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

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Presentation on theme: "“How Genetic and Environmental Factors Conspire to Cause Autism” Richard Deth, PhD Northeastern University Boston, MA."— Presentation transcript:

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 H2OH2O H2SH2S

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

5 Methane CH 3 Hydrogen sulfideH 2 S AmmoniaNH 3 Carbon dioxideCO 2 NH 2 CHCOOH CH 2 SH Cysteine Primordial Synthesis of Cysteine From Volcanic Gases

6 NH 2 CHCOOH CH 2 SH NH 2 CHCOOH CH 2 SH + NH 2 CHCOOH CH 2 S NH 2 CHCOOH CH 2 S + 2 H + Cysteine Disulfide Two Antioxidant Reducing Equivalents Cysteine can function as an antioxidant Two Cysteines

7 O2O2 O2O2 O2O2 O2O2 Genetic Mutation Novel Antioxidant Adaptation Evolution = Adaptation to threat of oxidation Adaptive features of sulfur metabolism =

8 Evolution = Metabolic Adaptations to an Oxygen Environment Figure from Paul G. Falkowski Science (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. LANGUAGE SOCIAL SKILLS

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

13 Methionine Synthase HCY MET SAH SAM DNA Methylation ATPPP+P i Adenosine MethylTHF THF Cystathionine Cysteine Glutathione γ-Glutamylcysteine Transsulfuration Pathway Methionine Cycle Redox Buffering D4 HCY D4 SAM D4 SAH D4 MET ATP PP+P i MethylTHF THF Phospholipid Methylation Adenosine Dopamine (Attention) NORMAL REDOX STATUS

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

15 Methionine Synthase HCY MET SAH SAM DNA Methylation ATPPP+P i Adenosine MethylTHF THF Cystathionine Cysteine Glutathione γ-Glutamylcysteine Transsulfuration Pathway Methionine Cycle Oxidative Stress Inhibits Methionine Synthase D4 HCY D4 SAM D4 SAH D4 MET ATP PP+P i MethylTHF THF Phospholipid Methylation Adenosine Dopamine (Impaired Attention) ( - ) OXIDATIVE STRESS  gene expression

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

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

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

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

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

21 Hydroxycobalamin Cyanocobalamin Glutathionylcobalamin Methylcobalamin Methionine Synthase SAM GSH 5-MethylTHF Homocysteine Methionine Synthesis of bioactive methylcobalamin (methylB12) requires glutathione and SAM D4R HCY D4R MET

22

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

24 James et al. (In Press)

25

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

27 DNA RNA Pre-mRNA Protein

28 Cap Domain Exons Site of alternative splicing by mRNA-specific adenosine deaminase Cap Domain Absent Cap Domain Present HCY FOL COB SAM Pre-mRNA mRNA Alternative Splicing of MS Pre-mRNA

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

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

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

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

33 Cap Domain is Absent from Methionine Synthase mRNA in 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 Tallan HH, Moore S, Stein WH. L-cystathionine in human brain. J Biol Chem Feb;230(2): Levels of cystathionine are markedly higher in human cortex than in other species

39 Methionine Synthase HCY MET SAH SAM >150 Methylation Reactons ATPPP+P i Adenosine MethylTHF THF Cystathionine Cysteine GSH γ-Glutamylcysteine GSCbl D4 HCY D4 SAM D4 SAH D4 MET ATP PP+P i MethylTHF THF Phospholipid Methylation Adenosine Dopamine Cysteine ( - ) PI3-kinase ( + ) ↓ IN NEURONAL CELLS MeCbl EAAT3 Glial CellsCysteinylglycine GSH SAM GSSG H2SH2S

40 EAAT3 VIEWED FROM OUTSIDE THE CELL

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

42 Membrane Fatty Acid

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

44 [ 35 S]-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

48

49 Outside view of membrane with D4 receptor

50 Close-up view of membrane with D4 receptor

51 Molecular Model of the Dopamine D4 Receptor Dopamine Methionine 313

52 Structural features of the dopamine D4 receptor Seven repeats are associated with increased risk of ADHD

53

54 Dopamine-stimulated phospholipid methylation is reduced for the 7-repeat form of the D4 Receptor 7 Repeat

55 7-repeats 2 or 4-repeats

56 PHOTONS OF LIGHT e.g. Color Size Texture Brain regions consist of networks of neurons that process and combine information MEMORY e.g. Utility

57 Neuron in networks can fire together in synchrony at different rates Levy et al. J. Neuroscience 20: (2000)

58 Combined theta and gamma oscillations in neuronal firing THETA (5-10 Hz) GAMMA (30-80 Hz)

59 Dopamine causes an increase in gamma frequency as recorded in a patient with Parkinsonism Blue = with dopamine (l-DOPA) Engel et al. Nature Rev. 2005

60 Gamma frequency oscillations promote effective interaction between brain regions with dopamine

61

62 Early electrophysiological markers of visual awareness in the human brain

63 KLHL12 ROC1 Ubiquitin Ligase Cul3 D4 Dopamine Receptor Ubiquitin D4 Receptor Down-Regulation Sensitive to Redox Status Mercury binding?

64 Genetic and Environmental Factors Can Combine to Cause Autism FMR-1, RELN MeCP2, ADA RFC, TCN2 COMT, ATP10C, ADA PON1, GSTM1 MET, NLGN3/4 MTHFR, ASL Genetic Risk Factors Environmental Exposures Impaired Sulfur Metabolism Oxidative Stress D4 Receptor Phospholipid Methylation Neuronal Synchronization ↓ Attention and cognition Methionine Synthase Activity DNA Methylation  Gene Expression Developmental Delay AUTISM Genetic Risk Factors Environmental Exposures Impaired Sulfur Metabolism Oxidative Stress D4 Receptor Phospholipid Methylation Neuronal Synchronization Attention and cognition ↓ Methionine Synthase Activity DNA Methylation  Gene Expression Developmental Delay AUTISM FMR-1, RELN MeCP2, ADA RFC, TCN2 COMT, ATP10C, ADA PON1, GSTM1 MET, NLGN3/4 MTHFR, ASL ↓ ↓ ↓

65 SNPs in Single Methylation Genes Increase the Risk of Obesity

66 Odds of obesity are 16-fold greater if all three SNPs are present Combinations of SNPs in Methylation Genes Can Increase Risk of Obesity Up To 16-fold

67 Thanks for your Research Support!! Autism Research Institute SafeMinds Cure Autism Now


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