Fig. 1: CHIR99021 reduces mitochondrial damage and cell death in HD mouse- and patient-derived neuronal cells. | Nature Communications

Fig. 1: CHIR99021 reduces mitochondrial damage and cell death in HD mouse- and patient-derived neuronal cells.

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

Fig. 1: CHIR99021 reduces mitochondrial damage and cell death in HD mouse- and patient-derived neuronal cells.The alternative text for this image may have been generated using AI.

HdhQ7 and HdhQ111 cells were treated with vehicle (Veh, DMSO) or CHIR99021 at the indicated doses for 2 days. a Cells were stained with tetramethylrhodamine (TMRM) fluorescence dye. TMRM relative fluorescence density indicates the extent of mitochondrial membrane potential (MMP). b Cells were stained with MitoSOXTM dye to assess mitochondrial superoxide (ROS) production. Histogram: the relative fluorescence density of MitoSOXTM. c Cells were subjected to a 16 h serum starvation with cell death measured by lactate dehydrogenase (LDH) release. d Mitochondrial respiratory activity was measured using a Seahorse XFP analyzer and a Mito stress kit. OCR oxygen consumption rate. e Basal respiration rate, maximal respiration rate, and ATP production are shown. Mixed striatal neurons were differentiated from iPS cells of patients with HD (1: GM04693, 41 CAG; 2: ND41656, 57 CAG; 3: ND36998, 60 CAG) and control subjects (a: HDF; b: NN0003888; c: nHDF). Twenty days after neuronal differentiation, cells were treated with DMSO (Veh) or CHIR99021 (1 μM) for 5 days. f MMP was assessed using a TMRM fluorescence probe (n = 4 biologically independent samples). g Neuronal viability was measured by MTT assay after the withdrawal of brain-derived neurotrophic factor (BDNF) for 12 h (n = 4 biologically independent samples). h Neurons were stained with anti-MAP2 (green)/anti-GAD67 (red) or anti-Tau (green)/anti-DARPP-32 (red) to indicate dendritic and axonal morphology, respectively. Control neurons: Nor, HD neurons: HD. Scale bar = 30 μm. i Quantification of MAP2+ neuronal dendrite length (Con n = 11, HD Veh n = 12, HD CHIR n = 9 cells). j Quantification of Tau+ neuronal axon length (Con n = 16, HD Veh n = 12, HD CHIR n = 9 cells). All values are reported as mean ± SEM. Data are representative of at least three independent experiments. Data were compared using one-way ANOVA with Tukey’s post hoc test in a–e, i, j, and two-way ANOVA with Tukey’s post hoc test in f, g. Exact p values are shown in the figures.

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