“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 Primates
85 million yrs
Humans
Origin
2.5 million yrs
of
Life

3 Billion Years

Methane
Hydrogen sulfide
Ammonia Anaerobic Life Aerobic Life
Carbon dioxide
No Oxygen!! Oxygen
(electrophile)
 Primordial Synthesis of Cysteine
From Volcanic Gases

Methane CH3
NH2CHCOOH
Hydrogen sulfide H2S CH2
Ammonia NH3 SH
Carbon dioxide CO2
Cysteine
 Cysteine can function as an antioxidant

Two Antioxidant
Reducing Equivalents

NH2CHCOOH
NH2CHCOOH NH2CHCOOH CH2
CH2 CH2 S
+ + 2 H+
S
SH SH
CH2
NH2CHCOOH

Two Cysteines Cysteine Disulfide

 Evolution = Adaptation to threat of oxidation

O2

O2

Genetic
Mutation
O2

O2

Novel
Adaptive features of
Antioxidant =
sulfur metabolism
Adaptation
 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.

LA

SOCI
N

AL S
GU

KILL
S
AG
E
Oxidative Oxygen
Metabolism Radicals

Genetic
Oxygen Radicals Risk Factors
Redox
Buffer Capacity
Heavy Metals
Redox
Buffer Capacity OXIDATIVE +
[Glutathione] STRESS Xenobiotics

NORMAL Methylation Neuronal

REDOX Degeneration
BALANCE Neuronal
Synchronization
 NORMAL REDOX STATUS

Glutathione Redox
Transsulfuration
Buffering
Pathway
γ-Glutamylcysteine

Cysteine
Methionine
Cystathionine Cycle
Adenosine Adenosine
D4SAH D4HCY HCY SAH
MethylTHF MethylTHF
Phospholipid Methionine DNA
Methylation Synthase Methylation
THF THF

D4SAM D4MET MET SAM

PP+Pi ATP ATP PP+Pi

Dopamine (Attention)
Autism is associated with oxidative stress and impaired methylation

28%↓

36%↓

38%↓
 OXIDATIVE STRESS

Glutathione Oxidative Stress

Transsulfuration
Inhibits
Pathway
γ-Glutamylcysteine Methionine Synthase

Cysteine
Methionine
Cystathionine Cycle
Adenosine Adenosine
D4SAH D4HCY HCY SAH
(-)
MethylTHF MethylTHF
Phospholipid Methionine DNA
Methylation Synthase Methylation
THF THF

D4SAM D4MET MET SAM ∆ gene

expression
PP+Pi ATP ATP PP+Pi

Dopamine (Impaired Attention)

 Toxic exposures, inflammation,
Ideal Cellular infections, aging
Redox Setpoint
Loss of normal
Oxidative Stress cellular function,
reduced
methylation

Recovery

GSH GSH
GSSG
= 30 GSSG
= 10
 Toxic exposures, inflammation,
Ideal Cellular infections, aging
Redox Setpoint
Loss of normal
cellular function.
Oxidative Stress reduced methylation

GSH Utilization > Supply

GSH Utilization < Supply

Recovery

Autism?
GSH GSH
GSSG
= 30 GSSG
= 10

Less Oxidizing More Oxidizing

Environment Environment
 Cognitive
Status Nitric Oxide
Synthesis

Catecholamine
Methylation Arginine
Methylation Gene
Expression

REDOX Methylation ~ 200

STATUS: Status: Methylation DNA/Histone
GSH SAM Reactions Methylation
GSSH SAH

Serotonin
Phospholipid Methylation
Creatine Methylation
Synthesis
Melatonin
Membrane
Energy
Properties
Status Sleep
Methionine synthase has five domains + cobalamin (Vitamin B12)

HCY Domain

SAM Domain

Cobalamin
(vitamin B12)

5-methyl THF Domain

Cobalamin Cap
Domain Domain
Without SAM domain methionine synthase requires
GSH-dependent methylcobalamin for reactivation

5-methyl THF Domain

SAM Domain

Cobalamin
(vitamin B12)

Cobalamin HCY Domain

Cap
Domain Domain
Synthesis of bioactive methylcobalamin (methylB12)
requires glutathione and SAM

Hydroxycobalamin Cyanocobalamin

GSH GSH

Glutathionylcobalamin

SAM
5-MethylTHF
Methylcobalamin
Methionine Homocysteine
Methionine
Synthase
D4RMET D4RHCY
 a b
120 120
Hydroxo-B12 Hydroxo-B12

pmol/min/mg protein

pmol/min/mg protein
100 100
Methyl-B12 Methyl-B12

MS activity

MS activity
80 80

60 60

40 40

20 20

0 0
0 -11 -10 -9 -8 -7 -6 -5 0 -11 -10 -9 -8 -7 -6 -5
Log [Lead ] M Log [Arsenic] M
c d
140 120
Hydroxo-B12 Hydroxo-B12
pmol/min/mg protein

pmol/min/mg protein
120 100
Methyl-B12 Methyl-B12
100
MS activity

MS activity
80
80
60
60
40
40
20 20

0 0
0 -12 -11 -10 -9 -8 -7 -6 -5 0 -12 -11 -10 -9 -8 -7 -6 -5
Log [Aluminum] M Log [Mercury] M
e f
100 1750
Hydroxo-B12 Control
pmol/min/mg protein

1500 Le ad
80 Methyl-B12 nmole/mg protein
Arse nic
1250
MS activity

Aluminum
60 M ercury
1000
[GSH]

Thime rosal
40 750
500
20
250
0 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 Basal
Thimerosal

nmol/mg protein
30
GSH levels *
GSH
Thimerosal = 1 µM 20

for 60 min 10

100 Basal
Thimerosal
Percent Control

80
Methylcobalamin levels
Thimerosal = 0.1 µM 60

for 60 min 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 Control Autism Autism p valuea

Children Pre-treatmentb Post-treatment
(n = 42) (n = 40) (n = 40)
Methionine 24 ± 3 21 ± 4 22 ± 3c ns
S-adenosylmethionine (SAM) (nmol/L) 78 ± 22 66 ± 13 69 ± 12c ns
S-adenosylhomocsyteine (SAH) 14.3 ± 4.3 15.2 ± 5 14.8 ± 4 ns
(nmol/L)
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 ± 3c 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)
 Table 2. Scores from the Vineland Adaptive Behavior Scales at baseline before and after 3
months intervention with methylB12 and folinic acid

Vineland Baseline Post-Treatment Change in Score p value

Category Score Score (mean; 95% C
(mean ± SD) (mean ± SD) I)
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.

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 γ-Glutamylcysteine
Thimerosal
Hexokinase Glycine
Glutaredoxin
Glucose-6-Phosphate NADPH (reduced) GSH
GSSG ROS Inactivation
G6PD
Reductase Detoxification
(e.g. GPx)
Glutaredoxin
NADP
GSSG
+
6-Phospho-gluconolactone (oxidized)
 DNA

Pre-mRNA

RNA

Protein
Alternative Splicing of MS Pre-mRNA

Cap Domain
Cap Domain Exons 19-21 Present

HCY FOL COB SAM

Site of alternative splicing by Cap Domain

mRNA-specific adenosine deaminase Absent

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 Human Cortex

Early Alzheimer’s 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 Cap mRNA levels

40 Control 45 Control
Autism Autism

Amplification Cycles
Amplification Cycles

40
35
35
30
30

25
25

20 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

EAAT3
(+) GSCbl
GSSG GSH
PI3-kinase SAM
γ-Glutamylcysteine
MeCb
Cysteine H2S
l
↓ IN NEURONAL
CELLS Cystathionine
Adenosine Adenosine
D4SAH D4HCY HCY SAH
(-)
Phospholip MethylTH MethylTHF
F Methionine >150
id Synthase Methylati
Methylatio THF THF on
n
D4SAM D4MET MET SAM
Reactons
PP+Pi ATP ATP PP+Pi

Dopamine
EAAT3 VIEWED FROM OUTSIDE THE CELL
 Membrane Fatty Acid

Open

Covering Loop

Aspartic Acid Closed

Ready for Transport
Membrane Fatty Acid
 [35S]-Cysteine uptake in Human Neuronal Cells

Control
10-4M Dihydrokainate
20
10-4MThreo-β -hydroxyaspartate
L-[35S]cysteine Uptake

37°C 20
(nmol/mg protein)

L-[35S]cysteine Uptake
15

(nmol/ mg protein)
15

10
10

5 0°C 5

0 0
0 1 2 3 4 5 6 0 1 3 5
Time in minutes Time in minutes

10.0
Control
Cycloleucine 10-3M
L-[35S]Cysteiene

nmol/mg protein

7.5
Wortmannin 10-7M
Uptake

LY-compound 10-7M
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
on
[L t ro
ea l
d]
[A 10
rs -7
e ni M

***
[A c]
lu 10
m -7
in M
um

***
[M ]1
er 0 -7
cu M

***
[T ry
hi ]1
m 0 -7
er M
os

***
al
]1
0 -7
M

***,^
[35S]-Cysteine uptake in Human Neuronal Cells
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 GAMMA
(5-10 Hz) (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 Ubiquitin
Redox Status
KLHL12 Cul3 ROC1
Mercury binding? Ubiquitin
Ligase
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 ↓ DNA Methylation

MET, NLGN3/4 FMR-1, RELN

↓ Neuronal Synchronization ∆ Gene Expression

↓Attention
Attention and cognition 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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