The effects of galactose ingestion and selected pharmacological agents on lifespan, climbing ability, and the expression of superoxide dismutase and catalase in Drosophila melanogaster
Jordens, Robert (Rob) Gerard
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The research presented here involves behavioural, pharmacological, genetic, and molecular biological characterization of a D-galactose model of advanced aging in the fruitfly, Drosophila melanogaster. Oregon-R wild-type (WT), a heterozygous superoxide dismutase (Sod)-deficient mutant (MT) (cSodN108red/TM3,Sb,Ser), a heterozygous catalase (Cat)-deficient MT (cpCatN2/TM3,Sb,Ser), and a Cat control strain (cpCat+/TM3,Sb,Ser) were examined for the effects of D-galactose substitution for D-sucrose on lifespan and climbing ability, while the Oregon-R WT and Sod MT were examined for the effects of D-galactose on sod and cat expression. Results indicate that D-galactose substitution caused a significant reduction in lifespan of all strains relative to their D-sucrose-fed controls. Similarly, D-galactose substitution caused a substantial reduction in the climbing ability (used as an indicator of aging) of Sod MT and Cat+ MT strains relative to their D-sucrose-fed controls. Given that (R)-deprenyl, (R)-2-HMP, and their desmethyl metabolites, (R)-desmethyldeprenyl and (R)-2-HPA had been reported to exhibit neural rescue and life extension ((R)-deprenyl only) effects, these compounds were examined for their ability to restore D-galactose shortened lifespan. Drugs were administered at an approximate dose of 1 ng/fly/day. Results indicate that all compounds were able to restore lifespan in WT and Sod MT flies to varying degrees. Drugs were unable to restore lifespan in CatN2 MT and Cat+ MT flies. Northern blots were used to establish age-related trends in sod and cat mRNA expression. In WT flies, results indicated an apparent peak in cat expression at day 20 as well as an overall decrease in the level of its expression over the first 30 days of life in D-galactose-fed flies; D-sucrose-fed flies exhibited increased expression over the first 30 days of life. Due to difficulties with sod Northern blots, RT-PCR was used to assess the ability of D-galactose to affect D. melanogaster sod and cat mRNA expression. Both the WT and Sod MT strain were selected for RT-PCR analysis given their high sensitivity to Dgalactose ingestion. Flies fed on D-sucrose, D-galactose, and D-galactose plus (R)2-HMP were raised to their average lifespans and euthanized. RT-PCR optimization involved sod and cat linearity curves and an 18S internal control. Analysis of sod and cat expression was done in duplicate. Averaged data suggested increased sod expression in the Sod MT fed on D-galactose and D-galactose + (R)-2-HMP; cat expression was increased in D-galactose-fed WT and Sod MT fed on D-galactose and D-galactose-fed plus (R)-2-HMP. This work has shown an effect of D-galactose on the lifespan and climbing ability of Drosophila as well as the ability of selected drugs to prevent this effect (lifespan only). Further, cat expression was shown to peak earlier in D-galactose-fed flies, possibly indicating D-galactose-induced premature aging. RT-PCR results indicate that the D-galactose effect on Drosophila aging may occur via a free radical-mediated mechanism.