Abstract
Mitochondrial dysfunction is associated with a number of human diseases. As an example, we recently established in vivo Drosophila models of IBMPFD (Inclusion body myopathy, Paget disease, and frontotemporal dementia), and uncovered that human disease mutations of the p97/VCP (Valosin Containing Protein) gene behave as hyperactive alleles associated with mitochondrial defects. Pharmacologic inhibition of VCP strongly suppressed disease and mitochondrial pathology in these animal models. In this protocol, we describe a method to evaluate mitochondrial respiratory function in IBMPFD patient-derived fibroblasts, as well as investigate the role of pharmacologic treatments. These experiments complement work done in animal models by investigating mitochondrial biology and the pharmacologic response in a human cell-based model of the disease. In principle, this technique can be used to investigate mitochondrial respiratory function for any disease in which patient-derived fibroblasts are available.
Highlights
Missense mutations of p97/VCP are causative for inclusion body myopathy, Paget disease and frontotemporal dementia (IBMPFD) (Watts et al, 2004), a rare autosomal dominant disease associated with muscle weakness, bone pain and neurocognitive decline
VCP mutations are associated with other neuropathic diseases [sporadic amyotrophic lateral sclerosis (ALS), hereditary spastic paraplegia and Charcot-Marie-Tooth 2 neuropathy] (Abramzon et al, 2012; de Bot et al, 2012; Gonzalez et al, 2014)
The VCP gene encodes a highly conserved and abundant member of the AAA+ (ATPases associated with diverse cellular activities) protein family, which participates in multiple cellular processes, and potentially impacts mitochondrial biology (Meyer et al, 2012)
Summary
We describe the measurement of oxygen consumption rates in immortalized human fibroblasts derived from disease patients and age-matched controls. Trypsin-EDTA (Sigma Aldrich, catalog number: T4049) 8. Seahorse XFe96 FluxPak (Agilent Technologies, catalog number: 102416-100), including sensor cartridge, calibration utility plate, 96-well culture plate, loading guides, calibrant solution 12. Bicarbonate-free DMEM (Sigma-Aldrich, catalog number: D5030) 13. Sodium pyruvate (Sigma-Aldrich, catalog number: S8636) 15.
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