How Genetic and Environmental Factors Conspire to Cause

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

Overview - Sulfur metabolism and evolution - Oxidative stress as an adaptive response -Methionine synthase in autism - D 4 dopamine receptor-mediated PLM - Neuronal synchrony and attention

Earliest life appears to have arisen at hydrothermal vents emitting hydrogen sulfide and other gases at high temperature and pressure H 2 S H 2 O

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 Oxygen (electrophile) Aerobic Life

Primordial Synthesis of Cysteine From Volcanic Gases Methane Hydrogen sulfide Ammonia Carbon dioxide CH 3 H 2 S NH 3 CO 2 NH 2 CHCOOH CH 2 SH Cysteine

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

Evolution = Adaptation to threat of oxidation O 2 Genetic Mutation O 2 Novel Antioxidant Adaptation = Adaptive features of sulfur metabolism

Evolution = Metabolic Adaptations to an Oxygen Environment Figure from Paul G. Falkowski Science 311 1724 (2006)

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

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.

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. GE UA NG LA SOCI AL SK ILLS

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

NORMAL REDOX STATUS Transsulfuration Pathway Glutathione Redox Buffering γ-Glutamylcysteine Cysteine Methionine Cycle Cystathionine Adenosine D 4 SAH D 4 HCY Methyl. THF Phospholipid Methylation Adenosine Methionine Synthase THF D 4 SAM Methyl. THF DNA Methylation THF D 4 MET PP+Pi SAH HCY ATP Dopamine (Attention) SAM MET ATP PP+Pi

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

OXIDATIVE STRESS Transsulfuration Pathway Glutathione γ-Glutamylcysteine Oxidative Stress Inhibits Methionine Synthase Cysteine Methionine Cycle Cystathionine Adenosine D 4 SAH D 4 HCY Methyl. THF Phospholipid Methylation Adenosine Methyl. THF Methionine Synthase THF D 4 SAM ATP (-) DNA Methylation THF D 4 MET PP+Pi SAH HCY SAM gene MET ATP Dopamine (Impaired Attention) PP+Pi expression

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

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

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

Methionine synthase has five domains + cobalamin (Vitamin B 12) HCY Domain SAM Domain Cobalamin (vitamin B 12) 5 -methyl THF Domain Cobalamin Domain Cap Domain

Without SAM domain methionine synthase requires GSH-dependent methylcobalamin for reactivation 5 -methyl THF Domain SAM Domain Cobalamin (vitamin B 12) Cobalamin Domain Cap Domain HCY Domain

Synthesis of bioactive methylcobalamin (methyl. B 12) requires glutathione and SAM Hydroxycobalamin Cyanocobalamin GSH Glutathionylcobalamin SAM 5 -Methyl. THF Methylcobalamin Homocysteine Methionine D 4 RMET Methionine Synthase D 4 RHCY

Thimerosal decreases methylcobalamin levels to a much greater extent than GSH levels in SH-SY 5 Y human neuronal cells GSH levels Thimerosal = 1 M for 60 min Methylcobalamin levels Thimerosal = 0. 1 M for 60 min

James et al. (In Press)

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

DNA Pre-m. RNA Protein

Alternative Splicing of MS Pre-m. RNA Cap Domain Present Cap Domain Exons 19 -21 HCY FOL COB SAM Site of alternative splicing by m. RNA-specific adenosine deaminase Pre-m. RNA Cap Domain Absent m. RNA

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

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

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

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

Cap Domain is Absent from Methionine Synthase m. RNA in All Elderly Subjects (> 70 yrs) Human Cortex Controls Human Cortex Early Alzheimer’s Human Cortex Late Alzheimer’s

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

Representative comparison of methionine synthase cap domain m. RNA for autistic and control subjects

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

Conclusion: There are lower amounts of m. RNA 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 D 4 dopamine receptor-mediated phospholipid methylation.

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.

Cysteinylglycine Cysteine Glial Cells GSH EAAT 3 (+) GSSG PI 3 -kinase GSCbl GSH SAM γ-Glutamylcysteine Cysteine ↓ IN NEURONAL CELLS Cystathionine Adenosine D 4 SAH Adenosine D 4 HCY Methyl. THF Phospholipid Methylation Methyl. THF ATP Dopamine SAM MET ATP PP+Pi (-) >150 Methylation Reactons THF D 4 MET PP+Pi SAH HCY Methionine Synthase D 4 SAM Me. Cbl H 2 S

EAAT 3 VIEWED FROM OUTSIDE THE CELL

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

Membrane Fatty Acid

[35 S]-Cysteine uptake in Human Neuronal Cells Dependent upon PI 3 -kinase and MAT activity

[35 S]-Cysteine uptake in Human Neuronal Cells

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.

D 4 Dopamine Receptor-mediated Phospholipid Methylation

Side view of membrane with D 4 receptor

Outside view of membrane with D 4 receptor

Close-up view of membrane with D 4 receptor

Molecular Model of the Dopamine D 4 Receptor Dopamine Methionine 313

Structural features of the dopamine D 4 receptor Seven repeats are associated with increased risk of ADHD

Dopamine-stimulated phospholipid methylation is reduced for the 7 -repeat form of the D 4 Receptor 7 Repeat

2 or 4 -repeats 7 -repeats

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

Neuron in networks can fire together in synchrony at different rates Levy et al. J. Neuroscience 20: 7766 -7775 (2000)

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

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

Gamma frequency oscillations promote effective interaction between brain regions with dopamine

Early electrophysiological markers of visual awareness in the human brain

D 4 Dopamine Receptor D 4 Receptor Down-Regulation Sensitive to Redox Status KLHL 12 Cul 3 ROC 1 Mercury binding? Ubiquitin Ligase Ubiquitin

Genetic and Environmental Factors Can Combine to Cause Autism Genetic Risk Factors Environmental Exposures PON 1, GSTM 1 Impaired Sulfur Metabolism Oxidative Stress MTHFR, ASL RFC, TCN 2 ↓Methionine Synthase Activity COMT, ATP 10 C, ADA Me. CP 2, ADA ↓D 4 Receptor Phospholipid Methylation MET, NLGN 3/4 ↓DNA Methylation FMR-1, RELN Gene Expression ↓Neuronal Synchronization ↓Attention and cognition Developmental Delay AUTISM

SNPs in Single Methylation Genes Increase the Risk of Obesity

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

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