How Genetic and Environmental Factors Conspire to Cause Autism”
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“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
“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
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