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

Doxorubicin

Adriamycin

Doxorubicin: pharmacology vs. toxicology

Doxorubicin

ROS

Apoptosis

Caspase 3

Intercalation of DNA

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”

Antioxidants

Prooxidants

Oxidative StressOxidative Stress

O2.- H2O2

.OH

Major ROS Major ROS

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

Important Enzyme-Catalyzed ReactionsImportant Enzyme-Catalyzed Reactions

Biological Pathways for Oxygen Biological Pathways for Oxygen ReductionReduction

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Sources of ROS

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

Microsomes as a source of ROS (I)Microsomes as a source of ROS (I)

What are microsomes? (ER)

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

Activation of acetaminophen to radicals resulting to nephrotoxicity

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

Involvement of Mitochondria in PD

Mitochondrial bioenergetics

ROS generation

Autophage

Mitophagy

Substrates/cofactors ROS

ROS-mediated Regulation of Autophagy

Crosstalk between Autophagy and Apoptosis

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

Interaction between PINK1 and Parkin

Abeliovich A., 2010, Nature 463, 744-745

Gene expression/polymorphism vs PD

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?

Identification of increased oxidative stress and ROS

• Animal studies• Tissue cultures

More ?

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

Antioxidant and treatment of neurodengerative disease

Smith and Murphy, AAS 2010

Mitochondria-specific targeting with MitoQ

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

Why did Mito-Q fail in PD patients?

• Lessons of neuroprotective drugs

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

antioxidant to protect oxidative damage to neurons

Icariin

ROS based-Chinese herbal medicine

More ???

ROS based-Chinese herbal medicine

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

Thanks