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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

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Extension of Lifespan by Overexpression of Superoxide Dismutase in Drosophila melanogaster

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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


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