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Fig. 6 | Molecular Neurodegeneration

Fig. 6

From: PARIS induced defects in mitochondrial biogenesis drive dopamine neuron loss under conditions of parkin or PINK1 deficiency

Fig. 6

Dopaminergic neurotoxicity associated with Drosophila PARIS attenuated by PINK1/parkin pathway (a) Immunoblot analysis and quantification of Drosophila PARIS (dPARIS) in flies expressing the indicated transgenes under the control of TH-Gal4 driver, N = 3. b DA neuron counts in 30-day old flies. N = 10 flies per indicated genotype. c Climbing performance in 30-day old flies. N = 60 flies per indicated genotype. TH-Gal4/+ flies served as control. d Phosphorylation of dPARIS by PINK1 verified in Drosophila S2 cells. Similar results observed in three independent experiments. e Coimmunoprecipitation using FLAG antibodies show interaction of C-terminal V5-tagged dPARIS with N-terminal FLAG tagged parkin in Drosophila S2 cells. N = 3. f Reciprocal coimmunoprecipitation using V5-antibodies verify parkin interaction with dPARIS in Drosophila S2 cells transfected with the indicated constructs, N = 3. g Ubiquitination of dPARIS assessed in Drosophila S2 cells transfected with the indicated constructs. Immunoblot analysis shows ubiquitination of dPARIS is enhanced by ectopic expression of parkin and leads to decreased dPARIS protein levels. Quantification of ubiquitinated dPARIS normalized to dPARIS-V5 protein levels shown, N = 3. h Representative maximal Z-projections of confocal stack images, immunostained for mito-GFP (green) and TH (red) in the indicated genotypes. Scale = 25 μM. i Quantification of intensity ratio of mito-GFP to TH immunofluorescence in the indicated genotypes. TH > mito-GFP flies served as control, N = 10 flies per genotype. j Mitochondrial DNA copy number assessed in FACS sorted DA neurons from 30-day old flies of the indicated genotypes. Mean ratio of mtDNA to nuDNA from three independent FACS experiments shown. TH > GFP flies used as control. k Quantitative RT-PCR analysis of Drosophila homologs of PARIS (dPARIS), PGC-1α (Spargel), NRF1 (ewg), NRF-2 (Delg) and mitochondrial transcriptional factor A (TFAM) in FACS sorted DA neurons from 30-day old flies. Th > GFP flies served as control. Mean of three independent FACS experiments per genotype shown. Quantitative data = mean ± SEM. One-way ANOVA *p < 0.05, **p < 0.01, *** p < 0.001, ****p < 0.0001. See also Additional file 15, Figure S5

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