Journal: Molecular Neurodegeneration

Article Title: Mitochondrial CISD1/Cisd accumulation blocks mitophagy and genetic or pharmacological inhibition rescues neurodegenerative phenotypes in Pink1/parkin models

doi: 10.1186/s13024-024-00701-3

Figure Lengend Snippet: Drosophila Cisd is functionally more similar to CISD1. A Immunoblots of protein lysates from RPE1 cells ± YFP-Parkin overexpression (OE) treated with antimycin A (4 µM) and oligomycin (10 µM) for the indicated time to induce mitophagy, probed for mitophagy marker pUb, and degradation of TOM20 and CISD1/2 (CISD1, Proteintech, 16006-1-AP; and CISD2, Proteintech, 13318-1-AP), alongside respective loading control total Ub and Tubulin. Blot is representative of 3 replicate experiments. B Confocal micrographs of U2OS cells transfected with human CISD1-FLAG or CISD2-FLAG, counter-stained with antibodies against TOM20 (mitochondria) or Calnexin (ER). C Confocal micrographs of Drosophila larval neurons expressing transgenic mito-GFP and WT control, human CISD1-HA or CISD2-HA driven by nSyb -GAL4. D Immunoblots of protein lysates of 2- and 20-day-old whole flies expressing the indicated transgenes via da -GAL4 versus WT control, probed for pUb and CISD1/2 with (CISD2, Proteintech, 13318-1-AP). Scale bars = 10 μm

Article Snippet: A Immunoblots of protein lysates from RPE1 cells ± YFP-Parkin overexpression (OE) treated with antimycin A (4 μM) and oligomycin (10 μM) for the indicated time to induce mitophagy, probed for mitophagy marker pUb, and degradation of TOM20 and CISD1/2 (CISD1, Proteintech, 16006-1-AP; and CISD2, Proteintech, 13318-1-AP), alongside respective loading control total Ub and Tubulin.

Techniques: Western Blot, Over Expression, Marker, Control, Transfection, Staining, Expressing, Transgenic Assay

Journal: Molecular Neurodegeneration

Article Title: Mitochondrial CISD1/Cisd accumulation blocks mitophagy and genetic or pharmacological inhibition rescues neurodegenerative phenotypes in Pink1/parkin models

doi: 10.1186/s13024-024-00701-3

Figure Lengend Snippet: Cisd overexpression blocks mitophagy flux. A Immunoblot analysis of whole fly lysates from 2-day-old flies of the indicated genotypes, analysed for pUb, and Tubulin or total protein levels as loading controls. Blot is representative of 3 replicate experiments. Cisd overexpression was driven by da -GAL4. B Confocal microscopy analysis of adult Drosophila flight muscle from 2-day-old flies of WT control, parkin mutant and Cisd overexpression driven by da -GAL4 immunostained for mitochondria (ATP5A) and pUb. C – F Confocal analysis of mitophagy reporter mito -QC (OMM-localised tandem RFP-GFP) of WT or Cisd overexpression driven by nSyb -GAL4, in larval ( C , D ) or adult ( E , F ) neurons with ‘red-only’ mitolysosomes shown. D , F Number of mitolysosomes quantified shown in C and E. Data points indicate individual animals analysed. Statistical analysis: unpaired t-test; ** P < 0.01; **** P < 0.0001. Scale bars = 10 μm

Article Snippet: A Immunoblots of protein lysates from RPE1 cells ± YFP-Parkin overexpression (OE) treated with antimycin A (4 μM) and oligomycin (10 μM) for the indicated time to induce mitophagy, probed for mitophagy marker pUb, and degradation of TOM20 and CISD1/2 (CISD1, Proteintech, 16006-1-AP; and CISD2, Proteintech, 13318-1-AP), alongside respective loading control total Ub and Tubulin.

Techniques: Over Expression, Western Blot, Confocal Microscopy, Control, Mutagenesis

Journal: Molecular Neurodegeneration

Article Title: Mitochondrial CISD1/Cisd accumulation blocks mitophagy and genetic or pharmacological inhibition rescues neurodegenerative phenotypes in Pink1/parkin models

doi: 10.1186/s13024-024-00701-3

Figure Lengend Snippet: Cisd overexpression causes autophagosome accumulation and prevents autophagy. A Confocal micrographs of WT control versus Cisd overexpressing adult flight muscle via Mef2 -GAL4, immunostained for p62 alongside imaging mCherry-Atg8a autophagosome reporter. B Electron micrographs of flight muscle as in A, showing multiple autophagic vesicles (inset) in proximity to disrupted mitochondria (arrowheads). C Larval neurons of WT control versus Cisd overexpressing or Atg5 knockdown (via nSyb -GAL4 driver) animals immunostained for p62 alongside ATP5a (mitochondria) and DAPI. D Immunoblot analysis of protein lysates from whole flies overexpressing Cisd (via da -GAL4) or WT controls. Blots were probed with antibodies against p62, Atg8a (LC3), Cisd (CISD2, Proteintech, 13318-1-AP) and Tubulin. Quantification of replicate blots is shown in Fig. S A, B. E Quantification of the number of autolysosomes shown in F. Data points indicate individual animals analysed. Statistical analysis: unpaired t-test; *** P < 0.001. F Confocal microscopy analysis of adult flight muscle WT control versus Cisd overexpressing animals co-expressing the autophagy flux reporter GFP-mCherry-Atg8a driven by Mef2 -GAL4. G , H Locomotor climbing assay of 2-day-old adult flies expressing the indicated transgenes (via da -GAL4). I Immunoblot analysis of equivalent samples analysed in G and H, probed for autophagy markers (p62 and Atg8a), Cisd and Tubulin. Quantification of replicate blots is shown in Fig. S C. Statistical analyses: Kruskal-Wallis non-parametric test with Dunn’s post-hoc correction. *** P < 0.001; **** P < 0.0001. Scale bars = 10 μm for light microscopy, or indicated on image for EM

Article Snippet: A Immunoblots of protein lysates from RPE1 cells ± YFP-Parkin overexpression (OE) treated with antimycin A (4 μM) and oligomycin (10 μM) for the indicated time to induce mitophagy, probed for mitophagy marker pUb, and degradation of TOM20 and CISD1/2 (CISD1, Proteintech, 16006-1-AP; and CISD2, Proteintech, 13318-1-AP), alongside respective loading control total Ub and Tubulin.

Techniques: Over Expression, Control, Imaging, Knockdown, Western Blot, Confocal Microscopy, Expressing, Climbing Assay, Light Microscopy