion, autophagy inducer sestrin2 (SESN2) protein is also upregulated and promotes removal of
damaged mitochondria during inflammasome activation (M. J. Kim et al., 2016). Non-canonical
inflammasome components, caspase-11 can also promotes fusion of lysosomes and phagosomes
and will aid in clearance of bacteria during infections (S. Qian et al., 2017). Administration of
autophagy inducers like rapamycin leads to recruitment of pro-IL-1 beta in autophagosomes and
subsequent degradation (S. Qian et al., 2017). Administration of autophagy inhibitors like
revasterol and 3-MA can lead increased mitochondrial ROS production and IL-1 beta production
(Y. P. Chang et al., 2015; S. Qian et al., 2017; Rodgers et al., 2014). Similarly loss of autophagy
proteins like LC3, beclin and Atg5 can lead to increase caspase-1 activation and IL-1 beta
production in macrophages (Nakahira et al., 2011; S. Qian et al., 2017; R. Zhou et al., 2011).
38
Capase-1 activation can result in feedback inhibition of autophagy through cleavage of parkin thus
amplifying the inflammasome activation (Alvarez-Arellano et al., 2018; S. Qian et al., 2017; J. Yu
et al., 2014). NLRP3 inflammasome assembly can lead to dysfunctional of autophagy process via
downregulating PINK1 (S. Qian et al., 2017; Y. Zhang et al., 2014). Lysosomal cathepsin-B can
trigger autophagy dysfunction and NLRP3 inflammasome activation in manganese treated BV2
microglia (Diya Wang et al., 2017). Obviously, autophagy dysfunction leads to accumulation of
depolarized mitochondria and damaged ROS producing mitochondria, release of oxidized
mitochondrial DNA into cytosol thereby leading to NLRP3 inflammasome activation in microglia
(E. Elliott et al., 2015; Harris et al., 2017; Tatsuya Saitoh et al., 2016; C.-S. Shi et al., 2012).
ER Stress
ER stress can cause activation of NLRP3 inflammasome assembly and activation through
various mechanisms. ER stress proteins PERK and IRE1 alpha leads to upregulation of CHOP
ultimately leading to NLRP3 inflammasome activation (D. N. Bronner et al., 2015; E. I. Elliott et
al., 2015; Lebeaupin et al., 2015b). Furthermore, ER stress can lead to activation of thioredoxin
interacting protein (TXNIP) which triggers NLRP3 inflammasome activation and secretion of pro-
inflammatory cytokines (Lerner et al., 2012; J. Song et al., 2015; Yoshihara et al., 2014).
Moreover, ER stress induced cleavage of caspase-2 and release of pro-apoptotic factor Bid can
trigger mitochondrial ROS production and NLRP3 inflammasome activation (D. N. Bronner et al.,
2015). Additionally, as ER is a major reservoir of intracellular calcium, it plays a major role in
regulating intracellular calcium levels, calcium signaling, NLRP3 inflammasome assembly and
activation (Yuan He et al.; G. S. Lee et al., 2012). Another study mentions that, deficiency of
uncoupling protein (UCP) in astrocytes leads to exacerbation of ER stress and accompanying
NLRP3 inflammasome activation (M. Lu et al., 2014). Alternatively, ER stress can disrupt the
39
physiological steady state communication between ER and mitochondria thereby triggering
mitochondrial dysfunction, enhanced ROS production and NLRP3 inflammasome activation
(Menu et al., 2012a). Lastly, excessive activation of voltage dependent anion channels (VDCC)
which are major source of communication between between endoplasmic reticulum and
mitochondria results in increased ROS production and inflammasome activation in microglial
cells (R. Zhou et al., 2011).
NLRC4
Gram negative Bacteria like salmonella typhimurium, Pseudomonas Aeruginosa,
Kelbsiella pneumonia, shigella and Bukholderia pseudomammei triggers NLCR4 dependent
caspase-1 activation and pyroptosis as an innate immune response (Duncan & Canna, 2018;
Hafner-Bratkovic, 2017). NLCR4 is related to clinical syndromes such as enterocolitis, auto-
inflammation and macrophage activation syndrome (Duncan et al., 2018). NLCR4 plays an
important role for mounting an immune response against systemic and enteric pathogens (Duncan
et al., 2018). Release of bacterial proteins (flagellin and type III secreted system) into cytosol
occurs as a result of fusion of bacteria-containing phagosomes with lysosomes (Latz et al., 2013;
Vijay A. K. Rathinam et al.). Both intracellular and extra cellular bacterial flagellin and T3SS
trigger immune response via NLCR4 inflammasome pathway (Zhao & Shao, 2015). Needle and
rod proteins of flagellin and Type III secreted system (T3SS) released into cytosol will be detected
by NAIP proteins (NBD domain containing inhibitor of apoptosis proteins) (Hafner-Bratkovic,
2017). Phosphorylation of the NLCR4 proteins leads to activation and enables it to interact with
NAIP proteins (Duncan et al., 2018). These bacterial ligands along with NAIP proteins bind to
NLRC4 proteins and attract other dormant NLRC4 molecules ultimately leading to formation of
NLRC4 inflammasome (Haitao Guo et al., 2015). NLRC4 inflammasome leads to formation strong
40
and robust immune response against bacterial pathogens (Hafner-Bratkovic, 2017). Another
unique feature of NLCR4 is that it can interact with pro-caspase-1 even in the absence of ASC
protein (Apoptosis speck containing a CARD) (Duncan et al., 2018). NLRP4 inflammasome
differs from NLRP3 inflammasome in that it does not require priming for activation (Denes et al.,
2015; Luigi Franchi et al., 2006). The role of ASC speck in NLCR4 inflammasome includes, pro-
caspase-1 cleavage, IL-1 beta and IL-18 maturation and neutrophil migration (Hafner-Bratkovic,
2017). Additionally, NLCR4 inflammasome activation is capable of producing pro-inflammatory
prostaglandins and leukotrienes via activation of arachidonic acid metabolism (Duncan et al.,
2018; von Moltke et al., 2012). The end target effects of NLCR4 inflammasome activation includes
pyroptosis, apoptosis, expulsion, eicosanoid secretion and pro-inflammatory cytokine secretion
(Duncan et al., 2018; Hafner-Bratkovic, 2017). The main functional significance of NLCR4
includes mounting an immune response against enteric, oral mucosal and lung pathogens (Cai et
al., 2012; L. Franchi et al., 2012; Tomalka et al., 2011). A growing body of evidence suggests that,
NLCR4 induced systemic inflammatory response following enteric pathogens can lead to ischemic
brain injury in mice (Denes et al., 2015). Recent studies suggest that, NLCR4 inflammasome is
associated with accentuating breast cancer progression and attenuating melanoma progression
(Janowski et al., 2016; Kolb et al., 2016). The other functions of NLRC4 includes actin
reorganization and infected epithelial cell expansion (Hafner-Bratkovic, 2017).
Microglia and Neuro-inflammation in PD
Under physiological conditions, normal microglia are present in resting or quiescent state
in healthy human brain (S. U. Kim & de Vellis, 2005; Q. Li & Barres, 2017; Wolf et al., 2017).
Microglial cells are regarded as first line of defense in response to injury, infection or toxin
41
exposure (S. U. Kim et al., 2005; Q. Li et al., 2017; Wolf et al., 2017). Slight disturbance in CNS
homeostasis will alert the resting microglial cells, which will proliferate and migrate to the site of
injury to destroy and remove the pathogens or damage associated molecules (S. U. Kim et al.,
2005; Q. Li et al., 2017; Wolf et al., 2017). They secrete chemokines and cytokines as well as
nitric oxide and reactive oxygen species to mount an immune response to remove the damaged
cell (S. U. Kim et al., 2005; Q. Li et al., 2017; Wolf et al., 2017). Uncontrolled and excessive
microglial activation can be deleterious to neighboring neurons and can lead to pathologic
consequences (S. U. Kim et al., 2005; Q. Li et al., 2017; Wolf et al., 2017). Oxidative stress,
proteosomal dysfunction and mitochondrial dysfunction have been implicated in the pathogenesis
of PD (J. L. Eriksen et al., 2005b). Activation of microglia is regarded as a histological hallmark
of brain pathology (Dheen et al., 2007). Microglial activation, production of pro-inflammatory
cytokines and neuro-inflammation are regarded as most common predisposing factors in
pathogenesis of neurodegenerative diseases like PD (Y. S. Kim & Joh, 2006; Sudhakar R.
Subramaniam & Howard J. Federoff, 2017). Prolonged deleterious crosstalk between reactive
microglia and neurons can ultimately lead to neuronal damage through release of pro-
inflammatory cytokines and reactive oxygen intermediates (Dheen et al., 2007; Wolf et al., 2017).
Activated microglia exhibit morphological changes consistent with transition from
neuroprotective to neurotoxic subsets and ultimately progression of dopaminergic
neurodegeneration in Parkinson’s disease (Sawada et al., 2006). In neuropsychiatric lupus, Tweak
stimulation of microglia resulted in increased expression of pro-inflammatory cytokines like TNF,
IP-10, MMP-9 and iNOS 4-6 fold as compared to isotype control (Stock & Putterman, 2014).
There is substantial evidence that, pharmacological blocking of Tweak / Fn14 pathway has
afforded protection in multiple models of neuro-inflammation (T. Dohi & L. Burkly, 2012). The
42
potential causative factors those are responsible for microglial neuro-inflammation in PD are not
very known. Activated microglia, T-lymphocyes and pro-inflammatory markers are present in the
substantia nigra of PD patients (Brochard et al., 2009; Hirsch et al., 2012; Sudhakar R.
Subramaniam et al., 2017). Additionally, endogenous pathogenic antigens like alpha synuclein
can lead to activation of adaptive immunity as evidenced by the presence of CD8+ and CD4+
lymphocytes in the brain of PD patients (Brochard et al., 2009; Hirsch et al., 2012). Chronic
microglial activation leads to the production of pro-inflammatory cytokines like TNF-alpha, IL-1
beta, IL-6 and IFN gamma which eventually leads to dopaminergic neurodegeneration in PD (J.
L. Eriksen et al., 2005b; Carina Cintia Ferrari et al., 2006; McCoy et al., 2006). The intracellular
signaling pathways that are responsible for microglial activation and pro-inflammatory cytokine
production includes MAPK (Mitogen activated protein kinase) pathways (Kiernan et al., 2016). It
is interesting to note that, polymorphisms of genes like TNF-alpha, IL-1 beta and IFN gamma were
associated with increased risk of developing PD (Hirsch et al., 2012). Accumulating evidence
suggests that, autosomal recessive PD associated genes like LRRK2, Parkin, Pink, DJ-1 are also
associated with microglial activation and neuro-inflammation in PD (J. L. Eriksen et al., 2005b).
Neurotoxic factors like alpha-synuclein, MMP-3 and ATP released from degenerating neurons can
amplify the neuro-inflammatory response and thus can continuously accentuate the cycle of
dopaminergic neurodegeneration in PD (George et al., 2015; Y.-S. Kim et al., 2007; W. Zhang,
Wang, et al., 2005). Accumulating evidence suggests that there are two microglial phenotypes that
can influence the process of neuro-inflammation in PD (Sudhakar R. Subramaniam et al., 2017).
Microglial phenotype (M1) is pro-inflammatory or disease causing phenotype and their activation
leads to production of pro-inflammatory cytokines like TNF-α, IL-1β, IL-6, IL-12, NOS and
reactive oxygen species which can accelerate the pro-inflammatory response in PD (Pisanu et al.,
43
2014; Sudhakar R. Subramaniam et al., 2017). Microglial phenotype (M2) is anti-inflammatory or
neuroprotective phenotype and their activation leads to production of anti-inflammatory cytokines
like IL-4, IL-13, IL-10, and TGF-β which can offer neuroprotection in PD (Pisanu et al., 2014;
Sudhakar R. Subramaniam et al., 2017). In addition, peripheral immune cells like macrophages
can migrate to the brain parenchyma via a breach in BBB and can lead to the generation of
cytokines like IL-1β, TNF-α and IL-6 (Blatteis, 1992). Blood brain barrier disruption and
endothelial dysfunction have been implicated in PD (Kortekaas et al., 2005). As a result, peripheral
macrophages and lymphocytes can enter the brain parenchyma and initiate neuro-inflammatory
response thus contributing to the pathogenesis of dopaminergic neurodegeneration in PD
(Kortekaas et al., 2005). In this context, immune response mounted by peripheral infections has
been regarded as a risk factor for development and progression of PD (Bu et al., 2015).
The various mechanisms through which microglia induced neuro-inflammation causes
oxidative stress in neurons includes production of Reactive oxygen species (ROS),
cyclooxygenase (COX) and lipoxygenase pathways (Tansey et al., 2007). Prostaglandins produced
by the microglial COX-2 enzymes induce oxidative stress in neurons by perturbation of
intracellular signaling mechanisms like decrease in mitochondrial membrane potential, decrease
in GSH activity, excessive generation of lipid peroxidation products and regulation of pro-
inflammatory cytokine production (Blom et al., 1997; Tansey et al., 2007). Eicosanoids produced
by the microglial lipo-oxygenase enzymes produce oxidative stress in neurons through production
of peroxide radicals and activation of programmed cell death pathways (Maccarrone et al., 2001).
Neuron-microglia interactions have been shown to regulate in microglial activation and neuro-
inflammation in the brain. In this regard, some of protein fragments released from degenerating
neurons like neuromelanin (NM) will enhance the microglial phagocytic activity against dying
44
dopaminergic neurons facilitating their own removal. Some of the immunomodulators secreted by
the neuronal cells like CX3CL1, CD200, CD22, CD47, CD95 and neural cell adhesion molecule
(NCAM), bind to and help to maintain microglia in their resting state (Hoek et al., 2000; Mott et
al., 2004; Numakawa et al., 2004; Sheridan & Murphy, 2013). Deficiency or dysfunction of these
immunomodulators results in conversion of resting microglia to activated microglia thus leading
to neuro-inflammatory response and dopaminergic neurodegeneration in PD.
Since microglial activation is considered one of the important contributing factor for
dopaminergic neuronal death, a wide variety of pharmacological interventions have been assessed
to rescue the dopaminergic neurons from cell death. Accumulating evidence suggests that, PPAR
gamma (peroxisome proliferator activated receptor) agonists and glucocorticoids receptor agonists
can protect against microglial activation, neuro-inflammation and dopaminergic
neurodegeneration in animal models of PD (Nolan et al., 2013). Additionally, minocycline (Broad-
spectrum antibiotic) has been effective in offering protection against dopaminergic neuronal cell
death owing to its ability to reduce blood brain barrier leakiness (BBB), microglial activation,
leucocyte migration and TNF upregulation (Ryu & McLarnon, 2006; Tansey et al., 2007;
Wasserman & Schlichter, 2007; Yong et al., 2004). Furthermore, NSAIDS (Non-Steroidal Anti-
inflammatory Drugs) like ibuprofen can potentially decrease the PD risk in the population by 20%-
30% probably due to their various anti-inflammatory effects (N. P. Rocha et al., 2015). The various
potential mechanisms through which NSAIDS act are through blocking PGE2 production (Blom
et al., 1997), suppressing glutamate excitotoxicity (Pasinetti, 1998), reduction of free radical
production (Lehmann et al., 1997), activation of PPAR gamma receptors (Lehmann et al., 1997;
Ricote et al., 1998) and attenuating oxidative stress (Kimura, 1997). Finally, antibiotic drug like
rifampicin reduces rotenone induced inflammation through suppressing NLRP3 inflammasome
45
activation, microglial activation and neuro-inflammation (Y. Liang et al., 2015). Further research
should be done to characterize the detrimental effects of microglia on neuronal survival in animal
models to facilitate the identification of new molecular targets.
Astrocytes
Astrocytes are glial cells and are related to physiological and pathological functions in the
central nervous system. They perform many functions like neuronal development, CNS blood
flow, transmitter homeostasis, synapse formation, metabolism and blood brain barrier maintenance
(Sofroniew & Vinters, 2010). In case of CNS injury, astrocytes are involved in neuroprotection,
blood brain barrier repair, reducing vasogenic edema and limiting the spread of infection to
surrounding areas in the central nervous system (Sofroniew et al., 2010). Astrocytes are associated
activation of Parkinsonian neurotoxin MPTP into active form MPP+ through mono-amino
oxidases (Przedborski et al., 2000). Astrocyte dysfunction due to mutated familial PD genes can
lead to impairment in inflammatory response, lipid handling, mitochondrial health and lysosomal
function (Booth et al., 2017). DJ-1 and parkin mutations can lead to astrocyte dysfunction and
neuronal death via mitochondrial stress and ER stress respectively (Phatnani & Maniatis, 2015).
Reactive astrogliosis has been evidenced in many regions like substantia nigra, putamen and
hippocampus in LPS and MPTP animal model of PD (Cabezas et al., 2014). Additionally, excess
alpha-synuclein accumulation in astrocytes can lead to impairment of their physiological functions
thereby accentuating dopaminergic neurotoxicity in PD (Gu et al., 2010). Activated astroglial cells
can secrete pro-inflammatory cytokines like TNF-alpha, IL-1 beta and IL-6 which can potentiate
dopaminergic neuronal death (Rappold & Tieu, 2010). Activated astrocytes can also cause
microglial activation which can ultimately result in pro-inflammatory cytokine production and
dopaminergic neurodegeneration in mid brain (Gu et al., 2010). Overexpression of Nrf2 in
46
astrocytes has been shown to afford protection against MPTP induced neurodegeneration in animal
model of PD (P. C. Chen et al., 2009).
Animal Models of PD
LPS Animal Model
Microglial activation and neuro-inflammation are recognized as key contributing factors
for dopaminergic neurodegeneration in PD. Therefore, development of appropriate models to
study the mechanism of neuro-inflammation will lead to development of effective therapeutic
interventions. LPS (Lipopolysaccride) is the endotoxin found in the outer membrane of gram
negative bacteria that can cause wide variety of systemic responses ranging from pyrexia to septic
shock (M. Liu & Bing, 2011; Schletter et al., 1996). LPS indirectly influences dopaminergic
neuronal survival via its effects on glial activation. LPS injection into brain results in upregulation
of CD14 receptor and acts through Toll like receptor (TL4) in the microglia (Hoshino et al., 1999;
Lacroix et al., 1998). It is interesting that, there are regional difference in LPS induced
neurotoxicity in the rat brain. Using in vitro cell culture models such as mixed neuronal cultures
of substantia nigra, LPS was found to induce the production of pro-inflammatory cytokines and
death of dopaminergic neurons whereas hippocampus and cortex mixed cultures were relatively
unresponsive to LPS treatment (W. G. Kim et al., 2000). LPS induced microglial activation results
in pro-inflammatory mediators like TNF alpha, IL-1 beta, nitric oxide species and reactive oxygen
species which ultimately results in dopaminergic neurodegeneration and neuronal cell death in PD
models (Arai et al., 2004; Boje & Arora; M. Liu et al., 2011). A growing body of evidence supports
the involvement of MAPK pathway in the mechanism of microglial activation response (Bhatia et
al., 2016; Bhatia et al., 2017; S. H. Kim, Smith, et al., 2004; Santa-Cecilia et al., 2016; Xing et al.,
2011). Additionally, downstream signaling mediator such as NADPH oxidase has been implicated
in LPS induced microglial reactive oxygen species (ROS) production and pro-inflammatory gene
47
expression which is linked to dopaminergic neurotoxicity (Liya Qin et al., 2004; Liya Qin et al.,
2007). Moreover, LPS and neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP) can
act synergistically to elicit NADPH oxidase activation and NOS production leading to
dopaminergic neurodegeneration (H.-M. Gao et al., 2003).
Systemic LPS administration results in production of peripheral TNF alpha which crosses
the blood brain barrier and results in microglial mediated pro-inflammatory cytokine production
and delayed loss of dopaminergic neurons in substantia nigra in PD (Liya Qin et al., 2007). Along
with systemic administration of LPS, other invivo models were also developed to study the effect
of LPS in nigrostriatal dopaminergic system. In intra-nigral LPS model, LPS was stereotaxically
injected into substantia nigra (SN) of the mid brain and its effects were studied. In this context,
direct injection of LPS into SN of the mid brain resulted in microglial activation, reduction of
dopamine levels and tyrosine hydroxylase activity and ultimately resulting in dopaminergic cell
damage (Castaño et al., 1998). Furthermore, direct injection of LPS into SN of the mid brain
resulted in behavioral abnormalities, mitochondrial respiratory abnormalities, intracytoplasmic
inclusions and loss of dopaminergic neurons (D.-Y. Choi et al., 2009).
Another model that was used to study the LPS induced neuro-inflammatory response is
stereotaxic injection of LPS into striatum of midbrain (Intra-striatal LPS animal model).
Substantia nigra with relatively higher population of microglia is more susceptible than striatum
to stereotaxic injection of LPS as its main mode of action is via induction of strong microglial
activation and neuro-inflammation (Herrera et al., 2000). Stereotaxic LPS injection into substantia
nigra or striatum resulted in progressive dopaminergic degeneration and dopamine depletion via
production of production of proinflammatory cytokines like TNF-alpha, IL-6, IL-1 alpha and NO
both in substantia nigra and striatum (Castaño et al., 1998; M. Liu et al., 2011). Nigral stereotaxic
48
injection of LPS resulted in microglial activation, IL-1 beta release, caspase-11 activation and
decreased TH positive cells in substantia nigra of midbrain (Arai et al., 2004). In another study,
intrastrial stereotaxic LPS injection resulted in dopaminergic cell death mainly via NO induced
neuro-inflammation (L Hunter et al., 2009).
In another model, intranasal administration of LPS for 5 months resulted in behavioral
abnormalities, cytoplasmic inclusions, striatal dopamine loss and loss of dopaminergic neurons
(Q. He et al., 2013). In order to develop novel therapeutic interventions against LPS-induced
microglial activation response and associated dopaminergic neurodegeneration, studies were
performed in cell culture and animal models of PD. For example, revasterol (nonflavonoid
polyphenol) has been shown to be protective against LPS induced neurotoxicity by inhibiting
MAPK pathway, Nuclear factor-kB activating pathway and NADPH oxidase (F. Zhang et al.,
2010). Catalpol (an iridoid glycoside,contained richly in the roots of Rehmannia glutinosa) was
found to offer neuroprotection against LPS induced microglial activation by significantly reducing
the secretion pro-inflammatory mediators like TNF alpha, nitric oxide species (NOS) and reactive
oxygen species (ROS) (Tian et al., 2006). Other neuroprotective agents that were found to offer
protective against LPS induced neurotoxicity includes fluoxetine, 3-hydroxymorphinan, Gö6976
(selective protein kinase C (PKC) inhibitor and IL-10 through various mechanisms involving
inhibition of P38, MAPK pathway, NADPH oxidase and pro-inflammatory cytokine production
(Jeohn et al., 2002; Li Qian et al., 2006; F. Zhang et al., 2012; W. Zhang, Qin, et al., 2005).
MPTP Mouse Models
The most widely used experimental model of PD include the murine MPTP model. MPTP
(1-methyl-4-Phenyl-1, 2, 3, 6 tetrahydro-pyridine) is lipophilic and selectively destroys the
nigrostriatal neurons (G. Meredith & D. Rademacher, 2011). Despite the popularity of the model,
49
variable loss of nigral neurons, striatal DA depletion and neurobehavioral deficits have hampered
the utility of the model. Importantly, the MPTP models fails to reproduce the motor disturbance
evidenced in PD. Despite that it has been routinely used to better understand the pathological basis
of PD and cellular mechanisms that underlie the increased vulnerability of dopaminergic neurons
to the neutotoxic effects of MPTP. In this context, MPTP mouse models can display variable
degrees of nigral cell death, striatal dopamine loss and behavioral deficits depending upon the
dosage and timing of the MPTP regimen. The most commonly used MPTP models include acute,
subacute and chronic. MPTP can be co-administrated with probenecid which retards renal
clearance of MPTP thereby potentially increasing its neurotoxicity (Petroske et al., 2001). In
theacute MPTP model, C57black mice were injected with 4x 18 mg/kg of MPTP and sacrificed at
3h 6h 12h 24h and 72h after the last MPTP injection (N. Panicker, H. Saminathan, H. Jin, M. Neal,
D. S. Harischandra, et al., 2015). In subacute model, 15-30 mg/kg MPTP is given on a daily basis
for 5-10 days and animals are sacrificed 3 days after last injection (Anamitra Ghosh et al., 2012).
In chronic MPTP mouse model, 25 mg/kg MPTP is injected every 3.5 days for 5 weeks and
animals were sacrificed 1-3 weeks’ after injection (Meredith et al., 2008). A key aspect that is
evidenced in different MPTP mouse models include a reduction in dopamine uptake which
positively correlates with DA cell loss in substantia nigra (Meredith et al., 2008; Novikova et al.,
2006).
In Acute and Subacute mouse model, less than 50% of the nigral dopaminergic neurons
are destroyed whereas in chronic MPTP model there is wide spread destruction of nigral
dopaminergic neurons (Meredith et al., 2008). Acute MPTP model produces 40%
dopaminergic (DA) cell loss in substantia nigra (SN) and 90% loss of striatal DA 7 days after
50
treatment (Andrea Serra et al., 2008; G. Meredith et al., 2011). In Subacute MPTP model, death
of dopaminergic neurons occurs in apoptotic form 6 days after MPTP treatment and 59% striatal
DA depletion at 1day after MPTP treatment (Andrea Serra et al., 2008). Death of nigral neurons
occurs by non-apoptotic form in Acute MPTP model whereas it is apoptotic in subacute MPTP
model (G. Meredith et al., 2011).
Acute MPTP model regimen causes increase in DA release and its extracellular metabolites
like 3-methoxytyramine (3-MT) and decrease in dopamine metabolites like DOPAC (3, 4-
dihydroxyphenyl acetic acid) and HVA (homovanillic acid) in the dialysates of striatum (Andrea
Serra et al., 2008). In Sub-acute MPTP model, there is progressive day to day reduction in DA and
3-MT levels in the dialysates of striatum (Andrea Serra et al., 2008). It is interesting to know that
loss of striatal DA and reduction in dopamine uptake tend to spontaneously reverse over a period
of time in Acute and Subacute MPTP model (G. Meredith et al., 2011). In contrast, loss of striatal
DA and reduction in dopamine uptake tend to persist for 6 months in chronic MPTP model (G.
Meredith et al., 2011). Acute and Subacute MPTP models cause rapid transient neurotoxic insult
to the nigrostriatal system that does not exceed threshold for producing long lasting motor
abnormalities (Petroske et al., 2001). Whereas, in chronic MPTP model there is long lasting
neurotoxic injury to the nigrostriatal system which positively correlates with significant motor
abnormalities over a period of time (Petroske et al., 2001). In chronic MPTP model, mice show
significant disability as early as 3 days after MPTP treatment and impaired performance persists
for over 6 months (Novikova et al., 2006). Chronic MPTP model produces 50 -70% dopaminergic
(DA) cell loss in substantia nigra and 95-98% loss of striatal DA 3 weeks after treatment (Meredith
et al., 2008). Strong inflammatory responses with reactive microglia were visualized in SN as early
51
as 3 weeks after MPTP treatment and they persist for months in chronic MPTP model (Meredith
et al., 2008).
MPTP easily crosses the blood brain barrier due to its lipid solubility (Singer & Ramsay,
1990; Tieu, 2011). Once it enters the central nervous system, it is converted into its active MPP+
form by mono-amino oxidases (MAO-B) (Singer et al., 1990; Tieu, 2011). MPP+ is actively taken
up into nigral dopaminergic neurons by dopamine (DA) transporters (Singer et al., 1990; Tieu,
2011). Various mechanisms through which MPTP causes dopaminergic cell neurotoxicity includes
complex I inhibition, dopamine metabolism, disruption of calcium homeostasis, oxidative stress,
alteration of iron metabolism and GSH depletion. (Singer, T.P. et al 1990 & Sian, J. et al 1999).
Two most commonly proposed hypotheses for MPTP induced dopaminergic neurodegeneration
includes oxidative stress and mitochondrial impairment. According to the oxidative stress
hypothesis, there is generation of superoxide radicals and GSH depletion whereas in mitochondrial
impairment there is inhibition of NADH dehydrogenase, complex I and oxidative phosphorylation
(Singer, T.P. et al 1990).
Microglial activation is another most important feature that is present in both acute and
chronic MPTP models (G. Meredith et al., 2011). In MPTP treated mice there is significant
neuroinflammation characterized by upregulation of mRNA and proteins levels of TNF-alpha, IL-
1 beta and IL-6 cytokine and their receptor levels in substantia nigra and caudate-putamen regions
(Lofrumento et al., 2010). The neuroinflammation that develops in MPTP treated mice is not
limited to substantia nigra but can occur in other regions like olfactory bulb (Vroon et al., 2007).
Previous studies indicate that use of orally active apocynin and rosiglitazone loaded microspheres
(continuous dopaminergic stimulation) offered protection against MPTP induced neuro-
inflammation in mouse models of PD (Anamitra Ghosh et al., 2012; X. Yu et al., 2015).
52
There can be difference in the susceptibility of mouse strains to MPTP induced
dopaminergic cell loss, striatal DA depletion and behavioral abnormalities (G. Meredith et al.,
2011). The various underlying mechanisms underlying differential vulnerability of mouse strains
to MPTP induced neurotoxicity includes, uptake of MPTP into brain MAO-B expression, DA
transporter expression, metabolism of MPTP, number of TH positive neurons in the SN, and
neuroprotective mechanisms (Gainetdinov et al., 1997; Imai et al., 2001; Muthane et al., 1994;
Sedelis et al., 2000). Two most commonly used behavioral test that were used to assess the
dopaminergic cell loss and dopamine depletion in MPTP mouse models of PD include open rod
test, rotarod test (G. Meredith et al., 2011). In open rod test, the amount of time spent in moving
is measured whereas as rota rod test measures the duration spent by mice moving over the rod
(Rozas et al., 1997; Rozas et al., 1998; Sedelis et al., 2000). Another study used mice swim test
for measuring striatal dopamine depletion in MPTP model of Parkinson Disease (Haobam et al.,
2005).
Since MPTP treated mice develop dopaminergic neurodegeneration reminiscent of PD,
various interventions were studied and therapeutic benefit was monitored by measuring the
parameters like dopaminergic cell loss, DA depletion and behavioral abnormalities (G. Meredith
et al., 2011). Therapies that were targeting various mechanism of actions underlying MPTP
induced neurodegeneration has been developed and tested in the past. Therapeutic interventions
aimed at oxidative stress (Antioxidant agents), microglial activation (Apocynin, Minocycline,
PPAR gamma agonists), neuronal nitric acid synthase pathway (7-Nitroindazole), dopamine
deletion (Bromocriptine), caspase activation (Bcl-2), NADH linked oxidation have shown modest
therapeutic success (Breidert et al., 2002; S. Lin et al., 2003; Muralikrishnan & Mohanakumar,
1998; Nicklas et al., 1985; Perry et al., 1985; L. Yang et al., 1998).
53
6-OHDA model
The next most commonly used model for studying molecular pathogenesis in Parkinson
Disease is 6-OHDA (Hydroxy Dopamine) model. 6-OHDA model is used for testing the efficacy
of novel therapeutic interventions and for studying the mechanism of neurotransmission in
midbrain (Bové et al., 2005; Tieu, 2011). The most frequently used method of delivery of 6-OHDA
for developing animal model is intracerebral injection either into substantia nigra or striatum as it
cannot cross the blood brain barrier (Batassini et al., 2015; Bové et al., 2005; Tieu, 2011).
Intracerebral injection of 6-OHDA in substantia nigra produces dopaminergic cell death occurring
within 24h whereas delivery to striatum results in more protracted neurodegeneration with cell
death occurring within 1-3 weeks (Bové et al., 2005; Tieu, 2011). 6-OHDA striatum model
(progressive neuro degeneration) is regarded as a better model to study because it resembles
clinically progressive PD and is associated with non-motor symptoms (Tieu, 2011). It is important
to note here that, the success rate of intracerebral injection of 6-OHDA into smaller brain structure
like substantia nigra is only 30% whereas injection into bigger brain structure such as striatum has
a success rate is close to 90% (Simola et al., 2007; Tieu, 2011; Torres & Dunnett, 2012). In most
cases, 6-OHDA unilateral model is used and bilateral model is rarely used because it can lead to
severe neurological disabilities and ultimately death of the animal (Blesa et al., 2012). Another
advantage of unilateral 6-OHDA model is that, lesioned hemisphere can be compared with
contralateral non-lesioned hemisphere using behavioral tests (Blesa et al., 2012; Bové et al., 2005;
Tieu, 2011). 6-OHDA bears a structural resemblance with dopamine and thereby enters the
dopaminergic neurons through dopamine transporters (DAT) (Blesa et al., 2012; Bové et al., 2005;
Hanrott et al., 2006; Tieu, 2011). Due to its structural resemblance of 6-OHDA to catecholamine,
it can also pass through nor-epinephrine transporters and cause degeneration of central sympathetic
54
nervous systems (Bové et al., 2005; Hirsch et al., 2003; Tieu, 2011). The mechanism of 6-OHDA
induced neurotoxicity is debatable, but various mechanisms has been proposed such as oxidative
stress, mitochondrial dysfunction, caspase activation and ROS production (Bové et al., 2005;
Hanrott et al., 2006; Y. Saito et al., 2007). It is interesting to know that the 6-OHDA induced cell
death of dopaminergic neurons exhibit the features of both apoptosis and necrosis (Hanrott et al.,
2006). It has been proposed that, 6-OHDA induced apoptosis of dopaminergic neurons is due to
caspase-3 dependent cleavage of PKC-delta (Hanrott et al., 2006). Another mechanism by which
6-OHDA mediates dopaminergic cell death involves ERK activation which can be rescued by
parkin mediated P62 stabilization and upregulation (X. O. Hou et al., 2015; Kulich et al., 2007).
Astroglial cell involvement is also regarded as an important contributing factor for dopaminergic
degeneration in 6-OHDA model as evidenced by increase in CSF levels of GFAP and S100B
(Batassini et al., 2015; Bové et al., 2005). Lewy bodies, the hallmark pathological feature of PD
are not always present in 6-OHDA models (Bové et al., 2005). The two most commonly performed
behavioral tests for assessing 6-OHDA models include circular motor behavior and forelimb
akinesia whose severity is directly proportional to the degree of nigrostriatal degeneration (Blesa
et al., 2012; Deumens et al., 2002; Hanrott et al., 2006; Metz et al., 2005; Tieu, 2011). Additional
studies that can be done for studying 6-OHDA model include measuring dopamine levels in the
brain and examining the TH-positive neurons in the mid brain (De Jesús-Cortés et al., 2015; Stayte
et al., 2015). Nicotinamide adenine dinucleotide analogues (P7C3 compounds) are shown to be
neuroprotective against behavioral abnormalities, dopamine level changes and dopaminergic cell
death in 6-OHDA PD model in rats (De Jesús-Cortés et al., 2015).
55
Alpha-Synuclein Brain Infusion Model
Alpha-synuclein is found in Lewy bodies which are histological hallmark of Parkinson
Disease (Braak et al., 1999; Spillantini et al., 1998). Misfolded alpha-synuclein tends to aggregate
and is proposed to be involved in the dopaminergic neurodegeneration in PD (Braak et al., 1999;
Spillantini et al., 1998). Animal models were developed by delivering alpha-synuclein
intracerebrally either by injecting viral vectors or direct injection of mutant alpha-synuclein fused
to protein transduction domain (Bazzu et al., 2010; Kirik et al., 2002; Lo Bianco et al., 2004;
Recchia et al., 2008)
Lentiviral vectors are used for studying the effect of alpha-synuclein in triggering rapid
dopaminergic neurodegeneration and Lewy body pathology in substantia nigra in animal models
of PD (Chesselet, 2008). Rats are preferred over mice for lentiviral overexpression studies as they
have a larger brain size thus making it relatively easier to target substantia nigra for delivery of
alpha-aynuclein (Chesselet, 2008). Lentiviral overexpression models usually produce partial loss
of dopaminergic neurons as compared to toxin models which usually produce massive loss of
dopaminergic neurons due to differential expression of viral vectors witnin dopaminergic neuronal
cells (Kirik et al., 2002). The advantage of using lentiviral vectors overexpressing alpha-synuclein
is that, they can be administered unilaterally and contralateral untreated side can be used as a
control for comparison (Kirik et al., 2002).
Lentivirus overexpressing human alpha-synuclein injected stereotaxically into substantia
nigra resulted in 30-80% loss of dopaminergic neurons, 40-50% loss of striatal dopamine levels,
40-50% loss of tissue TH activity, significant behavioral abnormalities along with wide
distribution of alpha-synuclein positive inclusions within the dopaminergic neurons and
neurodegeneration (Kirik et al., 2002; Lo Bianco et al., 2002). In another model, direct stereotaxic
56
injection of alpha-synuclein fused to protein transduction domain (TAT-- AlphasynA30P) into
substantia nigra resulted in 26% loss of dopaminergic neurons and 78% loss of striatal dopamine
levels and 79% loss of DOPAC (Dihydroxyphenylacetic acid) (Recchia et al., 2008). In another
study Chung et al demonstrated that, AAV overexpressing alpha-synuclein injection into rat
substantia nigra resulted in changes in synaptic transmission, axonal transport and microtubules
along with neuroinflammation (Chung et al., 2009). Likewise, lentivirus over expressing mutant
alpha-synuclein injected into the striatum and substantia nigra of mice resulted in alpha-synuclein
aggregation within neurons, Lewy body pathology, alpha-synuclein positive neuronal varicosities
subsequently resulting progressive degeneration of alpha-synuclein positive neurons within one
year (Lauwers et al., 2003). Moreover, adenoviral overexpression of alpha-synuclein into rat
substantia nigra resulted in progressive dopaminergic neurodegeneration in SN over 13 weeks,
serine 129 phosphorylation of alpha-synuclein and caspase-9 activation with smilar changes in
cortical region (Mochizuki et al., 2006). In a similar fashion, adenovirus overexpression of alpha-
synuclein into rat substantia nigra resulted in progressive dopaminergic neurodegeneration,
activation of caspase-9 and upregulation of heat shock proteins at 24 weeks (St Martin et al., 2007).
Intrastriatal injection of pathogenic alpha-synuclein fibrils resulted in cell to cell
transmission, Lewy body pathology, loss of dopaminergic neurons, dopamine reduction in
substantia nigra pars compacta associated with behavioral deficits (K. C. Luk et al., 2012; Paumier
et al., 2015). Intrastriatal injection of Lewy bodies into monkeys and mice results in neuronal
uptake of alpha-synuclein, transmission of alpha-synuclein anterogradely and retrogradely
ultimately resulting in progressive dopaminergic neurodegeneration (Recasens, S. et al).
57
Spleen Tyrosine Kinase (SYK)
Structure and Expression
Syk is a non-receptor tyrosine kinase, which has two Src homology domains (SH2) and
kinase domain (Futterer et al., 1998; Mócsai et al., 2010; Taniguchi et al., 1991). These domains
are interconnected by inter-domains: A and B (Geahlen, 2014; Sada et al., 2001). Its Molecular
weight is 74 kDa (Mócsai et al., 2010). Alternative splicing can yield another form namely Syk-B
that lacks 29 AA from inter-domain B (Turner et al., 2000; L. Wang et al., 2003). There are some
structural resemblances between human and mouse Syk (Andres et al., 1995). Mammalian Syk or
Zeta chain associated protein kinase (ZAP70) bears close structural resemblance with Syk
although less homology with Syk amino acid sequences (Andres et al., 1995). Mostly, Syk is
located near the plasma membrane in the cytoplasm but, sometimes it is localized in the nucleus
too (Kulathu et al., 2009; H. Ma et al., 2001; S. Yanagi et al., 2001). Syk is expressed in wide
variety of cell types. Hematopoietic cells, macrophages, neuronal cells, fibroblasts, epithelial cells
and vascular endothelial cells express Syk in varying proportions (S. Yanagi et al., 2001; Shigeru
Yanagi et al., 1995).
Mechanism of Activation
The factors that can activate Syk include Low-Density Lipoprotein (LDL)(S. H. Choi et
al., 2009) Lipopolysaccharide (Miller et al., 2012), Pathogen-Associated Molecular Patterns
(PAMPs) (Rogers et al., 2005) and Damage-Associated Molecular Patterns (DAMPs) (Huysamen
et al., 2008; Sancho et al., 2009; Yamasaki et al., 2008). In the resting cells, syk is in inactive auto-
inhibited state due to linking of kinase domain with inter-domain A & B (Geahlen, 2009; Kulathu
et al., 2009). Phosphorylation of tyrosine residues in the inter domains A and B will result in release
of syk from auto-inhibited state to its active state (Geahlen, 2014; Mócsai et al., 2010). The most
58
crucial event for activation of Syk include phosphorylation of ITAMs (Immuno-receptor tyrosine-
based activation motifs) in immune cells (Hirose et al., 2004; C. A. Lowell, 2011). Phosphorylation
of ITAM tyrosine is through Src family kinases like Lyn (H. Ma et al., 2001). Next, syk binds to
the phosphorylated tyrosines of ITAM receptors which will result in phosphorylation of additional
tyrosine residues on syk kinase leading to activated spleen tyrosine kinase (Geahlen, 2009, 2014;
Mócsai et al., 2010; Tsang et al., 2008). Activated syk kinase will activate downstream molecules
to mediate various signaling events within the immune cells (Geahlen, 2009; Mócsai et al., 2010;
Tsang et al., 2008). Alternatively, another report mentions that Syk can also be activated by
binding to cytoplasmic tail of 3 integrin without interaction with phosphorylated ITAMs
(Woodside et al., 2001). Active Syk with its phosphorylated tyrosines can interact with
downstream signaling molecules like VAV, PLC-gamma, PI3K and SLP-76/65 (Geahlen, 2014;
C. A. Lowell, 2011; Mócsai et al., 2010; Ying et al., 2011). Furthermore, Syk regulates
physiological and pathological processes through interaction with intermediate signaling
molecules like NF-kB, CARD-BCL10-MALT complex, AKT, Ca+, MAP kinases, PKC and
protein kinase2 (Geahlen, 2014; C. A. Lowell, 2011; Mócsai et al., 2010).
Functions
Syk mediates cytoskeleton rearrangement, Reactive Oxygen Species (ROS) generation,
pro-inflammatory cytokine production and phagocytosis (Berton et al., 2005; S. H. Choi et al.,
2012; Jaumouille et al., 2014; M. J. Kim et al., 2012; Rogers et al., 2005; Y.-S. Yi et al., 2014a;
Yoon et al., 2013). Furthermore, it is involved in diverse biological processes like cell adhesion,
innate pathogen recognition, inflammatory responses and antibody dependent cytotoxicity (Endale
et al., 2013; Jeong et al., 2014; Mócsai et al., 2010; W. S. Yang et al., 2015).
59
Pathogen Recognition and Immune Response
Syk is required for P- Selectin Glycoprotein Ligand 1 (PSGL1) mediated leukocyte rolling
on the endothelium which is essential for transport of immune cells to the site of injury for
mounting an immune response (Mócsai et al., 2010; Newton & Dixit, 2012). Foreign pathogens
trigger Syk mediated phagocytosis, ROS and chemokine production in immune cells thus helping
in mounting an immune response in host cells in response to pathogens (Brungs et al., 2015;
Greenberg, 1999; Mócsai et al., 2010; Newton et al., 2012). Lastly, syk is responsible for Epstein-
barr virus transformation and lytic replication of Kaposi Sarcome virus within the infected B-cells
(Grywalska & Rolinski, 2015; Tomlinson & Damania, 2004).
NLRP3 Inflammasome
Spleen tyrosine kinase (Syk) is regarded as a major upstream regulating kinase responsible
for inflammasome activation in immune cells (Federica Laudisi et al., 2014). Nigericin, Mono
sodium urate and Alum has been known to cause Syk mediated inflammasome activation in LPS
primed peritoneal macrophages (Y. C. Lin et al., 2015). Syk has been shown to cause
phosphorylation of apoptosis associated speck-like protein containing a CARD (ASC) thus
promoting caspase-1 activation thereby, resulting in NLRP3 inflammasome mediated production
of pro-inflammatory cytokines like IL-1 beta and IL-18 (Gross et al., 2009; H. Hara, K. Tsuchiya,
I. Kawamura, R. Fang, E. Hernandez-Cuellar, Y. Shen, J. Mizuguchi, E. Schweighoffer, V.
Tybulewicz, et al., 2013). Syk triggers NLRP3 inflammasome mediated caspase-1 activation
through ASC polymerization and oligomerization in immune cells (Y. C. Lin et al., 2015).
Infection with fungal pathogens usually elicits NLRP3 inflammasome activation through Syk
regulated ROS production, K+ efflux and NF-kB signaling events (Gross et al., 2009). Syk
60
mediated signaling through CARD9-BCL10-MALT1 is essential in pathogen and tissue damage
recognition (Marakalala et al., 2010; Newton et al., 2012).
Platelet, Lymphocyte and Osteoclast Development
Syk mediated ITAM signaling is very important for osteoclast development and function
in bone metabolism through PLC-gamma2 (Phospho Lipase-C Gamma) and NFAT1 (Nuclear
Factor Activator of T-cells) (Mocsai et al., 2004; W. Zou et al., 2008). In platelets, Syk based
signaling is essential for development, function and arterial thrombus formation through PLC-
gamma2 and SLP76 (SH2 domain containing Leukocyte Domain) (Rivera et al., 2009). During
vascular development, Syk -ITAM signaling is useful for separation of blood and lymphatic
vessels via PLC-gamma2 and SLP76 (Abtahian et al., 2003). Syk is required differentiation of
pro-B cells to pre-B cells acting through intermediate signaling molecules like SLP76 (Herzog et
al., 2009).
Clinical Diseases
Spleen tyrosine kinase is associated with various inflammatory diseases like Rheumatic
arthritis, Allergic Asthma, Allergic Rhinitis, Gut ischemia reperfusion injury, Systemic Lupus
Erythematosis (SLE) and idiopathic thrombocytopenic purpura (ITP) (Bahjat et al., 2008; Bajpai
et al., 2008; Blease, 2005; Masuda & Schmitz, 2008; Podolanczuk et al., 2009; Wong et al., 2004).
Neurological Diseases
Numerous studies were done previously to implicate syk in neuro-inflammation after
subarachnoid hemorrhage in rats (Yue He et al., 2015). Previous findings indicate that,
phosphorylated-syk expression is increased in neurodegenerative diseases like Alzheimer disease
and Nasu-Hakola Disease (Satoh et al., 2012; Schweig et al., 2017). Increased Syk expression is
61
associated with neuroinflammation, gliosis, tau phosphorylation, amyloid beta accumulation and
pathological lesions of Alzheimer disease (Daniel Paris et al., 2014; Satoh et al., 2012; Schweig et
al., 2017). Fc receptor mediated Syk activation is also responsible for downstream signaling events
like cytokine secretion, phagocytosis and antigen presentation in Alzheimer Disease (Fuller et al.,
2014). In microglial cells, spleen tyrosine kinase is recruited to the stress granules leading to
phagocytic dysfunction and ROS & NOS production ultimately resulting in neuronal cell death (S.
Ghosh & Geahlen, 2015; Wolozin & Ikezu, 2015). Accumulating evidence suggests that, Syk
induced tyrosine phosphorylation of alpha-synuclein can be neuroprotective by decreasing its
oligomerization and aggregation (Lebouvier et al., 2008; A. Negro et al., 2002). In cerebral
ischemia, there is increased expression of Syk and phospho-syk 2h, 4h, 10h and 24h after
reperfusion (Yukiya Suzuki et al., 2013). Accordingly, Piceatannol (Syk inhibitor) protected
against cerebral via downregulation of phospho-syk, ICAM-1, MMP-9 and upregulation of
claudin-5 (Yukiya Suzuki et al., 2013). Furthermore, Syk inhibition resulted in resulted reduced
infarct size and offered neuroprotection in mice through decreased platelet activation (McKenzie,
2016). Additionally, mice treated with oral bioavailable syk inhibitor had reduced infarct size and
better neurological outcome 24h after transient middle cerebral artery occlusion (van Eeuwijk et
al., 2016).
Cancers
It can play a dual role in cancers. In hematological malignancies, it acts as a tumor promoter
whereas in cancers of non-immune cells it mediates tumor suppression (Krisenko & Geahlen,
2015). Spleen tyrosine kinase exhibits tumor suppressor activity by reducing metastasis and
promoting apoptosis in cancers such as breast cancer, hepatocellular carcinoma, melanoma and
pancreatic adenocarcinoma (Geahlen, 2014). In hematological malignancies like acute
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lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), Epstein Barr virus
associated lymphoma and non-hodgkins lymphoma, syk acts as a pro-survival factor through its
role in B-cell development and tonic signaling through B-cell receptor (Geahlen, 2014). In these
malignancies, it promotes tumor cell survival via activation of AKT, mTOR, PKC-delta, Mcl-1
and downregulation of GSK-3 beta (Geahlen, 2014). There is overexpression of Syk and tyrosine
Y525/526 phosphorylation in T-cell lymphomas (Feldman et al., 2008). In resistant diffuse B-cell
lymphomas, synthetic syk inhibitors resulted in G1-S cell cycle arrest via PLC-gamma and AKT
(Cheng et al., 2011). Accordingly, Syk inhibition would be an attractive therapeutic target for
therapeutic interventions in T-cell and B-cell lymphomas. So various Syk inhibitors like
fostamatinib, entospletinib and cerdulatinib are currently under development for various
hematological malignancies (D. Liu & Mamorska-Dyga, 2017). Retinoblastoma has high Syk
expression and it promotes survival via inactivation of GSK-3 beta and stabilization of anti-
apoptotic Mcl-1 (Myeloid cell leukemia sequence) (Geahlen, 2014; J. Zhang et al., 2012). In
human prostate and ovarian cancer, increased syk expression is associated with malignant
progression, recurrence and treatment resistance (Ghotra et al., 2015; Y. Yu et al., 2015).
Furthermore, it is associated with cancers like B-cell induced leukemia’s, Kaposi Sarcoma and
Epstein-Barr virus induced cancers (Mócsai et al., 2010).
Rheumatoid Arthritis
Rheumatoid arthritis (RA) is an autoimmune disease characterized by wide spread joint
destruction and bone inflammation due to hypersensitivity to self-antigens (Deng et al., 2016a;
Geahlen, 2014). It is an immune complex phenomenon mediated by type II and type III
hypersensitivity associated with IgG immune-complexes formed with self-antigens (Deng et al.,
2016a; Geahlen, 2014). Binding of IgG immune-complexes with Fc-gamma receptors on
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neutrophils and macrophages leads to activation of Syk-ITAM signaling resulting in production of
reactive oxygen species, nitric oxide and pro-inflammatory cytokines (Deng et al., 2016a; Geahlen,
2014; Mócsai et al., 2010). Thus deposition of these immune complexes on joints and tissues leads
joint destruction and other symptoms characteristic of RA (Boers, 2011; Deng et al., 2016a;
Geahlen, 2014). Accordingly, blocking of syk-ITAM signaling with syk inhibitors leads to
attenuating joint destruction in animal models and phase II clinical trials in humans (Boers, 2011;
Deng et al., 2016a; Flight, 2012; Geahlen, 2014; Nijjar et al., 2013; D. L. Scott, 2011; I. C. Scott
& Scott, 2014). Previous reports indicate that, there are higher levels of Phospho-Syk in peripheral
blood of rheumatic arthritis patients as compared to control patients (Deng et al., 2016b).
Treatment with Syk inhibitor resulted in clinical benefits in patients with Rheumatic arthritis
receiving methotrexate therapy (Weinblatt et al., 2008). Combined inhibition of Syk and JNK
kinases results greater clinical success than syk alone inhibition in chronic destructive arthritis of
mice (Llop-Guevara et al., 2015). Syk is essential for RANK-FcR-gamma signaling required for
osteoclast activation and bone damage in mouse models of RA (Geahlen, 2014). Moreover, syk is
also required for TNF-alpha induced production of proinflammatory cytokines and other degrading
enzymes responsible for bone damage in RA (Geahlen, 2014). Furthermore, Syk inhibition
resulted in downregulation of JNK induced expression of pro-inflammatory IL-6 and MMP-3 in
synoviocytes (Cha et al., 2006). Lastly, syk is also required for migration, rolling and adherence
of macrophages, neutrophils and monocytes to inflamed endothelium thus indirectly playing an
important role on pathogenesis of atherosclerosis in RA (Geahlen, 2014).
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Ischemic Reperfusion Injury
Syk activation has been linked to intestinal damage in intestinal ischemia reperfusion injury
model via Fcgamma receptor and complement signaling. In this context, blocking Syk mediated
Fcgamma receptor signaling in platelets resulted in reduced lung injury in intestinal mesentric
ischemia (Lapchak et al., 2012; Pamuk et al., 2010).
Tumor Necrosis Factor Weak stimulator of Apoptosis (TWEAK)
Introduction
Most of the members of the TNF family consist of ligands and receptors that activate
various signaling pathways to cause various effects ranging from cell survival, cell death and
development (Bodmer et al., 2002). Tweak is a member of TNF superfamily of cytokines. These
cytokines are synthesized as a type II trans membrane protein with a conserved C- terminal domain
(Bodmer et al., 2002). All the membrane bound forms can be cleaved by the certain CMK proteases
to a soluble form that can exert its actions in distant targets (Bodmer et al., 2002; BROWN et al.,
2003). The predisposing factors that regulate the processing of full length membrane tweak to
soluble tweak needs further investigation. The relative ratio of membrane tweak and soluble tweak
depends on the physiological and pathological state on the target tissues. The two forms of tweak
are functionally distinct and activate different downstream signaling pathways (Roos et al., 2010).
Tweak cDNA was accidently identified from mouse peritoneal macrophages during search
for erythropoietin messenger RNA. Later this cytokine was identified as Tweak due to its
proximity to TNF superfamily and its ability to weakly induce apoptosis (Chicheportiche et al.,
1997). After that, they identified complete human Tweak cDNA by performing PCR of human
tonsil and fetal liver tissues. Masters et al, also identified tweak cDNA from fetal kidney and
named it as APO3 ligand as it has affinity for death domain binding receptor Apo3 or DR3
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(Marsters et al., 1998). Apo3 ligand can cause caspase activation, Nf-kB activation and apoptosis
(Marsters et al., 1998). Further analysis showed that human Tweak gene and Fn14 gene is localized
to chromosome 17 (Chicheportiche et al., 1997; Marsters et al., 1998; Meighan-Mantha et al.,
1999). There is close structural resemblance between human tweak sequences and members of
TNF superfamily which explains which might explain the commonality with the characteristics of
TNF superfamily. Furthermore, Tweak is a multipotent cytokine that is associated with various
biological functions like growth, differentiation, migration, apoptosis, angiogenesis and cytokine
production. These responses tend to vary with cell type, receptor activated and pathway involved.
The wide variety of cellular responses that are mediated by Tweak / Fn14 pathway in different cell
types might be responsible for maintaining normal health status and mediating various
pathological conditions. Tweak binds to its receptor Fn14 and exerts many of its actions (Burkly
et al., 2011; Inoue et al., 2000).
Fibroblast growth factor inducible gene (Fn14) gene is regulated by growth factors and is
a type I trans membrane protein (Locksley et al., 2001). It does not share sequence similarity with
any other protein (Locksley et al., 2001). Fn14 is the major signaling receptor for Tweak. CD163
receptor appears to be scavenger receptor for tweak (Bover et al., 2007). CD163 receptor is mainly
present in the macrophages and monocytes and is mainly responsible for internalization and
degradation of soluble tweak (Bover et al., 2007). Thus, over expression of CD163 receptor can
sequester the soluble tweak and prevent it from exerting its biological actions. TNF super family
usually mediates downstream pathways through TRAF and Death Domain molecules (Fesik, 2000;
Inoue et al., 2000). Through these molecules, they activate pathways like NF-kB (nuclear kB) and
MAPK (mitogen activated protein kinase pathways) (Bover et al., 2007; BROWN et al., 2003;
Burkly et al., 2011; Locksley et al., 2001). Additionally, various kinases such as Glycogen
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Synthase kinase-3 and sphingomyelinase are also activated by TNF family members (Naismith et
al., 1995; Novoyatleva et al., 2010; Wajant et al., 1999). Tweak shares some of the above-
mentioned features with the TNF family.
One of the most important signaling pathways activated by Tweak / Fn14 interactions is
Nf-KB pathways which results in expression of target genes. Over expression of Fn14 receptor in
malignant glioma leads to increased migration rate through Rac1 / NF-kB dependent mechanism
which ultimately leads to poor patient survival rates (Tran et al., 2006). Tweak treatment or over
expression of fn14 in malignant glioma results in increased glioma cell survival and resistance to
cytotoxic therapy through NF-kB dependent upregulation of Bcl-xl / Bcl-w expression (Tran et
al., 2006). Evidence of activation of Nf-kB pathway by Tweak can be seen in glioblastoma cancer
(Tran et al., 2006), keratinocytes (L. Jin et al., 2004), renal tubular cells (A. B. Sanz et al., 2010),
and neurons (Polavarapu et al., 2005).
Tweak can activate Mitogen activated protein kinase (MAPK) pathway in some cell types.
Transforming growth factor activated kinase has been shown to be required for activation of
MAPK/ JNK pathway by Tweak / Fn14 interactions (Bhattacharjee et al., 2012; M. Kumar et al.,
2009). Tweak increases the expression of sclerotin through JNK and ERK pathways in osteoblasts
and thus reduces bone formation (Vincent et al., 2009). In addition, it down regulated bone
morphogenic protein expression (Alkaline phosphatase) and up regulated RANKL expression in
osteoblasts through activation of MAPK ERK pathway (Ando et al., 2006).
Tweak / Fn 14 pathway can be responsible for regulating normal health and physiological
status. There is evidence that, tweak causes hepatocyte proliferation which can cause liver
regeneration after injury (Burkly et al., 2011; Girgenrath et al., 2006; Jakubowski et al., 2005). It
can also regulate mesenchymal stem cell proliferation (Girgenrath et al., 2006). Moreover, it also
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acts on Fn14 receptor in mesenchymal stem cells and leads to increase in pro survival and cell
cycle responsive genes (Burkly et al., 2011; Girgenrath et al., 2006). Additionally, it can cause
blockade of myogenesis (Burkly et al., 2011; Dogra et al., 2006) adipogenesis (Burkly et al., 2011)
chrondrogenesis and osteogenesis (Perper et al., 2006). So abnormal function of this Tweak-Fn14
pathway in tissues might result in pathological tissue remodeling (Burkly et al., 2011; Zheng &
Burkly, 2008). This pathogenic tissue remodeling is important in regulating many autoimmune
and systemic disorders (W.-D. Xu et al., 2016; Zheng et al., 2008).
TNF family members are involved in wide variety of diseases like atherosclerosis,
osteoporosis, autoimmune diseases, allograft rejection and cancer (Locksley et al., 2001). Tweak
-Fn14 interactions were implicated in pathogenesis of many autoimmune disorders, systemic
diseases, cancers and neurological diseases (Burkly et al., 2011; Jeffrey A. Winkles, 2008). Tweak-
Fn14 signaling pathway in particular is associated with systemic lupus erythematosis (SLE), Lupus
nephritis, Rheumatoid arthritis, malignancies and skeletal muscle pathologies (Bodmer et al.,
2002; Burkly et al., 2011; Jeffrey A. Winkles, 2008). It also appears to be involved in various
neurological conditions like experimental autoimmune encephalitis and acute ischemic stroke and
multiple sclerosis (Burkly et al., 2011). It is quite possible that, Tweak / Fn14 pathway can be a
potential target for developing therapeutic interventions in the various systemic, autoimmune and
neurological disorders in the near future.
Tweak and Neurological Diseases
Tweak and its Fn14 receptor are expressed in tissues of central nervous system and are
found to be involved in pathogenesis of wide variety of neurological diseases. Tweak is also
expressed in neurons, astrocytes, microglia and endothelial cells (Desplat-Jego et al., 2009; Yepes,
2007; Yepes et al., 2005). Microglia but not astrocytes expressed membrane Tweak on them after
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stimulation with interferon gamma (Yepes et al., 2005). The functional roles of membrane and
secreted Tweak in glial cells are yet to be determined. Tweak acting on the endothelial cells seems
to control the permeability of blood brain barrier (Yepes, 2007). Tweak stimulates the endothelial
cells to secrete cytokines like IL-6 that can contribute to leakage of blood brain barrier (Lynch et
al., 1999). According to Polavarupu et al, Tweak contributes to disruption of BBB by NF-kB
activation and MMP-9 upregulation in brain (Polavarapu et al., 2005). Another study mentions
that, under cerebral ischemia, Tweak upregulation leads to increased expression of monocyte
chemo-attractant (MCP-1) in astrocytes, increased passage of neutrophils into ischemic area thus
causing BBB disintegration through MMP-9 production (Woldeab B. Haile et al., 2010) Tweak
can cause proliferation and gliosis which is a hallmark of many degenerative and malignant
neoplasms in central nervous system (Yepes et al., 2005). The higher expression of Fn14 in
neurons suggests that it might be having some functional / physiological role in them. According
to Tanabe et al, over expression of Fn14 receptor occurs in Dorsal root ganglions after their injury
to promote growth cone lamelipodial formation and neuronal over growth (Tanabe et al., 2003).
After neural injury, Fn14 mediates neuronal regeneration through RAC1 GTPase dependent
mechanism via its ligand Tweak (Tanabe, K. et al 2003). Apart from Fn14, its ligand Tweak also
seems to have physiological function in neurons. Tweak causes inhibition of proliferation of neural
progenitor cells from sub-ventricular region of adult mice through activation of Nf-kB pathway
and lowered expression of Hes1 transcription factor (Scholzke et al., 2011). It is interesting to note
that, Tweak induced blockage of neuronal cell proliferation is due to suppression of neuronal
differentiation but not due to neuronal cell death.
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Tweak and Cerebral Ischemia
Little is known about the predisposing factors that would enhance the expression of Tweak
and Fn14 receptor in neurons and glial cells. According to Haile et al, oxygen / glucose deprivation
was found to cause increased expression of Tweak and Fn14 in wild type neurons and wild type
astrocyte cultures (W. B. Haile, R. Echeverry, F. Wu, et al., 2010; Woldeab B. Haile et al., 2010;
Yepes et al., 2005). Oxygen / glucose deprived conditions can also cause increased Tweak / Fn14
expression in endothelial cell basement membrane – astrocyte interface (Woldeab B. Haile et al.,
2010). Tweak / Fn14 pathway in astrocytes – endothelial interface can lead to up regulation of
MCP-1 protein through activation of Nf-kB which is a strong chemotactic signal for macrophages
/ monocytes thus contributing to neuro inflammatory component in ischemic stroke (Woldeab B.
Haile et al., 2010). Tweak can stimulate endothelial cells, microglial cells and astrocytes to secrete
wide variety of pro-inflammatory cytokines that can also contribute to neuro inflammation in acute
stroke model (Bowerman et al., 2015; S. H. Kim, Kang, et al., 2004; Polavarapu et al., 2005;
Yepes, 2007; Manuel Yepes, 2013). Tweak can induce cell death by multiple mechanisms like
caspase activation, cathepsin-B mediated necrosis, TNF cooperation, MAPK, NF-kB activation
and JAK-STAT signaling (Chapman et al., 2013; Nakayama et al., 2002; Varfolomeev et al., 2012;
Vince et al., 2008; Harald Wajant, 2013). Acute Middle Cerebral Artery occlusion in mice leads
to up regulation of Tweak, Fn14, active caspase-3 with accumulation poly ADP ribose polymers
in ischemic areas as compared to contralateral normal side (Woldeab B. Haile et al., 2010). In
another experimental middle cerebral artery occulsion study, Tweak / Fn14 induced neuronal cell
death is due to activation of NF-kB pathway, caspase-3 activation and IKB alpha kinase activation
but is completely independent of TNF alpha cooperation (Woldeab B. Haile et al., 2010; Potrovita
et al., 2004). Previous studies indicate that, monitoring serum Tweak levels might be beneficial
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and their levels can correlate with prognosis and severity of neurological diseases like cerebral
ischemia. According to Inta et al, serum Tweak levels were elevated above normal within 24 h
within stroke onset (Inta et al., 2008). Within 24 h of cerebral ischemia, Tweak / Fn14 levels were
up regulated in primary cortical neurons (Potrovita et al., 2004). The elevated serum Tweak levels
within 24 h of stroke onset might represent release of excess Tweak from infarcted brain tissues
(Inta et al., 2008). Thus monitoring serum Tweak levels within 24h of stroke might indicate
severity of ischemia thereby warranting immediate therapy to rescue infarcted brain tissue.
Developing therapeutic interventions against Tweak / Fn14 receptor might afford protection
against and prevent worsening of cerebral ischemia. According to Yepes et al, administration of
soluble Fn14-Fc decoy receptor can be helpful in preventing neuronal cell death, microglial
activation and reducing infarct volume in middle cerebral artery occlusion (Yepes, 2007; Manuel
Yepes, 2013; Yepes et al., 2005). Likewise, Zhang et al, showed that administration of similar
Fn14-Fc decoy receptor within 1 hr of stroke onset can decrease NF-kB activation, MMP-9
production and blood brain barrier degradation (X. Zhang et al., 2007). Conversely other studies
propose that, Tweak / Fn14 pathway activation during cerebral ischemia might be beneficial and
offers ischemic tolerance to surviving neurons in the ischemic penumbra. According to Echeverry
et al, up regulation of Tweak / Fn14 during cerebral ischemia helps in promoting ischemic
tolerance thereby promoting survival via ERK activation, TNF alpha upregulation and inactivation
of B-cell lymphoma associated death promoter protein (Echeverry et al., 2012). Taken together,
the neuro inflammatory, neurotoxic and neuro protective role of Tweak / Fn14 pathway in acute
middle cerebral artery occlusion and cerebral ischemia still need to be investigated in the near
future.
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Tweak and Multiple Sclerosis
Multiple sclerosis is a demyelinating disease that usually has relapsing and remitting
pattern of clinical course. The main pathological hallmarks of multiple sclerosis include myelin
loss, oligodendrocyte destruction, plaque formation and accumulation of reactive astrocytes /
microglia (Iocca et al., 2008; Lucchinetti et al., 2000). Tweak is also expressed in spinal cord and
seems to be involved in the pathogenesis of diseases. Tweak and Fn14 expression is up regulated
in the spinal cord but not in encephalon in EAE and thus it can be potentially involved in
pathogenesis of experimental autoimmune encephalitis (Desplat-Jego et al., 2005; Iocca et al.,
2008; Mueller, Pedré, et al., 2005). Lymphocytes, macrophages and glial cells might be
responsible for the increased Tweak expression in spinal cord during experimental autoimmune
encephalitis (Mueller, Pedre, et al., 2005; B. Serafini et al., 2008). The exact mechanism by which
Tweak causes demyelination of the spinal cord neurons is not well known until now. In cuprizone
induced model of EAE, Tweak null mice tend to have decreased microglia accumulation and
significant delay in demyelination thus providing the evidence for involvement of Tweak in
pathophysiology of EAE (Iocca et al., 2008). Tweak can cause accumulation of reactive microglia
and decrease in oligodendrocytes in corpus callosum thus contributing to the pathogenesis of
experimental autoimmune encephalitis (EAE). Tweak / Fn14 pathway can activate multiple
signaling pathways to produce end target effects in EAE. The downstream signaling pathways that
are activated by Tweak / Fn14 interactions in EAE include NF-kB and MAPK pathways (A. Nazeri
et al., 2014). According to Nazeri et al, the various mechanisms by which Tweak mediates EAE
includes neuro degeneration, tissue remodeling, BBB disruption and astrogliosis (A. Nazeri et al.,
2014). So based on the above mentioned facts, Tweak and Fn14 would be potential targets in
reducing the severity of clinical and histological parameters in experimental autoimmune
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encephalitis. According to Desplat–Jego et al, administration of anti-Tweak monoclonal antibodies
resulted in decreased CNS inflammation, angiogenesis, neuronal loss and clinical severity on EAE
(Desplat-Jego et al., 2005). Tweak can cause endothelial cells and astrocytes to release CCL2 /
MCP-1 which is a potent chemo-attractant for inflammatory cells into CNS / spinal cord thus
amplifying neuro inflammation (Woldeab B. Haile et al., 2010; Mueller, Pedre, et al., 2005).
Vaccination with extracellular domain of Tweak / Fn14 decreased the mononuclear infiltration and
clinical severity in rat and murine variant of EAE (Mueller, Pedre, et al., 2005). T- lymphocytes
also seem to play important role in pathogenesis of MS and EAE. Administration of Fn14 – TRAIL
fusion protein reduced the clinical severity in EAE by dampening T- lymphocyte induced
inflammatory response in spinal cord and brain (Prinz-Hadad et al., 2013). Taken together, we can
assume that Tweak / Fn14 pathway can cause neuro degeneration, neuro inflammation, neuronal
loss and astrogliosis thereby perpetuating the extent of neurological injury in the mouse / rat model
of experimental autoimmune enchepalomyelitis. Further research should take place to explore the
regulation of Tweak expression and downstream signaling pathways activated to develop novel
therapeutic strategies to reduce clinical severity in MS and EAE.
Tweak and Cerebral Neoplasms
Tweak / Fn14 pathway is involved in regulation of many cancers including systemic and
brain neoplasms. Tweak and Fn14 gene expression is seen in brain tumors like Glioblastoma
Multiforme (Prinz-Hadad et al., 2013). Tweak can regulate physiological function of tumor cells.
Tweak is involved in cancer cell survival, proliferation, migration and apoptosis (Winkles et al.,
2006). Exploring the Tweak pathway can yield much more information regarding its role in cancer
cell physiology. Fn14 receptor expression tends to vary between cancerous and normal brain
tissue. Fn14 receptor is over expressed in high grade brain tumors like pilocytic astrocytoma,
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anaplastic astrocytoma, oligodendroglioma and Glioblastoma multiformae (Winkles et al., 2006).
Surprisingly, overexpression of Fn14 receptor is associated negative health outcomes. Over
expression of Fn14 receptor in malignant glioma leads to increased migration rate through Rac1 /
NF-kB dependent mechanism which ultimately leads to poor patient survival rates (Tran et al.,
2006). The capacity of cancer to metastasize and invade the neighboring tissues is usually
associated with poor clinical outcome. Angiogenesis or formation of new blood vessels is one the
most important predisposing factor for cancer metastasis (Winkles et al., 2006). Tweak can cause
endothelial cell proliferation and angiogenesis thus promoting tumor metastasis (Winkles et al.,
2006). Tumor cells produce fibroblast growth factor (FGF-2) and vascular endothelial cell growth
factor (VEGF) which can up regulate Fn14 gene expression (Donohue et al., 2003; Winkles et al.,
2006). Tweak, FGF-2 and VEGF can cooperate with each other in mediating vascular proliferation
thus causing tumor cell metastasis. The downstream signaling pathways that will be activated by
Tweak / Fn14 interactions to promote tumor migration will be a potential target for interventions
in the future. It was shown that, Tweak / Fn14 pathway in Glioblastoma causes tumor cell
migration by activating RhoG- specific guanine nucleotide specific exchange factor which is
overexpressed in these neoplasms (Fortin Ensign et al., 2013). Cancer progression is also
dependent on the ability of the tumor cells to survive and withstand cytotoxic therapy. Tweak /
Fn14 pathway tends to regulate the survival of cancerous cells by activating specific signaling
pathways. Tweak treatment or over expression of fn14 in malignant glioma results in increased
glioma cell survival and resistance to cytotoxic therapy through NF-kB dependent upregulation of
Bcl-xl / Bcl-w expression (Tran et al., 2006). Another mechanism that helps in cancer cell survival
would be local invasion of cancer cells to surrounding brain tissue that would reduce the success
rate of surgical resections. There is evidence that, Tweak can function as chemotactic factor that
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promotes Glioblastoma tumor cell migrate into surrounding brain tissue by activating Lyn Kinase
(Dhruv et al., 2014). Third mechanism by which Tweak / Fn14 pathway can increase Glioma cell
survival would be activation of Akt2 pathway (Fortin Ensign et al., 2013). Taken together, we can
assume that designing interventions against Tweak / Fn14 pathway can lead to better clinical
outcomes in high grade solid brain tumors by decreasing cancer cell survival, migration,
angiogenesis and metastasis. In one study, agonistic antiFn14 antibody is likely to be highly
efficient in hindering tumor burden in multiple xenograft models (Michaelson et al., 2011).
Another option would be to target downstream signaling pathways like NF-kB and Akt2 activated
with Tweak / Fn14 interactions that would increase clinical survival rate in these brain neoplasms.
Tweak and Blood Brain Barrier
The blood brain barrier limits the entry of large molecules from systemic circulation into
brain. It is formed collectively by microvascular endothelial cells, astrocytes, pericytes and
neurons (L. Liu et al., 2013; Rubin & Staddon, 1999). The capillary endothelial cells are bound
together tightly by tight junctions and zonula adherens which form a protective covering limiting
the entry of toxic substances into the brain circulation (Rubin et al., 1999). Tweak expressed in
endothelial cells is involved in endothelial cell proliferation and angiogenesis independent of
vascular endothelial growth factor (VEGF) (Lynch et al., 1999). Endothelial cells and astrocytes
present in BBB can secrete Tweak during inflammatory conditions thus leading to decreased
integrity of blood brain barrier (Yepes, 2007; Yepes et al., 2005). Tweak acting on the endothelial
cells controls the permeability of blood brain barrier (Yepes, 2007). Tweak stimulates the
endothelial cells to secrete cytokines like IL-6 that can contribute to leakage of blood brain barrier
(Lynch et al., 1999). According to Polavarupu et al, Tweak contributes to disruption of BBB by
NF-kB activation and MMP-9 up regulation in brain (Polavarapu et al., 2005). Another study
75
mentions that, under cerebral ischemia, increased Tweak leads to increased expression of
monocyte chemo-attractant protein (MCP-1) in astrocytes, increased passage of neutrophils into
ischemic area and increased MMP-9 production thereby mediating blood brain barrier
disintegration (Woldeab B. Haile et al., 2010). According to Stephan et al, Tweak can stimulate
capillary endothelial cells to secrete pro-inflammatory cytokines and cell adhesion molecules
through MAPK pathway thereby increasing blood brain barrier permeability (Stephan et al., 2013).
Any compromise in the integrity of the blood brain barrier can lead to entry of toxic substances,
immune cells and inflammatory cells from systemic circulation into brain circulation thereby
causing deleterious effects. According to Wen et al, Tweak induced leakage in blood brain barrier
can lead to entry of autoantibodies, activated lymphocytes and cytokines into brain thus causing
neuropsychiatric manifestations in lupus nephritis patients (Wen et al., 2013). Based on the above
mentioned facts we can conclude that, Tweak / Fn14 pathway plays an important role in regulating
the integrity of BBB. Developing therapeutic agents to block the Tweak / Fn14 pathway can
prevent leakage via NF-kB activation, MAPK activation, MMP-9 production, MCP-1 production,
basement membrane laminin degradation and neuropsychiatric manifestations in pathological
conditions of the brain (Woldeab B. Haile et al., 2010; Polavarapu et al., 2005; Stephan et al.,
2013; Wen et al., 2013; X. Zhang et al., 2007).
Tweak and Parkinson’s Disease
There are very few studies done to study the role of Tweak /Fn14 pathway in dopaminergic
neurodegeneration of PD. According to a recent study, Tweak protein expression is increased in
striatum but not in substantia nigra in MPTP treated mice (Mustafa et al., 2016a). Furthermore,
neutralizing the Tweak /Fn14 interactions with the help of anti-Tweak antibody resulted in
substantial reduction in MPTP induced neurotoxicity in Sub acute MPTP model (Mustafa et al.,
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2016a). The neutralizing effects of Tweak-FN14 blockade in subacute acute MPTP model used in
this study might be due to protective effects against apoptotic cell death or microglial activation.
In the absence of Tweak, MPTP induced dopaminergic neurodegeneration occurs via TNF alpha
induced apoptotic cell signaling events (Mustafa et al., 2016b; Ray et al., 2015; Sriram et al., 2002).
These studies indicate that, Tweak / Fn14 pathway plays a minor role in the pathogenesis of
dopaminergic neurodegeneration in PD. Further research should be done to study whether Tweak
level fluctuates in serum and CSF of patients of various stages of Parkinson disease.
Overall, interruption of Tweak / Fn14 pathway seems to have favorable outcomes in
neurological diseases like intervertebral disc degeneration, ischemic stroke, cerebral edema and
multiple sclerosis (Arash Nazeri et al., 2013; H. Wajant, 2013; W. D. Xu et al., 2016; Yepes et al.,
2005; Yepes & Winkles, 2006).
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CHAPTER 2. INVOLVEMENT OF MITOCHONDRIA MEDIATED OXIDATIVE
STRESS DEPENDENT CELL SIGNALING EVENTS AND SYK TYROSINE KINASE
ACTIVATION IN TUMOR NECROSIS LIKE WEAK STIMULATOR OF APOPTOSIS
(TWEAK) INDUCED DOPAMINERGIC CELL DEATH.
To be Submitted to Journal of Biological Chemistry
Sri Kanuri1,2, Vivek Lawana1, Huajun Jin1, Vellareddy Anantharam1, Anumantha Kanthasamy1 and
Arthi Kanthasamy 1 *
Parkinson's Disorder Research Laboratory, Iowa Center for Advanced Neurotoxicology, Dept. of
Biomedical Sciences, Iowa State University, Ames, IA.
Abstract
Neuro-inflammation, mitochondrial dysfunction, and impaired clearance of aggregate
prone proteins are implicated in Parkinson's Disease (PD) pathogenesis; however, the mechanism
by which inflammatory mediators increase the vulnerability of dopaminergic neurons to oxidative
stress-induced apoptotic cell death remain poorly characterized. Therefore, the goal of the present
study was to investigate the cell signaling events that underlie TWEAK induced apoptotic cell
death using an in vitro dopaminergic cell culture model, N27 cells. Herein, we show that N27
cells express both TWEAK and its receptor Fn14 and that TWEAK-elicited dose dependent
apoptotic cell death in N27 cells. Exposure of N27 cells to TWEAK evoked dissipation of
mitochondrial membrane potential (MMP), suppression of GSH levels, activation of caspase-8 and
3 and concomitant up regulation of Phospho-Tau and Phospho-NF-kB P65 levels. Moreover, these
changes were accompanied by the down regulation of Phospho-AKT, P-GSK3Beta (Ser9) and
LC3 levels in TWEAK treated dopaminergic neuronal cells. Intriguingly, upregulation of TWEAK
1 Department of Biomedical Sciences, Iowa State University, Ames, IA 50010
2 Primary researcher and author
*To whom correspondence should be addressed. E-mail: [email protected]
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was evidenced in MPP+ treated dopaminergic neuronal cells. Likewise TWEAK was upregulated
in the SNpc of MPTP treated mice. Consistent with the role of TWEAK in the induction of
oxidative stress response, pretreatment of N27 cells with varying concentration of quercetin, a
bioflavanoid abrogated TWEAK-induced apoptotic cell death. In a similar fashion NF-kB
inhibitor, SN50 ameliorated TWEAK-induced loss of dopaminergic cell viability. Together,
these data suggest that TWEAK exerts deleterious effects on dopaminergic neuronal survival via
aberrant activation of NF-kB and impaired mitochondrial function in an oxidative stress dependent
manner.
Keywords Tweak; Neurotoxicity; Mitochondrial dysfunction; GSK-3 beta; Tau phosphorylation;
Protesasome; Autophagy; NF-kB activation; Parkinsonism Disease.
Abbreviations: PD, Parkinson Disease; AD, Alzheimer Disease; Tweak, Tumor Necrosis Factor
Weak stimulator of apoptosis; TNF, Tumor Necrosis Factor; ALS, Autophagy Lysosomal
system; UPS, Ubiquitin Proteasomal system; SLE, Systemic Lupus Erythematosis; BBB, Blood
brain barrier; MMP, Mitochondrial membrane potential; LC3, Light Chain 3.
Introduction
PD is one of the most common neurodegenerative disease characterized by chronic
progressive dopaminergic neurodegeneration in substantia nigra. The underlying mechanisms that
predispose to demise of dopaminergic neurons are complex, but oxidative stress is hypothesized
to one important predisposing factor that accentuates dopaminergic neuronal death (Javier Blesa
et al., 2015; Vera Dias et al., 2013; Hwang, 2013; C. Zhou et al., 2008). The commonly molecular
mechanisms that perpetuate oxidative stress in PD include, dopamine metabolism, mitochondrial
dysfunction, iron metabolism, GSH depletion, ubiquitin-proteosome dysfunction and autophagy
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dysfunction (Greenamyre et al., 2001; Hwang, 2013; Juarez Olguin et al., 2016; J. Lee et al., 2012;
K. L. Lim, 2007; M. S. Medeiros et al., 2016; Smeyne & Smeyne, 2013). The markers of oxidative
stress that are present in substantia nigra of post mortem PD cases includes decrease in
polyunsaturated fatty acids (PUFA), increase in malondialdehyde, 4-hydroxy-2-nonenal and
upregulation of nitrotyrosine (C. Zhou et al., 2008). DA metabolism can induce production of
superoxide and hydrogen peroxide radicals thereby contributing to oxidative stress and cell death
(Juarez Olguin et al., 2016; Miyazaki & Asanuma, 2008). Impairment of mitochondrial complex I
of ETC (Electron Transport Chain) that is commonly associated with PD results in upregulation
of ROS production, oxidative stress and neuronal death(Greenamyre et al., 2001; Keeney et al.,
2006). Ubiquitin proteasome system dysfunction can cause accumulation of misfolded,
aggregated, damaged proteins ultimately resulting in oxidative stress and contributing to neuronal
demise (K. L. Lim, 2007). GSH depletion is noticed in 40% of postmortem substantia nigra (SN)
tissues of PD patients (Smeyne et al., 2013). Excessive iron accumulation in PD can lead to
production of oxygen radicals through fenton reaction which ultimately leads to lipid peroxidation,
oxidative stress and dopaminergic cell death (Poetini et al.). Accordingly, GSH deficiency results
in excessive ROS production, oxidative stress and dopaminergic neurodegeneration in PD
(Smeyne et al., 2013). Dysfunctional autophagy process will hasten the accumulation of damaged,
oxidized mitochondria thereby leading to excess ROS production and oxidative stress induced
demise of dopaminergic neurons in PD (J. Lee et al., 2012).
PKC-delta is a novel PKC isoform which is widely expressed in various cells and tissues
(Kikkawa et al., 2002). It is involved in numerous physiological cell functions like growth,
differentiation and apoptosis (Kikkawa et al., 2002). Members of PKC family usually have an N-
terminal regulatory region and a C-terminal catalytic region (Newton, A.C. et al 1995). It is
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activated via generation of catalytic active fragment or through tyrosine phosphorylation (Kaul et
al., 2005; Kikkawa et al., 2002). Dopaminergic neurotoxins like MPP+ and TNF-alpha can activate
the PKC-delta by proteolytic cleavage ultimately resulting in dopaminergic neurodegeneration in
cell culture and animal models of PD (Richard Gordon et al., 2012; Kaul et al., 2003). Caspase-3
induced cleavage of proapoptotic protein PKC-delta into its active form ultimately results in
dopaminergic neurodegeneration in MPP+ and Dieldrin treated N27 cells (Kanthasamy et al.,
2006; Kanthasamy et al., 2008; Y. Yang et al., 2004). Furthermore, H2O2 induced tyrosine-311
phosphorylation of PKC-delta leads to its activation of its catalytic fragment and results in
dopaminergic neurodegeneration in cell culture model of PD (Kaul et al., 2005). Another report
implicates the role of PKC-delta in NOX-1 expression, oxidative stress and dopaminergic
neurodegeneration with paraquat treatment in cell culture model of PD (Cristovao et al., 2013).
Additional studies done indicate that, PKC-delta can lead to downregulation of tyrosine
hydroxylase activity and dopamine synthesis by upregulation of protein phosphatase-2A in
dopaminergic neurons in cell culture and animal models of PD (D. Zhang et al., 2007). LPS
induced stimulation of primary microglial cells resulted in PKC delta upregulation which
subsequently resulted in dopaminergic neurodegeneration in experimental models of PD (R.
Gordon et al., 2016). Accordingly, previous studies done with resveratrol had demonstrated the
dopaminergic neuroprotection by altering PKC-delta signaling apoptotic signaling pathway and
downregulating microglial activation (Richard Gordon et al., 2010).
Tumor necrosis factor like weak stimulator of apoptosis (Tweak) is a recently
discovered cytokine belonging to TNF superfamily. Tweak is 262 amino acid type II
transmembrane protein, whereas its major receptor, Fn14 gene is located on chromosome17p13,
which is very close to P53 tumor suppressor gene (Burkly et al., 2007; Chicheportiche et al., 1997;
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Marsters et al., 1998). Cell associated Tweak exists in membrane bound form (30-35 kDa) and
soluble form (18kDa) (Chicheportiche et al., 1997; De Ketelaere et al., 2004). All the membrane
bound forms can be cleaved by specific proteases to a soluble form that can exert its actions in the
neighboring cells and distant targets (Trebing et al., 2014). Tweak expression is highest in heart,
pancreas, colon, small gut, lung, ovary, prostate, spleen, lymph nodes, and appendix
(Chicheportiche et al., 1997). Tweak exerts many of its actions via Fn14 activation (Burkly et al.,
2011; Inoue et al., 2000). Tweak / Fn14 interaction appears to be involved in angiogenesis,
inflammation, proliferation, migration, cytokine production, cytotoxicity and apoptosis (Burkly et
al., 2007; Inoue et al., 2000). Tweak / Fn14 interactions were implicated in pathogenesis of
Rheumatic arthritis, SLE and skeletal muscle disorders (Burkly et al., 2007; Trebing et al., 2014).
Tweak also appears to be involved in various neurological conditions like stroke, neuro-
inflammatory diseases and multiple sclerosis (Burkly et al., 2007). It is quite possible that, Tweak
/ Fn14 pathway can be a potential target for developing therapeutic interventions in systemic
diseases, autoimmune conditions and cancers.
Spleen tyrosine kinase (Syk) in involved in neuronal growth and differentiation
(Tsujimura et al., 2001). Numerous studies were done previously to implicate syk in neuro-
inflammation after subarachnoid hemorrhage in rats (Yue He et al., 2015; Y. He, L. Xu, B. Li, Z.
N. Guo, et al., 2015). Defective TREM2 receptor induced Syk mediated down signaling events
leads to defective microglial phagocytosis, accumulation of amyloid plaques and neuronal cell
death in Alzheimer disease (Dempsey, 2015). Syk mediated signaling events are responsible for
neuro-inflammation, gliosis, amyloid beta accumulation and tau phosphorylation in Alzheimer
disease (D. Paris et al., 2014). In microglial cells, spleen tyrosine kinase is recruited to the stress
granules leading to phagocytic dysfunction and ROS & NOS production ultimately resulting in
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neuronal cell death (S. Ghosh et al., 2015; Wolozin et al., 2015). Previous findings indicate that,
phosphorylated-syk expression is increased in neurodegenerative diseases like Alzheimer disease
and Nasu-Hakola Disease (Satoh et al., 2012; Schweig et al., 2017). Recent report suggests that
tyrosine phosphorylated Syk is found in neurofibrillary tangles of hippocampal neurons in mouse
model of Alzheimer disease that providing evidence for linking to spleen tyrosine kinase to
neurodegeneration (Köhler et al., 2017). Increased Syk expression is associated with tau
phosphorylation, amyloid beta accumulation and pathological lesions of Alzheimer disease (Satoh
et al., 2012; Schweig et al., 2017). Fc receptor mediated Syk activation is also responsible for
downstream signaling events like cytokine secretion, phagocytosis and antigen presentation in
Alzheimer Disease (Fuller et al., 2014). Recently, it was reported that, alpha-synuclein can be
phosphorylated by spleen tyrosine kinase thus implicating its role in the pathogenesis of Parkinson
Disease (Lebouvier et al., 2008). Syk induced tyrosine phosphorylation of alpha-synuclein can be
neuroprotective by decrease oligomerization and aggregation (A. Negro et al., 2002).
Although there were several studies relating Tweak / Fn14 pathway with neuro-
inflammatory diseases, stroke and neurological malignancies, thus far its role in neurodegenerative
diseases has not been vigorously investigated. However, thus far there are very few studies thus
far examining the involvement of Tweak /Fn14 pathway in in neurodegenerative disorders like
PD. Our goal is to identify the role of this Tweak / Fn14 pathway in the pathogenesis of Parkinson’s
disease. Therefore, in the present study we sought to investigate the role of TWEAK in
dopaminergic neurotoxicity a using in vitro cell culture models of PD and to further confirm and
extend our in vitro data in an MPTP mouse model of PD to identify the signaling mechanisms of
dopaminergic neurodegeneration. Here we report that TWEAK treatment-induce oxidative stress,
mitochondrial dysfunction and NF-kB activation and that inhibition of NF-kB activation and
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abrogation of mitochondrial dysfunction is associated with neuroprotection. Our results suggest
that TWEAK activation might be a critical intermediate in PD associated dopaminergic neuronal
demise and that alteration of downstream signaling mechanisms may be conducive for the
successful dismantling of the dopaminergic neuronal cells. Therefore, precise understanding of
pathogenic mechanisms and identification of new molecular targets pertaining to Tweak / Fn14
pathway would be helpful in designing novel therapeutic strategies that might attenuate or reverse
the disease progression and afford symptomatic relief in PD patients.
Materials and Methods
Chemicals and Reagents
Cell culture supplies including RPMI 1640 media, penicillin/streptomycin, L-glutamine,
fetal bovine serum were purchased from Invitrogen. Recombinant Tweak was obtained from
Pepro-Tech technologies. Caspase-3, 8, 9 & 2 substrates and MTT assay kit were purchased from
Sigma technologies, St. Louis, MO. NF-kB inhibitor (SN-50) and Quercitin were purchased from
Life Technologies. Mitochondrial membrane potential JC-1 dye was obtained from Molecular
Probes, Eugene, OR. GSH assay kit was purchased from Cayman chemicals. Tweak antibody was
purchased from Santa Cruz Biotechnology, Dallas, TX. β-actin antibody was obtained from Sigma
technologies, St. Louis, MO. LC3A/B, Beclin , Fn14, Phospho-AKT, GSK-3 Beta total, GSK-3
Beta Tyrosine 216, GSK-3 Beta Serine 9 and Phospho-Tau were purchased from Cell Signaling
Technology, MA, USA.
Cell culture and Treatment
Rat mesencephalic dopaminergic cell line, generally referred to as N27 dopaminergic
cells, was a generous donation from Dr. Kedar N. Prasad (University of Colorado Health Sciences
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Center, Denver, CO). N27 cells has been used for studying the neurotoxic mechanisms related to
Parkinson Disease in our laboratory previously (Kanthasamy et al., 2006; Kaul et al., 2003). N27
cells were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2mM L-
glutamine, 50 units penicillin and 50μg/ml streptomycin. Cell cultures were maintained under 5%
CO2 at 37°C as previously described (Y. Yang et al., 2004). MN9D cell line (Fusion of ventral
mesencephalic and neuroblastoma cells) was a kind gift from Dr. Syed Ali (National Center for
Toxicological Research/FDA, Jefferson, AR). MN9D cells were grown in Dulbecco’s modified
Eagle’s medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 50 U penicillin,
and 50 units streptomycin. N27 cells were stimulated with Tweak 100 ng for 3h, 6h, 12h and 24h
respectively. Post treatment, N27 cells were collected for caspase assay, mitochondrial membrane
potential, proteosomal assay and western blot experiments respectively.
Animal Studies.
Wild type C57 black mice were house under standard conditions at constant temperature
(22 C), 30% relative humidity with 12h light cycle as per IACUC protocol with ad libitum access
to food and water. 6-8 old week mice were used in this study. The well characterized acute MPTP
mouse model is used in this study (G. E. Meredith & D. J. Rademacher, 2011; Przedborski et al.,
2004). The mice in the MPTP treatment group received 4x 18 mg/kg of MPTP every 2h and the
control mice received equivalent saline injections. The mice were sacrificed at 3h 6h 12h 24h and
72h after the last MPTP injection. Later substantia nigra tissues were dissected out and tissues
lysates were immunoblotted against protein of interest.
MTT Assay
Cell death was determined by MTT (3-(4,5-dimethylthiazol-3-yl)-2,5-diphenyl tetrazolium
bromide) assay, in which the activity of mitochondrial dehydrogenase enzymes in cleaving
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tetrazolium ring to formazan crystals was assessed (Afeseh Ngwa et al., 2009; Kitazawa et al.,
2001; Sun et al., 2006; Sun et al., 2009). After Tweak treatment, cells were incubated in serum-
free medium containing 0.25 mg/ml MTT for 1 hr at 37°C. Supernatant was removed and MTT
formazan crystals were solubilized with 200 ul dimethyl sulfoxide (DMSO). Formation of
formazan from tetrazolium was measured at 570 nm with a reference wavelength at 630 nm using
a Spectra-Max microplate reader (Molecular Devices, Sunnyvale California).
Sytox Assay
The assessment of cell viability is done through Sytox Green dye (membrane impermeable
dye) that enters the dead cells and intercalates with DNA and produces fluorescence (Afeseh Ngwa
et al., 2009; Latchoumycandane et al., 2005). After Tweak treatment, N27 cells grown in 24 well
plate were incubated with 1 uM Sytox Green dye. DNA bound Sytox green dye produces green
fluorescence that can be detected at an excitation wavelength of 485 nm and emission wavelength
of 538 nm with the help of fluorescent plate reader (Bio-tek microplate reader)(H. Jin et al., 2011;
Sherer et al., 2007). Sytox stained cells can be observed using Nikon TE 200 fluorescence
microscope equipped with SPOT digital camera.
Western Blot Analysis of Whole Cells Lysates
Treated cells were collected and lysed with RIPA buffer (Sigma) in ice and sonicated for
15 seconds on ice for each sample. Samples were centrifuged at 14,000 g for 15 min at 4°C.
Supernatants were collected and stored at −80°C until use. Protein estimation was done using
Bradford reagent and samples with equal amount of protein were separated on 8–15% SDS-PAGE,
depending on the molecular weight of the target protein. Later they were transferred onto
nitrocellulose membranes by electro-blotting at 110V for 90 min at 4 C (Ice-cold transfer). Next,
membranes were blocked briefly for 1h by incubating them in blocking buffer and later incubated
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with primary antibodies overnight at 4°C. To ensure equal protein loading, β-actin (1:10,000) was
used as loading control. Membranes were washed several times and incubated with specific
fluorescent labelled secondary antibody for 1 h at room temperature. Membranes were scanned
using the Odyssey IR Imaging system (LICOR) and data were analyzed with Odyssey 2.0 software
(Kanthasamy et al., 2006; Kitazawa et al., 2001; Sun et al., 2006; Sun et al., 2009).
Measurement of Mitochondrial Membrane Potential (MMP).
Changes in the mitochondrial membrane potential was detected by 5,5’, 6.6’- tetrachloro-
1,1’, 3,3’-tetraethyl-benzimidazolcarbocyanineidide (JC-1 dye) (Molecular Probes, Thermo Fisher
Scientific) a cataionic fluorescent lipophilic dye. After treatment, cells were exposed with 2 ug/ml
of JC-1 dye for 20 min at 37C in a 24 well plate. Later, cells were incubated with 200 uL of 10
mM Tris-HCL containing 0.1% Triton X-100 for 30 min on an eppendorf tube shaker. Samples
were then transferred to 96-well plates and measured with fluorescence plate reader. Healthy
mitochondria form J-aggregates which displays strong red fluorescence at excitation and emission
wavelengths at 560 and 595 nm, respectively. In depolarized mitochondria, JC-1 exists as J
monomers which shows strong green fluorescent intensity with excitation and emission
wavelength at 485 and 535 nm respectively. The shift in ratio of fluorescent intensity of JC-1
aggregates (Red fluorescence) to fluorescent intensity of monomers (green fluorescence) is used
as an indicator of depolarization of mitochondrial membrane potential (M. Lin et al., 2012).
Proteasomal Assay
The UPS (Ubiquitin Proteosomal System) degradation was measured by monitoring the
proteasome activity as described in our previous publications (Sun et al., 2006; Sun et al., 2009).
Briefly, after treatment cells were re-suspended in buffer containing 50mM Tris–HCl, 1mM EDTA
and 10mM EGTA, and incubated at 37°C for 20 min. After incubation, lysates were centrifuged
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at 16,000 × g for 10 min. Supernatants were collected and incubated with fluoregenic substrate,
50μM Suc-LLVY-AMC, for determination of proteasomal activities. After 1 hour incubation with
the substrate, samples were analyzed with fluorescence plate reader (excitation 380 nm and
emission 460 nm) (Gemini Plate Reader, Molecular Devices Corporation) (M. Lin et al., 2012).
GSH assay
The mono-chlorobimane fluorometric method was used to determine the cellular GSH
levels. After treatment, cells were collected, washed with PBS, and sonicated in a lysis buffer
(50 mM Tris, pH 7.4, 5 mM EDTA and 0.001% 3, 5-di-tert-butyl-4-hydroxytoulene (BHT).
Samples were centrifuged at 14,000 g for 10 minutes at 4°C. 1 mM of mono-chlorobimane and
10 U/mL of glutathione S-transferase in 50 mM Tris, pH 7.4, were dissolved and added to the
resulting supernatant. 200 μL of the mixture was transferred to a black 96-well plate and incubated
at 24°C for 30 minutes. The fluorescence of samples was measured using a spectramax microplate
reader (Molecular Devices Corp., Sunnyvale, CA) with excitation at 485 nm and emission at
645 nm (Chandramani Shivalingappa et al., 2012)
Caspase Enzymatic Activity Assay
Caspases activity was measured using specific fluorescent substrates namely, caspase-2
(Z-VDVAD-AFC), caspase-3 (Ac-DEVD-AFC), caspase-8 (Ac-VETD-AMC) and caspase-9 (Ac-
LEHD-AMC). Briefly, after treatment, cells were lysed with lysis buffer (50mM Tris-HCl, 1mM
EDTA, and 10mM EGTA) containing 10mM digitonin for 30 mins at 37 °C. Lysates were
centrifuged at 10,000 × g for 5 mins and cell-free supernatants were collected. Cell free
supernatants were incubated with a 50 uM specific fluorescent substrate at 37 °C for 1 h. Caspases-
2, 3, 8 and 9 activity was then measured using SpectraMax Gemini XS Microplate Reader
(Molecular Devices, Sunnyvale, CA) with excitation at 400 nm and emission at 400 nm. The
150
caspase-3, 8 and 9 activities were calculated as fluorescence units per milligram of protein (M. Lin
et al., 2012).
Immunohistochemistry
Double Immuno-labelling studies in the Substantia Nigra sections were performed as per
previously published protocols (Richard Gordon et al., 2012; M. Lin et al., 2012) Briefly, sections
were grown on poly-d-lysine-coated coverslips and treated 24-48 h later. At the end of treatments,
sections were fixed with 4% PFA, washed in PBS for three times. Later sections were incubated
in blocking buffer (PBS containing 2% BSA, 0.5% Triton X-100 and 0.05% Tween 20) for 1 h at
room temperature. Next, the sections were incubated with respective primary antibodies (Tweak)
diluted in 5% serum solution overnight at 4°C. Samples were then washed several times in PBS
and incubated with Alexa-488 and -555 dye-conjugated secondary antibodies for 1h at room
temperature. After several washes, sections were dehydrated by rising concentration of ethanol
followed by xylenes and coverslips were mounted using the Prolong Gold antifade reagent
(Molecular Probes. The formation of immune-complexes were visualized using Nikon inverted
fluorescence microscope equipped with an X100 oil immersion objective and images were
captured with SPOT digital camera (R. Gordon et al., 2016).
Data Analysis
Results are presented (PRISM software, Graph Pad, San Diego) as fold induction as
compared with control group. Results represent mean ± S.E.M. Statistical analysis was
performed by using one-way ANOVA followed by Student Newman Keuls post hoc test
(PRISM software) in order to compare between groups. P values < 0.05 were considered
significant.
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Results
Expression Pattern of Tweak in the Mouse Brain.
Expression of both TWEAK and Fn14 expression has been detected in both neurons and
astrocytes; however astrocytes displayed higher levels of TWEAK expression while Fn14
expression was found to be more prominent in neurons (Yepes et al., 2005). Previous studies
have demonstrated the role of Tweak / Fn14 pathway in experimental models of various
neurological disorders including cerebral ischemia, multiple sclerosis, Encephalitis and Parkinson
Disease (Burkly et al., 2007; Mustafa et al., 2016a; Jeffrey A. Winkles, 2008), therefore, we
characterized the expression profile of membrane and soluble Tweak in various brain regions.
Tissue expression profiling as assessed via WB analysis revealed constitutive expression of
membrane bound full-length TWEAK in the hippocampus and olfactory bulb whereas prominent
expression of cleaved soluble TWEAK was evidenced in the striatum and substantia nigra.
Intriguingly cerebellum expressed very low levels of both soluble and native TWEAK. Taken
together pronounced expression of soluble TWEAK levels in the striatum and substantia nigra
suggest that TWEAK may be a key regulator of dopaminergic neuronal survival in these brain
regions. Indeed, TWEAK neutralizing body was found to rescue dopaminergic neurons in a sub-
acute MPTP mouse model further supporting the deleterious effects of TWEAK on dopaminergic
neuronal integrity (Mustafa et al., 2016a).
Tweak and Fn14 are Expressed in Dopaminergic Neurons in Culture
Tweak has been shown to be expressed in diverse cells including neurons, astrocytes and
microglia (Inoue et al., 2000). Given that rat dopaminergic neuronal (N27) cells and ventral
mesencephalic and neuroblastoma (Mn9D) cells are routinely used for the investigation of cell
signaling events underlying PD-toxin induced dopaminergic neurotoxicity we initially determined
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the Tweak protein expression levels in rat dopaminergic neuronal (N27) cells and ventral
mesencephalic and neuroblastoma (Mn9D) cells. Immunoblot analysis revealed marked
expression of soluble and relatively low levels of expression of membrane bound full-length
Tweak in N27 cells (Fig 2A). On the contrary, we found that there is increased membrane bound
full-length and relatively low Soluble levels of Tweak expression in Mn9D cells.
Tweak and its receptor Fn14 are abundantly expressed in Neurovascular Unit comprising
of perivascular astrocytes, pericytes and neurons (M. Yepes, 2013). Since Tweak’s action is
mediated via Fn14 receptor, we decided to determine its expression levels in dopaminergic
neuronal cells using immunoblot analysis. Our data revealed an inverse correlation between Fn14
expression levels and concentrations of TWEAK treatment. Specifically, increased expression of
Fn14 was observed at lower doses of Tweak (30ng) whereas decreased Fn14 expression was
observed at higher doses of Tweak (50ng and 100 ng) (Fig 2B) suggesting that TWEAK may be a
key regulator of the signaling mechanisms that are activated under injury conditions.
Upregulation of Tweak Levels in the Serum and CSF Samples of PD Patients.
Targeting Tweak/Fn14 pathway can be helpful for developing therapeutic interventions in
systemic and neurological disorders (Desplat-Jego et al., 2002; Yepes et al., 2005). Monitoring
CSF and serum Tweak levels may provide further information on the diagnostic and prognostic
potential of this inflammatory chemokine. In this context serum Tweak levels were found to be
elevated within 24 hrs following stroke onset (Inta et al., 2008). It is plausible that serum Tweak
levels can fluctuate with severity of neurodegenerative diseases and characterizing their levels
might be beneficial in monitoring disease severity and determining therapeutic efficacy of
antiparkinsonian drugs. For this purpose, we determined serum Tweak levels in human PD
patients and compared them with age- matched control subjects. We obtained human serum
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samples from Human Brain and Spinal Resource Center (VA Greater Los Angeles Health care
system). Serum samples were collected from PD patients (n=4) and healthy controls (n=4). The
levels of TWEAK were assessed via WB analysis of patients’ samples. Western blotting analysis
revealed a 2-3-fold increase in TWEAK levels in the serum of PD patients compared to age-
matched controls (Fig 3A). Likewise, a marked increase in the expression of TWEAK (controls-
4; PD samples-8) was evidenced in the CSF of PD patients as compared to age matched control
subjects (Fig3B). Further longitudinal studies should be conducted to determine whether
expression levels of TWEAK can be used to predict motor dysfunction or cognitive decline in PD
patients.
Impact of a Parkinsonian Toxin on TWEAK Expression in Dopaminergic Neuronal Cells
MPP+ is the active metabolite of MPTP that crosses the blood brain barrier and causes
death of dopaminergic neurons (Sawada et al., 2006). MPP+ is a Parkinsonian toxin that causes
distinct pattern of cell death characterized by apoptosis and necrosis in PC12 cells, cerebellar
granule cells & mesencephalic dopaminergic neurons (Sawada et al., 2006). Therefore we
evaluated whether the MPP+ altered the levels of TWEAK in N27 dopaminergic cells. N27cells
were exposed to MPP+ (300 uM) for varying duration (6, 12 & 24h). At the end of the
corresponding time points cell lysates were prepared and Tweak expression was determined using
WB analysis. Our results revealed that MPP+ induces a time dependent upregulation of Tweak in
N27 cells suggesting that TWEAK induction might be linked to dopaminergic neurotoxicity (Fig
4A).
Next, we validated our findings in an in vivo MPTP mouse model of PD. Administration
of MPTP causes selective degeneration of nigrostriatal dopaminergic neurons and leads to
pathologic changes similar to Parkinson’s disease (Sawada et al., 2006). Acute MPTP model is
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widely used to study neurodegeneration in nigrostriatal dopaminergic system in PD (N. Panicker,
H. Saminathan, H. Jin, M. Neal, & D. S. Harischandra, 2015). Recently, it was reported that
blocking Tweak / Fn14 pathway can attenuate the dopaminergic neurodegeneration in subacute
MPTP model (Mustafa et al., 2016a). With previous studies implicating the role of Tweak/Fn14
pathway in dopaminergic neurodegeneration, we next investigated whether there is an
upregulation of Tweak expression in the substantia nigra of mice subjected to acute MPTP
treatment (4x 18 mg/kg of MPTP every 2h and sacrificed at 3h 6h 12h 24h and 72h after the last
MPTP injection). Using immunoblotting studies, we found out that TWEAK levels in the mouse
substantia nigra were significantly upregulated following MPTP treatment. A time course study
revealed that the maximal TWEAK expression occurs at 3-6 h following MPTP treatment
suggesting that TWEAK may serve as an early biomarker when no motor deficits are observed
(Fig 4B).
Furthermore, we confirmed our WB data using IHC studies using an MPTP mouse model
of PD. We performed IHC studies to determine the levels of TWEAK expression in tyrosine
hydroxylase (TH) positive neurons in substantia nigra using a sub-acute MPTP model of PD. In
this model, C57black mice were injected with 25 mg/kg of MPTP once daily for 5 consecutive
days and sacrificed at 3 days after the last MPTP injection. Brains were harvested and successive
30 μm ventral midbrain sections were processed for Tweak expression. Consistent with WB
findings, immune-histochemical analysis further validated pronounced expression of TWEAK in
the TH positive neurons in the substantia nigra in MPTP treated mice suggesting that TWEAK
may serve as a biomarker for surviving dopaminergic neurons in the PD brain (Fig 4C).
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Tweak-induces Concentration-Dependent Cell Death in N27 Dopaminergic Neuronal Cells
Tweak has been shown to elicit cytotoxic effects in human peripheral blood monocytes,
human natural killer cells, murine cortical neurons, murine renal cortical cell lines, murine
mesangial cell lines and murine mammary epithelial cell lines (Burkly et al., 2011). We initially
determined whether TWEAK impacts dopaminergic neuronal viability using the well
characterized N27 dopaminergic neuronal cell culture model. N27 cells were exposed to increasing
concentration of Tweak and TNF 30 ng was used as a positive control. At the end of 24h following
drug treatment (Tweak or TNF), cell viability was assessed by MTT analysis. Our results indicate
that, Tweak at 50, 75, and 100 ng induced 10%, 30% 25% apoptotic cell death in N27 cells
indicating that it induces concentration dependent cytotoxicity in N27 dopaminergic neuronal cells
(Fig 5A). We further validated TWEAK induced dopaminergic neurotoxicity using the Sytox
staining. Sytox green dye uptake occurs following disruption of plasma membrane, usually in
apoptotic dying cells. As anticipated both MPP + (300 uM) and TWEAK (150 ng) treated N27
cells exhibited remarkable dye uptake as compared to vehicle treated cells further confirming the
cell death inducing effects of TWEAK on dopaminergic neuronal cells (Fig 5B).
Effect of Tweak on Mitochondrial Membrane Potential (MMP) and GSH
evels in N27 Cells
Tweak has been shown to alter mitochondrial membrane potential in endometrial cells and
HT29 adenocarcinoma cell lines (Sabour Alaoui et al., 2012; Wilson & Browning, 2002). It is well
established that mitochondrial dysfunction and ROS production initiates a cascade of downstream
signaling events that eventually leads to in caspase dependent apoptotic neuronal cell death (S.
Maharjan et al., 2014). Since TWEAK-induced apoptotic cell death in N27 cells we hypothesized
that mitochondrial dysfunction might contribute to dopaminergic neurotoxicity. Therefore, we
first we sought to determine whether mitochondrial dysfunction is an important upstream signaling
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event in Tweak induced apoptotic cell death in N27 dopaminergic neuronal cells. We treated N27
dopaminergic neuronal cells with Tweak for increasing duration and measured the mitochondrial
membrane potential (MMP) using the cationic lipophilic JC-1 dye. As shown in Fig 6A, a time
dependent increase in the collapse of MMP (depolarization of ΔΨm) was observed in Tweak
treated N27 dopaminergic neuronal cells. Next, we examined whether the Quercitin (Flavonoid
antioxidant) can protect dopaminergic cells from Tweak-induced mitochondrial depolarization.
N27 dopaminergic cells were pretreated for 1 hour with Quercitin (3 uM) and then subsequently
exposed to Tweak. Tweak-induced dissipation of MMP in N27 cells was attenuated in the presence
of Quercitin at later time points (12h and 24h) (Fig 6B) suggesting that mitochondrial dysfunction
may serve a critical determinant of TWEAK-induced apoptotic cell death.
Several neurological disorders are characterized by dramatic reductions in GSH levels
thereby sensitizing neurons to mitochondria associated oxidative stress insults (Mari et al., 2009).
Therefore, assessing total cellular GSH levels will provide us a partial glimpse of the antioxidant
status in TWEAK treated cells. N27 dopaminergic neuronal cells were exposed to Tweak or TNF
for 24h. At the end of the incubation period total cellular GSH levels were determined by the
mono-chlorobimane fluorometric method. As shown Fig 6 C, intracellular stores of GSH levels
were depleted by 25% at the end of 24 hours following Tweak or TNF alpha treatment. Taken
together, our studies demonstrate that mitochondrial dysfunction coupled with GSH depletion may
initiate the deleterious cascade of downstream signaling events that eventually leads to apoptotic
cell death in Tweak stimulated N27 dopaminergic neuronal cells.
Tweak-Mediated Activation of Caspase-2,3, 8 and Caspase-9 in N27 Cells
In human endometrial cells, Tweak causes apoptotic cell death through activation of
caspases 3, 7 and 9 (D. Wang et al., 2010). Caspase 2 has been linked to apoptotic cascade
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mechanism in several culture cells (S. Kumar, 2009; L. Wang et al., 1994). In fact knock out of
caspase-2 gene was found to confere resistance against MPTP-induced dopaminergic
neurotoxicity (Tiwari et al., 2011) Mitochondrial dysfunction is commonly associated with
caspase dependent neuronal death (Maharjan, S. et al 2014). Proteosomal impairment leads to
caspase-3 dependent programmed cell death in the cultured cortical neurons (Tuffy et al., 2010).
Therefore, we sought to investigate whether caspase activation occurs prior to apoptotic cell death
in Tweak stimulated N27 cells. N27 neuronal cells were exposed to Tweak for increasing duration
(3-24h). At the end of the treatment period caspase activity was measured with the help of
Fluorogenic substrates for caspase 2 (Ac-VDVAD-AFC) caspase 3 (Ac-DEVD-AMC), 8 (Ac-
LEHD-AFC) and caspase 9 (Ac-LEHD-AMC). Exposure to 100 ng Tweak resulted in a time
dependent increase in caspase 2,3,8 and 9 enzymic activity in N27 cells (Fig 7A-D). In fact,
maximal induction of caspase activity was observed at 24h post Tweak treatment in N27
dopaminergic neuronal cells. Together our studies suggest that caspase activation might be linked
to TWEAK-induced dopaminergic neurotoxicity.
Impaired Ubiquitin Proteasome system (UPS) and Auto-lysosomal system (ALS) in Tweak
treated N27 cells.
In neurodegenerative diseases, autophagy impairment leads to accumulation of damaged
organelles and pathogenic proteins thereby leading to neuronal cell death. In PD, impairment of
mitophagy leads to accumulation of damaged mitochondria and caspase mediated apoptotic cell
death in neurons (R. A. Nixon & D. S. Yang, 2012). Alpha synuclein induced blockage of
chaperone mediated autophagy (CMA) prevents its uptake and clearance by lysosomes ultimately
resulting in pathological consequences in PD (R. A. Nixon et al., 2012). Measuring the levels of
auto-phagosomal membrane associated LC3 II using western blot analysis is a reliable method of
determining autophagic activation in the cell (Tanida et al., 2008). Therefore, we sought to
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investigate whether autophagy alteration is associated with Tweak induced cell death in N27
dopaminergic neuronal cells by immunoblot analysis. N27 cells were exposed to TWEAK for
varying duration (3h, 6h, 12h and 24h), cell lysates were prepared at the end of the indicated time
period and LC3I and II levels were determined using WB analysis. We observed time dependent
decrease in LC3 II levels in N27 dopaminergic cells following exposure to Tweak (Fig 8A).
Beclin1 dysfunction is associated with neurodegenerative disorders and cancers (Kang et al.,
2011). Since beclin1 is regarded as an important factor for regulating autophagy, we next
proceeded to determine the beclin1 levels in Tweak treated N27 cells by immunoblot analysis.
N27 cells were exposed to Tweak for varying time points (3h, 6h, 12h and 24h). Post incubation,
cell lysates were prepared and beclin1levels were determined using WB analysis. Similar to LC3
II levels we observed a time dependent decrease in beclin1 expression in N27 dopaminergic cells
exposed to Tweak (Fig 8B). Thus, our studies raise the possibility that impairment in autophagic
mechanism might partially contribute to TWEAK-induced apoptotic cell death partly via
accumulation of aggregate prone proteins and damaged organelles.
Previously we demonstrated using N27 cells that proteosomal inhibition leads to
inadequate clearance and accumulation of ubiquitin positive protein aggregates within the cytosol
(Sun et al., 2005; Sun et al., 2006; Sun et al., 2007). Proteasomal dysfunction also activates caspase
dependent and caspase independent apoptosis inducing pathways in neurons (Tuffy et al., 2010).
Recent studies suggest that proteasomal inhibition promotes programmed cell death in
proliferating endothelial cells (Drexler et al., 2000). Since ubiquitin-proteasome system is
primarily involved in cellular protein degradation, assessing proteosomal activity will provide us
information regarding cellular proteostasis. Therefore, we investigated the proteosomal activity in
Tweak treated N27 dopaminergic neuronal cells. N27 cells were exposed to Tweak for increasing
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time period. At the end of the indicated time points (3h, 6h, 12h and 24h), cell lysates were
prepared for the assessment of proteosomal function. As shown in Fig 8 C, a time-dependent
decrease in proteasomal activity was observed in N27 dopaminergic neuronal cells exposed to
Tweak (100 ng). In this context, proteasomal activity was reduced as early as 3 h following Tweak
treatment, and it steadily declined in a time dependent fashion. Taken together, impairment in the
cellular degradation machinery can be regarded as a key event that predisposes dopaminergic
neurons to TWEAK-induced apoptotic cell death.
Tweak Induced NF-kb Activation in N27 Cells
Tweak has been shown to activate both canonical and non- canonical NF-kB signaling
pathways (Saitoh et al., 2003). Activation of NF-kB pathway by Tweak has been demonstrated in
glioblastoma cells (Tran et al., 2005), keratinocytes (L. Jin et al., 2004), and renal tubular cells (A.
B. Sanz et al., 2010), and neurons (Polavarapu et al., 2005). Persistent activation of NF-kB
signaling pathway has been identified as a critical signaling event in chronic inflammatory diseases
(Hoesel & Schmid, 2013). Exposure of PC12 cells to 6-OHDA has been shown to induce NF Kb
activation via IkB alpha degradation and subsequent translocation of NF-kB dimers into nucleus
thereby leading to the induction of pro-apoptotic effects (Tarabin & Schwaninger, 2004). Given
the importance of NF-kB pathway in cell death mechanisms we investigated the role of NF-kB
signaling pathway in Tweak induced neuronal cell death in N27 cells. For this purpose, initially
we measured Tweak induced IkB-alpha degradation in N27 dopaminergic neuronal cells by
immunoblot analysis. N27 cells were exposed to TWEAK for increasing time period (6h, 12h and
24h), and cell lysates were prepared at the end of the indicated time points and immunoblotted for
IKB-alpha. As anticipated a time dependent increase in IkB-alpha degradation was evidenced in
Tweak treated N27 cells indicative of NF-kB activation (Fig 9A).
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To further investigate the mechanism of NF-kB signaling pathway, we next examined
phosphorylation of P65 in Tweak treated N27 cells by immunoblot analysis. N27 cells were
subjected to the same experimental conditions as detailed above and the magnitude of p65
phosphorylation was assessed via WB analysis. Our data indicated a time dependent increase in
P65 phosphorylation in Tweak treated N27 cells (Fig 9B). As shown above SN50, NF-kB inhibitor
attenuated TWEAK-induced apoptotic cell death further supporting the role of NF-kB activation
in the mechanism of TWEAK-induced dopaminergic neurodegeneration (Fig 9C).
Tweak Induced Downregulation of Phospho-AKT is Associated With GSK-3 Beta Activation
and Tau Phosphorylation.
Tweak exerts diverts effects via the activation of P13K/ AKT pathway in a cell type
specific manner. For example, Tweak can cause skeletal muscle wasting through inhibition of
P13K/ AKT pathway (C. Dogra et al., 2007). On the contrary, it promotes tumor cell survival
through activation of AKT pathway (L. Wu et al., 2013). Given the importance of P13K/ AKT
pathway in regulation of cell survival, we determined the activation status of Akt. So, we first
assessed phospho-AKT levels in Tweak treated N27 cells using immunoblot analysis. N27
dopaminergic neuronal cells were exposed to Tweak for varying duration (6-24h) and at the end
of the incubation period cell lysates were immunoblotted for Phospho-AKT. We found that Tweak
induced the downregulation of Phospho-AKT in dopaminergic cells (Fig 10A). These results
suggest that Tweak induced downregulation of Phospho-AKT levels may increase the
vulnerability of dopaminergic neuronal cells to pro-apoptotic insults.
Dysregulation of PI3K/Akt signaling event has been linked to PD neuropathology Studies
conducted in post mortem PD brains revealed a reduction in the ratio of p-Akt/t-Akt (Timmons et
al., 2009). Several drugs that are prescribed for PD treatment exert their therapeutic effects via
Akt activation (Aleyasin et al., 2010; Fernandez-Gomez et al., 2006; L. A. Greene et al., 2011; J.
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H. Lim et al., 2008; Nakaso et al., 2008; Salinas et al., 2001; Tasaki et al., 2010). For example
rasagiline has been shown to protect nigral dopaminergic neurons via activation of PI3K/Akt
signaling mechanism (Weinreb et al., 2006). Therefore, in the present study we sought to
determine Akt activation in TWEAK treated cells. Tweak treatment evoked a marked down
regulation of p-Akt levels at all-time points tested. Given that PI3K/Akt is a key upstream
regulator of GSK-3b, the down regulation of Akt might be associated with GSK-3b activation and
resultant cell death (H. Zhang et al., 2011). Together our studies suggest that impairment in the
PI3K/Akt pathway may underlie TWEAK-induced dopaminergic neurotoxicity owing to
compromised cell survival machinery.
Aberrant GSK-3 activity has been linked to numerous neurodegenerative disorders
including AD and PD (Credle, George, et al., 2015). In fact GSK 3 activity has been shown to
induce neuronal apoptosis and conversely GSK inhibitors have been shown to elicit
neuroprotective and antiapoptotic effects in several cellular and mouse models (Beurel & Jope,
2006; Pap & Cooper, 1998). Furthermore, PI3/Akt signaling pathway has been linked to the
inactivation of GSK-3b activity via phosphorylation at the Ser9 site (M.-H. Liang & Chuang,
2006). Having demonstrated that TWEAK-induced Akt down regulation, we hypothesized that
the inhibitory state of GSK 3b will be down regulated (GSK-3b ser9) while the activated state of
GSK-3b will be upregulated thereby facilitating the mechanism of dopaminergic cell death in
TWEAK treated cells. First, we assessed the response of GSK-3b signaling events in TWEAK
treated N27 dopaminergic cells at various time points. An increase in phosphorylation of GSK 3b
(pGSK-3 Beta Tyrosine 216) was evidenced as early as 6h and remained elevated for the reminder
of the treatment period (Fig 10C). Conversely down regulation of pGSK-3b Serine 9 was observed
in TWEAK treated cells, consistent with neurodegeneration (Fig 10D). In the Tet-inducible GSK-
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3b models postnatal expression of tet-inducible expression of GSK-3b-S9A (kinase active)
transgene led to neurodegeneration in the cortex striatum and hippocampus (Credle, George, et al.,
2015) further confirming the neuroprotective role of this kinase inactive state of GSK-3b. GSK-
3b has been shown to cause abnormal Tau phosphorylation. In fact his kinase has been linked to
a-synuclein-induced tau phosphorylation. Consistent with this, inhibition of GSK-3b was found
to inhibit accumulation of a-synuclein and p-tau formation in an MPTP mouse model of PD (Duka
et al., 2006). Given that Tau is a substrate of GSK-3b we assessed the levels of pTau in N27 cells
treated with TWEAK. An increase in p-tau was evidenced as early as 6h post drug treatment and
maximal induction was observed at 12h post TWEAK treatment (Fig 10E). Consistent with
previous reports a positive correlation between GSK-3 b activation and hyperphosphorylation of
p-Tau further suggest that functional interaction between GSk-3b and Tau coupled with
proteasomal dysfunction may lead to excessive accumulation of Tau ultimately leading to
dopaminergic neurodegeneration.
Discussion
Tumor necrosis factor like weak stimulator of apoptosis (Tweak) is a recently discovered
cytokine belonging to TNF superfamily. TWEAK has been implicated in numerous neurological
conditions, including Stroke, Neuro-inflammatory diseases, Glioblastoma Multiforme and
Multiple sclerosis(Bodmer et al., 2002; Burkly et al., 2007). We identified that Tumor Necrosis
Factor like Weak inhibitor of apoptosis (TWEAK) through targeted qPCR array which is
significantly up regulated in immortalized dopaminergic neuronal cells (N27 cells) following
inflammatory insult. In our study, initially we demonstrated differential Tweak expression in
dopaminergic neuronal cells and distinct brain regions. Next, we showed that, Tweak induces dose
dependent neurotoxicity in rat dopaminergic neuronal cells. Furthermore, we demonstrated that,
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there is marked upregulation of TWEAK levels in the mouse substantia in MPTP treated mice.
Additionally, investigation into downstream signaling mechanisms revealed that, TWEAK-
induced dissipation of MMP and consequent reduction in GSH levels might linked to TWEAK-
induced dopaminergic neurotoxicity. Furthermore, upregulation of redox sensitive kinases namely
SYK and PKC delta was accompanied by a concomitant down regulation of UPS and autophagic
mechanisms further highlighting the importance of cellular redox machinery in the modulation of
cellular protein quality control machinery in TWEAK treated cells. In this context, our studies
raise the possibility that dysregulated Akt/GSK/Tau signaling events might atleast in part
contribute to dopaminergic cell death mechanism owing to accumulation of aggregate prone
proteins. Intriguingly SN-50, NF-kB inhibitor and Quercetin (Flavonoid) attenuated cell death
and improved mitochondrial function thereby resulting in increased survival of dopaminergic
neuronal cells. Together, these findings suggest that TWEAK induced dopaminergic
neuropathology may be linked to PD pathogenesis. A schematic of Tweak-induced dopaminergic
cell death is depicted in Fig 3.6.
Neurons, astrocytes and microglia exhibit differential TWEAK expression pattern
(Desplat-Jego et al., 2002). For example, Tweak is expressed in primary murine neurons and
upregulated following OGD (oxygen glucose deprivation) (J. A. Winkles, 2008). Consistent with
this report our study is the first to demonstrate higher basal levels of membrane bound full-length
TWEAK in the hippocampus and olfactory bulb while higher basal levels of cleaved soluble
TWEAK was evidenced in the striatum and substantia nigra of C57 black mice. This differential
expression of Tweak in various brain regions and different cell types might imply that Tweak /
Fn14 pathway might elicit differential effects under pathological and physiological conditions.
Fn14 receptor is upregulated in PC12 cells by nerve growth factor treatment and promotes neuritic
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overgrowth and lamellipodia formation (Tanabe et al., 2003). Upregulation of Fn14 is associated
many inflammatory, neurological diseases and malignancies (J. A. Winkles, 2008). We found that,
Tweak upregulates Fn14 expression at lower concentration (30ng) with subsequent
downregulation at higher doses. It is plausible that TWEAK might induce receptor desensitization
at higher concentrations. Thus, further studies are warranted to study the effects of TWEAK on
Fn14 levels. Serum Tweak levels positively correlate with disease activity in Psoriatic arthritis /
Rheumatic arthritis and confirms its pathogenic role in the production of pro-inflammatory
cytokines (M. C. Park et al., 2008; Xia et al., 2011). Interestingly, our studies revealed a 2-3-fold
increase in TWEAK levels in the serum of PD patients compared to age-matched control patients
suggesting that TWEAK may serve as a biomarker for disease progression.
Administration of MPTP to mice causes selective degeneration of nigrostriatal
dopaminergic neurons and leads to pathologic changes similar to Parkinson’s disease (W. S. Choi
et al., 1999). Changes in neuro-inflammatory markers in PD patients showed increased levels of
TNF-alpha and IL-6, which may be responsible for mediating neuronal death (Dexter & Jenner,
2013; Dobbs et al., 1999). Previously we demonstrated that TNF-alpha-induced dopaminergic
apoptotic cell death through PKC delta proteolytic cleavage mechanism (Richard Gordon et al.,
2012) further supporting the pro apoptotic effects of TNF-alpha on dopaminergic neurons
(Gahring et al., 1996). Moreover, in a recent report blocking Tweak / Fn14 pathway was found to
attenuate dopaminergic neurodegeneration in a subacute MPTP model (Mustafa et al., 2016a). In
contrast to this report, Western blot analysis revealed marked upregulation of Tweak levels in the
mouse substantia nigra occuring at 3-6 h following MPTP treatment. Likewise, upregulation of
Tweak expression was observed in the TH positive neurons in the substantia nigra as determined
by IHC. Together our studies support the notion that TWEAK may serve as an oxidative stress
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sensitive pro-apoptotic factor that increases the vulnerability of dopaminergic neurons to PD
pathogenesis.
NF-kB has been shown to be upregulated in post mortem PD brains (A. Ghosh et
al., 2007). For example selective inhibition of the activation of NF-kB was found to protect
dopaminergic neurons in MPTP-intoxicated mice (A. Ghosh et al., 2007). In addition it has been
demonstrated that PD neurotoxin such as 6_OHDA- induced NF-kB activation elicited a
proapoptotic effect in PC12 cells (Tarabin et al., 2004). NF-kB are activated in response to
cytokines, pathogens, and other cell injuries (Camandola & Mattson, 2007). NF-kB exists as a
homodimer and heterodimer and these dimers are retained in the cytoplasm in their inactive state
via IkB repressor protein. Signal transduction via the p50:p65 heterodimer is involved in the
canonical pathway. Following stimulation by diverse ligands including TNFa, a signaling complex
is formed which leads to the activation of IkB kinase (IKK). IKK catalyze the phosphorylation
IkB and subsequent degradation via the UPS which leads to the nuclear translocation of p50:p65
and transcriptional activation of downstream NF-kB target genes (Kopitar-Jerala, 2015). Tweak
has been shown to activate the canonical and non-canonical NF-kB pathways (T. Dohi & L. C.
Burkly, 2012; Polavarapu et al., 2005; Saitoh et al., 2003). Both pathways have been shown to
require cIAP1 which is a critical component of downstream signaling events mediated by TWEAK
(Enwere et al., 2014). TWEAK-Fn14 has been demonstrated to activate canonical NF-kB
signaling in numerous cell types (L. Jin et al., 2004; Polavarapu et al., 2005; A. B. Sanz et al.,
2010; Tran et al., 2005). For example, TWEAK was found to induce apoptosis of cortical neurons
subjected to OGD atleast in part via NF-kB activation (Potrovita et al., 2004) and has been
postulated to play a role in the post ischemic inflammatory response. In another study, activation
of the canonical NFkB signaling pathway was linked to TWEAK-induced muscle protein
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degradation (Al-Sawaf et al., 2014; Charu Dogra et al., 2007). Consistent with these reports
marked induction of IkB degradation and p65 phosphorylation was evidenced in TWEAK
stimulated cells and as anticipated inhibition of NFkB activation via SN50 resulted in abrogation
of TWEAK-induced cell death. Indeed in a recent report Nrf2 mediated repression of NfkB was
found to protect against TWEAK-induced cell death following ischemia reperfusion in Nrf2-KO
mice (Al-Sawaf et al., 2014). Taken together these data indicate a role for NFkB in TWEAK-
induced induced dopaminergic cell death.
In the intrinsic apoptotic pathway loss of mitochondrial membrane permeability
leads to cytochrome c release and formation of the apoptosome complex encompassing
cytochrome c, APAF, ATP, and procaspase 9 in the cytoplasm which finally leads to cell death in
a caspase-3 dependent manner (Ghavami et al., 2014). Alternatively, the extrinsic apoptotic
pathway involves the activation of death receptors located in the plasma membrane such as TNF-
R. Stimulation of this receptor leads to receptor trimerization to which FADD and procaspase-8
has been shown to bind. Subsequently, activation of caspase-8 leads to the activation of caspase-
3 thereby completing the process of apoptotic cell death. Among the caspases, caspase-2, -8, -9
and -10 constitute the initiator caspases whereas caspase-3, -6, and -7 represent the effector
(Gómez-Sintes et al., 2011). Initiator caspase participate in the cleavage of the effector caspase
and cleaved effector caspase, in turn, cleave other protein substrates that are involved in apoptotic
cell death such as PARP (Gómez-Sintes et al., 2011). In the present study we found a positive
association between that the activation of Caspase-2,-8,-9, and cell death which suggest that
caspase-3 mediated activation of PARP cleavage is probably involved in TWEAK-induced
dopaminergic cell death. In addition GSK-3 has been shown to activate the intrinsic apoptotic
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pathway via its effects on Bax (Linseman et al., 2004), Bim (Hongisto et al., 2003), VDAC
(Pastorino et al., 2005) thereby contributing to mitochondrial dysfunction. In another study
thapsigargin, and ER stress inducer not only induced caspase-3 activation but also GSK-3 activity
supporting the positive impact of GSK-3 on caspase-3 mediated intrinsic cell death signaling
events (L. Song et al., 2002). Consistent with these reports, in the present study loss of
mitochondrial membrane potential preceded both GSK-3b and caspase-3 activation suggesting
functional interaction between mitochondrial dysfunction and GSK-3b activation via a caspase-3
dependent mechanism in TWEAK-induced apoptotic cell death.
The PI3K/Akt is one among the well characterized cell survival signaling
mechanisms. Ketamine induced downregulation of phospho-AKT and upregulation of GSK-3
beta phosphorylation leads to apoptosis of cultured rat cortical neurons (Shang et al., 2007). On
the other hand, erythropoietin induced activation of AKT levels and associated downregulation of
GSK-3 beta phosphorylation enhances cellular survival in rat cortical neurons (Shang et al., 2007).
Consistent with previous findings, downregulation of Phospho-AKT is positively associated with
GSK-3 beta activation and Tau Phosphorylation in Tweak treated N27 dopaminergic neuronal
cells suggesting that down regulation of Akt activation coupled with activation of GSK-3b may
underlie the increased vulnerability of dopaminergic neurons to TWEAK-induced apoptotic cell
death. In line with our finding a previous report showed that TWEAK inhibited phosphorylation
of Akt and its downstream target GSK-3b, suggesting that inhibition of PI3K/Akt signaling
mechanism might contribute to skeletal muscle atrophy in response to TWEAK (Charu Dogra et
al., 2007). These results suggest the existence of a possible negative feedback loop between Akt
and GSK3b and that the lack of Akt dependent inactivation of GSK-3b (GSK-3bY265) might have
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contributed to the neurotoxic mechanisms and associated dopaminergic cell death in TWEAK
treated cells.
It is well established that impairment in GSK3b contribute to the induction of cell
death signaling events that ultimately promote neuropathology in numerous neurodegenerative
diseases including PD (Petit-Paitel, 2010). In this context, a previous study revealed that GSK-3
beta is upregulated in the substantia nigra and that GSK-3b is localized within the halo of Lewy
bodies in post mortem PD brains suggesting that GSK-3b plays a role in PD pathogenesis (Nagao
& Hayashi, 2009). Furthermore in a recent report impairment in GSK-3 beta function was found
to contribute to the accumulation of tau and a-synuclein which in turn was found to be associated
with Parkinsonian neuropathology in a brain region specific manner (Credle, George, et al., 2015).
Consistent with this report a positive association between GSK3b activation and Tau
hyperphosphorylation was evidenced in TWEAK treated cells. To what extent GSK3b mediated
Tau hyperphosphorylation contributes to TWEAK-induced neuronal cell death remains to be
investigated.
It is well accepted that disruption of cellular protein quality control
characterized by accumulation of damaged organelles and misfolded proteins in the dopaminergic
neurons is a critical component of PD pathogenesis (Daniel J. Klionsky, 2007). Accumulation of
aggregate prone proteins can result from impaired protein clearance mechanisms owing to
sequential dysfunction of the ubiquitin proteasome system (UPS) and the autophagy lysosomal
system (ALS). Oxidative stress has been identified as key factor facilitating impaired proteostasis
(Janda et al., 2012). Using disease models and PD brains it has been implicated that dysfunction
in the autophagic process and UPS may be linked to PD pathogenesis (Dehay et al., 2010;
Ebrahimi-Fakhari et al., 2012; Janda et al., 2012). Additionally, enhancement of autophagy has
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been shown to be beneficial in experimental models of PD (Spencer et al., 2009). Previously, we
reported in N27 dopaminergic neuronal cells, that methamphetamine (MA)-induced oxidative
stress via PKC delta activation contributes to impairment in the UPS and autophagy (M. Lin et al.,
2012). More recently we demonstrated that impairment in the autophagic mechanisms via c-Abl,
a redox sensitive tyrosine kinase, may contribute to enhanced microglial activation response
characterized the activation of NLRP3 related markers in dopamine enriched brain regions
(Lawana et al., 2017). Consistent with these reports in the present study we demonstrated that
depletion of autophagic markers and down regulation of UPS coincided with proteolytic cleavage
of redox sensitive kinases such as PKC delta and SYK activation supporting the role of elevated
oxidative stress upon impairment of cellular proteostasis. In this context, both MMP collapse and
ROS generation preceded the disruption of cellular protein quality control and activation of redox
sensitive kinases further highlighting the potential involvement of oxidative stress in impaired
cellular proteostasis upon TWEAK stimulation of N27 cells. This is in accordance with a previous
report from our lab demonstrating that proteosomal inhibition via MG132 in N27 cells is
associated with oxidative stress response and ultimately cell death. (Sun et al., 2006). Further,
uncovering the role of autophagy and UPS in TWEAK-induced neurodegeneration may lead to
improved understanding of cellular mechanisms underlying dopaminergic neuropathology in PD.
Immunoreceptors have been shown to act as either an activator when present in
immunoreceptor tyrosine-based activation motifs (ITAMs) or as an inhibitor when present with
immunoreceptor tyrosine based inhibition motifs (ITIMs). Coordinated activation of Src and SYK
kinase families have been implicated in ITAM based signaling cascade (C. A. Lowell, 2011). Syk
tyrosine kinase is expressed in numerous cells including hematopoietic cells (Shigeru Yanagi et
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al., 2001). In a previous report Syk expression has been demonstrated in the neuronal cytoplasm
and SYK was found to phosphorylate synuclein (Alessandro Negro et al., 2001). In another report
Syk was found to phosphorylate Tau on Y18, and the interaction between kinase and the substrate
was demonstrated (Lebouvier et al., 2008). Intriguingly, neurofibrillary tangle (NFT) has been
shown to contain a phosphorylated Y18 residue, suggesting that SYK may be linked to AD
pathophysiology (G. J. Ho et al., 2005; Williamson et al., 2002). Consistent with this report WB
analysis performed in cell lysates from TWEAK treated cells displayed increased SYK activation
that positively correlated with proteolytic cleavage of PKC delta at 24h. Moreover, Tyr 525/526-
phosphorylated SYK and pTyr18 was present in granulovacuolar inclusions and in neurons with
neuro fibrillary tau pathology (Köhler et al., 2017) further supporting the neurodegenerative role
of SYK. Conversely, in another report Paris et al., (D. Paris et al., 2014) demonstrated that SYK
inhibition reduces Aβ production and increases the clearance of Ab across the blood brain barrier
(BBB). Together our data highlight that SYK activation may be linked to disruption of protein
clearance machinery thereby leading to the accumulation of aggregate prone proteins and
ultimately dopaminergic neurodegeneration presumably via an oxidative stress dependent
mechanism.
In conclusion, we provide the first evidence that TWEAK-induced dopaminergic
neurodegeneration via mitochondria mediated and caspase-3 mediated mechanism as well as a
SYK dependent mechanism. TWEAK could serve as a potential biomarker for PD based on the
observation that TWEAK levels were elevated in the serum of PD patients. Additionally, SYK
could serve as a novel target for the development of novel therapeutics for PD.
171
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Figures
Fig 1: Expression pattern of Tweak in the mouse brain. C57 black mice were selected for determining
Tweak expression in various brain regions. Untreated C57 black mice were sacrificed and various brain
regions such as substantia nigra, striatum, hippocampus, olfactory bulb, cortex and cerebellum were
dissected out. Brain tissue lysates were prepared and probed using a rabbit polyclonal antibody to Tweak
that detects membrane and active cleaved form. Optical densities of protein bands were analyzed with the
Odyssey imaging system (LI-COR) and -actin was used a loading control. Band intensities of membrane
and active cleaved Tweak were quantified with densitometric analysis and are normalized to -actin.
Representative immunoblot of at least 3 independent experiments was shown. The densitometric analysis
of normalized band intensity for each treatment group expressed as percentage of control group was plotted
on histogram. Statistical analysis was carried out using Anova followed by Student Newman Keul’s post
test. Asterisk (* p < 0.05,** p < 0.01) represents significant differences between Tweak expression between
various brain regions.
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Fig 2. Tweak and Fn14 are expressed in dopaminergic neurons in culture. A. Differential Tweak
expression in N27 cells and Mn9D cells. N27 cells (Rat dopaminergic neuronal cell line) and Mn9D cells
(fusion of embryonic ventral mesencephalic and neuroblastoma cells) were used to determine Tweak
expression in dopaminergic neuronal cells. N27 cells and Mn9D cells were grown in culture separately. Later,
untreated N27 cells and Mn9D cells were collected and lysates were prepared. Next, whole cell lysates were
probed with rabbit polyclonal antibody to Tweak that detects membrane and cleaved soluble Tweak. Odyssey
imaging system (LI-COR) was used to analyze the protein bands. Band intensities were normalized with -
actin for equal loading and quantified with densitometric analysis. Representative immunoblot of three
independent experiment was shown. Histogram represents normalized band intensity for each treatment group
expressed as percentage of control group. Statistical analysis was carried out using Anova followed by Student
Newman Keul’s post test. Asterisks (* p < 0.05 and** p < 0.01) represent significant differences between
membrane and soluble Tweak expression in N27 cells and Mn9D cells. B. Fn14 (Tweak receptor) upregulation
in Tweak stimulated N27 cells. Rat dopaminergic N27 cells treated with 30 ng/ml, 50 ng/ml and 100 ng/ml
Tweak for 24h. Post incubation whole cell lysates were prepared and resolved with 15% SDS-PAGE. Later
they were subjected to immunoblot analysis using antibodies specific to Fn14 receptor. Membranes were re-
probed with -actin to ensure equal protein loading. Band intensity was analyzed with the Odyssey imaging
system (LI-COR). Representative immunoblot of at least 3 independent experiments was shown. Histogram
represents normalized band intensity for each treatment group expressed as percentage of control group.
Statistical analysis was carried out using Anova followed by Student Newman Keul’s post test. Asterisk (** p
< 0.01) represents significant differences in Fn14 expression between control and Tweak treated groups.
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Fig 3 Upregulation of Tweak levels in the serum and CSF samples of Human PD patients. A-
B. Serum and CSF samples of PD patients and age matched controls were obtained from Human
Brain and Spinal Resource Center (VA Greater Los Angeles Health care system). Representative
western blot for Tweak expression in serum (Fig A) and CSF (Fig B) from human PD patients and
age matched controls probed using rabbit polyclonal antibody directed against Tweak. Increased
Tweak levels were evident in PD patient samples as compared to age matched control patients.
Densitometric band intensities for Tweak determined from control and PD samples expressed as
fold of control. Statistical analysis was carried out using student T test.
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189
Fig 4 Impact of Parkinsonian toxin on Tweak expression in dopaminergic neuronal cells A.
Time dependent increase in Tweak expression in MPP+ treated N27 dopaminergic neuronal cells.
N27 cells were treated MPP 300 uM for 6h,12h and 24h. Post treatment, whole cell lysates were
prepared. 40 ug of protein was loaded and resolved with 15% SDS-PAGE. Later they were
subjected to immunoblot analysis with rabbit polyclonal Tweak antibody that detects membrane
and active cleaved Tweak. Membranes were also probed with -actin to ensure equal protein
loading. A time dependent increase in Tweak expression was evidenced in MPP+ treated
dopaminergic neuronal cells which peaked at 12h and 24h. Experiments were repeated three times
and a representative western-blot was shown. The bar graph represents band intensities for the
cleaved Tweak from three blots quantified using densitometric analysis and normalized to -actin.
Asterisk (* p < 0.05) represents significant differences in between MPP+ treated groups and
controls using one-way ANOVA with bonferroni post test comparision. B. Tweak upregulation in
nigral dopaminergic neurons in Acute MPTP animal model of Parkinson Disease. In this model,
C57 black mice were injected with four MPTP doses (18 mg/kg) intraperitoneally and were
euthanized at 3h, 6h, 12h, 24h and 72h post MPTP injection. Substantia nigra was dissected out
and tissue lysates were prepared from mice treated with either MPTP or saline. Later, they were
subjected to immunoblot analysis with Tweak antibody that detects membrane and soluble Tweak.
-actin was used a loading control for equal loading of samples. Cleaved Tweak band intensity
was quantified by densitometric analysis after normalization to β-actin. Experiment was repeated
for three times with 4 mice per group. Representative immuno-blot indicates maximum Tweak
expression occurs at 3h and 6h in Substantia Nigra (Fig B) of MPTP treated mice as compared to
control mice. Asterisk (*p<0.05) significant differences in between MPTP treated groups and
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control group using one-way ANOVA with bonferroni post test comparison. C. Double
immunolabelling IHC for Tweak and TH in mouse nigral sections in subacute MPTP animal
model of Parkinson Disease. In this model, C57black mice were injected with 25 mg/kg of MPTP
once daily for 5 consecutive days and sacrificed at 3 days after the last MPTP injection. Later
successive 30 μm ventral midbrain sections will be cut with a cryostat and processed for immuno-
histochemical analysis of Tweak and TH expression with double immuno labeling studies.
Experiments were repeated three times and representative images were shown. Marked Tweak
expression in the TH+ neurons along the nigral tract of the MPTP treated mice (Fig C) .
Fig 5. Tweak induces concentration dependent cell death in N27 dopaminergic neuronal
cells. A. Assessment of dopaminergic neuronal cell viability with Tweak and TNF with MTT
analysis. N27 cells were treated with Tweak 30 ng, Tweak 50 ng, Tweak 75 ng, Tweak 100 ng and
TNF 30 ng for 24h. Post incubation, cell viability was assessed by measuring the formation of
formazan crystals from tetrazolium inside the active mitochondria. The mitochondrial activity
decreased significantly after exposure Tweak and TNF for 24 h . Cell survival was presented as
percent of control. Bar graph values are representative of mean ± S.E.M. for each experiment
performed with n = 8. Asterisk (p***<0.001) represents significant differences between Tweak
treated groups and control groups as determined by ANOVA followed by Student Newman Keul’s
test. B. Visualization of MPP+ and Tweak toxicity in dopaminergic neurons by Sytox assay. N27
cells were treated with MPP 300 uM, Tweak 300 ng and Tweak 150 ng for 24h. Post treatment,
cells were incubated with with 1uM Sytox green dye and cell death was determined by Sytox green
imaging. Visualization of Sytox positive cells was done under fluorescence microscopy with
excitation of 485 nm and emission of 538 nm. Increased green florescence is evident with Tweak
and MPP+ treatment indicating significant neurotoxicity in dopaminergic neuronal cells.
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Fig 6. Effect of Tweak on Mitochondrial Membrane potential (MMP) and GSH levels in N27
cells A. Time course studies of the impact of Tweak with and without Quercitin on MMP using
JC-1 fluorometry. N27 dopaminergic neuronal cells were treated with Tweak 100 ng for 3h,6h,12h
and 24h with and without Quercitin 3 uM. Post treatment, lysates were collected and incubated
with JC-1 dye for 30 min. Post incubation, changes in mitochondrial membrane potential were
measured spectrophotometrically using redox sensitive JC-1 dye with the help of Gemini
Fluorescence plate reader. Reduction in red: green absorbance ratio of JC-1 dye indicate the loss
of mitochondrial membrane potential (Δψm
). Each experiment is repeated three times with n=8.
Significant time dependent dissipation of mitochondrial membrane potential was evidenced in
dopaminergic neuronal cells upon exposure to Tweak which was partially reversed with quercitin.
Asterisk (**p<0.01, p***<0.001) represents significant differences between Tweak treated groups
and control group as determined by ANOVA followed by Student Newman Keul’s test. B.
Quercitin (Flavonoid antioxidant) offers partial protection against Tweak induced apoptotic cell
death in N27 cells. N27 cells were treated with Tweak 100ng with or without Quercitin for 3h, 6h,
12h and 24h. Post incubation, cell viability was assessed using MTT analysis which is based on
formation of formazan crystals. Tweak induced cell death in dopaminergic neuronal cells is
partially reversed by Quercitin. Each experiment is repeated for three times with n = 8. Asterisks
6A
6B
6C
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(**p<0.01, p***<0.001) represent significant differences between Tweak / Quercitin treated
groups and control group as determined by ANOVA followed by Student Newman Keul’s test. C.
GSH depletion with Tweak and TNF treatment in N27 cells. Rat dopaminergic N27 cells treated
with 100 ng/ml Tweak and TNF 30 ng for 24h. Post-treatment, Tweak induced reduction in GSH
levels was measured by with Monochlorobimane fluometric method. Formation of MCB-GSH
complex was measured with spectrophotometer at Emission 460 nm and Excitation 360 nm to
measure the GSH levels in Tweak and TNF treated N27 cells. Each experiment is repeated three
times with n= 4. Values are expressed as percentage of GSH compared with untreated control.
Significant depletion of GSH levels (25%) were observed in dopaminergic neuronal cells upon
exposure with Tweak and TNF for 24h. Asterisk (*p<0.05) represents significant differences
between Tweak / TNF treated groups and control group as determined by ANOVA followed by
Student Newman Keul’s test.
Fig 7. Tweak induces extrinsic and intrinsic caspase activation in N27 cells. A-D. N27 cells were
treated with Tweak 100ng treatment for 3h, 6h, 12h and 24h. Post treatment, aliquots of cell extracts
were collected and the activities of caspase-8 (A), caspase-2 (B), caspase-9 (C) and caspase-3 (D)
were determined using fluorogenic substrates Ac-LEHD-AFC Ac-VDVAD-AFC Ac-LEHD-AMC
and Ac-DEVD-AMC, respectively with excitation of 380 nm and emission of 460 nm. Significant
increase in caspase-8 (n=6; p***<0.001), caspase-2 (n=6; p***<0.001), caspase-9 (n=6;
p***<0.001) and caspase-3 (n=6; p***<0.001) were observed after exposure to Tweak for 24h in
dopaminergic neuronal cells. Asterisk (p***<0.001) represents significant differences between
Tweak treated groups and control group as determined by ANOVA followed by Student Newman
Keul’s test.
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Fig 8. Impaired Auto-lysosomal system (ALS) and Proteosomal system (UPS) in Tweak treated N27
cells. A-B. Tweak causes Autophagy dysfunction in N27 cells. Immunoblot analysis of LC3 A/B and Beclin
in whole cell lysates of N27 dopaminergic neuronal cells treated with 100 ng/ml Tweak for 3h, 6h, 12h and
24h. Equal loading of protein in each lane is confirmed by probing the membrane with -actin. Densitometric
analysis of LC3 A/B and Beclin levels is represented next to western blot. Each experiment is repeated three
times and a representative immuno-blot representing LC3 A/B and beclin expression in Tweak treated N27
cells was shown. LC3 II and Beclin bands were quantified and expressed as percentage of control. Significant
downregulation of autophagy markers LC3 and beclin were evidenced in Tweak treated N27 cells. Asterisks
(* p < 0.05, *** p < 0.001) represents significant differences between Tweak treated control groups and
control group as indicated by Anova followed by Student Newman Keul’s post test. C. Proteosomal system
(UPS) in Tweak stimulated N27 rat dopaminergic neuronal cells. Rat dopaminergic N27 cells treated with
100 ng/ml Tweak for 3h,6h,12h to 24h. Post treatment, proteosomal activity was assayed by with the help
of substrate Suc-Leu-Val-Tyr-AMC (chymotrypsin like activity) cleavage in the Tweak stimulated cell
culture lysates. Enzymatic activity was normalized to protein concentration and expressed as percentage of
vehicle treated cells. Each experiment is performed three times with n=8. Significant time dependent
downregulation of proteosomal activity was noticed in dopaminergic neuronal cells upon Tweak treatment
as compared to controls cells. Asterisk (*p<0.05) represents significant differences between Tweak treated
groups and control group as determined by ANOVA followed by Student Newman Keul’s test.
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Fig 9. Tweak induced NF-kB activation precedes cell death in N27 rat dopaminergic neuronal
cells. A-B. NF-kB pathway activation in Tweak treated N27 cells. N27 dopaminergic neuronal
cells treated with Tweak for 6h, 12h and 24h. Post incubation, lysates were probed with NF-kB
phospho-P65 and IkB-alpha by western blotting. Significant time dependent IkB-alpha
degradation and NF-kB phospho-P65 activation was evidenced in dopaminergic neuronal cells
upon Tweak stimulation. Band intensities of NF-kB phsospho-P65 and IkB-alpha from three blots
were quantified using densitometric analysis after normalizing with -actin. Each experiment is
repeated three times and a representative western blot is shown. Asterisk (** p < 0.01) represents
significant differences between Tweak treated groups and control group as analyzed by ANOVA
followed by Student Newman Keul’s post test. C. SN-50 (NF-kB inhibitor) offers partial
neuroprotection against Tweak induced neurotoxicity in N27 cells. N27 cells were treated with
Tweak 100ng and 30 ng TNF with or without NF-kB inhibitor SN-50 for 24h. Post incubation, cell
viability was assessed using MTT analysis. Each experiment performed three times with n = 8.
Tweak induced dopaminergic neuronal cell death is partially reversed by SN-50 (NF-kB inhibitor).
Asterisk (p***<0.001) represent significant differences between Tweak/TNF treated groups and
control group as determined by ANOVA followed by Student Newman Keul’s test.
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Fig 10. Phospho-AKT, GSK-3 beta and Phospho-tau immunoblot analysis following Tweak
treatment. A-E. Rat dopaminergic N27 cells were treated with 100 ng/ml Tweak for 3h, 6h, 12h
and 24h. Post treatment, aliquots of cell extracts were collected and lysates were prepared. Equal
amounts of protein from treated cell lysates were separated on SDS-PAGE and transferred onto
nitrocellulose membrane for immunoblot analysis using antibodies for Phospho-AKT, GSK-3
parent, GSK-3 beta Serine9 and Phospho-tau. Equal loading in all the lanes is ascertained by re-
probing the membrane with -actin. All the experiments were repeated three times and a
representative western blot is shown. Bar graphs represent densitometric scanning analysis of
protein bands after normalization with -actin corresponding to the western blot. Downregulation
of Phospho-AKT is associated with GSK-3 beta activation and phospho-tau accumulation in
Tweak stimulated N27 rat dopaminergic neuronal cells. Asterisks (* p < 0.05,** p < 0.05)
represents significant differences between Tweak treated groups and control group as determined
by Anova followed by Student Newman Keul’s post test.
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CHAPTER 3. SYK CONTRIBUTES TO TWEAK-INDUCED INFLAMMATORY
RESPONSE THROUGH ACTIVATION OF NLRP3 INFLAMMASOME ACTIVATION,
ER STRESS RESPONSE and ALS DYSFUNCTION IN MICROGLIA
To be Submitted to Journal of Neuroscience
Sri Kanuri1,2, Neeraj Singh1, Vivek Lawana1, Huajun Jin1, Vellareddy Anantharam1, Anumantha
Kanthasamy1 and Arthi Kanthasamy1 *
Abstract
Accumulating evidence suggest a positive association between microglial activation
response and DAgic neurodegenerationin in the pathogenesis of Parkinson’s disease (PD). But
the precise innate immune mechanism that the microglia recruit to trigger or promote inflammatory
response during PD progression remain poorly understood. The present study explored the roles
of syk, a pro-inflamamtory kinase in the regulation of Tweak-induced microglial activation
response using BV2 microglial cell line and primary microglia in culture. In BV2 microglial cells,
TWEAK challenge for increasing duration resulted in a coincident increase in SYK activation and
stress granule formation as assessed via WB and enzymatic analysis. We further found that
TWEAK enhanced NLRP3 inflammasome activation, as reflected by increased generation of IL-
1b, IL-18 in Bv2 cells. Intriguingly, TWEAK-induced NLRP3 inflammasome activation coincided
with an upregulation of NOX, ER stress response and JNK as well as impairment of auto-
phagosomal lysosomal system (ALS) function in BV2 microglial cells. Pretreatment with RX788,
a selective SYK inhibitor or siRNA mediated gene silencing of SYK in BV2 microglial cells
attenuated TWEAK-induced NLRP3 inflammasome activation, ER stress response, NOX and JNK
1 Department of Biomedical Sciences, Iowa State University, Ames, IA 50010
2 Primary researcher and author
*To whom correspondence should be addressed. E-mail: [email protected]
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activation, as well as ALS dysfunction. Intriguingly, our in vivo studies using a TWEAK neuro-
inflammation mouse model (TWEAK infusion into the striatum) failed to show an alteration in the
expression levels of SYK or NLRP3 related markers in the striatum. Likewise, piceatannol, a Syk
inhibitor failed to elicit an anti-inflammatory response in the TWEAK neuro-inflammation model.
Taken together our studies suggest that at least in TWEAK stimulated BV2 microglial cells, SYK
mediated oxidative stress response, ER stress response and ALS dysfunction may contribute to
NLRP3 inflammasome activation. Further studies are needed to optimize the TWEAK neuro-
inflammation mouse model.
Introduction
Parkinson’s disease (PD) is most common neurodegenerative disorder characterized
by microglial activation and neuroinflammation and dopaminergic neurodegeneration in
substantia nigra (Machado et al., 2016; L. Qian & Flood, 2008). PD is characterized by motor
symptoms like tremor, rigidity, bradykinesia, gait disturbances, impaired handwriting, speech
deficits and non-motor symptoms like apathy, depression, cognitive dysfunction, hallucinosis,
orthostatic hypotension, urogenital dysfunction, constipation and rapid sleep movement disorder
(Moustafa et al., 2016; Poewe, 2008). Neuro-inflammation is regarded as an important
contributing factor for dopaminergic cell death in PD (Machado et al., 2016; Q. Wang et al., 2015).
Microglial mediated neuro-inflammatory is being increasingly recognized as a critical link in PD
pathology (Machado et al., 2016; L. Qian et al., 2008). Activated microglia has been demonstrated
in substantia nigra of post mortem brain tissues and positron emission tomography scan (PET) has
provided substantia evidence of microglial activation in mid brain tissues (Bartels et al., 2010;
McGeer et al., 1988). Sustained and chronic microglial activation in mid brain results in production
of pro-inflammatory cytokines like IL-1 beta, TNF-alpha, IL-6 and IFN-gamma which
198
subsequently leads to dopaminergic neurodegeneration (C. C. Ferrari et al., 2006; K. Kaur et al.,
2017; Nagatsu & Sawada, 2007; Q. Wang et al., 2015)
Innate immune response functions as a defense mechanism against invading pathogens and
damage associated molecules. Inflammasomes are multiprotein platforms that has been linked to
the pathogenesis of numerous diseases including AD (H. Guo et al., 2015; Lamkanfi & Dixit,
2012; M. K. Saito, 2011). The most commonly studied inflammasome nucleotide-binding
oligomerization domain, leucine rich repeat and pyrin domain containing inflammasome (NLRP3)
is an intracellular multi-meric protein complex that becomes activated by wide variety of
pathogens, environmental irritants, toxins and PAMPs (Pathogen associated molecular patterns)
(Kate Schroder et al., 2010). It consists of NLRP3 scaffold, ASC adaptor and caspase-1(Kate
Schroder et al., 2010). A wide variety of exogenous factors can activate the NLRP3 inflammasome
including ATP, Asbestos, Silica, uric acid and amyloid (Cassel et al., 2008; S. Mariathasan et al.,
2006; Martinon et al., 2006).
NLRP3 serves as a platform for the maturation and secretion of IL-1B and IL-18 in a
caspase-1 dependent manner (Gurung & Kanneganti, 2016; Shaw et al., 2011). Intriguingly, in a
recent report fibrillary alpha synuclein was found to activate IL-1B production in an NLRP3
dependent manner in monocytes (Codolo et al., 2013) Likewise the expression of NLRP3 related
markers were found to be upregulated around the site of injection in the LPS and 6-OHDA
intracerebral rat model. Notably, caspase-1 inhibitor, Ac-YVAD-CMK inhibited the mRNA and
protein expression of NLRP3 inflammasome related markers. The inhibition of NLRP3
inflammasome was found to positively correlate with the preservation of dopamine neurons in the
substantia nigra (Mao et al., 2017). Thus, these studies provide further support for the positive
immunomodulatory role of NLRP3 inflammasome in neurodegenerative diseases including PD.
199
Microglial cells mediate many inflammatory responses through activation of various
intracellular signaling molecules such as tyrosine kinases (Y. S. Yi et al., 2014a). Syk is a newly
identified non-receptor tyrosine kinase that belongs to SYK family that participates in various
physiological and pathological processes (Berton et al., 2005; S. H. Choi et al., 2009; Rogers et
al., 2005). Syk is a non-receptor tyrosine kinase, which has two Src homology domains (SH2) and
a kinase domain (Futterer et al., 1998; Mocsai et al., 2010; Taniguchi et al., 1991). These domains
are inter-connected by inter-domains: A and B (Futterer et al., 1998; Mocsai et al., 2010; Taniguchi
et al., 1991). Hematopoietic cells, macrophages, neuronal cells, fibroblasts, epithelial cells and
vascular endothelial cells express Syk in varying proportions (S. Yanagi et al., 2001; S. Yanagi et
al., 1995). It has been shown to play an important role in immune signaling, autoimmune diseases,
microglial activation, osteoclast maturation, platelet development and hematological malignancies
(Mocsai et al., 2010). Syk is regarded as a major upstream regulating kinase responsible for NLRP3
inflammasome activation in immune cells (F. Laudisi et al., 2014). Syk has been believed to cause
phosphorylation of apoptosis associated speck-like protein containing a CARD (ASC) thus
promoting caspase-1 activation thereby, resulting in NLRP3 inflammasome mediated production
of pro-inflammatory cytokines like IL-1 beta and IL-18 (Gross et al., 2009; H. Hara, K. Tsuchiya,
I. Kawamura, R. Fang, E. Hernandez-Cuellar, Y. Shen, J. Mizuguchi, E. Schweighoffer, & V.
Tybulewicz, 2013; Y. C. Lin et al., 2015). Nigericin, Mono sodium urate and Alum has been
known to cause Syk mediated inflammasome activation in LPS primed peritoneal macrophages
(F. Laudisi et al., 2014).
The recently characterized TNF superfamily member include the TNF-like weak inducer
of apoptosis (TWEAK) and its receptor fibroblast growth factor inducible 14 (Fn14) receptor
(Chicheportiche et al., 1997; Wiley et al., 2001).TWEAK is a cytokine that is widely expressed in
200
astrocytes, neurons, and macrophages and is upregulated in many neurological diseases, namely,
Stroke, Multiple Sclerosis Encephalomyelitis and Parkinson Disease (J. A. Winkles, 2008; Yepes,
2007). Moreover it has been implicated in variety of biological responses like inflammation,
migration, angiogenesis and apoptosis (Burkly et al., 2007). TWEAK-Fn14 signaling axis has been
linked to NF-kB activation via both canonical and non-canonical pathways involving cIAP1 and
increased transcription of the downstream mediator cyclooxygenase-2 which has been shown to
be upregulated in PD brains and in experimental models of PD (Teismann et al., 2003). In fact in
a recent report expression of TWEAK was found to be upregulated transiently in the striatum of
MPTP treated mice suggesting that TWEAK might an important component of MPTP-induced
neurotoxicity and thereby PD neuropathology (Mustafa et al., 2016a). Moreover, it is plausible
that TWEAK-induced inflammatory response may increase the vulnerability of dopaminergic
neurons to oxidative stress-induced neuronal injury. Thus far, TWEAK mediated downstream
signaling response upon microglial activation remain poorly characterized.
In the present study, we investigated the molecular basis of TWEAK-induced
inflammatory response. Our central hypothesis is that, Tweak causes NLRP3 inflammasome
activation via the activation of SYK kinase in an oxidative stress-dependent manner in the
microglia. Our results revealed that, Tweak causes microglia activation through Syk/NLRP3 axis.
Furthermore, our study highlights the anti-inflammatory effect of R788 in the TWEAK in vitro
cell culture model. Our results suggest that TWEAK induced Spleen Tyrosine Kinase activation
might be a critical upstream signaling event that elicits NLRP3 inflammasome activation
presumably via an ER stress (ERS) and autophagolysosomal system (ALS) dependent manner.
201
Materials and Methods
Chemicals and Reagents
Lipopolysaccharide (LPS) (Escherichia coli 0111: B4, endotoxin content 6.6000000
EU/mg) and β-actin were obtained from Sigma-Aldrich (St Louis, Mo, USA). R788 Syk inhibitor
was purchased from Santa Cruz Biotechnology, Dallas, TXBiotechnology). Recombinant Tweak
was obtained from Pepro-Tech (Rocke Hill, NJ, USA). IL-1 beta, IL-18 and TNF alpha ELISA
were purchased from eBiosciences (San Diego, California). PCR primers for IL-1 beta, IL-18 and
TNF alpha, DCFDA-H2 kit for ROS assay, RPMI, FBS, l-glutamine, penicillin, streptomycin,
were purchased from Invitrogen (Gaitherburg, MD). JC-1 Dye was purchased from Thermo-Fisher
Scientific (New Hampshire, USA). Bradford protein assay kit was purchased from Bio-Rad
Laboratories (Hercules, CA, USA). NLRP3, Caspase-1, IL-1 beta, ER stress markers (CHOP,
Phospho-PERK, Phospho-elf and GRP-94), Autophagy Markers (LC3, Beclin, P62, P-AMPK and
ATG5-12), Phospho-JNK and Phospho-P38 were purchased from Cell Signaling Technology,
MA, USA.
BV2 Cell Culture
The BV-2 immortalized cell line was obtained from Dr. Sanjay Maggirwar (University of
Rochester Medical Center, Rochester, NY, USA) and was grown and routinely maintained in 10%
RPMI at 37°C and 5% CO2. BV2 cells has been used for studying the neurotoxic
mechanisms related to Parkinson Disease in our laboratory. BV2 cells were grown in RPMI 1640
medium supplemented with 10% fetal bovine serum, 2mM L-glutamine, 50U penicillin and
50μg/ml streptomycin. Cell cultures were maintained under 5% CO2 at 37°C as previously
described. The media is changed every few days. After treatment, cells were collected by
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trypsinization, spun down at 200 g for 5 min, and washed with ice-cold phosphate-buffered saline
(PBS). The lysates from the cell pellets were appropriately used for various assays.
Western Blot Analysis
Treated cells were collected and lysed with RIPA buffer (Sigma) in ice and sonicated for
15 seconds on ice for each sample. Samples were centrifuged at 14,000 g for 15 min at 4°C.
Supernatants were collected and stored at −80°C until use. Protein estimation was done using
Bradford reagent and samples with equal amount of protein were separated on 8–15% SDS-PAGE,
depending on the molecular weight of the target protein. Later they were transferred onto
nitrocellulose membranes by electro-blotting at 110V for 90 min at 4 C (Ice-cold transfer). Next,
membranes were blocked briefly for 1h by incubating them in blocking buffer and later incubated
with primary antibodies overnight at 4°C. To ensure equal protein loading, β-actin (1:10,000) was
used as loading control. Membranes were washed several times and incubated with specific
fluorescent labelled secondary antibody for 1 h at room temperature. Membranes were scanned
using the Odyssey IR Imaging system (LICOR) and data were analyzed with Odyssey 2.0
software.
Mitochondrial Membrane Potential (MMP)
Changes in the mitochondrial membrane potential was detected by 5,5’, 6.6’- tetrachloro-
1,1’, 3,3’-tetraethyl-benzimidazolcarbocyanineidide (JC-1 dye) (Molecular Probes, Thermo Fisher
Scientific) a cataionic fluorescent lipophilic dye. In depolarized mitochondria, monomeric form
shows strong green fluorescence with excitation and emission wavelength at 485 and 535 nm
respectively. In healthy mitochondria, aggregated monomers usually display strong red
fluorescence at excitation and emission wavelengths at 560 and 595 nm, respectively. The shift in
ratio of fluorescent intensity of JC-1 aggregates (Red fluorescence) to fluorescent intensity of
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monomers (green fluorescence) is used as an indicator of depolarization of mitochondrial
membrane potential.
ELISA Assay
Microglia were plated in a 96-well plate. Later supernatant was collected and was used for
determination of IL-1β, IL-18 and TNF alpha concentrations using ELISA kit (ebiosciences) as
per manufacturer’s instructions.
ROS Assay
Changes in intracellular ROS was determined using CM-H2DCFDA, a fluorescent
probe, by following the protocols as suggested by manufacturer instructions (Molecular Probes,
Thermo Fisher Scientific). BV2 microglial cells were plated in 96-well plates, post treatment,
treatment media was removed and cells were washed with PBS and incubated with 1uM CM-
H2DCFDA dye dissolved in HBSS for 1 hr. Later, cells were washed with PBS and ROS
production was assessed using a microplate reader with excitation 485 nm and emission 530 nm
respectively.
Griess Assay
Nitric oxide production was assessed by quantification of nitrate in the supertanant with
the help of Griess assay. Cells were plated in a 96 well plate and treated with Tweak 100 ng, TNF
30 ng and LPS 1ug/ml. Post treatment, 100 ul of supernatant was collected and mixed with equal
volume of Griess agent and incubated in a plate shaker for color development for 15 min. The
absorbance at 450 nm was measured using microplate reader and nitrate concentration was
assessed from sodium nitrite standard curve (N. Panicker, H. Saminathan, H. Jin, M. Neal, & D.
S. Harischandra, 2015).
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qRT-PCR
RNA isolation from treated BV2 microglial cells was performed using the Absolutey RNA
Miniprep kit (Trizol Reagent). Later, extracted RNA was used for cDNA synthesis by reverse
transcription with the AffinityScript qPCR cDNA synthesis system (Agilent Technologies) as per
the manufacturer's instructions. Quantitative SYBR Green PCR assays for gene expression were
performed using the RT2 SYBR Green Master Mix with pre-validated primers (SA Biosciences).
Master Mix was used to perform qRT-PCR analysis with verified primers TNF-α (catalog
#PPM57735E), IL-1β (catalog #PPM57735E) and IL-18 (catalog #PPM57735E). The mouse 18S
rRNA gene (catalog #PPM57735E) was used as the housekeeping gene for normalization. The
fold change in gene expression was determined by the ΔΔCt method using the threshold cycle (Ct)
value for the housekeeping gene and the respective target gene of interest in each sample.
Lysosomal PH
Changes in Lysosomal pH was characterized using the ratiometric lysosomal pH probe
LysoSensor Yellow/Blue dextran (Invitrogen L22460). At the end of the treatment period, cells
were loaded with 1 mg/ml LysoSensor and incubated overnight. Cells were then washed,
trypsinized and re-suspended in PBS. The fluorescence was monitored using the fluorescent
microplate reader with emission wavelengths at 535 nm and 430 nm with excitation at 340 nm.
The ratio of emission at 535 nm/430 nm was used for assessing lysosomal pH for each sample.
Syk Kinase Assay
Post treatment, treated cell lysates were added to ice cold master-mix consisting of: 5 μl
10X kinase buffer (200 mM HEPES, pH 7.4, 10 mM MnCl2, 10 mM MgCl2, 10 mMDTT, 10 mM
Na3VO4, 10 mM NaATP), 5ug Syk Kinase peptide substrate biotin labelled (Ana-Spec) and final
volume adjusted with ddH2O to 50 μl. Recombinant Syk enzyme (1 μg/reaction) was used as a
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positive control. Reactions were incubated at 30°C for 30 min. Reaction was stopped by addition
of 950 μl 2% ice-cold BSA. Next, 100 ul of supertanant was added to streptavidin coated 96 well
plate and incubated for 3h and later plates were washed for 3 times. Furthermore, 4G10 anti-
phosphotyrosine antibody (Millipore) diluted 1:1000 in 5% milk/TBS was added to each well,
allowed to incubate for 1 h at room temperature on a shaker. Plates were washed for 3 times with
wash buffer, and anti-IgG2b HRP-conjugated secondary (Southern Biotech) diluted 1: 1000 in5%
milk/TBS was added to each well and incubated for 1 h at room temperature followed by washing
with wash buffer for 3 times. Lastly, plates were developed with Horseradish Peroxidase Substrate
Kit (Biorad) and color was allowed to develop for 15 min. Plates were read with an Infinite m200
plate reader (Tecan) at 405 nm (Schlatterer et al., 2011).
Animal Study
6-8-week-old C57 black mice were obtained from Charles River and housed under
standard conditions at 22 C with 30% relative humidity with 12h light cycle as per IACUC
protocol. Mice were randomly assigned into three different groups (Control, Tweak and Tweak
and Syk inhibitor). Piceatannol (100 mg/kg) was administered twice 12h before and 1h before
Tweak treatment (Y. He, L. Xu, B. Li, Z. N. Guo, et al., 2015; Yukiya Suzuki et al., 2013). Acute
intra-striatal Tweak induced neuroinflammation model of PD was used for this study (Polavarapu
et al., 2005). Mice were given intra-striatal injections of 2 ul Tweak (1 ug/ul) using stereotaxic
surgery (W. B. Haile, R. Echeverry, F. Wu, et al., 2010; Polavarapu et al., 2005). 24 h post
treatment, mice were sacrificed and striatum were collected and stored at 80 C. Later tissue lysates
were immunoblotted for the protein of interest.
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Syk SiRNA Transfection
Transfection of BV2 cells was done using Amaxa Nucleofector Kit (Lonza). Briefly, Bv2
cells were mixed with 100 ul transfection buffer (400 uM ATP-disodium, 600 uM magnesium
chloride, 100 uM potassium hypophosphate, 20 uM sodium bicarbonate and 5 uM glucose. Later
Syk SiRNA and control SiRNA (Santa Crux BioTechnology, CAT # SC-44328 and CAT # SC-
44230) were added to the transfection mixture. Next, transfection of cells was done with
electroporation using A23 program of Lonza Nucleofector 2b Devise. Finally, cell were incubated
for 48-72 h followed by treatment with Tweak for another 24h.
Data Analysis
Results are analyzed with PRISM 6.0 software (Graph Pad, San Diego). Results are
presented as mean ± S.E.M. P values were assessed using one-way ANOVA followed by post hoc
Tukey test (PRISM software) in order to compare between treatment groups. P values < 0.05 were
considered significant.
Results
Temporal Pattern of Spleen Tyrosine Kinase Activation and Stress Granule Formation in
Microglial Cells in Response to Inflammogens
A growing body of evidence supporta a role for SYK in the activation of inflammatory
cells including microglial cells. For example Spleen Tyrosine Kinase is involved in LPS induced
intracellular signaling pathways and pro-inflammatory responses in macrophages (Ulanova et al.,
2007). Additionally, in cell culture models of Alzheimer Disease amyloid fibrils activate spleen
tyrosine kinase in primary microglial cells to induce pro-inflammatory responses (Combs et al.,
1999; S. Ghosh et al., 2015).Therefore, we utilized the well characterized BV2 microglia model,
which has been routinely used for the study of PD associated neuro-inflammation (R. Gordon et
al., 2016). In the present study, we sought to investigate whether SYK is activated in LPS or
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TWEAK treated microglial cells. BV2 microglial cells were either exposed to LPS or TWEAK
for increasing duration (3h, 6h, 12h, 18h and 24h) and expression of spleen tyrosine kinase was
determined using WB analysis.
As shown in (Fig 1A), we found that both LPS and Tweak combination treatment
induced a time dependent increase in Spleen Tyrosine Kinase expression in Bv2 microglial cells.
A similar trend was evidenced in Tweak alone treated cells (Fig 1B). We further determined
whether the increase in SYK protein levels is accompanied by a concomitant increase in SYK
kinase activity. To this end we performed enzymatic kinase activity assays in BV2 cells stimulated
with TWEAK. For this purpose, R788, Syk inhibitor pretreated cells were stimulated with Tweak.
As anticipated TWEAK treated BV2 microglial cells showed a time dependent increase in the
activation of SYK kinase at the end of 12 and 24h post drug treatment. In fact, a pronounced
upregulation was observed at 24h post TWEAK treatment. Furthermore R788, a SYK kinase
inhibitor markedly attenuated SYK kinase activity (63 % reduction) in TWEAK treated cells
further confirming the role of SYK in inflammagen-induced microglial activation response (Fig
1C). Taken together, these results indicate that, Tweak-induced microglial activation response may
be linked to SYK activation.
In a previous report SYK was found to localize to stress granules (SGs) in primary
microglial cells stimulated with A beta or sodium arsenite (S. Ghosh et al., 2015). To examine
whether stress granules are formed in response to TWEAK, we treated BV2 microglia with
TWEAK for the indicated duration (0-24h). At the end of the drug treatment period cell lysates
were prepared and immunoblotted for the SG marker G3BP1. A marked time dependent increase
in G3BP1 was evidenced in TWEAK treated cells (Fig 1D). Notably a positive correlation between
SYK activation and G3BP1 upregulation was evidenced in TWEAK treated cells as compared to
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vehicle treated cells suggesting that SYK activation may promote the formation of SGs to
sequester aggregate prone proteins.
Temporal Pattern of Activation of NLRP3 Related Markers in LPS Primed TWEAK
Stimulated Microglial Cells
NLRP3 (Nucleotide-binding domain and leucine rich repeat containing family, pyrin
domain containing 3) has been shown to be a key regulator of innate immunity and activated in
response to diverse stimuli including microbial infection or exposure to sterile inflammatory
stimuli (H. Guo et al., 2015; Gurung et al., 2016; Lamkanfi et al., 2012; Kate Schroder et al.,
2010; Shaw et al., 2011). In this context caspase-1 mediated proteolytic processing of pro-IL-1b
and pro-IL 18 to their active forms has been linked to NLRP3 inflammasome activation. LPS is a
widely studied inflammagen that has been shown induce the expression of pro IL-1B via an NF-
kB dependent mechanism in microglial cells (C. Dostert et al., 2008). Therefore, microglial cells
were primed with LPS to promote NF-kB activation which has been shown to serve as signal-1for
NLRP3 inflammasome activation. Additionally, it recapitulates excessive microglial activation
response observed in PD brains (Machado et al., 2016; S. R. Subramaniam & H. J. Federoff, 2017;
Q. Wang et al., 2015). In the initial set of experiments, we determined the temporal pattern of
activation of NLRP3 related markers in either TWEAK or LPS/TWEAK treated cells. BV2 cells
were exposed to TWEAK (100 ng/ml) for increasing duration (3h, 6h, 12h and 24h) in the presence
or absence of LPS (1 ug/ml, 3h priming). As shown in Fig 2A-D, NLRP3, caspase-1, IL-1 beta
and IL-18 expression was upregulated in LPS and Tweak stimulated BV2 microglial cells.
Interestingly, we also noticed a similar trend in BV2 cells exposed to Tweak alone (Fig 2E-G).
Given that TWEAK alone elicited the activation of NRLP3 related markers in the absence of a
priming step we next studied the influence of TWEAK on pro-inflammatory factor secretion in
microglial cells. For this reason, we measured pro-inflammatory cytokine secretion after treatment
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of primary microglial cells with Tweak at various time points (8h and 24h) using ELISA analysis.
We noticed a significant increase in the secretion of pro-inflammatory cytokines namely IL-1 beta
and TNF-alpha (Fig 2H) in Tweak stimulated BV2 microglia at 8h and 24h, respectively. To
further assess whether increased pro-inflammatory cytokine secretion in Tweak treated BV2
microglial cells was due to increased gene transcription, we performed gene expression analysis
of pro-inflammatory cytokines using Real time PCR. We found that, Tweak significantly up-
regulated mRNA levels of IL-1 beta, IL-18 and TNF-alpha in a time dependent manner (Fig 2I) in
BV2 microglial cells. Taken together, our studies suggest that TWEAK by itself as well as LPS
and TWEAK combination results in a heightened inflammatory response characterized by marked
upregulation of pro-inflammatory markers including NLRP3 inflammasome related markers.
Activation of SYK and NLRP3 Related Markers in Primary Microglia Upon TWEAK
Stimulation
Next, we validated TWEAK-induced pro inflammatory response in primary microglial
cells. For these studies, primary microglia were isolated from brains of mice either post-natal day
(PND) 2 or 3. Primary microglial cells were treated with TWEAK for the indicated duration (3,
6, 12 &24h) and immunoblotted for SYK, NLRP3 related markers such as caspase-1, IL-1b,
NLRP3 as well as redox sensitive kinase PKC delta (PKCd). A time dependent upregulation of
SYK and accompanying enhancement of NLRP3 related markers and PKC delta was evidenced in
TWEAK treated cells (Fig 3A-E). Interestingly, a marked increase in PKC delta and NLRP3
inflammasome activation as well SYK was observed at 24h post drug treatment. Together, these
results demonstrate that redox sensitive kinases are upregulated upon microglial activation with
an inflammatory stimulus.
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Tweak Promotes ROS Generation and Dissipation of Mitochondrial Membrane Potential
(MMP) in BV2 Microglial Cells.
Mitochondria mediated ROS (Reactive Oxygen species) production and has been shown
to function as signal-2 for NLRP3 inflammasome activation in immune cells (E. K. Jo et al., 2016;
J. Yu et al., 2014). We next determined whether NLRP3 inflammasome activation is associated
with mitochondrial ROS generation in TWEAK treated cells. For this purpose, BV2 microglial
cells were treated with Tweak at the indicated time points and ROS production was examined
using redox sensitive CM-H2DCFDA dye. A time dependent increase in ROS generation was
evidenced whereby ROS generation was observed as early as 3h post TWEAK treatment and
remained elevated during the entire treatment duration (Fig- 4 A).
Previous studies have shown that, mitochondrial impairment is commonly associated
with NLRP3 inflammasome activation in immune cells. In this context, Caspase-1 induced
dissipation of mitochondrial membrane potential and associated mitochondrial damage and
dysregulation of mitophagy has been linked to the activation of the NLRP3 inflammasome (J. Yu
et al., 2014). In order to determine whether TWEAK induced ROS production is associated with
loss of mitochondrial membrane potential we assessed mitochondrial membrane potential using
redox sensitive JC-1 dye, a catatonic fluorescent lipophilic dye. As shown in the Fig 4B, Tweak
and LPS in combination induced a sustained reduction in MMP quantification of mitochondrial
membrane potential (MMP) as assessed via spectrometric plate reader analysis. Together these
data suggest that TWEAK-induced excessive ROS generation presumably via loss of MMP and
associated mitochondrial dysfunction may lead to the activation of NLRP3 inflammasome.
Previous reports have demonstrated that SYK dependent activation of NADPH oxidase is
critical for autophagosome formation which in turn enhances fungicidal activity in macrophages
(Tam et al., 2014). In another study SYK was found to regulate OxLDL-induced ROS generation
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in macrophages (S. H. Choi et al., 2015). We therefore examined whether NOX-2 activation
requires SYK. We depleted endogenous levels of SYK via siRNA mediated gene KD. NOX-2
activation in TWEAK stimulated BV2 microglial cells was reduced in SYK SiRNA transfected
cells as compared to scramble transfected cells (Fig 4C). Together these data provide new insights
into the mechanisms by which TWEAK regulates ROS generation in the microglia. Thus, hyper
activation of NOX via SYK may partially contribute to TWEAK-induced oxidative stress
response.
Tweak Induces ER Stress Response (ERS) in BV2 Microglial Cells
After having established the expression of SYK and increased ROS generation in the BV2
microglia, the next step was to test the ability of TWEAK to induce ER stress response (ERS)
Recent studies have demonstrated that ER stress can-induce NLRP3 inflammasome activation (D.
N. Bronner et al., 2015; Lerner et al., 2012). For example ER stress has been shown to cause the
release of mitochondria derived release of damage associated molecular patterns such as ROS
which in turn has been linked to NLRP3 inflammasome activation (Iyer et al., 2013; R. Zhou et
al., 2011). Unfolded protein response sensors such as IREa, ATF6 and PERK have been identified
as major sensors of ER stress response, an adaptive program that has been shown to regulate cell
fate in cells subjected to cellular stress. To determine whether ER stress response is induced during
TWEAK-induced NLRP3 inflammasome activation we investigated the levels of ER stress
markers in TWEAK treated microglial cells. Robust activation of ER stress markers including
CHOP, GRP-94, Phospho-Elf alpha, Phospho-PERK and ATF-4 expression were seen in TWEAK
treated BV2 microglial cells in a delayed manner (Fig 5 A-E). Thus Tweak-induced the activation
of ER stress markers in microglial cells as previously observed in cells subjected to microbial
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infection (Denise N. Bronner et al., 2015; Martinon, 2012). Overall, the results suggest that
TWEAK induces ERS in BV2 microglial cells.
Autophagosomal Lysosomal System (ALS) Modulation in TWEAK Stimulated BV2
Microglial Cells.
Autophagy represent a catabolic mechanism that sequesters portions of cytoplasm
including damaged organelles and macromolecules into double membrane vesicles known as
autophagic vacuoles and eventually delivered to the lysosome for degradation leading to the
recycling of macromolecules (D. J. Klionsky et al., 2016). Studies indicate that, autophagy
negatively regulates NLRP3 inflammasome activation and pro-inflammatory cytokine production
in immune cells (Tatsuya Saitoh et al., 2016; Yuk & Jo, 2013; Z. Zhong et al., 2016). It has been
proposed that impairment in autophagic mechanisms may lead sustained activation of NLRP3
inflammasome in immune cells (Tatsuya Saitoh et al., 2016; Yuk et al., 2013; Z. Zhong et al.,
2016).
To determine the relationship between TWEAK-induced inflammatory response and
autophagic mechanism, the BV2 microglial cell line was treated with TWEAK for increasing
duration and the induction of autophagic markers were assessed via WB analysis. The extent of
accumulation of autophagic markers including LC3 II, p62, but not beclin 1 (upregulation observed
in a delayed manner-24h) increased in a time dependent manner (FIG 6A-E) indicating an impaired
autophagic flux in microglial cells treated with TWEAK. Alternatively, the accumulation of AV
related markers may be attributed to lysosomal dysfunction. These studies indicate a positive
association between impairment in autophagic function and NLRP3 inflammasome activation in
microglia treated with TWEAK.
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Next, we determined whether lysosomal damage is linked to NLRP3 inflammasome
activation in Tweak treated microglial cells. Earlier studies indicate that, NLRP3 inflammasome
activation and IL-1 beta secretion in LPS treated macrophages occurs through the release of
cytoplasmic lysosomal Cathepsin B originating from altered lysosomal membrane permeability
(K. Weber & J. D. Schilling, 2014). Moreover lysosomal membrane damage and resultant release
of cathepsin-B into the cytosol has been implicated in NLRP3 inflammasome activation in
endothelial cells and immune cells exposed to an inflammatory stimuli (Y. Chen et al., 2015; E.
K. Jo et al., 2016). Therefore, we assessed the expression of lysosomal cathepsins in Tweak treated
BV2 microglial cells. For this purpose, BV2 microglia was incubated with Tweak for the indicated
duration (3h, 6h, 12h and 24h). Post incubation, expression of lysosomal cathepsins was
determined using WB analysis. As shown in the Fig 6 F-G we found out that Cathepsin-B,
Cathepsin-D is upregulated at 24h following Tweak treatment in Bv2 microglia. In fact, increase
in lysosomal PH results in release of cathepsin-B and subsequent activation of NLRP3
inflammasome in pneumococcal meningitis (Hoegen et al., 2011). Since lysosomal PH disruption
is associated with NLRP3 inflammasome activation in immune cells, we next characterized
lysosomal pH in Tweak treated microglial cells. BV2 microglial cells were treated with Tweak at
various time points (6h, 12h and 24h) and lysosomal pH was measured by spectro-photometric
plate reader analysis using a redox sensitive Lyso-Sensor dye. As shown in the Fig 6H, lysosomal
pH is increased in Tweak treated microglial cells in time dependent manner indicating that
lysosomal acidity is altered. Taken together, our studies suggest that impairment in lysosome
mediated clearance of autophagic vacuoles owing to loss of lysosomal acidity and associated loss
of membrane permeability might partly contribute to the activation of NLRP3 inflammasome in
microglial cells challenged with TWEAK.
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SYK Regulates The Activation of NLRP3 Inflammasome in Tweak Treated Microglial Cells
Studies had shown that Spleen tyrosine kinase is key player in NLRP3 inflammasome
activation in immune cells. Spleen tyrosine kinase is involved in NLRP3 inflammasome activation
in yeast cells, macrophages and neutrophils in response to microbial infection (Mocsai et al.,
2010). We therefore sought to investigate the role of SYK in NLRP3 regulation in TWEAK treated
macrophages. We investigated whether pharmacological inhibition via R788 could suppress
NLRP3 inflammasome activation in response to pro-inflammatory stimuli such as TWEAK.
Genetic deletion of SYK results in early embryonic lethality, precluding the inclusion of SYK -/-
mice in the current study (Jakus et al., 2010; Mocsai et al., 2010). Hence, we tested the anti-
inflammatory effects of R788, fostamatinib sodium, a small molecule of Syk inhibitor that has
shown efficacy in animal models of Asthma, Lymphoma, Glomerulonephritis and Diabetes
(McAdoo, 2011). To determine the effects of SYK inhibition on TWEAK-induced NLRP3
inflammasome activation, BV2 microglial cells were pretreated with R788 (Syk inhibitor) for 1h
followed by treatment with Tweak for 24h. Post treatment, NLRP3 inflammasome related markers
expression was determined using western blot analysis. As shown in Fig. 6A, R788 pretreatment
resulted in decreased activation of NLPR3 (Fig 7 A) and Caspase-1 (Fig 7 B) as well as IL-1 beta
(Fig 7 C) in TWEAK stimulated microglial cells. To further ascertain the regulatory role of syk in
inflammasome activation in microglial cells, we used RNAi approach. For these studies, BV2
microglial cells were initially transfected with scrambled or Syk siRNA. At the end of 72h
following transfection the SYK expression was evaluated using WB analysis. Greater than 70%
reduction in SYK levels was observed in SYK siRNA transfected cells as compared with
scrambled siRNA transfected microglial cells validating down regulation of SYK expression (Fig.
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7 D). BV2 microglial cells were treated with Tweak for another 24h following transfection with
the respective siRNA. Post treatment, NLRP3 inflammasome related markers expression were
determined using western blot analysis. Immunoblot analysis revealed that TWEAK-induced
induction of NLRP3 related markers as well as pro-inflammatory cytokine levels were diminished
by siRNA mediated knock down of SYK (Fig 7 E-F). Taken together, these results confirmed that
TWEAK-induced SYK regulated activation of NLRP3 related markers in BV2 cells.
Involvement of SYK in TWEAK-induced MAPK Activation
MAPK family include the well-studied ERK1/2, JNK, and p38 MAPK which has been
linked to the induction of numerous cellular functions including inflammatory response (Johnson
& Lapadat, 2002). In several cell types activation of MAPK family members have been linked to
the activation of iNOS, COX-2 and proinflammatory cytokines (Dai et al., 2011; Frazier et al.,
2009; Hevia et al., 2004; J. Y. Kim et al., 2006). We next examined whether TWEAK-induced
the activation of MAPK signaling pathway in BV2 microglial cells. As anticipated Tweak induced
the upregulation of all three MAPKs as early as 30 min in BV2 microglial cells (Fig. 8A-C). In
fact inhibition of MAPK via antioxidants such as resveratrol has been shown to inhibit NO, PGE2,
iNOS, COX-2 and pro-inflammatory cytokine (Zhong et al., 2012). Moreover, SYK-induced
MAPK8/JNK2 activation in response to OxLDL has been shown to play an important role in
MHC-II expression in macrophages (S. H. Choi et al., 2015). Furthermore, JNK and SYK have
been shown to be essential for NLRP3 inflammasome activation in an in vivo model of MSU and
alum-induced peritonitis (H. Hara, K. Tsuchiya, I. Kawamura, R. Fang, E. Hernandez-Cuellar, Y.
Shen, J. Mizuguchi, E. Schweighoffer, & V. Tybulewicz, 2013). To test whether the suppressive
effects of R788 on NLRP3 inflammasome activation occurred via MAPK signaling pathway in
TWEAK treated microglia, BV2 microglial cells were pretreated with the SYK inhibitor R788 and
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subsequently incubated with TWEAK for another 24h. As shown in (Fig. 8D-E), MAPK
activation markers pJNK, and p38MAPK in response to TWEAK were decreased in R788
pretreated cells. Thus, our data suggest that SYK mediated activation of MAPK elicits a pro-
inflammatory response via increased NLRP3 inflammasome activation in TWEAK treated
microglial cells.
SYK Regulates TWEAK-induced Impairment In Autophago Lysosomal Function and GSK
3b Activation
Because SYK has been shown to regulate autophagy in macrophages following microbial
infection (S. H. Choi et al., 2015), we next investigated whether it is involved in the regulation of
autophago lysosomal function in TWEAK treated BV2 microglial cells. For this purpose, BV2
microglial cells were transfection with the scrambled and Syk siRNA and subsequently incubated
with Tweak. Post treatment, ALS markers expression were determined using western blot analysis.
As shown in (Fig 9 A-B) siRNA mediated knock down of SYK attenuated TWEAK-induced
impairment in ALS function in BV2 microglial cells. These data indicate that SYK may contribute
to the regulation of ALS function in response to TWEAK in BV2 microglial cells.
Next, we explored whether Tweak induces lysosomal damage in Bv2 microglial cells
through a Syk dependent mechanism. SYK mediated lysosomal dysfunction has been implicated
in the response to infection in innate immune cells (J. He et al., 2005; Y. Q. Lu et al., 2017). For
this purpose, BV2 microglial cells were pretreated with R788 (Syk inhibitor) for 1h followed
treatment with Tweak for 24h. Post treatment, lysosomal cathepsin-B expression was determined
using western blot analysis. As shown in (Fig 9 C) R788 pretreatment resulted in decreased
activation of cathepsin-B in Tweak stimulated BV2 microglia. To further ascertain the regulatory
effects of Syk on lysosomal damage in inflammogen treated microglial cells, we used RNAi
approach. Immunoblot analysis revealed downregulation of cathepsin-B (Fig 9D) in TWEAK
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stimulated Syk Si RNA transfected cells as compared to scrambled siRNA transfected cells. Taken
together, our studies indicate that, Spleen tyrosine kinase is an important regulator of ALS function
in Tweak stimulated BV2 microglial cells.
Glycogen synthase kinase (GSK)-3 is a multifunctional kinase that is differentially
regulated by the phosphorylation of serine or Tyr. For example, phosphorylation of serine 9 in
GSK-3B has been shown to inhibit its activation whereas the enzymatic activity of GSK-3 beta is
enhanced by phosphorylation of TYR 216 (Jope et al., 2007). Furthermore numerous studies have
identified that ER stress is a positive regulator of GSK-3B which in turn can lead to an elevated
inflammatory response (Resende et al., 2008). Moreover, SYK has been shown to negatively
regulate TLR4-mediated IL-10 production while promoting the generation of pro-inflammatory
cytokines (TNFa and IL-6) via modulation of GSK 3b (H. Yin et al., 2016). In the present study,
we sought to determine whether SYK can modulate GSK-3B activation in TWEAK treated
microglial cells. As expected immunoblotting studies confirmed that TWEAK induced Tyr216
GSK-3B phosphorylation. Consistent with a previous study demonstrating that SYK deficiency
promotes anti-inflammatory response in dendritic cells (D. Paris et al., 2014; H. Yin et al., 2016).
SYK inhibition via R788 markedly down regulated TWEAK-induced GSK-3b activation (Fig 9E).
Likewise, siRNA mediated knockdown of SYK in Bv2 microglial cells resulted in reduced
expression of Tyr216 GSK-3B in response to TWEAK in Bv2 microglial cells (Fig 9F). Taken
together, our studies suggest that SYK is a critical determinant of GSK 3 beta activation and ALS
dysfunction in BV2 cells upon TWEAK stimulation.
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Evaluation Of The Therapeutic Efficacy of Syk Inhibitor (Piceatannol) In a TWEAK
Neuroinflammation Mouse Model.
To further investigate whether the data generated using in vitro models of TWEAK can be
validated in an in vivo scenario we utilized the intra striatal infusion neuro-inflammation model of
TWEAK. Additionally, we investigated the effects of SYK inhibitor Piceatannol in the afore
mentioned neuro-inflammation model. Previous studies have demonstrated the validity of using
the neuro-inflammation model of TWEAK to study TWEAK-induced inflammatory response (W.
B. Haile, R. Echeverry, J. Wu, et al., 2010; Polavarapu et al., 2005). Moreover, at the doses used
in the present study Piceatannol (Syk inhibitor) has been shown to downregulate Syk mediated
neuro-inflammation in mouse models of subarachnoid hemorrhage and ischemic stroke (Y. He, L.
Xu, B. Li, Z. Guo, et al., 2015; Y. Suzuki et al., 2013). To determine the effects of Piceatannol on
TWEAK-induced inflammatory response, mice were pretreated with Piceatannol (100 mg/kg
intraperitoneally) 15h and 1h before Tweak (2ul, 1 ug/ul) was stereotaxically injected unilaterally
into the striatum. Mice were sacrificed 24h after TWEAK administration and striatal tissues were
examined for the expression of neuro-inflammation related markers using Western blot analysis.
As shown in Fig 10 A-H, no significant difference in the striatal inflammatory markers between
control and TWEAK infused mice was evidenced. In a similar fashion Piceatannol pretreatment
treatment had no effect on TWEAK-induced modulation of Syk, Caspase-1, CHOP, LC3, Beclin,
GSK-3 beta Y216 and cathepsin-B expression in the striatum (Fig 10 A-H). At this time
optimization of the TWEAK neuro-inflammation model is needed and therefore we propose to
perform additional dose response studies to dissect TWEAK associated inflammatory response in
the striatum. Additionally, the dose of Piceatannol needs to be optimized to understand the
therapeutic effects of this drug in the in vivo TWEAK neuro-inflammation model.
219
Discussion
In the present study, we demonstrate the potential regulatory effects of SYK in TWEAK-
induced microglial activation response. Upon treatment of microglia with Tweak, a dramatic
upregulation of spleen tyrosine kinase coincided with NLRP3 inflammasome activation, pro-
inflammatory cytokine production, ER stress response (ERS), mitochondrial dysfunction, GSK-3
beta and ALS dysfunction. Interestingly, Syk inhibition downregulated NLRP3 inflammasome
activation and pro-inflammatory cytokine production in Tweak treated microglial cells. To further
identify the molecular mechanism of anti-inflammatory effects of R788 we investigated whether
R788 affected TWEAK-induced ERS and ALS dysfunction. Our studies revealed a positive
association between NLRP3 inflammasome inhibition and attenuation of ERS, ALS impairment
and mitochondrial dysfunction in cells that received a combination of TWEAK and R788. In our
in vivo studies, Piceatannol, a Syk inhibitor displayed a trend toward attenuation of TWEAK-
induced ALS modulation in an intracerebral Tweak neuro-inflammation mouse model although it
failed to reach statistical significance. Further studies are warranted to quantify the in vivo effects
of Piceatannol in the TWEAK Neuro-inflammation model. Our results highlight the pro-
inflammatory role of SYK in mediating TWEAK-induced NLRP3 inflammasome activation. A
potential mechanism by which TWEAK may induce NLRP3 inflammasome activation via SYK
by initiating downstream signaling events is illustrated in Fig 4.6.
Spleen Tyrosine Kinase is is 72 kDa non-receptor tyrosine kinase that plays an important
role in microglial activation and neuro-inflammation in immune cells (Y. S. Yi et al., 2014b). The
main trigger for activation of spleen tyrosine kinase is phosphorylation of ITAMs
(Immunoreceptor Tyrosine based Activation Motifs) (H. Hara, K. Tsuchiya, I. Kawamura, R.
Fang, E. Hernandez-Cuellar, Y. Shen, J. Mizuguchi, E. Schweighoffer, & V. Tybulewicz, 2013;
220
Turner et al., 2000; Y. S. Yi et al., 2014b). Its main functions include Innate pathogen recognition,
signaling tissue damage, inflammasome activation, bone metabolism, arterial thrombus formation,
vascular development, B-cell development, phagocytosis and gonadal development (Mocsai et al.,
2010; Tohyama & Yamamura, 2009; Y. S. Yi et al., 2014b). Consistent with a pro-inflammatory
role for SYK, TWEAK-induced robust activation of this enzyme in Bv2 and primary microglial
cells.
NLRP3 inflammasome is an intracellular multi-meric protein complex that becomes
activated by wide variety of pathogens, environmental irritants, toxins and PAMPs (Pathogen
associated molecular patterns) (Kate Schroder et al., 2010). It consists of NLRP3 scaffold, ASC
adaptor and caspase-1 (Kate Schroder et al., 2010). A wide variety of factors can activate the
NLRP3 inflammasome including ATP, Asbestos, Silica, uric acid and amyloid (Cassel et al., 2008;
Halle et al., 2008; S. Mariathasan et al., 2006; Martinon et al., 2006). NLRP3 plays an important
role in the generation of mature pro-inflammatory cytokines namely IL-1B and IL-18 in a caspase-
1 dependent manner in order to mount an innate immune response (Kate Schroder et al., 2010).
Accordingly, we demonstrated the upregulation of NLRP3, caspase-1, IL-18 and IL-1 beta via WB
analysis in Tweak and LPS treated BV2 microglial cells. A growing body of evidence supports a
role for SYK in the regulation of NLRP3 inflammasome upon exposure of inflammatory cells to
infectious agents (Gross et al., 2009; H. M. Lee et al., 2012; Shio et al., 2009). For example SYK
dependent ASC phosphorylation has been shown to promote NLRP3 inflammasome assembly and
activation (H. Hara, K. Tsuchiya, I. Kawamura, R. Fang, E. Hernandez-Cuellar, Y. Shen, J.
Mizuguchi, E. Schweighoffer, & V. Tybulewicz, 2013). Accordingly, Syk inhibitor (R788)
treatment resulted in attenuated expression of NLRP3 and caspase-1 in Tweak treated microglial
cells. Like-wise, siRNA mediated knockdown of Syk downregulated the expression of NLRP3
221
and caspase-1 in Tweak treated BV2 microglial cells. Syk induced NLRP3 activation is associated
with pro-inflammatory cytokine production including IL-1B and IL-18 in neutrophils and
macrophages (Feng et al., 2015; R. Lu et al., 2012; M. W. Ma et al., 2017; Miller et al., 2012).
Moreover, Syk inhibitors downregulated the pro-inflammatory cytokine production and
inflammatory responses in dendritic cells and immune cells (Aouar et al., 2016; Llop-Guevara et
al., 2015; Patterson et al., 2014). Accordingly, in the present study R788 induced the down
regulation of mRNA and protein levels of pro-inflammatory cytokines including IL-1B, IL-18,
and TNFa in Tweak treated primary microglial cells. To the best of our knowledge this is the first
study to demonstrate that SYK is a key regulator of NLRP3 inflammasome activation in microglial
cells in response to TWEAK.
The most common modes of NLRP3 activation includes K+ efflux, lysosomal damage and
ROS production (Cassel et al., 2008; C. Dostert et al., 2008; Halle et al., 2008; Hornung et al.,
2009; T. D. Kanneganti et al., 2006). Thus, in the present study, we investigated whether SYK
modulates the activation of ER stress, mitochondrial dysfunction, ALS dysfunction in Tweak
treated microglial cells. In innate immune cells such as microglia, SYK has been shown to play a
key role in receptor mediated generation of ROS and RNS (Y. S. Yi et al., 2014b). Moreover,
functional interaction of A beta with microglial cells has been shown to induce the phosphorylation
and activation of SYK thereby leading to ROS generation and neurotoxicity (Combs et al., 1999;
S. Ghosh et al., 2015; McDonald et al., 1997). Microglia stimulated with LPS trigger cytokine
secretion and ROS generation in a NOX2 dependent manner (S. Hong & Yu, 2017; L. Hou et al.,
2017; Kwon et al., 2017; L. Qin et al., 2004). Consistent with a role for SYK in oxidative stress
mechanisms, SYK inhibitors blocked the ability of chronically activated microglia to generate
ROS and RNS thereby blocking its neurotoxic effects (S. Ghosh et al., 2015). Moreover, ROS
222
triggered NLRP3 inflammasome activation have been identified as a proximal trigger although the
role of ROS remains controversial (Cassel et al., 2008; C. M. Cruz et al., 2007). Our results also
support a role for SYK in the regulation of NOX2 which is implicated in NLRP3 inflammasome
activation (Feng et al., 2015; M. W. Ma et al., 2017). Likewise, siRNA mediated KD of SYK
attenuated TWEAK-induced NOX2 activation suggesting that elevated NOX2 derived ROS may
be linked to NLRP3 inflammasome activation. Alternatively, we and others have demonstrated
that mitochondrial dysfunction leads to ROS production, release of mitochondrial DNA into the
cytosol eventually leading to NLRP3 inflammasome activation (L. Chen et al., 2015; Lawana et
al., 2017; Sarkar et al., 2017). Although, we demonstrated dissipation of mitochondrial membrane
potential, NOX2 activation and ROS production paralleled NLRP3 inflammasome activation in
Tweak treated BV2 microglial cells the contribution of mitochondrially derived ROS cannot be
entirely excluded. Thus, it is likely that concomitant activation of NOX2 and mitochondrial
dysfunction may lead to NLRP3 inflammasome activation in TWEAK treated microglial cells.
The expression levels of chaperone proteins including GRP78 and GRP 94 as well
as p-eiF2a are increased in response to unfolded protein response (UPR) and therefore have been
used as markers for ER stress response (Jain et al.). BV2 microglial cells treated with TWEAK
displayed an increase in the levels of the afore mentioned proteins, indicating that TWEAK induces
ER stress response (ERS). Owing to a positive correlation between increased SYK activity and
activation of ERS we hypothesized that TWEAK stimulation led to ERS via SYK activation.
Accordingly, our data indicate that TWEAK-induce ERS via SYK activation based on the fact
that, R788 and siRNA mediated knock down of SYK, both of which inhibited the induction of
ERS. ERS serves as an important triggering factor for NLRP3 inflammasome (Menu et al., 2012b;
J. Wang et al., 2015). ER stress sensor IRE1-alpha causes mitochondrial damage through caspase-
223
2 and Bid thereby leading to NLRP3 inflammasome activation and IL-1 beta production (D. N.
Bronner et al., 2015). Furthermore, in the UPR dependent pathway, ER stress related proteins IRE1
alpha and PERK leads to upregulation of Thioredoxin interacting protein (TXNIP) & CHOP;
thereby leading to NLRP3 inflammasome activation (D. N. Bronner et al., 2015; Lebeaupin et al.,
2015a; Lerner et al., 2012). A marked reduction in NLRP3 inflammasome markers positively
correlated with diminished ERS in a SYK dependent manner suggesting that TWEAK-induced
ER stress mechanism via SYK mediated mechanism may at least in part mediate NLRP3
inflammasome activation. Further study is required to clarify the impact of inhibition of ER stress
response on NLRP3 inflammasome activation under our experimental conditions.
Glycogen synthase kinase 3B (GSK-3b) has been shown to play a critical role in response
to bacterial infection. Depending on the cellular context, type of pathogen in which host bacteria
interaction occurs, GSK3B may elicits an inhibitory or stimulatory effect(Cortes-Vieyra et al.,
2012). For example, in LPS stimulated human monocytes GSK 3b was found to negatively
regulate the level of IL-1Ra, an anti-inflammatory cytokine while simultaneously increasing the
levels of IL-1B which was attributed to the modulation of extracellular signal related kinase 1/2
(ERK1/2) (Rehani et al., 2009). Furthermore, previous studies have demonstrated that inhibition
of GSK beta increased the production of anti-inflammatory cytokines while reducing the levels of
pro-inflammatory cytokines consistent with a model proposed by Martin et al (M. Martin et al.,
2005) whereby GSK-3b was found to promote pro inflammatory response (H. Wang et al., 2011;
Z. Wang et al., 2008). In line with these findings, TWEAK treatment elicited a marked increase
in the expression levels of tyrosine phosphorylated GSK B (Y2016 GSK-3B) while down
regulation of SYK activation via pharmacological inhibition or SYK siRNA resulted in profound
abrogation of pGSK 3B (Y2016) expression suggesting that SYK is a key regulator of GSK 3b
224
activation. Consistent with our finding SYK deficiency in dendritic cells was found to enhance
LPS-induced IFNb and IL-10 expression but suppressed pro-inflammatory cytokine levels, namely
TNF-a and IL-6 via PI3K-Akt and NF-kB signaling pathways suggesting a divergent influence of
SYK on pro-and anti-inflamamtory in TLR-mediated response (Hui Yin et al., 2016).
Complex interactions between cytoplasmic kinases including MAP kinase and generation
of proinflammatory cytokines via NF-kB dependent mechanisms have been suggested (Chiou et
al., 2011). In our previous study using LPS stimulated microglia we demonstrated that Fyn-PKCd
signaling axis is a critical regulator of MAP kinase activation in microglial cells (N. Panicker, H.
Saminathan, H. Jin, M. Neal, & D. S. Harischandra, 2015) suggesting that Fyn kinase is a major
upstream regulator of pro-inflammatory signaling events. The present results demonstrate a time
dependent increase in the activation of MAP kinase signaling events as demonstrated by increased
phosphorylations of p38, ERK1/2, and JNK following TWEAK stimulation. Moreover, R788
pretreatment in TWEAK stimulated microglial cells markedly down regulated MAPK activation.
Our results are consistent with a previous observation that pharmacological inhibition of SYK
attenuate MAPK, including P38MAPK and JNK signaling events (Clifford A. Lowell, 2011).
Contrary to our finding, a recent report demonstrated that Syk deficiency fails to alter the activation
of MAPK signaling in LPS stimulated dendritic cells (Hui Yin et al., 2016). Thus, it appears that
SYK may evoke cell type and context specific effects in regards to MAPK activation upon
inflammagen stimulation of myeloid cells. Further studies are warranted to study the impact of
MAPK inhibition on NLRP3 inflammasome activation in BV2 microglial cells subjected to
TWEAK stimulation.
Autophagic mechanisms involves the engulfment of damaged organelles and cellular into
double membrane vacuoles called autophagosomes. The autophagosomes ultimately fuse with the
225
lysosomes thereby facilitating recycling of cellular macromolecules. In fact, lysosomal
dysfunction is associated with the abnormal accumulation of autophagosomes in familial PD cases
(Dehay et al., 2010). Moreover, autophagy negatively regulates NLRP3 inflammasome activation
and pro-inflammatory cytokine production in immune cells (V. A. Rathinam et al., 2012; T. Saitoh
& S. Akira, 2016; Yuk et al., 2013; Z. Zhong et al., 2016). Conversely, genetic ablation of
autophagy related markers including LC3-II and BECN-1 was found to aggravate NLRP3
inflammasome activation in ATP stimulated macrophages (P. Gurung et al., 2015; Salminen et al.,
2012). In our study, we demonstrated increased expression of autophagy markers in Tweak treated
microglial cells. Given that mitochondrial dysfunction and ROS generation precedes NLRP3
inflammasome activation in TWEAK treated microglial cells our results raise the possibility that
excessive ROS generation via NOX2 activation alongside defective mitophagic mechanism owing
to impaired clearance of ROS generating damaged mitochondria might have partly contributed to
NLRP3 inflammasome activation. Indeed SYK has been shown to mediate ROS generation and
activation of MAPK which in turn activates autophagy via disassociation of BECN1 from Bcl2
(S. H. Choi et al., 2015; Hideki Hara et al., 2013; Kang et al., 2011). Our results underscore the
importance of SYK mediated autophagy upregulation in the induction of microglial pro
inflammatory response. Another NLRP3 inflammasome activating mechanism involves the
lysosomal membrane destabilization resulting in the cytosolic release of lysosomal hydrolases
including cathepsin B (R. Lu et al., 2012; Orlowski et al., 2015; Tseng et al., 2013; L. Wang et al.,
2016; K. Weber et al., 2014). Microbial toxins that trigger lysosomal membrane destabilization
leads to release of lysosomal cathepsin B into cytosol thereby causing NLRP3 inflammasome
activation (Tseng et al., 2013). Moreover, in LPS treated macrophages lysosomal Cathepsin B
release into cytosol results in NLRP3 inflammasome activation and IL-1 beta secretion (K. Weber
226
et al., 2014). Consistent with these reports, TWEAK-induced NLRP3 inflammasome activation
positively correlated with upregulation of cathespin B suggesting altered lysosomal function might
be partly responsible for NLRP3 inflammasome activation. Conversely, abrogation of cathepsin
B reduced caspase-1 mediated IL-1B release in response to MSU crystals (Kingsbury et al., 2011;
Välimäki et al., 2013). Intriguingly, Syk has been linked to autophagolysosomal system (ALS)
dysfunction upon microbial infection in macrophages (Krisenko, Higgins, et al., 2015).
Consistently, both R788 and Syk siRNA ameliorated TWEAK induced ALS impairment as
evidenced by reduced accumulation of LC3-II, Becn-1 and Cathepsin-B levels in BV2 microglial
cells. Together our studies suggest that SYK is a key upstream regulator of ALS function in
microglia in response to TWEAK. Further studies are warranted to determine whether ASC
phosphorylation is an important step in the mechanism of ALS dysfunction in TWEAK treated
cells.
Syk inhibitors are currently used for treatment of allergic rhinitis, autoimmune
thrombocytopenia,and B-cell malignancies (Mocsai et al., 2010). Fostamatinib sodium is a small
molecule of Syk inhibitor that has shown efficacy in animal models of Asthma, Lymphoma,
Glomerulonephritis and Diabetes (McAdoo, 2011). Based on the observation that R788 and siRNA
Syk known-down attenuated NLRP3 inflammasome activation, Caspase-1, CHOP, Cathepsin-B
and GSK-3 Beta activation in Tweak treated BV2 microglial cells we next sought to corroborate
our in vitro findings in an intra-striatal infusion mouse model of TWEAK that were pretreated with
the TWEAK inhibitor Piceatannol. In the in vivo TWEAK model although several of the
inflammation related markers displayed a trend toward an increase it failed to reach significance
as compared to vehicle treated mice. In a similar fashion Piceatannol failed to modulate TWEAK-
induced effects. Therefore, further studies are needed to confirm the inflammatory response
227
elicited following TWEAK infusion into the striatum, and the optimal dose of Piceatannol needed
to elicit an anti-inflammatory response.
Our present study emphasizes the pro-inflammatory role of SYK as assessed via the
activation of NLRP3 inflammasome in microglial cells in response to TWEAK in microglial cells.
Moreover, this is the first study demonstrating that inhibition of ROS generation, mitochondrial
dysfunction, ERS, and ALS dysfunction may partially account for the suppressive effect of SYK
inhibition on NLRP3 inflammasome activation. In conclusion, the study provides novel insights
into the anti-inflammatory role of R788 and in this context highlighted its ability to inhibit NLRP3
inflammasome via suppression of oxidative stress dependent mechanisms. Thus, selective
targeting of SYK may serve as a novel therapeutic intervention in the modulation of inflammatory
immune responses in several neuro-inflammation related disorders including PD.
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Figures
241
Fig 1. Increased Spleen Tyrosine Kinase and stress granules expression in Tweak treated BV2
microglia. A-B. Increased Syk expression in Tweak and LPS treated BV2 microglia. BV2 microglial cells
was treated with LPS 1ug/ml and Tweak 100 ng/ml for 3h, 6h, 12h, 24h. The control group was treated with
vehicle (1x PBS) used to prepare LPS and Tweak prior to treatment. Post treatment, 40 ug of protein that
was loaded was separated with 10% SDS-PAGE and probed with antibodies specific for Spleen Tyrosine
Kinase. To confirm equal protein in each lane, membranes were re-probed with Actin. The experiment
was repeated 2 times and a representative western blot was shown. Significant upregulation of spleen
tyrosine kinase expression was demonstrated in BV2 microglial cells upon stimulation with Tweak and LPS
using densitometric analysis after normalizing with -actin. Asterisk (**p < 0.01 or ***p < 0.001) represents
significant difference between Tweak / LPS treated groups and control group as determined by Anova
followed by Student Newman Keul’s post test. (C) Increased SYK Kinase activity in Tweak treated primary
microglial cells. The kinase assay was determined using a standard immunoprecipitation kinase assay.
Primary microglia were treated with Tweak 100 ng/ml for 12h and 24h with and without R788 Syk inhibitor
pretreatment. The SYK protein was immuno-precipitated from 500 ug of total protein and processed for in
vitro kinase assay with 5 ug biotin labelled SYK kinase substrate and ATP. Tyrosine phosphorylation of
SYK substrate was identified by monoclonal anti-phosphotyrosine antibody. The kinase activity is
quantified by densitometric analysis of phosphorylated SYK kinase substrate and expressed as percent
control. Recombinant SYK enzyme was used as a positive control in this experiment. SYK kinase assay is
repeated two times with N=6 and a representative graph is shown. The increase in SYK protein is
accompanied by increase in SYK kinase activity at 24h which was partially attentuated by pretreatment with
R788 (SYK inhibitor). Asterisk (**p < 0.01) represents significant differences between Tweak / R788
treated groups and control groups as determined by Anova followed by Student Newman Keul’s post test.
D) Increased stress granules expression in Tweak treated BV2 microglial cells. Immortalized BV2
microglial cells was treated with Tweak 100 ng/ml for 3h, 6h, 12h, 24h and then G3BP1 stress granules
levels were determined with western blotting. Actin was used as loading control for equal protein loading
in all the lanes. Representative western blots were shown and experiments were repeated two times.
Densitometric scanning analysis revealed increased G3BP1 expression in Tweak treated microglial cells at
12h and 24h. Asterisk (***p < 0.001) indicates significant differences between Tweak treated groups and
control group as calculated by Anova followed by Student Newman Keul’s post test.
242
243
Fig.2 Increased NLRP3 related markers and pro-inflammatory cytokine secretion in Tweak and
LPS treated BV2 microglial cells. A-D. BV2 microglial cells were primed with LPS (1ug/ml) for 3h
followed by stimulation with Tweak (100 ng/ml) for various time points (6h,12h & 24h). Post incubation,
lysates were separated with 12% SDS-PAGE and immunoblotted with antibodies specific for NLRP3
(A), Caspase-1 (B), IL-1 beta (C) and IL-18 (D). Membranes are re-probed with actin to ensure equal
protein loading in all the lanes. Densitometric scanning analysis revealed increased time dependent
increase in NLRP3 related markers in LPS and Tweak treated BV2 microglial cells. The experiment is
repeated two times and representative western blot is shown. Each data set is expressed as percentage of
control group and represents Mean ± S.E.M. Statistical differences were assessed with ANOVA followed
by Student Newman Keul’s post test. Asterisks (**p < 0.01 or ***p < 0.001) indicates significant
differences between Tweak treated groups and control groups. E–G Increased NLRP3 inflammasome
markers in Tweak stimulated BV2 microglia. BV2 microglia was treated with Tweak 100 ng/ml for 3h,
6h, 12h and 24h and lysates were probed with antibodies specific for NLRP3, Caspase-1 and IL-1 beta.
Band intensities of NLRP3, caspase-1 and IL-1 beta were quantified by densitometric analysis and
normalized to actin. Immunoblot analysis revealed that upregulation of NLRP3 (E), Caspase-1 (F) and
IL-1 beta (G) in Tweak treated BV2 microglial cells.The experiment was repeated two times and a
representative blot is shown. Asterisk (*p < 0.05) represents significant differences between Tweak
treated groups and control groups as assessed by ANOVA followed by Student Newman Keul’s post test.
H. Increased pro-inflammatory cytokine secretion in Tweak treated BV2 microglia. BV2 microglia were
treated with Tweak 100 ng for 8h and 24h. Cytokine levels in the supernatant were quantified at various
time points (8h and 24h) with the help of Luminex immunoassay system. Bar graph represents secreted
IL-1β and TNF-α in supernatant of Tweak treated microglial cells at various time points as determined
by ELISA immunoassay. The experiment is repeated two times and is represented as mean ± S.E.M with
N=6 in each treatment group. Statistical differences were assessed with ANOVA followed by Student
Newman Keul’s post test. Asterisks (**p < 0.01 or ***p < 0.001) indicates significant differences
between Tweak treated groups and control groups. I Increased pro-inflammatory cytokine mRNA
expression in Tweak treated BV2 microglia. SYBR Green real time quantitative PCR analysis of IL-1
beta, IL-18 and TNF mRNA in BV2 microglia treated with Tweak 100 ng/ml for 3h and 6h. After
treatment, RNA was extracted and RT-PCR was performed with 18s used as internal standard for
normalization. The normalized fold of increase in pro-inflammatory cytokine gene expression over the
control group was calculated by the ΔΔCt method. Each experiment is performed in triplicates at least
3 times independently. Statistical differences were assessed with ANOVA followed by Student Newman
Keul’s post test. Asterisks (**p < 0.01 or ***p < 0.001) indicates significant differences between Tweak
treated groups and control groups.
244
245
Fig 3. Increased NLRP3 inflammasome expression and Kinase expression in Tweak treated
BV2 microglial cells. A–C. Primary microglia was treated with Tweak 100 ng/ml for 3h, 6h,12h
and 24h. Lysates were probed for NLRP3, Caspase-1, IL-1 beta and -actin by western blotting.
Band intensities were quantified by densitometric analysis revealed upregulation of NLRP3 (A),
Caspase-1 (B), IL-1 beta (C) in Tweak treated primary microglial cells. Experiments were repeated
two times and a representative western blot is shown. Each data set is expressed as percentage of
control group. Asterisk (*p < 0.05 or **p < 0.01) represent significant differences between Tweak
treated groups and control groups as determined by ANOVA followed by Student Newman Keul’s
post test. D-E. Upregulation of SYK and PKC-delta kinase expression in Tweak treated primary
microglial cells. Following treatment of primary microglia with Tweak 100 ng/ml for 3h, 6h,12h
and 24h, redox sensitive kinases expression was evaluated when lysates were separated with SDS-
PAGE and subjected to western blot analysis with antibodies specific for SYK and PKC-delta.
Equal amount of protein was loaded in all the lanes with the help of -actin. Densitometric analysis
revealed upregulation of SYK (D) and PKC-delta (E) in Tweak treated primary microglial cells.
Experiment is repeated two times with representative blot shown. Asterisk (*p < 0.05 or **p < 0.01)
indicates significant differences between Tweak treated groups and control group as determined by
ANOVA followed by Student Newman Keul’s post test.
4A
4B
246
Fig.4 Tweak induced dissipation of Mitochondrial Membrane potential (MMP), ROS and
NOS production in BV2 microglial cells. A. Increased ROS production in Tweak treated BV2
microglia. Reactive oxygen species production was quantified in BV2 microglia treated with LPS
1ug/ml 6h, Tweak 50 ng, 250 ng and 500 ng for 3h and 6h. Total iROS generation was measured
by quantifying the redox sensitive CM-H2DCFDA spectrophotometrically using microplate
reader. Significant upregulation of ROS production was demonstrated in Tweak treated BV2
microglia that peaks at 3h and 6h. Experiment is repeated three times with N=6 in each treatment
group. Asterisk (p***<0.001) indicates significant differences between Tweak treated groups and
control group as determined by ANOVA followed by Student Newman Keul’s test. B. JC-1
fluorometry revealed significant time dependent dissipation of mitochondrial membrane potential
(MMP) in Tweak treated BV2 cells. BV2 microglial cells primed with LPS 1ug/ml for 3h were
treated with Tweak 100 ng for 24h prior to incubation with JC-1 dye for 30 min. Changes in
membrane potential (ΔΨm) were determined spectrophotometrically using Gemini Fluorescence
microplate reader. Reduction in red: green absorbance ratio of JC-1 dye indicate the loss of
mitochondrial membrane potential (Δψm). Significant time dependent dissipation of mitochondrial
membrane potential (MMP) was noticed in BV2 microglial cells upon Tweak and LPS treatment.
The experiment was repeated for three times with N=8 and a representative graph is shown.
Asterisk (p***<0.001) represents significant differences between Tweak / LPS treated groups and
control group as determined by ANOVA followed by Student Newman Keul’s test. C. SiRNA
mediated SYK knockdown suppressed in Tweak induced NOX2 production in BV2 microglia.
BV2 microglial cells were transfected with scrambled or SYK specific SiRNA for 72h and then
treated with or without Tweak (100 ng/ml) treatment for 24h. Whole cell lysates were probed with
NOX2 antibody using western blot with equal loading ascertained by -actin. Densitometric
analysis revealed that Syk knockdown downregulated NOX2 production Tweak stimulated BV2
microglial cells. Experiments were repeated four times and a representative sample was shown.
Asterisk (P***<0.001) represents significant differences between Tweak treated groups and
Tweak treated Syk SiRNA transfected as determined by ANOVA followed by Student Newman
Keul’s test.
4C
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Fig.5 Increased ER stress marker expression in Tweak treated BV2microglia. A-E. Western
blot analysis of cell lysates from BV2 microglial cells treated with Tweak (100 ng/ml) for 3h, 6h,
12h, 18h and 24h and probed for antibodies specific to CHOP, GRP-94, Phospho-elf, ATF-4 and
Phospho-PERK. -actin was used in ascertain equal protein loading in all the lanes. Band
intensities of ER stress markers were analyzed after normalization with -actin and were presented
next to western blot. The experiment is repeated three times and a representative blot was shown.
Each treatment group is represented as Mean ± S.E.M. Statistical differences between Tweak
treated groups and controls are analyzed using ANOVA followed by Student Newman Keul’s post
test. Asterisk (p < 0.01, *** p < 0.001) represents significant differences between Tweak treated
groups and control group.
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Fig.6 Autophagosomal lysosomal marker modulation in Tweak stimulated Bv2 microglial
cells. A-E. Increased Autophagy marker expression in Tweak treated BV2 microglia. Western blot
analysis of cell lysates treated from BV2 microglial cells were treated with Tweak (100 ng/ml) for
3-24h using antibodies specific to LC3, Beclin, P62 , ATG 5-12 and P-AMPK. All the lanes had
equal protein loading that were confirmed with -actin that was used as a internal loading control.
Bar graphs represent mean ± S.E.M with results are representative of at least 2 independent
experiments. Significant upregulation of autophagy markers was demonstrated in Tweak treated
BV2 microglia. Statistical differences between Tweak treated groups and controls was carried out
using ANOVA followed by Student Newman Keul’s post test. Asterisk (* p < 0.05,** p < 0.01,
*** p < 0.001) represents significant differences between Tweak treated groups as compared with
control. F-G. Lysosomal dysfunction with release of Cathepsin-B and Cathepsin-D in Tweak
treated BV2microglia. BV2 microglial cells were treated with 100 ng/ml Tweak for increasing
time periods (3-24h) and proteins are resolved by SDS-PAGE and immunoblotted for Cathepsin-
B and Cathepsin-D to detect Immature and mature active forms. Optical densities of protein bands
were analyzed with the Odyssey imaging system (LI-COR) and -actin was used a internal loading
control. Immunoblot analysis shows time dependent increase in secretion of active form of
cathepsin B and D in Tweak treated BV2 microglial cells. Results are representative of at least 2
independent experiments and a representative western blot is shown. Statistical analysis was
carried out using ANOVA followed by Student Newman Keul’s post test. Asterisk (*** p < 0.001)
represents significant differences between Tweak treated groups and control group. H. Increased
Lysosomal PH in Tweak treated BV2 cells. BV2 microglial cells were treated with 100 ng/ml
Tweak for 3h, 6h, 12h, 18h and 24h. Post treatment, lysosomal pH quantification was further
performed using the ratiometric lysosomal pH probe LysoSensor Yellow/Blue dextran (Invitrogen
L22460). Changes in lysosomal PH was determined by using the fluorescent microplate reader
with emission wavelengths at 535 nm and 430 nm with excitation at 340 nm. The experiment is
repeated three times with N=6 and expressed as percent of control. The data represent mean ±
S.E.M. Statistical analysis is performed with ANOVA followed by Student Newman Keul’s post
test. Asterisk (*** p < 0.001) represents significant differences between Tweak treated groups and
control groups
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Figure 7. Spleen Tyrosine Kinase regulates NLRP3 related markers in Tweak treated
microglial cells. A-C. R788 attenuates NLRP3 inflammasome related markers in the Tweak
treated BV2 microglial cells. BV2 microglial cells were pretreated with Syk inhibitor R788 (3uM
1hr) followed by treatment with Tweak (100ng/ml) stimulated BV2 microglial for 24h. Western
blot analysis of whole cell lysates from above treated microglial cells with NLRP3 related markers
showed that R788 pretreatment attenuated NLRP3, caspase-1 and IL-1 beta activation in Tweak
treated BV2 microglial cells. The band intensities of each treatment group were normalized with
-actin and presented as Mean ± S.E.M. The experiment is repeated for 2 times and a representative
blot is shown. Asterisk (* p < 0.05) represents significant differences between Tweak treated
groups and control groups as determined by ANOVA followed by Student Newman Keul’s post
test. D. SiRNA mediated SYK knockdown resulted in efficient knockdown of SYK expression in
BV2 cells. BV2 microglial cells were transfected with either scrambled or SYK siRNA. 72 hrs
post transfection, BV2 microglial cells were subjected to immunoblot analysis for SYK to confirm
efficient downregulation of SYK expression. Representative immunoblot image confirming the
SiRNA knockdown of SYK but not in scrambled SiRNA, reduces the SYK protein to 20% of
control levels in BV2 microglia (D). Asterisk (* p < 0.05) demonstrates significant differences
between Si RNA SYK transfected groups and scrambled SiRNA transfected groups as determined
by ANOVA followed by Student Newman Keul’s post test. E-F. Syk knockdown attenuates
NLRP3 inflammasome markers in Tweak treated microglial cells. 72h post-siRNA transfection
with either scrambled or the Syk siRNA, BV2 microglial cells were subjected to the Tweak (100
ng/ml) stimulation for 24h. Whole cell lysates were later probed with antibodies specific to NLRP3
and caspase-1 with equal loading ascertained with re-probing the membranes with -actin. Results
are representative of at least 3 independent experiments. Syk knockdown attenuated Tweak
mediated upregulated NLRP3 and Caspase-1 in BV2 microglia. Statistical differences between
treatment groups were determined by ANOVA followed by Student Newman Keul’s post test.
Asterisk (** p < 0.01 & *** p < 0.001) represents significant differences between as Tweak / SYK
SiRNA treated groups and Tweak treated groups.
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Figure 8. Involvement of Syk in MAPK activation in microglial cells. A-C. Increased MAP
kinases expression in Tweak treated BV2 microglia. BV2 microglial cells were treated with 100
ng/ml Tweak for 30 min, 60 min, 120 min and 180 min. Post treatment, whole cell lysates were
harvested and separated with SDS-PAGE. MAPK phosphorylation status was probed by western
blotting using antibodies specific to Phospho-P38, Phospho-JNK and Phospho-ERK. Values are
means ± S.E.M obtained from three independent experiments. Representative western blot
revealed significant upregulation of MAPKs Phospho- P38 (A), Phospho-C Jun ( B) and Phospho-
ERK (C) in Tweak treated BV2 microglial cells. Statistical differences between Tweak treated
groups and control was carried out using Anova followed by Student Newman Keul’s post test.
Asterisk (* p < 0.05,** p < 0.01) represents significant differences as compared with control group.
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D-E. R788 attenuates MAPK activation in the Tweak treated BV2 microglial cells. BV2 microglial
cells were pretreated with Syk inhibitor R788 (3uM 1hr) followed by treatment with Tweak
(100ng/ml) stimulated BV2 microglial for 1h. Western blot analysis of whole cell lysates from
above treated microglial cells showed that R788 pretreatment attenuated MAPK activation in
Tweak treated BV2 microglial cells. The band intensities of each treatment group were normalized
with -actin and presented as Mean ± S.E.M. The experiment is repeated for 2 times and a
representative blot is shown. Asterisk (* p < 0.05) represents significant differences between
Tweak treated groups and control groups as determined by Student Newman Keul’s post test.
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Figure 9. Syk regulates Tweak induced impairment of auto-lysosomal system and GSK-3
beta in Bv2 microglial cells. A-B. Syk knockdown attenuates Tweak induced impairment of auto-
lysosomal system (ALS) in Bv2 microglial cells. BV2 Cells were transfected with either scrambled
or the Syk siRNAs for 72 h. The transfected microglia were subsequently treated with and without
Tweak (100ng/ml) for 24 h. Post treatment, whole cell lysates were collected, separated with SDS-
PAGE and probed with antibodies specific for LC3 and Beclin. Equal protein loading in all the
lanes is ascertained with -actin. Results are representative of at least 4 independent experiments.
Syk knockdown attenuated Tweak mediated upregulated LC3 and Beclin (A-B) in BV2 microglia.
Asterisk (*** p < 0.001) represents significant differences between as Tweak / SYK SiRNA treated
groups and Tweak treated groups. C-D. SiRNA mediated SYK knockdown and SYK inhibitor
(R788) attenuates Tweak induced Cathepsin-B expression in BV2 microglial cells. For
knockdown studies, BV2 microglia were transfected with scrambed and Syk scrambled SiRNA
for 72h and then treated with or without Tweak (100 ng/ml) for 24h. For SYK inhibitor studies,
BV2 microglia were pre-incubated with SYK inhibitor R788 (3uM) or DMSO for 1hr and then
stimulated with or without Tweak (100 ng/ml) for 24h. Post treatment, whole cell lysates were
separated with SDS-PAGE and probed for Cathepsin-B using western blot with equal loading
ascertained by actin. The experiments were repeated for four times and a representative western
blot was shown. Si RNA mediated SYK knockdown or SYK inhibition by R-788 suppressed
Tweak mediated Cathepsin-B upregulation (C-D) in BV2 microglial cells. Asterisk (* p < 0.05,
*** p < 0.001) indicates significant differences between Tweak / SYK Si RNA / R788 treated
groups and Tweak treated groups as determined by Anova followed by Student Newman Keul’s
post test. E-F. SiRNA mediated SYK knockdown and SYK inhibitor (R788) attenuates Tweak
induced GSK-3 beta Y216 expression in BV2 microglial cells. For knockdown studies, BV2
microglia were transfected with scrambed and Syk scrambled SiRNA for 72h and then treated with
or without Tweak (100 ng/ml) for 24h. For SYK inhibitor studies, BV2 microglia were pre-
incubated with SYK inhibitor R788 (3uM) or DMSO for 1hr and then stimulated with or without
Tweak (100 ng/ml) for 24h. Post treatment, whole cell lysates were probed for GSK-3 beta Y216
using western blot with equal loading ascertained by actin. The experiments were repeated for
four times and a representative western blot was shown. Densitometric scanning analysis revealed
that, Si RNA mediated SYK knockdown or SYK inhibition by R-788 attentuated Tweak mediated
GSK-3 Beta Y 216 upregulation (E-F) in BV2 microglial cells. Asterisk (* p < 0.05, *** p <
0.001) indicates significant differences between Tweak / SYK Si RNA / R788 treated groups and
Tweak treated groups as determined by Anova followed by Student Newman Keul’s post test.
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Figure 10. Effect of Syk inhibitor Piceatannol on Tweak treated striatal brain tissues. A-G.
Mice were treated with 2 ul of PBS or Tweak (1ug/ml) injected stereotaxically into striatum with
and without 12h and 1h Piceatannol pretreatment. Mice were sacrificed after 24h Tweak treatment.
Striatal brain tissues were dissected out from PBS or Tweak with and without Piceatannol
pretreated mice 24h after Tweak treatment. Striatal tissues lysates were prepared, separated with
SDS-PAGE and probed by western blotting for Syk, GSK-3 beta Y216, Beclin, LC3 II, CHOP,
Cathepsin-B and Caspase-1. -actin is used to ensure equal protein loading in the lanes. A
representative western blot is shown and experiment is repeated two times. The normalized band
intensity for each treatment group expressed as percentage of control group was plotted on
histogram. No significant difference in the striatal inflammatory markers between control and
TWEAK infused mice was evidenced. In a similar fashion Piceatannol pretreatment treatment had
no effect on TWEAK-induced modulation of inflammation related marker expression in the
striatum Statistical analysis was carried out using Anova followed by Student Newman Keul’s post
test.
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CHAPTER 4. SPLEEN TYROSINE KINASE MEDIATES NLRP3 INFLAMMASOME
ACTIVATION IN LPS STIMULATED MICROGLIA VIA NF-KB ACTIVATION, MAP
KINASE AND ROS GENERATION
To be Submitted to Journal of Biological Chemistry
Sri Kanuri1,2, Wesley Schoo1, Huajun Jin1, Vellareddy Anantharam1, Anumantha Kanthasamy1 and
Arthi Kanthasamy1*
Abstract
Parkinson’s disease (PD) is a multifactorial neurodegenerative disorder that is
characterized by pronounced microglial activation response. Accumulating evidence indicates that
dopaminergic neurotoxicity may results from the cross talk between oxidative stress and the
activation of stress sensitive kinases in the microglia. The present study investigated the anti-
inflammatory effects of the spleen tyrosine kinase (Syk) inhibitor R788 in an in vitro N9 microglial
cell line challenged with LPS. Exposure of N9 microglial cells led to a time dependent increase in
Syk activation, ROS generation, MAPK and NF-kB activation. Further LPS induced NF-kB
activation preceded the activation of NLRP3 inflammasome as evidenced by the increased
expression levels of NLRP3 as well as mature IL-1β, and Cas-1. Intriguingly, inhibition of Syk via
R788 suppressed the activation of microglia via the suppression of iROS and nitric oxide
production, phosphorylation of NF-kB, mitogen activated protein kinases (MAPKs), extracellular
signal regulated kinases 1 and 2 (ERK1/2), p38 MAPK and glycogen synthase kinase-3β (GSK3β).
Notably the activation of the NLRP3 inflammasome was markedly inhibited by R788. In
conclusion, these data suggest that R788 exerts its anti-inflammatory response upon LPS
1 Department of Biomedical Sciences, Iowa State University, Ames, IA 50010
2 Primary researcher and author
*To whom correspondence should be addressed. E-mail: [email protected]
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stimulation by inhibiting NLRP3 inflammasome activation which might be partly attributed to
inhibition of NF-kB activation and MAPK activation as well as down regulation of ROS
production. Thus, R788 may serve as a useful therapeutic intervention for the treatment of
neuroinflammatory disorders including PD.
Introduction
Parkinson’s disease (PD) is the second most common neurodegenerative disorder affecting
approximately 1% of the population at age 55 and nearly 5% of the age of 85 (Block, Zecca, et al.,
2007; Hirsch & Hunot, 2009). It is characterized by the degeneration of dopaminergic neurons in
the substantia nigra resulting in the motor deficits recognized as the cardinal signs of the disease:
rigidity, bradykinesia, and tremor (Hirsch et al., 2012). Though the direct cause of this neuronal
loss is not fully understood, there has been evidence suggesting that neuroinflammation could be
involved in the progressive degeneration of these neurons (Block, Zecca, et al., 2007; L. Qin et al.,
2007).
Microglia, the resident innate immune cell, are present in varying concentrations
throughout the brain with the highest concentration in the substantia nigra (Block, Zecca, et al.,
2007). Normally, these cells exist in a resting, inactivated state but can be activated by
immunological stimuli (Block, Zecca, et al., 2007). When activated, microglia exhibit increased
expression of cellular receptors and molecules including pattern recognition receptors (PRR), Toll-
like receptors (TLR), and NADPH oxidase (Y. S. Kim et al., 2006; Rock et al., 2004); however,
upon chronic activation, these enzymes can potentially produce toxic amounts of their cytotoxic
products such as reactive oxygen species (ROS) and nitrous oxide (NO) (Hirsch et al., 2009)
thereby leading to neuronal death. Neuronal death or the release of damaging molecules is one
stimulus that can induce this over activated state in microglia, resulting in the phenomena known
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as reactive microgliosis (Block & Hong, 2007; Block, Zecca, et al., 2007). Lipopolysaccharide
(LPS) activation of microglia via the transmembrane receptor TLR4 is frequently used as a
neuroinflammatory model system and has been found to cause progressive dopaminergic
neurodegeneration reminiscent of PD (Block, Zecca, et al., 2007).
Spleen tyrosine kinase (Syk) is a cytoplasmic non-receptor tyrosine kinase that has been
shown to be involved in the receptor signaling pathways of a number of diverse cell types (Mocsai
et al., 2010). Structurally, Syk has two SRC homology 2 domains and a C-terminus kinase domain
with a molecular weight of 72 kDa (Mocsai et al., 2010; Y. S. Yi et al., 2014a). It has a well-
documented and crucial role in the intracellular signaling pathways of the classical
immunoreceptors; B cell receptors, T cell receptors and Fc receptors (Mocsai et al., 2010). Syk
has been shown to be important for proper development of both T and B cells, with varying
involvement at different stages in each cell type’s development (Mocsai et al., 2010). This adaptive
immune cell signaling acts via immunoreceptor tyrosine-based activation motifs (ITAMs) in which
the phosphorylation of the cytoplasmic tyrosine residues results in the recruitment of Syk (Mocsai
et al., 2010). Syk changes conformation when it binds to the phosphorylated ITAMs, allowing for
activation, autophosphorylation, and dissociation from the ITAM (Y. S. Yi et al., 2014a). It is
then able to activate downstream proteins in the signaling pathway via phosphorylation or direct
association (Y. S. Yi et al., 2014a). Syk has more recently been found to have a role in several
other cellular functions through activation by different upstream molecules and receptors (Mocsai
et al., 2010). Transmembrane integrins utilize ITAM-containing transmembrane adaptor
molecules to facilitate phosphorylation of Syk’s SH2 domain tyrosines, resulting in adhesion and
migration in leukocytes, activation of platelets, and promotes development of osteoclasts (Mocsai
et al., 2010). Syk has also been implicated in signaling pathways activated by P-selectin
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glycoprotein ligand-1 resulting in transcriptional activation via the ITAM related sequences of the
ezrin-radixin-moesin proteins (Berton et al., 2005).
Previous studies indicate that LPS is associated with chronic neuro-inflammation and
progressive neuro-degenration. Systemic administration of LPS in mice resulted in microglial
NLRP3 inflammasome activation, increased pro-inflammatory cytokines (TNF-alpha, IL-1 beta
and MCP-1) and reduction of dopaminergic neurons in substantia nigra (L. Qin et al., 2007; Z.-T.
Zhang et al., 2016; W. Zhu et al., 2017). Accumulating evidence suggests that, LPS induces neuro-
inflammatory responses through spleen tyrosine kinase in immune cells. In immune cells, LPS
induced activation of spleen tyrosine kinase resulted in NF-kB activation, NLRP3 inflammasome
formation, ROS generation and pro-inflammatory cytokines production (Miller et al., 2012).
Recent study reported that, Syk tyrosine kinase is essential for LPS mediated inflammation in
macrophages (Ulanova et al., 2007). Another study mentions that, spleen tyrosine kinase is
important for LPS induced regulation of inflammatory responses and endotoxin tolerance through
degradation of RIP140 in macrophages (P. C. Ho et al., 2012).
The multiple roles of Syk in leukocyte activation, function and inflammatory response has
increased interest in its possible role in the pathogenesis and progression of many immune-related
disorders and diseases (Y. S. Yi et al., 2014a). In this study, we propose to characterize the role
of Syk and its downstream signaling pathways in LPS induced proinflammatory signaling cascade,
including NLRP3 inflammasome activation in N9 microglia. Further characterization of these
signaling pathways would provide a better understanding of the therapeutic efficacy of Syk
inhibitors in modulating excessive immunomodulatory response as evidenced in Parkinson’s
disease.
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Materials and methods
Chemicals and Reagents
IMDM, fetal bovine serum (FBS), L-glutamine, IRDye®-tagged 2°antibodies, penicillin,
and streptomycin were purchased from Invitrogen (Gaithersburg, MD). 10X Tris glycine SDS
buffer, 10X Tris glycine, 10% Tween-20, and nitrocellulose membrane were purchased from Bio-
Rad (Hercules, CA). APS, TEMED, lauryl sulfate and Bromophenol Blue were purchased from
Sigma Chemicals (St. Louis, MO). Phospho-p38 and Phospho-ERK antibodies were purchased
from Cell Signaling Technology, MA. Syk, Phospho-p65 and Nox-2 antibodies were obtained
from Santa Cruz Biotechnology. NLRP3 antibody was obtained from Novus Biologicals. Caspase-
1 antibody was purchased from Adipogen. GSK3β Tyrosine 216 antibody was obtained from
Abcam.
N9 Microglial Cultures
The immortalized murine N9 microglial cells were maintained in Iscove's Modified
Dulbecco's Medium (IMDM) containing 10% FBS, 50 U/ml penicillin, 50µg/ml streptomycin, and
2mM L-glutamine and incubated in a humidified atmosphere of 5% CO2 at 37°C. Prior to
experiments cells were plated and allowed to settle for at least 24 hours at 37°C in 5% CO2. Cells
were treated with 1µg/ml LPS in IMDM with supplements, containing 0% FBS, for the duration
indicated in the experiments.
Immunoblotting analysis
N9 cell lysates were prepared using modified RIPA buffer before normalization for equal
amounts using the Bradford Protein Assay Kit. Equal amounts of protein were loaded and samples
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were separated on 12% SDS PAGE gels. Proteins were then transferred to a nitrocellulose
membrane and probed overnight at 4°C with their respective 1° antibodies; Caspase-1, Syk,
Phospho-p65, iNOS, Phospho-p38, Phospho-ERK & GSK3β Tyrosine 216. Membranes were then
washed three times with PBS containing 0.1% Tween-20, then IRDye®-tagged 2°antibodies (IR
Dye 800-conjugated anti-rabbit IgG (1:5000) or Alexa Flour 680-conjugated anti-mouse IgG
(1:10000; Molecular Probes, Invitrogen)) for 1hr at room temperature were added. Membranes
were visualized on the Odyssey infrared imaging system. β-actin (1:5,000) was used as a loading
control.
ROS Assay
Intracellular ROS was determined using 2′,7′-dichlorodihydrofluorescein diacetate assay
(DCFH-DA), a fluorescent probe, per previous published reports with minor alterations (L. Qian
et al., 2007). N9 microglial cells were plated in 96-well plates and treated with LPS 1ug/ml for
12h and 24h and 3 uM R788 for 1h. Post incubation, treatment media was removed and cells were
washed with PBS and incubated with 1uM CM-H2DCFDA dye dissolved in HBSS for 1 hr. Later,
cells were washed with PBS and ROS production was assessed using a microplate reader with
excitation 485 nm and emission 530 nm respectively using a Synergy2 multimode microplate
reader. Control culture fluorescence was subtracted as background and was interpreted as
increased iROS (L. Qian et al., 2007).
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Nitric oxide detection.
Nitric oxide production was determined by indirect quantification of nitrite, an end product
of NO oxidation, in the supernatant using the Griess reagent (sigma Aldrich). N9 microglial cells
were plated at a density of 105 cells per well in a 96-well plate before treating with 1µg/ml LPS
for 12hr and 24hr; or were pretreated with 3µM R788 for 1h. After the incubation, 50µl of the
supernatant was mixed with 50µl of Griess reagent in a new 96-well plate and incubated on a plate
shaker at room temperature for 15 minutes. Absorbance was measured at 450nm using a Synergy2
multimode microplate reader. Sample concentrations of nitrite were determined by comparison to
sodium nitrite standard curve.
Syk Inhibitor R788
Syk inhibitor (R788) was purchased from Santa Cruz Biotechnology. The Syk inhibitor is
dissolved in DMSO and stored at -20C for future use. We used the 3uM R788 pretreatment for 1h
in N9 microglia and later treated with LPS 1ug/ml for respective time points (Y. C. Lin et al.,
2010).
Data analysis
Data analysis was calculated using Prism 7.02 (GraphPad Software, San Diego, CA). Data
was analyzed using one-way ANOVA, expressed as mean ± SEM. Differences of p<0.05 were
considered statistically significant.
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Results
Upregulation of Syk and Activation of NLRP3 Inflammasome in N9 Microglial Cells
Exposed to LPS
It has been previously shown that exposure of neutrophils to LPS induced the
phosphorylation of Syk leading to the complex formation encompassing TLR4, p-Syk and p-
CEACAM1 (R. Lu et al., 2012). Furthermore LPS treatment was found to induce ROS generation,
caspase-1 induction, and IL-1β secretion in neutrophils (R. Lu et al., 2012) therefore we sought to
investigate the expression of Syk and inflammasome markers in N9 microglial cells exposed to
LPS by immunoblotting analysis. N9 microglial cells are a well characterized immortalized
microglial cell line that has been routinely used for examining the therapeutic efficacy of anti-
inflammatory agents. Briefly, N9 microglial cells were exposed to LPS for 3, 6, 12, and 24h in the
presence or absence of Syk inhibitor, R788, and the levels of Syk and NLRP3 related
proinflammatory markers were determined by Western blot analysis. As shown in Figure 1A Syk
expression increased at 6hr after LPS challenge, peaked at 12h, and remained elevated for the
reminder of the treatment period. An upregulation of Syk was accompanied by the upregulation
of proinflammatory markers as evidenced by the induction of NLRP3 related markers including
procaspase-1 and caspase-1respectively. The results demonstrated a time dependent increase in
the levels of NLRP3 markers whereby a maximal increase in the levels of procaspase-1, cleaved
caspase were evidenced at 12 and 24h respectively upon LPS challenge (Fig. 1A). To further
evaluate the influence of Syk on LPS-induced upregulation of NLRP3 markers we tested the anti-
inflammatory effects of Syk inhibitor R788 on LPS-induced upregulation of NLRP3 markers.
Remarkably, R788 suppressed LPS-induced upregulation of NLRP3 markers as measured by a
dramatic reduction in the levels of NLRP3, caspase-1, pro-IL-1β, and IL-1β respectively (Fig. 1B).
Therefore, our results suggest that Syk is a key upstream regulator of NLRP3 inflammasome and
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that R788 elicits its anti-inflammatory effects via suppression of NLRP3 inflammasome activation
in LPS stimulated microglia.
Fig 1. LPS-induces NLRP3 inflammasome activation via a Syk dependent mechanism. (A)
SYK and NLRP3 related markers expression in LPS treated N9 microglia. N9 microglia were
treated with 1µg/ml LPS for 3hr, 6hr, 12hr, and 24hr. The control group is treated with vehicle (1x
PBS) used to prepare LPS prior to treatment. Post incubation, whole cell lysates were separated
with SDS-PAGE, and probed by western blotting using antibody against SYK, NLRP3, caspase-1
(pro and cleaved) and β actin. -actin was used a internal loading control for equal loading. The
1A
1B
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experiment is repeated two times and a representative western blot is presented. Western blot
analysis demonstrated that is dramatic upregulation of SYK and NLRP3 related markers in LPS
treated N9 microglia. (B) R788 attenuates the LPS induced NLRP3 related markers in N9
microglia. N9 microglia were treated with 1µg/ml LPS with or without one hour pre-treatment
with 2.5 uM and 3.5 µM R788 for 24h and probed with antibodies specific to NLRP3, caspase-1,
IL-1 beta with western blot analysis. To confirm equal protein loading in all the lanes, membranes
were re-probed with -actin. Results are representative of at least 2 independent experiments.
Western blot analysis revealed that, R788 downregulated LPS induced upregulation of NLRP3,
caspase-1, IL-1 beta in N9 microglia.
R788 partially Inhibited The Generation of ROS And Nitrite Species via Syk in LPS-
Stimulated N9 Microglial Cells
To investigate the potential relationship between Syk and ROS generation we used Western
blot analysis (Fig 2A). N9 microglia were treated with 1µg/ml LPS for 12h and 24h in the presence
or absence of 3µM Syk inhibitor R788. The stimulation of N9 microglial cells with LPS resulted
in significant (p<0.001) increase in the levels of iROS at both the 12h and 24h time points
respectively. However pretreatment of N9 microglial cells with R788 significantly inhibited LPS
induced iROS generation (Fig 2A). This suggest that Syk is a critical regulator of intracellular
ROS generation.
Next, in order to assess how Syk activation is linked to nitrite generation we performed the
Griess assay. N9 cells were subjected to the same treatment conditions as detailed above. The
stimulation of N9 microglial cells with LPS led to a significant increase in the release of nitrite
species in the conditioned media; however pretreatment with R788 significantly suppressed nitrite
generation (Fig 2B). Next to investigate the mechanistic basis of R788 inhibitory effects on NO
generation we determined the levels of iNOS using Western blot analysis. LPS induced a rapid
and transient induction of iNOS (Fig 2C); however, R788 markedly reduced iNOS protein
expression in LPS stimulated N9 microglial cells (Fig. 2D). Taken together these results suggest
that Syk is involved in the generation of iROS and NO through the upregulation of iNOS in LPS
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stimulated microglia. This suggest that R788 might elicit its anti-inflammatory effects via its
antioxidant effects in LPS stimulated microglia.
2A
2B
2C
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Fig 2 Effect of R788 on LPS-induced iROS and NO generation in N9 microglial cells. (A).
R788 partially attenuates ROS production in LPS treated microglia. N9 cells were treated with
LPS (1µg/ml) for 12hr and 24hr with or without pretreatment with 3µM R788 for 1hr. Post-
treatment, reactive oxygen species production was quantified spectrophotometrically using redox
sensitive CM-H2DCFDA. The data is represented as percent of control and the experiment is
repeated 2 times with n=8 in each treatment group. Statistical differences were determined by
ANOVA followed by Student Newman Keul’s test. Asterisk (p***<0.001) represents significant
differences between LPS treated groups and control group. (B). R788 partially attenuates NOS
production in LPS treated N9 microglia. N9 cells were treated with LPS (1µg/ml) for 12hr and
24hr with or without pretreatment with 3µM R788 for 1hr. At the end of treatment times, 100 μl
of supernatant is mixed with equal volume of griess reagent. Later, the absorbance is measured at
540 nm using multimode microplate reader and nitrate concentration was determined from sodium
nitrite standard curve. The data is represented as percent of control and the experiment is repeated
2 times with n=8 in each treatment group. Statistical differences were determined by ANOVA
2D
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followed by Student Newman Keul’s test. Asterisk (p***<0.001) represents significant differences
between LPS treated groups and control group. (C) iNOS expression in LPS treated N9 microglia.
N9 microglia were treated with LPS (1µg/ml) for 30 min, 1h and 2h. Whole cell lysates were
separated with SDS-PAGE and subjected to western blot analysis by probing with iNOS antibody.
Equal protein is loaded in all the lanes with the help of -actin. The experiments are repeated two
times and a representative western blot is presented. Immunoblot analysis revealed that there is
dramatic upregulation of iNOS production in LPS treated N9 microglia.
Inhibition of ERK and p38 MAPK Activation By R788 in LPS Stimulated N9 Microglial
Cells.
It is well established that MAPK signaling events are linked to cytokine and LPS-simulated
upregulation of iNOS in microglial cells (Fiebich et al., 2002). Next we examined whether the
suppressive effect of R788 on the generation of proinflammatory mediators occurs via MAPK
signaling pathway. N9 cells were treated as described above and the levels of the phospho forms
of ERK and p38 MAPK were determined via Western blot analysis. Our studies revealed that LPS
induced a dramatic time dependent increase in the phosphorylation of p38 MAPK and ERK. Upon
LPS stimulation for increasing duration (30-6h) p38 MAPK phosphorylation was evidenced as
early as 30 min, peaked at 60’, and nearly diminishes at 2h following LPS challenge (Fig. 3A).
However the phosphorylation of ERK is evidenced at 30’ of LPS stimulation and remains elevated
for 60’ and a marked reduction was observed at 2hr (Fig. 3A). Furthermore, R788 remarkably
reduced the LPS-induced phosphorylation of p38 MAPK and ERK at 1h (Fig. 3B). These findings
suggest that R788 is an effective inhibitor of p38 MAPK and ERK in LPS-stimulated microglia
and that Syk may serve as a critical regulator of the activation of MAPK signaling pathways.
276
Fig 3. Involvement of Syk in LPS-induced upregulation of MAPK signaling pathways in N9
microglial cells. (A) Immunoblot analysis of LPS (1µg/ml) treated N9 microglia for 30min, 1h
and 2h and MAPK phosphorylation status was probed using antibodies specific for Phospho-ERK
and Phospho-P38. Membranes were re-probed with -actin which was used a loading control. The
experiments were repeated for 2 times and a representative immunoblot is shown. Immunoblot
analysis shows that is there is rapid and transient upregulation of MAP kinases Phospho-ERK and
Phospho-P38 in LPS treated microglia. (B) R788 attenuates the LPS induced MAPK activation in
N9 microglia. N9 microglia were treated with 1µg/ml LPS with or without one hour pre-treatment
2.5 uM and 3.5 µM R788 for 1h and subjected to immunoblot analysis using antibodies Phospho-
P38 and Phospho-ERK. To confirm equal protein loading in all the lanes, membranes were re-
probed with -actin. Results are representative of at least 2 independent experiments. Western blot
analysis revealed that, R788 downregulated LPS induced upregulation of Phospho-P38 and
Phospho-ERK in N9 microglia.
3B
3A
277
Suppression of LPS-Induced Activation of the Transcription Regulator NF-kB via R788 in
N9 Microglial Cells
It is well established that NF-kB serves as a critical intermediate in promoting
proinflammatory gene expression (Ulanova et al., 2007). Moreover, NF-kB has been identified as
a key contributor to the priming step during the activation of NLRP3 (Y. C. Lin et al., 2015).
Therefore, we asked whether R788-mediated inhibition of NF-kB activation may underlie its anti-
inflammatory responses. We used Western blot analysis to determine the effects of the compound
on LPS-induced phosphorylation of the p65 subunit of NF-kB in N9 microglial cells. As shown
in Fig. 4A treatment of N9 microglia with 1µg/ml LPS for 30 min, 1h, and 2h resulted in a rapid
and transient upregulation of pp65. This process was markedly inhibited by R788 (Fig. 4B). This
suggests that R788 via its inhibitory effects on NF-kB activation might not only attenuate NLRP3
inflammasome activation but also proinflammatory cytokine production in a Syk dependent
manner.
4A
278
Fig 4. LPS-induced activation of NF-kB inhibited by treatment with R788. A. Immunoblot
analysis of LPS (1µg/ml) treated N9 microglia for 30min, 1h and 2h and activation of NF-kB
signaling pathway was probed using antibodies against NF-kB P65. Membranes were re-probed
with -actin which was used a loading control. The experiments were repeated for 2 times and a
representative immunoblot is shown. Immunoblot analysis shows that is there is early activation
of NF-kB P65 pathway in LPS treated N9 microglia. B. R788 attenuates the LPS induced NF-kB
signaling pathways in N9 microglia. N9 microglia were treated with 1µg/ml LPS with or without
one hour pre-treatment 2.5 uM and 3.5 µM R788 for 1h and NF-kB immunoblot analysis was
performed with NF-kB P65. To confirm equal protein loading in all the lanes, membranes were re-
probed with -actin. Results are representative of at least 2 independent experiments. Western blot
analysis revealed that, R788 downregulated LPS induced upregulation of Phospho-P65 in N9
microglia.
4B
279
R788 Attenuates LPS-Induced GSK3β Activation
The role of glycogen synthase kinase 3β (GSK3β) in peripheral and CNS disorders are well
established (Bhat et al., 2004). However, the influence of Syk on GSK3β activation during LPS-
induced microglial activation remains poorly characterized therefore N9 microglial cells were
exposed to LPS for varying duration. At the end of the incubation period cell lysates were prepared
and probed for the phosphorylation status of GSK3β. As shown in Fig 5A, exposure of N9 cells
to LPS resulted in a time dependent increase in the phosphorylation status of GSK3β. GSK3β was
activated as early 2h peaked at 12h and remained elevated for the reminder of the treatment period.
We next determined whether inhibition of GSK3β activation may be linked to the anti-
inflammatory effects of R788 at 24h. R788 pretreatment in LPS treated cells caused a near
complete abrogation of GSK3β phosphorylation as compared to LPS only treated cells (Fig 5B).
Therefore our results suggest that R788 mediated inhibition of GSK3β activation might at least in
part contribute to its anti-inflammatory effects.
5A
280
Fig 5. R788 attentuates LPS induced GSK-3 beta activation in N9 microglial cells. A. GSK-3
beta Y 216 upregulated expression in LPS treated N9 microglia. N9 microglia were treated with
1µg/ml LPS for 30min, 1h, 2h, 6h, 12h and 24h. Whole cell lysates were separated with SDS-
PAGE and GSK-3 beta phosphorylation status was probed by using antibodies specific to for GSK-
3 beta Y216. Equal protein is loaded in all the lanes with the help of -actin. Band intensities
normalized to -actin reveal that there is dramatic time dependent increase in GSK-3 beta Y216
in LPS treated N9 microglia. The experiments are repeated two times and a representative western
blot is presented. Statistical differences between LPS treated groups and control groups were
assessed with ANOVA with followed by Student Newman Keul’s post test. B. R788 attenuates
the LPS induced GSK-3 beta Y 216 upregulation in N9 microglia. N9 microglia were treated with
1µg/ml LPS with or without one hour pre-treatment 2.5 uM and 3.5 µM R788 for 24h and GSK-3
beta immunoblot is performed as described. To confirm equal protein loading in all the lanes,
membranes were re-probed with -actin. Results are representative of at least 2 independent
experiments. Western blot analysis revealed that, R788 downregulated LPS induced upregulation
of GSK-3 beta Y216 in N9 microglia.
Discussion
LPS has previously been shown to activate microglia and lead to chronic activation similar
to that seen in neurodegenerative disorders like Parkinson’s disease (Block, Zecca, et al., 2007; H.
M. Gao et al., 2002). The excessive activation of the NLRP3 inflammasome has been suggested
to be linked to the induction of enhanced inflammatory response and has been identified as a
5B
281
critical component in the disease pathology of numerous disorders, including autoinflammatory
and neurodegenerative disorders (Gustin et al., 2015; Halle et al., 2008). In this study, we
investigated the involvement of Syk in the induction of the proinflammatory downstream signaling
pathway especially the activation of NLRP3 inflammasome upon stimulation of N9 microglial
cells with LPS. Herein we provide evidence that LPS enhanced Syk phosphorylation with a
concomitant activation of several positive mediators of the inflammasome, including ROS, ERK
and p38 MAP kinases, and NF-kB. Pharmacological inhibition of Syk by R788 suppressed the
activation of the afore mentioned proteins, suggesting an important role for Syk in NLRP3
inflammasome activation and downstream proinflammatory signaling mediators. This study, for
the first time, demonstrates the proinflammatory role of Syk during microglial activation response
following LPS treatment.
When immune cells are activated by treatment with lipopolysaccharide (LPS), a toll-like
receptor 4 (TLR4) ligand, Syk shows greatly increased activation in under 2 minutes, leading to
the activation of several downstream ligands, including the nod-like receptor family pyrin domain-
containing 3 (NLRP3) inflammasome and of nuclear factor-κB (NF-kB) (Miller et al., 2012; Y. S.
Yi et al., 2014a). This results in a range of cellular adaptations including the production of ROS,
cell differentiation and survival, and the increased expression of proinflammatory genes and
mediators (Miller et al., 2012; Y. S. Yi et al., 2014a). Consistent with these results we show a
similar pathway whereby microglia activation by LPS induces a time dependent increase in Syk
signaling events which is associated with the induction of proinflammatory response including
NLRP3 inflammasome activation pathway through ROS, MAPK and GSK3β signaling
mechanisms. Intriguingly pharmacological inhibition of Syk via R788 attenuated Syk dependent
downstream signaling events. Our findings are consistent with a recent report that demonstrated
282
that inhibition of Syk suppresses the inflammatory response in macrophages exposed to an
inflammatory stimuli (Y. S. Yi et al., 2014a).
The NLRP3 inflammasome is a multiprotein complex, composed of NLRP3, ASC,
and caspase-1, which are found in some cell types of the innate immune system and is responsible
for the maturation of cytokines including IL-1β and IL-18 (Guarda et al., 2011). NLRP3
inflammasome activation is a two-step process, requiring a first signal to induce the transcription
of NLRP3 and pro-IL-1β followed by a second signal leading to the activation of NLRP3
inflammasome and caspase-1 and the resultant maturation and secretion of IL-1β and IL-18 (Gross
et al., 2009). In this context a recent report demonstrated that microglial cells express the activated
NLRP3 inflammasome upon exposure to NLRP3 activating stimuli (Gustin et al., 2015). LPS is
frequently utilized as the priming signal for inflammasome activation, and some studies have
indicated that Syk plays a direct role in the formation of the inflammasome (Gross et al., 2009; Y.
C. Lin et al., 2015). Consistent with this, we found that LPS treatment of N9 microglia resulted in
the time dependent increased expression of Syk, NLRP3, pro-caspase-1, pro-IL-1β, cleaved
caspase-1 and IL-1β. When we blocked Syk, the increased expression of NLRP3, caspase-1
cleavage, and the maturation of IL-1β was not observed. This suggests a role for Syk in NLRP3
inflammasome activation. These findings are consistent with previous research in which LPS was
found to be sufficient for pro-IL-1β maturation and secretion by neutrophils (R. Lu et al., 2012).
Chronically activated microglia are a major source of ROS, chemokines and cytokines in
many neurodegenerative disorders (Block, Zecca, et al., 2007). The activation of NADPH oxidase
and iNOS in these microglia results in the excess production of free radicals that have been
implicated in the pathogenesis of neurodegenerative diseases (R. Gordon et al., 2016; Y. S. Yi et
al., 2014a). Syk has been shown to mediate NOX2 induction in macrophages after minimally
283
oxidized LDL binding to TLR4 and has been implicated in ROS production in human monocytes
(Bae et al., 2009; Miller et al., 2012). Furthermore, iNOS induction was found to be mediated in
a Syk dependent manner in LPS stimulated peritoneal macrophages and murine macrophages via
activation of MAP kinases (Chiou et al., 2011; S. H. Kim et al., 2015). Consistent with these
reports LPS stimulation of N9 microglia resulted in a marked increase in iNOS protein expression
with a concomitant increase in NO generation. Blockade of Syk resulted in an attenuated
expression iNOS, with an accompanying decrease in the generation of ROS and greatly diminished
NO release. Our studies demonstrate that Syk is an important regulator of ROS and NO release
which might be at least in part mediated via an NF-kB dependent mechanism (Chiou et al., 2011;
Y. S. Yi et al., 2014a).
MAP kinases play a crucial role as upstream regulators in the activation of several
transcription factors including NF-kB and several proinflammatory mediators, including iNOS,
IL-1β, and ROS production (Chaudhary et al., 2007; Y. S. Yi et al., 2014a). These proinflammatory
mediators have been linked to the proliferation of immune cells observed in neuroinflammatory
disorders. In addition, they have also been shown to be involved in the induction of NLRP3 and
pro-IL-1β, inflammasome components in LPS stimulated macrophages (Moon et al., 2015).
Moreover, Syk has previously been implicated as a positive mediator in both ERK and p38 MAP
kinase phosphorylation and activation (M. G. Ghonime et al., 2014; Miller et al., 2012). In the
present study, we found that LPS stimulation of microglia resulted in the rapid activation of both
ERK and p38 MAP kinase, suggesting Syk as a possible upstream regulator of MAPK activation.
As anticipated R788 inhibited LPS induced MAPK activation mechanisms further confirming that
Syk is a key upstream regulator of MAPK signaling events. Our results are in accordance with
previous findings in bone marrow derived macrophages exposed to Syk inhibitors (Eliopoulos et
284
al., 2006). Together our results indicate that Syk is a positive regulator of the MAP kinase
pathways which in turn presumably leads to the activation of NF-kB and resultant NLRP3
inflammasome activation, at least in LPS stimulated microglia cells.
GSK3β is a kinase involved in the regulation of very diverse functions, including cell cycle
control, apoptosis, cell differentiation and motility, and inflammation (Cortes-Vieyra et al., 2012).
GSK3β is regulated via autophosphorylation at tyrosine 216 resulting in kinase activation or
inhibited by phosphorylation of serine 9 (Cortes-Vieyra et al., 2012) resulting in a kinase-inactive
state. Furthermore, GSK3β has been shown to regulate the magnitude and duration of
inflammatory conditions as well as the progression of neurological disorders related to
neuroinflammation (Chan et al., 2009; Maixner & Weng, 2013; M. Martin et al., 2005; D. W. Park
et al., 2014). In this context, a recent report demonstrated that the dysregulation of GSK3β was
linked to Parkinsonian like pathophysiology with an accompanying brain region specific
phosphorylation and accumulation of tau and a-syn (Credle, George, et al., 2015). On the contrary,
siRNA mediated knock down of GSK3β or pharmacological inhibition of GSK3β was found to
diminish proinflammatory cytokine production in LPS stimulated monocytes (M. Martin et al.,
2005). In the same study, treatment of mice with GSK inhibitor SB216763 was found to
significantly attenuate LPS-induced mortality. Furthermore GSK3β has also been linked to the
suppression of anti-inflammatory cytokine generation, including IL-10 (Hu et al., 2006; M. Martin
et al., 2005). Consistent with these reports inhibition of GSK3β phosphorylation via R788
positively correlated with diminished proinflammatory mediator(s) generation suggesting a
positive modulatory role for GSK3β in the induction of proinflammatory signaling cascade. In
line with our findings, gene silencing of Syk was found to promote the generation of anti-
inflammatory cytokine IL-10 but suppress the levels of proinflammatory cytokines, TNF-a, IL-6
285
in a GSK3β dependent manner in dendritic cells (H. Yin et al., 2016). Together, our results suggest
that Syk and GSK3β cooperated with one and another via NF-kB activation to induce the
generation of proinflammatory mediators in LPS stimulated microglial cells.
Activation of NF-kB is a critical step in the transcription of many proinflammatory
mediators and microglial activation in response to LPS stimulation and Syk activation (Ulanova
et al., 2007; Y. S. Yi et al., 2014a). This activation is characterized by the phosphorylation and
activation of Inhibitory κB kinase (IKK) by MAP kinases, which then phosphorylates the
inhibitory protein inhibitory κB-α (IκBα) marking it for proteasomal degradation (N. Panicker, H.
Saminathan, H. Jin, M. Neal, D. S. Harischandra, et al., 2015). NF-kB subunit p65 subsequently
moves into the nucleus where it induces the transcription of proinflammatory genes and increased
NLRP3 and pro-IL-1β expression (Y. C. Lin et al., 2015). We showed increased Syk
phosphorylation was accompanied by phosphorylation of the p65 subunit of NF-kB during
stimulation of microglial cells with LPS in line with previous studies implicating Syk activation
in the induction of the NF-kB pathway in neutrophils and macrophages (Y. G. Lee et al., 2009;
Miller et al., 2012). Furthermore, blocking Syk activity in LPS treated cells also results in the
decreased activation of the NFkB reflected by decreased phosphorylation of p65 subunit of NF-
kB. Another study demonstrated decreased activation of NF-kB upon Syk inhibition in LPS
activated murine macrophages and peritoneal macrophages (Jeong et al., 2013). Consistent with
these studies we show that in R788 treated LPS challenged microglial cells a marked attenuation
of NFkB activation as indicated by decreased phosphorylation of the p65 subunit of NF-kB was
evidenced. These results suggest that Syk elicits its proinflammatory effect via an NF-kB
dependent cell signaling mechanisms at least in LPS stimulated microglial cells.
286
Taken together our results demonstrates how LPS stimulation of N9 microglia leads to the
activation of the NLRP3 inflammasome, GSK3β, MAP kinases and NF-kB, and ROS production
in a Syk dependent manner. Furthermore, the pharmacological inhibition of Syk by R788 results
in the inhibition of the afore mentioned proinflammatory mediators in LPS stimulated N9
microglial cells highlighting its anti-inflammatory effects. Taken together our results indicate that
R788 pretreatment attenuates LPS-induced NLRP3 inflammasome activation via suppression of
the maturation and release of IL-1β. Therefore, Syk may represent a novel therapeutic avenue for
he amelioration of neuroinflammation associated neurodegenerative disorders including PD.
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CHAPTER 5. CONCLUSIONS
This chapter summarizes the results presented in the individual chapters of this dissertation
with special emphasis on the role of Spleen Tyrosine Kinase in mediating dopaminergic
neurodegeneration and microglial activation following Tweak treatment and its relevance to
pathogenesis of Parkinson Disease. The major findings of each research chapter have been detailed
in the discussion section of the pertinent chapters.
Involvement of the Mitochondria Mediated Oxidative Stress Dependent Cell Signaling
Events and Syk Tyrosine Kinase Activation in Tumor Necrosis Factor Like Weak Stimulator
of Apoptosis (TWEAK) Induced Dopaminergic Cell Death
The primary finding from the chapter 3 of the thesis is that the Spleen Tyrsoine Kinase
(SYK) and mitochondrial oxidative stress events play a significant role in TWEAK induced
dopaminergic neurodegeneration in PD. Inflammation and oxidative damage have been implicated
in the pathogenesis of Parkinson’s disease (PD); however, key signaling events that potentiate the
oxidative stress response and resultant dopaminergic neurodegeneration remains to be established.
In this study, we sought to investigate the role of TWEAK and its downstream signaling events in
dopaminergic neurotoxicity using both in vitro and in vivo experimental model of PD. Initially
using a targeted qPCR array, we identified that TWEAK (Tweak Necrosis Factor Weak Inducer
of apoptosis) is significantly up regulated in dopaminergic neuronal cells (N27 cells) in response
to an inflammatory stressor. Later, we found that there is a 2-3-fold increase in TWEAK levels in
the serum of PD patients compared to age-matched control patients as assessed via western blot
analysis. Intriguingly, serum Tweak levels have been shown to fluctuate with disease severity and
that monitoring its levels have been shown to be beneficial in managing Rheumatoid Arthritis
(RA), Multiple Sclerosis (MS) and Peritoneal inflammation (Bertin et al., 2013; Ana Belen Sanz
et al., 2014). Furthermore, time course studies performed in an MPTP mouse model of PD
292
revealed that TWEAK is upregulated as early as 3-6h post drug treatment prior to the expression
of motor deficits suggesting that TWEAK may serve as an early biomarker for PD. A recent study
showed that TWEAK protein level was increased in the striatal tissue of MPTP treated mice
implicating TWEAK/FN14 pathway in dopaminergic neurodegeneration (Mustafa et al., 2016a).
In line with this finding, our results revealed that Tweak is differentially expressed in various brain
regions including substantia nigra, cortex, striatum, hippocampus, cerebellum and olfactory bulb
in control mice which might alteast in part explain the pathological significance of TWEAK
expression and its association to PD neuropathology. These studies highlight the utility of
TWEAK as a biomarker for the early detection of PD.
To dissect the mechanism of TWEAK induced dopaminergic neurodegeneration, we
utilized the well characterized rat dopaminergic neuronal cell line (N27 cells) that has been
routinely used to study PD associated neuropathological changes (G Kanthasamy et al., 2008).
Initially, we demonstrated the expression of Tweak and its receptor Fn14 expression in N27
dopaminergic cells. In agreement with our finding, TWEAK was previously shown to be expressed
in neurons, microglia and astrocytes in Multiple Sclerosis brain (Barbara Serafini et al., 2008). To
the best of our knowledge, we for the first time demonstrate that TWEAK and its receptor is
robustly expressed in N27 cells. Furthermore, we found that Tweak induced dose-dependent
increase in apoptotic cell death in N27 dopaminergic cells. Consistent with our finding, Tweak has
been shown to elicit toxic effects in cortical neurons, renal cortical cell lines, mesangial cell lines
and mammary epithelial cell lines (Burkly et al., 2007). To further characterize the mechanisms
underlying TWEAK induced dopaminergic neurotoxicity, we systematically examined
mitochondrial apoptotic signaling events, protein degradation machinery and NF-kB pathway. We
found a significant dissipation of mitochondrial membrane potential (MMP), suppression of GSH
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levels, caspase activation (2, 3, 8 & 9), impairment of protein degradation machinery, upregulation
of phospho-Tau, increased phospho-NF-kB P65 levels, depletion of phospho-AKT and GSK-3
beta activation in Tweak treated N27 dopaminergic neuronal cells. Moreover, our results also
revealed that these effects were accompanied by upregulation of redox sensitive kinases like
Spleen Tyrosine Kinase and PKC-delta in N27 dopaminergic neuronal cells treated with TWEAK.
Intriguingly, SN-50 (NF-kB inhibitor), Quercitin (Bioactive plant flavonoid) and R-788 (Syk
inhibitor) conferred resistance against Tweak induced neuronal cell death highlighting the
importance of NF-kB, mitochondrial stress and Syk signaling mechanisms in dopaminergic
neurodegeneration. Taken together, our results support a model whereby TWEAK exerts
dopaminergic neurotoxicity via SYK / NF-kB signaling mechanisms in a mitochondria dependent
manner.
SYK Contributes to TWEAK-Induced Inflammatory Response Through Activation of
NLRP3 Inflammasome Activation, ER Stress Response, and ALS Dysfunction in Microglia.
Emerging evidence suggests that microglial activation associated with NLRP3 activation
may be critically linked to PD associated neurodegeneration (Mao et al., 2017). Recent studies
support a role for Spleen Tyrosine Kinase (SYK) in NLRP3 inflammasome activation in immune
cells (Federica Laudisi et al., 2014; K. Neumann & Ruland, 2013). Additionally, Tweak is
implicated in the regulation of neuro-inflammatory responses in Multiple sclerosis, Stroke,
Neuropsychiatric Lupus, Glioma, and Schizophrenia (Boulamery & Desplat-Jégo, 2017).
However the key signaling events underlying Tweak induced microglial NLRP3 activation in
Parkinson’s disease remains speculative. Therefore in chapter 4, we sought to understand the role
of SYK in Tweak induced microglial activation response using an in vitro cell culture model and
neuroinflammation mouse model of PD. To dissect the mechanism of TWEAK induced microglial
NLRP3 activation, we utilized the well characterized microglial cell line (BV2 cells) that has been
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routinely used by our lab and others for the examination of neuro-inflammatory response (Nikhil
Panicker et al., 2015). Moreover, BV2 microglial cells can be considered as an efficient substitute
to primary microglial cells as they secrete substantial amounts of cytokines and NO upon
stimulation with LPS (Stansley et al., 2012). First, we demonstrated that there is increased SYK
expression and kinase activity in BV2 microglial cells in response to TWEAK stimulation. To
further elucidate the mechanistic basis of TWEAK induced inflammatory response we performed
time course studies. BV2 microglial cells were treated with TWEAK (100 ng/ml) for increasing
duration (3-24h) and NLRP3 inflammasome related markers were assessed by Western blot and
ELISA analysis. Our immunoblot analysis revealed that there is significant upregulation of NLRP3
related markers in BV2 microglial cells upon TWEAK treatment. ELISA analysis of BV2
microglial cells treated with TWEAK revealed the upregulation of pro-inflammatory cytokines
such as IL-1 beta and TNF-alpha. To further characterize the mechanisms underlying TWEAK-
induced microglial NLRP3 inflammasome activation we examined mitochondrial function, ER
stress response and autophagolysosomal system (ALS) function. Immunoblot analysis, revealed
ER stress response, mitochondrial dysfunction, lysosomal damage, MAPK activation, GSK-3 beta
activation in TWEAK stimulated BV2 microglial cells. Accumulating evidence suggests that, SYK
induced ROS production and cathepsin-B production is responsible for NLRP3 inflammasome
activation in LPS treated neutrophils (Rongze Lu et al., 2012). Therefore, in the next set of
experiments, we proceeded to examine the impact of SYK on oxidative stress, ALS dysfunction
and NLRP3 and associated signaling events in TWEAK treated microglial cells. Upon blockade
of SYK using a pharmacological inhibitor of SYK, RX788 or siRNA mediated gene silencing, we
found that attenuation of ALS dysfunction coincided with suppression of NLRP3 inflammasome
activation, ER stress response, NOX and JNK activation in TWEAK treated cells further
295
supporting the role of SYK in the regulation of redox mechanisms. To further validate our in vitro
findings we utilized the TWEAK neuroinflammation mouse model. For this purpose, TWEAK
was stereotaxically injected into the mouse striata in the presence or absence of Piceatannol (SYK
inhibitor) to further affirm the role of SYK in NLRP3 inflammasome activation. Intriguingly, our
vivo studies demonstrated that there was no significant difference in the expression levels of SYK
or NLRP3 related markers between Tweak infused mice and control mice. Likewise, piceatannol,
a Syk inhibitor had no effect in the TWEAK neuroinflammation model. At this stage TWEAK
related dose response studies are necessary to evaluate TWEAK associated neuroinflammatory
response in the mouse brain. Collectively, our findings highlight the importance of Syk mediated
oxidative stress, ER stress, ALS dysfunction and GSK-3 beta activation in Tweak induced
microglial activation response involving NLRP3 inflammasome activation. Taken together, our
results highlight the pivotal role of SYK in mediating heightened microglial activation response
upon TWEAK treatment.
Spleen Tyrosine Kinase Mediates NLRP3 Inflammasome Activation in LPS Treated N9
Microglial Cells via NF-kB, MAP Kinase and ROS Generation.
Previous studies indicate that, functional NLRP3 inflammasome is expressed in the
microglia of mouse brain (Gustin et al., 2015). NLRP3 inflammasome activation has been
recognized as a critical contributor to dopaminergic neurodegeneration in experimental models of
Parkinson’s disease (Mao et al., 2017). ERK signaling and proteasome function have been
implicated in the priming of NLRP3 inflammasome in LPS treated monocytes (Mohammed G.
Ghonime et al., 2014). However the underlying downstream signaling events that are responsible
for the activation of NLPR3 inflammasome activation remains to be established. Therefore in
chapter 5, we sought to examine the role of SYK in NLRP3 inflammasome activation in LPS
296
stimulated N9 microglial cells. To dissect the mechanism of LPS induced microglial NLRP3
activation, we utilized well established retroviral immortalized microglial cell line (N9 cells). N9
microglial cells can be considered as an efficient substitute to primary microglial cells to study
neuro-inflammatory mechanisms as they produce cytokines like TNF-alpha, IL-1 and IL-6 upon
LPS stimulation (Righi et al., 1989; Stansley et al., 2012). First using N9 microglial cell culture
model, we demonstrated that, Spleen Tyrosine Kinase (SYK) expression is increased in LPS
treated N9 microglial cells. Additionally, LPS has been previously shown to induce NLRP3
inflamamsome activation in neutrophils and macrophages (Y.-C. Lin et al., 2015; Rongze Lu et
al., 2012). To assess whether LPS is capable of inducing NLRP3 inflammasome activation in N9
microglial cells, we stimulated N9 microglial cells with LPS and quantified the neuro-
inflammatory responses using WB analysis and Luminex analysis. Our immunoblot analysis and
luminex cytokine analysis revealed upregulation of NLRP3 inflammasome and its markers. LPS
treated macrophages has been shown to generate pro-inflammatory cytokines via activation of NF-
kB, AKT and PDK1 (Y.-S. Yi et al., 2014b). Consistent with this report immunoblot analysis
revealed ROS generation, NOS production GSK-3 beta, MAPK and NF-kB activation in LPS
stimulated microglial cells. Additionally, pharmacological inhibition of SYK was found to
attenuate microglial activation response via inhibition of JNK and NF-kB signaling pathway (Y.
C. Lin et al., 2010). Therefore, in the next set of experiments, we examined whether blockade of
SYK attenuated NLRP3 inflammasome activation and proinflammatory marker (s) expression in
LPS treated microglial cells using RX788, a SYK inhibitor. Interestingly, SYK inhibition with
RX788 suppressed LPS induced microglial NLRP3 activation coincident with suppression of ROS
and nitric oxide production, phosphorylation of NF-kB, and activation of mitogen activated protein
kinases (MAPKs), extracellular signal regulated kinases 1 and 2 (ERK1/2), p38 MAPK and
297
glycogen synthase kinase-3β (GSK3β). In conclusion, our results support a model, whereby SYK
mediates microglial activation response in LPS stimulated cells via induction of NLRP3
inflammasome activation and ROS generation as well as NF-kB, MAPK and GSk-3 beta
activation. Taken together, our results highlight the therapeutic significance of targeting SYK for
the amelioration of neuroinflammation related disorders including PD.
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299
CHAPTER 6. FUTURE AREAS OF INVESTIGATION
What is The Role of ER Stress Mediated CXCL10/CXCR3 Signaling Axis In The Induction
of a Proinflamamtory Response and Neuronal Injury in Experimental Models of PD?
It is well established that activated microglial can elicit beneficial effects during the early
phase of neurodegeneration in PD (Norden et al., 2015) but excessive activation of microglia can
lead to neuronal damage owing to the buildup of neurotoxic mediators including TNF-a, IL-1b,
IL-6 (Y. S. Kim et al., 2006) in the vicinity of dopaminergic neurons. Excessive glial cell
activation is a critical feature in post mortem PD brains (McGeer et al., 1988) and animal models
of PD (Qinqin Wang et al., 2015). Moreover, in a recent study using neuroinflammation rat models
of PD (LPS and 6-OHDA), activation of inflammatory mediators including NLRP3 inflammasome
was found to accompany dopaminergic neurodegeneration in the substantia nigra compacta (Mao
et al., 2017) and inhibition of caspase-1 via Ac-YVAD-CMK was found to confer resistance
against neurotoxin-induced dopaminergic neurodegenerative effect. Nevertheless, the key
mediators that regulate microglial recruitment and activation remains elusive.
Chemokines represent inflammatory mediators that regulate microglial migration and
activation subsequent to inflammatory response by binding to specific receptors expressed on
target cells. In this context, CXCR3 serves as a receptor for CXC subclass members such as CXCL
9, 10, and 11 (Lacotte et al., 2009). CXCR3 participates in the recruitment of T cells and microglia
(de Jong et al., 2008). Moreover, CXCL10 expression has been shown to be increased in several
neurodegenerative diseases including AD (Galimberti et al., 2006). Furthermore functional cross
talk between CXCL10 and CXCR3 has been shown to induce microglial activation and induce
neuronal death in several models of neurodegeneration (N. Jo et al., 2003). Having demonstrated
that TWEAK stimulation of microglial cells elicits ERS and that it is positively associated with a
heightened microglial inflammatory response it would be important to understand to what extent
300
ER stress mediates CXCL10/CXCR3 signaling axis in TWEAK treated cells. In a previous study
the expression of CXCL10 and CXCR3 was found to be increased by two fold in response to ER
stress (ERS) in a retinal injury model (Ha et al., 2015). Furthermore, retinal cell apoptosis was
found to be elevated in WT mice as compared to CXCR3-deficient mice upon ERS further
supporting the idea that CXCR3 has a critical role in ER stress induced retinal neuronal injury.
We propose to assess the activation of CXCL10/CXCR3 signaling axis in response to diverse
inflammagens including TNF alpha, LPS and TWEAK and whether its upregulation is linked to
ER stress response in BV2 cells, primary microglial cells and in an in vivo neuroinflammation
model of PD. We hypothesize that ER stress mediated activation of CXCL10/CXCR3 signaling
axis contributes to microglial migration and resultant neuronal injury in neuroinflammation models
of PD. Also, to what extent mitochondria mediated oxidative stress contributes to the activation
of CXCL10/CXCR3 signaling axis and its impact on NLRP3 inflammasome, SYK and ALS
activation will provide novel insights into the role of mitochondria in the activation of chemokine
mediated signaling events and its influence on dopaminergic neuronal wellbeing.
What is The Contribution of Mitophagy Pathway During TWEAK-Induced Microglial
Activation Response?
Mitophagy refers to the selective removal of damaged mitochondria via the
autophagolysosomal degradative machinery. Numerous studies have demonstrated a pivotal role
for mitochondrial damage and subsequent release of mitochondrial DNA and mitochondrial ROS
in the induction of NLRP3 inflammasome activation (Prajwal Gurung et al., 2015). In this context
PARKIN, a E3 ubiquitin ligase is the best known player in the mitophagy process. The PINK1 and
PARKIN pathway contributes atleast in part to the clearance of damaged mitochondria via
mitophagy. Upon mitochondrial depolarization PINK1 accumulates in the outer mitochondrial
membrane (OMM) and recruits PARKIN and activates it by phosphorylation. Activated PARKIN
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ubiquitinates OMM which can be recognized by p62 an adapter protein which in turn recruits LC3
present on autophagosomes thereby facilitating the degradation of damaged mitochondria via
mitophagy. In a recent study Zhong et al found that p62 is increased by NF-kB signaling in
response to an inflammatory stimuli and that the p62 is recruited to the damaged mitochondria
which in turn is ubiquitinated by PARKIN thereby facilitating mitophagy. In another study
caspase-1 which is activated upon NLRP3 inflammasome activation was found to cause the
cleavage of PARKIN thereby leading to the accumulation of damaged mitochondria and
inflammasome activation via a vicious feed forward cycle (Jiujiu Yu et al., 2014). In the chapter
3 we demonstrated that TWEAK induced impairment in the autophagolysosomal system (ALS)
was associated with NLRP3 system. Therefore it would be interesting to investigate the cross talk
between PINK-PARKIN pathway and NLRP3 inflammasome activation in TWEAK-induced
heightened microglial activation response. Loss of PINK1 and PARKIN has been shown to
increase dopamine neurons susceptibility to stressors. Our studies raise the interesting possibility
as to whether dysfunctional mitophagy results in the generation of neurotoxic mediators thereby
facilitating the demise of dopaminergic neurons which are in a compromised bioenergetic state.
Therefore it would be beneficial to investigate the factors that regulate PINK-PARKIN to develop
novel therapeutics to mitigate aberrant inflammation associated disorders including PD.
Investigations Into The Role of SYK In Astrocytic Response Upon TWEAK Stimulation And
In Experimental Models of PD
Astrocytes are the most abundant type of glial cells in the brain. To date only few studies
have studied the role of astrocytes in neurodegenerative diseases including PD. Intriguingly,
several genes that have been shown to play a causative role in PD pathogenesis have been
expressed in astrocytes (Booth et al., 2017). Therefore improved understanding on their influence
in PD neuropathology might lead to the development of novel therapeutics for PD.
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Astrocytes provide structural and metabolic support and participates in the regulation of
synaptic transmission and blood flow within the brain. Moreover, astrocytes contribute to the
maintenance of blood brain barrier (BBB) which has been shown to be impaired in PD brains
(Gray & Woulfe, 2015). In fact survival of dopaminergic neurons are dependent on astrocyte
derived neurotrophic factor namely glial derived neurotrophic factor (GDNF) (F. H. Lin et al.,
1993; Saavedra et al., 2006; X. Wu et al., 2008). Emerging evidence indicate that dopaminergic
neurodegeneration in PD may be intimately linked to astrocyte dysfunction. In this context in post
mortem PD brains DJ-1 was found to be enhanced in the astrocytes (Bandopadhyay et al., 2004;
M. Neumann et al., 2004). Moreover DJ-1 has been linked to mitochondrial function and oxidative
stress in astrocytes exposed to rotenone (Mullett & Hinkle, 2009). Therefore we would like to test
whether there is a cross talk between SYK and DJ-1 in TWEAK stimulated cells. We would be
interested in addressing the following questions: 1) Is there a functional interaction between SYK
and DJ-1 in TWEAK stimulated astroglial cells? What is its relationship to the induction of an
inflammatory response? 2) What is the impact of SYK on astroglial mitochondrial function
especially mitochondrial dynamics in DJ-1 KO astrocytes? What is the role of SYK in
Parkinsonian toxin (rotenone)-induced mitochondrial bioenergetics deficits in astrocytes and how
does this compare with TWEAK-induced astrocytosis? If, so does blockade of SYK protect
astrocytes against bioenergetics deficits induced by Parkinsonian toxins. Successful completion
of the afore mentioned studies would provide novel insights into the role of SYK in the regulation
of astrocytic function in response to diverse stimuli. Furthermore it would improve our
understanding on the contribution of non-cell autonomous signaling mechanisms in the PD
associated dopaminergic neurodegeneration, which, in turn, may lead to improved therapeutic
outcome.
303
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ACKNOWLEDGMENTS
I would like to whole-heartedly thank my major Professor Dr. Arthi
Kanthasamy and Co-Major Professor Dr Anumantha Kanthasamy, for providing me of this
opportunity for pursuing my course work and research projects in their labs and supporting me
entirely during the Biomedical Sciences PHD graduate program. Their persistant encouragement,
assistance, guidance and optimism is the main reason to my successful completion of graduate
program and research projects. During the whole period of my graduate school in Iowa state, I had
developed skills so design research projects, perform experimental procedures, apply scientific
knowledge and investigate important research questions regarding pathophysiology of
neurological diseases. I would also like to appreciate and be very grateful of my committee
members Dr. Bellaire Bryan, Dr. Manju Reddy, Dr. Wilson Rumbeiha and Dr. Jesse Goff for their
constant support, encouragement, guidance regarding my research projects of the graduate PHD
program. Furthermore, I would like to extend my gratitude and acknowledgement to Dr
Anantharam Vellareddy for helping in ordering resources and animals for my research studies in
the lab. Additionally, I would like to thank the current lab members, Vivek Lawana and Neeraj
Singh for providing me great help in my experiments and I really enjoyed working with them as a
team. Also, I would like to thankful for my previous lab members- Drs. Huajun Jin, Richard
Gordon, Afeseh Ngwa Hilary and Anamitra Ghosh who helped me learning the research
techniques and helped me to adjusted to the lab. Also I would like to extend my gratitude to the
Department of Biomedical Sciences Staff – Loonan Rochelle, Kim Adams, William B. Robertson,
Amy Brucker and Hashman Emma for helping in the administrative tasks and duties during my
period of the PHD program.
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Finally, I would like to thank my loved ones - my grandparents, my parents, my wife, my
family and my friends for their persistant unconditional support and constant encouragement,
without which it would have been near impossible to finish this arduous task of complete PHD
graduate studies in Iowa state.