Abstract
Mutations in the gene encoding valosin-containing protein (VCP) lead to multisystem proteinopathies including frontotemporal dementia. We have previously shown that patient-derived VCP mutant fibroblasts exhibit lower mitochondrial membrane potential, uncoupled respiration, and reduced ATP levels. This study addresses the underlying basis for mitochondrial uncoupling using VCP knockdown neuroblastoma cell lines, induced pluripotent stem cells (iPSCs), and iPSC-derived cortical neurons from patients with pathogenic mutations in VCP. Using fluorescent live cell imaging and respiration analysis we demonstrate a VCP mutation/knockdown-induced dysregulation in the adenine nucleotide translocase, which results in a slower rate of ADP or ATP translocation across the mitochondrial membranes. This deregulation can explain the mitochondrial uncoupling and lower ATP levels in VCP mutation-bearing neurons via reduced ADP availability for ATP synthesis. This study provides evidence for a role of adenine nucleotide translocase in the mechanism underlying altered mitochondrial function in VCP-related degeneration, and this new insight may inform efforts to better understand and manage neurodegenerative disease and other proteinopathies.
Highlights
Mutations in the gene encoding valosin-containing protein (VCP) lead to multisystem proteinopathies including frontotemporal dementia
This study addresses the underlying basis for mitochondrial uncoupling using VCP knockdown neuroblastoma cell lines, induced pluripotent stem cells, and iPSC-derived cortical neurons from patients with pathogenic mutations in VCP
Our recent work has provided an insight into the observed mitochondrial pathologies through the utilization of a human dopaminergic neuroblastoma VCP knockdown cell line (SH-SY5Y), fibroblasts from patients carrying pathogenic mutations in the VCP gene and VCP knockdown mouse neurons, where we revealed an uncoupling of respiration from oxidative phosphorylation
Summary
Our previous study provided evidence of lower ATP levels as well as uncoupled mitochondria [4]. Due to the difference in charge between ATP and ADP, changes in ANT activity can affect mitochondrial membrane potential and respiration To test whether this is the underlying cause of the uncoupling event observed in VCP knockdown cells, we exposed SCR control and VCP KD cells to ANT inhibitors carboxyatractyloside (CATR; 5 M) or bongkrekic acid (BKA) (5 M) and RCR was assessed. The rate was significantly lower in neurons bearing the R155C mutation (n ϭ 17; p Ͻ 0.001; Fig. 4D) and R191Q mutation (n ϭ 12; p Ͻ 0.05) when compared with control iPSC-derived neurons (n ϭ 13) These results provide evidence that the ATP synthase in VCP mutant cells does not convert ADP to ATP as efficiently when compared with control cells. This approach does not distinguish between ATP synthase malfunction or a lower substrate (ADP) supply as the underlying basis for the difference in rate in ATP synthesis between control and VCP mutant cells
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