Redox Biology PDF

15 REDOX BIOLOGY 1  16. REDOX BIOLOGY I — CONTENTS — I. THE OXYGEN PARADOX 3 Sources of free radicals and oxidants superoxide anion radical I...

15 REDOX BIOLOGY 1  16. REDOX BIOLOGY I — CONTENTS — I. THE OXYGEN PARADOX 3 Sources of free radicals and oxidants superoxide anion radical II. FREE RADICALS AND OXIDATIVE STRESS hydrogen peroxide 1 What are free radicals mitochondrial sources a Definition 4 How free radicals mediate damage b Mechanism of formation lipid peroxidation c Electron-transfer reactions DNA oxidation superoxide anion radical 5 How cells protect themselves against hydrogen peroxide free radical damage hydroxyl radical Superoxide dismutases 2 Reactivity of free radicals Catalase superoxide anion radical Glutathione peroxidase hydrogen peroxide Peroxiredoxins hydroxyl radical Vitamin E Vitamin C Nrf2 and Friedreich Ataxia 2  15. REDOX BIOLOGY – LEARNING OBJECTIVES – Define free radicals and oxidants and the pathways for their formation in humans Discuss the reactivity of hydroxyl radical and whether or not there are cellular sources of this species Describe mitochondrial sources of superoxide radical and hydrogen peroxide and topography Define the steps of lipid peroxidation and identify the propagating species 3  15. REDOX BIOLOGY – LEARNING OBJECTIVES – Explain the function of specific antioxidant defenses or preventive antioxidants Distinguish the mechanism of the enzymes that remove hydrogen peroxide Discuss the mechanism of Amyotrophic Lateral Sclerosis as a function of mutated SOD1 Describe the mechanism of a-tocopherol transfer protein and clinical implications Explain the synergism between vitamins E and C Distinguish the role of free radicals and oxidants involved in oxidative eustress (health) and oxidative stress (disease) 4  I. THE OXYGEN PARADOX H2O CO2 NADP+ The element oxygen exists in air as a ADP + Pi RuBP diatomic molecule, O2. Except for certain photosystem II 3PGA electron-transport chain anaerobic and aerobic-tolerant unicellular photosystem I CALVIN CYCLE organisms, all animals, plants, and bacteria ATP require O2 for efficient production of energy by NADPH G3P the use of the O2-dependent electron transport chains, such as those in the mitochondria of sucrose eukaryotic cells. O2 appeared in significant 3PGA = 3-phospho-glyceric acid O2 G3P = glyceraldehyde-3-phosphate amounts in the earth's atmosphere over 2.5 x RuBP = ribulose-bis-phosphate 109 years ago, due to the evolution of photosyn- thesis by blue-green algae (cyanobacteria): as they split H2O to obtain the H needed to drive metabolic reductions, these bacteria released tones of O2 into the atmosphere. ! 5  I. THE OXYGEN PARADOX O2 appeared in significant amounts in the earth's atmosphere over 2.5 x 109 years ago, due to the evolution of photosynthesis by blue-green algae (cyanobacteria): as they split H2O, these bacteria released tones of O2 into the atmosphere. Other organisms began the evolutionary process of evolving antioxidant defense systems to protect against O2 toxicity. Organisms that tolerated the presence of O2 could evolved to use it for metabolism for efficient energy complex complex organisms production by using electron-transfer chains with O2 as the terminal acceptor, such as those organisms mitochondria complex present in mitochondria. mitochondria evolution organisms evolution mitochondria 21% atmosphere) evolution 21% (%ininatmosphere) oxygen oxygen utilization Oxygen photosynthetic utilization Oxygen 21% atmosphere) photosynthetic cells oxygen formation first cells utilization Oxygen formation of earth first cells photosynthetic of earth cells cells (% in(% formation first of earth cells 0 1 2 3 4 0 1 2 3 4 Time (billions of years) Time (billions of years) present 0 1 2 3 4 present day 6 day Time (billions of years)  I. THE OXYGEN PARADOX EVOLUTION OF COMPLEX ORGANISMS Among the evolutionary adaptations to the EFFICIENT OXIDATION OF FUELS CARBOHYDRATES appearance of O2 is the development of anti- oxidant defenses that allowed the evolution of O2 ENERGY + O2-using enzymes and electron-transfer chains FATS CO2 that enable the oxidation of food material more efficiently. PROTEINS THE OXYGEN PARAD RUST RUST! O2 is a double-edged sword: it promotes the efficient oxidation of fuel and production of energy and, at the same time, is involved in oxidation reactions in humans and in objects (common rust). 7  I. THE OXYGEN PARADOX O2 composes 21% of the atmosphere. Humans can take up to 40-50% O2 for several hours for medical treatment but will die from lung injury if exposed to 100% O2 for an extended period of time. Retinopathy of prematurity (ROP) – blindness of infants: it first arose in the 1940s when premature babies were being placed in incubators with high O2 levels. ROP has decreased in severity due to ! better O2 monitoring and supplementation of babies with vitamin E. 8  II. FREE RADICALS AND OXIDATIVE STRESS The formation of free radicals or oxidants is a well-established physio- logical event in aerobic cells, which convene enzymic and non-enzymic resources, known as antioxidant defenses, to remove these oxidizing species. An imbalance between oxidants and antioxidants, the two terms of the equation that defines oxidative stress, OXIDANTS ANTIOXIDANTS and the consequent damage to cell molecules constitutes the basic tenet of several pathophysiological states. 9  OXIDANTS ANTIOXIDANTS 1 What are oxygen radicals? 5 How do cells protect themselves against oxygen radicals 2 How reactive are oxygen radicals? 3 How are they generated in the cell? 4 How do they mediate cellular damage? 10  II. FREE RADICALS AND OXIDATIVE STRESS OXIDANTS ANTIOXIDANTS OXIDANTS ANTIOXIDANTS 1 What 1 What are oxygen are oxygen radicals? radicals? 11  II. FREE RADICALS AND OXIDATIVE STRESS 1 WHAT ARE OXYGEN RADICALS? a. Definition A free radical is defined as any species that contains one or more unpaired electron occupying an atomic or 2s 2p molecular orbital by itself. The box diagram configuration for O2 shows that in itself oxygen a diradical, because it possesses two unpaired electrons; the Lewis dot diagram shows also the diradical character of molecular oxygen. O O Based on the above definition, molecular oxygen is a radical, for it contains two unpaired electrons 12  II. FREE RADICALS AND OXIDATIVE STRESS 1 WHAT ARE OXYGEN RADICALS? c. Formation of oxidants by electron-transfer reactions The following scheme illustrates the sequential univalent reduction of O2 to H2O with formation of different intermediates: superoxide anion radical (O2.–), hydrogen peroxide (H2O2), and hydroxyl radical (HO.): a + 4e– O2 + 1e– O2.– + 1e– H2O2 + 1e– HO. + 1e– H2O 1 molecular superoxide hydrogen hydroxyl water oxygen anion peroxide radical 0n 02 05 Hoz Ho HO 13  II. FREE RADICALS AND OXIDATIVE STRESS 1 WHAT ARE OXYGEN RADICALS? c. Electron-transfer reactions: Superoxide anion radical The addition of one electron to molecular O2 results in the formation of superoxide anion radical (O2.–). O2.– is not a very reactive species and its chemical reactivity will depend on its site of generation in the cell, the possibility of being protonated to a stronger oxidant (perhydroxyl radical), and collision with suitable substrates. O2 O2.– H2O2 HO. H2O a molecular superoxide hydrogen hydroxyl water oxygen anion peroxide radical O O + 1 e– O O molecular superoxide oxygen anion O2 O2 +1 e– 14  II. FREE RADICALS AND OXIDATIVE STRESS 1 WHAT ARE OXYGEN RADICALS? 2e– 1e– O2 O2.– H2O2 HO. H2O a molecular superoxide hydrogen hydroxyl water oxygen anion peroxide radical c. Electron-transfer reactions: Hydrogen peroxide Hydrogen peroxide can be formed by: addition of two electrons to molecular oxygen O O + 2 e– O O molecular hydrogen oxygen peroxide addition of one electron to superoxide anion O O + 1 e– O O ( ) superoxide hydrogen anion peroxide 15  II. FREE RADICALS AND OXIDATIVE STRESS 1 WHAT ARE OXYGEN RADICALS? O2 O2.– H2O2 HO. H2O a molecular superoxide hydrogen hydroxyl water oxygen anion peroxide radical c. Electron-transfer reactions: Hydroxyl radical is formed upon one-electron reduction of hydrogen peroxide H O O H + 1 e– HO + O H hydrogen hydroxyl hydroxyl peroxide anion radical H2O2 HO. + HO– 16  II. FREE RADICALS AND OXIDATIVE STRESS 1 WHAT ARE OXYGEN RADICALS? c. Formation of oxidants by electron-transfer reactions + 4 e– + 1 e– + 1 e– + 1 e– + 1 e – O2 O2.– H2O2 HO. H2O molecular superoxide hydrogen hydroxyl water oxygen anion peroxide radical.........–....... O—O...... O—O....... O—O...... H.. O... has two has one has no has one unpaired electrons unpaired electron unpaired electrons unpaired electron it is a diradical it is a radical it is not a radical it is a radical radical not radios Dai al 17  II. FREE RADICALS AND OXIDATIVE STRESS 2 HOW REACTIVE ARE OXYGEN RADICALS AND OXIDANTS? OXIDANTS ANTIOXIDANTS OXIDANTS ANTIOXIDANTS 2 How 2 How reactive are reactive oxygen are oxygen radicals? radicals? 18  II. FREE RADICALS AND OXIDATIVE STRESS 2 HOW REACTIVE ARE OXYGEN RADICALS AND OXIDANTS? The chemical reactivity, whether of free radical character or not, varies substantially; in an appropriate setting all cell components –lipids, nucleic acids, proteins, and carbohydrates– are sensitive to damage by reactive species (encompassing oxygen-, nitrogen-, carbon-, and sulfur-centered radicals). O2 + 1e– O2.– + 1e– H2O2 + 1e– HO. + 1e– H2O molecular superoxide hydrogen hydroxyl water oxygen anion peroxide radical modest reactivity or damage to biomolecules no reactivity the most reactive radical in biological systems 19  II. FREE RADICALS AND OXIDATIVE STRESS 2 HOW REACTIVE ARE OXYGEN RADICALS AND OXIDANTS? a. Superoxide anion.– O2 O2 H2 O2 HO. H2 O a m olecular superoxide hydrogen hydroxyl water oxygen anion peroxide radical Two reactions of O2.– are important in a cellular setting: The reaction of O2.– with itself: dismutation modest reactivity or disproportionation. Products are hydrogen or damage to biomolecules peroxide and oxygen O2.– + O2.– + 2H+ ® H2O2 + O2 The protonation of O2.– to perhydroxyl radical in the vicinity of membranes, where the concentration of protons is higher: O2.– + H+ ® HO2. 20  II. FREE RADICALS AND OXIDATIVE STRESS 2 HOW REACTIVE ARE OXYGEN RADICALS AND OXIDANTS? a. Superoxide anion – O2 O2. H2 O2 HO. H2 O a molecular superoxide hydrogen hydroxyl water oxygen anion peroxide radical 2.1. Dismutation: O2.– + O2.– + 2H+ ® H2O2 + O2 O O + O O + 2H+ H O OH + O O superoxide superoxide hydrogen molecular anion anion peroxide oxygen 2.1. Protonation: O2.– + H+ ® HO2. H+ O O O OH superoxide perhydroxyl anion radical 21  II. FREE RADICALS AND OXIDATIVE STRESS 2 HOW REACTIVE ARE OXYGEN RADICALS AND OXIDANTS? b. Hydrogen peroxide – O2 O2. H2 O2 HO. H2 O a molecular superoxide hydrogen hydroxyl water oxygen anion peroxide radical Hydrogen peroxide is not a free radical and per se is very little reactive. Its reac- tivity in biological systems depends on: It can diffuse long distances and cross no reactivity membranes It can react with transition metals (e.g., Fe, Cu) and be cleaved to the highly reactive hydroxyl radical It can enter the cell by aquaporins or peroxyporins 22  II. FREE RADICALS AND OXIDATIVE STRESS 2 HOW REACTIVE ARE OXYGEN RADICALS AND OXIDANTS? c. Hydroxyl radical.– O2 O2 H2 O2 HO. H2 O molecular superoxide hydrogen hydroxyl water oxygen anion peroxide radical Hydroxyl radical is the most powerful oxidant n generated in biological systems and it reacts indiscriminately with all molecules. the most reactive The biochemical reactivity of hydroxyl radical radical in biological systems encompasses two type of reactions: Hydrogen abstraction state H ab stun Addition m 23  II. FREE RADICALS AND OXIDATIVE STRESS 2 HOW REACTIVE ARE OXYGEN RADICALS AND OXIDANTS? c. Hydroxyl radical Hydrogen abstraction Hydroxyl radical may react with any compound abstracting a hydrogen and yielding a free radical species of the compound and water: RH + HO. ® R. + H2O e.g., Hydroxyl radical-mediated H abstraction on DNA yields strand breaks Cu+ Cu++ Cu++ site-specific Fenton reaction H2O2 HO strand breaks Hydrogen abstraction Addition Hydroxyl radical adds to desoxigua- O O nosine and other DNA bases to yield 8- HN N + OH HN N OH hydroxydesoxyguanosine (8OHdG), which H2N N N H2N N N R R can be isolated in vivo; 8OHdG is a fingerprint of HO. attack on DNA. desoxy- 8-hydroxy- guanosine desoxyguanosine 24  II. FREE RADICALS, OXIDATIVE STRESS, AND DISEASES 2 HOW REACTIVE ARE OXYGEN RADICALS AND OXIDANTS? c. Hydroxyl radical Chemical Reactivity towards DNA H abstraction Addition strand breaks 8-dG 25  II. FREE RADICALS AND OXIDATIVE STRESS 3 CELLULAR SOURCES OF REACTIVE OXYGEN SPECIES OXIDANTS ANTIOXIDANTS OXIDANTS ANTIOXIDANTS 3 How are oxygen radicals 3 How are oxygen radicals generated in the cell? generated in the cell? 26  II. FREE RADICALS AND OXIDATIVE STRESS 3 CELLULAR SOURCES OF REACTIVE OXYGEN SPECIES Cells have sources of superoxide anion (O2.–) and hydrogen peroxide (H2O2). Hydroxyl radical (HO.) is generated from O2.– and H2O2. a. Superoxide anion radical The following table lists the most important reactions within the cell that generate superoxide anion (O2.–). Enzymatic reactions Cellular sources Environmental factors NADPH oxidase leukocytes and macrophages ultraviolet light NADPH-P450 reductase mitochondrial electron transfer X-rays xanthine oxidase microsomal monooxygenase toxic chemicals Hydroxylamines nitro compounds insecticides chemotherapeutic agents 27  II. FREE RADICALS AND OXIDATIVE STRESS 3 CELLULAR SOURCES OF REACTIVE OXYGEN SPECIES b. Hydrogen peroxide Hydrogen peroxide (H2O2) is generated within the cell by two distinct processes: non-radical or enzymic generation and radical or from superoxide anion disproportionation. Enzymic generation Dismutation of O2.– (non radical) (radical) glycolate oxidase O2.– + O2.– + 2H+ ® H2O2 + O2 acetyl-CoA oxidase D-amino acid oxidase NADH oxidase urate oxidase monoamine oxidase 28  II. FREE RADICALS AND OXIDATIVE STRESS 3 CELLULAR SOURCES OF REACTIVE OXYGEN SPECIES Mitochondria as major sources of O2.– and H2O2 I Mitochondria consume O2 associated with the electron leak electron leak process of oxidative phosphorylation. Under – O2 1-2% 2-3% Q e normal conditions, approximately 98-99% of the O2.–, H2O2 oxid III O2 is reduced to H2O as a consequence of aerobic oxidants respiration c metabolism; a small fraction of the O2 consumed O2 98-99% 95-98% IV (1-2%) is reduced univalently to O2.–, process H2O known as electron leak from the mitochondrial oxidative respiratory chain. damage 29  II. FREE RADICALS AND OXIDATIVE STRESS 3 CELLULAR SOURCES OF REACTIVE OXYGEN SPECIES Mitochondria as major sources of O2.– and H2O2 Superoxide anion and hydrogen peroxide are H2O2 formed in mitochondria at: The respiratory chain located in the mitochon- H2O2 dam ative a ge oxid drial inner membrane and through autoxidation O2.– nts 2 o xida 2-3% H2 O of ubisemiquinone. O2.– dismutates to H2O2 and O2 ants ak leak O2.–, irati oxid O2 dam tive on lectron age on a 8% oxid electr ele 95-9 e– resp H2 O O2 is released from mitochondria I Q III c IV MAO The monoamine oxidase activity (MAO) O2 located in the outer mitochondrial membrane and through oxidative deamination of biogenic H2O2 amines. 30  II. FREE RADICALS AND OXIDATIVE STRESS 3 CELLULAR SOURCES OF REACTIVE OXYGEN SPECIES c. Hydroxyl radical There are not enzymatic sources of hydroxyl radical. Most of the HO. generated in vivo originates from the breakdown of H2O2 via a Fenton reaction that entails a metal-dependent reduction of hydrogen peroxide (H2O2) to hydroxyl radical (HO.). Transition metals, such as copper (Cu), iron (Fe), and cobalt (Co), in their reduced form catalyze this reaction: Fe++ + H2O2 ® Fe+++ + HO– + HO. The reaction requires a reduced transition metal, process accomplished by O2.– Fe+++ + O2.– ® Fe++ + O2 The overall reaction, involving iron reduction by O2.– and iron oxidation by H2O2 Fe+++ + O2.– ® Fe++ + O2 Fenton Reaction Fe++ + H2O2 ® Fe+++ + HO– + HO. _________________________________________________ O2.– + H2O2 ® O2 + HO– + HO. Haber-Weiss Reaction 31  II. FREE RADICALS AND OXIDATIVE STRESS 3 CELLULAR SOURCES OF REACTIVE OXYGEN SPECIES Enzymatic reactions NADPH oxidase NADPH-P450 reductase xanthine oxidase Cellular sources leukocytes and macrophages mitochondrial electron transfer No cellular sources microsomal monooxygenase Fenton reaction from Environmental sources O2.– and H2O2 O2 → O2.– → H2O2 → HO. → H2O Dismutation Enzymic generation (non radical) glycolate oxidase acetyl-CoA oxidase D-amino acid oxidase NADH oxidase urate oxidase monoamine oxidase 32  II. FREE RADICALS AND OXIDATIVE STRESS 4 HOW DO OXIDANTS MEDIATE CELLULAR DAMAGE? OXIDANTS ANTIOXIDANTS 4 How 4 How do oxygen do oxidants mediate radicals cellular damage? mediate cellular damage? 33  II. FREE RADICALS AND OXIDATIVE STRESS 4 HOW DO OXIDANTS MEDIATE CELLULAR DAMAGE? Lipid Peroxidation Biomembranes and subcellular organelles are particularly vulnerable to oxidative attack due to the presence of polyunsaturated fatty acids (PUFA) in their membrane phospholipids. Lipid peroxidation consists of three steps: Initiation HO. can initiate lipid peroxidation by H abstraction of a fatty acid to yield an lipid alkyl radical: HO. + LH ® H2O + L. Propagation The lipid alkyl free radical (L.) reacts rapidly with O2 to form a lipid peroxyl radical (LOO.), which attacks a neighboring unsaturated fatty acid (RH) in the membrane to form hydroperoxides and a new lipid alkyl radical (L.): L. + O2 ® LOO.; LOO. + LH ® LOOH + L. Termination Termination occurs because of radical-radical interactions and depends on the intracellular oxygen concentration. 34  II. FREE RADICALS AND OXIDATIVE STRESS 4 HOW DO OXIDANTS MEDIATE CELLULAR DAMAGE? LH initiation Lipid Peroxidation (lipid) HO Initiation Oxidative impairment of biomembranes can L initiate a complex cascade of events leading to (alkyl radical) the formation of reactive, unstable oxidants, + O2 LOO long-lived toxic by-products or biologically (peroxyl radical) propagation active inflammatory mediators that have the LH potential of propagating damage beyond the L Propagation confines of the original focus. Free radical LOOH (lipid peroxide) attack of unsaturated fatty acids in membranes or lipoproteins is associated with important LO (alkoxyl radical) functional changes that result in cell dysfunc- LH tion or cell death. Lipid peroxyl radicals (LOO.) L are the propagating species LOH (alcohol) 35  II. FREE RADICALS AND OXIDATIVE STRESS 4 HOW DO OXIDANTS MEDIATE CELLULAR DAMAGE? DNA Oxidation The formation of hydroxyl radical (HO.) in the vicinity of DNA (site specific mechanism) results in H abstraction from the sugar in the DNA helix and addition to DNA bases; these lead to single strand breaks and nucleobase (8-hydroxydesoxy- guanosine) oxidation, respectively. Hydrogen abstraction: DNA strand breaks Cu+ Cu++ Cu++ site-specific Fenton reaction H2O2 HO strand breaks Hydrogen abstraction Addition: Nucleobase Oxidation O O HN N + OH HN N OH H2 N N N H2 N N N R R desoxy- 8-hydroxy- guanosine desoxyguanosine 36  II. FREE RADICALS AND OXIDATIVE STRESS 5 HOW DO CELLS PROTECT THEMSELVES AGAINST OXIDANTS? OXIDANTS ANTIOXIDANTS OXIDANTS ANTIOXIDANTS 5 How do cells protect themselves 5 How do cells protect themselves against oxygen radicals? against oxygen radicals? 37  II. FREE RADICALS AND OXIDATIVE STRESS 5 HOW DO CELLS PROTECT THEMSELVES AGAINST OXIDANTS? Mammalian cells are endowed with complex sets of protective mechanisms against free radicals, which are designed to prevent, limit, or repair oxidative damage. On the one hand, the cell convenes specific enzymic defenses against oxygen radicals, which are preventive antioxidants. OXIDANTS ANTIOXIDANTS On the other hand, small antioxidant molecules can 5 How do cells protect themselves react with a variety of free radicals and some of them against oxygen radicals? may be considered as chain-breaking antioxidants. 38  II. FREE RADICALS AND OXIDATIVE STRESS 5 HOW DO CELLS PROTECT THEMSELVES AGAINST OXIDANTS? a. Removal of superoxide anion: superoxide dismutases Superoxide dismutases (abbreviated SOD) catalyze the rapid dismutation of superoxide radical to hydrogen peroxide and oxygen. The rate of this reaction is 10,000-fold higher than that of the spontaneous dismutation. O2.– + O2.– + 2H+ H2O2 + O2 spontaneous, non-enzymatic dismutation 105 M–1s–1 O2.– + O2.– + 2H+ H2O2 + O2 SOD enzymatic dismutation 109 M–1s–1 39  II. FREE RADICALS AND OXIDATIVE STRESS 5 HOW DO CELLS PROTECT THEMSELVES AGAINST OXIDANTS? a. Removal of superoxide anion: superoxide dismutases All superoxide dismutases are metalloproteins O2 O2.– containing Cu,Zn or Mn. There are three types of superoxide dismutases in humans: Cu,Zn-superoxide dismutase (SOD1) (cytosol) Mn-superoxide dismutase (SOD2) (mitochondrial matrix SOD —Cu2+ SOD —Cu+ Cu,Zn-superoxide dismutase (SOD1) (mitochondrial intermembrane space) Cu,Zn-superoxide dismutase (SOD3) extracellular space O2.– H2O2 O2.– + O2.– + 2H+ → H2O2 + O2 40  II. FREE RADICALS AND OXIDATIVE STRESS 5 HOW DO CELLS PROTECT THEMSELVES AGAINST OXIDANTS? a. Removal of superoxide anion: superoxide dismutases The content of Cu,Zn- superoxide dismutase in human tissues: Tissue Cu,Zn-SOD µg/mg protein ________________________________________________________________________ Liver 4.7 Cerebral gray matter 3.7 Testis 2.2 Renal cortex 1.9 Cardiac muscle 1.8 Renal medulla 1.3 Pituitary 1.0 Lung 0.5 41  II. FREE RADICALS AND OXIDATIVE STRESS 5 HOW DO CELLS PROTECT THEMSELVES AGAINST OXIDANTS? a. Removal of superoxide anion: superoxide dismutases: Amyotrophic Lateral Sclerosis SOD1 and ALS – A defect in chromosome 21, which encodes for superoxide dismutase-1 (SOD1) accounts for approximately 20% of the familial cases of Amyotrophic Lateral Sclerosis (ALS) also known as Motor Neuron Disease and Lou Gehrig's disease. The disease affects motor neurons in the primary motor cortex, brainstem, and spinal cord, and results in both upper motor neuron (UMN) and lower motor neuron (LMN) signs. Once diagnosed, the median survival is 3-5 years. 42  AMYOTROPHIC LATERAL SCLEROSIS The disease affects motor neurons in the primary motor cortex, brainstem, and spinal cord, and results in both upper motor neuron (UMN) and lower motor neuron (LMN) signs. Once diagnosed, the median survival is 3-5 years. In people with amyotrophic lateral sclerosis (ALS), motor neurons, which help to send commands from the brain to muscle cells, become damaged, as depicted in this artist rendering. Credit: Kateryna Kon/Science Photo Library. Mullard A. Nature News Explainer 17 April 2023  TNF! receptor II. FREE RADICALS AND OXIDATIVE STRESS 5 Hi OW DO CELLS PROTECT THEMSELVES AGAINST OXIDANTS? a. SOD1 and ALS – Mechanism I#B nemo Mutant SOD1 hasactivated toxic effects on !the cell’s P DNA IKK SOD1 mRNA degradation machinery: the proteasomal pathway complex P " and autophagy. Misfolded mutant SOD1 cannot be mutant SOD1 targeted for ubiquitination and proteasomal degra- (misfolded) P dation and accumulates as oligomers and aggregates, autophagosome which lead to a stress response. The unfolded protein ubiquitination response entails toxicity that arises directly through oligomers proteasome aggregates the effects of the misfolded protein on processes such proteasome proteasom as mitochondrial respiration and axonal Thr transport 295–P and Thr295–P GSK3! PGC-1" indirectlyPGC-1" through disturbing normal proteostasis. PGC-1" ATP nuclear ADP TOXICITY membrane 44 NUCLEUS nucleus  II. FREE RADICALS AND OXIDATIVE STRESS 5 HOW DO CELLS PROTECT THEMSELVES AGAINST OXIDANTS? a. SOD1 and ALS – Diagnosis No definitive test for ALS exists, although Neurofilament Light chain (NfL) levels in cerebrospinal fluid and blood is used as a biomarker; the diagnosis is established by excluding other causes of progressive upper motor neuron and lower motor neuron dysfunction. However, advanced neuroimaging techniques help diagnose ALS: structural MRI, proton magnetic resonance spectroscopy, resting-state functional connectivity MRI, diffusion tensor imaging. a. SOD1 and ALS – Treatment Clinical care is based on symptom management and there is not effective treatment for patients with ALS. Physiotherapy - Respiratory function Nutritional support Palliative care and end-of-life decisions 45  II. FREE RADICALS AND OXIDATIVE STRESS 5 HOW DO CELLS PROTECT THEMSELVES AGAINST OXIDANTS? a. SOD1 and ALS – Treatment 릴구를 gma F F F G Riluzole remains the only evidence-based disease-modifying drug for ALS. Riluzole is an anti-glutamatergic drug proven to modify the O N course of ALS, but this treatment achieves only a modest improvement S N H 2 in survival. car O CH3 egg Edaravone – (Radicava) FDA approved in 2017 the use of edaravone for the treatment of ALS, which is administered as an intravenous N N infusion. The mechanism of action of edaravone is not clear and it might be based on its antioxidant properties. The drug might only be effective in early stages of the disease. 46  신계 a. SOD1 and ALS – Treatment Reducing the abundance of the pathogenic mutant protein has been attempted using anti-mutant superoxide dismutase 1 (SOD1) antibodies or antisense oligonucleotides targeting mutant SOD1 mRNA. Tofersen is an antisense oligonucleotide (ASO) being evaluated as a treatment for SOD1-ALS. 토정산 47  a. SOD1 and ALS – Treatment Reducing the abundance of the pathogenic mutant protein has been attempted using anti-mutant superoxide dismutase 1 (SOD1) antibodies or antisense oligonucleotides targeting mutant SOD1 mRNA. Tofersen (BIIB067; Biogen) blocks the production 一 of SOD1 protein using antisense oligonucleotide. In a phase III clinical trial, it was shown that early administration of Tofersen slowed decline in respiratory function, strength, and quality of life. Person with Biomarkers Used as Efficacy Indicators in Amyotrophic Person with ALS-Linked Gene Lateral Sclerosis (ALS) Trials ALS-Linked Gene Mutation + _________________________________________________________________ Healthy Person Mutation Antisense drug Biomarker Clinical trial Treatment response _________________________________________________________________ NfL plasma Phase 3 Tofersen Reduction of neuro- + Tofersen filament levels but change in clinical mRNA Mutated mRNA Mutated mRNA efficacy signals not significant compared with placebo at 6 months ____________________________________________________________________________ NfL plasma Phase 3 Tofersen Reduction of neurofila- ments levels and statisti- cally significant clinical efficacy signals observed at 12 months compared Normal Protein Toxic Protein Toxic Protein is with placebo group is made is made NOT made ALS Association 48  a. SOD1 and ALS – Treatment The use of stem cells for the treatment of ALS has attracted great interest. Several trials in which human neural stem cells are transplanted in the spinal cord of patients with ALS have been initiated (ClinicalTrials.gov identifiers: NCT01348451, NCT01640067). _________________________________________________________________________ Amyotrophic Lateral Sclerosis (ALS) Trials since 1996 with More than 100 Participants ________________________________________________________________________________________________ Mesenchymal stem Cudkowicz et al 2022 95/94 24 months (onset) Cells NfL CSF Phase 3 Nonsignificant reduction of NfL levels mesenchymal in patients receiving stem cell stem cell treatment and nonsignificant clinical efficacy signals _______________________________________________________________________________________________________________ NfL, Neurofilament Light chain. A biomarker of ALS 49  NEUROFILAMENT LIGHT CHAIN In the management of neurological diseases, the identification and quantification of axonal damage could allow for the improvement of diag- Neurofilament nostic accuracy. Neurofilament Light chain (NfL) is a neuronal cytosolic 10 nm Myelinated axon protein high expressed in large calibre myelinated axons. Its levels 60 nm -increase in cerebrospinal fluid (CSF) and blood proportionally to the degree of axonal damage in a variety of neurological disorders. Gaetani L. et al., J. Neurol Neurosurg Psychiatry (2019) 90, 870-881  II. FREE RADICALS AND OXIDATIVE STRESS 5 HOW DO CELLS PROTECT THEMSELVES AGAINST OXIDANTS? a. SOD1 and ALS – Treatment 51  II. FREE RADICALS AND OXIDATIVE STRESS 5 HOW DO CELLS PROTECT THEMSELVES AGAINST OXIDANTS? a. SOD1 and ALS – Treatment 52  II. FREE RADICALS AND OXIDATIVE STRESS 5 HOW DO CELLS PROTECT THEMSELVES AGAINST OXIDANTS? b. Removal of hydrogen peroxide: catalase Catalase is a heme-containing enzyme H2 와 Ortho H 2O present in all mammalian cells though liver HOOH and erythrocytes are particularly rich in catalase activity. This enzyme is located in the peroxisomes. Catalase is the most efficient enzyme in decomposing millions of H2O2 Fe3+ Fe4+=O resting compound molecules every second (107 M/s) into enzyme I molecular O2 and H2O: H2O2 + H2O2 ® 2H2O + O2 The two-stage mechanism of human catal- ase involves oxidation of Fe3+ to Fe4+=O, HOOH H 2O + O 2 which reacts with a second H2O2 molecule to produce H2O and O2. 53  II. FREE RADICALS AND OXIDATIVE STRESS 5 HOW DO CELLS PROTECT THEMSELVES AGAINST OXIDANTS? b. Removal of hydrogen peroxide: glutathione peroxidase γ-glu—cys—gly γ-glu—cys—gly | | Glutathione peroxidases (GPx) contain selenocysteine or SH S disulfide | cysteine in their active site. The enzyme occurs in cytosol S bond | γ-glu—cys—gly and the mitochondrial matrix and it requires glutathione (GSH), a tripeptide (glycine-cysteine–gglutamic), present GSH GSSG in the mM range in cells. The oxidized counterpart, GPx glutathione disulfide (GSSG) contains a disulfide bridge H2O2 H2O between two GSH molecules, and is present in the µM range. During this reaction, H2O2 is reduced to water and glutathione (GSH) is oxidized to glutathione disulfide (GSSG): NADPH GSSG H2O on H2O2 + 2GSH ® H2O + GSSG GSSG is reduced back to GSH by glutathione reductase (GR) at the expense of NADPH: GR GPx GSSG + NADPH ® 2GSH + NADP+ NADP+ GSH H2O2 54  II. FREE RADICALS AND OXIDATIVE STRESS 5 HOW DO CELLS PROTECT THEMSELVES AGAINST OXIDANTS? b. Removal of hydrogen peroxide: peroxiredoxins At variance with Glutathione Peroxidases (GPx), Peroxiredoxins (Prx) use thioredoxin instead of GSH. Thioredoxin is a small protein, 12 kDa, involved in thiol-disulfide exchange reactions and ! SH S is ubiquitously distributed in all tissues. Thioredoxins contain two Trx Trx vicinal cysteines that are involved in thiol-disulfide catalysis. SH S reduced oxidized Peroxiredoxins catalyze the reduction of H2O2 to H2O using Thioredoxin Thioredoxin T Trxred Trxox thioredoxin as the reducing compound. Thioredoxin Reductases S r recover back Trxox to Trxred: NADPH Trx H 2O H2O2 + Trxred ® H2O + Trxox S TR Prx SH NADP+ Trx H 2O 2 SH fred 55  II. FREE RADICALS AND OXIDATIVE STRESS 5 HOW DO CELLS PROTECT THEMSELVES AGAINST OXIDANTS? Specific Enzymatic Defenses CATALASE O2 + 1e– O2.– + 1e– H2O2 + 1e– HO. + 1e– H2O SOD GPX PRX 1Eur GSH GSSG TRXRED TRX OX Red ox 56  II. FREE RADICALS AND OXIDATIVE STRESS 5 HOW DO CELLS PROTECT THEMSELVES AGAINST OXIDANTS? Non-Specific Antioxidant Molecules §A second line of defense against free radical attack is constituted by small antioxidant molecules, such as vitamins E and C. §Some of these compounds are considered chain- OXIDANTS OXIDANTS ANTIOXIDANTS ANTIOXIDANTS breaking antioxidants because they effectively interrupt free radical propagation reactions. 5 How do cells protect 5 How themselves do cells protect themselves against oxygen radicals? against oxygen radicals? §The quenching or scavenging of a free radical by an antioxidant follows a general equation in which the free radical (R.) abstracts a hydrogen from the antioxidant (AH) with formation of an antioxidant-derived radical (A.): AH + R. ® A. + RH R TRY 57 A  14 II. FREE RADICALS AND OXIDATIVE STRESS 5 HOW DO CELLS PROTECT THEMSELVES AGAINST OXIDANTS? a. Small Antioxidant Molecules: Vitamin E Natural vitamin E is a mixture of tocopherols (a, b, and g) and tocotrienols (a, b, and g). It is a lipid soluble vitamin, which concentrates mainly in the interior of membranes and plasma lipoproteins. It is the major lipid soluble antioxidant in human blood lipid plasma. R1 HO CH3 CH3 CH3 CH3 R2 O CH3 Tocopherols R3 corn, soybean, olive oil R1 HO CH3 CH3 CH3 CH3 R2 O CH3 R3 Tocotrienols palm, rice brain, barley oils a = R1 = R2 = R3 = CH3 b = R1 = R3 = CH3; R2 = H g = R1 = H; R2 = R3 = CH3 58  II. FREE RADICALS AND OXIDATIVE STRESS 5 HOW DO CELLS PROTECT THEMSELVES AGAINST OXIDANTS? a. Small Antioxidant Molecules: Vitamin E Vitamin E, a lipid-soluble vitamin, reacts at considerable rates with a variety of free. radicals (ROO.) formed during lipid radical species, with emphasis on lipid peroxyl peroxidation. During the course of this reaction, as in any other antioxidant mechanism, a free radical of vitamin E is formed: the a-tocopheroxyl radical that has a chemical reactivity lower than that of the original free radicals scavenged. R1 R1 HO.. HO CH3 + ROO. CH3 + ROOH R2 O R R2 O R R3 R3 _________ antioxidant _________ ____ free____ ____ antioxidant-derived ____ __ non-radical __ radical radical product 59  II. FREE RADICALS AND OXIDATIVE STRESS 5 HOW DO CELLS PROTECT THEMSELVES AGAINST OXIDANTS? LH (lipid) initiation a. Small Antioxidant Molecules: Vitamin E. +X Vitamin E is a chain-breaking antioxidant because. L it quenches the chain propagating species, the lipid (alkyl radical) peroxyl radical (LOO.). + O2 α-TOH LOO. (peroxyl radical) propagation LH. L α-TO. LOOH (lipid peroxide) LO. (alkoxyl radical) LOH (alcohol) 60  II. FREE RADICALS AND OXIDATIVE STRESS 5 HOW DO CELLS PROTECT THEMSELVES AGAINST OXIDANTS? a. Small Antioxidant Molecules: Vitamin E Function Vitamin E was discovered in 1922 by Evans & Bishop as a dietary factor necessary for reproduction in rats. In humans, vitamin E deficiency increases early miscarriage risk, thus posing public health concerns. The major function of vitamin E is as a lipid-soluble antioxidant, i.e., a lipid peroxyl radical scavenger. Only a-tocopherol meets human vitamin E requirements and a-tocopherol is recognized by the hepatic a-toco- Three-dimensional structure of αTTP pherol transfer protein (a-TTP). 61  II. FREE RADICALS AND OXIDATIVE STRESS 5 HOW DO CELLS PROTECT THEMSELVES AGAINST OXIDANTS? a. Small Antioxidant Molecules: Vitamin E Function Vitamin E –complexed to lipoprotein particles– is internalized through the scavenger receptor (SR-B1); the vitamin within endocytic vesicles associates with a-TTP, that facilitates the transfer of its ligand (a-tocopherol) to transport vesicles. Vitamin E is secreted from the cell through the ABC-A1 transporter. a-TTP is the primary determinant of a-tocopherol plasma levels. In deficiencies of the a-TTP gene, vitamin E remains trapped within the endocytic vesicles. Go SECRETION INTERNALIZATION αTTP SR-B1 ABC A1 endocytic transport vesicles vesicles 62  II. FREE RADICALS AND OXIDATIVE STRESS 5 HOW DO CELLS PROTECT THEMSELVES AGAINST OXIDANTS? a. Small Antioxidant Molecules: Vitamin E Deficiency Human vitamin E deficiency was shown to be a defect in the gene for the a-TTP. enter restorer These patients showed the symptoms of peripheral neuropathies (Ataxia with Vitamin E Deficiency) (AVED). Vitamin E deficiency is associated with neurological abnormalities in humans; in addition to liver, aTTP mRNA is expressed in the human brain cerebral cortex and in cerebellum as well as in human placenta. The concentrations of placental aTTP mRNA are second only to the liver. 63  II. FREE RADICALS AND OXIDATIVE STRESS 5 HOW DO CELLS PROTECT THEMSELVES AGAINST OXIDANTS? Ataxia with Vitamin E Deficiency is associated with neurological abnormalities in humans: Peripheral neuropathy Loss of sensation in hands and feet Inability to walk: ataxia To keep her balance, the child with ataxia walks bent forward with feet wide apart. She takes irregular steps Treatment: Lifelong high-dose oral vitamin E supplementation to bring α-tocopherol plasma levels to a normal range 64  II. FREE RADICALS AND OXIDATIVE STRESS 5 HOW DO CELLS PROTECT THEMSELVES AGAINST OXIDANTS? Small Antioxidant Molecules: Vitamin E Deficiency Ataxia with Vitamin E Deficiency This is due to genetic defects in a-TTP and proceeds with neurologic abnormalities characterized by progressive peripheral neuropathy Fat Malabsorption Syndromes Occurs because vitamin E absorption requires biliary and pancreatic sections; example children with cholestatic liver disease; also neurologic abnormalities appear early unless vitamin E deficiency is corrected. Genetic Defects in Lipoprotein Synthesis Patients with low or non-detectable circulating chylomicrons, VLDL, or LDL; lipoproteins with apolipoprotein B are necessary for effective absorption and plasma vitamin E transport. Patients also develop a characteristic neurologic syndrome, progressive peripheral neuropathy. Severe Malnutrition Patients with protein energy malnutrition (PEM) who require also amino acids to synthesis a-TPP; supplementation with a-tocopherol improve neurologic abnormalities. 65  VITAMIN E DEFICIENCY INCREASES LIPID PEROXIDATION Pregnancy Fat Embryonic Malabsorption ¯Vit E Development Progressive ­ LIPID Dysregulation Malnutrition ¯Vit E Peripheral PEROXIDATION and Death Neuropathy Fatty Liver AVED ¯Vit E Eye Diseases 66  II. FREE RADICALS AND OXIDATIVE STRESS 5 HOW DO CELLS PROTECT THEMSELVES AGAINST OXIDANTS? b. Small Antioxidant Molecules: Vitamin C Vitamin C or ascorbic acid (AH–) is a water-soluble vitamin that reacts with many radical species producing semidehydroascorbic acid or ascorbyl radical (A.–) AH– + R. ® A.– + RH HO HO O O O O HO H. HO H R HO OH.O OH RH The ascorbyl radical (A.–) can react with itself at substantial rates (2 x 105 M–1s–1), thus providing a mechanism for self-regeneration of vitamin C: A.– + A.– ® AH– + A 67  CIE II. FREE RADICALS AND OXIDATIVE STRESS 5 HOW DO CELLS PROTECT THEMSELVES AGAINST OXIDANTS? Small Antioxidant Molecules: Vitamin C Vitamin C Content in Human Tissues Reaction of Vitamin C with Radicals Tissue Vitamin C Rate Constant (mg/kg) % Total Body (M–1s–1) Skeletal muscle 35 52 HO. hydroxyl 1.1 x 1010 Brain 140 11 RO. alkoxyl 1.6 x 109 Liver 125 9 ROO. Peroxyl 1.0 x 106 Skin 30 7 GS. Thiyl 6.0 x 108 Adipose tissue 10 7. UH – urate radical 1.0 x 106 Lungs 70 4 α-TO. Tocopheroxyl 2.0 x 105 Blood 9 2 A–. ascorbyl 2.0 x 105 Kidneys 55 2 CPZ –. chlorpromazine 1.4 x 109 Heart 55 1 O2 –. superoxide 1.0 x 105 68  II. FREE RADICALS AND OXIDATIVE STRESS 5 HOW DO CELLS PROTECT THEMSELVES AGAINST OXIDANTS? b. Small Antioxidant Molecules: Vitamin C Ascorbic acid can also reduce (recover) the tocopheroxyl radical of vitamin E: HO HO O O O O HO H. HO H α-TO HO OH.O OH α-TOH Vitamin E is a specific scavenger of lipid peroxyl radicals (LOO ) and the a-.. tocopheroxyl radical (a-TO ) can be recovered by vitamin C: ROO.. A– α-Toc-OH rooster. ROOH α-Toc-O AH– arrest 69  II. FREE RADICALS AND OXIDATIVE STRESS 5 HOW DO CELLS PROTECT THEMSELVES AGAINST OXIDANTS? b. Small Antioxidant Molecules: Vitamin C Mammals contain dehydroascorbate reductase that reduces ascorbyl radical back to ascorbate whilst oxidizing GSH to GSSG 2 A.– + 2 GSH free radical free quenching radical quenching and formation ––– and formationof of thethe ––– antioxidant derived radical antioxidant derived radical dehydro- ascorbate HO AH– GSSG reductase o dehydroascorbate 2 AH– + GSSG reductase H2O A GSH ––– recovery of antioxidant ––– recovery of the antioxidant 70  II. FREE RADICALS AND OXIDATIVE STRESS 5 HOW DO CELLS PROTECT THEMSELVES AGAINST OXIDANTS? b. Small Antioxidant Molecules: Vitamin C Ascorbic Acid (AA) is co-transported with sodium (Na+) across membranes by SVCT1- 2 (Sodium-dependent Vitamin C Transporters1-2, whereas the oxidized form, dehydro- ascorbate (DHA), is transported by glucose transporters (GLUT1-4) –2e– Na+ AA AA DHA DHA SVCT1-2 GLUT1-4,8,10 +2e– 71  II. FREE RADICALS AND OXIDATIVE STRESS 5 HOW DO CELLS PROTECT THEMSELVES AGAINST OXIDANTS? Small Antioxidant Molecules: Synergism between Vitamin C and Vitamin E Vitamin E is a lipid-soluble vitamin and antioxidant (a chain-breaking antioxidant). Vitamin E is present in membranes. Vitamin C is a water-soluble vitamin and antioxidant; the antioxidant-derived radical, ascorbyl radical, is recovered via dehydroascorbate reductase. Vitamin C is present in the cytosol. The compartmentalization of vitamins E and C provides a synergistic antioxidant mechanism by which the free radical character is transferred from the lipid phase (membrane) to the water phase (cytosol).Quenching Quenchingofofperoxyl peroxylradicals radicals Enzymatic Enzymaticrecovery recoveryofofthe the inInthe themembrane membraneby byvitamin vitamin EE vitamin vitaminCCradical radical ROOH α-TO. AH– GSSG ROO. α-TOH A.– GSH Lipidphase Lipid phase Waterphase Water phase Recovery of the vitamin Recovery vitamin EEradical radical in cytosol in cytosol by vitamin vitamin CC 72  VITAMINS C AND E DEFICIENCIES L-ASCORBIC ACID α-TOCOPHEROL (VITAMIN C) (VITAMIN E) DEFICIENCY DEFICIENCY CH3 HO CH3 H3C O CH3 O O HO H CH3 CH3 CH3 H3C HO OH CH3 SCURVY AVED 73  II. FREE RADICALS AND OXIDATIVE STRESS 5 HOW DO CELLS PROTECT THEMSELVES AGAINST OXIDANTS? — Nrf2 — SH Oxidation (H2O2)S –"S Sox SH SH Electrophiles Nrf2 (Nuclear factor erythroid 2–related factor 2 is a | | Electrophilic attack | | HO KEAP1 Keap1 2 2 Keap1 KEAP1 transcription factor that regulates cellular resistance Nrf2 SH Sox SH SH Oxidation (H2O2)S –"S | | Electrophilic attack | | to oxidants. Nrf2 remains in cytosol when bound to KEAP1 HO2 2 KEAP1 Keap1 ATP Keap1 PKC/ Keap 1. Oxidation of Keap1thiols by H2O2 or PI3KNrf2 ADP electrophiles results in release of Nrf2, which binds ATP PKC/ Prot PI3K Nrf2 P to the Antioxidant Responsive Element (ARE) Peroxiredoxins ADP mRNA Peroxire GPx ARE Mn-SOD facilitating the transcription of antioxidant systems, GPx Sulfiredoxin Cytoprotective Prot Nrf2 P GPxgenes Mn-SOD Heme Oxygenas Peroxiredoxins such as peroxiredoxins, glutathione peroxidases, HemeOxygenase-1 Sulfiredo GPx Peroxire ARE ARE mRNA Peroxiredoxins Mn-SOD SOD2 GPx Sulfiredoxin Cytoprotective Mn-SOD, and heme-oxygenase-1. genes Mn-SO Heme Oxygena Sulfired 가 NEL ARE 74  II. FREE RADICALS AND OXIDATIVE STRESS 5 HOW DO CELLS PROTECT THEMSELVES AGAINST OXIDANTS? — Nrf2 and Friedreich Ataxia — o SH Oxidation (H2O2)S –"S Sox Friedreich ataxia is a progressive neurodegenerative SH SH | | Electrophilic attack | | Omaveloxolone HO 2 2 KEAP1 KEAP1 Keap1 Keap1 disorder that causes a progressive loss of coordina- Nrf2 SH Sox SH SH Oxidation (H2O2)S –"S tion and muscle strength, eventually relegating | | Electrophilic attack | | HO KEAP1 Keap1 ATP 2 2 O KEAP1 HKeap1 O patients to the full-time use of a wheelchair. PKC/ PI3KNrf2 H N N H F F Omaveloxolone (SkyclarysTM), an Nrf2 activator, ADP ㅡㅡ ATP O H improves mitochondrial functions, restores redox PKC/ Omaveloxolone Prot PI3K Nrf2 P Peroxiredoxins balance, and reduces inflammation in models of ADP mRNA Peroxire GPx ARE Mn-SOD GPx Sulfiredoxin Friedreich ataxia. The safety and efficacy of Cytoprotective Pro Nrf2 P GPxgenes Mn-SOD Heme Oxygena Peroxiredoxins omaveloxolone (MOXIe Study)* as a therapeutic HemeOxygenase-1 Sulfired GPx Peroxir ARE ARE mRNA Peroxiredoxins Mn-SOD SOD2 GPx Sulfiredoxin Cytoprotective agent was further confirmed by its approval by the genes Mn-SO Heme Oxygena Sulfired FDA in February 2023. *Lynch D.R. et al (2021) Ann Neurol 89, 212-225.