Redox&and&Methyla.on&in&the&& Gut,BrainandImmuneSystem … · 2012. 5. 19. · GSR 1.72 Down DRD4...
Transcript of Redox&and&Methyla.on&in&the&& Gut,BrainandImmuneSystem … · 2012. 5. 19. · GSR 1.72 Down DRD4...
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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)
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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Deth, unpublished observations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Deth, unpublished observations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Deth, unpublished observations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
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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
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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
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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