In no cell type is their function more vital than in neurons, in which limited glycolysis causes the cell to rely on

oxidative phosphorylation for ATP production. In this study, we used dynamic imaging techniques to explore the mitochondrial pathophysiology in a VCP knockdown (VCP KD) human dopaminergic neuroblastoma cell line (SH-SY5Y) and in fibroblasts from patients carrying three independent pathogenic mutations in the VCP gene. We demonstrate that VCP deficiency induces Sotrastaurin the uncoupling of respiration from oxidative phosphorylation. This results in decreased mitochondrial membrane potential, leading to higher respiration and lower ATP levels due to reduced ATP production. These findings define a mechanism whereby VCP dysfunction may cause cell death and highlight pathophysiological events that may occur in IBMPFD. Mitochondrial membrane potential (ΔΨm) is an indicator of mitochondrial health and function. To study VCP implication in mitochondrial function, we transiently silenced the VCP gene using siRNA in CH5424802 research buy SH-SY5Y human neuroblastoma cells (see Figure S1A available online) and shRNA in mouse primary cortical cultures ( Figure 1D). Additionally, stable populations of VCP KD SH-SY5Y cells were generated using shRNA ( Figure S1B). ΔΨm was measured in VCP-deficient SH-SY5Y cells ( Figure 1A), in human fibroblasts from three patients with independent VCP mutations (R155C, R155H, and R191Q;

for donors’ details see Figure S1E and Table S1) and age-matched controls ( Figure 1B), and in primary neurons and astrocytes ( Figures 1C and 1D). A significant decrease in ΔΨm was observed in all VCP-deficient cell models studied (SH-SY5Y cells = 72% ± 8%, n > 20 cells in 3 independent experiments compared to either untransfected cells or cells transfected with scramble (SCR) control siRNA; primary neurons = 62% ± 9% and primary astrocytes = 74% ± 4%, n ≥ 5 cells in 3 independent experiments compared to cells transfected

with SCR control shRNA; fibroblasts from patient 1 = 86% ± 2%, n = 7; fibroblasts from patient 2 = 85% ± 2%, n = 8; fibroblasts from patient 3 = 91% ± 2%, n = 5, compared to age-matched control fibroblasts) ( Figures 1A–1C). Overexpression of R155H, Bumetanide R191Q, and R155C VCP mutants in SH-SY5Y cells is associated with a significant reduction in the TMRM signal (TMRM in cells overexpressing R155H VCP = 73% ± 3%; R191Q VCP = 65% ± 3%; R155C VCP = 62% ± 13% compared to overexpressed WT VCP; n ≥ 3), confirming that the three pathogenic VCP mutations have a dominant-negative effect ( Figure 1E). Re-expression of WT, but not mutant VCP, rescued the TMRM signal in a clonal population of stable VCP KD SH-SY5Y cells (untransfected cells = 61% ± 9%; WT VCP = 97% ± 7%; R155H VCP = 62% ± 4%; R191Q VCP = 60% ± 1%; R155C VCP = 74% ± 5% compared to SCR SH-SY5Y control cells; n ≥ 3) ( Figures 1F and S1D). In healthy cells, ΔΨm is maintained by mitochondrial respiration.