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“How Genetic and Environmental Factors Conspire to Cause Autism”
Richard Deth, PhD Northeastern University Boston, MA
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Overview Sulfur metabolism and evolution
Oxidative stress as an adaptive response Methionine synthase in autism D4 dopamine receptor-mediated PLM Neuronal synchrony and attention
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Earliest life appears to have arisen at hydrothermal vents emitting
hydrogen sulfide and other gases at high temperature and pressure H2S H2O
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Evolution 3 Billion Years Primates 85 million yrs Humans
Origin of Life 3 Billion Years Methane Hydrogen sulfide Ammonia Carbon dioxide No Oxygen!! Anaerobic Life Aerobic Life Oxygen (electrophile)
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Primordial Synthesis of Cysteine
From Volcanic Gases Methane CH3 Hydrogen sulfide H2S Ammonia NH3 Carbon dioxide CO2 NH2CHCOOH CH2 SH Cysteine
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Cysteine can function as an antioxidant
Two Antioxidant Reducing Equivalents NH2CHCOOH CH2 S NH2CHCOOH CH2 SH NH2CHCOOH CH2 SH + + 2 H+ Two Cysteines Cysteine Disulfide
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= Evolution = Adaptation to threat of oxidation O2 O2 Genetic Mutation
Novel Antioxidant Adaptation = Adaptive features of sulfur metabolism
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Metabolic Adaptations to an Oxygen Environment
Evolution = Metabolic Adaptations to an Oxygen Environment Figure from Paul G. Falkowski Science (2006)
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EVOLUTION = LAYER UPON LAYER OF USEFUL ADAPTIVE RESPONSES TO
ENVIRONMENTAL THREATS
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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.
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LANGUAGE SOCIAL SKILLS
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
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Oxidative Metabolism Heavy Metals + Xenobiotics OXIDATIVE STRESS
Oxygen Radicals Oxidative Metabolism Genetic Risk Factors Oxygen Radicals Redox Buffer Capacity Heavy Metals + Xenobiotics Redox Buffer Capacity [Glutathione] OXIDATIVE STRESS NORMAL REDOX BALANCE Methylation Neuronal Degeneration Neuronal Synchronization
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NORMAL REDOX STATUS D4SAH D4HCY D4SAM D4MET Methionine Synthase Redox
Buffering Glutathione Transsulfuration Pathway γ-Glutamylcysteine Cysteine Methionine Cycle Cystathionine Adenosine Adenosine D4SAH D4HCY HCY SAH MethylTHF Methionine Synthase MethylTHF Phospholipid Methylation DNA Methylation THF THF D4SAM D4MET MET SAM PP+Pi ATP ATP PP+Pi Dopamine (Attention)
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Autism is associated with oxidative stress and impaired methylation
38%↓ 28%↓ 36%↓ 14
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D4HCY D4SAM D4SAH D4MET OXIDATIVE STRESS Methionine Synthase gene
DNA Methylation ATP PP+Pi Adenosine MethylTHF THF Cystathionine Cysteine Glutathione γ-Glutamylcysteine Transsulfuration Pathway Cycle Oxidative Stress Inhibits Methionine Synthase D4HCY D4SAM D4SAH D4MET Phospholipid Dopamine (Impaired Attention) ( - ) OXIDATIVE STRESS gene expression
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Toxic exposures, inflammation,
infections, aging Ideal Cellular Redox Setpoint Loss of normal cellular function, reduced methylation Oxidative Stress Recovery GSH GSSG GSH GSSG = 30 = 10
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Toxic exposures, inflammation,
infections, aging Ideal Cellular Redox Setpoint Loss of normal cellular function. reduced methylation Oxidative Stress GSH Utilization > Supply GSH Utilization < Supply Recovery Autism? GSH GSSG GSH GSSG = 30 = 10 Less Oxidizing Environment More Oxidizing Environment 17
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REDOX STATUS: GSH GSSH 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
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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
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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
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Synthesis of bioactive methylcobalamin (methylB12)
requires glutathione and SAM Hydroxycobalamin Cyanocobalamin GSH GSH Glutathionylcobalamin SAM 5-MethylTHF Methylcobalamin Methionine Synthase Homocysteine Methionine D4RMET D4RHCY
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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
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James et al. (In Press)
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6-Phospho-gluconolactone
DETERMINANTS OF THE GSH/GSSH RATIO Cellular uptake Transsulfuration 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) NADP+ GSSG 6-Phospho-gluconolactone
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DNA Pre-mRNA RNA Protein
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Site of alternative splicing by mRNA-specific adenosine deaminase
Alternative Splicing of MS Pre-mRNA Cap Domain Present Cap Domain Exons 19-21 HCY FOL COB SAM Site of alternative splicing by mRNA-specific adenosine deaminase Cap Domain Absent Pre-mRNA mRNA
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SAM domain is present in MS mRNA from human cortex, but CAP Domain is absent
80 year old subject HCY FOL CAP COB SAM
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SAM domain is present in MS mRNA from human cortex, but CAP Domain is absent
HCY FOL CAP COB SAM Control Subject: Age 80 yrs 30
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CAP Domain is present in MS mRNA from 24 y.o. subject
HCY FOL CAP COB SAM Partial splicing product
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CAP Domain is present in MS mRNA from 24 y.o. subject
Control Subject: Age 24 yrs HCY FOL CAP COB SAM 32
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Human Cortex Late Alzheimer’s
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
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mRNA for methionine synthase is
2-3 fold lower in cortex of autistic subjects as compared to age-matched controls
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Representative comparison of methionine synthase cap domain
mRNA for autistic and control subjects 35
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No age-dependent trend was observed
for either Cobalamin or Cap domains in individuals 30 years or younger
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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.
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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 Feb;230(2):
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D4SAH D4HCY D4SAM D4MET Cysteine Cysteinylglycine GSH Glial Cells
EAAT3 ( + ) GSH GSCbl GSSG PI3-kinase SAM γ-Glutamylcysteine MeCbl Cysteine H2S ↓ IN NEURONAL CELLS Cystathionine Adenosine Adenosine D4SAH D4HCY HCY SAH ( - ) MethylTHF Phospholipid Methylation MethylTHF >150 Methylation Reactons Methionine Synthase THF THF D4SAM D4MET MET SAM PP+Pi ATP ATP PP+Pi Dopamine 39
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EAAT3 VIEWED FROM OUTSIDE THE CELL
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Membrane Fatty Acid Open Covering Loop Aspartic Acid Ready for Transport Closed
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Membrane Fatty Acid
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[35S]-Cysteine uptake in Human Neuronal Cells
Dependent upon PI3-kinase and MAT activity
![[35S]-Cysteine uptake in Human Neuronal Cells](http://slideplayer.com/slide/3310040/11/images/43/%5B35S%5D-Cysteine+uptake+in+Human+Neuronal+Cells.jpg "Dependent upon PI3-kinase and MAT activity.")
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[35S]-Cysteine uptake in Human Neuronal Cells
![[35S]-Cysteine uptake in Human Neuronal Cells](http://slideplayer.com/slide/3310040/11/images/44/%5B35S%5D-Cysteine+uptake+in+Human+Neuronal+Cells.jpg "[35S]-Cysteine uptake in Human Neuronal Cells")
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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.
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D4 Dopamine Receptor-mediated Phospholipid Methylation
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Side view of membrane with D4 receptor
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Outside view of membrane with D4 receptor
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Close-up view of membrane with D4 receptor
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Molecular Model of the Dopamine D4 Receptor
Methionine 313
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Structural features of the dopamine D4 receptor
Seven repeats are associated with increased risk of ADHD
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7 Repeat Dopamine-stimulated phospholipid methylation is reduced
for the 7-repeat form of the D4 Receptor 7 Repeat
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2 or 4-repeats 7-repeats
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VISION PHOTONS OF LIGHT MEMORY
Brain regions consist of networks of neurons that process and combine information PHOTONS OF LIGHT e.g. Color Size Texture VISION MEMORY e.g. Utility
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Neuron in networks can fire together in synchrony at different rates
Levy et al. J. Neuroscience 20: (2000)
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Combined theta and gamma oscillations in neuronal firing
(5-10 Hz) GAMMA (30-80 Hz) 58
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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 59
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Gamma frequency oscillations promote effective
interaction between brain regions with dopamine 60
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Early electrophysiological markers of visual awareness in the human brain
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D4 Dopamine Receptor D4 Receptor Down-Regulation Sensitive to Redox Status Ubiquitin KLHL12 ROC1 Ubiquitin Ligase Cul3 Mercury binding?
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Genetic and Environmental Factors Can Combine to Cause Autism
Genetic Risk Factors Genetic Risk Factors Environmental Exposures Environmental Exposures PON1, GSTM1 PON1, GSTM1 Impaired Sulfur Metabolism Impaired Sulfur Metabolism Oxidative Stress Oxidative Stress MTHFR, ASL MTHFR, ASL RFC, TCN2 RFC, TCN2 ↓ Methionine Synthase Activity Methionine Synthase Activity COMT, ATP10C, ADA COMT, ATP10C, ADA MeCP2, ADA MeCP2, ADA ↓ D4 Receptor Phospholipid Methylation D4 Receptor Phospholipid Methylation ↓ DNA Methylation DNA Methylation MET, NLGN3/4 MET, NLGN3/4 FMR FMR - - 1, RELN 1, RELN ↓ Neuronal Synchronization Neuronal Synchronization D D Gene Expression Gene Expression ↓ Attention and cognition Attention and cognition Developmental Delay Developmental Delay AUTISM AUTISM
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SNPs in Single Methylation Genes Increase the Risk of Obesity
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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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Thanks for your Research Support!!
Autism Research Institute SafeMinds Cure Autism Now
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