Redox and Methyla.on in the Gut, Brain and Immune System. Part I: General Principles. Viewing Life Through Redox Glasses

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1 Overview Redox and in the Gut, Brain and Immune System Part I: General Principles Richard Deth, PhD Northeastern University Boston, MA - 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 Viewing Life Through Redox Glasses Earliest life appears arose at hydrothermal vents emitting hydrogen sulfide. As oxygen became more plentiful, evolution was driven by the ability to adapt and to resist oxidation. Oxidation Reduction H 2 S Oxidation: Loss of an electron Reduction: Gain of an electron From Paul G. Falkowski; Science (2006) Life: The evolved ability to resist oxidation Death: The inevitable outcome of oxidation Primordial Synthesis of From Volcanic Gases 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 CH 3 Hydrogen sulfide H 2 S Ammonia NH 3 Carbon dioxide CO 2 NH 2 CHCOOH CH 2 SH 1

2 The Glutathione Redox Equilibrium EVOLUTION = LAYER UPON LAYER OF USEFUL ADAPTIVE RESPONSES TO OFFSET THE THREAT OF OXIDATION H The ability to control oxidation is at the core of evolution REDUCED 2 G OXIDIZED Each addition is strengthened because it builds on the solid core already in place. - 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. The available level of antioxidant () controls the level of aerobic metabolism and formation = Redox Signaling Glucose NADH + NADPH Selenoproteins Glutathione Antioxidant Supply LANGUAGE SOCIAL SKILLS O 2 + 4H + Glutathionylation of Complex I Reactive Oxygen Species 2H 2 O + formation ROS Oxidative Cellular Damage [Antioxidant] No Damage Mitochondrial respiration is a primary source of reactive oxygen species Oxygen is taken up from the GI tract and is distributed to the body and the brain GI Tract Blood Brain GI Epithelial Cell Blood Brain Barrier Astrocytes + H 2 O Reactive Oxygen Species (ROS) Dietary Protein Liver Neurons From: Kiley P J & Storz G;

3 The brain extracellular environment (CSF) has more than 100- fold less cysteine than plasma!! Neurons [] = 0.21mM [CYS] [CYS] [] = 0.3 μm BRAIN [CysGly] CSF Astrocytes [] = 0.91mM [] [CYS] = 2.2 µm Blood- Brain Barrier BLOOD [] = 3.5 μm [CYS] =282 μm for glutathione synthesis can be provided by either transsulfuration of homocysteine or by uptake from outside the cell NEURONAL CELL Transsulfuration Cysteinylglycine Glutathione γ- Glutamylcysteine Synthesis Cystathionine Adenosine >150 Reactons Glial Cells (Astrocytes) 12-fold lower 125-fold lower Dietary protein Data from: Castagna et al. Neurology, 45: (1995) and Sun et al. J Biol Chem, 281: (2006). Cognitive Status Nitric Oxide Synthesis synthase provides redox-sensitive global coordination of metabolism: HOMEOSTASIS REDOX STATUS: GSSH Catecholamine Status: Arginine ~ 200 Reactions Gene Expression DNA/Histone HIONINE SYNTHASE REGULATION DNA MS DNA TNF- α Alternative Splicing MS pre- RNA MS RNA t- RNA Amino Acids GLOBAL REGULATION Ubiquitination Protein Translation Energy Status Creatine Synthesis Phospholipid Membrane Properties Serotonin Melatonin Sleep Cellular Redox Status MS Cystathionine [] [] Global Metabolic Coordination RNA Splicing DNA Transcription of DNA and histones is fundamental to epigenetic regulation of gene expression during development Regulation of gene expression during development TranscripEon Factor RegulaEon: Growth Factors Neurotransmi:ers Hormones CpG TF CpG TF binding region DNA EpigeneEc RegulaEon: Start site for mrna synthesis RNA polymerase X Gene sequence TranscripEon mrna TranslaEon Protein (e.g. enzyme) DNA DNMT MBDP (e.g. MeCP2) Me Me CpG CpG TF binding region HMT Me Me Me Me Histone proteins X No mrna DNA + Histone = HeterochromaEn Genes are silenced and transcripeon is blocked 3

4 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 will interfere with the many roles of epigenetic regulation. PrEP Prenatal Epigenetic Programming Maternal Metabolism Maternal Nutrition Maternal Toxic Exposures Placental Function Genetic Factors BIRTH PEP Postnatal Epigenetic Programming Breast Milk (Formula) Toxic Exposures Physical Experiences Emotional Experiences Antioxidant Availability (i.e. cysteine and ) Metabolic Activity (Antioxidant demand) Odds of autism decrease with increasing duration of breastfeeding Homeostatic Equilibrium Metabolic Activity (Higher antioxidant demand) Antioxidant Availability (Low cysteine and ) Oxidative Stress Adaptive Epigenetic Changes 4

5 GI tract absorption of cysteine is critical for postnatal epigenetic programming (PEP) GI TRACT BLOOD BRAIN Dietary Protein GI Epithelial Cells LIVER Blood Brain Barrier Astrocytes Neurons REDOX & HYLATION PATHWAYS" D4-" Phospholipid" " Transsulfuration! Pathway" D4-Receptor " PLM Cycle" D4-" Adenosine" Methyl-" PP + Pi" " " D4-" D4-" Dopamine (Attention)" Glutathione ()" #-Glutamylcysteine" " " " Cystathionine" " " Adenosine" Methyl-" "! " PP + Pi" Redox" Buffering" " Cycle" " > 150" " Reactions" " ( - )" Neurons have impaired transsulfuration and low levels that depend upon growth factor-stimulated cysteine uptake Neurotrophic Growth Factors PI3- kinase Phospholipid D4 D4 PARTIALLY BLOCKED IN NEURONAL CELLS Cystathionine Adenosine Dopamine Cysteinylglycine D4 D4 γ- Glutamylcysteine Astrocytes Cys.ne Adenosine >1,000 Reac.ons NEURON Levels of cystathionine are markedly higher in human cortex than in other species [Cystathionine] mg / 100 gm wet wt Human Brain Monkey Rat Guinea Pig Cat Cow Chicken Duck Human Liver Human Kidney Human Muscle Tallan HH, Moore S, Stein WH. L-cystathionine in human brain. J Biol Chem Feb;230(2): Growth factor regulation of cysteine uptake is a powerful mechanism to regulate redox and methylation in the brain REDOX SIGNALING: Neurotrophic growth factors increase -mediated uptake of cysteine. Neurotrophic Growth Factors PI3- kinase Cysteinylglycine γ- Glutamylcysteine Glial Cells (Astrocytes) Leading to: -Increased / -Increased methionine synthase activity -Increased DNA methylation and epigenetic consequences Cystathionine Adenosine >150 Reactons 5

6 IGF-1 stimulates methionine synthase activity and increases DNA methylation in human neuronal cells By increasing cysteine uptake, neurotrophic growth factors can increase methionine synthase activity and regulate gene expression Neurotrophic Growth Factors PI3- kinase Cysteinylglycine γ- Glutamylcysteine Epigene.c regula.on Cystathionine of gene Adenosine expression DNA Glial Cells (Astrocytes) REDOX SIGNALING: TNF-alpha decreases cysteine uptake Tumor necrosis factor alpha (TNF-alpha), a pro-inflammatory cytokine, decreases -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 causes a large and prompt decrease in methionine synthase mrna Despite inhibition of cysteine uptake, cysteine and levels do not decrease after TNF-alpha, indicating that it increases transsulfuration 6

7 TNF-alpha decreases cysteine uptake and decreases methionine synthase activity, but increases transsulfuration TNF- alpha ( ) ( ) ( ) Cysteinylglycine γ- Glutamylcysteine Cystathionine Adenosine Glial Cells (Astrocytes) Epigene.c regula.on of gene expression DNA NEUROTROPHIC GROWTH FACTORS" GROWTH FACTORS" " -Mediated Influx" Epigenetic" Regulation" ( - )" CYSTEINE" " Redox Status" TNF-alpha (Inflammatory Cytokine)" " Transsulfuration" " " " " " " " HYLATION" ( > 150 Reactions )" Myelination" Neural Network" Synchronization" Oxygen, sulfur and selenium are chemically similar in the periodic table. Selenium and selenoproteins in redox regulation Water Oxygen e.g ROS e - Sulfur e.g. Reducing equivalents are passed from selenium to sulfur and from sulfur to oxygen Reducing electrons move from NADPH through the selenoprotein thioredoxin reductase to thioredoxin and glutathione Thioredoxin Reductase e - Selenium e.g. Thioredoxin Reductase Illustrations from: Bentor, Yinon. Chemical Element.com NADPH Hydrogen (Reducing Equivalent) Electron flow 7

8 Selenoproteins provide an.oxidant electrons The selenoprotein Thioredoxin reductase (TrxR) is directly or indirectly responsible for keeping vitamin E, ascorbic acid, glutathione, thioredoxin and ubiquinone reduced. Glucose e - e - Selenocysteine- based e - NADPH Redox Proteins e - (TrxR,TGR, GPx) e GR - e - e - Grx e - - based Redox Proteins (Trx, Prx) e - - Reduc.on of oxidized proteins and phospholipids. - Reduc.on of glutathionylated proteins GR = glutathione reductase Trx = thioredoxin = reduced glutathione TrxR = thioredoxin reductase = oxidized glutathione TGR = thioredoxin- glutathione reductase GPx = glutathione peroxidase Prx = peroxiredoxin Grx = glutaredoxin e - = electron Glucose is the major source of reducing power for maintaining reduced glutathione Selenoprotein Glucose NADP + Glucose- 6- P G6PD NADPH Thioredoxin Reductase 6- P- gluconolactone Thioredoxin CpG CpG CpG G6PD gene (on) status DNA Demethylase CpG CpG CpG CH 3 CH 3 CH 3 G6PD gene (off) DNA Methyltransferase Ac.vity SULFUR AND SELENIUM AMINO ACIDS H N 3 H C COO CH SH CYSTEINE Binding Constant = Hg 2+ H N 3 H C COO CH 2 Se SELENOCYSTEINE Binding Constant = (million-fold higher affinity) From Dr. Nicholas Ralston Univ. of North Dakota 8

9 Mercury gradually migrates to highest affinity targets (i.e. selenoproteins) Selenoproteins Thioredoxin fold proteins (dual stable thiols) Protein thiols (mono thiol sites) Thiol metabolites (, cysteine) Hg 2+ Selenoprotein func.ons relevant to au.sm Selenocysteine Hg 2+ Highest levels of are in selenium-rich ependymal cells which are stem cells for astrocytes and neurons Selenoprotein Ac.vi.es Redox Regula.on and An.oxidant Ac.vity (e.g. TrxR) Thyroid Hormone Synthesis (e.g. DIO) ROS Inac.va.on and Oxida.on Repair (e.g. GPx) Selenoprotein P Is high in Ependymal cells Ac.vity Intracellular Redox Status DNA and Histone Epigene.c Regula.on of Gene Expression Brain Development Astrocyte [] = 0.91 mm Neuron [] = 0.21 mm *Mercury inhibi.on of selenoproteins could disrupt redox regula.on and epigene.c regula.on during brain development Ependymal [] = 2.73 mm Sun et al. J BIOL CHEM. VOL. 281, pp , 2006 Scharpf et al. J. NEURAL TRANS.VOL 114, , 2007 Adult neurogenesis occurs in the subventricular zone (SVZ) of the cortex and the subgranular zone (SGZ) of the hippocampus Prevailing redox conditions determine the proportion of neurons vs. astrocytes which develop from neuronal stem cells Pluripotent Stem Cells (Ependymal Cells) Oxidized State Pluripotent Stem Cells (Ependymal Cells) Normal State Pluripotent Stem Cells (Ependymal Cells) Reduced State Less More Neurons Astrocytes Neurons Astrocytes More Less Astrocytes Neurons Vescovi et al. Nature Reviews Cancer 6, (June 2006) 9

10 Sperm are rich in selenium Does Redox Control Development Via Epigenetic Effects? Eggs are rich In cysteine Gene Expression DNA and Histone Neuronal cells juanv.wordpress.com/ Redox signaling plays a central role in the immune response REDOX SIGNALING IN THE IMMUNE SYSTEM: Dendritic cells release, similar to astocytes in brain Antigen Effector T-cells take up cysteine, similar to neurons in brain Antibody production Regulatory T-cells compete with T eff for cysteine and restrict the immune response provides uptake is expressed in T cell lymphocytes and highest expression is in Treg cells Autoimmune-prone SJL/J mice have lower expression in mixed T cell lymphocytes 10

11 Autoimmune-prone SJL/J mice have lower levels of in brain, unaffected by postnatal thimerosal treatment Autoimmune-prone SJL/J mice have lower methionine synthase activity in brain, unaffected by postnatal thimerosal treatment ACKNOWLEDGEMENTS 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 11