Fig. 7: CAST-dependent Drp1-mediated mitochondrial fragmentation mediates CHIR99021 protection in HD. | Nature Communications

Fig. 7: CAST-dependent Drp1-mediated mitochondrial fragmentation mediates CHIR99021 protection in HD.

From: Small-molecule suppression of calpastatin degradation reduces neuropathology in models of Huntington’s disease

Fig. 7

a HdhQ7 and HdhQ111 cells were treated with DMSO (vehicle) or CHIR99021 (3 μM) for 2 days, with cells stained with anti-Tom20 antibody to assess mitochondrial morphology. Left: representative images. Scale bar = 30 μm. Right: histogram showing the percentage of cells with fragmented mitochondria. At least 100 cells/group were counted (n = 3). b Total levels of the indicated proteins classified into three pathways (mitochondrial dynamics, biogenesis, and autophagy) were examined and quantified. The mean relative protein level value was presented in a dual-color heat map. **p = 0.0055, Q111-CHIR99021 versus Q111-Veh. Relative protein abundance scale: 0.5–2.0. c Mitochondria were isolated from the striatum of 12-week-old R6/2 and WT littermates treated with vehicle or CHIR99021 and subjected to western blotting. The relative abundance of mitochondrial Drp1 was quantitated (histogram). d Mitochondrial fractions were isolated from HdhQ7 cells expressing control shRNA (shCon) or CAST shRNA (shCAST) after treatment with 3-nitroproponic acid (3-NP) and/or CHIR99021. Representative immunoblots and quantification of Drp1 relative levels (n = 4). e Representative images of anti-Tom20 staining (green) in shCon- or shCAST-expressing cells treated with Veh or CHIR99021. Scale bar = 30 μm. f Histogram shows the percentage of cells with fragmented mitochondria. At least 100 cells/group were counted (n = 5). g Representative images of anti-Tom20 staining (green) in shCon- or calpain I shRNA (shCAPN1)-expressing HdhQ111 cells treated with Veh or CHIR99021. Scale bar = 30 μm. h Histogram shows the percentage of cells with fragmented mitochondria. At least 100 cells/group were counted (n = 6). All values are reported as mean ± SEM and compared using one-way ANOVA with Tukey’s post hoc test. Data are representative of at least three independent experiments. Exact p values are shown in the figures. i Summary graphic depicting the ability of CHIR99021 to stabilize CAST and promote mitoprotection and neuroprotection.

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