Redox and Methylation in the Gut, Brain and Immune System Part I: General Principles
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Transcript of Redox and Methylation in the Gut, Brain and Immune System Part I: General Principles
Redox and Methylation in the Gut, Brain and Immune System
Part I: General Principles
Richard Deth, PhD
Northeastern University
Boston, MA
Overview
- Oxidation and antioxidant metabolism
-Epigenetic regulation of gene expressionPrenatal epigenetic programming (PREP)Postnatal epigenetic programming (PEP)
- Brain-specific redox features
-Role of selenium and selenoproteins
-Effects of heavy metals on redox and methylation
-Redox signaling in the immune response
Viewing Life Through Redox Glasses
Oxidation
Reduction
Oxidation
Reduction
Oxidation: Loss of an electron
Reduction: Gain of an electron
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)
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
Methane CH3
Hydrogen sulfideH2SAmmonia NH3
Carbon dioxide CO2
NH2CHCOOHCH2
SH
Cysteine
Primordial Synthesis of CysteineFrom Volcanic Gases
G
The Glutathione Redox Equilibrium
H
2
REDUCED GSH OXIDIZED GSSG
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
- New capabilities are added in the context of the particular environment in which they are useful and offer a selective advantage.
- Recently added capabilities are the most vulnerable to loss when and if there is a significant changes in the environment.
- Cognitive abilities of the human brain are particularly vulnerable.
LAN
GU
AG
E
SOCIAL SKILLS
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
Mitochondrial respiration is a primary source of reactive oxygen species
Oxygen
ATP+
H2O
ReactiveOxygen Species(ROS)
From: Kiley P J & Storz G; 2004
GI Tract Blood BrainBloodBrain
Barrier
GIEpithelial
Cell
Liver Cysteine Cystine
Cysteine CysteineCysteine
Cystine Cystine
Astrocytes
Cystine GSH
Neurons
Cysteine
CysteineGSHDietaryProtein
Cysteine is taken up from the GI tract and is distributed to the body and the brain
GSH
The brain extracellular environment (CSF) has more than 100-fold less cysteine than plasma!!
Blood-BrainBarrierBRAIN BLOOD
[GSH] = 3.5 μM
[CYS] =282 μM
[GSH] = 0.91mM[GSH] = 0.21mM
[CYS] = 2.2 μM
[CYS]
[CYS]
[GSH] = 0.3 μMCSF
Neurons Astrocytes
[GSH][CysGly]
125-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).
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
REDOXSTATUS:
GSHGSSH
MethylationStatus:
SAMSAH
~ 200 MethylationReactions
Nitric OxideSynthesis
PhospholipidMethylation
DNA/HistoneMethylation
GeneExpression
ArginineMethylation
MembraneProperties
CreatineSynthesis
CognitiveStatus
EnergyStatus
CatecholamineMethylation
SerotoninMethylation
Melatonin
Sleep
MS DNA
MS pre-RNA
MS RNA
MSHCY
MET SAM
SAH
[SAM]
[SAH]
CystathionineCysteineGSH
CellularRedox Status
DNATranscription
MethylationRNA
Splicing
TranslationMET t-RNA
Protein
Ubiquitination
TNF-α( - )
Methylation( - )
( + )Amino Acids
Global Metabolic
Coordination
METHIONINE SYNTHASE REGULATION
GLOBAL REGULATION
AlternativeSplicing
DNA Methylation( - )
Methionine synthase provides redox-sensitiveglobal coordination of metabolism: HOMEOSTASIS
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
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
Odds of autism decrease with increasing duration of breastfeeding
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)
Transsulfuration
Pathway
REDOX & METHYLATION PATHWAYSGlutathione
(GSH) RedoxBufferingγ-
Glutamylcysteine
Cysteine
Cystathionine
HCY
Methionine
Cycle
SAH
Adenosine
> 150Methylatio
nReactions
SAM
( - )
ATP PP + Pi
THF
Methyl-THF
MethionineSynthase
MET
Adenosine
D4-SAH
ATPPP + Pi
Dopamine (Attention)
PhospholipidMethylation
D4-HCY
D4-MET
D4-SAM
Methyl-THF
THF THF
D4-Receptor PLM Cycle
Neurons have impaired transsulfuration and low GSH levels that depend upon growth factor-stimulated cysteine uptake
MethionineSynthase
HCY
MET
SAH
SAM
>1,000Methylation Reactions
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
Tallan HH, Moore S, Stein WH. L-cystathionine in human brain. J Biol Chem. 1958 Feb;230(2):707-16.
Levels of cystathionine are markedly higher inhuman cortex than in other species
REDOX SIGNALING:
Neurotrophic growth factors increaseEAAT3-mediated uptake of cysteine. Leading to:-Increased GSH/GSSG-Increased methionine synthase activity-Increased DNA methylation and epigenetic consequences
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 factor regulation of cysteine uptake is a powerful mechanism to regulate redox and methylation in the brain
EAAT3
IGF-1 stimulates methionine synthase activity andincreases DNA methylation in human neuronal cells
MethionineSynthase
HCY
MET
SAH
SAM
DNAMethylation
ATP PP+Pi
Adenosine
MethylTHF
THF
Cystathionine
Cysteine
GSH
γ-Glutamylcysteine
Cysteine
( - )
PI3-kinase ( + )
EAAT3
Glial Cells(Astrocytes)
Cysteinylglycine GSH
GSSG
By increasing cysteine uptake, neurotrophic growth factors canincrease methionine synthase activity and regulate gene expression
Epigenetic regulation of gene expression
( + )
( + )
Neurotrophic Growth Factors
EAAT3
REDOX SIGNALING:
Tumor necrosis factor–alpha (TNF-alpha), a pro-inflammatory cytokine, decreases EAAT3-mediated uptake of cysteine.
Leading to: - Decreased methionine synthase mRNA- Increased transsulfuration
- TNF-alpha levels are elevated in brain and CSF of autistic subjects
TNF-alpha decreases cysteine uptake
TNF-alpha causes a large and prompt decrease in methionine synthase mRNA
Despite inhibition of cysteine uptake, cysteine and GSH levels do not decrease after TNF-alpha, indicating that it increases transsulfuration
MethionineSynthase
HCY
MET
SAH
SAM
↓ DNAMethylation
ATP PP+Pi
Adenosine
MethylTHF
THF
Cystathionine
Cysteine
GSH
γ-Glutamylcysteine
Cysteine
EAAT3
Glial Cells(Astrocytes)
Cysteinylglycine GSH
GSSG
TNF- alpha
TNF-alpha decreases cysteine uptake and decreases methionine synthase activity, but increases transsulfuration
Epigenetic regulation of gene expression
( )
( )
( + )
( )
NEUROTROPHIC GROWTH FACTORS
EAAT3-Mediated Influx
GROWTH FACTORS
Redox Status
EpigeneticRegulation
( > 150 Reactions )
Transsulfuration
TNF-alpha (Inflammatory Cytokine)
GSH
GSSG
METHYLATION
Neural NetworkSynchronization
Myelination
CYSTEINE
( + )( - )
MethionineSynthase
HCY
MET SAH
SAM
( + )
Selenium and selenoproteins in redox regulation
Oxygen, sulfur and selenium are chemically similar in the periodic table.
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
Thioredoxin Reductase
Electron flow
Reducing electrons move from NADPH through the selenoprotein thioredoxin reductase to thioredoxin and glutathione
Glucose NADPH Selenocysteine-basedRedox Proteins
(TrxR ,TGR, GPx)
Grx
GR
GSSG GSH
Cysteine-basedRedox Proteins
(Trx , Prx)
e- e- e-
e-
e-
e--Reduction of oxidized proteins and phospholipids.
-Reduction of glutathionylated proteins
e-
e- e-
GR = glutathione reductase Trx = thioredoxinGSH = reduced glutathione TrxR = thioredoxin reductaseGSSG = oxidized glutathione TGR = thioredoxin-glutathione reductaseGPx = glutathione peroxidase Prx = peroxiredoxinGrx = glutaredoxin e- = electron
Selenoproteins provide antioxidant electrons
The selenoprotein Thioredoxin reductase (TrxR) is directly or indirectly responsible for keeping vitamin E, ascorbic acid, glutathione, thioredoxin and ubiquinone reduced.
CpG CpG CpGG6PD gene (on)
G6PD
NADP+
6-P-gluconolactone Glucose-6-P
NADPH Thioredoxin Reductase
Thioredoxin
DNA MethyltransferaseCpG CpG CpG
G6PD gene (off)
CH3 CH3 CH3
MethionineSynthaseActivity
SAMSAH
GSH status
DNADemethylase
Selenoprotein
Glucose
Glucose is the major source of reducing powerfor maintaining reduced glutathione
CH
SH
N
H
H COOC3
2CH
Se
N
H
H COOC3
SELENOCYSTEINE
2
CYSTEINE
SULFUR AND SELENIUM AMINO ACIDS
From Dr. Nicholas Ralston Univ. of North Dakota
Hg2+
Binding Constant = 1045Binding Constant = 1039
(million-fold higher affinity)
Selenoproteins
Thioredoxin fold proteins(dual stable thiols)
Protein thiols (mono thiol sites)
Thiol metabolites (GSH, cysteine)
Hg2+
Mercury gradually migrates to highestaffinity targets (i.e. selenoproteins)
*Mercury inhibition of selenoproteins could disrupt redox regulation and epigenetic regulation during brain development
Selenoprotein Activities
Hg2+( - )
Redox Regulation and Antioxidant Activity
(e.g. TrxR)
EpigeneticRegulation of
Gene Expression
BrainDevelopment
ROS Inactivation and Oxidation Repair
(e.g. GPx)
Thyroid HormoneSynthesis(e.g. DIO)
DNA and HistoneMethylation
Intracellular Redox Status
Selenocysteine
Selenoprotein functions relevant to autism
Methionine Synthase Activity
Astrocyte [GSH] = 0.91 mM
Neuron [GSH] = 0.21 mM
Ependymal [GSH] = 2.73 mM
Selenoprotein P Is high inEpendymal cells
Highest levels of GSH are in selenium-rich ependymal cellswhich are stem cells for astrocytes and neurons
Sun et al. J BIOL CHEM. VOL. 281, pp. 17420–17431, 2006Scharpf et al. J. NEURAL TRANS.VOL 114, 877-884, 2007
Vescovi et al. Nature Reviews Cancer 6, 425–436 (June 2006)
Adult neurogenesis occurs in the subventricular zone (SVZ) of the cortex and the subgranular zone (SGZ) of the hippocampus
Pluripotent Stem Cells(Ependymal Cells)
Oxidized State
MoreAstrocytes
LessNeurons
Pluripotent Stem Cells(Ependymal Cells)
Normal State
AstrocytesNeurons
Pluripotent Stem Cells(Ependymal Cells)
Reduced State
LessAstrocytes
MoreNeurons
Prevailing redox conditions determinethe proportion of neurons vs. astrocyteswhich develop from neuronal stem cells
Eggs are richIn cysteine
Sperm are rich in selenium
GSH
GSH
GSH
GSH
GSH
GSH
∆ DNA and Histone Methylation
∆ GeneExpression
Does Redox ControlDevelopment ViaEpigenetic Effects?
juanv.wordpress.com/
Neu
ron
al c
ells
REDOX SIGNALING IN THEIMMUNE SYSTEM:
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
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
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