Neuroscience

Parkinson’s disease-associated Pink1 loss disrupts vesicle trafficking in Ensheathing glia causing dopaminergic neuron synapse loss

Lorenzo Ghezzi
Ulrike Pech
Nils Schoovaerts
Suresh Poovathingal
Kristofer Davie
Jochen Lamote
Roman Praschberger author has email address
Patrik Verstreken author has email address

VIB-KU Leuven Center for Brain & Disease Research; Leuven, Belgium
KU Leuven, Department of Neurosciences, Leuven Brain Institute, Leuven, Belgium
VIB Flow Core Leuven, VIB Technologies, Leuven, Belgium
Medical University of Innsbruck, Institute of Human Genetics; Innsbruck, Austria

https://doi.org/10.7554/eLife.105386.1

Open access
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Figures and data

(A) tSNE of the cells of Pink1^P399L knock-in mutants (5 days old). Cell types are labeled with colors indicating the number of deregulated genes compared to control. EG are encircled and labeled. (B-B”) (B) SUM intensity of confocal images of fly brains (5 ± 1 days old) stained with anti-GFP (Green) and anti-Brp (Magenta), where anti-GFP marks EG and anti-Brp marks presynaptic sites of the antennal lobes. Scale bar: 20 µm (B’) SUM intensity of confocal images of control flies vs control flies 24 hours after ORNs severing. (B”) SUM intensity of confocal images of Pink1^KO-WS vs Pink1^KO-WS flies 24 hours after ORNs severing. (C) Quantification of GFP intensity with the glomeruli of the antennal lobe area in 5 ± 1 days old flies (as in B) relative to the control. ANOVA with Dunnet’s multiple comparison test, * is p<0.05, ** is p<0.01, *** is p<0.001. Bars: mean ± SD; points are individual animals N≥13 per genotype. (D-D”) SUM intensity of confocal images of fly brains (5 ± 1 days old) stained with anti-GFP (Green) and anti-Brp (Magenta), where anti-GFP marks EG and anti-Brp marks presynaptic sites of the antennal lobes. Scale bar: 20 µm. (D’) SUM intensity of confocal images of w1118 with Pink1 downregulation in EG. (D”) SUM intensity of confocal images of Pink1^KO-WSwith Pink1 rescued in EG. (E) Quantification of GFP intensity within the glomeruli of the antennal lobe area in 5 days old flies (as in D) relative to the control. ANOVA with Tukey’s multiple comparison test, * is p<0.05, ** is p<0.01, *** is p<0.001. Bars: mean ± SD; points are individual animals N≥10 per genotype.

(A) Representative ERG traces: ON peak is highlighted by the arrow. (B) Normalized ON peak response of flies (5 ± 1 days old). ANOVA with Tukey’s multiple comparison test, * is p<0.05, ** is p<0.01, *** is p<0.001. Bars: mean ± SD; points are individual animals N≥11 per genotype. (C-C”) (C) Maximum projection confocal image of control and Pink1^KO-WS in Mushroom Bodies (MB) of aged flies (22 ± 2 days old), stained with anti-TH (cyan) and anti-DLG (magenta) antibodies, DLG is used to mark post-synaptic site of MB. The black and white image is the middle Z-plan within the region of interest of the MB(ROI, yellow), it is used to represent the thresholded TH area (white). Scale bar: 20 µm. (C’) Maximum projection confocal image of w1118 with Pink1 downregulation in EG. (C”) Maximum projection confocal image of Pink1^KO-WS with Pink1 rescued in EG. (D) Quantification of dopaminergic synaptic area within MB in aged flies (22 ± 2 days old). ANOVA with Tukey’s multiple comparison test, * is p<0.05, ** is p<0.01, *** is p<0.001. Bars: mean ± SD; points are individual animals N≥12 per genotype.

(A) Scheme of cell-type specific transcriptomics. Panel created using BioRender.com/p56u250. (B) Scaled gene expression of representative genes for EG and neurons after sorting EG or neurons using the protocol described in figure A. Figure generated using R studio (R Core Team, 2022). (C) Differentially expressed genes in EG in Pink1^KO-WScompared to control flies, plotted according to their Log2 Fold change and the -Log10 of the adjusted p-value. Intercept in RED (-Log10 adjusted p-value = 4.31). *Two data points are outside the boundaries of the plot. (D) ERG ON peak value differences to Pink1^KO-WS (red) flies (5 ± 1 days old) of Pink1^KO-WS flies (5 ± 1 days old) with DEGs downregulated, or upregulated, specifically in EG. ON peak value are expressed as the difference to Pink1^KO-WS. ANOVA with Dunnett’s test, * is p<0.05, ** is p<0.01, *** is p<0.001. Bars: mean ± SD; points are individual animals N≥3 per genotype. *One data point is outside the boundaries of the plot.

(A-A’) (A) Representative ERG traces. (A’) Representative ERG traces of genes Pink1^KO-WS flies with Vps13, mon1, and rab7 downregulated in EG (B) Normalized ON peak response of flies (5 ± 1 days old). Kruskal-Wallis with Dunn’s multiple comparison test, * is p<0.05, ** is p<0.01, *** is p<0.001. Bars: mean ± SD; points are individual animals N≥8 per genotype. (C) Maximum projection confocal image of control and Pink1^KO-WS in Mushroom Bodies (MB) of aged flies (22 ± 2 days old), stained with anti-TH (cyan) and anti-DLG (magenta) antibodies, DLG is used to mark post-synaptic site of MB. The black and white image is the middle Z-plan within the region of interest of the MB(ROI, yellow), it is used to represent the thresholded TH area (white). Scale bar: 20 µm. (D) Quantification of dopaminergic synaptic area within MB in aged flies (22 ± 2 days old). ANOVA with Dunnett’s multiple comparison test, * is p<0.05, ** is p<0.01, *** is p<0.001. Bars: mean ± SD; points are individual animals N≥13 per genotype.

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