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Transcript of Reactive Oxidative Species Generation and Neurodegenerative Disorders Shaoyu Zhou, Ph.D. Department...
Reactive Oxidative Species Generation and Neurodegenerative Disorders
Shaoyu Zhou, Ph.D.
Department of PharmacologyZunyi Medical College
11. 5. 2014
Outline
• Reactive oxidative species (ROS) and oxidative stress
• ROS sources (mitochondria)• Neurodegeneration: vulnerability to ROS• Antioxidants based treatment of
neurodegenerative disorders
Definitions
Oxidative stress (Kemp et al. 2008) “An imbalance in prooxidants and antioxidants with
associated disruption of redox circuitry and macromolecular damage”
Antioxidant (Halliwell and Gutteridge, 2007)“ A substance that, when present at a low
concentration compared with that of an oxidizable substrate, inhibits oxidation of the substrate”
Hydroxyl radical (Hydroxyl radical (..OH)OH)
O2.- + Fe3+ O2 + Fe2+ (ferrous)
H2O2 + Fe2+ OH- + .OH + Fe3+ (ferric)
O2.- + H2O2 OH- + O2 + .OH
Haber-Weiss
Fenton
•Transition metal catalyzed
•Other reductants can make Fe2+ (e.g., GSH, ascorbate, hydroquinones)
•Fe2+ is an extremely reactive oxidant
Biological Pathways for Oxygen Biological Pathways for Oxygen ReductionReduction
Fro
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Fu
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Endogenous sources of ROS and RNSEndogenous sources of ROS and RNS
Mitochondria
Lysosomes
Peroxisomes
Endoplasmic Reticulum
Cytoplasm
Microsomal Oxidation, Flavoproteins, CYP enzymes
Myeloperoxidase(phagocytes)
Electron transport
Oxidases,Flavoproteins
Plasma MembraneLipoxygenases,Prostaglandin synthase
NADPH oxidase
Xanthine Oxidase,NOS isoforms
FeCu
Transition metals
Mitochondria as a source of ROSMitochondria as a source of ROS
Turrens, J Physiol, 2003
Localization of the main mitochondrial sources of superoxide anion
Mitochondrial electron chain
Quinone cycle
Chandel & Budinger, Free Radical Biol Med, 2007
Peroxisomes as a source of ROS and RNSPeroxisomes as a source of ROS and RNSFatty Acid
Acyl-CoA
Enoyl-CoA
Hydroxyacyl-CoA
Ketoacyl-CoA
Acetyl-CoA Acyl-CoA shortened by two carbons
Fatty acyl-CoA synthetase
Acyl-CoA oxidase
Enoyl-CoA hydrolase
Hydroxyacyl-CoAdehydrogenase
Thiolase
H2O2
Fatty Acid
Acyl-CoA
Enoyl-CoA
Hydroxyacyl-CoA
Ketoacyl-CoA
Acetyl-CoA Acyl-CoA shortened by two carbons
Fatty acyl-CoA synthetase
Acyl-CoA oxidase
Enoyl-CoA hydrolase
Hydroxyacyl-CoAdehydrogenase
Thiolase
H2O2
Enzymes in mammalian peroxisomes that generate ROS
Schader & Fahimi, Histochem Cell Biol, 2004
NADPH oxidaseNADPH oxidasePresent mainly in neutrophils (oxidative burst), but also in many other cell types
xanthine oxidase xanthine oxidase
Cytoplasmic sources of ROS and RNSCytoplasmic sources of ROS and RNS
NO•
Nitric Oxide Synthases (NOS):
neuronal nNOS (I)
endothelial eNOS (III)
inducible iNOS (II)
Lysosome as a source of ROS and RNSLysosome as a source of ROS and RNS
Myeloperoxidase undergoes a complex array of redox transformations and produces HOCl, degrades H2O2 to oxygen and water, converts tyrosine and other phenols and anilines to free radicals, and hydroxylates aromatic substrates via a cytochrome P450-like activity
Davydov, Trends Biochem Sci, 2001
Microsomes as a source of ROS (II)Microsomes as a source of ROS (II)
Coupling in cytochrome-P450-containing monooxygenases
Exogenous sources of free radicalsExogenous sources of free radicals
• RadiationUV light, x-rays, gamma rays
• Chemicals that react to form peroxidesOzone
• Chemicals that promote superoxide formationQuinones
• Chemicals that are metabolized to radicals
e.g., polyhalogenated alkanes, phenols, aminophenols
• Chemicals that release iron ferritin
UV radiationUV radiation
H2O2 OH. + OH.UVB
UVA = 320-400 nmUVB = 290-320 nmUVC = 100-290 nm
•Primarily a concern in skin and eye•Can also cause DNA damage•Can form singlet oxygen in presence of a sensitizer
Ionizing radiationIonizing radiation
-rays2H2O H2O + e- + H2O*
H2O* H + .OH
Activation of benzene to myelotoxic metabolites by P450 and myeloperoxidase
Reductive dehalogenation of carbon tetrachloride to trichlorimethyl radical initiating lipid peroxidation
Oxidative stress and cell damage
•High doses:directly damage/kill cells
•Low doses/chronic overproduction of oxidants: activation of cellular pathwaysstimulation of cell proliferationdamage to cellular proteins, DNA and lipids
Oxidative stress mediated damage to macromolecules
•DNA-- oxidative DNA damage (8-OhdG)-- DNA mutations
Consequences of lipid peroxidationConsequences of lipid peroxidation
• Structural changes in membranesalter fluidity and channelsalter membrane-bound signaling proteinsincreases ion permeability
• Lipid peroxidation products form adducts/crosslinks with non lipids
e.g., proteins and DNA• Disruptions in membrane-dependent signaling
Protein targets for ROSProtein targets for ROS
HS CH2CHCOOH
NH2
S CH2CH2H3C C
H
NH2
COOH HO CH2CHCOOH
NH2
HN CH2CHCOOH
NH2
CH2CHCOOH
N
HN
NH2
Cysteine Methionine Tyrosine
Histidine
Tryptophan
Oxidized proteins and amino acids found in biological systems
The Brain is Uniquely Vulnerable to Oxidative Damage
• Intolerance for blood flow interruptions• Limited regeneration-although neurogenesis and gliogenesis
can be stimulated• Aging sensitive
The Brain is Uniquely Vulnerable to Oxidative Damage
• Multiple sources of ROS generation (e.g. MAO, Aconitase, Nox(s), Complex I, P450s, neurotrophic factor withdrawal
• Redox active metal-rich (catalytic iron)• Resident immune cells (microglia) produce ROS and cytokines• Limited antioxidant and repair capacity (low catalase)
Common Mediators of Neurodegeneraton
• Reactive species and oxidative/nitrative damage
• Mitochondrial dysfunction• Proteosomal dysfunction• Abnormal protein aggregates• Inflammation
Mitochondria“the power plant of the cell”
• Produce ATP by coupling of oxidative phosphorylation to respiration
• Major source of energy and endogenous reactive oxygen species (ROS)
• Mitochondrial genome is highly susceptible to oxidative damage, lacks histone packaging
• Critical role in apoptosis via release of soluble factors (cytochrome c)
Mitochondrial DNA
Mitochondria is the important biological source and target of reactive oxygen species (ROS) and free radicals.
Mitochondrial DNA (16,569 bp) is a small, circular genome, encoding 13 essential proteins of respiratory chain as well as 2 rRNA and 22 tRNA genes.
mtDNA point mutation, deletion, insertion.
Mitochondrial DNA mutation increases with age
Association of PD proteins with mitochondria
Moura et al, 2010. Environmental and Molecular Mutagenesis 51, 391-405
Association of gene sets (share common biological function) and PD
Meta-analysis of 522 gene sets identified 12 set genes associated with PD.
Source: Zheng et al, 2010. Science Translational Medicine
Coordinated defects in cellular energetics:
mitochondrial electron transport chain gene set (95 genes)Based on 410 microarrays (221 cases and 189 controls)
ROS and antioxidant therapy of neurodegenerative diseases
Does a disease have a strong rationale for reactive species involvement?
Classification of Antioxidants
• Direct Antioxidants– SOD/O2
-.
– Catalase/H2O2
• Indirect Antioxidants– Inhibitors of cellular sources of oxidants
(chelators/metals)– Inducers of cellular antioxidants
(sulforaphane/Nrf2 targets-GSH)
Natural Antioxidant and Mimics
• Directly scavenging peroxyl and hydroxyl radicals, peroxynitrite, and hypochlorous acid.
• Major antioxidant mechanisms include the ability to delocalize charge, semi-quinone formation.
• May induce endogenous antioxidants through nrf2 activation
• Vitamin E and/or C, thiols, CoQ, polyphenols
Antioxidant Enzyme Mimics
• Two major classes based on endogenous enzymes that scavenge superoxide and hydrogen peroxide.– SOD mimics that are selective and non-selective and
some that contain a redox active metal.– Peroxidase mimics that are selenium based or contain a
redox active metal.• Selenium-based compounds need to be stable and usually
require endogenous antioxidants like GSH to recycle compounds to active state.
• Metal-based compounds need to have good affinity for metal and can form high oxygen states that can be pro-oxidant under low endogenous antioxidant conditions.
• Efficacy (high rate constant with ROS)• Stability• Safety• Favorable pharmacokinetic properties• Cell and mitochondria permeable• Non-toxic metabolites
• Efficacy, potency • Stability• Safety• Favorable pharmacokinetic properties• Blood-brain-barrier permeability• Oral bioavailability
Desirable Properties of Compounds
Neurodegenerative DiseasesAntioxidants
Mito-Q
Lack of efficacy in clinical trial of Parkinson’s disease (PROTECT study)
• Efficacious in preclinical studies• Stable• Well tolerated• Favorable pharmacokinetic properties• Blood-brain-barrier permeable• Cell and mitochondria permeable
Potential reasons for negative result:
• Lack of efficacy may be related to timing of drug administration (too late)
• Lack of correlation with appropriate biomarker(s) of oxidative damage
Clinical trial: prevention of inflammation caused by Hepatitis C virus
Oxidative stress response
ROS/RNSROS/RNSROS/RNSROS/RNS
Adaptation ResponsesAdaptation ResponsesAdaptation ResponsesAdaptation Responses
e.g. Neurotrophic factors,e.g. Neurotrophic factors,Neurogenesis, DNA repair etcNeurogenesis, DNA repair etce.g. Neurotrophic factors,e.g. Neurotrophic factors,Neurogenesis, DNA repair etcNeurogenesis, DNA repair etc
Oxidation of proteins,Oxidation of proteins, lipids and DNAlipids and DNAOxidation of proteins,Oxidation of proteins, lipids and DNAlipids and DNA
Calcium dysregulationCalcium dysregulationCalcium dysregulationCalcium dysregulationOrganelle dysfunctionOrganelle dysfunctionOrganelle dysfunctionOrganelle dysfunction
NecrosisNecrosisNecrosisNecrosisApoptosisApoptosisApoptosisApoptosis
Failure to adaptFailure to adapt
Is oxidative stress a “druggable” target for brain disorders?
• Should ROS be a target for brain disorders? – Drugs targeting sources of ROS may work better
• Dual roles of ROS: Signaling vs damage– Do antioxidant compounds interfere with physiological processes?
Does redox signaling role interfere with antioxidant efficacy?• Are ROS merely associated with the disease process or play a causative
role?– Criteria for assigning a causative role of ROS
Need biomarker-guided clinical studies to verify antioxidant efficacy
• Lack of verification of oxidative damage using appropriate biomarkers may explain failure of antioxidant clinical trials
• Biomarkers for monitoring antioxidant efficacy– Need organ specific biomarkers
Summary
• Sources of ROS
• Mitochondrial mediated neuron degeneration
• Antioxidant and intervention/treatment