Abnormal Redox System In Autism

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 DECREASED ANTI-OXIDANTS Endogenous

• NO • Xanthine oxidase • Homocysteine

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.

Malondialdehyd e

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 Autism Siblings

Same sex

Age (Years) (Mean ± S.E.)

Different sex

Plasma MDA (nmol / ml) (Mean ± S.E.)

Autism

4.4 ± 0.3

0.497 ± 0.025*, φ

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 φ

p < 0.005, unpaired t-test

Antioxidant Defense Mechanisms • Enzymatic antioxidants - Superoxide dismutase (SOD) - Glutathione peroxidase (GSH-Px) - Catalase (CAT) • Non-enzymatic antioxidants - Glutathione, α-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, No. 2, 2008

Editor: Abha Chauhan, Ph.D.

Associate Editors: Ved Chauhan, Ph.D. George Perry, Ph.D.

Vol. 4,

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.

MITOCHONDRIAL MEMBRANE POTENTIAL (MMP) IN LYMPHOBLASTS FROM AUTISM AND CONTROL SUBJECTS BY RHODAMINE (RH) 123 FLUORESCENCE ASSAY Fluorescence (arbitrary unit)

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 endogenous anti- oxidants

Environmental factors

Genetic factors

Impaired neuronal development

Oxidative stress & Mitochondrial dysfunction

Decreased prostaglandin production

Increased inflammatory response

Clinical symptoms of autism

Alte red immune response Membrane lipid abnormalities

Impaired energy production Increased excitotocity

Abnormal signal transduction

Pathogenesis of autism

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. (Reproduced, 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)

Phosphatidylserine (PS)

Phosphatidylinositol (PI)

Sphingomyelin (SPG)

COMPOSITION OF PHOSPHOLIPIDS IN ERYTHROCYTES MEMBRANES OF CHILDREN WITH AUTISM AND THEIR NONAUTISTIC SIBLINGS Phospholipid fractions

Phospholipid (µg/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

Mean ± SE

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

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 µg)

PE (0.08 µg)

SPG PS + PI

Lipids

Lipids + Cu2+

● PE is oxidized in the presence of copper.

Effect of metal cations on the oxidation of PE in the lymphoblasts

100 90 80 70

*

60 50 40 30 20 10 Control

Copper

Iron

Zinc

Calcium Cadmium

% effect of copper on PE oxidation

110

0

110

120

% effect of copper on PE oxidation

Percent oxidation of PE by metal cations

120

110 100 90

*

80 70

*

60

*

50

*

40 30 20 10 0 0.0

100

80 70 60

5.0

7.5

10.0

12.5

^

50

^

^

40 30 20 10 0

2.5

*

90

0

CuCl2 [µM]

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

Effect of copper on the PE of lymphoblasts from autism and control subjects 100

a

90

c, e

80

b

70 60

d,f 50 40 30 20

Control

2+

2+

200 µM Cu

400 µM Cu

No copper

2+

400 µM Cu

No copper

0

2+

10

200 µM Cu

% efffect of copper on oxidation of PE in lymphoblasts

110

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.

ol

tr

(to ta pe lP r+ E) to ta lP C E on tr ol C op (dia cy pe lP r+ E) di ac yl C PE on tr ol C (a op lk en pe yl r+ PE al ) ke ny lP E

C op

C on

% 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

0

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). • ↑ ω-9 fatty acids in autism (Bu et al., 2006), ↓ ω-3 fatty acids in Attention Deficit Disorder, Alzheimer’s disease, Schizophrenia and depression (Young and Conquer, 2005). • Dietary ω-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

Increased Ca2+-ATPase activity in the lymphoblasts of autistic subjects

Ca 2+ /Mg 2+ ATPase activity (µ g phosphrus / mg protein / min)

50

40

30

20

10

0

Autism

Siblings Control

Chauhan et al. J. Neurochem. 108, Suppl. 1, 33 (2009)

280

Phopshorus released ( µ g)/mg protein/h

Phosphorus released (µ g)/mg protein/h

Increased activities of Ca2+-ATPase and Na+/K+ ATPase in the cerebellum of autistic subjects

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

4.5

80

4.0

70

Membrane Protein kinase C (Optical density/mg protein)

CytosolicProtein kinase C (Optical density/mg protein)

Cytosolic

3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0

Autism

Control

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