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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 expression Prenatal epigenetic programming (PREP)

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Redox and Methylation in the Gut, Brain and Immune System Part I: General Principles

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  1. Redox and Methylation in the Gut, Brain and Immune System Part I: General Principles Richard Deth, PhD Northeastern University Boston, MA

  2. Overview • Oxidation and antioxidant metabolism • Epigenetic regulation of gene expression • Prenatal 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

  3. Viewing Life Through Redox Glasses • Oxidation: Loss of an electron • Reduction: Gain of an electron

  4. As oxygen became more plentiful, evolution was driven by the ability to adapt and to resist oxidation. Earliest life appears arose at hydrothermal vents emitting hydrogen sulfide. H2S From Paul G. Falkowski; Science311 1724 (2006)

  5. 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

  6. NH2CHCOOH CH2 SH Primordial Synthesis of Cysteine From Volcanic Gases Methane CH3 Hydrogen sulfide H2S Ammonia NH3 Carbon dioxide CO2 Cysteine

  7. The Glutathione Redox Equilibrium G H 2 OXIDIZED GSSG REDUCED GSH

  8. EVOLUTION = LAYER UPON LAYER OF USEFUL ADAPTIVE RESPONSES TO OFFSET THE THREAT OF OXIDATION The ability to control oxidation is at the core of evolution Each addition is strengthened because it builds on the solid core already in place.

  9. - 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. SOCIAL SKILLS LANGUAGE

  10. The available level of antioxidant (GSH) controls the level of aerobic metabolism and ATP formation = Redox Signaling Selenoproteins Antioxidant Supply Glucose NADPH Glutathione NADH+ O2 + 4H+ 2H2O + ATP formation Reactive Oxygen Species ROS [Antioxidant] Oxidative Cellular Damage No Damage Glutathionylation of Complex I

  11. Mitochondrial respiration is a primary source of reactive oxygen species Oxygen ATP + H2O Reactive Oxygen Species (ROS) From: Kiley P J & Storz G; 2004

  12. Cysteine is taken up from the GI tract and is distributed to the body and the brain GSH

  13. The brain extracellular environment (CSF) has more than 100-fold less cysteine than plasma!! Blood-Brain Barrier BRAIN BLOOD Neurons Astrocytes [GSH] = 0.21mM [GSH] = 0.91mM [GSH] = 3.5 μM [CYS] [CYS] =282 μM [CYS] [CysGly] [GSH] CSF [GSH] = 0.3 μM [CYS] = 2.2μM 12-fold lower 125-fold lower Data from: Castagna et al. Neurology, 45:1678-83 (1995) and Sun et al. J Biol Chem, 281:17420-31 (2006).

  14. Cysteine for glutathione synthesis can be provided by either transsulfuration of homocysteine or by uptake from outside the cell NEURONAL CELL

  15. Cognitive Status Nitric Oxide Synthesis Catecholamine Methylation Arginine Methylation Gene Expression REDOX STATUS: GSH GSSH Methylation Status: SAM SAH ~ 200 Methylation Reactions DNA/Histone Methylation Serotonin Methylation Phospholipid Methylation Creatine Synthesis Melatonin Membrane Properties Energy Status Sleep

  16. Methionine synthase provides redox-sensitive global coordination of metabolism: HOMEOSTASIS

  17. Methylation of DNA and histones is fundamental to epigenetic regulation of gene expression during development

  18. Regulation of gene expression during development X Transcription Factor Regulation: Growth Factors Start site for mRNA synthesis Neurotransmitters Hormones TF CpG CpG TF binding region Gene sequence DNA RNA polymerase mRNA Transcription Translation Protein (e.g. enzyme) Epigenetic Regulation: Me SAM Me MBDP (e.g. MeCP2) HMT Histone proteins Me Me SAM CpG CpG DNMT Me X No mRNA Me TF binding region DNA DNA + Histone = Heterochromatin Genes are silenced and transcription is blocked

  19. Epigenetic changes in gene expression are the Primary mechanism underlying development

  20. Epigenetic marks change in response to the environment and contribute to adaptive capabilities gained through evolution

  21. 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 will • interfere with the many roles of • epigenetic regulation.

  22. PrEP PEP Postnatal Epigenetic Programming Prenatal Epigenetic Programming Maternal Metabolism Maternal Nutrition Maternal Toxic Exposures Placental Function Genetic Factors Breast Milk (Formula) Toxic Exposures Physical Experiences Emotional Experiences BIRTH

  23. Antioxidant Availability (i.e. cysteine and GSH) Metabolic Activity (Antioxidant demand) Homeostatic Equilibrium ↑Metabolic Activity (Higher antioxidant demand) ↓Antioxidant Availability (Low cysteine and GSH) Adaptive Epigenetic Changes Oxidative Stress →

  24. Odds of autism decrease with increasing duration of breastfeeding

  25. GI tract absorption of cysteine is critical for postnatal epigenetic programming (PEP)

  26. Redox Buffering Glutathione (GSH) TranssulfurationPathway γ-Glutamylcysteine Cysteine Methionine Cycle D4-Receptor PLM Cycle Cystathionine Adenosine Adenosine • REDOX & METHYLATION PATHWAYS D4-SAH D4-HCY HCY SAH ( - ) Methyl-THF Methyl-THF Phospholipid Methylation Methionine Synthase > 150 Methylation Reactions THF THF THF D4-MET D4-SAM SAM MET PP+ Pi PP+ Pi ATP ATP Dopamine (Attention)

  27. Neurons have impaired transsulfuration and low GSH levels that depend upon growth factor-stimulated cysteine uptake Neurotrophic Growth Factors Astrocytes Cysteine Cysteinylglycine Cystine GSH GSH EAAT3 ( + ) PI3-kinase GSH GSSG EAAT3 γ-Glutamylcysteine Cysteine PARTIALLY BLOCKED IN NEURONAL CELLS Cystathionine Adenosine Adenosine D4SAH D4HCY SAH HCY ( - ) MethylTHF MethylTHF Phospholipid Methylation • >1,000 • Methylation • Reactions Methionine Synthase THF THF D4SAM D4MET SAM MET ATP PP+Pi ATP PP+Pi Dopamine NEURON

  28. Levels of cystathionine are markedly higher in human cortex than in other species Tallan HH, Moore S, Stein WH. L-cystathionine in human brain. J Biol Chem. 1958 Feb;230(2):707-16.

  29. REDOX SIGNALING: Neurotrophic growth factors increase EAAT3-mediated uptake of cysteine. Leading to: -Increased GSH/GSSG -Increased methionine synthase activity -Increased DNA methylation and epigenetic consequences

  30. Growth factor regulation of cysteine uptake is a powerful mechanism to regulate redox and methylation in the brain Glial Cells (Astrocytes) Neurotrophic Growth Factors Cysteinylglycine Cysteine GSH EAAT3 GSH GSSG ( + ) PI3-kinase γ-Glutamylcysteine Cysteine EAAT3 Cystathionine Adenosine SAH HCY ( - ) MethylTHF • >150 • Methylation • Reactons Methionine Synthase THF SAM MET ATP PP+Pi

  31. IGF-1 stimulates methionine synthase activity and increases DNA methylation in human neuronal cells

  32. By increasing cysteine uptake, neurotrophic growth factors can increase methionine synthase activity and regulate gene expression Glial Cells (Astrocytes) Cysteinylglycine Cysteine GSH Neurotrophic Growth Factors EAAT3 GSH GSSG ( + ) PI3-kinase γ-Glutamylcysteine ( + ) • Epigenetic regulation • of gene expression Cysteine EAAT3 Cystathionine Adenosine ( + ) SAH HCY ( - ) MethylTHF • DNA • Methylation Methionine Synthase THF SAM MET ATP PP+Pi

  33. 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

  34. TNF-alpha decreases cysteine uptake

  35. TNF-alpha causes a large and prompt decrease in methionine synthase mRNA

  36. Despite inhibition of cysteine uptake, cysteine and GSH levels do not decrease after TNF-alpha, indicating that it increases transsulfuration

  37. TNF-alpha decreases cysteine uptake and decreases methionine synthase activity, but increases transsulfuration Glial Cells (Astrocytes) Cysteinylglycine TNF- alpha Cysteine GSH ) ) ) ( ( ( EAAT3 GSH GSSG γ-Glutamylcysteine • Epigenetic regulation • of gene expression Cysteine ( + ) Cystathionine Adenosine SAH HCY MethylTHF • ↓ DNA • Methylation Methionine Synthase THF SAM MET ATP PP+Pi

  38. NEUROTROPHIC GROWTH FACTORS GSH GSSG Methionine Synthase EAAT3-Mediated Influx Transsulfuration CYSTEINE MET SAM SAH HCY GROWTH FACTORS TNF-alpha (Inflammatory Cytokine) ( + ) ( + ) ( - ) Redox Status METHYLATION ( > 150 Reactions ) Epigenetic Regulation Neural Network Synchronization Myelination

  39. Selenium and selenoproteins in redox regulation

  40. Oxygen, sulfur and selenium are chemically similar in the periodic table.

  41. Water Oxygen e.g ROS Reducing equivalents are passed from selenium to sulfur and from sulfur to oxygen e- Sulfur e.g. GSH e- NADPH Selenium e.g. Thioredoxin Reductase Hydrogen (Reducing Equivalent) Illustrations from: Bentor, Yinon. Chemical Element.com

  42. Reducing electrons move from NADPH through the selenoprotein thioredoxin reductase to thioredoxin and glutathione Thioredoxin Reductase Electron flow

  43. Selenoproteins provide antioxidant electrons e- e- e- Selenocysteine-based Redox Proteins (TrxR ,TGR, GPx) Cysteine-based Redox Proteins (Trx , Prx) Glucose NADPH e- e- e- GR e- -Reduction of oxidized proteins and phospholipids. -Reduction of glutathionylated proteins e- e- GSSG GSH Grx GR = glutathione reductase Trx = thioredoxin GSH = reduced glutathione TrxR = thioredoxin reductase GSSG = oxidized glutathione TGR = thioredoxin-glutathione reductase GPx = glutathione peroxidase Prx = peroxiredoxin Grx = glutaredoxin e- = electron

  44. The selenoprotein Thioredoxin reductase (TrxR) is directly or indirectly responsible for keeping vitamin E, ascorbic acid, glutathione, thioredoxin and ubiquinone reduced.

  45. Glucose is the major source of reducing power for maintaining reduced glutathione Selenoprotein NADPH Thioredoxin Reductase NADP+ 6-P-gluconolactone Glucose-6-P Glucose G6PD Thioredoxin CpG CpG CpG G6PD gene (on) GSH status DNA Demethylase Methionine Synthase Activity G6PD gene (off) CpG CpG CpG SAM SAH DNA Methyltransferase CH3 CH3 CH3

  46. SULFUR AND SELENIUM AMINO ACIDS H H H N C COO H N C COO 3 3 CH CH 2 2 Se SH CYSTEINE SELENOCYSTEINE Binding Constant = 1045 Binding Constant = 1039 Hg2+ (million-fold higher affinity) From Dr. Nicholas Ralston Univ. of North Dakota

  47. Mercury gradually migrates to highest affinity targets (i.e. selenoproteins) Selenoproteins Thioredoxin fold proteins (dual stable thiols) Protein thiols (mono thiol sites) Thiol metabolites (GSH, cysteine) Hg2+

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