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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

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ABNORMAL REDOX SYSTEM IN AUTISM

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  1. 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

  2. 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

  3. 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

  4. 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

  5. GENETICSOF 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

  6. ENVIRONMENTAL FACTORS IN AUTISM Thalidomide, Valproic acid Heavy metals (lead, mercury) Bisphenol A Air pollutants Chemicals and toxins Pathogenic bacteria Viral infection

  7. BIOCHEMICAL ABNORMALITIES IN AUTISM  Increased blood levels of serotonin (neurotransmitter) Increased oxidative stress     Abnormalities in membrane structure and function  Aberrant signal transduction

  8. OXIDATIVE STRESS IN NEUROLOGICAL DISEASES Alzheimer’s disease Down’s syndrome Parkinson disease Schizophrenia

  9. 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 .

  10. Potential mechanism of oxidative stress and mitochondrial abnormalities in autism Chauhan and Chauhan, Pathophysiology 13 (2006) 171–181

  11. 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.

  12. Malondialdehyde Malondialdehyde (MDA) is an end product of peroxidation of polyunsaturated fatty acids, and is a marker of lipid peroxidation.

  13. 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

  14. INCREASED LIPID PEROXIDATION IN AUTISM Increased MDA in 87% autism as compared to normal siblings * p < 0.005, paired t-test  p < 0.005, unpaired t-test

  15. 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)

  16. REDUCED SERUM TRANSFERRIN & CERULOPLASMIN LEVELS IN AUTISM 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

  17. 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

  18. 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).

  19. 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

  20. 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.

  21. 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

  22. 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)

  23. Mitochondrial abnormalities in the lymphoblasts from autism Chauhan et al. J. Neurochem. 108, Suppl. 1, 33 (2009)

  24. 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.

  25. Involvement of oxidative stress and environmental factors in mitochondrial dysfunctions Mitochondrial dysfunction may result in inflammation, decreased mitochondrial membrane potential and altered energy metabolism.

  26. Mitochondrial ROS in lymphoblasts from autism and control subjects by dihydrorhodamine 123 fluorescence assay 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.

  27. MITOCHONDRIAL MEMBRANE POTENTIAL (MMP) IN LYMPHOBLASTS FROM AUTISM AND CONTROL SUBJECTS BY RHODAMINE (RH) 123 FLUORESCENCE ASSAY 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.

  28. MITOCHONDRIAL MEMBRANE POTENTIAL IN LYMPHOBLASTS FROM AUTISM AND CONTROL SUBJECTS BY JC-1 FLUORESCENCE ASSAY 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.

  29. Confocal microscopic analysis of JC-1 showing decreased mitochondrial membrane potential in autistic lymphoblasts Control Autism 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).

  30. · Increased endogenous Environmental factors Genetic factors pro - oxidants · Decreased end ogenous anti - oxidants Impaired neuronal development Oxidative stress Decreased prostaglandin production & Clinical symptoms Mitochondrial Increased inflammatory response of autism dysfunction Alte red immune response Impaired energy production Membrane lipid abnormalities Pathogenesis Increased excitotocity of autism Abnormal signal transduction Cell death Decreased synaptic efficiency Neuronal membrane dysfunction Impaired serotonin recep tor 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).

  31. Membrane abnormalities in autism

  32. Phospholipids in the membrane Phosphatidylcholine (PC) Phosphatidylethanolamine (PE) Phosphatidylinositol (PI) Sphingomyelin (SPG) Phosphatidylserine (PS)

  33. COMPOSITION OF PHOSPHOLIPIDS IN ERYTHROCYTES MEMBRANES OF CHILDREN WITH AUTISM AND THEIR NONAUTISTIC SIBLINGS ● 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)

  34. Trinitrobenzene sulfonic acid (TNBS) reacts with amine-containingmolecules such as PE and PS B A C 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)

  35. INCREASED AMINOGLYCEROPHOSPHOLIPIDS LEVELS IN PLASMA OF CHILDREN WITH AUTISM Mean  SE 0.2022  0.021 0.1575  0.017 Siblings Autism

  36. Levels of ceruloplasmin (copper-binding protein) are decreased in autism Chauhan et al. (2004) Life Sci. 75, 2539-2549

  37. COPPER DECREASES THE LEVELS OF PHOSPHATIDYLETHANOLAMINE IN THE LYMPHOBLASTS Thin layer chromatogram PE PC SPG PS + PI Lipids Lipids + Cu2+ ● PE is oxidized in the presence of copper.

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

  39. Effect of copper on the PE of lymphoblasts from autism and control subjects Copper oxidizes more PE in the lymphoblasts from autism than from control

  40. Phosphatidylethanolamine: C1 & C2- Acyl groups (Non-plasmalogenic) C1- O-alkenyl group (plasmalogen), C2-acyl group (Plasmalogenic) C1 C2 Copper oxidizes both plasmalogenic and non-plasmalogenic PE equally.

  41. Copper-mediated oxidation of PE-plasmalogen and non-plasmalogenic PE Copper oxidizes both plasmalogenic and non-plasmalogenic PE

  42. 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.

  43. DECREASED MEMBRANE FLUIDITY IN AUTISM Membrane fluidity is inversely proportional to DPH fluorescence polarization. * p < 0.015, unpaired t-test Chauhan et al. J. Neurochem. 108, Suppl. 1, 33 (2009)

  44. RELATIONSHIP BETWEEN MEMBRANE FLUIDITY AND SEVERITY OF AUTISM Correlation coefficient: r = 0.72, p < 0.02 Membrane fluidity decreases with severity of autism

  45. 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.

  46. 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.

  47. Action of phospholipase A2 on phospholipids Phosphatidylcholine Phospholipase A2 Lysophosphatidylcholine Arachidonic acid

  48. 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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