Redox and Methylation in the Gut, Brain and Immune System

Part II: Autism-specific Aspects

Richard Deth, PhD

Northeastern University

Boston, MA

Overview

-Methionine synthase in human brain -Autism: An epigenetic disorder triggered by

oxidative stress

-Gluten and casein-derived opiate peptides

- D4 dopamine receptors, methylation and ADHD

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

SAM Domain

CobalaminDomain

CapDomain

Cobalamin(vitamin B12)

SAM Domain

CobalaminDomain

CapDomain

5-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

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

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

700

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

R2 = .94

Age (years)

MS

Cap

mR

NA

(ar

bitr

ary

units

)

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

HCY FOL CAP COB SAM

Deth, unpublished observations

CAP Domain is absent from methioninesynthase mRNA in elderly human cortex

HCY FOL CAP COB SAM

80 year old subject

Deth, unpublished observations

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

HCY FOL CAP COB SAM

80 year old subject

Under 2

0

Over 6

00.0

0.5

1.0

1.5M

S m

RN

A C

ap/C

ob

Rat

io

Deth, unpublished observations

115

180

84

115

180

84

In human neuronal cells, MS exists as two molecular weight bands.

Both are smaller than the expected full-size molecular weight of 140 kDa.

125 kDaExons 16-18are absent

110 kDaExons 16-20are absent

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-mRNA

38%↓

28%↓

36%↓

Autism is associated with oxidative stress 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

Data from Waly et al. (In Prep)

Oman Autism Collaboration

Drs. Mostafa Waly and Yahya Al-Farsi

Data from Waly et al. (In Prep)

Gender differences between autistic subjects in Oman

Data from Waly et al. (In Prep)

Folate and B12 Levels are Markedly Lower

Data from Waly et al. (In Prep)

Odds of autism decrease with increasing duration of breastfeeding

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)

mRNA for methionine synthase is 2-3 fold lower in cortex of autistic subjects

as compared to age-matched controls

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

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 ROLE OF GUT INFLAMMATION IN AUTISM:

GLUTEN AND CASEIN-DERIVED PEPTIDES

INHIBIT CYSTEINE UPTAKE

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

( - )

EAAT3-mediated cysteine/selenocysteine uptake in the terminal ileum

EAAT3

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

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

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%

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

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)

Opiate peptides are released from gluten and casein by enzymes in the GI tract and regulate cysteine uptake

↓Cysteine Uptake ↓ Antioxidant Capacity

GlutenCasein

OpiatePeptides GI Inflammation

(celiac disease)

Autoimmunity(Type 1 diabetes)

Autism

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

42

CH3

DOPAMINE

DOPAMINE –STIMULATED PHOSPHOLIPID METHYLATION

MethylfolateMethionineSynthase

Structural features of the dopamine D4 receptor

Seven repeats areassociated withincreased risk ofADHD

7-repeats

2 or 4-repeats

Gamma frequency responses are stronger for 7-repeat D4 receptor or 10-repeat dopamine transporter

Dopamine causes an increase in gamma frequencyin a patient with Parkinsonism

Blue = with dopamine (l-DOPA)

Csibra et al. Science 290 1582-1585 (2000)

Increased gamma frequency activity in response to a visual task at eight months of age vs. six months

Broca’sarea

Wernicke’sarea

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

ADULT DIFFICULTIES:Violent or non-violent criminal convictionSubstance dependence or psychiatric diagnosisAggression against partners or minorsNo high school diplomaOut of wedlock parenthoodGovernment welfare recipientUnemployed for > six months

Presence of the 7-repeat D4 receptor or the 10-repeat form of the dopamine uptake transporter was associated with “Adult Difficulties”

Autism vulnerability genes

THF

5,10-CH2-THF

5-CH3-THF

B12

Cystathionine

DMG

Methionine

Homocysteine

SAM Methyl Acceptor

Methyltransferase

Methylated ProductMTHFR

TC II

SAH

Cysteine

Glutathione

Adenosine

Autism-associated Genetic Polymorphisms in the Methionine Cycle

GST

COMT

From Dr. Jill James

RFC-1

Increased Frequency of Risk-inducing Polymorphisms in Autism:Many can be found in Sulfur Metabolism-related Genes

Reduced folate carrier

Catechol-O-methyltransferase

Transcobalamin 2

Glutathione-S-transferase M1

MethylenetetrahydrofolateReductase

Methionine SynthaseReductase

Particular Combinations of Polymorphisms are Associated with Even Higher Risk

Selected Genes Implicated in Autism

GENE NAME FUNCTION PON1 Paraoxonase 1 Hydrolyzes homocysteine thiolactone to homocysteine Inactivates pesticides GSTM1 Glutathione-S-transferase M1 Detoxifies xenobiotics MTRR Methionine synthase reductase Reactivates methionine synthase MTHFR Methyltetrahydrofolate reductase Provides methylfolate for methionine synthase ADSL Adenylylsuccinate lyase de novo purine synthesis RFC Reduced folate carrier Transports folate into cells TCN2 Transcobalamin 2 Transports vitamin B12 into cells COMT Catechol-O-methyltransferase Inactivation of catecholamines ATP10A Phospholipid translocase Maintains phosphatidylethanolamine (PE) at inner membrane surface ADA Adenosine deaminase Converts adenosine to inosine

Selected Genes Implicated in Autism

GENE NAME FUNCTION MET MET protooncogene Tyrosine kinase-linked receptor Hepatocyte growth factor receptor NLGN3/4 Neuroligin 3/4 Synapse formation and modulation; partner to neurexin NRXN1 Neurexin 1 Synapse formation and modulation; partner to neuroligin FRM1 Fragile-X 1 Protein synthase; neuronal spine length RELN Reelin Synapse formation MECP2 Methyl-CpG binding protein Regulation of gene transcription UBE3A Ubiquitin-protein ligase 3A Protein turnover DRD3 D3 Dopamine receptor Receptor for dopamine, Gi-linked ALAD Aminolevulinic acid dehydratase Heme synthesis pathway enzyme

59

Genetic and Environmental Factors Can Combine to Cause Autism

FMR-1, RELN

MeCP2, ADA

RFC, TCN2

PON1, GSTM1

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, ATP10A, ADA

PON1, GSTM1

MET, NLGN3/4, NRXN1

MTRR, MTHFR, ADSL

↓

↓ ↓

Neuroinflammation

JESSIE

NATE

Christina

MalavGirish

Vidya

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