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Redox  and  Methyla.on  in  the    Gut,  Brain  and  Immune  System  

 

Part  II:  Au.sm-‐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

Cobalamin Domain Cap Domain

Cobalamin (vitamin B12)

SAM Domain

Cobalamin Domain Cap Domain

5-Methyl THF Domain

HCY Domain

Cobalamin (vitamin B12)

1

2 3

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)

28 weeks of fetal 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

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

R2 = .94

Age (years)

MS

Cap

mR

NA

(arb

itrar

y un

its)

2

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Deth, unpublished observations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 methionine synthase 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Deth, unpublished observations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

Unde

r 20

Over

600.0

0.5

1.0

1.5

MS

mR

NA

Cap

/Cob

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Deth, unpublished observations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 kDa Exons 16-18 are absent

110 kDa Exons 16-20 are 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

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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

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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

MethylatedDNA

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

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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 BCM7 A2 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 Brain Blood Brain Barrier Gluten/Casein

GI Epithelial

Cell

Liver ↓Cysteine ↓Cys.ne

Cysteine ↓Cysteine Cysteine

↓Cys.ne ↓Cys.ne

Astrocytes

↓Cys.ne ↓GSH

Neurons

↓Cysteine

↓Cysteine ↓GSH

Wheat/Milk

Gluten/Casein Opiate  Pep.des

Oxida.ve  Stress  ↓  Methyla.on

↓GSH

Opiate    Pep.des

(  -‐  )  

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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 ileum Data from Drs. Steve Walker and Arthur Krigsman

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

r = 0.92

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

Breast Milk Constituents Can Exert Epigenetic Effects

Casein    

Beta  Casomorphin      

             GI    Pep.dases    

Modula.on  of  GI  cysteine  uptake  

GI  Tract  

Δ  Cellular  Redox  Status  

Systemic  availability  of  cysteine  

Immune  System  

Brain  

BREAST  MILK    

Growth    Factors    

DHA  

Δ  Methyla.on  Status  

Δ  Gene  Expression  

The GI tract is a critical site for immune cell activation, especially during postnatal development. Decreased cysteine uptake can promote oxidative stress and inflammation, resulting in epigenetic effects which can last 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

Gluten Casein

Opiate Peptides 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

Methionine  Synthase  

HCY  

MET  

SAH  

SAM  

         >150  Methyla.on      Reactons  

ATP   PP+Pi  

Adenosine  

MethylTHF  

THF  

Cystathionine  

Cysteine  

GSH  

γ-‐Glutamylcysteine    

D4HCY  

D4SAM  

D4SAH  

D4MET  ATP  PP+Pi  

MethylTHF  

THF  

Phospholipid  Methyla.on  

Adenosine  

Dopamine    

Cysteine  

(  -‐  )  

PI3-‐kinase  

(  +  )  

PARTIALLY  BLOCKED  IN  NEURONAL  CELLS  

EAAT3  

Healthy  Glial  Cells  (Astrocytes)  

Cysteinylglycine   GSH  

GSSG  

Growth Factors

GSCbl  

MeCbl  

SAM  OHCbl  

42

CH3

DOPAMINE

DOPAMINE –STIMULATED PHOSPHOLIPID METHYLATION

Methylfolate Methionine Synthase

8

Structural features of the dopamine D4 receptor

Seven repeats are associated with increased risk of ADHD

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 frequency in 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’s area Wernicke’s area

9

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

Methionine  Synthase  

HCY  

MET  

SAH  

SAM  

         >150  Methyla.on      Reactons  

ATP   PP+Pi  

Adenosine  

MethylTHF  

THF  

Cystathionine  

Cysteine  

GSH  

γ-‐Glutamylcysteine    

D4HCY  

D4SAM  

D4SAH  

D4MET  ATP  PP+Pi  

MethylTHF  

THF  

Phospholipid  Methyla.on  

Adenosine  

Dopamine    

Cysteine  

(  -‐  )  

PI3-‐kinase  

(  +  )  

PARTIALLY  BLOCKED  IN  NEURONAL  CELLS  

EAAT3  

Healthy  Glial  Cells  (Astrocytes)  

Cysteinylglycine   GSH  

GSSG  

Growth Factors

GSCbl  

MeCbl  

SAM  OHCbl  

ADULT DIFFICULTIES: Violent or non-violent criminal conviction Substance dependence or psychiatric diagnosis Aggression against partners or minors No high school diploma Out of wedlock parenthood Government welfare recipient Unemployed 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 Product MTHFR

TC II

SAH

Cysteine

Glutathione

Adenosine

Autism-associated Genetic Polymorphisms in the Methionine Cycle

GST

COMT

From Dr. Jill James

RFC-1

10

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 Methylenetetrahydrofolate

Reductase

Methionine Synthase Reductase

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  

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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

Δ Gene Expression

Developmental Delay

AUTISM

↓

Δ

AUTISM

FMR - 1, RELN

MeCP2, ADA

RFC, TCN2

COMT, ATP10A, ADA

PON1, GSTM1

MET, NLGN3/4, NRXN1

MTRR, MTHFR, ADSL

↓

↓ ↓

Neuroinflammation

JESSIE

NATE Christina

Malav Girish

Vidya

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Brain Samples: Autism Tissue Program Harvard Brain Tissue Resource Center Tissue Resource Center (Australia) Stanley Medical Research Foundation and donor families. Collaborators: Antonio Persico Suzanne De la Monte Hamid Abdolmaleky Sultan Qaboos University

Mostafa Waly and Yahya Al-Farsi Grant Support: Autism Research Institute SafeMinds National Autism Association Autism Speaks

ACKNOWLEDGEMENTS