Interactions Among Models Of Amyotrophic Lateral Sclerosis, Parkinson Disease And Ageing In Drosophila melanogaster

2020-04-20T23:05:56Z (GMT) by Emily P. Hurley Brian E. Staveley

This study aims to investigate the effects of genes directly related to ALS and Parkinson Disease and to study the biological outcomes of the altered expression of such significant genes that associate and interact with them. Experiments were conducted which examined the consequences that altered gene expression has upon Drosophila melanogaster. This study aims to generate models of human neurodegenerative disease in the fly. All stocks were obtained from the Bloomington Drosophila Stock Center at Indiana University (IN, USA) and stored at room temperature (~ 21º C). Stocks were maintained on a standard media comprised of 65 g/L cornmeal, 50 ml/L fancy grade molasses, 10 g/L yeast and 5.5 g/L agar which was then treated with 2.5 ml propionic acid and 5 ml of 0.1 g/ml methylparaben. Longevity experiments were conducted to examine the survival of experimental flies in comparison to control flies. Critical class male progeny were collected daily and placed in vials with fresh medium. A sample size of approximately 300 males was collected in total and stored at 25º C for the duration of the experiment. The flies were scored every two days to examine if any death had occurred. A fly was considered dead when no movement was observed. Males were transferred onto fresh media every four days to obtain a healthy environment. The data was analyzed using the Graphpad Prism 8 software (Graphpad Software Inc.) with a comparison of the survival curves analyzed by the Log-rank (Mantel-Cox) test. Significance was determined at 95%, at a P-value less than or equal to 0.05 with Bonferroni correction. Locomotor experiments were conducted to examine the motor function of the flies. This analysis required 70 critical class male progeny to be collected from each cross within 24 hours. Critical class males were maintained in vials with ten flies per vial, stored at 25º C, and placed on new medium once per week throughout the experiment. The analysis began one week after collection and then every seven days after until flies had a minimum climbing score for two consecutive weeks, or less than ten flies remained alive. For each genotype, the climbing ability of five cohorts was analyzed. For each cohort of 10 flies, ten trials were then carried out, which resulted in a total of 500 trials per genotype per week. To score the climbing ability of the flies a 30 cm glass tube with a 1.5 cm diameter was used which was marked with five 2 cm sections starting from the bottom with the remainder of the glass tube left as a buffer zone. The flies were scored based on the height that was reached on the tube after a ten second time period. A climbing index was then calculated as Climbing index = Σ nm/N, where n represents the number of flies at a given level, m is the score of the level (between 1 and 5), and N is the total number of flies climbed in that trial. The data was analyzed using the Graphpad Prism 8 software (Graphpad Software Inc.) A nonlinear regression curve was produced with a 95% confidence interval, with the slope of each curve representing the rate of decline in climbing ability and the Y-intercept representing the initial climbing ability. Curves were considered to be significantly different if P < 0.05. Eye analysis of D. melanogaster was used to determine the effects of gene manipulation. The GMR-Gal4 transgenic line was used, which allowed expression of the paternally contributed transgenes in the eye of the fly. Critical class male progeny from each cross was collected, aged for 3 to 5 days post eclosion, and frozen at -80 °C, in order to sacrifice and preserve the flies before being placed on SEM studs. Studs were placed in a desiccator for at least 48 hours to dry. Using either the Mineral Liberation Analyzer FEI 650F or the FEI Quanta 400 Scanning Electron Microscope, ten different eye images for each genotype were taken to visualize the left eye of the fly. Images were then examined using the National Institutes of Health (NIH) ImageJ software for the extent of eye development, counting both the number of ommatidia and bristles, this data was then analyzed using Graphpad Prism 8 (Graphpad Software Inc.).