abnormal redox system in autism ved chauhan head, cellular neurochemistry laboratory nys institute...
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ABNORMAL REDOX SYSTEM IN AUTISM
Ved Chauhan
Head, Cellular Neurochemistry Laboratory
NYS Institute for Basic Research in Developmental Disabilities, Staten Island, New York
Autism One, 2009
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AUTISM
Severe neurodevelopmental disorder in children
Onset before the age of 3 years
Affects 1 in 150 children
Characterized by
- impaired social interaction
- delayed speech development
- limited verbal communication
- stereotyped and repetitive behavior patterns
- abnormal eye contact
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PERVASIVE DEVELOPMENTAL DISORDERS (PDD)
Autism
Asperger’s disorder (not associated with language delay or general intellectual impairments)
Childhood disintegrative disorder
Rett’s disorder
PDD – not otherwise specified
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DIAGNOSTIC TESTS FOR AUTISM
Behaviorally defined disorder
- Autism Diagnostic Interview Revised (ADI-R) Criteria
- Autism Diagnostic Observation Schedule (ADOS) Criteria
Severity of autism
- Childhood Autism Rating Scale (CARS)
- Pervasive Developmental Disorder Behavior Inventory (PDDBI) Scale
No biochemical or genetic test
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GENETICS OF AUTISM
Polygenetic disorder with involvement of genes mainly on chromosome 7, 15 and 16
Twin studies: high concordance of ~ 90 % among identical twins as compared to ~5 % for fraternal twins and other siblings
Males are 3-4 times more commonly affected than females
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ENVIRONMENTAL FACTORS IN AUTISM
Thalidomide, Valproic acid
Heavy metals (lead, mercury)
Bisphenol A
Air pollutants
Chemicals and toxins
Pathogenic bacteria
Viral infection
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BIOCHEMICAL ABNORMALITIES IN AUTISM
Increased blood levels of serotonin (neurotransmitter)
Increased oxidative stress
Abnormalities in membrane structure and function
Aberrant signal transduction
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OXIDATIVE STRESS IN NEUROLOGICAL DISEASES
Alzheimer’s disease
Down’s syndrome
Parkinson disease
Schizophrenia
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Under normal conditions, a dynamic equilibrium exists between the production of reactive oxygen species (ROS) and the antioxidant capacity of the cell.
Oxidative stress and injury to cells occur when ROS generation overpowers the biochemical defense mechanism of the cell to neutralize and eliminate ROS. These ROS are highly toxic and react with lipids, proteins and nucleic acids, and lead to impaired cell functions and cell death .
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Chauhan and Chauhan, Pathophysiology 13 (2006) 171–181
Potential mechanism of oxidative stress and mitochondrial abnormalities in autism
INCREASED PRO-OXIDANTS DECREASED ANTI-OXIDANTS Endogenous Exogenous (Environmental factors)
NO Xanthine oxidase Homocysteine
Heavy metals (Hg, Pb) Thalidomide, Valproic
acid, Retinoic acid Air pollutants Chemicals (BPA) and Toxins Pathogenic bacteria Viral infection
Production of free radicals
Lipid peroxidation Protein oxidation DNA oxidation
OXIDATIVE STRESS IN AUTISM
Mitochondrial damage Impaired energy production Increased excitotoxicity
Antioxidant enzymes (SOD, GPx, catalase)
Ceruloplasmin Transferrin Abnormal Cu/Fe
metabolism
Glutathione
Genetic factors
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Lipid peroxidation reflects a chain reaction between polyunsaturated fatty acids and ROS providing a continuous supply of free radicals. It results in the formation of lipid peroxides and hydrocarbon polymers that are highly toxic to the cell, and leads to loss of membrane functions and integrity.
LIPID PEROXIDATION
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MalondialdehydMalondialdehydee
Malondialdehyde (MDA) is an end product of peroxidation of polyunsaturated fatty acids, and is a marker of lipid peroxidation.
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CONTROLS
Developmentally normal (non-autistic) siblings
Variables such as race, diet, socio-economic status and genetic background would be similar between normal siblings and autistic children
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Autism* Siblings Autism Siblings0.0
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Same sex Different sex
Pla
sma
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INCREASED LIPID PEROXIDATION IN AUTISM
Age (Years)(Mean S.E.)
Plasma MDA (nmol / ml)(Mean S.E.)
Autism 4.4 0.3 0.497 0.025*,
Siblings 6.0 0.9 0.396 0.019
Increased MDA in 87% autism as compared to normal siblings
* p < 0.005, paired t-test
p < 0.005, unpaired t-test
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Antioxidant Defense MechanismsAntioxidant Defense Mechanisms
Enzymatic antioxidants
- Superoxide dismutase (SOD)
- Glutathione peroxidase (GSH-Px)
- Catalase (CAT)
Non-enzymatic antioxidants
- Glutathione, -tocopherol, ascorbic acid
- Transport & Storage proteins
(Transferrin, Ferritin, Ceruloplasmin)
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No. of subjects
Age (Years) Transferrin(mg / ml)
Ceruloplasmin(mg / ml)
Autism 19 6.0 0.45 2.456 0.664a, b 0.2996 0.0138c
Siblings 19 6.8 0.87 2.699 0.093 0.3296 0.0182
Reduced transferrin levels in 16 / 19 (84 %) of autism
Reduced ceruloplasmin levels in 13 / 19 (68 %) of autism
a p < 0.005, paired t-test
b p < 0.05, unpaired t-test
c p < 0.02, paired t-test
REDUCED SERUM TRANSFERRIN & CERULOPLASMIN LEVELS IN AUTISM
These results suggest abnormal iron and copper metabolism in autism
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RELATIONSHIP BETWEEN CERULOPLASMIN / TRANSFERRIN LEVELS AND LOST ACQUIRED LANGUAGE SKILLS IN AUTISM
Reduced ceruloplasmin / transferrin levels were observed most strongly in children who had shown a loss of previously acquired language skills
Children with autism who had not lost language skills had ceruloplasmin / transferrin levels similar to that seen in the normal siblings
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OTHER STUDIES ON OXIDATIVE STRESS IN AUTISM
• Increased TBA-reactive substances in erythrocytes (Zoroglu et al. 2004).
• Increased excretion of 8-isoprostane-F2 alpha in the urine (Ming et al. 2005).
• Increased NO levels in RBCs (Sogut et al. 2005).
• Increased plasma levels of nitrites/nitrates (Sweeten et al. 2004).
• Elevated cerebellar 3-nitrotyrosine levels (Sajdel-Sulkowska et al. 2008).
• Increased density of lipofuscin (matrix of oxidized lipid and cross-linked protein) in language-related cortical brain areas in autism (Lopez-Hurtado, and Prieto, 2008).
• Increased levels of lipid-derived oxidative proteins modifications in autism (Zhu et al., 2008).
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Review
Oxidative stress in autism
Abha Chauhan,* and Ved Chauhan
NYS Institute for Basic research in Developmental Disabilities, Staten Island, NY, 10314
Pathophysiology 13 (2006) 171 - 181
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Special Issue On
AUTISM SPECTRUM DISORDERS
American Journal of Biochemistry and Biotechnology, Vol. 4, No. 2, 2008
Editor:
Abha Chauhan, Ph.D.
Associate Editors:
Ved Chauhan, Ph.D.
George Perry, Ph.D.
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EFFECT OF OXIDATIVE STRESSEFFECT OF OXIDATIVE STRESS
Increased lipid peroxidation
Cell membrane damages
Alterations in membrane fluidity and permeability
Oxidative changes in proteins
Cytotoxicity
Damage to mitochondrial and nuclear DNA
Enzyme modification
Cell death
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INCREASED LIPID PEROXIDATION IN THE CEREBELLUM AND TEMPORAL CORTEX FROM AUTISM SUBJECTS
MDA levels were significantly increased in the cerebellum by 124%, and in the temporal cortex by 256% in autism as compared to control subjects.
Chauhan et al. J. Neurochem. 108, Suppl. 1, 33 (2009)
Cerebellum Temporal cortex
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Mitochondrial abnormalities in the lymphoblasts from autism
Chauhan et al. J. Neurochem. 108, Suppl. 1, 33 (2009)
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Energy metabolism in the cell. Glucose is prime source of energy in the cell. Acetyl CoA is produced from glucose, amino acids and fatty acids. Acetyl CoA enters TCA cycle (Fig. B) and provides NADH (reduced electron carrier) for complex I of electron transport chain (ETC), and succinate for complex II (Fig. A). ETC produces proton gradient (membrane potential) that eventually leads to ATP production.
B
A
Mitochondria
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Involvement of oxidative stress and environmental factors in mitochondrial dysfunctions
Mitochondrial dysfunction may result in inflammation, decreased mitochondrial membrane potential and altered energy metabolism.
Oxidative stress
Mitochondrial dysfunctions
Altered membrane potential
Altered energy metabolism
Environmental factors
Inflammation
Oxidative stress
Mitochondrial dysfunctions
Altered membrane potential
Altered energy metabolism
Environmental factors
Inflammation
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DHR 123 is an oxidation-sensitive lipophilic dye that enters the cell, and fluoresces when it is oxidized by ROS to rhodamine 123.
Mitochondrial ROS levels were significantly higher in autistic lymphoblasts as compared to control lymphoblasts.
C ontrol Autism0
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Mitochondrial ROS in lymphoblasts from autism and control subjects by dihydrorhodamine 123 fluorescence assay
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Rh 123, a cell-permeable cationic dye, preferentially partitions into mitochondria because of highly negative MMP.
Mitochondrial membrane potential was significantly lower in autistic lymphoblasts as compared to control lymphoblasts.
C ontrol Autism0
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MITOCHONDRIAL MEMBRANE POTENTIAL (MMP) IN LYMPHOBLASTS FROM AUTISM AND CONTROL SUBJECTS BY
RHODAMINE (RH) 123 FLUORESCENCE ASSAY
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JC-1 exists as a green fluorescent monomer at lower MMP and as red fluorescent aggregates at higher MMP.
Mitochondrial membrane potential (Red / green fluorescence ratio ) was significantly lower in autistic lymphoblasts than in control lymphoblasts.
C ontrol Autism0.0
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MITOCHONDRIAL MEMBRANE POTENTIAL IN LYMPHOBLASTS FROM AUTISM AND CONTROL SUBJECTS BY JC-1 FLUORESCENCE ASSAY
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Autism ControlJC-1 exists as a green fluorescence monomer at lower membrane potential and as a red fluorescence dimer at higher membrane potentials. Autistic lymphoblasts shows lower membrane potential (more green) than in control lymbhoblasts (more red).
Confocal microscopic analysis of JC-1 showing decreased mitochondrial membrane potential in autistic lymphoblasts
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Schematic depiction of potential mechanisms that may mediate neuronal dysfunction and clinical symptoms in autism. (Reproduced, in part, from Chauhan, A. and Chauhan, V. Pathophysiology 2006: 13, 171-181).
Oxidative stress & Mitochondrial dysfunction
Membrane lipid abnormalities
Neuronal membrane dysfunction
Decreased prostaglandin production
Cell death
Decreased synaptic efficiency
Impaired serotonin receptor functions
Alte red immune response
Increased inflammatory response
Impaired neuronal development
Impaired energy production
Increased excitotocity
Clinical symptoms of autism
Pathogenesis of autism
Abnormal signal transduction
Genetic factors Environmental factors Increased endogenous pro- oxidants Decreased endogenous anti- oxidants
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Membrane abnormalities in autism
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Phospholipids in the membrane
Phosphatidylcholine (PC)
Phosphatidylethanolamine (PE)
Phosphatidylserine (PS)Phosphatidylinositol (PI) Sphingomyelin (SPG)
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PE and PS ratio of erythrocytePE and PS ratio of erythrocyte
AutismAutism Normal siblingsNormal siblings
0.85 0.85 0.198* 0.198* 1.42 1.42 0.106 0.106
COMPOSITION OF PHOSPHOLIPIDS IN ERYTHROCYTES MEMBRANES OF CHILDREN WITH AUTISM AND THEIR NONAUTISTIC SIBLINGS
Phospholipid Phospholipid fractions fractions
Phospholipid (Phospholipid (g/mg protein)g/mg protein)
AutismAutism
(N = 8)(N = 8)
Non-autistic Non-autistic siblingssiblings
(N = 8)(N = 8)
PEPE 5.12 5.12 0.54*0.54* 6.00 6.00 0.630.63
PCPC 6.40 6.40 0.370.37 6.60 6.60 0.400.40
SPGSPG 5.29 5.29 0.460.46 5.26 5.26 0.460.46
PI + PSPI + PS 5.89 5.89 0.710.71 5.26 5.26 0.460.46
● Levels of PE are decreased and that of PS are increased in the erythrocyte membrane of children with autism.
● Ratio of membrane PE/PS is decreased in autism as compared to control subjects.
Chauhan et al. Life Sci. 74, 1635-1646 (2004)
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Trinitrobenzene sulfonic acid (TNBS) reacts with amine-containing molecules such as PE and PS
0 25 50 75 100 1250.0
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Concentration of lipids (nmol)
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A: Wavelength scans of PE-TNBS (dotted line) and PS-TNBS (solid line).B: Wavelength scan of plasma lipid-TNBS. C: Standard curves of PS and PE as measured by TNBS assay
Chauhan et al. Life Sci. 74, 1635-1646 (2004)
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Autism Siblings0.000.010.020.030.040.050.060.070.080.090.100.110.120.130.140.150.160.170.180.190.200.210.220.230.240.250.260.270.280.290.300.310.320.33
Ab
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INCREASED AMINOGLYCEROPHOSPHOLIPIDS LEVELS IN PLASMA OF CHILDREN WITH AUTISM
Mean SE 0.2022 0.021 0.1575 0.017
Autism Siblings
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Levels of ceruloplasmin (copper-binding protein) are decreased in autism
Chauhan et al. (2004) Life Sci. 75, 2539-2549
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PE
PC
SPG
PS + PI
Lipids Lipids + Cu2+
COPPER DECREASES THE LEVELS OF PHOSPHATIDYLETHANOLAMINE IN THE LYMPHOBLASTS
Thin layer chromatogram
LipidsLipids Lipids + CuLipids + Cu2+2+
PE (0.56 PE (0.56 g)g) PE (0.08 PE (0.08 g)g)
● PE is oxidized in the presence of copper.
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Control Copper Iron Zinc Calcium Cadmium0
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Chauhan et al. Am. J. Biochem. Biotech. 4:95-100 (2008).
Effect of metal cations on the oxidation of PE in the lymphoblasts
● Among the metal cations, only copper oxidized PE
● Copper-mediated oxidation of PE was dependent on copper concentration and incubation time period.
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Effect of copper on the PE of lymphoblasts from autism and control subjects
No
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C ontro l Autism
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s
Copper oxidizes more PE in the lymphoblasts from autism than from control
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Copper oxidizes both plasmalogenic and non-plasmalogenic PE equally.
Phosphatidylethanolamine:
C1 & C2- Acyl groups (Non-plasmalogenic)
C1- O-alkenyl group (plasmalogen), C2-acyl group (Plasmalogenic)
C1
C2
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0
10
20
30
40
50
60
70
80
90
100
110
*
*****
% e
ffe
ct
of
co
pp
er
on
ox
ida
tio
n o
f P
E(t
ota
l, d
iac
yl P
E a
nd
alk
en
yl P
E)
Copper-mediated oxidation of PE-plasmalogen and non-plasmalogenic PE
Copper oxidizes both plasmalogenic and non-plasmalogenic PE
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Biological membrane
● Hydrophobic core of membrane is maintained by fatty acid chains of phospholipids.● The movement of fatty acids provides the fluid environment. ● Unsaturated fatty acids enhance membrane fluidity. ● Peroxidation of lipids decreases membrane fluidity.
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Age (Years)Age (Years)
(Mean (Mean S.E.) S.E.)
DPH Fluorescence DPH Fluorescence PolarizationPolarization
(Mean (Mean S.E.) S.E.)
Autism (N = 10)Autism (N = 10) 6.09 6.09 0.38 0.38 0.2225 0.2225 0.0045* 0.0045*
Siblings (N =16)Siblings (N =16) 6.56 6.56 1.08 1.08 0.2077 0.2077 0.0026 0.0026
DECREASED MEMBRANE FLUIDITY IN AUTISM
* p < 0.015, unpaired t-test
Autism Siblings0.18
0.19
0.20
0.21
0.22
0.23
0.24
0.25D
PH
Ste
ady
Sta
teF
luo
resc
ence
Po
lari
zati
on Membrane fluidity is inversely
proportional to DPH fluorescence polarization.
Chauhan et al. J. Neurochem. 108, Suppl. 1, 33 (2009)
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RELATIONSHIP BETWEEN MEMBRANE FLUIDITY AND SEVERITY OF AUTISM
Correlation coefficient: r = 0.72, p < 0.02
Membrane fluidity decreases with severity of autism
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Maintenance of optimum membrane fluidity is critical to biological functions
It has a marked effect on membrane properties.
It modulates the activity of membrane - bound enzymes, ion channels and receptors.
The activity of integral membrane proteins are markedly affected by the physical state of the lipids in which they are embedded.
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The phospholipids make up the bulk of all internal and external neuronal membranes. Alteration in membrane lipids can result in defective membrane functions and therefore, may have wide impact on learning and behavior.
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Phosphatidylcholine
LysophosphatidylcholineArachidonic acid
Phospholipase A2
Action of phospholipase A2 on phospholipids
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Unsaturated fatty acids and autism
•Physical state of the membrane affects the functions of membrane- associated proteins, e.g., unsaturated fatty acids of neuronal phospholipid affect functions such as neuronal transmission, ion channels, enzyme regulation & gene expression (Young and Conquer, 2005), insulin receptors in fluid membrane (Neufeld and Corbo, 1984).
• -9 fatty acids in autism (Bu et al., 2006), -3 fatty acids in Attention Deficit Disorder, Alzheimer’s disease, Schizophrenia and depression (Young and Conquer, 2005).
• Dietary -3 supplementation affects the behavior abnormalities: hyperactivity and stereotypic features (Amminger et al.,2007).
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Polyunsaturated fatty acids are decreased in the erythrocyte membranes of autism as compared to normal control (Bell et al. 2000, 2004).
Chromosomal linkage studies in autism points to a locus where PLA2 gene is located (Lamb et al. 2000).
Bell et al. (2004) reported that PLA2 activity is increased in
the erythrocytes of autism as compared to controls.
Increased levels of PLA2 in the erythrocytes of patients with
schizophrenia (Ward et al. 2000) and dyslexia (MacDonell et al. 2000).
Phospholipase A2 and autism
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Lymphoblasts PLA2 activity (cpm/4h)
Autism 8634 1704
Control 3061 1437
Increased PLA2 activity in lymphoblasts from autism
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Activities of Ca2+/Mg2+-ATPase, Na+/K+-ATPase, protein kinase C and protein kinase A in autism
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Autism Siblings C ontrol0
10
20
30
40
50
Ca
2+/M
g2+
AT
Pas
e ac
tivity
(g
ph
osp
hru
s / m
g p
rote
in /
min
)
Increased Ca2+-ATPase activity in the lymphoblasts of autistic subjects
Chauhan et al. J. Neurochem. 108, Suppl. 1, 33 (2009)
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C ontrol Autism80
100
120
140
160
180
200
220
240
260
280
Ph
osp
ho
rus
rele
ased
(
g)/
mg
pro
tein
/hIncreased activities of Ca2+-ATPase and Na+/K+ ATPase in the cerebellum of autistic subjects
Ca2+-ATPase
C ontrol Autism80
100
120
140
160
180
200
220
240
260
Ph
op
sho
rus
rele
ased
g
)/m
g p
rote
in/h
Na+/K+-ATPase
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Autism C ontrol0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
Cyt
oso
licP
rote
in k
inas
e C
(Op
tica
l d
ensi
ty/m
g p
rote
in)
Autism C ontrol0
10
20
30
40
50
60
70
80
Mem
bra
ne
Pro
tein
kin
ase
C(O
pti
cal
den
sity
/mg
pro
tein
)
Decreased activity of Protein kinase C (PKC) in the lymphoblasts of autism
Cytosolic Membrane-bound
Activity of PKC in the cytosol and membrane fraction of lymphoblasts from autism are decreased as compared to controls
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Autism C ontrol0
10
20
30
40M
emb
ran
e P
rote
in K
inas
e A
(Op
tica
l d
ensi
ty /
mg
pro
tein
)
Increased Protein kinase A activity in the lymphoblasts of autism
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Membrane-associated proteins and their involvement in the etiology of autism
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Pten controls development of neuronal and synaptic function (Fraser et al. 2008).
Pten mutations have been reported in autistic individuals with macrocephaly (Butler et al. 2005; Goffin et al. 2001; Zori et al. 1998).
Decreased levels of Akt are associated with schizophrenia (Emamian et al. 2004), and in individuals with TSC mutations exhibiting central nervous system disorders including autism (Wiznitzer 2004).
Reelin plays a pivotal role in migration of neurons and in the development of neuronal connections.
Dysregulation of reelin has been reported in the brain of individuals with autism (Fatemi et al. 2001, 2005; Serajee et al. 2006)
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Neuroligins are postsynaptic transmembrane proteins that bind to neurexins.
Neuroligins maintain the functionality of synaptic circuitry.
Both neurxins and neuroligins have been identified as candidate genes for autism.
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Serotonin plays role in anger, aggression, mood, sleep, appetite, and metabolism.
Hyperserotonemia is reported in platelets of autistic subjects.
Serotonin transporter (SERT)-binding capacity is disturbed in autism (Makkonen et al. 2008; McDougle 2008).
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CONCLUSIONS Lipid peroxidation is increased in the plasma of autistic children as compared to their developmentally normal non-autistic siblings.
The levels of transferrin (iron-transport protein) and ceruloplasmin (copper-transport protein), the major antioxidant proteins, are decreased in the serum of children with autism as compared to their normal siblings. These effects were seen most strongly in autistic children who had shown a loss of previously acquired language skills.
Lipid peroxidation is increased in the cerebellum and temporal cortex from autism as compared to control subjects.
The levels of free radicals i.e. ROS in the mitochondria are significantly higher in the autism lymphoblasts as compared to control lymphoblasts.
The mitochondrial membrane potential is significantly decreased in autism lymphoblasts as compared to control lymphoblasts.
Levels of aminoglycerophospholipids are altered in the plasma and Levels of aminoglycerophospholipids are altered in the plasma and erythrocyte membrane of autism.erythrocyte membrane of autism.
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Levels of ceruloplasmin are decreased in the plasma of autism.Levels of ceruloplasmin are decreased in the plasma of autism.
Decreased levels of PE in the membrane could be due to copper- mediated oxidation of PE in autism.
Copper-mediated oxidation of PE is higher in lymphoblasts from autistic subjects as compared to control subjects.
Fluidity of the membrane is decreased in autism.Fluidity of the membrane is decreased in autism.
Activity of phospholipase AActivity of phospholipase A22 is increased in the lymphoblasts of is increased in the lymphoblasts of
autism.autism.
Activity of CaActivity of Ca2+2+-ATPase is increased in the lymphoblasts and brains of -ATPase is increased in the lymphoblasts and brains of autism.autism.
Activity of NaActivity of Na++/K/K++-ATPase is increased in the brains of autism.-ATPase is increased in the brains of autism.
Activities of membrane- bound and cytosolic PKC are decreased in the Activities of membrane- bound and cytosolic PKC are decreased in the lymphoblasts of autism.lymphoblasts of autism.
Activity of PKA is increased in the lymphoblasts of autism.Activity of PKA is increased in the lymphoblasts of autism.
These results suggest that mitochondrial dysfunction, oxidative stress, membrane abnormalities and aberrant cell signaling may contribute to pathophysiology of autism.
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Acknowledgements
SPONSORS: New York State Office of Mental Retardation and Developmental Disabilities, NYS Legislative Funds for Autism Research, Autism Speaks, Autism Research Institute, and Department of Defense
Lina Ji
Abha Chauhan Balu MuthaiyahEssa Mohamed
Ira Cohen Maripez Gonzalez
Ed Jenkins
Jerzy Wegiel
Ted Brown