Fig. 4. mISR and altered mitochondrial morpho-dynamics are associated with CHCHD2 dysregulation in mHTT-carrying neural cells.

a, b Representative immunostaining and related quantifications of CHCHD2 protein colocalization with nuclear staining Hoechst in NPCs and in cerebral organoids (COs) at day 49. Dots represent individual images collected from at least two biological replicates for NPCs (n = at least 35 individual images per sample) and COs (n = at least 77 individual images per sample). **p < 0.01, ****p < 0.0001; two-tailed Mann-Whitney U test. Scale bar: 100 µm. c Heatmap of log-fold change (LFC) comparisons of genes involved in mitochondrial integrated stress response (mISR) and mitochondrial dynamics. Data obtained from RNA sequencing of iPSCs, NPCs, and COs; *p < 0.05, **p < 0.01, ***p < 0.001; 70Q/70Q and WT/70Q vs. WT/WT, HD neuruloids vs. WT neuruloids²⁵; two-sided likelihood ratio test, without multiple comparison adjustments. d Isoform usage in NPCs determined with long-read transcriptomics. Percentage of use in WT/WT NPCs and 70Q/70Q NPCs and related transcript model are shown. e, f Representative immunostaining and related quantification of mitochondrial footprint (comprising both small and large mitochondrial structures) based on TOM20 signal in cerebral organoids from 70Q/70Q and WT/WT. Dots represent individual images collected over three independent experiments. **p < 0.01, ***p < 0.001, unpaired two-tailed t test (AU = arbitrary units). Scale bar: 100 µm. g Quantifications of protein abundance of TOM20 based on immunostaining performed in cerebral organoids and NPCs. The amounts of positive TOM20 signal per image was normalized to Hoechst signal. Dots represent individual images collected from at least two biological replicates for NPCs (n = at least 35 individual images per sample) and COs (n = at least 77 individual images per sample) (AU = arbitrary units). **p < 0.01, ****p < 0.0001, ns: not significant; two-tailed Mann-Whitney U test.