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Oxidative Stress in Caenorhabditis elegans: Protective Effects of Spartin.

Truong T, Karlinski ZA, O'Hara C, Cabe M, Kim H, Bakowska JC - PLoS ONE (2015)

Bottom Line: These results suggest an age-dependent decline in motor function in mutant animals.In the behavioral assays, spg-20 mutant animals showed a significant decrease in both crawling speed and thrashing frequency compared with wild-type animals.These data suggest that the protein F57B10.9/SPG-20 might have a protective role against oxidative stress.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Pharmacology and Therapeutics, Loyola University Chicago, Maywood, Illinois, United States of America.

ABSTRACT
Troyer syndrome is caused by a mutation in the SPG20 gene, which results in complete loss of expression of the protein spartin. We generated a genetic model of Troyer syndrome in worms to explore the locomotor consequences of a mutation of the Caenorhabditis elegans SPG20 orthologue, F57B10.9, also known as spg-20. Spg-20 mutants showed decreased length, crawling speed, and thrashing frequency, and had a shorter lifespan than wild-type animals. These results suggest an age-dependent decline in motor function in mutant animals. The drug paraquat was used to induce oxidative stress for 4 days in the animals. We measured survival rate and examined locomotion by measuring crawling speed and thrashing frequency. After 4 days of paraquat exposure, 77% of wild-type animals survived, but only 38% of spg-20 mutant animals survived. Conversely, animals overexpressing spg-20 had a survival rate of 95%. We also tested lifespan after a 1 hour exposure to sodium azide. After a 24 hour recovery period, 87% of wild type animals survived, 57% of spg-20 mutant animals survived, and 82% of animals overexpressing spg-20 survived. In the behavioral assays, spg-20 mutant animals showed a significant decrease in both crawling speed and thrashing frequency compared with wild-type animals. Importantly, the locomotor phenotype for both crawling and thrashing was rescued in animals overexpressing spg-20. The animals overexpressing spg-20 had crawling speeds and thrashing frequencies similar to those of wild-type animals. These data suggest that the protein F57B10.9/SPG-20 might have a protective role against oxidative stress.

No MeSH data available.


Related in: MedlinePlus

Deletion of spg-20 in transgenic animals.(A) Schematic diagram shows primers annealing on both WT and spg-20 mutant genes. (B) Deletion of spg-20 was confirmed by PCR genotyping. Two sets of primers were used to confirm complete deletion of spg-20 in mutant strains. Primer pairs AB amplified the wild-type (WT) genome (600 bp), and primer pairs CB amplified the mutant genome (500 bp). (C) PCR confirms deletion of spg-20 as well as presence of spg-20 containing plasmid in transgenic animals expressing GFP. The presence of a band at 600 bp using primer pairs A and B and a band at 1700 bp using primers C and B are indicative of wild-type animals. A 500 bp band using primer pairs C and B along with the absence of a band using primer pairs A and B are indicative of the animals containing the spg-20(tm5514) knockout mutation. The presence of a band at 600 bp using primer pairs A and B, along with both a 500 bp and a 1700 bp band using primer pairs C and B is indicative of animals overexpressing spg-20, which contain both the spg-20(tm5514) knockout mutation and the spg-20 fosmid.
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pone.0130455.g002: Deletion of spg-20 in transgenic animals.(A) Schematic diagram shows primers annealing on both WT and spg-20 mutant genes. (B) Deletion of spg-20 was confirmed by PCR genotyping. Two sets of primers were used to confirm complete deletion of spg-20 in mutant strains. Primer pairs AB amplified the wild-type (WT) genome (600 bp), and primer pairs CB amplified the mutant genome (500 bp). (C) PCR confirms deletion of spg-20 as well as presence of spg-20 containing plasmid in transgenic animals expressing GFP. The presence of a band at 600 bp using primer pairs A and B and a band at 1700 bp using primers C and B are indicative of wild-type animals. A 500 bp band using primer pairs C and B along with the absence of a band using primer pairs A and B are indicative of the animals containing the spg-20(tm5514) knockout mutation. The presence of a band at 600 bp using primer pairs A and B, along with both a 500 bp and a 1700 bp band using primer pairs C and B is indicative of animals overexpressing spg-20, which contain both the spg-20(tm5514) knockout mutation and the spg-20 fosmid.

Mentions: The spg-20 gene in C. elegans contains 8 exons, and the spg-20(tm5514) mutation spans exons 1 through 6 (Fig 1B). To confirm the mutation in the spg-20(tm5514) animals, we performed PCR using two sets of primers. Primers A and B were used to amplify the wild-type genome (600 bp), and primers C and B were used to amplify the both the wild type and the spg-20(tm5514) mutant genomes, which are distinguished by the length of the fragment (500 bp and 1700 bp, respectively) (Fig 2A). Fig 2B illustrates the annealing of the first and second primer sets onto the respective wild-type and mutant genomes. When DNA from wild-type animals was used, we observed a band at approximately 600 bp with primers A and B and a band at approximately 1700 bp with primers C and B, as predicted. When using spg-20 mutant DNA, we observed no band with primers A and B and a band at approximately 500 bp with primers C and B, as predicted (Fig 2B). The absence of a band with primers A and B and the presence of a smaller band with primers C and B confirmed the deletion of spg-20 in the mutants.


Oxidative Stress in Caenorhabditis elegans: Protective Effects of Spartin.

Truong T, Karlinski ZA, O'Hara C, Cabe M, Kim H, Bakowska JC - PLoS ONE (2015)

Deletion of spg-20 in transgenic animals.(A) Schematic diagram shows primers annealing on both WT and spg-20 mutant genes. (B) Deletion of spg-20 was confirmed by PCR genotyping. Two sets of primers were used to confirm complete deletion of spg-20 in mutant strains. Primer pairs AB amplified the wild-type (WT) genome (600 bp), and primer pairs CB amplified the mutant genome (500 bp). (C) PCR confirms deletion of spg-20 as well as presence of spg-20 containing plasmid in transgenic animals expressing GFP. The presence of a band at 600 bp using primer pairs A and B and a band at 1700 bp using primers C and B are indicative of wild-type animals. A 500 bp band using primer pairs C and B along with the absence of a band using primer pairs A and B are indicative of the animals containing the spg-20(tm5514) knockout mutation. The presence of a band at 600 bp using primer pairs A and B, along with both a 500 bp and a 1700 bp band using primer pairs C and B is indicative of animals overexpressing spg-20, which contain both the spg-20(tm5514) knockout mutation and the spg-20 fosmid.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4482654&req=5

pone.0130455.g002: Deletion of spg-20 in transgenic animals.(A) Schematic diagram shows primers annealing on both WT and spg-20 mutant genes. (B) Deletion of spg-20 was confirmed by PCR genotyping. Two sets of primers were used to confirm complete deletion of spg-20 in mutant strains. Primer pairs AB amplified the wild-type (WT) genome (600 bp), and primer pairs CB amplified the mutant genome (500 bp). (C) PCR confirms deletion of spg-20 as well as presence of spg-20 containing plasmid in transgenic animals expressing GFP. The presence of a band at 600 bp using primer pairs A and B and a band at 1700 bp using primers C and B are indicative of wild-type animals. A 500 bp band using primer pairs C and B along with the absence of a band using primer pairs A and B are indicative of the animals containing the spg-20(tm5514) knockout mutation. The presence of a band at 600 bp using primer pairs A and B, along with both a 500 bp and a 1700 bp band using primer pairs C and B is indicative of animals overexpressing spg-20, which contain both the spg-20(tm5514) knockout mutation and the spg-20 fosmid.
Mentions: The spg-20 gene in C. elegans contains 8 exons, and the spg-20(tm5514) mutation spans exons 1 through 6 (Fig 1B). To confirm the mutation in the spg-20(tm5514) animals, we performed PCR using two sets of primers. Primers A and B were used to amplify the wild-type genome (600 bp), and primers C and B were used to amplify the both the wild type and the spg-20(tm5514) mutant genomes, which are distinguished by the length of the fragment (500 bp and 1700 bp, respectively) (Fig 2A). Fig 2B illustrates the annealing of the first and second primer sets onto the respective wild-type and mutant genomes. When DNA from wild-type animals was used, we observed a band at approximately 600 bp with primers A and B and a band at approximately 1700 bp with primers C and B, as predicted. When using spg-20 mutant DNA, we observed no band with primers A and B and a band at approximately 500 bp with primers C and B, as predicted (Fig 2B). The absence of a band with primers A and B and the presence of a smaller band with primers C and B confirmed the deletion of spg-20 in the mutants.

Bottom Line: These results suggest an age-dependent decline in motor function in mutant animals.In the behavioral assays, spg-20 mutant animals showed a significant decrease in both crawling speed and thrashing frequency compared with wild-type animals.These data suggest that the protein F57B10.9/SPG-20 might have a protective role against oxidative stress.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Pharmacology and Therapeutics, Loyola University Chicago, Maywood, Illinois, United States of America.

ABSTRACT
Troyer syndrome is caused by a mutation in the SPG20 gene, which results in complete loss of expression of the protein spartin. We generated a genetic model of Troyer syndrome in worms to explore the locomotor consequences of a mutation of the Caenorhabditis elegans SPG20 orthologue, F57B10.9, also known as spg-20. Spg-20 mutants showed decreased length, crawling speed, and thrashing frequency, and had a shorter lifespan than wild-type animals. These results suggest an age-dependent decline in motor function in mutant animals. The drug paraquat was used to induce oxidative stress for 4 days in the animals. We measured survival rate and examined locomotion by measuring crawling speed and thrashing frequency. After 4 days of paraquat exposure, 77% of wild-type animals survived, but only 38% of spg-20 mutant animals survived. Conversely, animals overexpressing spg-20 had a survival rate of 95%. We also tested lifespan after a 1 hour exposure to sodium azide. After a 24 hour recovery period, 87% of wild type animals survived, 57% of spg-20 mutant animals survived, and 82% of animals overexpressing spg-20 survived. In the behavioral assays, spg-20 mutant animals showed a significant decrease in both crawling speed and thrashing frequency compared with wild-type animals. Importantly, the locomotor phenotype for both crawling and thrashing was rescued in animals overexpressing spg-20. The animals overexpressing spg-20 had crawling speeds and thrashing frequencies similar to those of wild-type animals. These data suggest that the protein F57B10.9/SPG-20 might have a protective role against oxidative stress.

No MeSH data available.


Related in: MedlinePlus