Fig. 4: Formation of Httex1 72Q inclusions induces mitochondrial alterations and the reduction of ER-exit sites. | Nature Communications

Fig. 4: Formation of Httex1 72Q inclusions induces mitochondrial alterations and the reduction of ER-exit sites.

From: Nuclear and cytoplasmic huntingtin inclusions exhibit distinct biochemical composition, interactome and ultrastructural properties

Fig. 4

a Electron micrographs of mitochondria in HEK cells overexpressing empty vector (EV) or Httex1 72Q. The insets depict higher magnification of the mitochondria found at the periphery of the Httex1 72Q inclusions or in EV controls. Scale bars = 1 μm. b Measurement of the mitochondrial length with FIJI reveals a significant reduction in the size of the mitochondrial profile located in the proximity of the inclusions. An unpaired t-test was performed (two-sided) resulting in a p-value of 0.0006. An unpaired t-test was performed (two-sided) resulting in a p-value of 0.0006. c HEK cells from 4 independent experiments were gently detached for high-resolution respirometry (HRR) 48 h after transfection with indicated constructs. HRR was performed in respiration media. After the measurements of routine respiration, cells were permeabilized by digitonin, and different respirational states were subsequently induced using a substrate-uncoupler-inhibitor titration (SUIT) protocol. Routine respiration, NADH-driven, or complex 1-linked respiration after the addition of ADP (OXPHOS state) (NP), NADH- and succinate driven, or complex 1 and 2-linked respiration in the OXPHOS state (NSP), and in the uncoupled electron transport system (ETS) capacity (NSE), as well as succinate driven, or complex 2-linked respiration in the ETS state (SE) were assessed. The graphs represent the mean ± SD of 4 independent experiments. Two-way ANOVA showing a significant interaction between the respirational states and the polyQ repeat length (p-value = 0.004). d Representative confocal images of HeLa cells transfected with Httex1 16Q, 39Q, or 72Q or EV. Cells were fixed 48 h after transfection and immunostained. Httex1 was detected with the MAB5492 Htt antibody (grey), and ER exit sites (ERES) were detected with Sec13 (red). Scale bars = 20 μm. e, f ERES number (e) and size (f) quantifications from confocal imaging were performed using FIJI. The graphs represent the mean ± SD of 3 independent experiments represented as a relative percentage to Httex1 16Q control. One-way ANOVA followed by a Tukey honest significant difference [HSD] post hoc test was performed (p-values < 0.0001 for panels e and f). *P < 0.05, **P < 0.005, ***P < 0.001 for multiple comparisons.

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