1. Reactive Oxidative Species Generation and Neurodegenerative Disorders
Shaoyu Zhou, Ph.D.
Department of Pharmacology
Zunyi Medical College

2. Outline Reactive oxidative species (ROS) and oxidative stress
ROS sources (mitochondria)
Neurodegeneration: vulnerability to ROS
Antioxidants based treatment of neurodegenerative disorders

3. Doxorubicin
Adriamycin

4. Doxorubicin: pharmacology vs. toxicology
ROS
Apoptosis
Caspase 3
Intercalation of DNA

5. 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”

6. Oxidative Stress
Antioxidants
Prooxidants

7. Major ROS
O H2O OH

8. Hydroxyl radical (.OH) O2.- + Fe3+ O2 + Fe2+ (ferrous)
H2O2 + Fe2+ OH- + .OH + Fe3+ (ferric)
O2.- + H2O2 OH- + O2 + .OH
Fenton
Haber-Weiss
Transition metal catalyzed
Other reductants can make Fe2+ (e.g., GSH, ascorbate, hydroquinones)
Fe2+ is an extremely reactive oxidant

9. Important Enzyme-Catalyzed Reactions

10. Biological Pathways for Oxygen Reduction
From: McMurry and Castellion “Fundamentals of general, organic and biological chemistry”

11. Sources of ROS

12. Prostaglandin synthase
Endogenous sources of ROS and RNS
Mitochondria
Lysosomes
Peroxisomes
Endoplasmic Reticulum
Cytoplasm
Microsomal Oxidation,
Flavoproteins,
CYP enzymes
Myeloperoxidase
(phagocytes)
Electron transport
Oxidases,
Flavoproteins
Plasma Membrane
Lipoxygenases,
Prostaglandin synthase
NADPH oxidase
Xanthine Oxidase,
NOS isoforms Fe Cu
Transition metals

13. Localization of the main mitochondrial sources of superoxide anion
Mitochondria as a source of ROS
Mitochondrial electron chain
Localization of the main mitochondrial sources of superoxide anion
Quinone cycle
Turrens, J Physiol, 2003
Chandel & Budinger, Free Radical Biol Med, 2007

14. Peroxisomes as a source of ROS and RNS
Enzymes in mammalian peroxisomes that generate ROS
Schader & Fahimi, Histochem Cell Biol, 2004

15. NADPH oxidase
Present mainly in neutrophils (oxidative burst), but also in many other cell types

16. Cytoplasmic sources of ROS and RNS
xanthine oxidase
xanthine oxidase
Nitric Oxide Synthases (NOS):
neuronal nNOS (I)
endothelial eNOS (III)
inducible iNOS (II)
NO•

17. Lysosome 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

18. Microsomes as a source of ROS (I)
What are microsomes? (ER)

19. Microsomes as a source of ROS (II)
Coupling in cytochrome-P450-containing monooxygenases
Davydov, Trends Biochem Sci, 2001

20. Exogenous sources of free radicals
Radiation
UV light, x-rays, gamma rays
Chemicals that react to form peroxides
Ozone
Chemicals that promote superoxide formation
Quinones
Chemicals that are metabolized to radicals
e.g., polyhalogenated alkanes, phenols, aminophenols
Chemicals that release iron ferritin

21. UV radiation Ionizing radiation H2O2 OH. + OH. g-rays
UVB
UVA = nm
UVB = nm
UVC = nm
Primarily a concern in skin and eye
Can also cause DNA damage
Can form singlet oxygen in presence of a sensitizer
Ionizing radiation
g-rays
2H2O H2O + e- + H2O*
H2O* H + .OH

22. Activation of benzene to myelotoxic metabolites by P450 and myeloperoxidase
Reductive dehalogenation of carbon tetrachloride to trichlorimethyl radical initiating lipid peroxidation

23. Activation of acetaminophen to radicals resulting to nephrotoxicity

24. Oxidative stress and cell damage
High doses:
directly damage/kill cells
Low doses/chronic overproduction of oxidants:
activation of cellular pathways
stimulation of cell proliferation
damage to cellular proteins, DNA and lipids

25. Oxidative stress mediated damage to macromolecules
DNA
-- oxidative DNA damage (8-OhdG)
-- DNA mutations

26. Consequences of lipid peroxidation
Structural changes in membranes
alter fluidity and channels
alter membrane-bound signaling proteins
increases ion permeability
Lipid peroxidation products form adducts/crosslinks with non lipids
e.g., proteins and DNA
Disruptions in membrane-dependent signaling

27. Protein targets for ROS
Cysteine Methionine Tyrosine
Oxidized proteins and amino acids found in biological systems
Histidine
Tryptophan

28. The Brain is Uniquely Vulnerable to Oxidative Damage
Intolerance for blood flow interruptions
Limited regeneration-although neurogenesis and gliogenesis can be stimulated
Aging sensitive

29. 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)

30. Common Mediators of Neurodegeneraton
Reactive species and oxidative/nitrative damage
Mitochondrial dysfunction
Proteosomal dysfunction
Abnormal protein aggregates
Inflammation

31. Involvement of Mitochondria in PD
Mitochondrial bioenergetics
ROS generation
Autophage

32. Mitophagy

33. ROS-mediated Regulation of Autophagy
Substrates/cofactors
ROS

34. Crosstalk between Autophagy and Apoptosis

35. 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)

36. 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

37. Association of PD proteins with mitochondria
Moura et al, Environmental and Molecular Mutagenesis 51,

38. Interaction between PINK1 and Parkin
Abeliovich A., 2010, Nature 463,

39. Gene expression/polymorphism vs PD

40. 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, Science Translational Medicine

41. Coordinated defects in cellular energetics: mitochondrial electron transport chain gene set (95 genes) Based on 410 microarrays (221 cases and 189 controls)

42. ROS and antioxidant therapy of neurodegenerative diseases
Does a disease have a strong rationale for reactive species involvement?

43. Identification of increased oxidative stress and ROS
Animal studies
Tissue cultures
More ?

44. 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)

45. 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

46. 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.

47. Neurodegenerative Diseases
Desirable Properties of Compounds
Antioxidants
Neurodegenerative Diseases
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

48. Antioxidant and treatment of neurodengerative disease

49. Mitochondria-specific targeting with MitoQ
Smith and Murphy, AAS 2010

50. Mito-Q Efficacious in preclinical studies Stable Well tolerated
Favorable pharmacokinetic properties
Blood-brain-barrier permeable
Cell and mitochondria permeable
Lack of efficacy in clinical trial of Parkinson’s disease (PROTECT study)

51. Why did Mito-Q fail in PD patients?
Lessons of neuroprotective drugs

52. 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

53. ROS based-Chinese herbal medicine
antioxidant to protect oxidative damage to neurons
Icariin

54. ROS based-Chinese herbal medicine
More ???

55. Oxidative stress response
e.g. Neurotrophic factors,
Neurogenesis, DNA repair etc
Adaptation Responses
Failure to adapt
ROS/RNS
Apoptosis
Necrosis
Oxidation of proteins,
lipids and DNA
Organelle dysfunction
Calcium dysregulation

56. 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

57. 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

58. Summary Sources of ROS Mitochondrial mediated neuron degeneration
Antioxidant and intervention/treatment

59. Thanks