Unique Aspects of Redox Regulation in Human

Brain and Their Implications for Autism

Richard Deth, PhD

Northeastern University
Boston, MA
Overview

- Oxidation and Evolution

- Regulation of Redox Status

- Brain-specific Redox Features

- Methionine synthase in human cortex

- across the lifespan
- in autism

- Selenoproteins and mercury toxicity

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
 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 Xenobiotics
[Glutathione] STRESS

NORMAL Methylation Neuronal

REDOX Degeneration
BALANCE Neuronal
Synchronization
 Cysteine for glutathione synthesis can be provided by either
transsulfuration of homocysteine or by uptake from outside the cell
Cysteine

GSSG GSH
Glutathione
γ-Glutamylcysteine
Synthesis
Cysteine

Cystathionine
Adenosine
HCY SAH
(-)
MethylTHF
Methionine >150
Synthase Methylati
THF on
MET SAM
Reactons
ATP PP+Pi

Dietary protein
 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
 During oxidative stress methionine synthase is turned off,
allowing more homocysteine to flow toward GSH synthesis,
while methylation activity is decreased
Cysteine

GSSG GSH
Glutathione
γ-Glutamylcysteine
Synthesis
Cysteine

Cystathionine
Adenosine
OXIDATIVE
STRESS HCY SAH
(-)
MethylTHF >150
Methionine
Synthase Methylati
THF on
MET SAM
Reactons
ATP PP+Pi

Dietary protein
 Inflammation is a metabolic state of oxidative stress,
normally occurring in response to environmental challenges
Infection, allergy,
trauma, chronic illness Inflammatory
State
- Survival mode

- Loss of normal
function

Recovery: - Impaired
Adaptive responses methylation
to oxidative stress

GSH GSH
GSSG
= 30 GSSG
= 10
Normal
Oxidative Stress
Redox Setpoint
 Aging is associated with increased oxidative stress,
as adaptive responses fail to restore normal redox status

↑ Inflammatory
Aging Diseases:

- Alzheimer’s disease
- Parkinson’s disease
- Diabetes
- Heart Failure

GSH GSH
GSSG
= 30 GSSG
= 10
Normal
Oxidative Stress
Redox Setpoint
Exposure to persistent environmental toxins promotes
oxidative stress and impairs the ability to recover

Heavy Metal and

Xenobiotic Exposure Inflammatory
State
- Survival mode

- Loss of normal
function

- Impaired
methylation

- Autism??

GSH GSH
GSSG
= 30 GSSG
= 10
Normal
Oxidative Stress
Redox Setpoint
Autism is associated with oxidative stress and impaired methylation

28%↓

36%↓

38%↓
he Brain Compartment (CSF) has low Thiol leve
and maintains an Oxidative Stress environmen
trocytes provide Cysteine to Neurons for surv
Blood-Brain
Barrier
BRAIN BLOOD
Neurons Astrocytes

[GSH] = 0.21mM [GSH] = 0.91mM

[GSH] = 8μM
[CYS]
[CYS] =200μM
[CYS] [CysGly] [GSH]

CSF
[GSH] = 1 μM [CYS] = 2
μM
 Neurons obtain cysteine from GSH released by Glial cells,
via a growth factor-controlled transporter (EAAT3)

Healthy
Growth Cysteine Cysteinylglycine GSH Glial Cells
Factors (Astrocytes)
EAAT3
(+)
GSSG GSH
PI3-kinase
γ-Glutamylcysteine

Cysteine

Cystathionine
Adenosine
HCY SAH
(-)
MethylTHF
Methionine >150
Synthase Methylati
THF on
MET SAM
Reactons
ATP PP+Pi
 Transsulfuration of homocysteine (HCY) to cysteine is restricted in
human neuronal cells, increasing importance of cysteine uptake
Healthy
Growth Cysteine Cysteinylglycine GSH Glial Cells
Factors (Astrocytes)
EAAT3
(+)
GSSG GSH
PI3-kinase
γ-Glutamylcysteine

Cysteine
PARTIALLY
BLOCKED IN Cystathionine
NEURONAL CELLS Adenosine
HCY SAH
(-)
MethylTHF
Methionine >150
Synthase Methylati
THF on
MET SAM
Reactons
ATP PP+Pi
Methionine synthase in human neuronal cells requires methylB12 (MeCbl),
whose synthesis is glutathione-dependent
Healthy
Growth Cysteine Cysteinylglycine GSH Glial Cells
Factors (Astrocytes)
EAAT3
(+) GSCbl
GSSG GSH
PI3-kinase OHCbl SAM
γ-Glutamylcysteine
MeCb
Cysteine H2S
PARTIALLY l
BLOCKED IN Cystathionine
NEURONAL CELLS Adenosine
HCY SAH
(-)
MethylTHF
Methionine >150
Synthase Methylati
THF on
MET SAM
Reactons
ATP PP+Pi
 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.
In neurons, D4 dopamine receptors carry out phospholipid methylation,
which requires methionine synthase to supply methyl groups
Healthy
Growth Cysteine Cysteinylglycine GSH Glial Cells
Factors (Astrocytes)
EAAT3
(+) GSCbl
GSSG GSH
PI3-kinase OHCbl SAM
γ-Glutamylcysteine
MeCb
Cysteine
PARTIALLY l
BLOCKED IN Cystathionine
NEURONAL
Adenosine CELLS 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
DOPAMINE –STIMULATED PHOSPHOLIPID METHYLATION

DOPAMINE

CH3 Methionine
Synthase
Methylfolate
25
2 or 4-repeats

7-repeats
Methionine Synthase
Structure and function

Brain levels
Across the lifespan
In autism
Methionine synthase has five domains + cobalamin (Vitamin B12)
Domains alternate interacting with cobalamin during turnover

HCY Domain
SAM Domain

3 2

1
Cobalamin
(vitamin B12)

Cobalamin
Cap 5-Methyl THF Domain
Domain
Domain
3' HCY FOL CAP COB SAM 5'

187 bp 197 bp 419 bp

Exon 19 20 21 22 23 24 25

188 bp 122 bp
Decrease of Cob domain mRNA
with increasing age, 40 subjects
600
MS Cob mRNA (arbitrary units)

T1/2fast = 3.4 years

500 T1/2slow = 29.4 years
400 R2 = .91

300

200

100

0
0 10 20 30 40 50 60 70 80 90 100
Age (years)
Decrease of Cap domain with
increasing age, 40 subjects
700
MS Cap mRNA (arbitrary units)

T1/2fast = 2.2 years

600
T1/2slow = 20 years
500 R2 = .94
400
300
200
100
0
0 10 20 30 40 50 60 70 80 90
Age (years)
 CAP Domain is present in
MS mRNA from 24 y.o. subject

HCY FOL CAP COB SAM

 CAP Domain is absent from methionine
synthase mRNA in elderly human cortex

80 year old subject

HCY FOL CAP COB SAM
Age-dependent decrease in the
ratio of Cap to Cobalamin mRNA

80 year old subject

1.5
MS mRNA Cap/Cob Ratio

HCY FOL CAP COB SAM

1.0

0.5

0.0
0

0
2

r6
er

ve
nd

O
U
 Alternative Splicing of MS Pre-mRNA
leads to age-dependent exon skipping
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
 Exons 16-18 are
deleted in
fetal human brain
MS exists as two lower MW bands in SH-SY5Y cells

Normal full size MW = 140 kDa

125 kDa 180

Exons 16-18
are absent
115
110 kDa
Exons 16-20 84
are absent
 Methionine synthase activity can be regulated via
multiple levels of control in response to oxidative stress

DNA
Transcription

Pre-mRNA
(introns + exons)
Splicing

RNA
(exons only)
Translation

Protein B12 Cofactor

 mRNA for methionine synthase is
2-3 fold lower in cortex of autistic subjects
as compared to age-matched controls
Age-dependent trend of methionine synthase
CAP domain mRNA is absent in autism
MS Cap levels (arbitrary

400 Controls
T1/2 = 2.7 yrs
300 r2 = 0.94 Autistic
units)

200

100

0
0 10 20 30
Age (years)
 Paired comparisons of CAP domain
mRNA to age-matched controls
(Same samples as Vargas et al. 2005)

400
1-5 yrs
Methionine Synthase
CAP domain mRNA

6-10 yrs
(arbitrary units)

300
11-15 yrs
16-20 yrs
200
21-25 yrs
26-30 yrs
100

0
Control Autistic
 Paired comparisons of Cob domain
mRNA to age-matched controls
(Same samples as Vargas et al. 2005)

400
1-5 yrs
Methionine Synthase
COB domain mRNA

6-10 yrs
(arbitrary units)

300
11-15 yrs
16-20 yrs
200
21-25 yrs
26-30 yrs
100

0
Control Autistic
 Age-dependent changes in Cap and
Cobalamin mRNA in Control vs. Autism

80 year old subject

HCY 5/4
FOL CAP 11/9COB 30/30
SAM
{
{
{
C A C A C A
Selenoproteins, mercury and redox status
Glucose is the major source of reducing power
for maintaining reduced glutathione
NADP+ NADPH Thioredoxin Reductase
Glucose Glucose-6-P 6-P-gluconolactone
G6PD Thioredoxin

GSH status
 Thioredoxin reductase is a selenoprotein

NADP+ NADPH Thioredoxin Reductase

Glucose Glucose-6-P 6-P-gluconolactone
G6PD Thioredoxin

CpG CpG
CpG G6PD gene (on)
GSH status

DNA Methionine
Demethylase Synthase
G6PD gene (off) Activity

CpG CpG
CpG DNA Methyltransferase SAM
CH3 CH3 CH3 SAH
Hg
 SULFUR AND SELENIUM AMINO ACIDS

H H

H3 N C COO H3 N C COO

CH 2 CH 2

SH Se

CYSTEINE SELENOCYSTEINE

Binding Constant = 1039 Binding Constant = 1045

Hg2+ (million-fold higher affinity)

From Dr. Nicholas Ralston Univ. of North Dakota

Mercury gradually migrates to highest
affinity targets (i.e. selenoproteins)
Selenoproteins

Thioredoxin fold proteins

(dual stable thiols)

Protein thiols
(mono thiol sites)

Thiol metabolites (GSH, cysteine)

Hg2+
Highest levels of GSH are in selenium-rich ependymal cells
which are stem cells for astrocytes and neurons

Selenoprotein P
Is high in
Ependymal cells

Astrocyte [GSH] = 0.91 mM

Neuron [GSH] = 0.21 mM

Ependymal [GSH] = 2.73 mM

Sun et al. J BIOL CHEM. VOL. 281, pp. 17420–17431, 2006
Scharpf et al. J. NEURAL TRANS.VOL 114, 877-884, 2007
 Prevailing redox conditions determine
the proportion of neurons vs. astrocytes
which develop from neuronal stem cells

Pluripotent Stem Cells Pluripotent Stem Cells Pluripotent Stem Cells

(Ependymal Cells) (Ependymal Cells) (Ependymal Cells)
Oxidized State Normal State Reduced State

Less More Neurons Astrocytes More Less

Neurons Astrocytes
Astrocytes Neurons
Neuronal cells
 In human neuronal cells thimerosal partially inhibits
the selenoprotein thioredoxin reductase with high potency,
but inhibits thioredoxin with only low potency

Thioredoxin reductase Thioredoxin

0.03

0.010

Enzyme Activity
Enzyme Activity

0.02

0.005
0.01

0.000 0.00
0 -12 -11 -10 -9 -8 -7 -6 -5 -4 0 -12 -11 -10 -9 -8 -7 -6 -5 -4
[Thimerosal] M [Thimerosal] M

55
Thimerosal-induced reduction of GSH levels
in SH-SY5Y human neuroblastoma cells

900
800
(nmol / mg protein)

700
600
[GSH]

500
400
300
200
100
0
0 -12 -11 -10 -9 -8 -7 -6 -5
Log [Thimerosal] M

56
 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
Genetic and Environmental Factors Can Combine to Cause Autism

Genetic Risk Factors Environmental Exposures

PON1, GSTM1

Impaired Sulfur Metabolism

Neuroinflammation
Oxidative Stress
MTRR, MTHFR, ADSL 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 and cognition Developmental Delay

AUTISM 58
“… and a child shall lead
them.”

Disorders sharing metabolic features with autism:

Attention-deficit hyperactivity disorder

Alzheimer’s disease
Schizophrenia
Parkinson’s disease
Chronic fatigue syndrome
Amyotrophic lateral sclerosis
Multiple sclerosis
Type 2 diabetes
Obesity
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