Unique Aspects of Redox Regulation in Human Brain and Their Implications for Autism

1. Unique Aspects of Redox Regulation in Human Brain and Their Implications for Autism Richard Deth, PhD Northeastern University Boston, MA

2. Overview • Oxidation and Evolution • Regulation of Redox Status • Brain-specific Redox Features • Methionine synthase in human cortex • - across the lifespan • in autism • Selenoproteins and mercury toxicity

3. Earliest life appears to have arisen at hydrothermal vents emitting hydrogen sulfide and other gases at high temperature and pressure H2S H2O

4. NH2CHCOOH CH2 SH Primordial Synthesis of Cysteine From Volcanic Gases Methane CH3 Hydrogen sulfide H2S Ammonia NH3 Carbon dioxide CO2 Cysteine

5. NH2CHCOOH NH2CHCOOH CH2 CH2 S SH SH CH2 NH2CHCOOH NH2CHCOOH CH2 S Cysteine can function as an antioxidant Two Antioxidant Reducing Equivalents + + 2 H+ Two Cysteines Cysteine Disulfide

6. Evolution = Adaptation to threat of oxidation O2 O2 Genetic Mutation O2 O2 Novel Antioxidant Adaptation Adaptive features of sulfur metabolism =

7. Evolution = Metabolic Adaptations to an Oxygen Environment Figure from Paul G. Falkowski Science311 1724 (2006)

8. EVOLUTION = LAYER UPON LAYER OF USEFUL ADAPTIVE RESPONSES TO ENVIRONMENTAL THREATS

9. The ability to control oxidation is at the core of evolution Each addition is strengthened because it builds on the solid core already in place.

10. New capabilities are added in the context of the particular environment in which they are useful and offer a selective advantage. Recently added capabilities are the most vulnerable to loss when and if there is a significant changes in the environment. Humans cognitive abilities are particularly vulnerable. SOCIAL SKILLS LANGUAGE

11. Neuronal Synchronization Oxygen Radicals Oxidative Metabolism Genetic Risk Factors Oxygen Radicals Redox Buffer Capacity Heavy Metals + Xenobiotics Redox Buffer Capacity [Glutathione] OXIDATIVE STRESS Methylation NORMAL REDOX BALANCE Neuronal Degeneration

12. Cysteine for glutathione synthesis can be provided by either transsulfuration of homocysteine or by uptake from outside the cell Cysteine GSH GSSG Glutathione Synthesis γ-Glutamylcysteine Cysteine Cystathionine Adenosine SAH HCY ( - ) MethylTHF • >150 • Methylation • Reactons Methionine Synthase THF SAM MET ATP PP+Pi Dietary protein

13. Cognitive Status Nitric Oxide Synthesis Catecholamine Methylation Arginine Methylation Gene Expression REDOX STATUS: GSH GSSH Methylation Status: SAM SAH ~ 200 Methylation Reactions DNA/Histone Methylation Serotonin Methylation Phospholipid Methylation Creatine Synthesis Melatonin Membrane Properties Energy Status Sleep

14. During oxidative stress methionine synthase is turned off, allowing more homocysteine to flow toward GSH synthesis, while methylation activity is decreased Cysteine GSH GSSG Glutathione Synthesis γ-Glutamylcysteine Cysteine Cystathionine Adenosine OXIDATIVESTRESS SAH HCY ( - ) MethylTHF • >150 • Methylation • Reactons Methionine Synthase THF SAM MET ATP PP+Pi Dietary protein

15. Inflammation is a metabolic state of oxidative stress, normally occurring in response to environmental challenges Infection, allergy, trauma, chronic illness Inflammatory State - Survival mode - Loss of normal function - Impaired methylation Recovery: Adaptive responses to oxidative stress GSH GSSG GSH GSSG = 30 = 10 Normal Redox Setpoint Oxidative Stress

16. Aging is associated with increased oxidative stress, as adaptive responses fail to restore normal redox status ↑ Inflammatory Diseases: - Alzheimer’s disease - Parkinson’s disease - Diabetes - Heart Failure Aging GSH GSSG GSH GSSG = 30 = 10 Normal Redox Setpoint Oxidative Stress

17. Exposure to persistent environmental toxins promotes oxidative stress and impairs the ability to recover Heavy Metal and Xenobiotic Exposure Inflammatory State - Survival mode - Loss of normal function - Impaired methylation - Autism?? GSH GSSG GSH GSSG = 30 = 10 Normal Redox Setpoint Oxidative Stress

18. 28%↓ 36%↓ 38%↓ Autism is associated with oxidative stress and impaired methylation

19. Blood-Brain Barrier BRAIN BLOOD Neurons Astrocytes [GSH] = 0.21mM [GSH] = 0.91mM [GSH] = 8μM [CYS] [CYS] =200μM [CYS] [CysGly] [GSH] CSF [GSH] = 1 μM [CYS] = 2μM The Brain Compartment (CSF) has low Thiol levels and maintains an Oxidative Stress environment; Astrocytes provide Cysteine to Neurons for survival

20. Neurons obtain cysteine from GSH released by Glial cells, via a growth factor-controlled transporter (EAAT3) Healthy Glial Cells (Astrocytes) Growth Factors Cysteinylglycine Cysteine GSH EAAT3 ( + ) GSH GSSG PI3-kinase γ-Glutamylcysteine Cysteine Cystathionine Adenosine SAH HCY ( - ) MethylTHF • >150 • Methylation • Reactons Methionine Synthase THF SAM MET ATP PP+Pi

21. Transsulfuration of homocysteine (HCY) to cysteine is restricted in human neuronal cells, increasing importance of cysteine uptake Healthy Glial Cells (Astrocytes) Growth Factors Cysteinylglycine Cysteine GSH EAAT3 ( + ) GSH GSSG PI3-kinase γ-Glutamylcysteine Cysteine PARTIALLY BLOCKED IN NEURONAL CELLS Cystathionine Adenosine SAH HCY ( - ) MethylTHF • >150 • Methylation • Reactons Methionine Synthase THF SAM MET ATP PP+Pi

22. Methionine synthase in human neuronal cells requires methylB12 (MeCbl), whose synthesis is glutathione-dependent Healthy Glial Cells (Astrocytes) Growth Factors Cysteinylglycine Cysteine GSH EAAT3 ( + ) GSCbl GSH GSSG OHCbl PI3-kinase SAM γ-Glutamylcysteine MeCbl Cysteine H2S PARTIALLY BLOCKED IN NEURONAL CELLS Cystathionine Adenosine SAH HCY ( - ) MethylTHF • >150 • Methylation • Reactons Methionine Synthase THF SAM MET ATP PP+Pi

23. Levels of cystathionine are markedly higher in human cortex than in other species Tallan HH, Moore S, Stein WH. L-cystathionine in human brain. J Biol Chem. 1958 Feb;230(2):707-16.

24. Healthy Glial Cells (Astrocytes) Growth Factors Cysteinylglycine Cysteine GSH EAAT3 ( + ) GSCbl GSH GSSG OHCbl PI3-kinase SAM γ-Glutamylcysteine MeCbl Cysteine PARTIALLY BLOCKED IN NEURONAL CELLS Cystathionine Adenosine Adenosine D4SAH D4HCY SAH HCY ( - ) MethylTHF MethylTHF Phospholipid Methylation • >150 • Methylation • Reactons Methionine Synthase THF THF D4SAM D4MET SAM MET ATP PP+Pi ATP PP+Pi Dopamine In neurons, D4 dopamine receptors carry out phospholipid methylation, which requires methionine synthase to supply methyl groups

25. DOPAMINE –STIMULATED PHOSPHOLIPID METHYLATION DOPAMINE CH3 Methylfolate Methionine Synthase

26. 2 or 4-repeats 7-repeats

27. Methionine Synthase Structure and function Brain levels Across the lifespan In autism

28. HCY Domain SAM Domain SAM Domain 3 2 1 Cobalamin Cobalamin (vitamin B12) (vitamin B12) Cobalamin Cobalamin Cap Cap 5-Methyl THF Domain Domain Domain Domain Domain Methionine synthase has five domains + cobalamin (Vitamin B12) Domains alternate interacting with cobalamin during turnover

30. Decrease of Cob domain mRNA with increasing age, 40 subjects

31. Decrease of Cap domain with increasing age, 40 subjects

32. CAP Domain is present in MS mRNA from 24 y.o. subject FOL SAM HCY CAP COB

33. CAP Domain is absent from methioninesynthase mRNA in elderly human cortex 80 year old subject FOL SAM HCY CAP COB

34. Age-dependent decrease in the ratio of Cap to Cobalamin mRNA 80 year old subject FOL SAM HCY CAP COB

35. Alternative Splicing of MS Pre-mRNA leads to age-dependent exon skipping Cap Domain Present Cap Domain Exons 19-21 FOL SAM COB HCY Site of alternative splicing by mRNA-specific adenosine deaminase Cap Domain Absent Pre-mRNA mRNA

36. Exons 16-18 are deleted in fetal human brain

37. MS exists as two lower MW bands in SH-SY5Y cells Normal full size MW = 140 kDa 125 kDa Exons 16-18 are absent 110 kDa Exons 16-20 are absent

38. Methionine synthase activity can be regulated via multiple levels of control in response to oxidative stress

39. mRNA for methionine synthase is 2-3 fold lower in cortex of autistic subjects as compared to age-matched controls

40. Age-dependent trend of methionine synthase CAP domain mRNA is absent in autism

41. Paired comparisons of CAP domain mRNA to age-matched controls (Same samples as Vargas et al. 2005)

42. Paired comparisons of Cob domain mRNA to age-matched controls (Same samples as Vargas et al. 2005)

43. Age-dependent changes in Cap and Cobalamin mRNA in Control vs. Autism 80 year old subject FOL SAM HCY CAP COB

44. Selenoproteins, mercury and redox status

45. Glucose is the major source of reducing power for maintaining reduced glutathione NADPH Thioredoxin Reductase NADP+ 6-P-gluconolactone Glucose-6-P Glucose G6PD Thioredoxin GSH status

46. Thioredoxin reductase is a selenoprotein NADPH Thioredoxin Reductase NADP+ 6-P-gluconolactone Glucose-6-P Glucose G6PD Thioredoxin CpG CpG CpG G6PD gene (on) GSH status DNA Demethylase Methionine Synthase Activity G6PD gene (off) CpG CpG CpG SAM SAH DNA Methyltransferase CH3 CH3 CH3

47. Hg

48. SULFUR AND SELENIUM AMINO ACIDS H H H N C COO H N C COO 3 3 CH CH 2 2 Se SH CYSTEINE SELENOCYSTEINE Binding Constant = 1045 Binding Constant = 1039 Hg2+ (million-fold higher affinity) From Dr. Nicholas Ralston Univ. of North Dakota

49. Mercury gradually migrates to highest affinity targets (i.e. selenoproteins) Selenoproteins Thioredoxin fold proteins (dual stable thiols) Protein thiols (mono thiol sites) Thiol metabolites (GSH, cysteine) Hg2+