Project description:Cigarette smoking is associated with reduced risk of developing Parkinson’s disease (PD). To identify genes that interact with nicotine/smoking, we performed hypothesis-free genome-wide experiments in a paraquat-induced Drosophila model and in a case-control study of PD. We demonstrated that nicotine extends life-span in paraquat-treated Drosophila (P=4E-30). Brain tissue from flies treated with combinations of paraquat and nicotine revealed elevated expression of CG14691 with paraquat which was restored with nicotine co-treatment (P(interaction)=2E-11, P(FDR-adjusted)=4E-7). Independently, variants in the 5’ region of SV2C, a human ortholog of CG14691, gave the strongest signal for interaction with smoking (P(interaction)=9E-8). The effect of smoking on PD risk varied six-fold by SV2C genotype (P(heterogeneity)=4E-10). Moreover, SV2C variants identified here were associated with SVC2 gene-expression in the HapMap data. Present results suggest synaptic vesicle protein SV2C plays a role in PD pathogenesis, and that the SV2C genotype may be useful for clinical trials of nicotine for treating PD.
Project description:Cigarette smoking is associated with reduced risk of developing Parkinson’s disease (PD). To identify genes that interact with nicotine/smoking, we performed hypothesis-free genome-wide experiments in a paraquat-induced Drosophila model and in a case-control study of PD. We demonstrated that nicotine extends life-span in paraquat-treated Drosophila (P=4E-30). Brain tissue from flies treated with combinations of paraquat and nicotine revealed elevated expression of CG14691 with paraquat which was restored with nicotine co-treatment (P(interaction)=2E-11, P(FDR-adjusted)=4E-7). Independently, variants in the 5’ region of SV2C, a human ortholog of CG14691, gave the strongest signal for interaction with smoking (P(interaction)=9E-8). The effect of smoking on PD risk varied six-fold by SV2C genotype (P(heterogeneity)=4E-10). Moreover, SV2C variants identified here were associated with SVC2 gene-expression in the HapMap data. Present results suggest synaptic vesicle protein SV2C plays a role in PD pathogenesis, and that the SV2C genotype may be useful for clinical trials of nicotine for treating PD. Drosophila female heads were dissected after exposure to plain food, food with paraquat, food with nicotine, or food with both paraquat and nicotine, and genome-wide expression was quantified using Affymetrix microarrays. 20 heads were pooled per replicate, with each treatment in triplicate, and all flies were dissected at the same timepoint, after 10 days of pretreatment (with 0.1 mg/ml nicotine or no nicotine, depending on treatment group), and then a further 6 days of treatment with either 5 mM paraquat or no paraquat, continuing on the pretreatment dose of nicotine. One biological replicate in the paraquat-only group showed RNA degradation and was not included in the normalization or subsequent analyses.
Project description:dFOXO targets in adult Drosophila melanogaster females, and the effect of insulin signalling and stress on binding. The experimets determined the binding locations of dFOXO in the whole adult female fly using ChIP-chip. The protocol was validated using mock conditions: pre-immune serum or IP on chromatin from foxo null flies. The response of this binding to stress induced by treatment of flies with paraquat or by their exposure to starvation, as well as the response to an insulin-signalling-reducing genetic manipulation (over-expression of dominant negative form of the insulin receptor), was determined.
Project description:We investigate the biological effects of radiation using Drosophila Melanogaster as a model organism, focusing on gene expression and lifespan analysis to determine the effect of different radiation doses. Our results support a threshold effect in response to radiation: no effect on lifespan and no permanent effect on gene expression is seen at doses below 10,000 Roentgens.
Project description:Exposure to Paraquat and RNA interference knockdown of mitochondrial superoxide dismutase (Sod2) are known to result in significant lifespan reduction, locomotor dysfunction, and mitochondrial degeneration in Drosophila melanogaster. Both perturbations increase the flux of superoxide, a progenitor reactive oxygen species, but the molecular underpinnings of the resulting phenotypes are poorly understood. Improved understanding of such processes could lead to advances in the treatment of numerous age-related disorders. Superoxide toxicity can act through protein carbonylation. Analysis of carbonylated proteins is attractive since reactive carbonyl groups are not present in the twenty canonical amino acids and are amenable to labeling and enrichment strategies. Here, carbonylated proteins were labeled with biotin hydrazide and enriched on streptavidin-coated beads. On-bead digestion was used to release carbonylated protein peptides, with relative abundance ratios versus controls obtained using the iTRAQ mass spectrometry-based proteomics approach. While Paraquat exposure and Sod2 knockdown have similar phenotypes, differences in protein carbonylation were anticipated because Paraquat exposure was expected to increase the concentration of superoxide throughout the cell while Sod2 knockdown was only expected to raise the concentration of superoxide in the mitochondrial matrix. Paraquat exposure resulted in widespread increases in carbonylated protein relative abundance: the median Paraquat-exposed to control carbonylated protein relative abundance ratio was 1.53. For Sod2 knockdown, in contrast, the median carbonylated protein relative abundance ratios were 1.13 versus the RNA interference driver control and 1.05 versus the RNA interference transgene control. However, some proteins did show large increases in carbonylated protein relative abundance on Sod2 knockdown, most notably cytochrome c oxidase subunit Vb, possibly providing some indication of the molecular basis of the Sod2-knockdown phenotype.
Project description:Thermal acclimation study on Drosophila melanogaster reared at 3 different temperatures (12, 25, and 31oC). The proteomic profiles of D. melanogaster under these different temperatures were analyzed and compared using label-free tandem mass spectrometry.