Redox and Methylation in the Gut, Brain and Immune System

Part I: General Principles

Richard Deth, PhD

Northeastern University

Boston, MA

Overview

- Oxidation and antioxidant metabolism

-Epigenetic regulation of gene expressionPrenatal epigenetic programming (PREP)Postnatal epigenetic programming (PEP)

- Brain-specific redox features

-Role of selenium and selenoproteins

-Effects of heavy metals on redox and methylation

-Redox signaling in the immune response

Viewing Life Through Redox Glasses

Oxidation

Reduction

Oxidation

Reduction

Oxidation: Loss of an electron

Reduction: Gain of an electron

Earliest life appears arose at hydrothermal vents emitting hydrogen sulfide.

H2S

As oxygen became more plentiful, evolution was driven by the abilityto adapt and to resist oxidation.

From Paul G. Falkowski; Science 311 1724 (2006)

Life: The evolved ability to resist oxidation

Death:The inevitable outcome of oxidation

Evolution:Gradual acquisition and manifestation of novel adaptive strategies to survive oxidation

Development:Programmed and progressive changes in gene expression, driven by changes in oxidation status, via epigenetic mechanisms

Methane CH3

Hydrogen sulfideH2SAmmonia NH3

Carbon dioxide CO2

NH2CHCOOHCH2

SH

Cysteine

Primordial Synthesis of CysteineFrom Volcanic Gases

G

The Glutathione Redox Equilibrium

H

2

REDUCED GSH OXIDIZED GSSG

The ability to controloxidation is at thecore of evolution

Each addition isstrengthened because

it builds on thesolid core already

in place.

EVOLUTION = LAYER UPON LAYER OF USEFUL ADAPTIVE RESPONSES TO OFFSET THE THREAT OF OXIDATION

- 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.

- Cognitive abilities of the human brain are particularly vulnerable.

LAN

GU

AG

E

SOCIAL SKILLS

O2 + 4H+ 2H2O + ATP

formationReactiveOxygenSpecies

ROS

No Damage

[Antioxidant]

NADH+

Glucose

Oxidative Cellular Damage

The available level of antioxidant (GSH)controls the level of aerobic metabolismand ATP formation = Redox Signaling

NADPHSelenoproteins

GlutathioneAntioxidant

Supply

Glutathionylation of Complex I

Mitochondrial respiration is a primary source of reactive oxygen species

Oxygen

ATP+

H2O

ReactiveOxygen Species(ROS)

From: Kiley P J & Storz G; 2004

GI Tract Blood BrainBloodBrain

Barrier

GIEpithelial

Cell

Liver Cysteine Cystine

Cysteine CysteineCysteine

Cystine Cystine

Astrocytes

Cystine GSH

Neurons

Cysteine

CysteineGSHDietaryProtein

Cysteine is taken up from the GI tract and is distributed to the body and the brain

GSH

The brain extracellular environment (CSF) has more than 100-fold less cysteine than plasma!!

Blood-BrainBarrierBRAIN BLOOD

[GSH] = 3.5 μM

[CYS] =282 μM

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

[CYS] = 2.2 μM

[CYS]

[CYS]

[GSH] = 0.3 μMCSF

Neurons Astrocytes

[GSH][CysGly]

125-fold lower12-fold lower

Data from: Castagna et al. Neurology, 45:1678-83 (1995) and Sun et al. J Biol Chem, 281:17420-31 (2006).

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

MethionineSynthase

HCY

MET

SAH

SAM

>150Methylation

Reactons

ATP PP+Pi

Adenosine

MethylTHF

THF

Cystathionine

Cysteine

GSH

γ-Glutamylcysteine

Cysteine

( - )

GSSG

Dietary protein

GlutathioneSynthesis

Transsulfuration

Glial Cells(Astrocytes)

Cysteinylglycine GSH

MethionineSynthase

HCY

MET

SAH

SAM

>150Methylation

Reactons

ATP PP+Pi

Adenosine

MethylTHF

THF

Cystathionine

Cysteine

GSH

γ-Glutamylcysteine

Cysteine

( - )

GSSG

Dietary protein

GlutathioneSynthesis

Transsulfuration

Glial Cells(Astrocytes)

Cysteinylglycine GSH

NEURONALCELL

REDOXSTATUS:

GSHGSSH

MethylationStatus:

SAMSAH

~ 200 MethylationReactions

Nitric OxideSynthesis

PhospholipidMethylation

DNA/HistoneMethylation

GeneExpression

ArginineMethylation

MembraneProperties

CreatineSynthesis

CognitiveStatus

EnergyStatus

CatecholamineMethylation

SerotoninMethylation

Melatonin

Sleep

MS DNA

MS pre-RNA

MS RNA

MSHCY

MET SAM

SAH

[SAM]

[SAH]

CystathionineCysteineGSH

CellularRedox Status

DNATranscription

MethylationRNA

Splicing

TranslationMET t-RNA

Protein

Ubiquitination

TNF-α( - )

Methylation( - )

( + )Amino Acids

Global Metabolic

Coordination

METHIONINE SYNTHASE REGULATION

GLOBAL REGULATION

AlternativeSplicing

DNA Methylation( - )

Methionine synthase provides redox-sensitiveglobal coordination of metabolism: HOMEOSTASIS

Methylation of DNA and histones is fundamental to epigenetic regulation of gene expression during development

Regulation of gene expression during development

X

Gene sequence

Start site for mRNA synthesis

TF

Growth Factors

Neurotransmitters Hormones

RNA polymerase mRNA

Protein(e.g. enzyme)

DNA

TranscriptionTranslation

DNADNA + Histone = HeterochromatinGenes are silenced and transcription is blocked

Me Me

MBDP(e.g. MeCP2) Histone

proteins

HMT

Me Me

Me MeDNMT

SAM

SAMCpG CpG

Transcription Factor Regulation:

Epigenetic Regulation:

TF binding region

TF binding region

CpG CpG

No mRNAX

Epigenetic changes in gene expression are the Primary mechanism underlying development

Epigenetic marks change in response to the environment and contribute to adaptive capabilities gained through evolution

Essentially:

Epigenetics = A memory system

• linked to gene expression• responsive to redox status• driver of development• active in all cell types• active at all ages• capable of transgenerational effects

Agents which interrupt antioxidant and/or methylation pathways willinterfere with the many roles of epigenetic regulation.

PrEP PEPPrenatal

EpigeneticProgramming

Maternal Metabolism

Maternal Nutrition

MaternalToxic Exposures

Placental Function

Genetic Factors

PostnatalEpigenetic

Programming

BIRTH

Breast Milk (Formula)

Toxic Exposures

Physical Experiences

Emotional Experiences

Metabolic Activity(Antioxidant demand)

Antioxidant Availability(i.e. cysteine and GSH)

Homeostatic Equilibrium

↑ Metabolic Activity(Higher antioxidant demand)

↓Antioxidant Availability(Low cysteine and GSH)

Oxidative Stress →Adaptive

Epigenetic Changes

Odds of autism decrease with increasing duration of breastfeeding

GI TRACT BLOOD BRAIN

BloodBrain

Barrier

GIEpithelial

Cells

LIVER Cysteine Cystine

Cysteine CysteineCysteine

Cystine Cystine

Astrocytes

Cystine GSH

Neurons

Cysteine

CysteineGSHDietaryProtein

Methionine

GSH GSHEAAT3

EAAT3

GI tract absorption of cysteine is critical forpostnatal epigenetic programming (PEP)

Transsulfuration

Pathway

REDOX & METHYLATION PATHWAYSGlutathione

(GSH) RedoxBufferingγ-

Glutamylcysteine

Cysteine

Cystathionine

HCY

Methionine

Cycle

SAH

Adenosine

> 150Methylatio

nReactions

SAM

( - )

ATP PP + Pi

THF

Methyl-THF

MethionineSynthase

MET

Adenosine

D4-SAH

ATPPP + Pi

Dopamine (Attention)

PhospholipidMethylation

D4-HCY

D4-MET

D4-SAM

Methyl-THF

THF THF

D4-Receptor PLM Cycle

Neurons have impaired transsulfuration and low GSH levels that depend upon growth factor-stimulated cysteine uptake

MethionineSynthase

HCY

MET

SAH

SAM

>1,000Methylation Reactions

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

AstrocytesCysteinylglycine GSH

GSSG

NeurotrophicGrowth Factors

GSH

NEURON

Cystine

EAAT3

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

REDOX SIGNALING:

Neurotrophic growth factors increaseEAAT3-mediated uptake of cysteine. Leading to:-Increased GSH/GSSG-Increased methionine synthase activity-Increased DNA methylation and epigenetic consequences

MethionineSynthase

HCY

MET

SAH

SAM

>150Methylation Reactons

ATP PP+Pi

Adenosine

MethylTHF

THF

Cystathionine

Cysteine

GSH

γ-Glutamylcysteine

Cysteine

( - )

PI3-kinase ( + )

EAAT3

Glial Cells(Astrocytes)

Cysteinylglycine GSH

GSSG

Neurotrophic Growth Factors

Growth factor regulation of cysteine uptake is a powerful mechanism to regulate redox and methylation in the brain

EAAT3

IGF-1 stimulates methionine synthase activity andincreases DNA methylation in human neuronal cells

MethionineSynthase

HCY

MET

SAH

SAM

DNAMethylation

ATP PP+Pi

Adenosine

MethylTHF

THF

Cystathionine

Cysteine

GSH

γ-Glutamylcysteine

Cysteine

( - )

PI3-kinase ( + )

EAAT3

Glial Cells(Astrocytes)

Cysteinylglycine GSH

GSSG

By increasing cysteine uptake, neurotrophic growth factors canincrease methionine synthase activity and regulate gene expression

Epigenetic regulation of gene expression

( + )

( + )

Neurotrophic Growth Factors

EAAT3

REDOX SIGNALING:

Tumor necrosis factor–alpha (TNF-alpha), a pro-inflammatory cytokine, decreases EAAT3-mediated uptake of cysteine.

Leading to: - Decreased methionine synthase mRNA- Increased transsulfuration

- TNF-alpha levels are elevated in brain and CSF of autistic subjects

TNF-alpha decreases cysteine uptake

TNF-alpha causes a large and prompt decrease in methionine synthase mRNA

Despite inhibition of cysteine uptake, cysteine and GSH levels do not decrease after TNF-alpha, indicating that it increases transsulfuration

MethionineSynthase

HCY

MET

SAH

SAM

↓ DNAMethylation

ATP PP+Pi

Adenosine

MethylTHF

THF

Cystathionine

Cysteine

GSH

γ-Glutamylcysteine

Cysteine

EAAT3

Glial Cells(Astrocytes)

Cysteinylglycine GSH

GSSG

TNF- alpha

TNF-alpha decreases cysteine uptake and decreases methionine synthase activity, but increases transsulfuration

Epigenetic regulation of gene expression

( )

( )

( + )

( )

NEUROTROPHIC GROWTH FACTORS

EAAT3-Mediated Influx

GROWTH FACTORS

Redox Status

EpigeneticRegulation

( > 150 Reactions )

Transsulfuration

TNF-alpha (Inflammatory Cytokine)

GSH

GSSG

METHYLATION

Neural NetworkSynchronization

Myelination

CYSTEINE

( + )( - )

MethionineSynthase

HCY

MET SAH

SAM

( + )

Selenium and selenoproteins in redox regulation

Oxygen, sulfur and selenium are chemically similar in the periodic table.

Oxygen e.g ROS

Sulfure.g. GSH

Seleniume.g. Thioredoxin Reductase

Illustrations from: Bentor, Yinon. Chemical Element.com

Reducing equivalents are passed from selenium to sulfur and from sulfur to oxygen

Hydrogen(Reducing Equivalent)

Water

e-

e-

NADPH

Thioredoxin Reductase

Electron flow

Reducing electrons move from NADPH through the selenoprotein thioredoxin reductase to thioredoxin and glutathione

Glucose NADPH Selenocysteine-basedRedox Proteins

(TrxR ,TGR, GPx)

Grx

GR

GSSG GSH

Cysteine-basedRedox Proteins

(Trx , Prx)

e- e- e-

e-

e-

e--Reduction of oxidized proteins and phospholipids.

-Reduction of glutathionylated proteins

e-

e- e-

GR = glutathione reductase Trx = thioredoxinGSH = reduced glutathione TrxR = thioredoxin reductaseGSSG = oxidized glutathione TGR = thioredoxin-glutathione reductaseGPx = glutathione peroxidase Prx = peroxiredoxinGrx = glutaredoxin e- = electron

Selenoproteins provide antioxidant electrons

The selenoprotein Thioredoxin reductase (TrxR) is directly or indirectly responsible for keeping vitamin E, ascorbic acid, glutathione, thioredoxin and ubiquinone reduced.

CpG CpG CpGG6PD 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

Selenoprotein

Glucose

Glucose is the major source of reducing powerfor maintaining reduced glutathione

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)

*Mercury inhibition of selenoproteins could disrupt redox regulation and epigenetic regulation during brain development

Selenoprotein Activities

Hg2+( - )

Redox Regulation and Antioxidant Activity

(e.g. TrxR)

EpigeneticRegulation of

Gene Expression

BrainDevelopment

ROS Inactivation and Oxidation Repair

(e.g. GPx)

Thyroid HormoneSynthesis(e.g. DIO)

DNA and HistoneMethylation

Intracellular Redox Status

Selenocysteine

Selenoprotein functions relevant to autism

Methionine Synthase Activity

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

Vescovi et al. Nature Reviews Cancer 6, 425–436 (June 2006)

Adult neurogenesis occurs in the subventricular zone (SVZ) of the cortex and the subgranular zone (SGZ) of the hippocampus

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

Eggs are richIn cysteine

Sperm are rich in selenium

GSH

GSH

GSH

GSH

GSH

GSH

∆ DNA and Histone Methylation

∆ GeneExpression

Does Redox ControlDevelopment ViaEpigenetic Effects?

juanv.wordpress.com/

Neu

ron

al c

ells

REDOX SIGNALING IN THEIMMUNE SYSTEM:

Redox signaling plays a central role in the immune response

Dendritic cells release GSH,similar to astocytes in brain

Effector T-cells take up cysteine,similar to neurons in brain

Regulatory T-cells competewith Teff for cysteineand restrict the immune response

Antigen Antibodyproduction

EAAT3 providesCysteine uptake

EAAT3 is expressed in T cell lymphocytes and highest expression is in Treg cells

Autoimmune-prone SJL/J mice have lower EAAT3 expression in mixed T cell lymphocytes

Autoimmune-prone SJL/J mice have lower levels of GSH in brain, unaffected by postnatal thimerosal treatment

Autoimmune-prone SJL/J mice have lower methionine synthase activity in brain, unaffected by postnatal thimerosal treatment

Brain Samples:Autism Tissue ProgramHarvard Brain Tissue Resource CenterTissue Resource Center (Australia)Stanley Medical Research Foundationand donor families.

Collaborators:Antonio PersicoSuzanne De la MonteHamid Abdolmaleky

Sultan Qaboos UniversityMostafa Waly and Yahya Al-Farsi

Grant Support:Autism Research InstituteSafeMindsNational Autism AssociationAutism Speaks

ACKNOWLEDGEMENTS