Citations

Mitochondrial dysfunction and fragmentation precede neuronal death in many neurodegenerative disorders, including those involving motor neurons. In other cell types, pro‐inflammatory agents such as TNF‐α cause marked mitochondrial fragmentation. These inflammatory insults may underlie the vulnerability of specific neurons during age‐associated conditions, such as sarcopenia. We hypothesize that motor neuron exposure to pro‐inflammatory cytokines induces mitochondrial fragmentation. Experiments were conducted using a mouse motor neuron‐like cell line, NSC‐34. These cells were differentiated by serum depletion until neurite structures (>50 um) were observed, ~24 h. The differentiated NSC‐34 cells were then treated with mouse TNF‐α at varying concentrations; control (0 ng), 10 ng/ml, and 20 ng/ml for 24 h. The cells were then loaded with MitoTracker Green to image mitochondria using a Nikon A1R confocal microscope (60x oil immersion 1.4 NA). Images were analyzed using MatLab macro to calculate form factor, aspect ratio and mitochondrial volume density. TNF‐α exposure caused mitochondrial fragmentation as reflected by decreased form factor (27% reduction) and aspect ratio (12 % reduction). These results are consistent with inflammatory cytokine exposure leading to mitochondrial fragmentation in motor neurons which may increase their vulnerability mitochondrial dysfunction and cell death. Support or Funding Information NIH: RO1AG044615 This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Glycine (Gly) is a key inhibitory neurotransmitter at phrenic motor neurons (PhMN). In development, Gly is excitatory, transitioning to inhibitory action in the postnatal period. In adult conditions such as amyotrophic lateral sclerosis (ALS), excessive excitatory signaling onto motor neurons results in excitotoxic motor neuron death. However, it is not known how an imbalance of neurotransmission early in development impacts PhMNs at maturity. This is a knowledge gap, as some congenital hypertonia conditions exhibit an imbalance in neurotransmitter signaling, with these individuals suffering significant morbidity and mortality from respiratory disorders. Using an animal model of congenital hypertonia ( spa mouse [B6.Cg‐ Glrb spa /J] with a Gly receptor mutation), we evaluated PhMN and motor unit physiology. Similar to previous work in other Gly mutants, we hypothesized that spa mice will have excessive developmental PhMN loss with preserved PhMNs having a smaller somal size compared to wildtype (WT) mice. We also hypothesized that fewer PhMNs in spa mice will result in an increased diaphragm (DIAm) innervation ratio (number of DIAm fibers innervated by a PhMN) and increased DIAm neuromuscular transmission failure (NMTF). S pa and WT mice from our colony, ages 6 to 16 weeks, underwent unilateral retrograde labeling of PhMNs via rhodamine phrenic nerve dip. After three days, mice were euthanized, perfused with 4% paraformaldehyde, cervical spinal cord excised and processed for longitudinal cryosectioning (70 µm) and prepared for confocal imaging. Absolute PhMN counts were obtained using mosaic images of adjacent serial sections. Somal size measurements (i.e. surface area) were obtained using ImageJ. DIAm force was determined from DIAm strip preparations stretched to optimal length, and force was evoked by phrenic nerve stimulation at 40 Hz in 330 ms duration trains repeated each s (33% duty cycle) for 120 s. To assess NMTF, force evoked by phrenic nerve stimulation was compared to force evoked by direct DIAm stimulation superimposed every 15 s. Total DIAm fiber number was estimated in H&E stained cross‐sections. S pa mice had 30% fewer PhMNs compared to WT ( P =0.005) with somal size being 22% smaller in spa mice ( P =0.019). Spa mice had 20% NMTF during the initial stimulus train compared to 8% in WT mice with spa mice having 82% NMTF and WT having 40% NMTF at end of stimulation. Spa mice had a 39% greater innervation ratio of DIAm motor units compared to WT. Developmental PhMN loss and somal size is significantly disrupted in congenital hypertonia due to impaired Gly signaling and may impact performance of high‐force expulsive DIAm activities. In addition, the increased innervation ratio of DIAm motor units in spa mice, likely contributes to the NMTF due to branch point failure. Overall, this results in less effective airway defense and straining behaviors in congenital hypertonia. Our next step is to identify crucial developmental milestones when excessive PhMN loss occurs and the impact on large versus small PhMNs.

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.