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“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