Superoxide Dismutase: Baseline for Characterizing Oxidative Stress in Drosophila

Rolan Milutinovic, Northeastern Illinois University

Elyse Bolterstein is the faculty sponsor of this poster.

Description

Free radicals such as reactive oxygen species (ROS) can change DNA, lipid, and protein structure in organisms through oxidative stress. Superoxide dismutase (SOD) is a protein that combats oxidative stress through the catalyzation of unstable superoxide molecules to hydrogen peroxide, which is then turned into water and eliminated. Mutations in SOD are hallmark factors of familia amyotrophic lateral sclerosis (ALS), a neurodegenerative disorder. Drosophila melanogaster has homologs to all three human SOD genes making it a suitable model organism with which to study the attributes of oxidative stress. Drosophila SOD1 is the most abundant of the three homologs and is located in the cytoplasm. Multiple SOD1 mutant stocks are available, including the alleles SOD1 n1 , SOD n64 , SOD1 X-39 , SOD1 Df(3L)ED4470 , and SOD1 Df(3L)BSC753 . Both SOD1 n1 and SOD n64 contain point mutations, G49S and G42E respectively, while SOD1 X-39 contains a 395 bp deletion. SOD1 Df(3L)ED4470 and SOD1 Df(3L)BSC753 are deleted fragment strains containing large deletions (310 and 710 base pairs, respectively) in the SOD1 gene locus on the third left chromosome. Previous research has shown that SOD1 mutant flies are sensitive to paraquat, an herbicide and oxidative stress inducer. Also, reduced SOD1 expression decreases fertility, adult emergence, and lifespan. Because the current available mutant alleles are homozygous lethal, we created viable trans homozygous mutants: SOD1 n1 /SOD1 X-39 , SOD1 n1 /SOD1 Df(3L)ED4470 , and SOD1 n1 /SOD1 Df(3L)BSC753 . The goal of my project was to characterize phenotypes of the trans homozygote mutants so that we can use them to establish a baseline for studying the effects of oxidative stress in our lab. We found that our SOD1 n1 /SOD1 X-39 larvae were sensitive to 20 mM paraquat (44.6% relative survival to their heterozygous counterparts). These results differ from past studies showing mutant sensitivity to paraquat at doses as low as 0.5 mM, suggesting that the trans homozygous mutants may still have some SOD1 gene expression. To assess the impacts of SOD1 mutations on fertility, we used hatching frequency assays. We found that when mutant SOD1 females were mated to wild type males, they produced eggs with lower hatching frequencies: 25.0% for SOD1 n1 /SOD1 X-39 females compared to 95% for wildtype females. This suggests that maternally loaded SOD1 is critical during development. When mutant males were mated with wild type females, we observed the following hatching frequencies: 32.5% for SOD1 n1 / SOD1 Df(3L)ED4470 , 40.9% for SOD1 Df(3L)BSC753 and 45.1% for SOD1 n1 /SOD1 X-39 . My future plans include treating SOD1 mutants with vitamin C (ascorbic acid) to enhance antioxidant abilities of SOD1 deficient flies and therefore “rescue” the hatching defect.

 
Apr 19th, 11:00 AM

Superoxide Dismutase: Baseline for Characterizing Oxidative Stress in Drosophila

Free radicals such as reactive oxygen species (ROS) can change DNA, lipid, and protein structure in organisms through oxidative stress. Superoxide dismutase (SOD) is a protein that combats oxidative stress through the catalyzation of unstable superoxide molecules to hydrogen peroxide, which is then turned into water and eliminated. Mutations in SOD are hallmark factors of familia amyotrophic lateral sclerosis (ALS), a neurodegenerative disorder. Drosophila melanogaster has homologs to all three human SOD genes making it a suitable model organism with which to study the attributes of oxidative stress. Drosophila SOD1 is the most abundant of the three homologs and is located in the cytoplasm. Multiple SOD1 mutant stocks are available, including the alleles SOD1 n1 , SOD n64 , SOD1 X-39 , SOD1 Df(3L)ED4470 , and SOD1 Df(3L)BSC753 . Both SOD1 n1 and SOD n64 contain point mutations, G49S and G42E respectively, while SOD1 X-39 contains a 395 bp deletion. SOD1 Df(3L)ED4470 and SOD1 Df(3L)BSC753 are deleted fragment strains containing large deletions (310 and 710 base pairs, respectively) in the SOD1 gene locus on the third left chromosome. Previous research has shown that SOD1 mutant flies are sensitive to paraquat, an herbicide and oxidative stress inducer. Also, reduced SOD1 expression decreases fertility, adult emergence, and lifespan. Because the current available mutant alleles are homozygous lethal, we created viable trans homozygous mutants: SOD1 n1 /SOD1 X-39 , SOD1 n1 /SOD1 Df(3L)ED4470 , and SOD1 n1 /SOD1 Df(3L)BSC753 . The goal of my project was to characterize phenotypes of the trans homozygote mutants so that we can use them to establish a baseline for studying the effects of oxidative stress in our lab. We found that our SOD1 n1 /SOD1 X-39 larvae were sensitive to 20 mM paraquat (44.6% relative survival to their heterozygous counterparts). These results differ from past studies showing mutant sensitivity to paraquat at doses as low as 0.5 mM, suggesting that the trans homozygous mutants may still have some SOD1 gene expression. To assess the impacts of SOD1 mutations on fertility, we used hatching frequency assays. We found that when mutant SOD1 females were mated to wild type males, they produced eggs with lower hatching frequencies: 25.0% for SOD1 n1 /SOD1 X-39 females compared to 95% for wildtype females. This suggests that maternally loaded SOD1 is critical during development. When mutant males were mated with wild type females, we observed the following hatching frequencies: 32.5% for SOD1 n1 / SOD1 Df(3L)ED4470 , 40.9% for SOD1 Df(3L)BSC753 and 45.1% for SOD1 n1 /SOD1 X-39 . My future plans include treating SOD1 mutants with vitamin C (ascorbic acid) to enhance antioxidant abilities of SOD1 deficient flies and therefore “rescue” the hatching defect.