Unique Aspects Of Redox Regulation In Human Brain And Their Implications For Autism

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 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 SH

+

CH2 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.

N LA GU

SOCI

AL S

E

AG

KILL

S

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

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

SAH

HCY Methionine Synthase

MethylTHF

THF MET ATP

>150 Methylati on SAM Reactons

PP+Pi

Dietary protein

(-)

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

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

OXIDATIVE STRESS

Adenosine

SAH

HCY Methionine Synthase

MethylTHF

THF MET ATP

>150 Methylati on

SAM Reactons

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 - Impaired methylation

Recovery: Adaptive responses to oxidative stress

GSH GSSG

= 30

Normal Redox Setpoint

GSH GSSG

= 10

Oxidative Stress

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

Aging

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

GSH GSSG

= 30

Normal Redox Setpoint

GSH GSSG

= 10

Oxidative Stress

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 GSSG

= 30

Normal Redox Setpoint

GSH GSSG

= 10

Oxidative Stress

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 BRAIN

Blood-Brain Barrier

Neurons

Astrocytes

[GSH] = 0.21mM

[GSH] = 0.91mM

[CYS] [CYS] [CysGly] [GSH]

CSF

[GSH] = 1 μM [CYS] = 2 μM

BLOOD

[GSH] = 8μM [CYS] =200μM

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

Cysteine Cysteinylglycine (+)

PI3-kinase

Healthy Glial Cells (Astrocytes)

GSH

EAAT3 GSSG

GSH

γ-Glutamylcysteine Cysteine Cystathionine Adenosine

HCY Methionine Synthase

SAH

MethylTHF

THF MET ATP

(-)

>150 Methylati on SAM Reactons

PP+Pi

Transsulfuration of homocysteine (HCY) to cysteine is restricted in human neuronal cells, increasing importance of cysteine uptake Growth Factors

Cysteine Cysteinylglycine (+)

PI3-kinase

Healthy Glial Cells (Astrocytes)

GSH

EAAT3 GSSG

GSH

γ-Glutamylcysteine Cysteine

PARTIALLY BLOCKED IN Cystathionine NEURONAL CELLS Adenosine HCY Methionine Synthase

SAH

MethylTHF

THF MET ATP

(-)

>150 Methylati on SAM Reactons

PP+Pi

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

Cysteine Cysteinylglycine (+)

PI3-kinase

Healthy Glial Cells (Astrocytes)

GSH

EAAT3 GSSG

GSCbl

GSH

OHCbl γ-Glutamylcysteine

SAM

Cysteine H2S

PARTIALLY BLOCKED IN Cystathionine NEURONAL CELLS Adenosine HCY Methionine Synthase

SAH

MethylTHF

THF MET ATP

MeCb l

(-)

>150 Methylati on SAM Reactons

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 Growth Factors

Cysteine Cysteinylglycine

Healthy Glial Cells (Astrocytes)

GSH

EAAT3

(+)

GSSG

GSCbl

GSH

OHCbl γ-Glutamylcysteine

PI3-kinase

SAM

Cysteine

PARTIALLY BLOCKED IN Cystathionine NEURONAL Adenosine CELLS Adenosine

D4SAH Phospholip id Methylatio n

D4HCY

MethylTH F

Methionine Synthase

THF

D4SAM PP+Pi

D4MET ATP

Dopamine

HCY

SAH

MethylTHF

THF MET ATP

MeCb l

(-)

>150 Methylati on SAM Reactons

PP+Pi

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 Domain

Cap Domain

5-Methyl THF Domain

3'

HCY

FOL

187 bp

Exon 19

COB

197 bp

20 188 bp

CAP

5'

SAM

419 bp

21

22

23

24

25 122 bp

MS Cob mRNA (arbitrary units)

Decrease of Cob domain mRNA with increasing age, 40 subjects 600 T1/2fast = 3.4 years T1/2slow = 29.4 years R2 = .91

500 400 300 200 100 0

0

10 20 30 40 50 60 70 80 90 100 Age (years)

MS Cap mRNA (arbitrary units)

Decrease of Cap domain with increasing age, 40 subjects 700 T1/2fast = 2.2 years T1/2slow = 20 years R2 = .94

600 500 400 300 200 100 0

0

10

20

30 40 50 60 Age (years)

70

80

90

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

1.5

FOL

CAP

COB

1.0

0.5

r6 0 ve O

er 2

0

0.0 U nd

HCY

MS mRNA Cap/Cob Ratio

80 year old subject SAM

Alternative Splicing of MS Pre-mRNA leads to age-dependent exon skipping 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

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 Exons 16-18 are absent 110 kDa Exons 16-20 are absent

180 115 84

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)

Protein

Translation B12

Cofactor

mRNA for methionine synthase is 2-3 fold lower in cortex of autistic subjects as compared to age-matched controls

MS Cap levels (arbitrary units)

Age-dependent trend of methionine synthase CAP domain mRNA is absent in autism

400

Controls

T1/2 = 2.7 yrs r2 = 0.94

300

Autistic

200 100 0

0

10

20

Age (years)

30

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

Methionine Synthase CAP domain mRNA (arbitrary units)

400

1-5 yrs 6-10 yrs

300

11-15 yrs 16-20 yrs 21-25 yrs 26-30 yrs

200 100 0

Control

Autistic

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

Methionine Synthase COB domain mRNA (arbitrary units)

400

1-5 yrs 6-10 yrs

300

11-15 yrs 16-20 yrs 21-25 yrs 26-30 yrs

200 100 0

Control

Autistic

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

80 year old subject A

11/9COB

C

A

30/30 SAM

{

C

CAP

{

5/4 FOL

{

HCY

C

A

Selenoproteins, mercury and redox status

Glucose is the major source of reducing power for maintaining reduced glutathione NADP+ Glucose

Glucose-6-P

NADPH Thioredoxin Reductase 6-P-gluconolactone

G6PD

Thioredoxin

GSH status

Thioredoxin reductase is a selenoprotein

NADP+ Glucose

Glucose-6-P

NADPH Thioredoxin Reductase 6-P-gluconolactone

G6PD

CpG CpG CpG

DNA Demethylase

CpG CpG CpG CH3 CH3 CH3

Thioredoxin

G6PD gene (on)

G6PD gene (off) DNA Methyltransferase

GSH status Methionine Synthase Activity SAM SAH

Hg

SULFUR AND SELENIUM AMINO ACIDS

H

H H3 N

C

H3 N

COO

C

COO

CH 2

CH 2

Se

SH CYSTEINE

SELENOCYSTEINE Binding Constant = 1045

Binding Constant = 1039

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 (Ependymal Cells) Oxidized State

Pluripotent Stem Cells (Ependymal Cells) Normal State

Less Neurons

Neurons

More Astrocytes

Astrocytes

Pluripotent Stem Cells (Ependymal Cells) Reduced State

More Neurons

Less Astrocytes

Neuronal cells

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

Thioredoxin reductase 0.010

Enzyme Activity

Enzyme Activity

0.03

0.005

0.02

0.01

0.00

0.000 0

-12 -11 -10

-9

-8

-7

[Thimerosal] M

-6

-5

-4

0

-12 -11 -10

-9

-8

-7

-6

-5

[Thimerosal] M

55

-4

[GSH] (nmol / mg protein)

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

900 800 700 600 500 400 300 200 100 0

0

-12

-11

-10

-9

-8

-7

-6

-5

Log [Thimerosal] M

56

b

a

120

Hydroxo-B12

100

MS activity pmol/min/mg protein

MS activity pmol/min/mg protein

120

Methyl-B12

80 60 40 20 0 -11

-10

-9

-8

-7

-6

20 -11

-10

-9

-8

-7

-6

-5

Log [Arsenic] M

d 120

Hydroxo-B12 Methyl-B12

120 100

MS activity pmol/min/mg protein

MS activity pmol/min/mg protein

40

0

140

80 60 40 20 0

-12

-11

-10

-9

-8

-7

-6

Hydroxo-B12

100

Methyl-B12

80 60 40 20 0

-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 60 40 20

0

-12

-11

-10

-9

-8

-7

Log [Thimerosal] M

-6

-5

[GSH] nmole/mg protein

MS activity pmol/min/mg protein

60

-5

Log [Lead ] M

c

0

Methyl-B12

80

0 0

0

Hydroxo-B12

100

1500 1250 1000 750 500 250 0

Control Le ad Arse nic Aluminum M ercury Thime rosal

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 MET, NLGN3/4

↓DNA Methylation

FMR-1, RELN

↓Neuronal Synchronization ↓ Attention and cognition

AUTISM

∆Gene Expression Developmental Delay

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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