Fig. 5

PD gene candidates harboring LoF variants enhance α-synuclein toxicity in Drosophila. Conserved fly orthologs of human genes discovered from WES analysis were targeted with RNAi (IR) and screened for enhancement of α-synuclein pathology using the pseudopupil assay (a top row). For each line evaluated, the severity of retinal degeneration was scored based on penetrance of the α-synuclein pseudopupil phenotype and enhancers required consistent results for at least two independent RNAi lines (see Additional file 1: Table S8). Representative results from the primary screen are shown for controls (Rh1-GAL4 / +; UAS-α-synuclein / +) and one IR line each for the implicated enhancers [Human Gene-Fly Ortholog (experimental genotype shown)]: ARSB-CG32191 (Rh1-GAL4 / +; UAS-α-synuclein / UAS-CG32191.IR.v14294), TMEM134-CG12025 (Rh1-GAL4 / UAS-CG12025.IR.v104336; UAS-α-synuclein / +), PTPRH-Ptp10D (Rh1-GAL4 / UAS-Ptp10D.IR.v1102; UAS-α-synuclein / +), and VPS13-Vps13 (Rh1-GAL4 / UAS-Vps13.IR.HMS02460; UAS-α-synuclein / +). At the 15-day-old time point, Rh1 > α-synuclein causes a weakly-penetrant pseuodopupil phenotype and mild histopathologic changes which are amenable to modifier screening (compare with Fig. 4, panels c and e). Enhancers identified in the primary screen were confirmed based on retinal histology (a middle row) and demonstrated increased tissue destruction and disorganization. Activation of RNAi was not associated with any significant retinal degeneration in the absence of α-synuclein co-expression (a bottom row, Rh1-GAL4 / IR transgene). Scale bars: 20 μm. b Enhancement of α-synuclein-induced retinal degeneration was quantified based on the extent of vacuolar changes (area occupied by vacuoles / total retinal area). For quantification, three animals were examined per genotype. For PTPRH, additional confirmation was obtained by evaluating flies doubly heterozygous for strong alleles of the paralogs Ptp10D and Ptp4e (see also Additional file 2: Figure S5). Statistical comparisons were made using unpaired t-tests. Error bars are based on Standard Error of the Mean. *p < 0.05; **p < 0.01