Ddc gal4

The following flies were used in this study: dopa decarboxylase (ddc)-GAL4 and yellow white (yw) (Bloomington Drosophila Stock Center). Human VPS35-expressing flies were created by generating transgenic human VPS35 wild type or mutant (P316S, D620N and L774M) cDNA containing a Hemagglutinin (HA) tag at the C-terminus and inserted into the pUAST-attB plasmid, which will allow the UAS constructs to land into a chosen attP site in the fly genome during microinjection. Constructs were then sent for microinjection into Drosophila embryos (BestGene). Flies were raised on standard yeast-cornmeal-agar medium at 25°C with 12-hour light and dark cycle.

Promoter lines containing ddc-GAL4 were obtained from Bloomington Drosophila Stock Center (Bloomington, IN, USA). The human A30P α-syn transgenic fly line (P{UAS-Hsap\SNCA.A30P}40.1, deposited by Prof. Nancy Bonini, University of Pennsylvania) expressing UAS controlled human mutant A30P α-syn is purchased from Bloomington Drosophila Stock Center (Bloomington, IN, USA). Yellow white control or transgenic α-syn A30P mutant flies were crossed with ddc-GAL4 lines to induce overexpression of α-syn specifically in DA neurons in transgenic fly heads. Flies were routinely raised at 25 °C on cornmeal media for 30 days in the presence or absence of 2 mM NAC, GA, TA, and l-cys before assessments of fly climbing behavior and analysis of DA content and TH positive DA neuron numbers in fly heads. For the climbing assay, motor ability was assessed at 15 min interval using the negative geotaxis assay. Briefly three cohorts of 20 female and age-matched control flies were anesthetized and placed in a vertical plastic column (length, 25 cm; diameter, 1.5 cm). After a 2 h recovery period, flies were tapped to the bottom and the percentage of flies that climb to or above the top column line in 1 min was calculated. Triplicate trials were performed in each experiment at 15 min interval.

Flies carrying the UAS-Rippase followed by a short PEST sequence (aa 422–461 of the mouse ornithine decarboxylase gene) to decrease its half-life and potential toxicity (pJFRC165-20XUAS-IVS-R::PEST in attP2) were from Gerald Rubin (Janelia Farms, Ashburn, VA, USA); Alrm-Gal4 was from Marc Freeman (UMASS Medical School, Worcester, MA, USA), C380-Gal4 and C57-Gal4 were from Vivian Budnik (UMASS Medical School, Worcester, MA, USA), UAS-GFP-Rab5 from Marcos Gonzalez-Gaitan (University of Geneva, Switzerland) and GFP-Rab5 knock-in flies (Fabrowski et al., 2013 (link)) from Stefano De Renzis [European Molecular Biology Laboratory (EMBL) Heidelberg, Germany]. YFP-HA-Rab11 knock-in flies (Dunst et al., 2015 (link)) were from Marko Brankatschk (Max Planck Institute of Molecular Cell Biology and Genetics, Germany), Hemese-Gal4 (#8699), Repo-Gal4 (#7415), ddc-Gal4 (#7009) and w; Df(exel)6078/CyO (#7558) were from the Bloomington Drosophila Stock Center.