Oxidative stress and aging: is intervention possible?

1. Oxidative stress and aging: is intervention possible? Simon Melov Ph.D. Buck Institute for Age Research

2. The Buck Institute for Age Research

3. Aging research approaches and opinions of the past • Grind & Find • Correlations, correlations, correlations….. • Too complex • Programmed death • Thousands of genes involved • Dogma

4. 1990’s - THE decade of invertebrate aging research • Johnson, T.E. Increased life-span of age-1 mutants in Caenorhabditis elegans and lower Gompertz rate of aging. Science 249, 908-12 (1990) • Kenyon, C., Chang, J., Gensch, E., Rudner, A. & Tabtiang, R. A C. elegans mutant that lives twice as long as wild type. Nature 366, 461-4 (1993) • Vanfleteren, J.R. Oxidative stress and ageing in Caenorhabditis elegans. Biochem J 292 ( Pt 2), 605-8 (1993) ; Larsen, P.L. Aging and resistance to oxidative damage in Caenorhabditis elegans. Proc Natl Acad Sci U S A 90, 8905-9 (1993) • Lithgow, G.J., White, T.M., Melov, S. & Johnson, T.E. Thermotolerance and extended life-span conferred by single-gene mutations and induced by thermal stress. Proc Natl Acad Sci U S A 92, 7540-4. (1995) • Kimura, K.D., Tissenbaum, H.A., Liu, Y. & Ruvkun, G. daf-2, an insulin receptor-like gene that regulates longevity and diapause in Caenorhabditis elegans [see comments]. Science277, 942-6 (1997) • Lin, Y.J., Seroude, L. & Benzer, S. Extended life-span and stress resistance in the Drosophila mutant methuselah [see comments]. Science 282, 943-6 (1998) • Sun, J. & Tower, J. FLP recombinase-mediated induction of Cu/Zn-superoxide dismutase transgene expression can extend the life span of adult drosophila melanogaster flies. Mol Cell Biol 19, 216-28 (1999)

5. Trivial to extend lifespan in invertebrates

6. Demonstrable success in aging research • Known mechanisms of action are key (bias) • Worm, fly, mouse • New mutants are no longer novel (caveats) • Linkage of gene action to physiology • Inferred therapeutics

7. What are the effects of oxidative stress? Exogenous studies toxicological studies, dose response etc. correlative Endogenous studies ROS produced as a result of normal metabolism Genetics: transgenic as well as Knockouts

8. Efficacy of EUK antioxidants in biology • Autoimmune disease • Stroke • Alzheimers disease • Parkinsons disease • ALS • Apoptosis • Mitochondrial dysfunction • Radiation damage • Aging

9. Euk-189 N N M n O O O C H O C H 2 3 O O C H C H C H C H 2 3 2 3 Euk-8 Euk-134 M n M n N N N N C l C l O O O O O C H H C O 3 3 Catalytic antioxidants tested in Sod2-/- mice, and in aging paradigms

10. EUK-134 protects dopaminergic neurons from toxicity by MPP+ Control MPP+ * MPP+ EUK-134 (0.5 µM) EUK-134 (0.5 µM) (MPP+ 10 µM) K. Pong et al., Brain Res. (2000)

11. Caenorhabditis elegans • Advantages as an aging model • Small size, complete genome, AGE mutants (e.g. age-1), short lifespan • Pharmacological screening • (disadvantage - pharmacological screening) • Advantages as a mitochondrial model • Metabolic mutants • respiratory chain • increased lifespan • mev-1 • Oxidative damage, • shortened lifespan

12. Extension of lifespan in C.elegans through antioxidant treatment Fraction alive

13. EUK-134 inhibits nitration of tyrosine hydroxylase in DA neurons 1 2 3 4 1. Control 2. EUK-134 3. MPP+ 4. MPP+ and EUK-134 a-NTYR 1 2 3 4 a-TH K. Pong et al., Brain Res. (2000)

14. Reactive Oxygen species and the Respiratory Chain cyt c O2 O2- H2O2 cyt c CoQ .OH- Succinate O2- H2O2 O2 NADH SOD1 IMS + H FeS C1 a a3 Q0 FeS N-2 b b CuB SDH Qi III FeS N-1, N-3, N-4 FMN MATRIX Fe2+ SOD2 Complex I Complex II Complex III Complex IV

15. WEAK FORM Oxygen free radicals generated as a function of metabolic rate cause cumulative oxidative damage, resulting in structural degeneration, functional decline, and age-related diseases. MODERATE FORM Oxygen free radicals generated as a function of metabolic rate cause cumulative oxidative damage, resulting in structural degeneration, functional decline, and age-related diseases.Oxidative stress is the predominant cause of age-associated degenerative change. STRONG FORM Oxygen free radicals generated as a function of metabolic rate cause cumulative oxidative damage, resulting in structural degeneration, functional decline, and age-related diseases.Oxidative stress is the predominant cause of age-associated degenerative change, and thus the determinant of MLSP.

16. Rate-of-Living Hypothesis (1906, 1928) Radiation Biology: Morphology EPR Spectroscopy: •OH, O2•– Denham Harman Free Radical Theory (1956) Rubner Pearl Sies & Boveris Stochastic (“wear and tear”) Theories (1930s-1950s) H2O2 Mitochondrial Superoxide (1979) Cell Biology Superoxide Dismutase (1969) Analytical Biochemistry SOD Central Dogma (mid-1980s) O2•– H2O2 Fridovich & McCord Brief History

17. Neurodegeneration Vascular Tone Cardiac Output Sensory Acuity Skin Thickness Bone Density Endocrine Function Immune Function Cancer The Mechanistic Abyss (circa mid-1980s) ??? Oxidative Stress ???

18. A Simplifying Hypothesis Oxidative DNA Damage Mitochondrial Oxidant Generation Metabolic Rate Oxidative Mutagenesis Carcinogenesis High High High High High Rats: Low Low Low Low Low Humans:

19. Neurodegeneration Vascular Tone Cardiac Output Sensory Acuity Skin Thickness DNA Damage Bone Density Endocrine Function Oncogenesis Immune Function Cancer The Mechanistic Canyon (circa 1995) Oxidative Stress

20. Neurodegeneration Vascular Tone Cardiac Output Sensory Acuity Skin Thickness Cell Death Bone Density Endocrine Function Immune Function Cancer The Mechanistic Canyon (circa 1995) Oxidative Stress

21. Neurodegeneration Energy Crisis Vascular Tone Mitochondrial Damage Cardiac Output Sensory Acuity Skin Thickness Bone Density Endocrine Function Immune Function Cancer The Mechanistic Canyon (circa 1995) Oxidative Stress

22. Neurodegeneration Energy Crisis Vascular Tone Mitochondrial Damage Cardiac Output Sensory Acuity Skin Thickness Cell Death DNA Damage Bone Density Endocrine Function Oncogenesis Immune Function Cancer The Mechanistic Canyon (circa 1995) Oxidative Stress

23. 4 e– reduction to water e– e– e– e– O2 H2O O2• – H2O2 • OH Unreactive at STP, but a great electron acceptor Biological activation via radicals, transition metals Generally, radical intermediates are enzyme-bound Reacts with virtually any molecule at diffusion-limited rates The molecule that makes ionizing radiation toxic Actually a chemical reductant Not so terribly reactive with most biomolecules Mitochondrial superoxide the major source of active oxygen Maintained at very low concentration Superoxide dismutases Not so terribly reactive with most biomolecules Maintained at very low concentration Catalases, peroxidases, GSH, etc…

24. 4 e– reduction to water e– e– e– e– O2 H2O O2• – H2O2 • OH Spontaneous Fe2+ /Fe3+ SODs CAT + POXs fast s l o w Fe2+ /Fe3+ Fe•S

25. Types of Evidence Oxidative Phenomenology Dietary Restriction Rate-of-Living/Oxygen Tension Dietary Supplementation Pharmacological Intervention Comparative Biology Classical and Population Genetics Transgenic Models Human Degenerative Disease

26. AP sites carbonylation 8-oxoguanine thymine glycol mixed disulfides thymidine glycol loss of Fe•S (aconitase, glutamine synthetase) MIXED dsDNA breaks lipofuscin DNA P-S-S-H PROTEIN glycooxidation products etheno adducts aldehydes TBARS exhaled ethanes isprostanes LIPIDS Oxidative Biomarkers GSH SUGARS Oxidative Phenomenology

27. Endocrine function The Problem With Biomarkers 1. Increased damage or decreased repair? damage 2. Cause of consequence of aging? 3. False negatives? steady-state pool 10-4 - 10-5 ? 4. What does it all mean? repair Oxidative Phenomenology

28. Oxidative Phenomenology Evidence in favor of the FRTA: “Expected” changes in 100s of studies. Many tissues, many species. Specific repair systems for many end-products characterized. BUT… Methodological problems with most such work. Negative studies buried? Specific repair systems for many end-products characterized. Absolute and relative magnitude of increases underwhelming. HOWEVER… Grind-and-find studies necessary to establish baselines. Biomarkers most useful in comparative and intervention studies. Oxidative Phenomenology

29. Dietary Restriction Note: dietary restriction does not generally decrease metabolic rate or activity in mammals. Evidence in favor of the FRTA: Generally: decreased age-specific accumulation of biomarkers. Generally: decreased sensitivity to oxidative stress. Sometimes: increased antioxidant activities. BUT… Almost all age-related alterations are slowed by DR. Hence: cause and effect are hopelessly entangled. HOWEVER… DR is a litmus test, and the FRTA has “passed” it. Dietary Restriction

30. Rate-of-Living/Oxygen Tension • Evidence in favor of the FRTA: • Models: • Physical restraint of insects. • Thermal manipulation of poikilotherms. • Increased/decreased oxygen tension of invertebrates. • Results largely supportive of the FRTA. BUT… • Applicability of models to other phyla? • Decreased life span is not a powerful phenotype. HOWEVER… • Negative results would have been robust, so positive results are important. Rate-of-Living/Oxygen Tension

31. Dietary Supplementation • Evidence in favor of the FRTA: • Some amelioration of age-related degenerative change: • ALCAR/lipoic acid in rat. • Phenolic antioxidants in rodents (blueberries). • Data appear to support the weak form of FRTA. BUT… • Most experiments have been negative. • Extension of life span: virtually no evidence. HOWEVER… Dietary supplementation is a flawed approach -- physiology restricts degrees of experimental freedom and potency. Falsification is problematic. Dietary Supplementation

32. Pharmacological Intervention • Evidence in favor of the FRTA: • Some amelioration of age-related degenerative change: • PBN in gerbils • Extension of life span: • Euk-134 in nematodes • Efficacy in Mammals • Euk-189 in mice? BUT… • Many experiments have been negative. HOWEVER… Falsification of the FRTA with drugs will be difficult. Pharmacological Intervention

33. Comparative Biochemistry Comparative Biochemistry

34. Oxidative DNA damage rates correlate with metabolic rate Comparative Biology

35. Mitochondrial Lipid Content Comparative Biology

36. Genetics • Evidence in favor of the FRTA: • Many long-lived mutants demonstrate increased antioxidant defenses and better tolerance of oxidative stress. • Population selection for increased life span sometimes (not always) associated with increased SOD activity in long-lived strains. • Short-lived mutants often associated with decreased antioxidant defenses, increased ROS generation. BUT… • Long-lived mutants possess generally better resistance against many stressors. HOWEVER… Many stressors may act via oxidative mechanisms. Genetics

37. Transgenic Models • Evidence in favor of the FRTA: • Life span extensions with transgenic SOD Drosophila. BUT… • Negative results with various Tg KO and heterozygous organisms. HOWEVER… • Many stressors may act via oxidative mechanisms. • Oxidative defenses are both redundant and interconnected -- crude genetic engineering is likely to be often compromised. • Overexpression of SOD prohibited in vitro. Transgenic Models

38. Value of Evidence Oxidative Phenomenology - LOW Dietary Restriction - MODERATE Rate-of-Living/Oxygen Tension - MODERATE Dietary Supplementation - LOW Pharmacological Intervention - HIGH Comparative Biology - VERY HIGH Genetics - VERY HIGH Transgenic Models - VERY HIGH Human Degenerative Disease - LOW Functional and Comparative Genomics VERY HIGH

39. e– e– e– e– O2 H2O O2• – H2O2 • OH Amplification Mechanisms? Chromosomal Architecture Lipofuscin/ Lysosomes Cellular Redox Telomeres NO• Fe•S Protein S-Nitrosation Signal Transduction Gene Expression Apoptosis

40. Homeostasis: Is Oxidative Stress Special? Degenerative Change Stresses/Damage Defenses/Repair Mutagenesis (cancer) Oxidative DNA damage Base-excision repair DNA polymerase errors Nucleotide-excision repair Endothelial dysfunction (heart disease) DNA proof-reading Depurination/deamination Immune dysfunction (rheumatoid arthritis) Lipid repair/turnover Lipid oxidation Catalytic antioxidants Non-enzymatic glycation Connective tissue dysfunction (osteoarthritis) Radical scavenging systems Protein denaturation Neuroendocrine dysfunction (muscle loss, wasting) Protein chaperones Protein oxidation Protein repair/turnover Chromosomal demethylation Amyloidosis (Alzheimer’s disease) Lysosomal turnover Cellular garbage accumulation Myopathies (weakness) Mitochondrial maintenance Mitochondrial oxidant generation Cellular proteolysis/turnover Lysosomal fragility Etc… Etc… Etc… Evolutionary Pressure

41. Questions/Answers Yes. No. ??? • WEAK: Are oxygen free radicals important in aging? • STRONG: Do oxygen free radicals determine MLSP? • MODERATE: Are oxygen free radicals predominant in aging? • energetics • cell division • cell arrest • cell death • chromosomal stability • gene expression • signal transduction

42. Questions/Answers Does CO2 determine plant growth? No… Does transcription determine embryogenesis? No… CO2 has “nothing to do with” the determination of plant growth. Transcription has “nothing to do with” embryogenesis. Mechanisms — not measurement.

43. The FRTA is no longer theoretical in the “weak” form. The FRTA is unintelligible in the “strong” form. The Free Radical “Perspective” on Aging has been productive, and is an object lesson for homeostasis. Oxidative stress is ubiquitous, and may be the single most significant category of cellular stress. This is clearly something which can be therapeutically targeted It’s not a question of whether, but rather of when, how, and how much.

44. Euk-189 N N M n O O O C H O C H 2 3 O O C H C H C H C H 2 3 2 3 Euk-8 Euk-134 M n M n N N N N C l C l O O O O O C H H C O 3 3 Catalytic antioxidants tested in Sod2-/- mice, and in aging paradigms

45. Neurodegeneration Energy Crisis Vascular Tone Mitochondrial Damage Cardiac Output Sensory Acuity Skin Thickness Cell Death DNA Damage Bone Density Endocrine Function Oncogenesis Immune Function Cancer Potential sites of intervention Oxidative Stress