Brown, Alyssa

Vitamin D deficiency and vitamin D receptor (VDR) polymorphisms are associated with adverse pulmonary and diaphragm muscle (DIAm)-associated respiratory outcomes. We used a skeletal muscle-specific tamoxifen-inducible VDR knockout to investigate DIAm dysfunction following reduced VDR signaling. Marked DIAm weakness and atrophy of type IIx and/or IIb fibers are present in muscle-specific tamoxifen-induced VDR knockout mice compared with controls. These results show that the VDR has a significant biological effect on DIAm function independent of systemic effects on mineral metabolism.

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 O 2 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 (perimeter 2 /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 O 2 consumption. A quantitative histochemical method was used to measure the maximum velocity of the succinate dehydrogenase (SDH max ) 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, O 2 consumption per mitochondrion (normalized for mitochondrial volume density) decreased. This was consistent with a TNFα‐induced decrease in SDH max . Despite the decrease in O 2 consumption and SDH max 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 O 2 consumption per mitochondrion thereby damping ROS formation, while maintaining overall O 2 consumption to meet ATP demand. Support or Funding Information Supported by NIH grants AG44615 and HL105355.

Inflammation triggers a homeostatic response that is mediated by pro‐inflammatory cytokines such as TNFα. We hypothesize that in a motor neuron‐like cell line, NSC‐34, TNFα decreases mitochondrial function, specifically succinate dehydrogenase (SDH) activity, which is a key enzyme in the tricarboxylic acid (TCA) cycle as well as complex II in the electron transport chain (ETC). Experiments were performed on cultured NSC‐34 cells that were differentiated by serum depletion until neurite structures (>50 μm) were observed. The differentiated cells were treated with TNFα (20 ng/ml) for 24 h. To measure the maximum velocity of the SDH reaction (SDH max ), cells were incubated in a solution containing 80 mM succinate and 1.5 mM nitro blue tetrazolium (NBT), the reaction indicator. As the SDH reaction proceeded, images were acquired every 15 s using a 40× objective on an Olympus IX71 inverted microscope. Linearity of the SDH reaction was confirmed across the 8‐min period, and SDH max was determined and expressed as mM fumarate/L tissue/min. To determine mitochondrial volume density, the cells were loaded with MitoTracker Green and imaged in 3D using a confocal microscope. The cell volume occupied by labeled mitochondria was determined from 3D reconstructions of 1 mm optical slices. In NSC‐34 cells exposed to TNFα, we found that mitochondrial volume density increased, and mitochondria were fragmented as compared to untreated controls. We also found that TNFα exposure decreased SDH max when normalized for mitochondrial volume density. In other cell types, we found that TNFα‐induces an increase in PGC1a and Drp1 expression and a decrease in Mfn2 expression, all of which affect mitochondrial structure. In related studies, we also found that TNFα exposure decreases O 2 consumption per mitochondrion in NSC‐34 cells. We conclude that by reducing the redox potential (SDH max ) of mitochondria, O 2 consumption per mitochondrion is reduced, thereby damping ROS formation. The impact of this homeostatic response is offset by mitochondrial biogenesis and an increase in mitochondrial volume density, thereby maintaining overall cellular O 2 consumption and ATP production but at reduced cost of ROS formation. Support or Funding Information Supported by NIH grants AG44615 and HL105355.

Neuroinflammation triggers a homeostatic response that is mediated by pro‐inflammatory cytokines such as TNFα. We hypothesize that in a motor neuron‐like cell line, NSC‐34, TNFα decreases mitochondrial O 2 consumption. NSC‐34 cells were differentiated by serum depletion (~24 h) until neurite structures (>50 μm) were observed. The cells were then exposed to TNFα (20 ng/ml) for 24 h. To determine mitochondrial volume density, the cells were loaded with MitoTracker Green and imaged in 3D using a Nikon A1R confocal microscope (60x oil immersion 1.4 NA). After treatment with TNFα, mitochondria appeared fragmented but mitochondrial volume density increased by ~50%, as compared to control untreated cells. A Agilent Seahorse XF Analyzer was used to determine the O 2 consumption of treated and untreated cells during a stress test that determines basal respiration, ATP production and maximum respiration. O 2 consumption was normalized to mitochondrial volume density in both treated and untreated groups. After TNFα exposure, basal respiration, ATP production and maximum respiration decreased per mitochondrion. However, overall O 2 consumption of the cells was unchanged after TNFα exposure due to an offsetting increase in mitochondrial volume density. Using MitoSox, we also measured reactive oxygen species (ROS) formation in treated and untreated NSC‐34 cells. ROS is a byproduct of mitochondrial O 2 consumption, and can cause cellular damage. After TNFα exposure, ROS formation increased compared to untreated cells. We conclude that mitochondrial biogenesis and an increase in mitochondrial volume density represent a homeostatic mechanism to reduce O 2 consumption the per mitochondrion, thereby damping ROS formation while maintaining overall cellular O 2 consumption and ATP production. Support or Funding Information Supported by NIH grant AG44615 and HL105355.

Phrenic motor neuron (PhMN) development in early onset hypertonia is poorly understood. Yet, respiratory disorders are a common cause of morbidity and mortality. In spa mice, an animal model of early onset hypertonia, we found ~30% fewer PhMNs, compared with controls. This PhMN loss disproportionately affected larger PhMNs. Thus, the number and heterogeneity of the PhMN pool are decreased in spa mice, likely contributing to the hypertonia, impaired neuromotor control, and respiratory disorders.