“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

Thanks for your Research Support!!

Autism Research Institute

SafeMinds

Cure Autism Now