TNFα Increases Mitochondrial Biogenesis in Motor Neurons

Abstract

Pro‐inflammatory cytokines, like TNFα, mediate a cellular stress response to inflammation that may target mitochondria. In the present study, we hypothesize that in motor neuron‐like NSC‐34 cells, TNFα induces mitochondrial fragmentation and biogenesis in order to maintain overall O2 consumption of these cells. NSC‐34 cells were differentiated by serum depletion (~24 h) until neurite structures (>50 μm) were observed. Cells were then exposed to TNFα (20 ng/ml) for 24 h. Mitochondria were labeled using MitoTracker Green and imaged using confocal microscopy. Mitochondrial morphometry was assessed by measuring aspect ratio (ratio of long to short axis) and form factor (perimeter2/4p·Area). Expression of mitofusin 2 (Mfn2 – fusion protein) and dynamin related protein 1 (Drp1 – fission) was determined by Western blot. From 3D reconstruction of optical slices of NSC‐34 cells, cell and mitochondrial volumes were determined and mitochondrial volume density calculated. Mitochondrial biogenesis was assessed by measuring mitochondrial DNA, and expression of PGC1α was measured. An Agilent Seahorse XF Analyzer was used to measure cellular O2 consumption. A quantitative histochemical method was used to measure the maximum velocity of the succinate dehydrogenase (SDHmax) reaction within NSC‐34 cells to determine the functionality of the tri‐carboxylic acid (TCA) cycle and the electron transport chain (ETC). Finally, reactive oxygen species (ROS) formation was measured using MitoSox fluorescence. After exposure to TNFα, aspect ratio and form factor of mitochondria decreased by ~15% compared to untreated controls with consistent changes in Mfn2 and Drp1 expression. After TNFα exposure, mitochondrial DNA expression increased as did mitochondrial volume density, together indicating increased mitochondrial biogenesis. After TNFα exposure, O2 consumption per mitochondrion (normalized for mitochondrial volume density) decreased. This was consistent with a TNFα‐induced decrease in SDHmax. Despite the decrease in O2 consumption and SDHmax per mitochondrion after TNFα exposure, ROS formation increased reflecting mitochondrial dysfunction. We conclude that mitochondrial fragmentation, biogenesis and increased mitochondrial volume density are a homeostatic mechanism to decrease O2 consumption per mitochondrion thereby damping ROS formation, while maintaining overall O2 consumption to meet ATP demand.Support or Funding InformationSupported by NIH grants AG44615 and HL105355.