Abstract
Mitochondrial dysfunction is a recognized hallmark of neurodegenerative diseases and abnormal mitochondrial fusion-fission dynamics have been implicated in the pathogenesis of neurodegenerative disorders. This study characterizes the effects of metabolic flux inhibitors and activators on mitochondrial fusion dynamics in the neuronal cell culture model of differentiated PC12 cells. Using a real time confocal microscopy assay, it was found that the carnitine palmitoyltransferase I (CPTI) inhibitor, etomoxir, reduced mitochondrial fusion dynamics in a time-dependent manner. Etomoxir also decreased JO2, ΔΨm and reactive oxygen species (ROS) production rates. The mitochondrial pyruvate carrier (MPC) inhibitor, UK5099, reduced fusion dynamics and in combination with etomoxir these inhibitory effects were amplified. Use of the pyruvate dehydrogenase (PDH) kinase inhibitor dichloroacetate, which is known to increase metabolic flux through PDH, reversed the etomoxir-induced effects on fusion dynamics, JO2, ΔΨm but not ROS production rates. Dichloroacetate also partially reversed inhibition of mitochondrial fusion dynamics caused by the parkinsonian-inducing neurotoxin, MPP+. These results suggest that dichloroacetate-induced activation of metabolic flux in the mitochondrion may be a mechanism to restore normal mitochondrial fusion-fission dynamics in metabolically challenged cells.
Highlights
An imbalance of mitochondrial fusion-fission dynamics is present in models of neurodegeneration and Parkinson’s disease (PD; Knott and Bossy-Wetzel, 2008; Poole et al, 2008; Yang et al, 2008; Büeler, 2009; Kamp et al, 2010; Santos et al, 2015)
Differentiated PC12 cells transfected with pDsRed2-mito and PA-GFP-mito were analyzed by confocal microscopy over a 45 min period (Figures 1A–E) and the PA-GFP-mito mean pixel intensities decreased in a time-dependent manner (Figure 1F), indicative of mitochondrial fusion
Addition of the CPT1 inhibitor, etomoxir, resulted in a dose-dependent inhibition of mitochondrial fusion rates in differentiated PC12 cells (Figures 2A,B). These results suggest a potential role for fatty acid oxidation in maintaining mitochondrial dynamics homeostasis
Summary
An imbalance of mitochondrial fusion-fission dynamics is present in models of neurodegeneration and Parkinson’s disease (PD; Knott and Bossy-Wetzel, 2008; Poole et al, 2008; Yang et al, 2008; Büeler, 2009; Kamp et al, 2010; Santos et al, 2015). Fused mitochondrial networks are generated in cell types when increased respiratory capacity and bioenergetics function is required (Westermann, 2012). Nutrient excess increases respiration rates and a robust fragmentation of the mitochondrial network (Molina et al, 2009), while low availability of nutrient sources elongates mitochondria by inhibiting recruitment of fission-inducing Drp to the mitochondria (Gomes et al, 2011). Fatty acids are a source of cellular energy and fatty acid import into mitochondria is sourced directly by lipid droplets (LDs) in close proximity to highly fused mitochondria (Rambold et al, 2015).
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