Fig. 5: Inhibition of GCase activity disrupts mitochondria–lysosome contact untethering in dopaminergic neurons. | Nature Communications

Fig. 5: Inhibition of GCase activity disrupts mitochondria–lysosome contact untethering in dopaminergic neurons.

From: Dysregulation of mitochondria-lysosome contacts by GBA1 dysfunction in dopaminergic neuronal models of Parkinson’s disease

Fig. 5

a Healthy control WT human iPSC-derived dopaminergic neurons were treated with vehicle (Ctrl) or CBE (50 μM; 7 days) to inhibit GCase activity. Representative time-lapse confocal images of contacts between mitochondria (red, Mito-RFP) and lysosomes (green, Lyso-GFP) in Ctrl (left) and CBE-treated (right) human neurons. Yellow arrows mark stable M–L contacts. White arrows mark the site of M–L contacts after contact untethering. Black line shows duration of contacts. Scale bar = 500 nm. b Representative confocal images of lysosomes (LAMP1–GFP) in the Ctrl (left) and CBE-treated (right) human neurons. Scale bar = 1 μm. c The effects of CBE treatment on lysosomal morphology were confirmed by quantification of the percentage of lysosomes that were enlarged (diameter >0.5 μm) in M–L contacts (n = 12 neurons per condition, N = 3 independent experiments). Unpaired two-sided Student’s t-test; ***p = 0.0001. d CBE treatment in control neurons increased the average minimum duration of stable M–L contacts (n = 60 contacts per condition, N = 3 independent experiments). Unpaired two-sided Student’s t-test; ***p = 0.0001. e Representative confocal images of immunocytochemistry of GlcCer in the Ctrl (left) and CBE-treated (right) human neurons. Scale bar = 5 μm. f Quantification of CBE treatment leading to increased GlcCer levels as measured by immunofluorescence signal of GlcCer (n = 21 neurons per condition, N = 3 independent experiments). g Exogenous GlcCer treatment in control neurons increased the average minimum duration of stable M–L contacts (n = 93 contacts from Ctrl, n = 94 contacts from +GlcCer neurons, N = 3 independent experiments). f, g Unpaired two-sided Student’s t-test; ***p < 0.0001. h–k Western blot analysis of i TBC1D15, j Rab7, and k Fis1 levels in Ctrl and CBE-treated neurons. Protein levels were normalized to loading control GAPDH. Values are expressed as fold-change compared to Ctrl (N = 5 independent experiments). Paired two-sided Student’s t-test; *p = 0.0104. l, m GST-RILP pull-down to measure GTP-bound Rab7 levels in Ctrl and CBE-treated neurons. m Rab7-GTP levels were normalized to total Rab7 normalized to GAPDH. Values are expressed as fold-change compared to Ctrl (N = 3 independent experiments). Paired two-sided Student’s t-test; *p = 0.0242. For all quantifications, data are means ± S.E.M., *p ≤ 0.05, ***p ≤ 0.001.

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