“Prenatal and Postnatal Epigenetic Programming (PreP and PEP):

Implications for GI, Immune and Neuronal Function in Autism”

Richard Deth, PhD

Northeastern University

Boston, MA

Overview

- Oxidation and antioxidant metabolism

- Autism: An epigenetic disorder Prenatal epigenetic programming (PREP) Postnatal epigenetic programming (PEP)

-Brain-specific redox features

- Redox signaling in the immune response

- Gluten and casein-derived opiate peptides

Viewing Life Through Redox Glasses

Oxidation

Reduction

Oxidation

Reduction

Oxidation: Loss of an electron

Reduction: Gain of an electron

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

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)

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

Ergo:

1.Life, from beginning to end, depends upon sufficient levels of antioxidant.

2.Disorders and diseases commonly reflect an interference with the capacity to resist oxidation.

3.Agents or organisms which interfere with antioxidant capacity will disrupt normal development and cause diseases.

4.Metabolic treatments that restore and sustain antioxidant capacity will be effective in reversing many disorders and diseases.

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

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

38%↓

28%↓

36%↓

Autism is associated with inadequate levels of glutathione and impaired methylation

Authors(Reference)

Control n Autistic n

GSH status

Additional related findings

James et al.(2004)

33 20 46% ↓ ↓GSH/GSSG, ↓SAM/SAH, ↓cysteine

James et al.(2006)

73 80 32% ↓ ↓GSH/GSSG, ↓SAM/SAH, ↓cysteine

Geier and Geier(2006)

Lab-based normal values

10 36%↓ ↓cysteine

Adams et al. (2011)

55 43 21% ↓ ↓SAM, ↓cysteine

Pasca et al.(2009)

13 15 33% ↓ ↓cysteine

Pastural et al.(2009)

12 15 35% ↓ ↓cysteine

Al-Gadani et al.(2009)

30 30 27%↓ ↑lipid peroxides, ↓vitamin E, ↓SOD, ↓GPx

Melnyk et al.(2011)

40 40 29%↓ ↓GSH/GSSG, ↓SAM/SAH, ↓cysteine, ↓DNA methylation

James et al.(2009)

42 40 28%↓ ↓GSH/GSSG, ↓SAM/SAH, ↓cysteine

Geier et al.(2009)

120 28 24% ↓ ↓cysteine

Geier et al.(2009)

Lab-based normal values

28 24% ↓ ↑GSSG, ↓cysteine, ↓taurine, ↓sulfate

Eleven studies showing significantly lower GSH in autism

GLUTATHIONE (GSH)

[Cysteine]

The available level of intracellular cysteine is rate-limiting for GSH synthesis

γ- Glutamylcysteine

Glycine

Glutamate

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

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

Absorption and distribution of cysteine/cystine

The GI tract is a critical site forimmune cell activation, especiallyduring postnatal development.

Decreased cysteine uptake canpromote oxidative stress andinflammation, resulting inepigenetic effects which canlast throughout life.

Postnatal Epigenetic Programming (PEP)

Prenatal Epigenetic Programming (PREP)

METHYLATION IS INHIBITED BY OXIDATIVE STRESS

HCY

MET

SAH

SAM

~1,000MethylationReaction

svia 209

MTases

( - )

ATP PP + Pi

DIETARY PROTEIN

THF

MethylTHF

MethionineSynthase

Adenosine

Vitamin B12(Cobalamin)

A reversible reaction!

( + )

Global methylation =

SAMSAH( )

Oxidative Stress

( - )

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

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)

Odds of autism decrease with increasing duration of breastfeeding

Data from Waly et al. (In Prep)

Folate and B12 Levels are Markedly Lower

Data from Waly et al. (In Prep)

Thus malnutrition-based autism in Oman shares a similar metabolic profile to autism in the U.S.

i.e. Oxidative stress and impaired methylation are fundamental features of autism

Data from Waly et al. (In Prep)

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 factors stimulate cysteine uptake, providing a powerful mechanism to regulate redox and methylation

EAAT3

Tyr-Pro-Phe-Val-Glu-Pro-Ile

β-Casein ( 40 % of milk protein)

Human breast milk

Human β-Casomorphin -7

α- gliadin-7

Tyr-Pro-Gln-Pro-Gln-Pro-Phe

Gliadins

Wheat, barley, rye

Beta-Casein

59 66 -67

-L- V- Y- P- F- P- G- P- I -X

A1 A2(His at 67) (Pro at 67)

Bovine β-Casomorphin -7(0.4 g/L milk)

Tyr-Pro-Phe-Pro-Gly-Pro-Ile

FOOD-DERIVED OPIATE PEPTIDES

A1 casein contains HIS at position 67 and is readily hydrolyzed to BCM7A2 casein contains PRO at position 67 and is resistant to hydrolysis

62 nM

14 nM

16 nM

4 nM

7.022 nm

IC50 values

Bovine casomorphin 7 decreases cysteine, glutathione, and methionine levels in neuronal cells.

Homocysteine and cystathionine levels are increased.

This is consistent with:

1. Decreased cysteine uptake

2. Decreased GSH synthesis

3. Lower activity of methionine synthase

4. Increased transsulfuration

Opiate peptides can inhibit cysteine uptake and promote oxidative stress

( - )

GI Tract Blood BrainBloodBrain

Barrier Gluten/Casein

GIEpithelial

Cell

Liver ↓Cysteine ↓Cystine

Cysteine ↓CysteineCysteine

↓Cystine ↓Cystine

Astrocytes

↓Cystine ↓GSH

Neurons

↓Cysteine

↓Cysteine↓GSH

Wheat/Milk

Gluten/CaseinOpiate Peptides

Oxidative Stress↓ Methylation

↓GSH

Opiate Peptides

( - )

Effects of morphine and bovine and human casomorphin-7 peptides on redox/methylation gene expression in human neuronal cells

Breast Milk Constituents Can Exert Epigenetic Effects

Casein

Beta Casomorphin

GI Peptidases

Modulation of GI cysteine uptake

GI Tract

Δ Cellular Redox Status

Systemic availability of cysteine

ImmuneSystem

Brain

BREAST MILK

Growth Factors

DHA

Δ Methylation Status

Δ Gene Expression

Body

CYSSe-CYS

CYS Se-CYS

GSH

Se-Proteins

GSSG

Redox Status

Epigenetic Regulation

EAAT3

GI Epithelial Cell

GSHCYSExtracellular

RedoxStatus

TerminalIleum

GI tractLumen

Food-derivedamino acids

Terminal Ileum is critical site for absorption of cysteine and selenocysteine via the EAAT3 transporter

Body

B12/Intrinsic Factor

Redox Status

Epigenetic Regulation

GI Epithelial Cell

TerminalIleum

GI tractLumen

Food-derivedB12+ Folate

GS-B12 (?)B12/Transcobalamin II

RFC-1

Folic acid

RFC-1

Folic acid

MRP1

Folic acid

Terminal ileum is also critical for vitamin B12 and folic acid absorption

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

fold change directionCBS 2.37 DownGSS 1.52 DownTNFA 1.63 UpGSR 1.72 DownDRD4 2.02 DownGCLC 1.58 DownCTH 2.63 DownGCLM 1.7 DownEAAT3 2.19 DownMTHFR 1.55 DownNRF2 3.12 Down

Terminal Ileum

Autism-associated changes in gene expression in terminal ileumData from Drs. Steve Walker and Arthur Krigsman

Methionine Adenosyl

Transferase 2A (MAT 2A)

Cysteine

EAAT3

SAH

SAM

HCY

MET

Cystathionine

Cysteine

GSH

γ-Glutamylcysteine

GSSG

Cystine

Homocystine

MethylTHF

THF

ATP PP+P

MethionineSynthase

Adenosine

DNA

Methylated DNA

GSH GSCblSAM

OHCbl

MeCbl

Gamma-glutamylcysteine synthetase (GCLC/GCLM)

Cystathionine gamma-lyase (CGL/CTH)

Methyl Transferases

Ex. DNA (cytosine-5-)-methyltransferase (DNMT3A)

Ex. Catechol-O-methyltransferase (COMT)

Methionine Synthase

(MS)

Cystathionine beta-synthase (CBS)

Glutathione Synthetase (GSS)

Glutathione Reductase

(GSR)

Excitatory amino acid Transporter

Figure:2

A preliminary qRT-PCR evaluation of redox/methylation-related gene expression In blood-derived RNA of autistic vs. control subjects

The brain extracellular environment (CSF) has more than 10-fold less cysteine than plasma!!

Blood-BrainBarrierBRAIN BLOOD

[GSH] = 3.5 μM

[CYS] =22 μM

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

[CYS] = 2.2 μM

[CYS]

[CYS]

[GSH] = 0.3 μMCSF

Neurons Astrocytes

[GSH][CysGly]

10-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).

Redox and Methylation Pathways in Neurons

MethionineSynthase

HCY

MET

SAH

SAM

>1,000MethylationReactions

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

Methionine synthase mRNA in cortex progressively decreases with increasing age in normal subjects

0 10 20 30 40 50 60 70 80 90 1000

100

200

300

400

500

600

T1/2fast = 3.4 yearsT1/2slow = 29.4 years

R2 = .91

Age (years)

MS

Cob

mR

NA

(ar

bitr

ary

units

)

28 weeks offetal development

400-fold

The level of methionine synthase mRNA is reduced by 50% in postmortem brain of autistic subjects vs. age-matched controls

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

EAAT3-mediated cysteine/selenocysteine uptake in the terminal ileum

EAAT3

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

A1 Beta Casein Consumption is correlated with increased risk of juvenile-onset diabetes

r = 0.92

Using A1 cow milk formula with hydrolyzed casein lowered autoimmunity and incidenceof type I diabetes by ~ 50%

GI uptake of cysteine is critical throughout life, but especially during postnatal epigenetic programming (PEP)

↓Cysteine Uptake ↓ Antioxidant Capacity

GlutenCasein

OpiatePeptides GI Inflammation

(celiac disease)

Autoimmunity(Type 1 diabetes)

Autism

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

Collaborators:Sultan Qaboos University

Mostafa Waly and Yahya Al-Farsi

Grant Support:Autism Research InstituteSafeMindsNational Autism AssociationAutism Speaks

ACKNOWLEDGEMENTS