The Free Radical Theory of Aging

1
The Free Radical Theory of Aging

• Tomas A. Prolla Ph.D
• Associate Professor
• Dept. of Genetics Medical Genetics
• University of Wisconsin-Madison

2
(No Transcript)
3
Aging Theories

• Shortening of telomeres
• Oxidative stress to macromolecules
• Glycation
• Dysdifferentiation
• rDNA
• DNA damage and mutations
• Membrane dysfunction
• Impaired protein synthesis
• Disturbances in calcium metabolism
• Autoimmunity

4
The Free Radical Theory of Aging

• Originally proposed by Denham Harman in 1956
• Harman D. Aging a theory based on free radical
  and radiation chemistry. Journal of Gerontology
  11298-300.
• Key postulates
• There is a relationship between the length of the
  life cycle (growth, decline and death) and
  metabolic rates
• The universality of aging suggests that
  biochemical mechanisms may be similar in
  different organisms
• Irradiation of living organisms causes mutations,
  cancer and aging, through formation of .OH and
  O2.- radicals
• These may arise in living organisms, perhaps at
  respiratory enzymes, and cause damage and aging
  in the cell over time.

5
Sources of Free Radicals in the Cell

• Oxidase-catalyzed reactions, such as
  macrophage-neutrophil catalyzed reactions
  (inflammation). The membrane-bound form of NADPH
  oxidase, which generates superoxide, is well
  characterized. Isoforms of NADPH oxidase exist
  in membranes nearly of every cell
• Auto-oxidation of reduced forms of
  NADP-dehydrogenase-linked flavoproteins.
• Mixed-function oxidases (MFOs) reactions
• Cytochrome-P450 proteins
• Auto-oxidation of the semiquinone form of CoQ
  (mitochondrial electron transport)

6
Coenzyme Q The major source of free radicals in
the cell?

• Coenzyme Q (CoQ) or ubiquinone is a redox-active
  lipophilic substance present in cellular
  membranes, consisting of a quinone head and a
  chain of isoprene units numbering 9 or 10
  depending on the species.
• CoQ transfers electrons in the mitochondrial
  electron transport chain from complexes I and II
  to complex III
• CoQ acts as a potent antioxidant in the inner
  mitochondrial membrane (Kagan et al, Biochem.
  Biophys. Res. Commun. 169851-857, 1990 and Lass
  and Sohal, Arch. Biochem. Biophys.
  352229-236,1998.
• Auto-oxidation of the CoQ semiquinone may be the
  major intracellular source of superoxide (Boveris
  et al. Biochem J., 156435-444, 1976)

7
Mitochondrial Pathways of ROS Generation
8
Mitochondrial Mutations and AgingA new version
of the Free radical theory of aging Fleming et
al., Gerontology 2844-53, 1982.

• Cells are dependent on molecular oxygen and
  oxidative phosphorylation to provide high energy
  compounds needed for biological functions.
• The mitochondrial genome is a double-stranded
  circular DNA molecule that encodes 13 components
  of the electron transport system (ETS). There
  are 2 to 10 copies of the mitochondrial genome
  per mitochondrion, and up to hundreds of
  mitochondria/cell
• The base modification 8-OH-Dg increases as a
  function of age in various species, including
  humans. Base substitution mutations and deletions
  also increase in frequency , reaching up to 89
  of total mtDNA in heart (Hayakawa et al. Biochem.
  Biophys. Res. Comm. 226369-77, 1996). Mutations
  also increase in fibroblasts from humans (Wang et
  al, Proc. Natl. Acad. Sci USA 984022-7,2001)
• Cells deficient in Cytochrome C Oxidase activity
  increase in number in skeletal muscle and heart
  of monkeys, suggesting mutations in mtDNA
  (Muller-Hocker, Mech. Ageing Dev
  86197-213,1996).
• In rats, aging is associated with reduced
  mitochondrial membrane potential. (Hagen, et al.
  Proc. Natl. Acad. Sci USA 991870-5,2002)

9
Oxidative Stress and Aging A critical Role for
Mitochondria?
10
Biochemical Evidence for the Hypothesis

• Proteins can be converted to carbonyl derivatives
  through reaction with lipid peroxidation products
  . In general, protein carbonyls increase with age
  in multiple tissues Beal et al, Free. Rad. Biol.
  Med. 32797-803, 2002.
• Reactive oxygen species (ROS) can react with DNA,
  generating a wide variety of modified bases. The
  base-modification 8-OH-dG increases with aging in
  multiple tissues (liver, kidney, brain, heart,
  muscle) of C57Bl6 mice (Hamilton et al, Proc.
  Natl. Acad. Sci USA 9810469-74,2001).
• Glutathione is one of the most abundant
  intracellular antioxidants. The ratio of
  reduced/oxidized glutathione (GSH/GSSG) declines
  with aging in multiple rodent tissues.Rebrin et
  al, Free. Rad. Biol. Med. 35626-35, 2003 and
  Suh, Arch. Bioche. Biophys. 423126-135,2004)
• Increases in lipid peroxidation, as determined by
  both isoprostane and MDA assays, are common but
  not universal.

11
Caloric Restriction (CR) and Aging

• CR (30 - 50? caloric intake w/o malnutrition)
  is the only intervention shown in mammals to
  extend maximum lifespan and retard the
  development of a broad spectrum of age-associated
  pathophysiological changes. Three topics are
  being actively studied
• Mechanisms by which CR retards aging in
  rodents
• Effects of CR on aging and diseases in
  primates
• Development of CR mimetics (e.g.,
  nutraceuticals)

12
Adapted from Hursting et al, Annual Reviews of
Medicine, 54131-152,2003.
13
Diseases Inhibited by CR in Laboratory Rodents

• Cancers (breast, lymphoma, GI, prostate, etc)
• Diabetes
• Hypertension
• Heart Failure
• Kidney Disease
• Autoimmune Diseases (lupus, anemia, etc.)
• Muscle Wasting

14
(No Transcript)
15
(No Transcript)
16
Impact of caloric restriction on oxidative stress

• Reduces age-related increases in DNA damage
  (Hamilton et al, Proc. Natl. Acad. Sci USA
  9810469-74,2001).
• Reduces tissue lipid peroxidation (Zeinal et al,
  FASEB J. 141825-36, 2000)
• Increases resistance to oxidative stressors such
  as Paraquat and heat (Hall et al, FASEB
  J.1478-86,2000).
• Reduces tissue protein oxidation (Zeinal et al,
  FASEB J. 141825-36, 2000,and Leeuwenburgh et al,
  Arch. Biochem. Biophys, 34674-80, 1997)
• Reduces mitochondrial ROS production (Bevilacqua
  et al, Am. J. Physiol Endocrinol. Metab. In
  press)

17
Summary of gene expression profiling procedure
Young tissue
Old tissue
Biological data analysis
mRNA
mRNA
Statistical data analysis
cDNA
cDNA
Image/Data analysis using Affymetrix algorithm
cRNA
cRNA
18
Experimental Design

• To study aging compare 5-month-old and
  30-month-old normally fed mice
• C57Bl6J males
• Individually-housed
• n 3 (leading to 9 pairwise comparisons)
• To study CR compare 30-month-old normally fed
  mice to age-matched mice fed 26 fewer calories
  from 6 weeks of age.

19
(No Transcript)
20
Gene Expression Profile of Skeletal Muscle from
Old Mice

• Stress Response
• Heat shock response genes, DNA damage response
  genes, Oxidative stress
• Inducible genes
• Energy Metabolism
• Reduced glycolysis, mitochondrial dysfunction
• Neuronal Injury
• Reinnervation induced genes, muscle injury
  induced genes, neurite extension
• And sprouting

Most alterations (80) were completely or
partially Prevented by caloric restriction.
21

• Do Dietary Antioxidants Retard the Aging
  Process?
• Previous studies have failed to show increases in
  maximum lifespan with several
• antioxidants, including 2-Mercaptoethylamine,
  Vitamin E and combinations of
• Dietary antioxidants
• Study design
• Mouse B6C3F1 males
• ? Dietary manipulation started at 14 months of
  age
• - CR group 58 kcal/week (41 reduction from
  the control group)
• - LA group supplementation of ?-lipoic acid
  (600 mg/kg) to control diet
• - CQ group supplementation of coenzyme Q10
  (100 mg/kg) to control diet

22
Lee et al, in press.
23
Why do antioxidants fail to extend lifespan?

• Histopathological findings of mice receiving
  a-lipoic Acid, CoQ10 or under CR
• suggest that unlike CR, dietary antioxidants do
  not prevent spontaneous tumors
• Feeding Hep. Carcinoma Lymphoma
• Control Fed 45.5 67.3
• a-Lipoic Acid 50.0 60.0
• CoQ10 53.4 72.4
• CR 27.6 24.1

24
Genetic data does not provide strong support to
the free radical theory of aging

• Mice that lack one copy of the MnSOD gene display
  increased oxidative damage but do NOT display
  accelerated aging (Van Rammen et al., Physiol.
  Genomics 1629-37, 2003).
• Overexpression of SOD and Catalase in Drosophila
  does not result in consistent increases in
  lifespan (Orr and Sohal, Exp. Gerontol.
  383227-230, 2003)
• However.
• Mice that lack p66shc display reduced oxidative
  stress levels, and an increase in lifespan
  (Migliaccio et al., Nature 402309-313, 1999)
• Mice that are deficient in IGFI-receptor live
  longer and are more resistant to oxidative stress
  (Holzenberger. et al 421182-187, 2003)

25
The Genetic Control of Aging Mutations that
reduce size prolong life
SCH9 yeast Chico Flies GRH/BP Mice
26
Pathways that Impact Mammalian Aging
27
Postmitotic tissues may be the critical ROS
targets in aging

• Dramatic increases in age-related mortality
  involve diseases of the heart and nervous system
• Affected cells include Neurons, cardiomyocytes
  and skeletal muscle fibers
• Loss of cells and subsequent tissue dysfunction
  may contribute to Alzheimers and Parkinsons
  Diseases, heart failure and muscle wasting
• Antioxidants retard and reverse brain aging, Liu
  et al., Proc. Natl. Acad. Sci. USA 1008526-8531,
  2003)