Figure 6.
Augmentation of mitochondrial protein import rescues neurons from mutant Htt-induced death. (a) DIV 5 cortical neurons were transduced with WT Httex1-25Q (Htt25Q) or mutant Httex1-72Q (Htt72Q) lentivirus and at DIV 6 were cotransduced with lentivirus expressing three subunits of the TIM23 complex, Tim23, Tim50 and Tim17a. Mitochondria isolated from transduced neurons at DIV 10 were subjected to pOTC import assay (30 min). Overexpression of the TIM23 complex subunits increased pOTC import in Htt72Q neurons (*P = 0.007 compared to GFP-expressing Htt72Q neurons, F2,27 = 14.05, n = 10 samples per condition from 5 independent experiments). (b) Primary cortical neurons transduced as in a were subjected to MTS assays at DIV 14. Overexpression of the TIM23 complex subunits partially but significantly metabolic activity (*P = 0.006 compared to GFP-expressing Htt72Q neurons, F2,45 = 59.39, n = 17 wells per group for GFP-expressing Htt25Q and GFP-expressing Htt72Q neurons, n = 14 wells for TIM23-expressing Htt72Q neurons from 4 independent experiments). (c) Primary cortical neurons transduced as in a were assessed for cell death by scoring nuclear morphology at DIV 14. Htt72Q–expressing neurons showed increased cell death compared to Htt25Q–expressing neurons (#P < 0.0001, GFP-expressing Htt72Q compared to GFP-expressing Htt25Q neurons). Overexpression of the TIM23 complex subunits in Htt72Q neurons inhibited neuronal death (*P < 0.0001 compared to GFP-expressing Htt72Q neurons). F3,51 = 29.47, n = 16 (GFP-expressing Htt25Q neurons), 15 (GFP-expressing Htt72Q neurons) and 12 (TIM23-expressing Htt72Q and TIM23-expressing Htt25Q neurons) wells per group from 3 independent experiments; 200 neurons were counted per well. (a–c) Data are presented as mean + s.e.m. One-way ANOVA, Bonferroni t-test.