Extension of Lifespan by Overexpression of Superoxide

Dismutase in Drosophila melanogaster

Orr and Sohal

Background

• Hypothesis is that oxygen free radicals/reactive oxygen species (ROS) cause of aging

• Main assumption of this theory is that normal antioxidant defense levels are not sufficient, so that some ROS escape elimination.

• ROS cause molecular damage, some of which is irreparable, accumulates with age

• A direct causal link between ROS and aging has not been established.

• If ROS cause aging, then enhanced defense against ROS should– Reduce oxidative stress– Decrease the rate of aging– Extend lifespan

• Orr and Sohal decided to test the theory.

• Examine effects of over-expressing Cu-Zn superoxide dismutase (SOD) and catalase in flies

• SOD and catalase are the major defenses against ROS

• SOD converts superoxide anion radical to H2O2

• catalase breaks down H2O2 into water and oxygen

Methods

• Created transgenic flies that had two copies of the SOD and catalase genes

• Compared lifespan to controls

• Compared metabolism, activity to controls

Results

• Flies that overexpressed SOD and catalase – Lived ~ 30% longer than controls (median and

maximum lifespan)– Had lower levels of damage due to ROS– Had higher metabolic rates at older ages– Had delayed loss of motor ability

• The results were different from an earlier study by the authors, in which only SOD or only catalase were overexpressed.

• In that study, average lifespan was extended up to 10%, but maximum lifespan was unchanged.

• The combination of SOD and catalase overexpression is critical.

Does Over-expression of SOD1 Extend Lifespan in

Drosophila?Orr, Sohal

• This paper discusses the shortcomings of previous experiments (including those of the authors).

• Lifespan has been extended in short-lived flies.• This was achieved by bolstering antioxidant

defenses.• But is this possible only with flies with

compromised genetic backgrounds?• We don’t really know the effect of SOD1 on

robust backgrounds.

• At first glance, evidence supports the oxidative stress hypothesis.

• Aerobic cells generate ROS as a by-product of oxidative metabolism.

• Macromolecules are oxidized in the steady state of even young animals.

• The rate of ROS damaged molecules increases with age.

• So we must infer that:– cells normally exist under a certain level

of oxidative stress– oxidative stress increases with age– accumulation of molecular damage is

major factor in senescence.

• It follows that decreased oxidative stress:– should delay age-related accumulation

of oxidative damage– should extend lifespan.

• The over-expression of SOD1 (CuZn-SOD) is shown to extend lifespan.

• But efforts to extend lifespan in wildtype fruit flies only partly successful.

• Perhaps, antioxidative defences in wildtype have achieved an optimal balance.

Over-expression of SOD1 in flies

• Three groups (including Orr and Sohal) report positive findings.

• But did not prove SOD1 had a beneficial effect on a robust genetic background.

Transgenic effects on longevity only obvious in short-lived strains.

• One study found 48% extension of lifespan in one strain.

• This strain lives about 37 days.

• The control group lived 25 days.

• Even normal lab flies live 50-70 days.

• So antioxidative intervention may be beneficial in genetically compromised flies.

• Previous experiments have not recorded metabolic data.

• Their results equally support the hypothesis that metabolic defects lead to longevity.

Extension of Drosophila Lifespan by Over-expression of Human

SOD1 in Motorneurons Parkes, Elia, Dickinson, Hilliker

et al.

Hypotheses

• 1. Chronic and unrepaired oxidative damage to motor neurons may be a factor in aging.

• 2. Sod mutants can be rescued by restoring Sod activity in motorneurons alone.

• 3. Lifespan extension involves the catalytic activity of SOD in motorneurons.

• 4. Lower metabolic rate contributes to extended lifespan.

Materials

• Expression of Human Sod1 transgene (HS) in fruit flies achieved by using yeast GAL4/UAS system.

• D42-GAL4 activator used here is:– expressed broadly in embryogenesis– but restricted to motorneurons & interneurons

in the adult CNS.

Method for each hypothesis

• 1. Chronic and unrepaired oxidative damage to motor neurons may be a factor in aging.– Overexpress HS on motorneurons.

• 2. Sod mutants can be rescued by restoring Sod activity in motorneurons alone.– Selectively overexpress Sod in Sod mutants.

• 3. Lifespan extension involves the catalytic activity of SOD in motorneurons.– Apply oxidative stress via paraquat and

ionized radiation.

• 4. Lower metabolic rate contributes to extended lifespan.– Measure respiration rates in transgenic and

control flies.

Results

• Targeting motorneurons causes dramatic life extension.– HS increased lifespan 40%– doubles survival (95%) between 27-50 days of

age.

• Note: Previous studies show increased Sod levels in many tissues had little effect unless combined with an increase in catalase.

• Note: In situ hybridization shows that expression of HS is limited to adult motorneurons. (including those controlling flight muscles)

• Selective expression of Sod1 in motorneurons restored lifespan in a dose dependent manner in Sod mutant flies.

• Resistance to oxidants was increased

• Metabolic rate not reduced in transgenic flies.

Conclusions

• Overexpressing human SOD1 in adult motorneurons:– extends lifespan 40%– rescues lifespan of short lived Sod null

mutants– elevates resistance to oxidative stress.

• Motorneuron dysfunction due to lack of Sod is one cause of reduced lifespan in Sod mutants.

• Supports the idea that elevated ROS metabolism is involved in extended lifespan.

• Changes in overall metabolism not responsible for effects on lifespan.

• ROS metabolism determines lifespan in critical cell types like motorneurons.

• This is a refinement of the free radical theory of aging.

Targeted Neuronal Gene Expression and Longevity in

Drosophila Phillips, Parkes, Hilliker

• Most organisms have two (or more) types of superoxide dismutase (SOD):– SOD1 throughout the cell (CuZnSOD)– SOD2 in the mitochondria (MnSOD)

• SOD1 in motorneurons previously shown to extend lifespan by 140%.

• SOD1 activated in motorneurons also rescues the lifespan of Sod1 null mutants

• 1. Is lifespan affected by the expression of mitochondrial– catalase (CAT) and/or– SOD2 (MnSOD)?

• 2. Can SOD2 rescue SOD1 null mutants?

• 3. Is lifespan affected by ROS metabolism in cells other than motorneurons?

• Expression of SOD2 in motorneurons:

• wildtype showed lifespan increase of 30%

• SOD1 null mutants partially rescues adult lifespan

• Expression of SOD1 in skeletal muscle showed no effect on lifespan.

Expression of catalase in motorneurons

• catalase null mutants show massive mortality in first 2-3 days of adulthood

• the few that survive have nearly normal lifespan

• restoration of catalase reduces mortality and leads to normal lifespan

• wildtype showed no change in lifespan with catalase overexpression

Co-expression of SOD1 & CAT in motorneurons

• SOD1 alone extends lifespan by 40%

• Combination of catalase with SOD1 negates effect of SOD1; normal lifespan

• elevated CAT activity probably offset the effect of SOD1

Conclusions

• Motorneurons limit normal lifespan of fruit flies.

• ROS part of the mechanism.

Extension of Lifespan with Superoxide

Dismutase/Catalase Mimetics in Worms

Melov, Ravenscroft, Malik et al.

Background

• If ROS contributes to aging, then aging can be slowed by reducing the effects of ROS.

• This can be done in 2 ways:– reduce the amount of ROS generated– increase the amount of antioxidant

repair activities.

• Genetic mutations and manipulations that resist oxidation also extend lifespan.

Hypothesis

• Synthetic superoxide dismutase/catalase mimetics can :– Extend lifespan in wildtype worms– Restore lifespan in short-lived worm mutants

that lack mitochondrial SOD.

Materials

• 2 mimetics were tested:– EUK-8 (has SOD & catalase-like

activity)– EUK-134 (an analog of EUK-8 with

more catalase activity).

• Adult worms

• Divide worms into several groups:– untreated wildtype control– worms treated with EUK drugs– short-lived mutants (lacking

mitochondrial SOD) .

• Introduce varying concentrations of mimetics into the medium.

• Mimetics entered worms by ingestion

Results: wildtype worms

• SOD/catalase mimetic increased lifespan of wildtype 54%.– no overall dose response observed

• aging worms eat less, mimetic levels decline

• fertility unchanged

• body size unchanged

Results: mutant worms

• restored normal lifespan (up 67%)

Conclusions

• Findings are consistent with amelioration of chronic endogenous oxidative stress.

• Mimetics extend lifespan by bolstering natural antioxidant defenses.

Reversal of Age-related Learning Deficits and Brain Oxidative

Stress in Mice with Superoxide Dismutase/catalase Mimetics

Liu, Liu, Bi, Thompson

Background

• Loss of learning and memory function from 8-11 months in aging mice is associated with increases in markers of brain oxidative stress.

• Contextual fear learning and levels of protein oxidation in brain show strong negative correlation.

Hypothesis

• Clinical application of synthetic catalytic scavengers of ROS are beneficial in reversing age-related learning deficits.

Treat mice with SOD mimetics

• SOD mimetics from Eukarion: EUK-189 and EUK-207.

• Female mice at 8 months old randomly assigned to 6 groups (16-18 per group)– control– untreated control– low dose EUK-189– high dose EUK-189– low dose EUK-207– high doseEUK-207

• Minipumps implanted in anesthetized mice.

• drug delivered for 28 days

• low rate ~9nmol/day

• high rate ~ 0.09 mol/day

• Pumps replaced twice of over 3 months of treatment.

Behavioral testing after 3 months

• Plexiglass cages.

• Videocam to record freezing behaviour (index of fear conditioning).

• Computer to control events.

• Mice placed alone in clean chamber.

• Day 1: 3 minutes elapse, 3 tones sound, footshock, 1 minute later mouse removed.

• Day 2: testing for conditioning to context, no sound, no footshock, 8 minutes later mouse removed

• Day 3: testing for conditioning to tone, mice placed in different chamber,1 minute elapses, 1 tone sounds, 7 minutes later mouse removed

Behavioral Analysis

• Fear conditioning measured as % of time mice exhibited a freezing response (absence of all movement except breathing)

• Measured for auditory (startle threshold) and visual functions.

• Measured for nociception ( onset latency to tail flick at 51C hotplate)

Effects of SOD/catalase mimetic on fear conditioning learning

• control mice show low levels of freezing for both tone and context (indicates impaired learning and memory at middle age)

• SOD mimetic mice show increased tone and context freezing at both doses

• Compared EUK-207 mice at 11 months old to control mice at 8 months old: drug appears to reverse impairment of older mice

Effects of SOD/catalase mimetic on brain oxidative stress

• Mimetics decreased age-related free-radical damage: – decreased lipid peroxidation– decreased protein oxidation