“How Genetic and Environmental Factors Conspire to Cause Autism”Richard Deth, PhD Northeastern University Boston, MA Overview - Sulfur metabolism and evolution - Oxidative stress as an adaptive response -Methionine synthase in autism - D4 dopamine receptor-mediated PLM - Neuronal synchrony and attention rli est lif e ap pea rs t o h ave aris en at hydr othe rmal v en ts e mit ting dr oge n sulfi de and o ther ga ses at h igh t emp erat ure and pr essur H2S H2O Evolution Origin of Life Primates 85 million yrs Humans 2.5 million yrs 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 CH3 H2S NH3 CO2 NH2CHCOOH CH2 SH Cysteine Cysteine can function as an antioxidant Two Antioxidant Reducing Equivalents NH2CHCOOH CH2 SH NH2CHCOOH + CH2 SH NH2CHCOOH CH2 S + 2 H+ S CH2 NH2CHCOOH Cysteine Disulfide Two Cysteines Evolution = Adaptation to threat of oxidation O2 O2 Genetic Mutation O2 O2 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. N LA GU AG E SOCI AL S KILL S Oxidative Metabolism Oxygen Radicals Oxygen Radicals Genetic Risk Factors Redox Buffer Capacity Redox Buffer Capacity [Glutathione] OXIDATIVE STRESS Heavy Metals + Xenobiotics NORMAL REDOX BALANCE Methylation Neuronal Synchronization Neuronal Degeneration NORMAL REDOX STATUS Transsulfuration Pathway Glutathione γ-Glutamylcysteine Cysteine Cystathionine Adenosine Adenosine HCY MethylTHF THF DNA Methylation SAM ATP PP+Pi SAH Methionine Cycle Redox Buffering D4SAH Phospholipid Methylation D4HCY MethylTHF Methionine Synthase MET THF D4SAM PP+Pi D4MET ATP Dopamine (Attention) Autism is associated with oxidative stress and impaired methylation 28%↓ 36%↓ 38%↓ OXIDATIVE STRESS Transsulfuration Pathway Glutathione γ-Glutamylcysteine Cysteine Cystathionine Adenosine Adenosine HCY MethylTHF THF SAH (-) DNA Methylation SAM ∆ gene ATP PP+Pi Methionine Cycle Oxidative Stress Inhibits Methionine Synthase D4SAH Phospholipid Methylation MethylTHF D4HCY Methionine Synthase MET THF D4SAM PP+Pi D4MET ATP expression Dopamine (Impaired Attention) 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 GSH GSSG = 10 Less Oxidizing Environment More Oxidizing Environment Cognitive Status Catecholamine Methylation Nitric Oxide Synthesis Arginine Methylation Gene Expression REDOX STATUS: GSH GSSH Methylation Status: SAM SAH ~ 200 Methylation Reactions DNA/Histone Methylation Serotonin Methylation Creatine Synthesis Energy Status Phospholipid Methylation Melatonin Membrane Properties Sleep Methionine synthase has five domains + cobalamin (Vitamin B12) HCY Domain SAM Domain Cobalamin (vitamin B12) 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 B12) Cobalamin Domain Cap Domain HCY Domain Synthesis of bioactive methylcobalamin (methylB12) requires glutathione and SAM Hydroxycobalamin GSH Glutathionylcobalamin SAM 5-MethylTHF Methylcobalamin Methionine Methionine Synthase D4RHCY Homocysteine Cyanocobalamin GSH D4RMET a 120 b 120 100 80 60 40 20 0 0 -11 -10 -9 -8 -7 -6 -5 MS activity pmol/min/mg protein MS activity pmol/min/mg protein Hydroxo-B12 Methyl-B12 100 80 60 40 20 0 0 -11 -10 -9 -8 Hydroxo-B12 Methyl-B12 -7 -6 -5 c 140 Log [Lead ] M Hydroxo-B12 Methyl-B12 d 120 Log [Arsenic] M Hydroxo-B12 Methyl-B12 MS activity pmol/min/mg protein 120 100 80 60 40 20 0 0 -12 -11 -10 -9 MS activity pmol/min/mg protein 100 80 60 40 20 0 0 -12 -11 -10 -9 -8 -7 -6 -5 -8 -7 -6 -5 e 100 Log [Aluminum] M Hydroxo-B12 Methyl-B12 f 1750 Log [Mercury] M Control Le ad Arse nic Aluminum M ercury Thime rosal MS activity pmol/min/mg protein [GSH] nmole/mg protein 80 60 40 20 0 1500 1250 1000 750 500 250 0 0 -12 -11 -10 -9 -8 -7 -6 -5 Log [Thimerosal] M Thimerosal decreases methylcobalamin levels to a much greater extent than GSH levels in SH-SY5Y human neuronal cells 40 GSH nmol/mg protein Basal Thimerosal GSH levels Thimerosal = 1 µM for 60 min 30 20 10 0 * 100 Percent Control Basal Thimerosal Methylcobalamin levels Thimerosal = 0.1 µM for 60 min 80 60 40 20 0 * Efficacy of methylcobalamin and folinic acid treatment on glutathione redox status and core behaviors in children with autism James et al. (In Press) Table 1. Mean plasma metabolite concentrations (± SD) in age-matched control children, children with autism at baseline before intervention, and after 3 months intervention with methylcobalamin and folinic acid Plasma Metabolite Concentration Methionine S-adenosylmethionine (SAM) (nmol/L) S-adenosylhomocsyteine (SAH) (nmol/L) SAM/SAH (µmol/L) Homocysteine (µmol/L) Cysteine (µmol/L) Cysteinylglycine (µmol/L) Total Glutathione (tGSH) (µmol/L) Free Glutathione (fGSH) (µmol/L) GSSG (µmol/L) tGSH/GSSG fGSH/GSSG a Control Children (n = 42) 24 ± 3 78 ± 22 14.3 ± 4.3 5.6 ± 2.0 5.0 ± 1.2 210 ± 18 45 ± 6 7.5 ± 1.8 2.8 ± 0.8 0.18 ± 0.07 47 ± 18 17 ± 6.8 Autism Autism Pre-treatmentb Post-treatment (n = 40) (n = 40) 21 ± 4 22 ± 3c 66 ± 13 69 ± 12c 15.2 ± 5 14.8 ± 4 4.7 ± 1.5 4.8 ± 1.8 191 ± 24 40 ± 9 5.4 ± 1.3 1.5 ± 0.4 0.28 ± 0.08 21 ± 6 6±2 5.0 ± 2.0 5.3 ± 1.1 215 ± 19 46 ± 9 6.2 ± 1.2c 1.8 ± 0.4 c 0.22 ± 0.06 c 30 ± 9 c 9 ± 3c p valuea ns ns ns ns 0.04 0.001 0.002 0.001 0.008 0.001 0.001 0.001 Pre- and Post-treatment comparison All pre-treatment values were significantly different from control with the exception of Hcy and SAH (p<0.005). c Post-treatment values significantly different from control (p< 0.01) ns = not significant (> 0.05) b Table 2. Scores from the Vineland Adaptive Behavior Scales at baseline before and after 3 months intervention with methylB12 and folinic acid Vineland Category Communication Daily Living Skills Socialization Motor Skills Composite Score Baseline Score (mean ± SD) 65.3 ± 12.9 67.0 ± 76 68.2 ± 9.3 75.6 ± 9.7 66.5 ± 9.2 Post-Treatment Score (mean ± SD) 72.0 ± 15.5 76.0 ± 17.7 75.7 ± 14.7 79.0 ± 14.7 73.9 ± 17.0 Change in Score (mean; 95% C I) 6.7 (3.5, 10) 9.0 (4.0, 14) 7.5 (3.5, 11) 3.3 (0, 8) 6.6 (2.3, 11) p value <0.001 <0.007 <0.005 0.12 <0.003 Table 3. Magnitude of Vineland score increase after intervention with methylcobalamin and folinic acid for three months by quartile. Children whose baseline pre-treatment score was within the lowest quartile are compared to children whose pre-treatment score was in the upper quartile. Score Increase Score Increase Vineland Category Lowest Quartile Upper Quartile Communication 4 13 Daily Living 4 12 Socialization 3 10 Motor Skills 1 1 Composite Score 3 9 DETERMINANTS OF THE GSH/GSSH RATIO Cellular uptake Transsulfuration Cysteine Glutamate Glucose Hexokinase Glucose-6-Phosphate G6PD NADP Glutaredoxin (oxidized) NADPH Thimerosal Glutaredoxin (reduced) γ-Glutamylcysteine Glycine GSH GSSG Reductase ROS Inactivation Detoxification (e.g. GPx) 6-Phospho-gluconolactone + GSSG DNA Pre-mRNA RNA Protein Alternative Splicing of MS Pre-mRNA Cap Domain Present Cap Domain Exons 19-21 HCY FOL COB SAM Cap Domain Absent Site of alternative splicing by mRNA-specific adenosine deaminase Pre-mRNA mRNA SAM domain is present in MS mRNA from human cortex, but CAP Domain is absent 80 year old subject HCY FOL CAP COB SAM SAM domain is present in MS mRNA from human cortex, but CAP Domain is absent Control Subject: Age 80 yrs HCY FOL CAP COB SAM CAP Domain is present in MS mRNA from 24 y.o. subject HCY FOL CAP COB SAM Partial splicing product CAP Domain is present in MS mRNA from 24 y.o. subject Control Subject: Age 24 yrs HCY FOL CAP COB SAM 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 mRNA 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 mRNA for autistic and control subjects No age-dependent trend was observed for either Cobalamin or Cap domains in individuals 30 years or younger Cobalamin Domain 40 Cap mRNA levels Amplification Cycles Control Autism 45 40 35 30 25 20 Amplification Cycles Control Autism 35 30 25 20 0 10 20 30 40 0 10 20 30 40 Age Age 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. 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. Cysteine Cysteinylglycine (+) GSH Glial Cells GSCbl SAM MeCb l EAAT3 GSSG GSH γ-Glutamylcysteine ↓ IN NEURONAL Cystathionine CELLS Adenosine PI3-kinase Cysteine H2S Adenosine D4SAH Phospholip id Methylatio n D4HCY HCY SAH MethylTH F THF Methionine Synthase MethylTHF D4SAM PP+Pi D4MET ATP THF MET ATP PP+Pi >150 Methylati on SAM Reactons (-) Dopamine EAAT3 VIEWED FROM OUTSIDE THE CELL Membrane Fatty Acid Open Covering Loop Aspartic Acid Ready for Transport Closed Membrane Fatty Acid [35S]-Cysteine uptake in Human Neuronal Cells Control 20 L-[35S]cysteine Uptake (nmol/mg protein) 15 10 5 0 L-[35S]cysteine Uptake (nmol/ mg protein) 37°C 20 15 10 5 0 10-4M Dihydrokainate 10-4MThreo-β -hydroxyaspartate 0°C 0 1 2 3 4 5 6 0 1 3 5 Time in minutes Time in minutes 10.0 Control Cycloleucine 10-3M Wortmannin 10-7M LY-compound 10-7M L-[35S]Cysteiene Uptake nmol/mg protein 7.5 5.0 2.5 0.0 Dependent upon PI3-kinase and MAT activity L-[35S]-cysteine uptake nmol/ mg protein 10 0 2 4 6 8 C [L ea d] 10 -7 on t ro l M [A rs e *** [A lu m [M in um 10 -7 ni c] M *** *** *** [35S]-Cysteine uptake in Human Neuronal Cells ]1 0 -7 er M cu [T ry hi ]1 m 0 -7 er M os al ]1 0 -7 M ***,^ 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. D4 Dopamine Receptor-mediated Phospholipid Methylation Side view of membrane with D4 receptor Outside view of membrane with D4 receptor Close-up view of membrane with D4 receptor Molecular Model of the Dopamine D4 Receptor Dopamine Methionine 313 Structural features of the dopamine D4 receptor Seven repeats are associated with increased risk of ADHD Dopamine-stimulated phospholipid methylation is reduced for the 7-repeat form of the D4 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 D4 Dopamine Receptor D4 Receptor Down-Regulation Sensitive to Redox Status KLHL12 Cul3 ROC1 Mercury binding? Ubiquitin Ligase Ubiquitin Genetic and Environmental Factors Can Combine to Cause Autism Genetic Risk Factors Environmental Exposures PON1, GSTM1 Impaired Sulfur Metabolism Oxidative Stress MTHFR, ASL RFC, TCN2 ↓Methionine Synthase Activity COMT, ATP10C, ADA MeCP2, ADA ↓ D4 Receptor Phospholipid Methylation MET, NLGN3/4 FMR-1, RELN ↓ DNA Methylation ∆ Gene Expression Developmental Delay ↓ Neuronal Synchronization ↓Attention and cognition Attention 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 Thanks for your Research Support!! Autism Research Institute SafeMinds Cure Autism Now
Report "How Genetic and Environmental Factors Conspire to Cause Autism”"