“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
H2S
H2O
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
CH3
H2S
NH3
CO2
NH2CHCOOH
CH2
SH
Cysteine
Cysteine can function as an antioxidant
Two Antioxidant
Reducing Equivalents
NH2CHCOOH
NH2CHCOOH
CH2
+
CH2
SH
SH
Two Cysteines
NH2CHCOOH
CH2
S
+ 2 H+
S
CH2
NH2CHCOOH
Cysteine Disulfide
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.
Oxidative
Metabolism
Oxygen
Radicals
Genetic
Risk Factors
Oxygen Radicals
Redox
Buffer Capacity
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
D4SAH
Adenosine
D4HCY
MethylTHF
Phospholipid
Methylation
Methionine
Synthase
THF
D4SAM
MethylTHF
DNA
Methylation
THF
D4MET
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
D4SAH
Adenosine
D4HCY
MethylTHF
Phospholipid
Methylation
DNA
Methylation
THF
THF
D4MET
PP+Pi
(-)
MethylTHF
Methionine
Synthase
D4SAM
SAH
HCY
ATP
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 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
Cyanocobalamin
GSH
GSH
Glutathionylcobalamin
SAM
5-MethylTHF
Methylcobalamin
Homocysteine
Methionine
Methionine
Synthase
D4RMET
D4RHCY
b
a
120
120
100
MS activity
pmol/min/mg protein
MS activity
pmol/min/mg protein
Hydroxo-B12
Methyl-B12
80
60
40
20
0
-11
-10
-9
-8
-7
-6
60
40
20
-5
Log [Lead ] M
c
0
-11
-10
-9
-8
-7
-6
-5
Log [Arsenic] M
d
140
120
Hydroxo-B12
Methyl-B12
120
100
Hydroxo-B12
MS activity
pmol/min/mg protein
MS activity
pmol/min/mg protein
Methyl-B12
80
0
0
80
60
40
20
100
Methyl-B12
80
60
40
20
0
0
0
-12
-11
-10
-9
-8
-7
-6
-5
Log [Aluminum] M
e
0
-12
-11
-10
-9
-8
-7
-6
-5
Log [Mercury] M
f
100
1750
Hydroxo-B12
Methyl-B12
80
[GSH]
nmole/mg protein
MS activity
pmol/min/mg protein
Hydroxo-B12
100
60
40
20
0
0
-12
-11
-10
-9
-8
-7
Log [Thimerosal] M
-6
-5
1500
1250
1000
750
500
250
0
Control
Lead
Arsenic
Aluminum
Mercury
Thimerosal
Thimerosal decreases methylcobalamin levels
to a much greater extent than GSH levels
in SH-SY5Y human neuronal cells
Basal
Thimerosal
GSH levels
Thimerosal = 1 M
for 60 min
GSH
nmol/mg protein
40
30
20
*
10
0
Basal
Thimerosal
Methylcobalamin levels
Thimerosal = 0.1 M
for 60 min
Percent Control
100
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
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
Control
Children
(n = 42)
24 ± 3
78 ± 22
14.3 ± 4.3
Autism
Pre-treatmentb
(n = 40)
21 ± 4
66 ± 13
15.2 ± 5
Autism
Post-treatment
(n = 40)
22 ± 3c
69 ± 12c
14.8 ± 4
p valuea
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
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
ns
0.04
0.001
0.002
0.001
0.008
0.001
0.001
0.001
ns
ns
ns
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
Thimerosal
Hexokinase
Glucose-6-Phosphate
NADPH
Glutaredoxin
(reduced)
Glycine
GSH
GSSG
Reductase
G6PD
6-Phospho-gluconolactone
γ-Glutamylcysteine
NADP+
Glutaredoxin
(oxidized)
GSSG
ROS Inactivation
Detoxification
(e.g. GPx)
DNA
Pre-mRNA
RNA
Protein
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
Pre-mRNA
Cap Domain
Absent
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
Cap mRNA levels
Cobalamin Domain
Amplification Cycles
35
30
25
Control
Autism
45
Amplification Cycles
Control
Autism
40
40
35
30
25
20
20
0
10
20
Age
30
40
0
10
20
Age
30
40
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
45
[Cystathionine]
mg / 100 gm wet wt
40
35
30
25
20
15
10
5
0
t
t
in key Ra Pig Ca ow ken uck ver ey cle
a
C ic D Li dn s
a
Br on
e
h
n Ki Mu
n
C
i
a
an M
u
m an an
m
G
u
u
H um um
H
H H
Data from: Tallan et al. J. Biol. Chem. 230, pp 707-716 (1958)
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
EAAT3
(+)
GSSG
PI3-kinase
GSCbl
GSH
SAM
γ-Glutamylcysteine
Cysteine
↓ IN NEURONAL CELLS
Cystathionine
Adenosine
D4SAH
Adenosine
D4HCY
MethylTHF
Methionine
Synthase
THF
D4SAM
ATP
Dopamine
SAM
MET
ATP
PP+Pi
(-)
>150
Methylation
Reactons
THF
D4MET
PP+Pi
SAH
HCY
MethylTHF
Phospholipid
Methylation
MeCbl
H2S
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
10-4M Dihydrokainate
10-4MThreo- -hydroxyaspartate
20
37C
L-[35S]cysteine Uptake
(nmol/ mg protein)
L-[35S]cysteine Uptake
(nmol/mg protein)
20
15
10
5
0C
0
15
10
5
0
0
1
2
3
4
5
0
6
1
3
5
Time in minutes
Time in minutes
L-[35S]Cysteiene
Uptake
nmol/mg protein
Control
-3
7.5
5.0
2.5
0.0
Dependent upon PI3-kinase and MAT activity
Cycloleucine 10 M
Wortmannin 10-7M
LY-compound 10-7M
L-[35S]-cysteine
Uptake
(nmol/ mg protein)
6
10.0
5
4
3
2
1
0
0.0
0.1
0.2
0.3
0.4
0.5
0.6
ETOH % (V/V)
L-cysteine uptake was assessed at 1 minut
[35S]-Cysteine uptake in Human Neuronal Cells
Dose-Dependent Effect of Dopamine (DA) on
of LY and Wortmannin on DA-stimulated Cysteine Uptake
L-[35S]Cysteine uptake usingEffect
SY cells
150
Cysteiene Uptake
nmol/ mg protein
Cysteine Uptake
nmol/mg protein
150
100
50
0
-9
-8
-7
-6
-5
-4
10-5M DA
10-5M DA + 10-7M Wort
10-5M DA + 10-7M LY
100
50
0
Log [DA] M
Dose-Dependent Effect of thimerosal on
L-[35S]Cysteine uptake using SY cells
6
4
***
***
***
2
***
***,^
[M
in
um
]1
0 -7
er
M
cu
[T
ry
hi
]1
m
0 -7
er
M
os
al
]1
0 -7
M
M
[A
lu
m
ni
c]
10
-7
M
-7
rs
e
[A
[L
e
ad
]1
0
on
tr
ol
0
Cysteine Uptake
nmol/mg protein
8
C
L-[35S]-cysteine uptake
nmol/ mg protein
10
50
45
40
35
30
25
20
15
10
5
0
-13 -12 -11 -10
-9
-8
-7
-6
Log [Thimerosal]M
-5
-4
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
↓ DNA Methylation
FMR-1, RELN
↓ Neuronal Synchronization
↓Attention
Attention and cognition
 Gene Expression
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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