Effect of Superoxide DismutaseCatalase Mimetics on Lifespan and by yurtgc548


									    Effect of Superoxide
Dismutase/Catalase Mimetics
 on Lifespan and Oxidative
     Stress in Housefly
        Bayne, Sohal
• SOD/catalase mimetics extend lifespan in
• EUK-8 and EUK-134
• Houseflies (musca domestica)
• Divide flies into groups:
  –   controls
  –   low dose
  –   medium dose
  –   high dose
• Administer mimetics at various
  concentrations via drinking water
• effects on lifespan
• amounts of protein carbonyls under
  different conditions:
  – normoxic
  – hyperoxic (100% ambient oxygen at 10-12
   days for 48 hrs)
    EUK-8 under normoxic conditions:

•   flies drank less water
•   shortened lifespan 67% at high doses
•   no effect at low doses
•   no effect on protein carbonyl content
EUK-8 under hyperoxic conditions:
• shortened lifespan 13%
• no effect on protein carbonyl content
      EUK-134 under normoxic
• shortened lifespan at high doses
• no effect at low doses
• no effect on protein carbonyl content
• The effects of SOD/catalase mimetics appear to
  be species specific.
• SOD/catalase mimetics do not extend lifespan in
  house flies.
  – (contrast with results in worms)
• SOD/catalase mimetics do not affect protein
  carbonyl levels in house flies.
• SOD/catalase mimetics do shorten lifespan
  during oxidation stress.
  – (possibly due to toxicity or its opposite, low intake)
 Effects of caloric restriction on
 mitochondrial reactive oxygen
     species production and
bioenergetics: reversal by insulin

     Lambert, A.J. and Merry, B.J.
• Putting it all together: CR, insulin,
  mitochondria, and free radicals
• Study the effects of CR and insulin on
  mitochondrial reactive oxygen species
• Caloric restriction extends mean and
  maximum lifespan in model organisms.
• CR delays the onset and incidence of age-
  related diseases.
• CR ameliorates the age-related decline in
  DNA repair and protein turnover.
• The exact mechanisms of how CR causes
  these effects are not known.
• Attenuation of the rate of accrual of tissue
  oxidative damage by decreased
  generation of free radicals is a plausible
• Mitochondria are the main producers of
  free radicals.
• Mitochondrial DNA is particularly
  susceptible to free radical damage.
• Mitochondria from CR animals show
  reduced rates of free-radical generation.
• Male Brown Norway rats, housed singly
• Intake of food was limited such that body
  weights were maintained at 55% of the
  age-matched controls (fully-fed rats).
• Insulin miniosmotic pumps implanted
• Give insulin (0.55 microL/hr) to group of
  adult rats continuously for 2 weeks.
• Measure production of free radicals,
  specifically hydrogen peroxide (H2O2).
• Measure activity in specific components of
  mitochondrial energetic pathways (proton
  gradient, substrate oxidation, other).
• Plasma insulin levels were significantly lower in
  CR than in control rats.
• Hydrogen peroxide production rate significantly
  lower in CR (0.25 nmol/min/mg) than in fully-fed
  rats (0.60 nmol/min/mg)
• Decrease in hydrogen peroxide production rate
  was partially reversed (0.40 nmol/min/mg) by 2
  weeks of 0.55 microL/hr insulin treatment of CR
• CR decreases insulin levels and
  decreases free-radical levels in the
• Increasing insulin levels counteract the
  reduction of free-radical levels in the
Q and Extension of Life
    Span in Worms
      Larsen, Clarke
• Coenzyme Q (ubiquinone) is sold in drug
  stores as an anti-oxidant.
• Many people take Q as a life-extension
• Coenzyme Q is a carrier of electrons in the
  mitochondrial Electron Transport Chain.
• Electron transport in complexes I & III
  create a proton gradient across the inner
• This is coupled to the synthesis of ATP by
  complex V (Fo/F1 ATPase).
              Q functions:
• antioxidant (scavenges electrons)
• prooxidant (generates superoxide)
• a redox-active component of plasma-
  membrane electron transport
• uridine synthesis
• a cofactor for proton-pumping activity in
  uncoupling proteins in mitochondria.
    Q6, Q7, Q8, Q9, and Q10
• Coenzyme Q can have a variable length
  side chain, with typically 6 to 10 subunits,
  hence Q6, Q7, Q8, Q9, and Q10.
• Different species tend to produce Q with a
  particular length side chain
  – Q10 in human
  – Q9 in worm
  – Q8 in bacteria
         Q mutants in worms
• Clk-1 mutants in worms lack endogenous
  – relies instead on Q8 from bacterial diet.
• Clk-1 mutants live twice as long as
  wildtype worms.
• The missing clk-1 gene encodes a di-iron
  carbolxylate enzyme:
  – responsible for final hydroxylase step in Q
• A test to see if dietary Q alters lifespan of
  – Normal diet is OP50 (Q8 replete E.coli
  – Q-less diet is GDI (Q-less E.coli bacteria)
             Experiment 1
• Wild worms switched to Q-less diet during
  larval stage 4
  – avoids developmental interference

• Wildtype lifespan extended 59%.
• Lack of Q8 extends lifespan.
             Experiment 2
• Wild type fed Q-less diet from egg to old
• A small fraction died earlier than normal.
• Surviving worms lived longer than normal
• Had reduced medial lifespan compared to
  worms switched at L4.
    Experiment 2 Conclusions
• Q is beneficial in early development.
• Q contributes to short lifespan.
            Q vs. daf genes
• The daf-2 gene in worms regulates both:
  – development
  – lifespan.
• The longevity pathway includes 2 genes:
  – daf-2
  – age-1
• that extend lifespan when mutated.
      Suppression Analysis
• Used to determine the gene products
  required for a specific phenotype
• Example:
  – The longevity phenotype of daf-2 is
   considered to be suppressed by daf-16
   because the double mutant daf-16/daf-2 is
   short lived
• Thus, daf-2 mutants longevity requires
  wildtype daf-16 activity.
                 Analysis 1
• Suppression tests were performed on the Age
  phenotype with these gene products:
•     daf-16
•     daf-12.
• On a Q-replete diet, daf-16 and daf-12 mutants
  live shorter than wildtype.
• On a Q-less diet they live longer than wildtype.
• Neither mutation suppressed the lifespan
  extension produced by the Q-less diet.
• Therefore, these genes are not required for
               Analysis 2
• Effects of Q-less diet on longlived daf-2
  – e1370
  – m41.
• Both had longer median and max lifespans
  on Q-less compared to Q-replete diet.
               Analysis 3
• In C.elegans, daf-2 (e1370) is the longest
  lived when that mutation is combined with
  a mutation in either:
•     daf-12
•     or
•     clk-1.
• The daf-12/ daf-2 double mutant had no
  extended lifespan on either diet.
• The clk-1/ daf-2 double mutant had
  extended lifespans on a Q-less diet.
• The longevity mechanism here is additive:
  long-lived mutant + Q-less diet =even more
• The combination of mechanisms that
  reduce ROS generation and increase ROS
  scavenging result in decrease in total
  cellular ROS and apparently allows for an
  extended lifespan.
  Silencing of Ubiquinone
Biosynthesis Genes Extends
     Lifespan of Worms

 Asencio, Rodrigues-aguilera et al
• Identify genes that synthesize Coenzyme
  Q in C. elegans
• Determine their effect on lifespan
         This study observes

• 1. the effect of double-stranded RNA (dsRNA)
  as it interferes (RNAi) with gene sequences that
  are homologous to those of Q6 biosynthesis in
  saccharomyces cerevisiae during Q9
  biosynthesis and Q8 intake.
• 2. the respiratory chain properties in
  mitochondrial of silenced worms and some
  aspects of phenotype (especially lifespan)
• Using dsRNA interference, 8 genes were
  identified that participate in Q9
  biosynthesis in worms.
• RNA interference (RNAi) of Q9
  biosynthesis genes extends lifespan.
• Worms treated with RNAi produce less
  superoxide anions (30-50% less).
• At least 8 genes participate in Q9 biosynthesis.
• Silencing the genes results in:
  –   lowered Q9 levels
  –   lower superoxide production (in ETC)
  –   extended lifespan
  –   less damage to macromolecules in mitochondria.
• Findings support the endogenous oxidative
  stress hypothesis.
 Small Molecule Activators of
Sirtuins Extend Yeast Lifespan
      Howitz, Bitterman, et al.
• In budding yeast, CR extends lifespan by
  increased activity of Sir2 gene.
• Other members of this sirtuin (NAD+
  dependent protein deacetlylases) family
  – Sir2 in yeast (extends lifespan)
  – Sir2.1 in worms (extends lifespan)
  – Sirt1 in human (promotes cell survival).
• Sirtuins are thought to be part of an
  evolutionarilly conserved longevity
  pathway because:
  – they occur in rodents, flies, worms, yeast and
  – appear to promote survival by inducing a
    response to stress (drought etc.)
   Compounds that affect Sir
• resveratrol (found in red wine) activated
  Sirt1 2fold.
• quercetin (protein kinase inhibitor)
  activated Sirt1 5fold.
• piceatannol (protein kinase inhibitor)
  activated Sirt1 8fold.
• STACs (14 small molecule sirtuin
  activators) activated Sirt1 2fold.
• In yeast, resveratrol mimics CR by:
  – stimulating Sir2 (by not inhibiting it)
  – increasing DNA stability
  – extending lifespan by 70%
             Stabilizing DNA
• Sir2 probably extends lifespan of yeast by
  stabilizing repetitive DNA.
• Recombination of ribosomal RNA (rRNA) can
  generate an extrachromosomal molecule.
• This error is replicated to toxic levels in old cells.
• Resveratrol in combination with Sir2 reduce the
  frequency of rRNA recombination 60%.
• Suggests aging in yeast caused by genomic
  instability – not gene dysregulation.
• Preliminary experiments indicate
  resveratrol extends the lifespan of mulit-
  cell animals (flies and worms)
         resveratrol in humans
• lowers the Michaelis Constant of Sirt1 for both:
   – the acetylated substrate
   – and NAD+.
• is associated with health benefits including the
  mitigation of:
   – neurodegeneration
   – carcinogenesis
   – atherosclerosis.
• increases cell survival by stimulating Sirt1-
  dependent deacetylation of gene p53.
   – protect cultured human cells from radiation
• Glucose restricted yeast had no extension
  of longevity when treated with resveratrol.
• Resveratrol had no effect on lifespan of
  Sir2 null mutants.
• Indicates that resveratrol probably acts
  through CR pathway.

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