marco natali master thesis
TRANSCRIPT
UNIVERSITA' DEGLI STUDI DI PISA
Facoltà di Scienze Matematiche, Fisiche e Naturali
Corso di Laurea Specialistica in Scienze e Tecnologie Biomolecolari
TESI DI LAUREAHow would it be possible to promote neural regeneration
in adult brain? A lesson from urodele amphibian
Anno Accademico 2009/2010
Relatori: Candidato:
Prof.ssa Renata Batistoni, Università di Pisa Marco Natali
Ph.Dott. Andras Simon, Karolinska Institutet Matricola: 301288
En los centros adultos las vías nerviosasson casi fijas, terminadas, inmutables.
Todo puede morir, nada puede ser regenerado.Queda para la ciencia futura cambiar,
si es posible, este severo decreto.
Santiago Ramon y Cajal, 1928
INDEX
Abstract1 Introduction1.1 Neural Regeneration in XX century1.2 Adult mammalian regeneration1.2.1 Study on stimulated neurogenesis1.3 Salamander adult regeneration1.4 Neurodegeneration: the Parkinson's Disease1.4.1 Treatments1.5 Aim of the thesis1.5.1 Gene candidates2 Materials and Methods2.1 Buffers preparation2.2 Animals2.3 6-OHDA injury2.4 Tissues harvesting2.5 In situ hybridization2.5.1 Probes2.6 Immunostaining2.7 Antibodies2.8 Mounting2.9 Microscope2.9.1 Image handling for pixel counting quantitative analysis2.9.2 Other microscope3 Results3.1 Optimisation of in situ protocol3.2 Primary screen of genes of interest3.3 nRAD3.4 Jar23.4.1 More detailed analysis of the role of JARID23.5 Notch4 Discussion4.1 Notch4.2 nRAD4.3 Jar24.4 ConclusionReferences
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I
Abstract
One of the medicine's dogmas in the last century assumed impossible to generate new neurons by an adult mammalian. However, in the second half of the century, active neurogenesis of Neural Stem Cells was found in some brain regions (subventricular zone and subgranular zone). Regenerative capabilities of neural structures in an adult organism are extended in other animal species, for example in urodele amphibians. Salamanders can regenerate various tissues and organs, it is hypothesized that salamander cells are able to de-differentiate in a stem-like condition and proliferate in order to rescue lost tissues.It is possible, thanks to neurotoxins such as 6-hydroxydopamine, to induce Parkinsonian-like injuries in salamander midbrain, and these animals can rescue the lesions in 30 days.The Parkinson's Disease (PD) is a disorder characterized by progressive neural degeneration with slow course, typical of aged people and by primary symptoms involving the motorial system. The causes of PD are not completely understood yet, and this increases the difficulty of studies aimed to fight the disorder and to discover a standard therapy. Various therapeutic approaches are thought out in order of reverting Parkinsonian neurodegeneration and repairing injured tissues, among these is the induction of adult neurogenesis at the midbrain level.Aim of my thesis work is to assess the involvement of some candidate genes in neurogenesis of adult newts (Nothophtalmus viridescens), in order to get an insight into the gene pattern that control regeneration in midbrain and to allow future comparative studies with mammals. This work was performed in Dr. Andras Simon's laboratory at CMB Department of Karolinska Institutet in Stockholm (Sweden) from January 2010 to October 2010.Expression of these genes were tested by in situ hybridization technique in regenerating salamander model with midbrain lesioned by 6-hydroxydopamine. Control samples were done with the sense probe of candidate gene in place of the antisense one. The in situ protocol needed to be set up and adapted to salamander brain slices before to work, so a probe for Sonic Hedgehog was used since its expression in newt was already known.- Notch, codifies for a receptor protein that suppresses neural differentiation and is thought to mantain cells in an undifferentiated state. This gene was chosen as marker of stem-like cells. Although results presented expression of Notch only in cells known to be stem-like, the control samples showed some colouration. It was hypothesized the presence in the tissues of the opposite strand of RNA. Probes for other genes did not show this phenomenon.- CKM, codifies for a creatine kinase involved in ATP production for energy supply in muscles, it has also an homologue in brain. This gene was chosen as negative control expected to not be expressed. Results do not show any
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expression.- Annexin1, in mammals codifies for a membrane protein involved in anti-inflammatory processes in the Central Nervous System, so to preserve the CNS from neuropathologic worsening. This gene was chosen in order to understand if anti-inflammatory processes have an important role in regeneration of brain. Results did not show any expression.- Jarid2, in various species codifies for a transcription factor involved in epigenetic activation of developmental genes by Polycomb Complexes. This gene is known to be fundamental for differentiation of embryonic stem cells and of mouse neural embryonic development, so was the main candidate for this study. Results showed down-regulation in regenerating tissues and an indefinite localization.A further experiment, Jarid2 immunostaining, was also performed with a double aim: a) to compare the protein localization related with glial and neural cells; b) to check with a quantitative analysis if the down-regulation of Jarid2 during regeneration was effective. Results confirmed this hypothesis and showed a spread localization in neuronal cells, but a strong and definite expression in the stem-like cells.- RAD, in various species codifies for a small GTPase (variant of RAS) involved in muscle regeneration, so to be used as marker for regenerating muscles. This gene was chosen in order to compare regeneration between muscle and brain. Results show a strong expression in regenerating tissues strictly localized in few cells.As further experiment, the defined expression of RAD was compared with the presence of PCNA+ cells in order to understand if it was possible to exclude an eventual relationship between RAD and proliferation. The obtained result not confirms this possibility.
III
1 Introduction
1.1 Neural Regeneration in XX century
It was long thought that the mammalian brain was a static fixed structure:
"In the adult centers the nerve paths are something fixed, ended and
immutable. Everything may die, nothing may be regenerated. It is for the
science of the future to change, if possible, this harsh decree." (The Nobel
prize winner Ramon y Cajal, 1928)
This changed in the second part of '60s, when Joseph Altman used tritiated
thymidine to mark DNA and check mitotic activity. He discovered
constitutive proliferation in the hippocampus (Altman & Das, 1965) and
olfactory bulb (Altman, 1969) in rats. Scientific community did not accept
this discovery as prove of adult neurogenesis untill two decades after,
because was not possible to understand if neurons or other types of cells
were involved in this proliferation.
In '90s the evidence was found that precursor cells isolated from the
forebrain could differentiate in vitro into neurons (Reynolds and Weiss,
1992; Richards, 1992). In the following years, using immunofluorescent
labeling for bromodeoxyuridine (that labels DNA during the S phase of
proliferation) and for one of the neuronal markers, adult constitutive
neurogenesis was demonstrate in fishes, reptiles, birds, mammals and also
humans (Eriksson, 1998) injecting BrdU in cancer patients before death.
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1.2 Adult mammalian regeneration
Today, constitutive neurogenesis in adult human is known to involve only
the sub-ventricular zone of the olfactory bulb (SVZ) and the sub-granular
zone in dentate gyrus of the hippocampus (SGZ).
In the SGZ there are two cell types, identified as type 1 and 2, that form
new neurons with different frequency. While in SVZ there are two other cell
types, named type B and C, that present similar characteristic and form
new neurons.
Types 1 and B express glial fibrillary acidic protein (GFAP), they are
astrocytes and they divide infrequently. Instead types 2 and C does not
show glial characteristic and they divide continuously.
Type C cells are identified as transit amplifying cells (Belluzzi 2003), able
to give rise to immature neuroblasts.
Type B cells, localized inside the ependymal cell layer, are able to give rise
to type C cells when destroyed (Doetsch 1999), then they are identified as
adult neural stem cells of SVZ also if they has characteristics similar to
differentiated glia cells.
Other areas of the brain are generally considered non-neurogenetic,
although some studies suggest that low levels of neurogenesis may occur. In
2001 Gould observed new neurons BrdU positive in the white matter
(neocortex) of monkey, the possibility of migration from SVZ in the rostral
flux was considered (Gould 2001).
In 2003 it was reported that constitutive neurogenetic activity was present
in the mouse substantia nigra, and produced dopamingeric neurons.
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Figure 1Schematic of progenitor types and lineages in the adult brain SVZ. NSCs in the wall of the lateral ventricles of adult rodents correspond to type B cells (SVZ astrocytes). These cells retain epithelial properties, including extension of a thin apical process that ends on the ventricle and a basal process ending on blood vessels. B cells give rise to C cells, which correspond to nIPCs. B cells also generate oligodendrocytes through oIPCs. Dashed arrows illustrate hypothetical modes of division: blue for asymmetric and red for symmetric divisions. Investigators do not currently know how many times C cells divide.SVZ, sub-ventricular zone;NSCs, neural stem cells;nIPCs, neurogenic intermediate progenitor cells;oIPCs, oligodendrocytic intermediate progenitor cells.
This activity also seemed to increase after ablation of dopaminergic
neurons (Evidence for neurogenesis in the adult mammalian substantia
nigra. Zhao 2003). However, the following year a different research group
published that there was no evidence for new dopaminergic neurons in the
adult mammalian substantia nigra (Frielingsdorf 2004) generating a
controversy on this matter.
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1.2.1 Study on stimulated neurogenesis
Promoting neural regeneration after an injury can be defined as to
stimulate neural stem cells to proliferate, migrate, differentiate and thus
lead to tissue repair. To be able to undergo neural regeneration would
means that there is a possibility to rescue adult brain tissue after an injury
and also to cause the regression of neurodegenerative diseases.
It is hard to study adult neurogenesis within humans with the current tools
available. In order to gain a better understanding of adult neurogenesis
without invasive study on adult human brain, animal models are used.
Mices and rats are the two most used mammalian model, the first to study
genetic correlations and linkage, the second to study drug response.
Although a lot of work has been in mammalian models of neurogenesis,
regeneration of complex structures, such as brain tissue has not been
achieved. In particular, we have controversial evidences of regeneration in
midbrain related to Parkinson's Disease (Zhao 2003; Frielingsdorf 2004) or
its induction due to neurotoxins.
Stimulating the proliferation of neural cells in mammalian adult forebrain
is possible. It was proven that Shh protein (Sonic Hedgehog), is involved in
various organogenetic processes. In forebrain it affects periventricular
GFAP+ astrocytes and GFAP- early precursors, and stimulates
proliferation raising the number of neurons. Moreover, blocking Shh
function results in decrease of the neurons' number (Sonic hedgehog
controls stem cell behavior in the postnatal and adult brain. Palma et al.
2004. Development).
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Also the neurotransmitter dopamine may also have a role in neurogenesis,
because the loss of dopaminergic neurons or blockage of dopamine
production, decrease the proliferation of neural stem cells both in sub-
ventricular zone and in sub-granular zone (Höglinger et al. 2004).
1.3 Salamander adult regeneration
Salamander's regeneration abilities include the tail (Iten 1976), limbs
(Benzo 1975), the heart (Bader 1978), ocular structures (Keele 1973) and
parts of their brain (Parish 2007). Salamander is an useful animal model in
order to study adult regeneration. The brain of the highly regenerative
salamander, the red spotted newt (Notophthalmus viridescens), in
physiological condition shows similar distribution of active germinal niches
with mammalians. Proliferation zones are essentially restricted to the
forebrain (telencephalon and rostral diencephalon's areas) of the newt
under normal homeostatic condition (Berg 2010).
Unlike mammalians, salamanders do not have ependymal cells, instead the
ventricles are lined by radial glia-like cells so called ependymoglial layer.
The ependymoglia shows stem proprieties and is GFAP positive (Benraiss
1996, Lazzari 1997).
The midbrain of the newt, after a parkinsonian injury induced by
neurotoxin, regenerate the lost tissue in 30 days (Parish, 2007).
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Figure 2Immunostaining of PCNA in adult salamander brain identifies constitutive proliferation zones. in telencephalon and rostral diencephalon.(A, A') longitudinal sections, schematic representation of the newt brain. (B-J) telencephalic transversal section. (B) No proliferation cells are detected in the rostral olfactory bulb (OB). (C) PCNA+ cells line the medial wall of the lateral ventricles in the accessory olfactory bulb.(D,E) Proliferating cells line the lateral walls of the lateral ventricle adjacent to the dorsal and lateral pallium (Dp and Lp, respectively). (F,G) Ventral accumulation of proliferation situated ventrally to the striatum (Str) in the region of the bed nucleus of the stria terminalis (Bst). (H) A ventrally located proliferation zone in the area of suprachiasmatic nucleus (Sc), and a medial zone situated adjacent to the ventral thalamic nucleus (Vtn). In the most caudal region of the lateral ventricles, proliferating cells are scattered on both the medial and lateral wall. Note unspecific cytoplasmic labelling between medial and ventral proliferation zone (arrowhead). (I,J) Lack of proliferating cells in the midbrain and hindbrain, respectively. Tm, midbrain tegmentum; Tc, tectum; LDT, laterodorsal tegmental nucleus; Ra, Raphe nuclei. Scale bar: 100 µm
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Figure 3ATime course of TH+ cell regeneration following 6-OHDA injection. Sham, n=4-14 animals per group per time point (d3, n=4; d5, n=6, d7, n=5; d10, n=6; d17, n=5; d24, n=4; d30, n=14). Lesioned, n=5-18 animals per group per time point (d3, n=5; d5, n=6; d7, n=5; d10, n=6; d17, n=5; d24, n=6; d30, n=18). Mean±s.e.m.; Student’s t-test; *, P<0.05; **, P<0.01.(Parish 2007)
Figure 3BTotal number of BrdU+ TH+ cells following lesioning. Sham-lesioned animals pulsed days 0-30, n=11; lesioned animals pulsed days 0-30, n=8. Lesioned animals pulsed days 0-3, n=8. Lesioned animals pulsed days 4-23, n=8. Mean±s.e.m.; ANOVA with Tukey post-hoc test; ***, P<0.001.(Parish 2007)
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1.4 Neurodegeneration: the Parkinson's Disease
One of the most common diseases characterized by neurodegeneration is
the Parkinsons's Disease, which affects about 2% of human population over
65 years old. Parkinson's Disease is characterized by three classical
symptoms: resting tremors, rigidity, hypokinesia; but later it leads to
disturbances in gait, balance and sometimes also automic disturbances,
dementia, depression. The rest tremor is maximal when the limb is at rest
and disappears during voluntary movements and sleep. Rigidity comes
from joint stiffness and increased muscle tone and is often related to pain.
The muscle is passively shaken and it is due to the loss of regulation
between flexor muscles and antagonist muscles (Jankovic 2008).
Figure 4Human midbrain samples: parkinsonian on the left, normal on the right.Visible reduction of the substantia nigra in the parkinsonian sample.
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In normal conditions gamma circuit feels muscle tone and the
extrapyramidal circuit (composed by basal nuclei) regulates its activity. In
Parkinson's Disease this regulation is lost and the subsequent increase of
muscle tone causes movement problems.
Hypokinesia can develop at different severity level, slowness of movements
called bradykinesia, or in the worst cases absence of movement called
akinesia, such as freezing episodes. This problem comes from the planning
and initiation of the movements, more than from its execution. Sometimes
immobile Parkinsonian patients are capable to perform rapid movements if
they get excited. This suggests that their motor programmes could be
intact, but patients have difficulties in controlling them.
At histopathological level, Parkinson's Disease is characterized by a
progressive loss of midbrain substantia nigra dopaminergic neurons that
project to the striatum. This loss results in a disequilibrium between
excitatory and inhibitory mechanisms which control movements, so it is
responsible for the main motor symptoms in the disease. In healthy brains,
dopamine released by dopaminergic neurons in striatum regulates the
activity of GABAergic neurons, these neurons then release GABA in order
to inhibit motorial neurons. In Parkinson's Disease, dopaminergic neurons
die and thus dopamine levels in the striatum drop, causing deregulation of
GABAergic neurons, which leads to motor symptoms typical of the disease.
Another histological feature of Parkinson's Disease is the presence of Lewy
neurites and Lewy bodies within neurons. Lewy neurites are inclusions
composed of abnormally phosphorylated neurofilaments, while Lewy bodies
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Figure 5Organization of the basal ganglia. The cortex projects to the striatum that is GABAergic and project to the Gpi and to the SNr (both GABAergic). Gpi and SNr are efferent and project to the thalamus that is excitatory on the cortex. The striatum also projects to the Gpe (GABAergic) that project to the subtalamic nucleus (glutaminergic), projecting to the efferent nuclei.Gpe = external globus pallidus, Gpi = internal globus pallidus, SNc = substantia nigra pars compacta, SNr = substantia nigra pars reticolata, STN = subthalamic nucleus, PPN = pedunculopontine nucleus; Thalamus: CM = centromedian nucleus, VA = ventral anterior nucleus, VL = ventral lateral nucleus
are aggregates of proteins containing misfolded alpha-synuclein fibrils
together with other proteins such as ubiquitin. The reason why cells fail to
eliminate the misfolded alpha-synuclein through ubiquitination and
proteasome recycling is not known. These histological features appear
before any early symptoms of Parkinson's Disease and before any neuronal
loss has occurred, but unfortunately there is no diagnosis can be done
without an autopsy.
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The cause of Parkinson's Disease is largely unknown, because the majority
of cases are sporadic, however, several implied genes and susceptibility
factors have been identified in the last decade. According to these studies,
an abnormal increased oxidative stress and mitochondrial dysfunction,
together with protein misfolding and impairments in the ubiquitin-
proteasome and autophagy-lysosomal systems, contribute to Parkinson's
Disease.
Primary symptoms of Parkinson's Disease can be also induced by
neurotoxins: in 1980s some Californian heroin addicts used a drug and
developed symptoms of Parkinson's Disease.
The drug was MPPP, desmethylprodine similar to morphine, but its
synthesis under improper condition produced MPTP as a major product.
MPTP can pass the hematoencephalic barrier and, and once in the glial
cells, it is converted into MPP+ that can be transferred by dopamine
transporter inside dopaminergic neurons, where it blocks mitochondrial
NADH dehydrogenase leading neuronal death (Chiueh 2006).
1.4.1 Treatments
A common therapy of Parkinson’s disease is to artificially increase the
dopamine levels caused by the loss of the dopamingeric neurons. This can
be done through administering Levo-Dopa, a precursor of dopamine that
can cross the hematoencephalic barrier. However this treatment is only
useful to temporary stop motor symptoms of Parkinson's Disease, and it can
not stop the neural degeneration. Moreover patients cannot take L-Dopa for
long periods because this induces dyskinesias, another motorial disorder
(Simuni 1999).11
There is a surgical treatment, based on the inserting electrodes to stimulate
the internal segment of globus pallidus, the subthalamic nucleus or the
pedunculopontine nucleus (Silberstein, 2002). A device send continuously
electrical pulses to the target area inside the brain, this obtain to block the
impulses that cause tremors in a reversible way respect to a surgical
elimination of the brain areas involved.
However this is just a symptomatic therapy, it cannot stop the further
neuronal degeneration and have no effect on secondary symptoms of
advanced Parkinson's Disease.
The immunity system can be involved in degeneration of parkinsonian
neural tissue (Qian 2010). Microglia are considered to be the Central
Nervous System counterparts of peripheral macrophages, they quickly
respond to immunological stimuli with a burst of production of pro-
inflammatory mediators. An excess of these pro-inflammatory mediators
can aggravate the neuropathology and neuronal death, so other factors are
needed to regulate inflammatory response and decrease neural
degeneration.
Another method to increase dopamine levels within the striatum is based
on cell therapy. This strategy requires harvesting fetal or embyonic stem
and progenitor cells, differentiating them into dopaminergic neurons,
grafting these neurons into the striatum of patients and integrate them in
order to rebuild the lost connections.
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Some problems can rise with this treatment. In patients that used L-Dopa
for long time, dyskinesias get worse with the graft (Hagell 2002). In
patients that suspend immunosuppression, the innate immune system is
newly activated and functional improvement of the graft disappear.
However recent studies have shown that grafted neurons survive at
maximum as long as 16 years in brain of parkinsonian patients after
transplantation (Mendez 2008).
There are two source of cells for these experiments, from cell lines and from
embryos, but in humans only cells directly isolated from the embryos have
been used.
The fetus is known to be a great source of cells so it is not strange to use the
same mesencephalic tissue of embryo as a pool in order to harvest cells for
the graft. However there are some problems associated with this method.
First of all ethical issues that limit the availability of the tissue according
to quantity, quality and abortion time, so it is not simple to obtain the
correct cells for the graft. Moreover, the survival of fetal tissue after
transplantation seems to be less than 10% in the best of the cases
(Sortwell 2007).
It is possible to induct stem cells to differentiate in dopaminergic neurons
thanks to transcription factors, morphogens and survival factors, according
to defined protocols (Zhang 2010).
This strategy is based on understanding the development of midbrain
dopaminergic neurons and the ability to identify dopaminergic
differentiation factors.
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The alternative to cell replacement therapy would be to stimulate
endogenous neurogenesis. Neurogenesis could help to stop degenerative
processes by replacing the lost tissue.
1.5 Aim of the Thesis
This work characterizes itself as a study about involvement of the genes in
regulation of adult neurogenesis, focusing on a non-mammalian vertebrate
model, the red spotted newt, because its extensive regeneration abilities.
The chosen model is the newt with midbrain injured by 6-OHDA
neurotoxin, that eliminates dopaminergic neurons and mimes parkinsonian
symptoms.
1.5.1 Gene candidates
In order to promote neural regeneration, it must be understood the process.
To check the variation of expression and localization of candidate genes
after an injury, allows to understand which genes are implied in promoting
neural regeneration, to study neurogenic processes and to test the
regenerative potential of the vertebrate brain.
Recently the transcription profile of regenerating cells was analyzed thanks
to a micro array work (Berg, 2010) and from this list several candidate
genes that could have significant roles in the regeneration process were
identified.
Three different genes were chosen as candidates to start this study: AnxA1,
JARID2, nRAD; and two more genes, CKM and Notch, were chosen
respectively as negative control and stem marker.
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Notch encodes a receptor protein that participates in many developmental
cell fate decisions. Notch suppresses neuronal differentiation and is thought
to maintain precursor cell properties. In postnatal mammalian sub-
ventricular zone cells, Notch prevents migration to the olfactory bulb,
suppresses neuronal differentiation and decreases proliferation (Chambers,
2001).
Notch is involved in a lot of different processes and tissues, but in each case
its role seems to be related to the maintenance of stemness.
CKM (Creatine kinase of muscle) is expressed in skeletal muscle, in heart
and in vessels, and it has an homologue expressed in brain. Its role is
related to energy supply for cells and there are no evident reasons to relate
its role with neurogenesis, so it was chosen as negative control.
AnxA1 encodes a protein, Annexin A1 or Lipocortin 1, which is associated
with many cellular components, including plasma membrane
phospholipids, vesicles and cytoskeletal proteins. This protein plays a role
in differentiation, proliferation, plasma membrane repair, apoptosis and
several anti-inflammatory processes out of the cell (McKanna 1995; Solito
1998; Lim 1998; de Coupade 2000; Solito 2001; McNeil 2006).
In mammals AnxA1 appears to be constitutively expressed in the cells of
the innate immune system of the normal brain. Microglia are considered to
be the Central Nervous System counterparts of peripheral macrophages,
they rapidly respond to immunological stimuli with a burst of production of
pro-inflammatory mediators and are capable of phagocytosis.
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An excess of these pro-inflammatory mediators can aggravate the
neuropathology and neuronal death (Carey 1990; Mogi 1994; Muller 1998).
Annexin A1 inhibits several of these factors and suppresses the activity of
polymorphonucleates and macrophages, regulating in this way the
inflammatory response and promoting neuroprotection (Solito 2008).
Studying AnxA1 in Parkinson's Disease models allows to understand the
role of neuroprotection as rescue mechanism for degenerating brain tissue.
JARID2 (Jar2) encodes a transcription factor of the ARID family (AT rich
interaction domain), involved in several biological processes, also called
Jumonji. The Jumonji name, japanese word for "cruciform", derives from
the mutant mice jar2-/- that showed an abnormal brain morphology at fetal
stage E13.5 (Takahashi 2004).
JARID2 is part of PRC2 (polycomb repressive complex 2), a complex that
regulates developmental genes expression pattern in embryonic stem cells
and also during development. PRC2 contains three core subunits, all of
which are necessary for trimethylation of lysine 27 on histone 3
(H3K27met3) in order to silence gene expression. The role of JARID2 is
identified in recognition of DNA sequences and recruitment of the complex.
In xenopus, JARID2 depletion results in aberrant increase of H3K27
methylations, failure of differentiation and developmental gene induction,
so that development stops at gastrulation (Peng & Wysocka 2009).
In mouse embryonal stem cells, Jar2 depletion results in failure of
trascriptional activation related to differentiation marker genes and
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abnormal epigenetic pattern (Pasini & Helin 2010).
In mammalian heart, JARID2 binds to the promoter of cyclin D1 and
represses the transcriptional activity in order to suppress cardiac myocyte
proliferation. However its role is closely related with cyclin D1 and cell
cycle exit, also in brain tissues. During neurogenesis in particular, JARID2
seems to be involved in neuronal differentiation and migration of neural
progenitor cells (Takahashi et Takeuchi 2007).
RAD (Ras associated with diabetes) is a variation of a RAS signaling small
GTPase expressed in skeletal muscle of diabetic patients, and constitutively
in hearth and lung (Reynet et Kahn 1993).
RAD interacts with calmodulin, protein kinase II (Moyers et Bilan 1997)
and betatropomyosin (Zhu et Bilan 1996) and it seems to play a role in
muscle regeneration, but its role is not clear yet. In rats, RAD is found to be
expressed in vascular smooth cells where there is a vascular lesion
(Fu et Zhang 2005).
In newt, nRAD is expressed in skeletal muscle cells within 4 hours after
limb amputation. The presence of retinoic acid, that causes an enhanced de-
differentation of myotubes in regenerating limbs, can increase the
expression of nRAD after amputation
(Shimuzu-Nishikawa, Tsuji et Yoshizato 2001).
nRAD is than used as marker for regeneration in muscle and its presence
as gene candidate has the aim to discover an eventual parallelism between
brain and muscle regeneration.
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2 Materials and Methods
2.1 Buffers preparation
0.2 M phosphate buffer
165.3 g Na2HPO4 x 7H2O (268.07 Da)
25.6 g NaH2PO4 x H2O (137.99 Da)
Water to 4 liters
PBS
2 liters 0.2 M phosphate buffer
35 g NaCl
Water to 4 liters
4% Paraformaldehyde
200 ml water
16 g PFA
Heat to 65°C and check pH between 7.2 and 7.4 adding 10M NaOH
200 ml 0.2M phosphate buffer
3 g NaCl
Filter to 0.2 µm
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Prehybridization Buffer
10 ml deionized formamide
5 ml 20x SSC
2 ml 50x Denhardt's
5 mg yeast RNA (sigma R6759)
10 mg salmon sperm DNA
Water to 20 ml
B1
100 ml 1 M Tris-HCl pH 7.5
30 ml 5 M NaCl
Water to 1 liter
B3
100 ml 1 M Tris-HCl pH 9.5
20 ml 5 M NaCl
10 ml 5 M MgCl2
Water to 1 liter
Filter to 0.45 µm
B4
4.5 µl (NBT) Nitroblue Tetrazolium (Boehringer 1 383 213)
3.5 µl (BCIP) 5-Br-4-Cl-3-indolylphosphate (Boehringer 1 383 221)
0.24 µg Levamisole
B3 to 1 ml
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2.2 Animals
Adult red spotted newts, Notophtalmus viridescens (Charles Sullivan Co.,
Nashville TN), were mantained in a humified room at 25°C and fed weekly.
All experiments were performed according to European Community and
Local ethics committee guidelines.
2.3 6-OHDA injury
Newts were anaesthetized by placing them in an aqueous solution of 0.1%
Tricane for 20 minutes. Animals were placed in a neonatal stereotaxic head
frame. 200 nl of a solution consisting in 6-OHDA (6 µg/µl) and ascorbic acid
(0.2 mg/ml) was injected into the third ventricle with a glass micropipette
through a small hole drilled in the skull.
Subsequently the surgery hole in the skull was sealed with dental cement
and animals were left to recover overnight in a shallow container of water
before being placed back into a 25°C water environment.
2.4 Tissues harvesting
Animals were anaesthetized by immersion in an aqueous solution of 0.1%
Tricane for 20 minutes and perfused with 4% formaldehyde in PBS.
Animals were dissected and brains were rapidly placed in 4%
formaldehyde. After 1 hour of post fixation, brains were cryo protected in
20% sucrose in PBS for 12 hours and then embedded in OCT compound.
16 µm coronal sections were collected alternating on seven slides and stored
at -80°C.
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2.5 In situ hybridization
In situ allow to study the macroscopic distribution and cellular localization
of DNA and RNA sequences in a heterogeneous cell population. In this
study all the protocol was set to put in evidence RNA localization, so to
understand the expression of candidate genes.
Two digoxigenin-marked RNA probes were sinthetized for each candidate
gene, one sense and one antisense. The antisense probe annealates
specifically with mRNA of the gene during hybridization, so its expression
is revealed; while the sense probe has not any mRNA counterpart and
cannot annealate specifically, so nothing else than background is revealed
and this sample could be used as reference.
The first part of in situ technique require an RNAse free enviroment to
avoid the destruction of RNA strands.
Brain slices were completely fixated in 4% formaldehyde in PBS for 10
minutes, quickly washed in PBS and than permealized in a buffer 1.333%
triethanolamine, 0.175% HCl, 0.25% acetic anhydride in DEPC-treated
water for 15 minutes.
Slices were washed 3 times in PBS and treated with Prehybridization
Buffer at 37°C for 2 hours in a chamber humified with a solution of 50%
formamide and 5x SSC, than heat-denaturated RNA probe was added at
Prehybridization Buffer, slides were covered and let O/N at 70°C in the
humid chamber. Once the hybridization was done, the RNA strands can not
be attacked by RNAses.
Coverslips were removed and slides were put in 0.2x SSC at 70°C for 2
21
hours, than cooled at room temperature before to prepare slices with B1.
Slices were treated with B1 containing 10% blocking reagent for 1 hour in a
chamber humified with B1 itself, than 1:5000 Anti-digoxigenin antibody
coniugated with alcaline phosphatase enzyme was added to B1 and slices
let O/N at 4°C.
Slices were washed with B1 and equilibrated with B3, before to treat them
with B4 reagent and let it work at room temperature in a chamber
humified with B3 for a long time (from 2 hours to 2 days) untill the dye
produced by the alcaline phospatase antibody-coniugated marked tissue
visibly.
Slides were than washed in PBS to stop reaction and to reduce background
signal, then mounted in mounting media (DAKO) added with DAPI.
2.5.1 Probes
Clones of E. coli inserted with plasmid (pCR-BLUNT II-TOPO) for the
required gene sequence, stored at -80°C were thawed out and let to grow in
Luria-Bertani broth medium over night. The DNA was purified from the
growth thanks to MiniPrep kit by Qiagen and than digested to linearize the
plasmid.
Digestion with BamH1 restriction enzyme cuts the plasmid on the target
sequence of SP6 primer, meanwhile digestion with XHO1 cuts the plasmid
on the target sequence of T7 primer.
Figure 6Gel to check linearization of the plasmid.L = ladder; SP6 = plasmid digested by XHO1;T7 = plasmid digested by BamH1; C = control
22
Figure 7Map of the plasmid pCR-Blunt II-TOPO
Digested DNA was purified with Qiagen spinning column and its
concentration checked with NanoDrop 2000 spectrophotometer (Thermo
scientific).
RNA was synthesized from this DNA template using NTPs coniugated with
digoxigenin as marker, and treated with DNAse.
23
Sequence of Newt-Shh
1 AGTGGCCCTG GAGTCAAGCT GCGGGTGACC GAGGGTTGGG
41 ATGAGGATGG CCACCACTCT GAGGAGTCCC TGCACTACGA
81 GGGTCGGGCA GTGGACATCA CCACCTCGGA CCGGGACCGC
121 AGCAAGTATG GCATGCTGGC CCGCCTGGCC GCGGAGGCTG
161 GCTTCGACTG GGTCTACTTT GAGTCCAAGG CCCACATCCA
201 CTGCTCAGTG AAAGCAGAGA ACTCGGTAGC TGCAAAATCG
241 GGAGGCTGCT TCCCAGGCTC TGCCACGGTG ACCCTGGAGA
281 AAGGCATAAG GATGCCAGTG AAGGACCTGA GGCCGGGGGA
321 CAGGGTGCTC GCCGCAGATG GACAGGGCCG GCTGGTCTAC
361 AGCGACTTCC TCTTGTTCAT GGATAAAGAG GCGACGGCCA
401 GGAAAGTCTT CTACGTGATA GAGACCTCTC AGCCCCGGGA
441 GAGGCTCCGC CTGACCGCCG CTCACCTCCT CTTCGTAGCC
481 CAGGCGCACC CAGGAAACGC CAGTGGGGGC AACTTCCGGT
521 CCATGTTTGG CAGCGCAGGC TTCCGCTCCA TGTTCGCCAG
561 CAGCGTGCGG CCGGGGCACC GGGTGCTCAC AATGGACCGG
601 GAAGGCCGGG GGCTAANGGG AAGCCACAGT GGAGCGAGTG
641 TACCTGGAGG AGGCCACGGG GGCCTACGCT CCCGTCACTG
681 CACACGGGAC CGTGGTCATA GACAGGATGC TGGCCTCCTG
721 CTACGCGGTC ATAGAGGAAC ACAGGTGGGC ACACTGGGCC
761 TTCGCCCCTC TGAGAGTGGG CTTCGGGGTC TTGTCTTTCT
801 TCTCCCCCC
24
Sequence of Notch
1 TGACCAATCG GAGAAGTTGG AATGAGGAGA AGAGCTCGAC
41 CATTGGTCTG GGGATTCAGG AGATGGAGTC AAAAAGGGGT
81 GATCAGACAC CTGAAGTTGG TGGCTGGGAG TGTTGTCCAT
121 TGGAGAAGAG TAGCTATGCT GAGAAGGCGG
Sequence of CKM
1 CTTCCTGGTC TGGGTCAACG AGGAGGATCA TCTCCGTGTC
41 ATCTCTATGC AGAAGGGCGG CAACATGAAG GAGGTGTTCA
81 GGCGCTTCTG TGTTGGATTG CAGAAGATTG AAGAGATCTT
121 CAAGAAAGCT GGCCATCCCT TCATGTGGAA CGAGCATCTT
161 GGTTACGTGT TGACCTGCCC CTCCAACTTG GGCACTGGAC
201 TACGTGGTGG TGTCCACGTC AAACTCCCCA ACCTCTGCAA
241 GCACCCCAAG TTTGAGGAAA TTCTGACCAG ACTGCGCCTA
281 CAGAAACGTG GTACAGGTGG TGTGGATACC GCAGCTGTTG
321 GTGGTGTCTT TGACATCTCA AATGCTGACC GTCTGGGGTT
361 CTCTGAGGTA GAGCAGACAC AGATGGTGGT GGATGGCGTG
401 AAGCTCATGA TTG
25
Sequence of Jar2
1 GATTTCCTTA CGTTTCTATG TCTTCGAGGT TCTCCAGCCT
41 TGCCCAGCTC CATTGCGTGT CTTGGAACCT CGCTAGATGA
81 AGACGACGTG GAGGAGGAGG AGGATGAAAC ATGTTCTCAA
121 CGGACATGTG TTTAATGGGT ACAGCAAATC ACCAAGGGAA
161 AAAGAATCCG CCCAGAAACA CAAAAGTAAA GAAGCCACAC
201 CAGGGAAGGA GCGGAATGCT GAACAGAGGG TTGAGAGGCG
241 AAGAGAGCGG GCCGCTGCTG CTGCTAACCA CACTCCTGCT
281 CCACATACTG TCTCCTCCGC CAAAGGTCTC GCTGCTAGCC
321 ACCATACACT TCACAGATCG GCTCAGGACT TAAGGAAA
Sequence of AnxA1
1 TAGTCTCCTT TGGTTTCATC CAAAATAGCC TGGCGGAGTG
41 ATATGCCATA CATTCGCTTG TAATGTGCCT TGATCTCATC
81 CATATCAATT CCGGAACGGG AAACCATGAT TCTGATTAAT
121 TGCTTGTCAC GAGTTCCAGA TCCCTTCATA GACAAGAATA
161 GTCGTTCAGC AAAGTAAGAT GGCTTGCTTA CTGCACATTT
201 CACAATAGCG GTCAGGCATT TTTCAATATC ACCCTTCAGC
241 TCAAGATCCA GGGCCTTGTT CATGTCATGT TTACTGTAGC
281 TGGTATACCT CTGAAAGACC TTACGAAGGT GTGGACCACT
321 TCTTGTAGTA AGAAGGTCGA TGAACACCTT AACATCGGTT
361 CCTTTTCTCT TTTCTCCAGC TTCGTA
26
Sequence of nRAD
1 GCGTTGTCTT CTTTGCTGTC CTTGCGGAGT CGGATCTGTC
41 GGACGATGCC CTCAAACAGG TCCTTAACGT TATGGTGAAG
81 GGCGGCTGAC GTCTCGATGA ACTTGCAGTC AAACACTACC
121 GCGCATGCGC GGCCTTCTTC CACAGACACT TCTCGTGATC
161 TCACCAGATC ACTCTTGTTC CCCACGAGGA TGATTGGAAT
201 GTCTTCGCTC TGCCGCGCTC TCCTCGGCTG TATCCTCAGC
241 TCCGAGGCCT TCTCAAAGCT GGACTTGTCC GTCACAGAGT
261 ACACAATGAC GTATGCATCC CCCATTTTCA TGCACTGATT
301 CTGAAGCCAG TGGGTCTCAT CCTGCTCCCA TATGTCATA
2.6 Immunostaining
Slides with brain slices were fixed in 4% PFA for 5 minutes at room
temperature and then rapidly washed in PBS. Tissue slices were
permeabilized with 0.2% Triton-X in PBS for 15 minutes on a rocker and
then quickly washed again in PBS.
Slices were protected by aspecific antibodies binding in blocking serum (5%
goat serum, 0.2% Triton-X, PBS) for 1 hour at room temperature and than
primary antibodies were substituted to serum and let bind for 2 hours.
After primary antibodies binding, tissue slices were washed in PBS 3 times
and secondary antibodies fluorecrome-coniugated were placed on slides and
incubated for 2 hours at room temperature.
After secondary antibodies binding, slices were washed in PBS 3 times and
mounted in mounting media (DAKO) added with DAPI.
27
2.7 Antibodies
rabbit anti-JARID2 (1:2000, AbCam)
mouse anti-GFAP (1:1000, Chemicon)
mouse anti-PCNA (1:800, Chemicon)
mouse anti-Neun (1:200, AbCam)
Alexa 448 goat anti-mouse (1:500, Molecular Probes)
Alexa 594 goat anti-rabbit (1:500, Molecular Probes)
2.8 Mounting
After both in situ hybridization or immunostaining, slides were quickly
washed in pure water at room temperature before to be mounted with Dako
Flourescent Mounting Medium added with DAPI, and fixed by coverslips.
2.9 Microscope
Image capturing was performed using "Microscope Axioplan 2 imaging"
linked with "Axiocam HRm" (Carl Zeiss Microscopy) and the Apple
executable "OpenLab".
All quantitative analysis were based on pictures from this microscope.
2.9.1 Image handling for pixel counting quantitative analysis
Image handling was perfomed by "gIMP image editor 2.6" working on RGB
channels.
For every picture of immunofluorescence, was established an highlight
defined area thanks to the command “Colors > Threshold”.
28
29
The “Threshold” effect equalizes the measured brightness, so to make
impossible recognize down-regulation and up-regulation inside cells, but
put in evidence if the signal is present or not.
Pictures of the marker to quantify, were then merged on DAPI pictures
related to the same sample in order to show the area of co-expression of the
two signals, quantifiable in number of pixels thanks to the command
“Colors > Info > Histogram”.
The ratio between the co-expression area and the total DAPI area is an
approximation of the relative number of marked cells.
2.9.2 Other Microscope
Other pictures for in situ hybridization samples were captured later in the
University of Pisa by "Microscope Nikon Eclipse DSU2" linked with "Nikon
Digital Sight" camera.
3 Results
3.1 Optimization of in situ protocol
The In Situ Hybridization technique was chosen to determine the
expression pattern of candidate genes and to localize it cell by cell in the
sample tissues.
Because Sonic Hedgehog (Shh) is an important gene also involved in
forebrain neurogenesis and its expression in salamander midbrain was
already observed in the same lab (Berg 2010), a probe for Shh was initially
used in order to establish a correct protocol for in situ hybridization. The
known expression of Shh could be used as reference to chose the better
condition of work.
Figure 8In situ hybridization of Shh antisense probe in adult salamander midbrain.(A) uninjured, (B) 7 days after 6-OHDA injection.(Berg, 2010)
30
The first attempt to optimize the in situ protocol was to decrease the time of
reaction for the production of the dye signal, in order to block
overproduction. The kinetic in production of dye seemed to be the same for
specific signal and background, because using different time of reaction
affected all the coloration in the same way. This showed that saturation
was not responsible of the background signal.
The second attempt to optimize the protocol was to increase the annealing
temperature to hybridize RNA, in the hypothesis of an unspecific probe
binding. Annealing temperature was set at 80°C, but no differences were
evidenced comparing the result to the one of the usual protocol.
An alternative explanation for this background excess could be due to an
unspecific antibody binding. To monitor this, it was preformed the in situ
hybridization bypassing some step of the protocol, so to expect as result no
signal at all, when the unspecific antibody is eliminated from the protocol,
or only background, when the unspecific antibody binds unsought epitopes.
In the in situ experiment without the Shh probe, as in the negative control,
no hybridization could happen and the primary antibody could not be bound
to the probe, but washed away or bound in unspecific way.
The result showed the same background signal already seen in previous
experiments and confirmed that background was not due to a lack of probe
specificity.
In the in situ hybridization where the use of primary antibody was
bypassed, secondary antibody could not be bound to the primary one, but
washed away or bound in unspecific way.
31
The result revealed that background was present and put in evidence that
primary antibody was not responsible of any lack of specificity.
In the in situ hybridization without the use of secondary antibody, alkaline
phosphatase could not be present in the sample, so the reaction of dye
production could not be catalyzed.
The result showed that background signal was still present and underlined
that coloration was not produced by alkaline phosphatase enzyme
conjugated with secondary antibody.
It was then hypothesized the presence of an endogenous enzyme activity in
the tissue that could react with the substrate of reaction and catalyze it, so
an in situ hybridization with regular protocol was repeated adding an
inhibitor (Levamisole) and the background signal was eliminated.
3.2 Primary screen of genes of interest
Each candidate gene was shown in a micro array experiment (Berg, 2010)
to be differently expressed in lesioned tissues comparing with control.
nRAD and AnxA1 appeared up-regulated in 6-OHDA injured animals,
while JARID2 appeared down-regulated.
To confirm this data and to localize the gene expression, in situ
hybridizations were performed on gene fragments that had been cloned.
The first aim was to determine if these cloned fragments were sufficient to
produce functional probes. Sense and antisense probes were prepared for
every gene fragment and checked on injured material, harvested 7 days
after the 6-OHDA injection.
32
While it was not possible to see any signal for CKM and AnxA1 probes,
indicating probably they did not work; Notch, nRAD and Jar2 showed some
signal localized around the 3rd ventricle. The working probes were
analyzed further comparing expression between injured and control
material.
3.3 nRAD
In situ was done for the candidate gene nRAD, in order to find eventual
correlation between muscular and neuronal regeneration.
The result put in evidence strong expression strictly localized in ventral
layer of ependymoglial cells in the 3rd ventricle of injuried animals. Then a
parallelism between muscolar and neuronal regeneration could be possible.
The signal marks a little number of cells close each other and disappears at
short distance along the rostro-caudal axis.
Figure 9In situ hybridization of nRAD antisense probe in adult salamander midbrain
33
With the purpose to see if RAD positive cells are proliferating cells, an
other experiment was set up. It was supposed the population of cells in two
consecutive slices from the same brain to be similar, since every slice was
16-20 µm thin. Slides were numbered, in situ for RAD was performed on
slides with even numbers, while immunostaining for PCNA was done on
slides with odd numbers.
The result shows the expression of RAD in injured tissues on the ventral
diencephalic part of the third ventricle, close to PCNA expression.
34
Figure 10Consecutive slices treated with different techniques in order to show the localization of PCNA and nRAD.
In particular, nor PCNA nor RAD were present 3 days after injection; 7
days after injection the expression of RAD was strictly localized in the cells
edging the third ventricle and the expression of PCNA was localized in
some cells spread around; 14 days after injection the expression of RAD was
disappeared, instead PCNA positive cells were diminished in number.
The similarity among two slides is not enough to demonstrate that RAD
positive cells are proliferative, but the result suggests a possible correlation
in the regenerative path between PCNA and RAD.
3.4 Jar2
For the candidate gene JARID2 were set up both an in situ hybridization
and an immunostaining, in order to check the expression level of Jar2
mRNA and protein JARID2, since the role of this gene in epigenetic
regulation identifies it as an important candidate to study differentiation
processes.
In situ result showed an evident signal in uninjured antisense-hybridized
sample and a light decreasing of signal in lesioned tissue, both localized
close to the ventricle cell layer.
This result indicate the repressive and anti-proliferating activity of Jar2
has to be down-regulated during regeneration in order to allow proliferation
and subsequent neural differentiation.
Immunostaining result showed a strong expression in the cells bordering
the third ventricle, and some other isolated cells with lower expression.
35
Figure 11In situ hybridization of Jar2 antisense probe in adult salamander midbrain
This distribution appeared similar between control and injured tissues.
Cause the variable expression of JARID2 protein in cells not edging the
third ventricle, next it was sought to determine which cells expressed
JARID2 in the midbrain.
It was performed some co-immunostaining with cell type specific markers:
one for GFAP and one for NeuN.
GFAP marks ependymoglia cells, which are stem cells in salamander brain,
while NeuN is a pan-neuronal marker.
The immunostaining result for JARID2 and GFAP double labelling
revealed co-localization in the cell layer bordering the third ventricle. All
the cells with strong expression of JARID2 were GFAP positive, and all cell
bodies of GFAP positive cells showed a strong expression of JARID2.
Double immunostaining result for JARID2 and NeuN did not show any
36
Figure 12Co-immunostaining for JARID2 and GFAP in adult salamander midbrain.(up) Uninjured, (down) 7 days after 6-OHDA injection.
37
Figure 13Co-immunostaining for JARID2 and NeuN in adult salamander midbrain.(up) Uninjured, (down) 7 days after 6-OHDA injection.
38
overt correlation. NeuN positive cells appear to be reduced in the injured
tissue, due to the neuronal loss induced by 6-OHDA.
3.4.1 More detailed analysis of the role of JARID2
A detailed quantitative analysis was performed in order to check if JARID2
protein expression presented differences between control and injured
samples: pictures of immunofluorescence were edited to individuate
highlight defined area of signals expression and gain a ratio.
A definite threshold was set for each fluorescent signal, and all pixels under
that value were not considered to individuate the area of expression as
signal.
JARID2 expression signal was considered only when co-localized with DAPI
signal in order to isolate background from cell expression.
Figure 14Picture handled for the quantitative analysis. Down the third ventricle.(blue) DAPI = nuclea; (purple) co-localization JARID2-DAPI = Jar2+ cells
39
Areas were calculated with pixel counting, and the ratio between JARID2
area and DAPI area represents a good approximation about which cells are
JARID2 positive.
Results show a small but significative decrease of JARID2 expression
respect to DAPI (P=3.44%) in the injured sample. This result seems to
confirm the one of in situ hybridization about down-regulation of Jar2
during adult brain regeneration.
Figure 15Percentual of JARID2+ cells in adult salamander midbrain.(left) Uninjured, (right) 7 days after 6-OHDA injectionMu=0.660 Dev.st.u=0.187 nu=14M7=0.555 Dev.st.7=0.201 n7=12ld=24 t=1.3779 P=0.0344
40
3.5 Notch
In situ for the candidate gene Notch was performed with the purpose to
check neural stem cells, since GFAP is only a marker for glial
characteristics.
The result showed expression of Notch only in the cells boarding the third
ventricle as expected, but there was also a relevant increase of signal in the
injured tissue, seemingly in contradiction with the known role of Notch.
Morover, in the injured samples tested by sense probe was found
widespread signal. An eventual unspecific binding of the probe is expected
to present the same spread distribution in all similar samples, while Notch
presented it only for the sense probe in the injured material.
It was supposed that Notch transcription involves also the complementary
strand with the production of an antisense mRNA. These possibility was
however not evaluated further because of time constrictions.
Figure 16In situ hybridization of Notch antisense probe in adult salamander midbrain
41
4 Discussion
On three antisense probes for the candidate genes, two of them worked and
permitted to analyze the mRNA expression of the related genes. Both the
probes, nRAD and Jar2, permitted to show a differential expression
between the control and the injured samples.
Some consideration about the result obtained by stem marker Notch
compared with its role are needed.
4.1 Notch
Notch is known to decrease neuronal differentiation and proliferation
(Chambers 2001) and so to maintain stemness, so it would not be expected
to discover up-regulation of Notch in a regenerating tissue where
proliferation and neuronal differentiation are in act. Instead the result of in
situ hybridization seems to contradict this role. Further experiments are
required to confirm the up-regulation of Notch, expecially a quantitative
analysis such as qRT-PCR.
However the regulation of Notch in the injured tissue appears to be
different zone by zone, in fact there is not expression of Notch in the cells
far from the third ventricle where neuronal differentiation is in act, but the
42
43
signal increases in the cells edging the ventricle that are known to have
stem and glial properties.
Since the ependymoglial cells are stem cells, it is hypothesized that Notch
might play a role in gliogenesis, promoting proliferation of the
ependymoglia during tissue repair. PCNA expression in tissues 7 days after
6-OHDA injection could be a confirmation of this hypothesis, since a
relevant number of PCNA positive cells were from the ependymoglial layer.
4.2 nRAD
The evident nRAD expression in tissue 7 days after lesion, indicates the
involvement of this gene in adult brain regeneration, as muscle
regeneration, so to let think a parallelism between regenerative pattern of
the two structures can really exist.
The role of nRAD in regeneration (muscular or neuronal) is not yet known,
so it is not possible to speculate about experimental results compared with
its role. Anyway the exact localization of nRAD positive cells in injured
tissue so close with PCNA positive cells, suggests this gene could be
involved in proliferation of new cells. Also the little size of PCNA positive
cells gathered in the expression area of nRAD let think about a massive
proliferation of new cells directly linked with nRAD gene, supporting this
hypothesis.
However, to test it needs dedicated studies.
44
4.3 JARID2
Decreasing of Jar2 expression in injured tissues revealed by in situ
hybridization, indicates the involvement of JARID2 in the regenerative
pattern of adult vertebrate brain.
Quantitative analysis of JARID2 protein expression induces to confirm the
same result as in situ: there is a significative down-regulation in injured
tissues.
Moreover a qRT-PCR analysis performed in the same laboratory by a
parallel research individuated the same down-regulation of JARID2 in
6-OHDA treated tissues (personal communications).
A decrease of JARID2 expression in regenerating tissues is probably
necessary in order to avoid silencing of genes involved in proliferation
and/or differentiation by Polycomb repressive group 2, and to allow in this
way the correct processes for tissue repair.
The epigenetic regulation pattern could present differences between
uninjured and regenerating tissues, and show eventual targets of
transcription activated by epigenetic modifications in 6-OHDA injured
brains.
It would be required a Chromatine Immunoprecipitation in order to study
the epigenetic methylation pattern and check if some genes, known to be
up-regulated or activated 7 days after 6-OHDA injection, are influenced.
However this experimentation is subordinated to the study about genes
identified by micro array transcription assay.
Instead, focusing the attention on genes transcriptional levels at different
stages of the regenerating process, would allow to individuate which genes
regulate expression and activity of JARID2.
Then, after would be possible to individuate the genes involved in
recognizing neuronal injuries and consequent activation of regenerating
processes leading to complete tissue repair.
4.4 Conclusion
The obtained results and the perspectives born from them, represent the
work path open toward the possibility to promote neural regeneration in
adult brain of vertebrates and to understand why physiologically it does not
happen in mammals.
45
46
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53
I wish to say thanks
To Andras Simon who accepted me in his lab in Stockholm giving me the opportunity to know scientific
research from a different point of view;
To Matthew Kirkham, my supervisor, who took care of me and put up with all my several questions even while he
was concentrate in working on different problems;
To Heng Wang who received me when I arrived in Stockholm and found an accomodation for my first
months in Sweden so that I didn't freeze in the winter;
To Daniel Berg, Paula Borg and Sara Loof who were always present in the office to solve any doubts or just to
take a break with conversation and swedish fika;
To Prof.ssa Renata Batistoni who helped me in relating to international research community and encouraged me
continuously;
To my father and my mother who granted me economical support to survive during all period of my education, and
above all taught me to be independent, a very precious lesson useful to live alone in a foreign country;
To Alvaro, Rob, Nancy, Johanna, Olga, Anders and all other friends known in Sweden who made my stay period
more funny, comfortable and coloured;
To Pisa, all professors, associates and students who were part of my education or just of the concurrent life time,
also the ones who never came to visit me in Sweden;
To all my family and my friends that concurred in my realisation as now I am and I work.
Marco Natali