Computational protocol: Downregulation of DmMANF in Glial Cells Results in Neurodegeneration and Affects Sleep and Lifespan in Drosophila melanogaster

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Protocol publication

[…] Adult males (7–10 days old) were frozen in liquid nitrogen (30 flies of each genotype) at the beginning of the day (1 h after lights on). Heads were cut off on ice and manually crushed in liquid nitrogen using a handheld homogenizer (LLG). Then, they were homogenized using an ultrasonic homogenizer (Dr. Hielscher) in 2× Laemmli Buffer with protease inhibitors (Boehringer). Samples were gently shaken, frozen at −20°C, thawed and centrifuged at 13,200 rpm for 1 h at 4°C. The supernatant was denatured at 80°C for 5 min. The protein concentration was measured using a Quant-iT Protein Assay Kit and a Qubit fluorometer (Invitrogen). Electrophoresis was performed using a NuPAGE SDS-PAGE Gel System. A 4–12% Bis-Tris gel (Invitrogen) was loaded with 10 μg of protein. The proteins were blotted by electrotransfer onto a PVDF membrane (Invitrogen) followed by blocking with 5% dry milk in PBS with 0.1% Tween-20 for 1 h at room temperature. Membranes were incubated with rabbit anti-DmMANF (1:5000) and anti-α tubulin (1:20000, Abcam 4074) antibodies as a loading control. For secondary antibody, we used goat anti-rabbit HRP (1:10000, Abcam 6721). The ECL Western blotting detection kit (Advansta) was applied for immunodetection. Densitometric quantification was performed using ImageJ software. The Western blot analysis was repeated in four independent experiments. [...] Lifespan was examined for male experimental flies with silenced expression of DmMANF in glia (w1118; UAS-DmMANFRNAi/+; P{GAL4}repo/+, referred to as repo-Gal4>UAS-DmMANFRNAi in the text below) and in neurons (P{GAL4-elav.L}2/UAS-DmMANFRNAi, referred to as elav-Gal4>UAS-DmMANFRNAi in the text below), as well as for adequate control groups (w1118; P{GAL4}repo/+ and P{GAL4-elav.L}2/+ referred to as repo-Gal4/+ and elav-Gal4/+ in the text below, respectively). The lifespan examination of each group was conducted every 2 days, and each experiment was repeated three times. The significance of differences between Kaplan-Meier survival plots for the studied groups of flies was analyzed by log-rank test using STATISTICA (Statsoft) software. The median survival of each group indicates the day at which 50% of flies were dead. […]

Pipeline specifications

Software tools ImageJ, Statistica
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Drosophila melanogaster, Homo sapiens
Chemicals Dopamine, Histamine, Potassium, Sodium