Effect of Superoxide Dismutase/Catalase Mimetics

on Lifespan and Oxidative Stress in Housefly

Bayne, Sohal

Hypothesis

• SOD/catalase mimetics extend lifespan in houseflies.

Materials

• EUK-8 and EUK-134

• Houseflies (musca domestica) 200/cage/group

Methods

• Divide flies into groups:– controls– low dose– medium dose– high dose

• Administer mimetics at various concentrations via drinking water

Observe

• 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 conditions:

• shortened lifespan at high doses

• no effect at low doses

• no effect on protein carbonyl content

Conclusions

• 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 production.

Background

• 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 explanation.

• 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.

Methods

• 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 subcutaneously.

• 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).

Results

• 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 rats.

Conclusions

• CR decreases insulin levels and decreases free-radical levels in the mitochondria

• Increasing insulin levels counteract the reduction of free-radical levels in the mitochondria

Q and Extension of Life Span in Worms

Larsen, Clarke

Background

• Coenzyme Q (ubiquinone) is sold in drug stores as an anti-oxidant.

• Many people take Q as a life-extension supplement.

• 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 membrane.

• 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 Q9 – 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

synthesis

• A test to see if dietary Q alters lifespan of worms.– Normal diet is OP50 (Q8 replete E.coli

bacteria)– 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 age.

• 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

longevity.

Analysis 2

• Effects of Q-less diet on longlived daf-2 mutants:– 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

longevity

• 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

Aims

• 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)

Results

• 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).

Conclusions

• 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.

Background

• In budding yeast, CR extends lifespan by increased activity of Sir2 gene.

• Other members of this sirtuin (NAD+ dependent protein deacetlylases) family are:– 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 humans– 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.

Resveratrol

• 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.