Neuroscience

Parkinson’s disease-associated Pink1 loss disrupts ensheathing glia and causes dopaminergic neuron synapse loss

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

VIB-KU Leuven Center for Neuroscience, 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.105386.2

Open access
Copyright information

Figures and data

EG are affected non-cell autonomously by Pink1 loss-of-function (with supplementary fig 1)
(A) tSNE of the cells of Pink1^P399L knock-in mutants (5-day-old). Cell types are labeled with colors indicating the number of deregulated genes compared to control. EG are encircled and labeled. (B-B”) Maximum intensity projections of confocal images of fly brains (5 ± 1-day-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 in flies where CD8GFP is expressed via the EG driver MZ709-Gal4. Scale bar: 20 µm (B’) Maximum intensity projection of confocal images of controls vs. controls 24 hours after ORN-severing (injury). (B”) Maximum intensity projection of confocal images of Pink1^KO-WS vs Pink1^KO-WS 24 hours after ORNs severing (injury). (C) Quantification of GFP intensity within the glomeruli of the antennal lobe area in 5 ± 1-day-old flies (as in B) relative to controls. ANOVA with Dunnet’s multiple comparison test, * is p<0.05, ** is p<0.01, *** is p<0.001. Effect size: η =0.23. Bars: mean ± SD; points are individual animals N≥13 per genotype, 4 replicates. (D-D”) Maximum intensity projection of confocal images of fly brains (5 ± 1-day-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 in flies where CD8GFP is expressed via the EG driver MZ709-Gal4. Scale bar: 20 µm. (D’) Maximum intensity projection of confocal images of control (w¹¹¹⁸) and Pink1^KO-WS animals (D”) Maximum intensity projection of confocal images of animals with Pink1 downregulation in EG and Pink1^KO-WS with Pink1 rescued in EG. (E) Quantification of GFP intensity within the glomeruli of the antennal lobe area in 5-day-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. Effect size: η =0.36 Bars: mean ± SD; points are individual animals N≥10 per genotype, 4 replicates.

Pink1 in EG is necessary to support synaptic integrity (with Supplementary figure 2)
(A) Representative ERG traces of indicated genotypes: ON peak is highlighted by the arrow. (B) Normalized ON peak amplitude of flies (5 ± 1-day-old). ANOVA with Tukey’s multiple comparison test, * is p<0.05, ** is p<0.01, *** is p<0.001. Effect size: η =0.55 Bars: mean ± SD; points are individual animals N≥11 per genotype, 3 replicates. (C-C”) (C) Maximum intensity projection of confocal images of control and Pink1^KO-WS in Mushroom Bodies (MB) of aged flies (22 ± 2-day-old), stained with anti-TH (cyan) and anti-DLG (magenta) antibodies - DLG is used to mark post-synaptic sites of MB. The black and white image is the middle Z-plane within the region of interest of the MB (ROI, yellow), which is used to represent the thresholded TH area (white). Scale bar: 20 µm. (C’) Maximum intensity projection of confocal images of w¹¹¹⁸ with Pink1 downregulation in EG. (C”) Maximum intensity projection of confocal images of Pink1^KO-WS with Pink1 rescued in EG. (D) Quantification of the dopaminergic synaptic area at MB neuropil in aged flies (22 ± 2-day-old). ANOVA with Tukey’s multiple comparison test, * is p<0.05, ** is p<0.01, *** is p<0.001. Effect size: η = 0.38. Bars: mean ± SD; points are individual animals N≥12 per genotype, 3 replicates.

Cell type-specific transcriptomics reveals modifiers of neuronal dysfunction
(A)Scheme of cell-type specific transcriptomics (created using BioRender.com). (B)Scaled gene expression of representative genes for EG and neurons after sorting EG or neurons using the protocol described in (A) (R Core Team, 2022). N=2, 2 replicates. (C)Differentially expressed genes in EG in Pink1^KO-WS compared 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); light green dots are all detected genes, dark green are the 50 most deregulated genes, and the pink dot is from a gene positive in the genetic screen (D). *Two data points are outside the boundaries of the plot. To determine the transcriptomic profile of each genotype, a N=3 was used, and 3 replicates were performed. (D)ERG ON peak value differences to Pink1^KO-WS flies (5 ± 1-day-old) of control (red) and of Pink1^KO-WS flies with DEGs downregulated, or upregulated, specifically in EG (5 ± 1 days old). ON peak values 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. Effect size: η = 0.44. Bars: mean ± SD; points are individual animals N≥3 per genotype. *One data point is outside the boundaries of the plot.

Vps35 and Vps13 downregulation in EG rescues synaptic deficits in Pink1^KO-WS flies (with supplementary figure 3)
(A)Representative ERG traces of control,Pink1^KO-WS flies and Pink1^KO-WS flies with Vps35 or Vps13 downregulated in EG. (B)Quantification of the normalized ON peak response of flies with the genotypes in (A) (5 ±1-day-old). ANOVA with Dunnett’s multiple comparison test, * is p<0.05, ** is p<0.01, *** is p<0.001. Effect size: η = 0.48. Bars: mean ± SD; points are individual animals N≥10 per genotype, 3 replicates. (C)Maximum intensity projection of confocal images of Mushroom Bodies (MB) of aged flies (22 ± 2-day-old) of control, Pink1^KO-WS flies and Pink1^KO-WS flies with Vps13 downregulated in EG, labeled with anti-TH (cyan) and anti-DLG (magenta), DLG is used to mark the MB neuropil. The black and white image is the middle Z-plane within the region of interest of the MB (ROI, yellow), which is used to represent the thresholded TH area (white). Scale bar: 20 µm. (D)Quantification of the dopaminergic synaptic area within MB of aged flies (22 ± 2-day-old). ANOVA with Dunnett’s multiple comparison test, * is p<0.05, ** is p<0.01, *** is p<0.001. Effect size: η =0.23. Bars: mean ± SD; points are individual animals n≥22 per genotype, 5 replicates.

Modulation of ER–mitochondria contact sites and lipid transfer in ensheathing glia (EG) rescues Pink1-dependent neuronal dysfunction.
Schematic representation of the suggested model (created using BioRender.com). (A)Loss of Pink1 leads to an abnormal increase in endoplasmic reticulum (ER)–mitochondria contact sites (represented by blue thick lines), resulting in enhanced ER-to-mitochondria lipid transfer and dysregulation of ER lipid composition (represented by yellow lipids). Increased organelle membrane contacts and lipid flux in EG contribute to neuronal dysfunction in a non–cell autonomous manner. (B)Genetic downregulation of ER–mitochondria contact and lipid transfer regulators in EG rescues Pink1-induced neuronal phenotypes through two convergent mechanisms. Reduction of Vps35 decreases the number of ER–mitochondria contact sites, likely via MUL1-mediated Mitofusin (Mfn) turnover, leading to normalization of calcium and lipid homeostasis. In parallel, downregulation of Vps13, a lipid transfer facilitator at organelle contact sites, limits ER-to-mitochondria lipid transfer capacity, counteracting the excessive lipid flux induced by Pink1 loss. Both interventions restore organelle homeostasis in EG and result in rescue of neuronal dysfunction through a non–cell autonomous mechanism.

Representative confocal image of a (5 ± 1-day-old) MZ709-Gal4/+;; UAS-HisTageGFP/+ brain stained with anti-GFP (Cyan) and anti-ELAV (Magenta).

Scale bar: 50µm; N= 3, 1 replicate.

Examples of confocal Z-stacks of anti-DLG labeled fly brains (22 ± 2-day-old) of the indicated genotypes.

Such images were used to delineate ROIs to quantify DAN innervation onto the MBs. (A) control (w1118/y;; MZ709-Gal4/+); (B) Pink1KO-WS/Y;; MZ709-Gal4/+; (C) flies where Pink1 is downregulated in EG (w1118/y;; MZ709-Gal4/ Pink1RNAi); (D) Pink1KO-WS flies with expression of wild type Pink1 in EG (Pink1KO-WS/Y;; MZ709-Gal4/UAS-Pink1).

Examples of confocal Z-stacks of anti-DLG labeled fly brains (22 ± 2-day-old) of the indicated genotypes.

Such images were used to delineate ROIs to quantify DAN innervation onto the MBs. (A) control (w1118/y;; MZ709-Gal4/+); (B) Pink1KO-WS/Y;; MZ709-Gal4/+; (C) Pink1KO-WS flies with Vps13 downregulation in EG (Pink1KO-WS/Y;; Vps13RNAi/MZ709-Gal4).

Sign up for email alerts