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Mitochondria, Antioxidants and Aging

Mitochondria, Antioxidants and Aging. Holly Van Remmen, Ph.D. GRECC , South Texas Veterans Health Care System Department of Cellular and Structural Biology Barshop Institute for Longevity and Aging Studies University of Texas Health Science Center San Antonio.

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Mitochondria, Antioxidants and Aging

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  1. Mitochondria, Antioxidants and Aging Holly Van Remmen, Ph.D. GRECC , South Texas Veterans Health Care System Department of Cellular and Structural Biology Barshop Institute for Longevity and Aging Studies University of Texas Health Science Center San Antonio

  2. Review of the Role of Mitochondria in Aging • Mitochondrial Function and Oxidative Stress • Mitochondrial Theory of Aging • Studies Manipulating Mitochondrial Oxidative Stress in Animal Models-what can we learn?

  3. Apoptosis O2- H2O2 cytc cytc cytc cytc cytc cytc O2- O2- ROS Production H2O2 H2O2 ATP Production Electron Transport Chain I III IV II ATP Synthase H2O O2 ATP NADH TCA Cycle FADH Fatty Acid Oxidation Ca++ Regulation Signaling

  4. Sources of Reactive Oxygen Species Non mitochondrial: NADPH Oxidases Microsomal cytochrome P-450 Cyclooxygenases Monoamine oxidases Peroxisomal b oxidation of fatty acids Phagocytes >90% is mitochondrial electron transport chain contains several redox centers that may leak electrons to oxygen

  5. I II IV H2O2 GPX cytoplasm CAT SOD1 O2 O2●¯ H2O2 Intermembrane space III GPX H2O2 Matrix SOD2 O2 O2●¯ Mitochondrial ROS generation ROS production is at Complex I and Complex III

  6. Enzyme Target O2- O2- H2O2 lipid peroxides H2O2 Pr –S2 MnSOD CuZnSOD Gpx1 Gpx4 PrxIII Trx2 PH PH PHGPx PHGPx PHGPx PHGPx PH H2O H2O H2O H2O Non-enzymatic: Ascorbate Glutathione (GSH) Tocopherols PH Mitochondrial Antioxidant Defense: CuZnSOD ATP V III II IV I H2O2 O2- H2O2 Trx2 MnSOD Peroxisomes GPx H2O PrxIII H2O2 Catalase H2O Trx1 + O2 H2O2 O2- Nucleus 2H2O CuZnSOD GPx nDNA

  7. Oxidative Damage to Lipids Membrane peroxidation Decreased membrane fluidity PROTEIN oxo8dG Oxidative Damage to DNA Mutations Deletions H N N H O N H N N H 2 Sugar Oxidation of sulfhydryl groups Reactions with aldehydes Protein aggregation Oxidative Damage to Proteins Protein carbonyls Consequences of mitochondrial oxidative stress Marker F2-Isoprostanes 8-iso-PGF2a

  8. The problem: Mitochondria are required for energy production Mitochondria produce potentially harmful reactive oxygen species

  9. mitochondria What is the link? AGING

  10. Some Current Theories of Aging: • Rate of living theory Energy consumption is limiting for longevity • Evolutionary Theories Aging is a programmed event Mutation accumulation Antagonistic Pleiotropy • Genomic Instability • Cell Senescence/Telomere Shortening • GH/IGF-1 axis • Free Radical Theory

  11. Free Radical Theory of Aging “Aging: a theory based on free radical and radiation chemistry” 1956, D. Harman J.Gerontology 11(3):298-300 A single common process, modified by genetics and environmental factors, is responsible for the aging and death of all living things. “Aging and the degenerative diseases associated with it are attributed basically to the deleterious side attacks of free radicals on cell constituents and on the connective tissues…….. The free radicals probably arise largely through reactions involving molecular oxygen catalyzed in the cell by oxidative enzymes and in the connective tissues by traces of metals such as iron, cobalt, and manganese."

  12. Antioxidant Defense Oxidative Stress Theory of Aging Mitochondrial (ETC) Extra-mitochondrial (NADPH oxidase) Exogenous Sources (chemicals,radiation) ROS NO Imbalance between pro-oxidants and antioxidants. O2- H2O2 OH. ONOO- Chronic state of oxidative stress

  13. DNA Protein Lipid Oxidative Stress Theory of Aging Mitochondrial (ETC) Extra-mitochondrial (NADPH oxidase) Exogenous Sources (chemicals,radiation) Steady-state accumulation of oxidative damage that increases during aging ROS NO O2- H2O2 Antioxidant Defense ONOO- OH. Progressive loss in efficiency of cellular processes Net Result

  14. Mitochondria are major source of ROS

  15. Mitochondrial Theory of Aging Harman,D. 1972 “The biologic clock: the mitochondria?” J. Am Ger Society 20:145-57 Free radicals escaping from the respiratory chain …would be expected to produce deleterious effects mainly in the mitochondria …Are these effects mediated in part by mitochondrial DNA functions?

  16. -Alterations in ETC function -mtDNA deletions/mutations Damaged mitochondria produce more ROS Accumulation of oxidative damage with age Decreased ATP Cell Death/Apoptosis Functional Decline Aging Mitochondria Theory of Aging Increased Production of ROS

  17. Mitochondria Theory of Aging -Alterations in ETC function -mtDNA deletions/mutations Increased Production of ROS Damaged mitochondria produce more ROS Accumulation of oxidative damage with age Decreased ATP Cell Death/Apoptosis Functional Decline Aging

  18. Early studies supported an increase in ROS with age Housefly flight muscle (submitochondrial particles) Sohal and Sohal (1991) Mech of Aging and Dev. 187-202. Mouse kidney, heart and brain mitochondria (submitochondrial particles) Sohal et al (1994) Mech.Ageing Dev.121-133 Rat vastus lateralis or soleus muscle (homogenates/isolated mitochondria) Bejma and .Ji (1999) J.Appl.Physiol. 465-470

  19. Other studies show no increase….. Rat Muscle Capel et al. Soleus Mech. Aging Dev., (2004) 367-373. Tibialis Anterior Drew et al Am.J.Physiol Gastrocnemius Regul.Integr.Comp Physiol, (2003) R474-R480 Rat Heart Hansford et al J.Bioenerg.Biomembr., (1997) 89-95 Rat Liver Lopez-Torres et al. Free Radic.Biol.Med., (2002) 882-889

  20. * 120 120 100 100 80 80 pmol H2O2/min/mg mitochondrial protein 60 60 * 40 40 20 20 0 0 State 1 Glutamate malate 3-5 month 26-29 month 3-5 month 26-29 month 150 100 pmoles H2O2/min/mg 50 0 State 1 Pyruvate/Malate 9 month 29 month 9 month 29 month Increase may be tissue specific?..... Mouse hindlimb Skeletal muscle Mouse heart

  21. -Alterations in ETC function -mtDNA deletions Mitochondria Theory of Aging Increased Production of ROS Some tissues?

  22. Aging and ETC function Complex I II III IV V Muscle Tissue Human muscle Trounce et al., (1989) Lancet (8639):637-9 Human muscle Boffoli et al. (1994) BBA 1226:73-82 Sugiyama et al. (1993) Biochem Mol Biol Int 30:937-44 Rat muscle Muller-Hocker et al. (1990) J Neurol Sci 100:14-21 Human muscle Desai et al. (1996) Arch Bioc Biophys 333:145-151 B6C3F1 mice Muller-Hocker et al. (1996) Mech Aging Dev 86:197-213 monkeys Kwong et al. (2000) Arch Bioc Biophys 373: 16-22 C57Bl6 mice

  23. More recent studies in humans, mice Barrientos 1996 Rasmussne 2003 Chretien 1998, Kwong and Sohal 2000 No consistent change Current evidence suggests that there is no consistent overall defect in the ETC with age.

  24. Mitochondria Theory of Aging -Alterations in ETC function (?) -mtDNA mutations/deletions Increased Production of ROS Some tissues?

  25. MtDNA ~16549 bp -Double-stranded, circular molecule -2 to 10 copies in each mitochondrion > 1,000 in each cell mtDNA encodes for 37 genes: 22tRNA 13 mitochondrial peptides (ETC subunits) 16S and 26S rRNA (Remaining > 67 ETC subunits are nuclear encoded)

  26. mtDNA is more sensitive to oxidative damage than nuclear DNA Estimated at 1/8000 bases for mtDNA vs 1/130,000 for nDNA (Richter et al 1988 PNAS) -high metabolism -proximity to generation of ROS -lack of histones -repair systems less efficient

  27. mtDNA Deletions • Tissues that turn-over more slowly (skeletal muscle, heart) have more mtDNA deletions than more rapidly dividing tissues (liver) • Deletions increase with age in muscle heart and brain (identified using PCR) • Common deletion mtDNA4977 increases by a factor of 10,000 in muscle during a normal human lifespan reaching 0.1% of total muscle mtDNA by 84 yrs • Evidence indicates focal accumulation of deletions in some tissues

  28. Damaged mitochondria produce more ROS Accumulation of oxidative damage with age (mitochondrial and non-mitochondrial) Mitochondrial Theory of Aging Alterations in ETC function (?) mtDNA mutations/deletions YES Increased Production of ROS Some tissues?

  29. Age related changes in mitochondrial DNA oxidative damage Liver Mitochondrial DNA Liver Nuclear DNA 0.8 2.5 0.6 2.0 oxo8dG/2dG x105 0.4 1.0 0.2 0.0 0.0 3 – 6 months 26 months 3 – 6 months 26 months Van Remmen et al., Physiol. Genomics, 2003

  30. Plasma Free Isoprostanes Carbonyl Groups in Liver Protein 0.12 200 0.10 150 0.08 Carbonyl Levels (U/mg protein) 0.06 8-isoprostane (pg/ml) 100 0.04 50 0.02 0 0 Young (4-6 mths) Old (24-28 mths) Young (10 mths) Old (28-29 mths) Asish Chaudhuri, San Antonio Jack Roberts, Vanderbilt University

  31. Decreased ATP Cell Death Functional Decline ? Aging Mitochondrial Theory of Aging Alterations in ETC function (?) mtDNA mutations/deletions YES Increased Production of ROS Damaged mitochondria produce more ROS Some tissues? Accumulation of oxidative damage with age (mitochondrial and non-mitochondrial) YES

  32. Effects of Manipulating Mitochondrial Oxidative Stress in Animal Models

  33. MnSOD Tg control Altered expression of MnSOD in Drosophila Sun et al., Genetics. (2002) 161(2):661-72. Induced overexpression of mitochondrial Mn-SOD extends the life span of adult Drosophila melanogaster. Mean life span increased by an average of 16% Another study finds no increase in lifespan in MnSOD Tg flies (Orr et al., 2003 JBC)

  34. Increased Expression of Mitochondrial Uncoupling Protein 2 Targeted expression of the human uncoupling protein 2 (hUCP2) to adult neurons extends life span in the fly. Alterations in Mitochondrial Function and Oxidative Stress • decrease in ROS production • decrease in oxidative damage • increased resistance to paraquat Increase in lifespan Fridell et al., Cell Metab. 2005 1(2):145-52

  35. Increased expression of Mitochondrial genes Effect on Lifespan MnSOD UCP2 Seem to be consistent with the theory…..

  36. What can we learn about mitochondrial theory of aging from mouse models? • MnSOD-mitochondrial antioxidant • mtDNA polymerase mutant mice • Mitochondrial targeting of catalase

  37. MnSod (Sod2+/-) Knockout mice • Mitochondrial antioxidant enzyme (O2- + O2- H2O2 ) • Homozygous mutant is lethal (survival is less than 2 weeks) • 50% decrease in MnSOD activity in all tissues studied • No compensation by other major antioxidant enzymes 30 Wt Sod2 +/- 20 MnSOD Activity (relative units) 10 0 (Li et al. (1995) Nature Genetics. liver kidney heart brain muscle spleen

  38. PH PH PHGPx PHGPx PHGPx PHGPx PH H2O H2O H2O H2O Nucleus nDNA PH Sod2+/- Knockout Mice CuZnSOD H2O2 ATP II V III I IV O2- X H2O2 Trx2 Peroxisomes MnSOD GPx GPx b -Oxidation pathway H2O 2H2O PrxIII H2O2 Catalase H2O + Trx1 O2 H2O2 O2- 2H2O CuZnSOD GPx 2GSH GSSG

  39. Accumulation of oxidative damage with age Mitochondrial Theory of Aging Alterations in ETC function mtDNA mutations/deletions Increased Production of ROS Sod2+/- mice (Reduced Antioxidant Protection) Decreased ATP Cell Death Functional Decline Increased Aging

  40. Skeletal Muscle 300 500 250 400 * Aconitase Activity (munits/mg protein) 200 CoQ Reductase Activity (mmoles/min/mg protein) 300 150 200 100 * 100 50 0 0 • NADH • Oxidoreductase • Aconitase MnSOD Heterozygous Knockout Mice Oxidative damage to specific mitochondrial proteins aconitase NADH oxidoreductase

  41. Liver 6 5 * 4 * * 3 RCR 2 1 0 I III II MnSOD Heterozygous Knockout Mice Altered Mitochondrial Function /Decreased activities of ETCs

  42. MnSOD Heterozygous Knockout Mice Increased oxidative damage to DNA Nuclear DNA 6 month 26-28 month c 0.8 0.6 oxo8dG/2dG x105 0.4 0.2 0.0 WT Sod2+/- WT Sod2+/- WT Sod2+/- WT Sod2+/- Heart Muscle Brain Liver

  43. Accumulation of oxidative damage with age Mitochondrial Theory of Aging Alterations in ETC function mtDNA mutations/deletions Increased Production of ROS Sod2+/- mice (Reduced Antioxidant Protection) Aging

  44. 100 90 80 70 PercentSurvival 60 Sod2+/+ (n = 69) 50 Sod2+/- (n = 70) 40 30 20 10 0 0 5 10 15 20 25 30 35 40 45 Age (months) MnSOD Heterozygous Knockout Mice No change in survival Increase in Cancer

  45. Premature ageing in mice expressing defective mitochondrial DNA polymerase Nature, May 27, 2004, Trifunovic et al Homozygous knock-in mice for a proof reading deficient PolgA-mtDNA polymerase • 3-5 fold increase in point mutations and deletions in mtDNA • Normal appearance until ~6 months (25 weeks) -Weight loss, kyphosis, alopecia, decreased fat,osteoporosis • Decreased ETC activity and ATP production in heart

  46. mtDNA mutations -- Shortened lifespan • Median lifespan, ~48 weeks, or 336 days • All died by 61 weeks, or ~430 days a link between mtDNA mutations and ageing phenotypes in mammals? PolgA-mtDNA polymerase mutants Nature, May 27, 2004, Trifunovic et al

  47. Mitochondrial DNA Mutations, Oxidative Stress, and Apoptosis in Mammalian Aging Kujoth et al Science 259, 2005 -Targeted mutation of PolgA Mitochondrial DNA polymerase -Residue substitution in the exonuclease domain that impairs proof reading ability • increased mutations in mtDNA • No increase in oxidative damage • Apoptotic markers were increased during aging [increased caspase 3 activity]

  48. Phenotype shows age-associated characteristics -Indistinguishable in young age -Phenotype evident at ~9 months • Hair loss, graying, and kyphosis. • Thymic involution • Testicular atrophy associated with the depletion of spermatogonia • Loss of bone mass • Loss of intestinal crypts • Decrease in circulating red blood cells • Weight loss • Hearing loss • Loss of muscle mass Kujoth et al Science 259, 2005

  49. Maximal survival, 460 days. • Median survival, 416 days • Wildtype, both are > 850 days Are mtDNA mutations linked to apoptosis and aging? Mitochondrial DNA polymerase mutant Kujoth et al Science 259, 2005

  50. CuZnSOD ATP V III II PH PH IV I H2O2 O2- H2O2 Trx2 MnSOD Peroxisomes GPx GPx 2H2O H2O PrxIII H2O2 Catalase H2O Trx1 + O2 PHGPx PHGPx PHGPx PHGPx H2O2 O2- Nucleus PH H2O H2O H2O H2O 2H2O CuZnSOD GPx nDNA PH Extension of Murine Life Span by Overexpression of Catalase Targeted to Mitochondria CATALASE Increased expression in heart muscle and brain Increased over 50x in heart mitochondria Science, 2005- Schriner et al.

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