Fig. 6: 13-cis RA rescues mitophagic defects and reverses neurodegeneration in Kansl1+/− mice. | Nature Communications

Fig. 6: 13-cis RA rescues mitophagic defects and reverses neurodegeneration in Kansl1+/− mice.

From: Kansl1 haploinsufficiency impairs autophagosome-lysosome fusion and links autophagic dysfunction with Koolen-de Vries syndrome in mice

Fig. 6

a Phenotypic screening for small molecules that enhance the mitophagic activity of Kansl1fl/fl/CAG-cre neurons. b mitoKeima imaging in primary neurons treated with tamoxifen (1 μM, 5 days) and13-cis RA (20 μM, 24 h). Scale bar, 10 μm. c Quantification of mitoKeima signal in (b). The ratio of acidic/neutral mitoKeima signal in control cells was normalized to ‘1’. n = 48, 52, 43, 49 from left to right. d Quantification of the percentage of LC3B+ Lyso+ puncta to total LC3B puncta in primary neurons (DMEM, 12 h) treated with 13-cis RA (20 μM, 24 h) (n = 22, 21, 46, 27 cells examined in 3 independent experiments from left to right). e Quantification of MitoSOX mean intensity by flow cytometry. Primary neurons were treated with tamoxifen (1 μM, 5 days) and cultured with 13-cis RA (2 μM, 7 days). MitoSOX intensity of WT is normalized to ‘1’ (n = 3 mice in each group). f Western blot analysis of Biotin-13-cis RA pulldown assay. HeLa cells cultured with Biotin-13-cis RA (5 μM, 24 h) were treated with 2 h EBSS. g Western blot analysis of in vitro mapping assays among Biotin-13-cis RA and recombinant His-tagged indicated proteins. h Western blot analysis of co-precipitation of the Flag-Snap29 with other SNARE proteins in HeLa cells with KANSL1 knockdown. Cells cultured with Biotin-13-cis RA (5 μM, 24 h) were treated with 2 h EBSS. i Representative images of dendritic spine of hippocampus CA1 regions visualized with Golgi-cox staining. Scale bar, 5 μm. j, Quantification of spine density in (i). n = 25, 26, 33, 31 (from 2 to 3 mice in each group). k Novel object recognition test. For discrimination index, see details in “Methods”. n = 7, 5, 6, 5 mice from left to right. l Morris water maze test. Mean escape latencies time to find the platform. n = 7, 5, 6, 5 mice from left to right. Mice were about 6 months old. Source data are provided as a Source data file. All data are means ± SEM. c, d, e, j, k One-way ANOVA with Tukey’s multiple hoc test; l by two-way ANOVA test with Dunnett’s multiple hoc test.

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