Download Autism One

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
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
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
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
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
ENVIRONMENTAL FACTORS
IN AUTISM
Thalidomide, Valproic acid
Heavy metals (lead, mercury)
Bisphenol A
Air pollutants
Chemicals and toxins
Pathogenic bacteria
Viral infection
BIOCHEMICAL ABNORMALITIES IN AUTISM

Increased blood levels of serotonin (neurotransmitter)

Increased oxidative stress
 Abnormalities in membrane structure and function

Aberrant signal transduction
OXIDATIVE STRESS IN NEUROLOGICAL DISEASES
Alzheimer’s disease
Down’s syndrome
Parkinson disease
Schizophrenia
 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 .
Potential mechanism of oxidative stress and mitochondrial abnormalities in autism
INCREASED PRO-OXIDANTS
Endogenous
 NO
 Xanthine oxidase
 Homocysteine
DECREASED ANTI-OXIDANTS
Exogenous
(Environmental factors)
 Heavy metals (Hg, Pb)
 Thalidomide, Valproic
acid, Retinoic acid
 Air pollutants
 Chemicals (BPA) and
Toxins
 Pathogenic bacteria
 Viral infection
Antioxidant enzymes
(SOD, GPx, catalase)
Glutathione
 Ceruloplasmin
 Transferrin

Abnormal Cu/Fe
metabolism
 Production of free radicals
Mitochondrial damage
Impaired energy production
Genetic factors
 Lipid peroxidation
 Protein oxidation
 DNA oxidation
Increased excitotoxicity
OXIDATIVE STRESS
IN AUTISM
Chauhan and Chauhan, Pathophysiology 13 (2006) 171–181
LIPID PEROXIDATION
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.
Malondialdehyde
Malondialdehyde (MDA) is an end product of peroxidation of
polyunsaturated fatty acids, and is a marker of lipid
peroxidation.
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
INCREASED LIPID PEROXIDATION IN AUTISM
0.7
Plasma MDA (nmole/ml)
0.6
0.5
0.4
0.3
0.2
0.1
0.0
Autism* Siblings
Same sex
Age (Years)
(Mean  S.E.)
Autism Siblings
Different sex
Plasma MDA (nmol / ml)
(Mean  S.E.)
Autism
4.4  0.3
0.497  0.025*, 
Increased MDA in 87% autism
as compared to normal siblings
Siblings
6.0  0.9
0.396  0.019
* p < 0.005, paired t-test

p < 0.005, unpaired t-test
Antioxidant 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)
REDUCED SERUM TRANSFERRIN & CERULOPLASMIN
LEVELS IN AUTISM
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
These results suggest abnormal iron and copper metabolism in autism
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
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).
Pathophysiology 13 (2006) 171 - 181
Review
Oxidative stress in autism
Abha Chauhan,* and Ved Chauhan
NYS Institute for Basic research in Developmental Disabilities, Staten Island, NY, 10314
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.
EFFECT 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
INCREASED LIPID PEROXIDATION IN THE CEREBELLUM
AND TEMPORAL CORTEX FROM AUTISM SUBJECTS
Cerebellum
Temporal cortex
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)
Mitochondrial abnormalities in
the lymphoblasts from autism
Chauhan et al. J. Neurochem. 108, Suppl. 1, 33 (2009)
Mitochondria
A
B
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.
Involvement of oxidative stress and environmental
factors in mitochondrial dysfunctions
Oxidative stress
Environmental factors
Mitochondrial dysfunctions
Inflammation
Altered membrane
potential
Altered energy
metabolism
Mitochondrial dysfunction may result in inflammation,
decreased mitochondrial membrane potential and altered
energy metabolism.
Fluorescence (arbitrary Unit)
Mitochondrial ROS in lymphoblasts from autism and control
subjects by dihydrorhodamine 123 fluorescence assay
150
125
100
75
50
25
0
Control
Autism
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.
Fluorescence (arbitrary unit)
MITOCHONDRIAL MEMBRANE POTENTIAL (MMP) IN
LYMPHOBLASTS FROM AUTISM AND CONTROL SUBJECTS BY
RHODAMINE (RH) 123 FLUORESCENCE ASSAY
80
70
60
50
40
30
20
10
0
Control
Autism
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.
Red/green fluorescence ratio
MITOCHONDRIAL MEMBRANE POTENTIAL IN
LYMPHOBLASTS FROM AUTISM AND CONTROL
SUBJECTS BY JC-1 FLUORESCENCE ASSAY
10.0
7.5
5.0
2.5
0.0
Control
Autism
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.
Confocal microscopic analysis of JC-1 showing decreased
mitochondrial membrane potential in autistic lymphoblasts
Autism
Control
JC-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).
 Increased endogenous
pro-oxidants
 Decreased end ogenous
anti-oxidants
Environmental factors
Genetic factors
Impaired neuronal development
Oxidative stress
&
Mitochondrial
dysfunction
Decreased prostaglandin production
Increased inflammatory response
Clinical symptoms
of autism
Altered immune response
Membrane lipid
abnormalities
Impaired energy production
Increased excitotocity
Pathogenesis
of autism
Abnormal signal transduction
Cell death
Neuronal membrane
dysfunction
Decreased synaptic efficiency
Impaired serotonin receptor functions
Schematic depiction of potential mechanisms that may mediate neuronal
dysfunction and clinical symptoms in autism. (Re produced, in part, from Chauhan, A. and
Chauhan, V. Pathophysiology 2006: 13, 171 -181).
Membrane abnormalities in autism
Phospholipids in the membrane
Phosphatidylcholine (PC)
Phosphatidylethanolamine (PE)
Phosphatidylinositol (PI)
Phosphatidylserine (PS)
Sphingomyelin (SPG)
COMPOSITION OF PHOSPHOLIPIDS IN ERYTHROCYTES MEMBRANES
OF CHILDREN WITH AUTISM AND THEIR NONAUTISTIC SIBLINGS
Phospholipid
fractions
Phospholipid (g/mg protein)
Autism
(N = 8)
Non-autistic
siblings
(N = 8)
PE
5.12  0.54*
6.00  0.63
PC
6.40  0.37
6.60  0.40
SPG
5.29  0.46
5.26  0.46
PI + PS
5.89  0.71
5.26  0.46
PE and PS ratio of erythrocyte
Autism
Normal siblings
0.85  0.198*
1.42  0.106
● 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)
Trinitrobenzene sulfonic acid (TNBS) reacts with
amine-containing molecules such as PE and PS
A
B
Absorbance at 410 nm
0.8
PS-TNBS
PE-TNBS
0.7
0.6
0.5
0.4
0.3
C
0.2
0.1
0.0
0
25
50
75
100
125
Concentration of lipids (nmol)
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)
Absorbance at 410 nm
INCREASED AMINOGLYCEROPHOSPHOLIPIDS
LEVELS IN PLASMA OF CHILDREN WITH AUTISM
0.33
0.32
0.31
0.30
0.29
0.28
0.27
0.26
0.25
0.24
0.23
0.22
0.21
0.20
0.19
0.18
0.17
0.16
0.15
0.14
0.13
0.12
0.11
0.10
0.09
0.08
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0.00
Autism
Mean  SE
0.2022  0.021
Autism
Siblings
0.1575  0.017
Siblings
Levels of ceruloplasmin (copper-binding protein)
are decreased in autism
Chauhan et al. (2004) Life Sci. 75, 2539-2549
COPPER DECREASES THE LEVELS OF
PHOSPHATIDYLETHANOLAMINE IN THE LYMPHOBLASTS
Thin layer chromatogram
PE
PC
Lipids
Lipids + Cu2+
PE (0.56 g)
PE (0.08 g)
SPG
PS + PI
Lipids
Lipids + Cu2+
● PE is oxidized in the presence of copper.
120
110
110
110
100
100
90
80
70
*
60
50
40
30
20
10
0
Control
Copper
Iron
Zinc
Calcium Cadmium
% effect of copper on PE oxidation
120
% effect of copper on PE oxidation
Percent oxidation of PE by metal cations
Effect of metal cations on the oxidation of PE in the lymphoblasts
100
90
*
80
70
*
60
*
50
*
40
30
20
10
0
0.0
*
90
80
70
60
^
50
^
^
40
30
20
10
0
2.5
5.0
7.5
10.0
12.5
0
CuCl2 [M]
1
2
3
4
5
6
7
Time (h)
Chauhan et al. Am. J. Biochem. Biotech. 4:95-100 (2008).
● Among the metal cations, only copper oxidized PE
● Copper-mediated oxidation of PE was dependent on copper
concentration and incubation time period.
8
9
110
100
a
90
c, e
80
b
70
60
d,f
50
40
30
20
Control
400M Cu2+
200M Cu2+
No copper
400M Cu2+
0
200M Cu2+
10
No copper
% efffect of copper on oxidation of PE in lymphoblasts
Effect of copper on the PE of lymphoblasts from autism and control
subjects
Autism
Copper oxidizes more PE in the lymphoblasts from autism than from
control
Phosphatidylethanolamine:
C1 & C2- Acyl groups (Non-plasmalogenic)
C1- O-alkenyl group (plasmalogen), C2-acyl group
(Plasmalogenic)
C1
C2
Copper oxidizes both plasmalogenic and nonplasmalogenic PE equally.
nt
ro
0
Co
l(
to
ta
pp
lP
er
E)
+
to
ta
lP
Co
E
nt
ro
l(
Co
di
ac
pp
yl
er
PE
+
di
)
ac
yl
Co
PE
nt
ro
l(
Co
al
ke
pp
ny
er
lP
+
al
E)
ke
ny
lP
E
Co
% effect of copper on oxidation of PE
(total, diacyl PE and alkenyl PE)
Copper-mediated oxidation of PE-plasmalogen and
non-plasmalogenic PE
110
100
90
80
70
60
50
30
*
40
**
***
20
10
Copper oxidizes both plasmalogenic and non-plasmalogenic PE
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.
DECREASED MEMBRANE FLUIDITY IN AUTISM
DPH Steady State
Fluorescence Polarization
0.25
Membrane fluidity is inversely
proportional to DPH
fluorescence polarization.
0.24
0.23
0.22
0.21
0.20
0.19
0.18
Autism
Siblings
Age (Years)
(Mean  S.E.)
DPH Fluorescence
Polarization
(Mean  S.E.)
Autism (N = 10)
6.09  0.38
0.2225  0.0045*
Siblings (N =16)
6.56  1.08
0.2077  0.0026
* p < 0.015, unpaired t-test
Chauhan et al. J. Neurochem. 108, Suppl. 1, 33 (2009)
RELATIONSHIP BETWEEN MEMBRANE FLUIDITY AND
SEVERITY OF AUTISM
Correlation coefficient: r = 0.72, p < 0.02
Membrane fluidity decreases with severity of autism
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.
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.
Action of phospholipase A2 on phospholipids
Phosphatidylcholine
Phospholipase A2
Lysophosphatidylcholine
Arachidonic acid
Unsaturated fatty acids and autism
•Physical state of the membrane affects the functions of membraneassociated 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).
Phospholipase A2 and autism
 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).
Increased PLA2 activity in lymphoblasts from autism
Lymphoblasts
PLA2 activity (cpm/4h)
Autism
8634  1704
Control
3061  1437
Activities of Ca2+/Mg2+-ATPase,
Na+/K+-ATPase, protein kinase C
and protein kinase A in autism
Ca2+/Mg2+ ATPase activity
(g phosphrus / mg protein / min)
Increased Ca2+-ATPase activity in the
lymphoblasts of autistic subjects
50
40
30
20
10
0
Autism
Siblings Control
Chauhan et al. J. Neurochem. 108, Suppl. 1, 33 (2009)
Phopshorus released
 g)/mg protein/h
Phosphorus released
protein/h
(
g)/mg
Increased activities of Ca2+-ATPase and Na+/K+
ATPase in the cerebellum of autistic subjects
280
260
240
220
200
180
160
140
120
100
80
Control
Autism
Ca2+-ATPase
260
240
220
200
180
160
140
120
100
80
Control
Autism
Na+/K+-ATPase
Decreased activity of Protein kinase C (PKC)
in the lymphoblasts of autism
Membrane-bound
Membrane Protein kinase C
(Optical density/mg protein)
CytosolicProtein kinase C
(Optical density/mg protein)
Cytosolic
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
Autism
Control
80
70
60
50
40
30
20
10
0
Autism
Control
Activity of PKC in the cytosol and membrane fraction of
lymphoblasts from autism are decreased as compared to controls
Membrane Protein Kinase A
(Optical density / mg protein)
Increased Protein kinase A activity in the
lymphoblasts of autism
40
30
20
10
0
Autism
Control
Membrane-associated proteins
and their involvement in the
etiology of autism
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)
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.
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).
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
erythrocyte membrane of autism.
 Levels of ceruloplasmin are decreased in the plasma of autism.
 Decreased levels of PE in the membrane could be due to coppermediated 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.
 Activity of phospholipase A2 is increased in the lymphoblasts of autism.
 Activity of Ca2+-ATPase is increased in the lymphoblasts and brains of
autism.
 Activity of Na+/K+-ATPase is increased in the brains of autism.
 Activities of membrane- bound and cytosolic PKC are decreased 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.
Acknowledgements
Lina Ji
Abha Chauhan
Balu Muthaiyah
Essa Mohamed
Ira Cohen
Maripez Gonzalez
Ed Jenkins
Jerzy Wegiel
Ted Brown
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