“How Genetic and Environmental Factors Conspire to Cause Autism”

1. “How Genetic and Environmental Factors Conspire to Cause Autism” Richard Deth, PhD Northeastern University Boston, MA

2. Overview • Sulfur metabolism and evolution • Oxidative stress as an adaptive response • Methionine synthase in autism • D4 dopamine receptor-mediated PLM • Neuronal synchrony and attention

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

4. Evolution Primates 85 million yrs Humans 2.5 million yrs Origin of Life 3 Billion Years Methane Hydrogen sulfide Ammonia Carbon dioxide No Oxygen!! Anaerobic Life Aerobic Life Oxygen (electrophile)

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

6. 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

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

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

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

10. 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.

11. 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

12. 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

13. NORMAL REDOX STATUS Redox Buffering Glutathione Transsulfuration Pathway γ-Glutamylcysteine Cysteine Methionine Cycle Cystathionine Adenosine Adenosine D4SAH D4HCY SAH HCY MethylTHF MethylTHF Methionine Synthase Phospholipid Methylation DNA Methylation THF THF D4SAM D4MET SAM MET ATP PP+Pi ATP PP+Pi Dopamine (Attention)

14. 28%↓ 36%↓ 38%↓ Autism is associated with oxidative stress and impaired methylation

15. OXIDATIVE STRESS Oxidative Stress Inhibits Methionine Synthase Glutathione Transsulfuration Pathway γ-Glutamylcysteine Cysteine Methionine Cycle Cystathionine Adenosine Adenosine D4SAH D4HCY SAH HCY ( - ) MethylTHF MethylTHF Methionine Synthase Phospholipid Methylation DNA Methylation THF THF D4SAM D4MET SAM MET ATP PP+Pi ATP PP+Pi Dopamine (Impaired Attention)  gene expression

16. Toxic exposures, inflammation, infections, aging Ideal Cellular RedoxSetpoint Loss of normal cellular function, reduced methylation Oxidative Stress Recovery GSH GSSG GSH GSSG = 30 = 10

17. Toxic exposures, inflammation, infections, aging Ideal Cellular RedoxSetpoint Loss of normal cellular function. reduced methylation Oxidative Stress GSH Utilization > Supply GSH Utilization < Supply Recovery Autism? GSH GSSG GSH GSSG = 30 = 10 More Oxidizing Environment Less Oxidizing Environment

18. 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

19. Methionine synthase has five domains + cobalamin (Vitamin B12) HCY Domain HCY Domain SAM Domain SAM Domain Cobalamin Cobalamin (vitamin B12) (vitamin B12) 5 5 - - methyl THF Domain methyl THF Domain Cobalamin Cobalamin Cap Cap Domain Domain Domain Domain

20. Without SAM domain methionine synthase requires GSH-dependent methylcobalamin for reactivation 5-methyl THF Domain SAM Domain Cobalamin (vitamin B12) HCY Domain Cobalamin Domain Cap Domain

21. Synthesis of bioactive methylcobalamin (methylB12) requires glutathione and SAM Hydroxycobalamin Cyanocobalamin GSH GSH Glutathionylcobalamin SAM 5-MethylTHF Methylcobalamin Methionine Synthase Homocysteine Methionine D4RMET D4RHCY

23. Thimerosal decreases methylcobalamin levels to a much greater extent than GSH levels in SH-SY5Y human neuronal cells GSH levels Thimerosal = 1 M for 60 min Methylcobalamin levels Thimerosal = 0.1 M for 60 min

24. James et al. (In Press)

26. DETERMINANTS OF THE GSH/GSSH RATIO Transsulfuration Cellular uptake Cysteine Glutamate Glucose γ-Glutamylcysteine Thimerosal Hexokinase Glycine Glutaredoxin (reduced) GSH Glucose-6-Phosphate NADPH GSSG Reductase ROS Inactivation Detoxification (e.g. GPx) G6PD Glutaredoxin (oxidized) GSSG NADP+ 6-Phospho-gluconolactone

27. DNA Pre-mRNA RNA Protein

28. Alternative Splicing of MS Pre-mRNA 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

29. SAM domain is present in MS mRNA from human cortex, but CAP Domain is absent 80 year old subject FOL SAM HCY CAP COB

30. SAM domain is present in MS mRNA from human cortex, but CAP Domain is absent Control Subject: Age 80 yrs FOL SAM HCY CAP COB

31. CAP Domain is present in MS mRNA from 24 y.o. subject FOL SAM HCY CAP COB Partial splicing product

32. CAP Domain is present in MS mRNA from 24 y.o. subject Control Subject: Age 24 yrs FOL SAM HCY CAP COB

33. Cap Domain is Absent fromMethionine Synthase mRNAin All Elderly Subjects (> 70 yrs) Human Cortex Controls Human Cortex Early Alzheimer’s Human Cortex Late Alzheimer’s

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

35. Representative comparison of methionine synthase cap domain mRNA for autistic and control subjects

36. No age-dependent trend was observed for either Cobalamin or Cap domains in individuals 30 years or younger

37. Conclusion: There are lower amounts of mRNA for methionine synthase in the cortex of autistic subjects and levels of the enzyme are also likely to be lower. Lower expression levels may reflect an adaptation to oxidative stress. This implies an impaired capacity for methylation, including D4 dopamine receptor-mediated phospholipid methylation.

38. 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.

39. Cysteinylglycine Glial Cells Cysteine GSH EAAT3 ( + ) GSCbl GSH GSSG PI3-kinase SAM γ-Glutamylcysteine MeCbl Cysteine H2S ↓ 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

40. EAAT3 VIEWED FROM OUTSIDE THE CELL

41. Membrane Fatty Acid Open Covering Loop Aspartic Acid Ready for Transport Closed

42. Membrane Fatty Acid

43. [35S]-Cysteine uptake in Human Neuronal Cells Dependent upon PI3-kinase and MAT activity

44. [35S]-Cysteine uptake in Human Neuronal Cells

45. Why put neurons at higher risk of oxidative stress? One possible explanation: Oxidative stress stops cells from dividing. Neurons have to avoid cell division, otherwise they would lose all their connections and all of their information value. Thus neurons must balance on the precarious knife-edge of oxidative stress.

46. D4 Dopamine Receptor-mediated Phospholipid Methylation

47. Side view of membrane with D4 receptor

49. Outside view of membrane with D4 receptor

50. Close-up view of membrane with D4 receptor