Fig. 4: Targeted neuronal overexpression of dDOR is neuroprotective in Drosophila models of Parkinson’s disease.
From: TP53INP1 exerts neuroprotection under ageing and Parkinson’s disease-related stress condition
A Survival curves on a 70-h period, showing the percent of survival of female flies fed with 2% sucrose (white disks) or poisoned with 20 mM Paraquat (PQ) diluted in 2% sucrose (black boxes, disks and triangles). UAS-dDOR transgenes (dDOR-F but not dDOR-L contains EcR binding motif) were overexpressed using a pan-neuronal GAL4 driver (elav-GAL4) and compared to the survival rate of elav-GAL4 flies crossed to w1118 controls. Survival assay was performed on three groups of ten flies in each condition. Log-rank test results: **P < 0.01, ***P < 0.0001. B Locomotor index determined by negative geotaxis tests performed on 3- and 10-day old adult male flies grown at 29 °C. Genotypes analyzed: hemizygous elav-GAL4 control flies (progeny of elav-GAL4 females and w1118 males), flies overexpressing dDOR isoforms in neurons (elav-GAL4 UAS-dDOR-F and L), flies overexpressing human α-synuclein gene p.Ala30Pro in neurons (elav-GAL4 UAS-SNCA-A30P), flies overexpressing both dDOR isoforms and human α-synuclein gene p.Ala30Pro in neurons (elav-GAL4 UAS-SNCA-A30P UAS-dDOR-F and L). Mean of 9 races involving 30 flies in total (±SD). Mann–Whitney U test results: elav-GAL4 UAS-SNCA-A30P versus elav-GAL4 control ***P < 0.001; elav-GAL4 elav-GAL4 UAS-SNCA-A30P versus elav-GAL4 UAS-SNCA-A30P UAS-dDOR-F or UAS-dDOR-L ###P < 0.001. C Z-projections of confocal image stacks showing DA neurons of the protocerebral anterior medial cluster (PAM) after TH immunolabelling of whole mounted brains from 15 days old adult male flies. Genotypes analyzed: hemizygous elav-GAL4 control males (progeny of elav-GAL4 females and w1118 males), flies overexpressing human α-synuclein gene p.Ala30Pro in neurons (elav-GAL4 UAS-SNCA-A30P), flies overexpressing dDOR-L and human α-synuclein gene p.Ala30Pro in neurons (elav-GAL4 UAS-SNCA-A30P UAS-dDOR-L). The color code represents the Z-depth of the stack on the anterior (A)–posterior (P) brain axis. D Quantification of TH-positive neurons in PAM clusters imaged in brains of 15 days old adult male flies. Number of clusters analyzed in each genotype: 15 for elav-GAL4, 16 for elav-GAL4 UAS-SNCA-A30, 15 for elav-GAL4 UAS-SNCA-A30P UAS-dDOR-L. Mann–Whitney U test results: elav-GAL4 UAS-SNCA-A30P versus elav-GAL4 control **P < 0.01; elav-GAL4 elav-GAL4 UAS-SNCA-A30P versus elav-GAL4 UAS-SNCA-A30P UAS-dDOR-L ##P < 0.01. E Locomotor index determined by negative geotaxis tests performed on 3- and 15-day old adult male flies grown at 29 °C. Genotypes analyzed: heterozygous TH-GAL4 control flies (progeny of TH-GAL4 females and w1118 males), flies overexpressing RNAi against Drosophila dPINK1 or dParkin in DA neurons (elav-GAL4 UAS-dPINK1_IR or UAS-dParkin_IR), and flies overexpressing both dDOR-F and RNAi against dPINK1 or dParkin in DA neurons (elav-GAL4 UAS-dPINK1_IR or UAS-dParkin_IR UAS-dDOR-F). All genetic conditions include a UAS-Dicer2 transgene. Mean of 9 races involving 30 flies in total (±SD). Mann–Whitney U test results: TH-GAL4 UAS-dPINK1_IR or UAS-dParkin_IR versus TH-GAL4 control **P < 0.01; TH-GAL4 UAS-dPINK1_IR or UAS-dParkin_IR versus TH-GAL4 UAS-dPINK1_IR or UAS-dParkin_IR UAS-dDOR-F ##P < 0.01.
