“How Genetic and Environmental Factors Conspire to Cause Autism”
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Transcript of “How Genetic and Environmental Factors Conspire to Cause Autism”
“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
Earliest life appears to have arisen at hydrothermal vents emitting hydrogen sulfide and other gases at high temperature and pressure
H2O
H2S
Originof
Life
Evolution
Anaerobic Life Aerobic Life
3 Billion Years
MethaneHydrogen sulfideAmmoniaCarbon dioxideNo Oxygen!! Oxygen
Humans2.5 million yrs
Primates85 million yrs
(electrophile)
Methane CH3
Hydrogen sulfide H2SAmmonia NH3
Carbon dioxide CO2
NH2CHCOOHCH2
SH
Cysteine
Primordial Synthesis of CysteineFrom Volcanic Gases
NH2CHCOOH
CH2
SH
NH2CHCOOH
CH2
SH
+
NH2CHCOOHCH2
S
NH2CHCOOH
CH2
S+ 2 H+
Cysteine Disulfide
Two AntioxidantReducing Equivalents
Cysteine can function as an antioxidant
Two Cysteines
O2
O2
O2
O2
GeneticMutation
NovelAntioxidantAdaptation
Evolution = Adaptation to threat of oxidation
Adaptive features of sulfur metabolism
=
Evolution = Metabolic Adaptations
to an Oxygen Environment
Figure from Paul G. FalkowskiScience 311 1724 (2006)
EVOLUTION = LAYER UPON LAYER OF USEFUL ADAPTIVE RESPONSES TO ENVIRONMENTAL THREATS
The ability to controloxidation is at thecore of evolution
Each addition isstrengthened because
it builds on thesolid 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 andif there is a significant changes in the environment.
Humans cognitive abilities are particularly vulnerable.
LA
NG
UA
GE
SOCIAL SKILLS
NORMALREDOX
BALANCE
Redox Buffer Capacity
[Glutathione]
Oxygen Radicals
OxygenRadicals
Redox Buffer Capacity
OXIDATIVE STRESS
Methylation
GeneticRisk Factors
Heavy Metals+
Xenobiotics
OxidativeMetabolism
Neuronal Synchronization
Neuronal Degeneration
MethionineSynthase
HCY
MET
SAH
SAM
DNAMethylation
ATP PP+Pi
Adenosine
MethylTHF
THF
Cystathionine
Cysteine
Glutathione
γ-Glutamylcysteine
TranssulfurationPathway
Methionine Cycle
RedoxBuffering
D4HCY
D4SAM
D4SAH
D4MET
ATPPP+Pi
MethylTHF
THF
PhospholipidMethylation
Adenosine
Dopamine (Attention)
NORMAL REDOX STATUS
38%↓
28%↓
36%↓
Autism is associated with oxidative stress and impaired methylation
MethionineSynthase
HCY
MET
SAH
SAM
DNAMethylation
ATP PP+Pi
Adenosine
MethylTHF
THF
Cystathionine
Cysteine
Glutathione
γ-Glutamylcysteine
TranssulfurationPathway
Methionine Cycle
Oxidative Stress Inhibits
Methionine Synthase
D4HCY
D4SAM
D4SAH
D4MET
ATPPP+Pi
MethylTHF
THF
PhospholipidMethylation
Adenosine
Dopamine (Impaired Attention)
( - )
OXIDATIVE STRESS
geneexpression
Oxidative Stress
GSHGSSG = 30 GSH
GSSG = 10
Ideal Cellular Redox Setpoint
Loss of normalcellular function,
reducedmethylation
Toxic exposures, inflammation,infections, aging
Recovery
Oxidative Stress
GSHGSSG = 30 GSH
GSSG = 10
Ideal Cellular Redox Setpoint
Loss of normalcellular function.
reduced methylation
Toxic exposures, inflammation,infections, aging
GSH Utilization > Supply
GSH Utilization < Supply
More OxidizingEnvironment
Less OxidizingEnvironment
Recovery
Autism?
REDOXSTATUS:
GSHGSSH
MethylationStatus:SAMSAH
~ 200 Methylation
Reactions
Nitric OxideSynthesis
PhospholipidMethylation
DNA/HistoneMethylation
GeneExpression
ArginineMethylation
MembraneProperties
CreatineSynthesis
CognitiveStatus
EnergyStatus
CatecholamineMethylation
SerotoninMethylation
Melatonin
Sleep
Methionine synthase has five domains + cobalamin (Vitamin B12)
SAM Domain
CobalaminDomain
CapDomain
5-methyl THF Domain
HCY Domain
Cobalamin(vitamin B12)
SAM Domain
CobalaminDomain
CapDomain
5-methyl THF Domain
HCY Domain
Cobalamin(vitamin B12)
SAM Domain
CobalaminDomain
CapDomain
5-methyl THF Domain
HCY Domain
Cobalamin(vitamin B12)
Without SAM domain methionine synthase requires GSH-dependent methylcobalamin for reactivation
Hydroxycobalamin Cyanocobalamin
Glutathionylcobalamin
Methylcobalamin
MethionineSynthase
SAM
GSHGSH
5-MethylTHF
HomocysteineMethionine
Synthesis of bioactive methylcobalamin (methylB12)requires glutathione and SAM
D4RHCYD4RMET
0
20
40
60
80
100
120
Methyl-B12
0 -11 -10 -9 -8 -7 -6 -5
Log [Lead ] M
Hydroxo-B12
MS
act
ivit
yp
mo
l/m
in/m
g p
rote
in0
20
40
60
80
100
120
Methyl-B12
0 -11 -10 -9 -8 -7 -6 -5
Hydroxo-B12
Log [Arsenic] M
MS
act
ivit
yp
mo
l/m
in/m
g p
rote
in
-12 -11 -10 -9 -8 -7 -6 -500
20
40
60
80
100
120
140
Hydroxo-B12
Methyl-B12
Log [Aluminum] M
MS
act
ivit
yp
mo
l/m
in/m
g p
rote
in
-12 -11 -10 -9 -8 -7 -6 -500
20
40
60
80
100
120Hydroxo-B12
Methyl-B12
Log [Mercury] M
MS
act
ivit
yp
mo
l/m
in/m
g p
rote
in
-12 -11 -10 -9 -8 -7 -6 -500
20
40
60
80
100
Hydroxo-B12Methyl-B12
Log [Thimerosal] M
MS
act
ivit
yp
mo
l/m
in/m
g p
rote
in
0
250
500
750
1000
1250
1500
1750ControlLeadArsenicAluminumMercuryThimerosal
[GS
H]
nm
ole
/mg
pro
tein
a b
c d
e f
0
20
40
60
80
100ThimerosalBasal
*
Perc
en
t C
on
tro
l
0
10
20
30
40 BasalThimerosal
*
GS
Hn
mo
l/m
g p
rote
in
Thimerosal decreases methylcobalamin levels to a much greater extent than GSH levels
in SH-SY5Y human neuronal cells
Methylcobalamin levelsThimerosal = 0.1 Mfor 60 min
GSH levels Thimerosal = 1 Mfor 60 min
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
Control
Children (n = 42)
Autism
Pre-treatmentb
(n = 40)
Autism
Post-treatment (n = 40)
p valuea
Methionine 24 ± 3 21 ± 4 22 ± 3c ns S-adenosylmethionine (SAM) (nmol/L)
78 ± 22 66 ± 13 69 ± 12c ns S-adenosylhomocsyteine (SAH) (nmol/L)
14.3 ± 4.3 15.2 ± 5 14.8 ± 4 ns
SAM/SAH (µmol/L) 5.6 ± 2.0 4.7 ± 1.5 5.0 ± 2.0 ns Homocysteine (µmol/L) 5.0 ± 1.2 4.8 ± 1.8 5.3 ± 1.1 0.04 Cysteine (µmol/L)
210 ± 18 191 ± 24 215 ± 19 0.001 Cysteinylglycine (µmol/L) 45 ± 6 40 ± 9 46 ± 9 0.002 Total Glutathione (tGSH) (µmol/L) 7.5 ± 1.8 5.4 ± 1.3 6.2 ± 1.2c 0.001 Free Glutathione (fGSH) (µmol/L) 2.8 ± 0.8 1.5 ± 0.4 1.8 ± 0.4 c 0.008 GSSG (µmol/L)
0.18 ± 0.07 0.28 ± 0.08 0.22 ± 0.06 c 0.001 tGSH/GSSG 47 ± 18 21 ± 6 30 ± 9 c 0.001 fGSH/GSSG 17 ± 6.8 6 ± 2 9 ± 3 c 0.001 a Pre- and Post-treatment comparison
b 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)
Efficacy of methylcobalamin and folinic acid treatment on glutathione
redox status and core behaviors in children with autism
James et al. (In Press)
Table 2. Scores from the Vineland Adaptive Behavior Scales at baseline before and after 3 months intervention with methylB12 and folinic acid
Vineland Category
Baseline Score (mean ± SD)
Post-Treatment Score (mean ± SD)
Change in Score (mean; 95% C
I)
p value
Communication 65.3 ± 12.9 72.0 ± 15.5 6.7 (3.5, 10) <0.001 Daily Living Skills 67.0 ± 76 76.0 ± 17.7 9.0 (4.0, 14) <0.007 Socialization 68.2 ± 9.3 75.7 ± 14.7 7.5 (3.5, 11) <0.005 Motor Skills 75.6 ± 9.7 79.0 ± 14.7 3.3 (0, 8) 0.12 Composite Score 66.5 ± 9.2 73.9 ± 17.0 6.6 (2.3, 11) <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.
Vineland Category
Score Increase Lowest Quartile
Score Increase Upper Quartile
Communication 4 13 Daily Living 4 12 Socialization 3 10 Motor Skills 1 1 Composite Score 3 9
GSH
GSSG
ROS InactivationDetoxification
(e.g. GPx)
GSSG Reductase
Glutaredoxin (reduced)
Glutaredoxin (oxidized)
NADPH
NADP+
Glucose-6-Phosphate
6-Phospho-gluconolactone
Glucose
Hexokinase
G6PD
γ-Glutamylcysteine
Glycine
GlutamateCysteine
TranssulfurationCellular uptake
DETERMINANTS OF THE GSH/GSSH RATIO
Thimerosal
DNA
RNA
Pre-mRNA
Protein
Cap Domain Exons 19-21
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
SAM domain is present in MS mRNA from human cortex, but CAP Domain is absent
HCY FOL CAP COB SAM
80 year old subject
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
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
HCY FOL CAP COB SAM
Control Subject: Age 24 yrs
Cap Domain is Absent fromMethionine 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 ofmethionine 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
Cap mRNA levels
0 10 20 30 4020
25
30
35
40
45 ControlAutism
Age
Am
pli
fica
tio
n C
ycle
s
Cobalamin Domain
0 10 20 30 4020
25
30
35
40AutismControl
Age
Am
pli
fica
tio
n C
ycle
s
Conclusion:
There are lower amounts of mRNA formethionine synthase in the cortex ofautistic subjects and levels of theenzyme are also likely to be lower.
Lower expression levels may reflect anadaptation to oxidative stress.
This implies an impaired capacity formethylation, including D4 dopaminereceptor-mediated phospholipid methylation.
Tallan HH, Moore S, Stein WH. L-cystathionine in human brain. J Biol Chem. 1958 Feb;230(2):707-16.
Levels of cystathionine are markedly higher inhuman cortex than in other species
MethionineSynthase
HCY
MET
SAH
SAM
>150Methylation Reactons
ATP PP+Pi
Adenosine
MethylTHF
THF
Cystathionine
Cysteine
GSH
γ-Glutamylcysteine
GSCbl
D4HCY
D4SAM
D4SAH
D4METATPPP+Pi
MethylTHF
THF
PhospholipidMethylation
Adenosine
Dopamine
Cysteine
( - )
PI3-kinase
( + )
↓ IN NEURONAL CELLS
MeCbl
EAAT3
Glial CellsCysteinylglycine GSH
SAMGSSG
H2S
EAAT3 VIEWED FROM OUTSIDE THE CELL
Aspartic Acid Ready for Transport
Membrane Fatty Acid
Covering Loop
Open
Closed
Membrane Fatty Acid
0 1 2 3 4 5 60
5
10
15
20
37C
0C
Time in minutes
L-[
35S
]cys
tein
e U
pta
ke(n
mo
l/m
g p
rote
in)
0 1 3 5
5
10
15
20
0
Control
10-4MThreo- -hydroxyaspartate
10-4M Dihydrokainate
Time in minutes
L-[
35S
]cys
tein
e U
pta
ke(n
mo
l/ m
g p
rote
in)
0.0
2.5
5.0
7.5
10.0Control
Cycloleucine 10-3MWortmannin 10-7MLY-compound 10-7M
L-[
35S
]Cys
teie
ne
Up
take
nm
ol/
mg
pro
tein
[35S]-Cysteine uptake in Human Neuronal Cells
Dependent upon PI3-kinase and MAT activity
[35S]-Cysteine uptake in Human Neuronal Cells
Control M-7
[Lea
d]10
M-7
[Ars
enic
] 10
M-7
[Alu
min
um] 1
0M-7
[Mer
cury
] 10
M-7
[Thim
erosa
l] 10
0
2
4
6
8
10
*** *** ****** ***,^
L-[
35S
]-cy
stei
ne
up
take
nm
ol/
mg
pro
tein
Why put neurons at higher risk of oxidative stress?
One possible explanation:
Oxidative stress stops cells from dividing. Neuronshave to avoid cell division, otherwise they would loseall their connections and all of their information value.
Thus neurons must balance on the precarious knife-edgeof oxidative stress.
D4 Dopamine Receptor-mediatedPhospholipid 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 areassociated withincreased risk ofADHD
Dopamine-stimulated phospholipid methylation is reduced for the 7-repeat form of the D4 Receptor
7 Repeat
7-repeats
2 or 4-repeats
PHOTONS OF LIGHT
e.g. Color Size
Texture
Brain regions consist of networks of neurons that process and combine information
MEMORYe.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 frequencyas recorded in a patient with Parkinsonism
Blue = with dopamine (l-DOPA)
Engel et al. Nature Rev. 2005
Gamma frequency oscillations promote effectiveinteraction between brain regions
with dopamine
Early electrophysiological markers of visual awareness in the human brain
KLHL12 ROC1Ubiquitin
Ligase
Cul3
D4 DopamineReceptor
Ubiquitin
D4 ReceptorDown-Regulation
Sensitive toRedox Status
Mercury binding?
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
↓
↓ ↓
SNPs in Single Methylation Genes Increase the Risk of Obesity
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
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