Unique Aspects of Redox Regulation in Human Brain and Their Implications for Autism

Richard Deth, PhDNortheastern 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

Earliest life appears to have arisen at hydrothermal vents emitting hydrogen sulfide and other gases at high temperature and pressure

H2O

H2S

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

NH2CHCOOHCH2

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.

LANGUAG

E

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

>150Methylation Reactons

ATP PP+Pi

Adenosine

MethylTHF

THF

Cystathionine

Cysteine

GSH

γ-Glutamylcysteine

Cysteine

( - )

GSSG

Dietary protein

Cysteine for glutathione synthesis can be provided by either transsulfuration of homocysteine or by uptake from outside the cell

GlutathioneSynthesis

REDOXSTATUS:

GSHGSSH

MethylationStatus:SAMSAH

~ 200 Methylation

Reactions

Nitric OxideSynthesis

PhospholipidMethylation

DNA/HistoneMethylation

GeneExpression

ArginineMethylation

MembraneProperties

CreatineSynthesis

CognitiveStatus

EnergyStatus

CatecholamineMethylation

SerotoninMethylation

Melatonin

Sleep

MethionineSynthase

HCY

MET

SAH

SAM

>150Methylation Reactons

ATP PP+Pi

Adenosine

MethylTHF

THF

Cystathionine

Cysteine

GSH

γ-Glutamylcysteine

Cysteine

( - )

GSSG

Dietary protein

During oxidative stress methionine synthase is turned off,allowing more homocysteine to flow toward GSH synthesis,

while methylation activity is decreased

GlutathioneSynthesis

OXIDATIVESTRESS

Oxidative Stress

GSHGSSG = 30 GSH

GSSG = 10Normal

Redox Setpoint

- Survival mode

- Loss of normal function

- Impaired methylation

Infection, allergy,trauma, chronic illness

Recovery:Adaptive responsesto oxidative stress

InflammatoryState

Inflammation is a metabolic state of oxidative stress, normally occurring in response to environmental challenges

Oxidative Stress

GSHGSSG = 30 GSH

GSSG = 10Normal

Redox Setpoint

↑ Inflammatory Diseases:

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

Aging

Aging is associated with increased oxidative stress, as adaptive responses fail to restore normal redox status

Oxidative Stress

GSHGSSG = 30 GSH

GSSG = 10Normal

Redox Setpoint

- Survival mode

- Loss of normal function

- Impaired methylation

- Autism??

Heavy Metal and Xenobiotic Exposure Inflammatory

State

Exposure to persistent environmental toxins promotes oxidative stress and impairs the ability to recover

38%↓

28%↓

36%↓

Autism is associated with oxidative stress and impaired methylation

The Brain Compartment (CSF) has low Thiol levels and maintains an Oxidative Stress environment;

Astrocytes provide Cysteine to Neurons for survival

Blood-BrainBarrierBRAIN BLOOD

[GSH] = 8μM

[CYS] =200μM

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

[CYS] = 2 μM

[CYS]

[CYS]

[GSH] = 1 μMCSF

Neurons Astrocytes

[GSH][CysGly]

MethionineSynthase

HCY

MET

SAH

SAM

>150Methylation Reactons

ATP PP+Pi

Adenosine

MethylTHF

THF

Cystathionine

Cysteine

GSH

γ-Glutamylcysteine

Cysteine

( - )

PI3-kinase

( + )EAAT3

HealthyGlial Cells

(Astrocytes)Cysteinylglycine GSH

GSSG

Growth Factors

Neurons obtain cysteine from GSH released by Glial cells,via a growth factor-controlled transporter (EAAT3)

MethionineSynthase

HCY

MET

SAH

SAM

>150Methylation Reactons

ATP PP+Pi

Adenosine

MethylTHF

THF

Cystathionine

Cysteine

GSH

γ-Glutamylcysteine

Cysteine

( - )

PI3-kinase

( + )

PARTIALLY BLOCKED IN NEURONAL CELLS

EAAT3

HealthyGlial Cells

(Astrocytes)Cysteinylglycine GSH

GSSG

Growth Factors

Transsulfuration of homocysteine (HCY) to cysteine is restricted inhuman neuronal cells, increasing importance of cysteine uptake

MethionineSynthase

HCY

MET

SAH

SAM

>150Methylation Reactons

ATP PP+Pi

Adenosine

MethylTHF

THF

Cystathionine

Cysteine

GSH

γ-Glutamylcysteine

GSCbl

Cysteine

( - )

PI3-kinase

( + )

PARTIALLY BLOCKED IN NEURONAL CELLS

MeCbl

EAAT3

HealthyGlial Cells

(Astrocytes)Cysteinylglycine GSH

SAMGSSG

H2S

OHCbl

Growth Factors

Methionine synthase in human neuronal cells requires methylB12 (MeCbl),whose synthesis is glutathione-dependent

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

In neurons, D4 dopamine receptors carry out phospholipid methylation,which requires methionine synthase to supply methyl groups

MethionineSynthase

HCY

MET

SAH

SAM

>150Methylation Reactons

ATP PP+Pi

Adenosine

MethylTHF

THF

Cystathionine

Cysteine

GSH

γ-Glutamylcysteine

D4HCY

D4SAM

D4SAH

D4METATPPP+Pi

MethylTHF

THF

PhospholipidMethylation

Adenosine

Dopamine

Cysteine

( - )

PI3-kinase

( + )

PARTIALLY BLOCKED IN NEURONAL CELLS

EAAT3

HealthyGlial Cells

(Astrocytes)Cysteinylglycine GSH

GSSG

Growth Factors

GSCbl

MeCbl

SAMOHCbl

25

CH3

DOPAMINE

DOPAMINE –STIMULATED PHOSPHOLIPID METHYLATION

MethylfolateMethionineSynthase

7-repeats

2 or 4-repeats

Methionine SynthaseStructure and function

Brain levelsAcross the lifespanIn autism

Methionine synthase has five domains + cobalamin (Vitamin B12)Domains alternate interacting with cobalamin during turnover

SAM Domain

CobalaminDomain Cap

Domain

Cobalamin(vitamin B12)

SAM Domain

CobalaminDomain Cap

Domain5-Methyl THF Domain

HCY Domain

Cobalamin(vitamin B12)

1

23

HCY FOL CAP COB SAM

187 bp 197 bp 419 bp

3' 5'

Exon 19 252420

188 bp 122 bp

21 22 23

HCY FOL CAP COB SAM

187 bp 197 bp 419 bp

3' 5'

Exon 19 252420

188 bp 122 bp

21 22 23

Decrease of Cob domain mRNA with increasing age, 40 subjects

0 10 20 30 40 50 60 70 80 90 1000

100

200

300

400

500

600T1/2fast = 3.4 years

T1/2slow = 29.4 yearsR2 = .91

Age (years)

MS

Cob

mR

NA

(arb

itrar

y un

its)

Decrease of Cap domain with increasing age, 40 subjects

0 10 20 30 40 50 60 70 80 900

100

200

300

400

500

600

700T1/2fast = 2.2 yearsT1/2slow = 20 years

R2 = .94

Age (years)

MS

Cap

mR

NA

(arb

itrar

y un

its)

CAP Domain is present in MS mRNA from 24 y.o. subject

HCY FOL CAP COB SAM

CAP Domain is absent from methioninesynthase mRNA in elderly human cortex

HCY FOL CAP COB SAM

80 year old subject

Age-dependent decrease in the ratio of Cap to Cobalamin mRNA

HCY FOL CAP COB SAM

80 year old subject

Under 20

Over 6

00.0

0.5

1.0

1.5M

S m

RN

A C

ap/C

ob R

atio

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-mRNAleads to age-dependent exon skipping

Exons 16-18 aredeleted in

fetal human brain

115

180

84

115

180

84

MS exists as two lower MW bands in SH-SY5Y cells

Normal full size MW = 140 kDa

125 kDaExons 16-18are absent

110 kDaExons 16-20are absent

DNA

RNA(exons only)

Pre-mRNA(introns + exons)

Protein

Transcription

Splicing

Translation

B12 Cofactor

DNA

RNA(exons only)

Pre-mRNA(introns + exons)

Protein

Transcription

Splicing

Translation

DNA

RNA(exons only)

Pre-mRNA(introns + exons)

Protein

Transcription

Splicing

Translation

B12 Cofactor

Methionine synthase activity can be regulated via multiple levels of control in response to oxidative stress

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

0 10 20 300

100

200

300

400

Autistic

ControlsT1/2 = 2.7 yrs r2 = 0.94

Age (years)

MS

Cap

leve

ls (a

rbitr

ary

units

)

Paired comparisons of CAP domain mRNA to age-matched controls

(Same samples as Vargas et al. 2005)

0

100

200

300

400

11-15 yrs

26-30 yrs21-25 yrs

1-5 yrs6-10 yrs

16-20 yrs

Control Autistic

Met

hion

ine

Synt

hase

CAP

dom

ain

mR

NA

(arb

itrar

y un

its)

Paired comparisons of Cob domain mRNA to age-matched controls

(Same samples as Vargas et al. 2005)

0

100

200

300

400

11-15 yrs

26-30 yrs21-25 yrs

1-5 yrs6-10 yrs

16-20 yrs

Control Autistic

Met

hion

ine

Synt

hase

CO

B d

omai

n m

RN

A(a

rbitr

ary

units

)

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

HCY FOL CAP COB SAM

80 year old subject{ { {C A C A C A

5/4 11/9 30/30{ { {C A C A C A

5/4 11/9 30/30{ { {C A C A C A

5/4 11/9 30/30

C A C A C A

5/4 11/9 30/30

Selenoproteins, mercury and redox status

G6PD

NADP+

6-P-gluconolactone Glucose-6-P

NADPH Thioredoxin Reductase

Thioredoxin

GSH status

Glucose is the major source of reducing powerfor maintaining reduced glutathione

Glucose

CpG CpG CpG G6PD gene (on)

G6PD

NADP+

6-P-gluconolactone Glucose-6-P

NADPH Thioredoxin Reductase

Thioredoxin

DNA MethyltransferaseCpG CpG CpG

G6PD gene (off)

CH3 CH3 CH3

MethionineSynthaseActivity

SAMSAH

GSH status

DNADemethylase

Thioredoxin reductase is a selenoprotein

Glucose

Hg

CH

SH

N

H

H COOC3

2CH

Se

N

H

H COOC3

SELENOCYSTEINE

2

CYSTEINE

SULFUR AND SELENIUM AMINO ACIDS

From Dr. Nicholas Ralston Univ. of North Dakota

Hg2+Binding Constant = 1045Binding Constant = 1039

(million-fold higher affinity)

Selenoproteins

Thioredoxin fold proteins(dual stable thiols)

Protein thiols (mono thiol sites)

Thiol metabolites (GSH, cysteine)

Hg2+

Mercury gradually migrates to highestaffinity targets (i.e. selenoproteins)

Astrocyte [GSH] = 0.91 mM

Neuron [GSH] = 0.21 mM

Ependymal [GSH] = 2.73 mM

Selenoprotein P Is high inEpendymal cells

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

Sun et al. J BIOL CHEM. VOL. 281, pp. 17420–17431, 2006Scharpf et al. J. NEURAL TRANS.VOL 114, 877-884, 2007

Pluripotent Stem Cells(Ependymal Cells)

Oxidized State

MoreAstrocytes

LessNeurons

Pluripotent Stem Cells(Ependymal Cells)

Normal State

AstrocytesNeurons

Pluripotent Stem Cells(Ependymal Cells)

Reduced State

LessAstrocytes

MoreNeurons

Prevailing redox conditions determinethe proportion of neurons vs. astrocyteswhich develop from neuronal stem cells

Neu

rona

l cel

ls

55

In human neuronal cells thimerosal partially inhibits the selenoprotein thioredoxin reductase with high potency,

but inhibits thioredoxin with only low potency

Thioredoxin

-12 -11 -10 -9 -8 -7 -6 -5 -40.00

0.01

0.02

0.03

0[Thimerosal] M

Enzy

me

Activ

ity

Thioredoxin reductase

-12 -11 -10 -9 -8 -7 -6 -5 -40.000

0.005

0.010

0[Thimerosal] M

Enzy

me

Activ

ity

56

Thimerosal-induced reduction of GSH levelsin SH-SY5Y human neuroblastoma cells

-12 -11 -10 -9 -8 -7 -6 -500

100200300400500600700800900

Log [Thimerosal] M

[GSH

](n

mol

/ m

g pr

otei

n)

0

20

40

60

80

100

120

Methyl-B12

0 -11 -10 -9 -8 -7 -6 -5

Log [Lead ] M

Hydroxo-B12

MS

activ

itypm

ol/m

in/m

g pr

otei

n0

20

40

60

80

100

120

Methyl-B12

0 -11 -10 -9 -8 -7 -6 -5

Hydroxo-B12

Log [Arsenic] M

MS

activ

itypm

ol/m

in/m

g pr

otei

n

-12 -11 -10 -9 -8 -7 -6 -500

20

40

60

80

100

120

140Hydroxo-B12Methyl-B12

Log [Aluminum] M

MS

activ

itypm

ol/m

in/m

g pr

otei

n

-12 -11 -10 -9 -8 -7 -6 -500

20

40

60

80

100

120Hydroxo-B12Methyl-B12

Log [Mercury] M

MS

activ

itypm

ol/m

in/m

g pr

otei

n

-12 -11 -10 -9 -8 -7 -6 -500

20

40

60

80

100Hydroxo-B12Methyl-B12

Log [Thimerosal] M

MS

activ

itypm

ol/m

in/m

g pr

otei

n

0

250

500

750

1000

1250

1500

1750ControlLeadArsenicAluminumMercuryThimerosal

[GSH

]nm

ole/

mg

prot

ein

a b

c d

e f

58

Genetic and Environmental Factors Can Combine to Cause Autism

FMR-1, RELN

MeCP2, ADA

RFC, TCN2

COMT, ATP10C, ADA

PON1, GSTM1

MET, NLGN3/4

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

↓

AUTISM

FMR-1, RELN

MeCP2, ADA

RFC, TCN2

COMT, ATP10C, ADA

PON1, GSTM1

MET, NLGN3/4

MTRR, MTHFR, ADSL

↓

↓ ↓

Neuroinflammation

“… and a child shall lead them.”Disorders sharing metabolic features with autism:

Attention-deficit hyperactivity disorderAlzheimer’s diseaseSchizophreniaParkinson’s diseaseChronic fatigue syndromeAmyotrophic lateral sclerosis Multiple sclerosisType 2 diabetesObesity

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