Citations

In human airway smooth muscle cells, TNFα induces reactive oxygen species (ROS) generation and promotes endoplasmic reticulum (ER) stress and mitochondrial fragmentation. Importantly, treatment with the ROS scavenger tempol effectively attenuated TNFα-induced oxidative stress, suppressed ER stress and mitochondrial fragmentation, and improved the intrinsic respiratory capacity and functional efficiency of individual mitochondria. These findings identify oxidative stress as a central mediator linking TNFα signaling to ER stress and mitochondrial fragmentation, highlighting tempol as a potential therapeutic strategy.

Abstract Striated muscle weakness and motor neuron (MN) death are associated with aging. These alterations affect both brainstem and spinal motor pools, including hypoglossal MNs innervating the tongue and lumbar MNs innervating the lower/hindlimbs. Both MNs and striated muscles exhibit type‐selective motor unit vulnerability, where larger MNs (likely fast fatigable motor units) and type IIx/b muscles are vulnerable to death and atrophy, respectively. These findings have been cemented in humans to some degree and in Fischer 344 and Wistar rats. However, MN survival in old age has not been evaluated in Sprague–Dawley rats. Here, in 6‐ (young) and 24‐month‐old (old) female and male Sprague–Dawley rats, we performed stereological MN counts on hypoglossal and lumbar MNs using 16 μm Nissl‐stained sections. We observed a 35% loss of hypoglossal and lumbar MNs in old age. Although mean MN somal surface areas were moderately reduced in hypoglossal and unchanged in lumbar MNs with age, frequency histogram analyses revealed a disproportionate loss of larger MN in old age hypoglossal and lumbar pools. These findings reveal reliable and robust observations of MN death in rats. This neurogenic aspect of neuromotor impairment with age may be a key driver of sarcopenia and frailty in the elderly.

Brain-derived neurotrophic factor (BDNF) signals through its high-affinity full length tropomyosin receptor kinase-B (TrkB.FL). One key downstream target of BDNF/TrkB.FL signaling is phosphorylation of the transcription factor cyclic AMP response element-binding (CREB) protein at serine 133 (pCREBS133). We hypothesized that a positive feedback loop exists in the spinal cord in which BDNF/TrkB.FL induces pCREBS133 which then transcriptionally targets pro-BDNF expression. To test this hypothesis, we employed a loss of function approach using a novel chemogenetic rat model (TrkBF616A) with a knock-in allele sensitive to the selective kinase inhibitor 1NMPP1 to inhibit TrkB.FL kinase activity. In female and male TrkBF616A rats, we then examined the effects of 1NMPP1 (orally through drinking water) induced TrkB.FL kinase inhibition on BDNF levels in the cervical (CSC) and lumbar (LSC) spinal cord. The spinal cord was collected at two time points at 0700 and 010 reflecting the trough and peak timepoints of endogenous BDNF expression. BDNF levels were quantified using ELISA. We found in the CSC, BDNF levels did not vary across timepoints in either 1NMPP1 or vehicle treated groups. However, 1NMPP1-induced TrkB.FL inhibition significantly reduced BDNF levels at both timepoints (P=0.004). This reduction suggests that cervical circuits rely on continuous CREB-dependent support of BDNF expression and that inhibiting TrkB.FL disrupts this loop throughout the day. In the LSC, control rats displayed time of day-dependent variations in BDNF with lower levels at 0700 compared to 0100 (P=0.02). In contrast, 1NMPP1-induced inhibition of TrkB.FL reduced BDNF levels at 0100—when BDNF normally peaks in the LSC (P=0.01). This nighttime specific deficit supports the idea that activity-driven pCREBS133 activation is necessary to elevate BDNF during the active phase and that blocking TrkB.FL signaling disrupts this pCREBS133-mediated positive feedback. Overall, these results support a positive feedback that is region- and time-specific and involves BDNF/TrkB.FL signaling inducing pCREBS133 phosphorylation which then targets pro-BDNF expression in the spinal cord. Supported by NIH grants HL146114 and AG44615. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Neuromotor control of diaphragm muscle (DIAm) involves size-dependent recruitment of phrenic motor neurons (PhMNs). To accomplish breathing, only smaller PhMNs innervating slow (type S) and fast, fatigue resistant (type FR) DIAm motor units are recruited. Thus, smaller PhMNs and type S and FR DIAm motor units are very active in sustaining ventilation throughout life. Larger PhMNs innervating more fatigable fast (type FF) motor units are very infrequently recruited for higher force, expulsive behaviors. Previously, we reported that smaller PhMNs have higher mitochondrial volume densities (MVD) reflecting their more frequent activation. Mitochondria are essential for maintaining energetic support and cellular homeostasis. We hypothesized that to sustain higher MVD, mitochondrial biogenesis is higher in smaller compared to larger PhMNs. In female and male Sprague Dawley rats (~3 months of age) PhMNs were retrogradely labeled by intrapleural injection of cholera toxin B (CTB). After 3 days, mitochondria in the cervical spinal cord were labeled by subdural injection of MitoTracker Red. The cervical spinal cord was collected and labeled PhMNs and mitochondria were imaged by high-resolution confocal microscopy and reconstructed in 3D. As previously reported, we found a significant negative correlation between MVD and PhMN somal surface area. By immunohistochemistry, we found an increased nuclear to cytosolic ratio of pCREBS1333 in smaller PhMNs compared to larger PhMNs. PGC1α and TFAM expression was also higher in smaller PhMNs. These findings support size-dependent differences in pCREBS133–PGC1α–TFAM pathway promoting mitochondrial biogenesis. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Brain derived neurotrophic factor (BDNF) signals through its high-affinity full-length tropomyosin receptor kinase-B (TrkB.FL) and plays role in maintaining the survival and function of phrenic motor neurons (PhMNs), neuromuscular transmission and synaptic activity. Disruption of this pathway is a hallmark of several neurodegenerative diseases associated with dysfunction and death of PhMNs with impaired mitochondrial function. BDNF/TrkB.FL signaling phosphorylates CREB at serine 133 (pCREBS133) and promotes its nuclear localization in cervical ventral horn of rats, however its downstream molecular pathway has not yet been established. Recently, we demonstrated that in human airway smooth muscle cells, pCREBS133, as a transcription factor, binds to the CRE sequence in the PGC1a promoter and activates its expression. PGC1a co-transcriptionally activates mitochondrial transcription factor A (TFAM) expression promoting mitochondrial DNA (mtDNA) replication and mitochondrial biogenesis. In the present study, we hypothesized that BDNF/TrkB.FL signaling promotes pCREBS133 phosphorylation and transcriptional activation of PGC1a and downstream expression of TFAM, leading to mitochondrial biogenesis. In this study, we employed a loss of function approach using a novel chemogenetic rat model (TrkBF616A) with a knock-in allele sensitive to the selective kinase inhibitor 1NMPP1 to inhibit TrkB.FL kinase activity. One group of female and male rats were treated intrathecally at C4 with either artificial cerebrospinal fluid (aCSF) or BDNF (450 ng/ 300 g b.w.; diluted in aCSF) for 40 mins, followed by collection of C3-C6 cervical ventral horn. In a second group, female and male rats were treated with 1NMPP1 (25 uM; i.p.) or the vehicle DMSO for 2 h. After 2 h, both groups were treated intrathecally with BDNF for 40 min and the C3-C6 cervical ventral horn collected. Intrathecal BDNF increased the pTrkB.FLY817 autophosphorylation and downstream phosphorylation of pCREBS133. Bioinformatic analysis identified a putative binding site of pCREBS133 on PGC1a promoter, which was confirmed by Chromatin immunoprecipitation (ChIP) assay. Consistent with this, BDNF treatment increased the expression of PGC1a and downstream TFAM, with an increase in mtDNA copy number. 1NMPP1 inhibition of TrkB.FL kinase (blunted BDNF-induced pTrkB.FLY817 phosphorylation) inhibited downstream activation of the pCREBS133/PGC1a/TFAM signaling cascade. Additionally, 1NMPP1 treatment blunted the BDNF-induced increase in mtDNA copy number. We conclude that BDNF/TrKB.FL signaling mediates mitochondrial biogenesis in the cervical ventral horn suggesting that interventions targeting this pathway could be a therapeutic strategy for neurodegenerative diseases. This research was funded by NIH Grants HL146114 & AG44615. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Airway hyperreactivity and hASM cell proliferation (hyperplasia) are hallmark characteristics of asthma. The results of the present study showed that short-term exposure of nonasthmatic hASM to TNFα (a proinflammatory cytokine) can also induce hASM cell proliferation via activation of an ER stress pathway. Identification of the ER stress signaling pathway as important in mediating hASM hyperplasia may provide an important therapeutic target for treating asthma or acute inflammation.

Metabolic dysfunction–associated steatohepatitis (MASH) is a progressive liver disease characterized by complex interactions between lipotoxicity, ER stress responses, and immune-mediated inflammation. We identified enrichment of the proinflammatory alarmin S100 calcium-binding protein A11 (S100A11) on extracellular vesicles stimulated by palmitate-induced lipotoxic ER stress with concomitant upregulation of hepatocellular S100A11 abundance in an IRE1A-XBP1s–dependent manner. We next investigated the epigenetic mechanisms that regulate this stress response. Publicly available human liver ChIP-Seq GEO datasets demonstrated a region of histone H3 lysine 27 (H3K27) acetylation upstream of the S100A11 promoter. H3K27 acetylation ChIP-qPCR demonstrated a positive correlation between lipotoxic ER stress and H3K27 acetylation of the region, which we termed the lipotoxicity-influenced enhancer (LIE) domain. CRISPR-mediated repression of the LIE domain reduced palmitate-induced H3K27 acetylation and corresponding S100A11 upregulation in Huh7 cells and immortalized mouse hepatocytes. Silencing of the murine LIE in 2 independent steatohepatitis models demonstrated reduced S100a11 upregulation and attenuated liver injury. We confirmed H3K27 acetylation and XBP1s occupancy at the LIE domain in human MASH liver samples and an increase in hepatocyte-derived S100A11-enriched extracellular vesicles in MASH patient plasma. Our studies demonstrate a LIE domain that mediates hepatic S100A11 upregulation. This pathway may be a potential therapeutic target in MASH.

Phrenic motor neurons (PhMNs) innervate the diaphragm muscle (DIAm), the primary pump muscle involved in breathing. It has been shown that brain-derived neurotrophic factor (BDNF) signals through its high-affinity, full-length tropomyosin receptor kinase B (TrkB.FL) to promote PhMN survival in embryonic and early postnatal development. Reduced BDNF/TrkB.FL signaling also appears to be involved in the age-related loss of PhMNs. Previously, we developed a novel chemogenetic rat model ( TrkB F616 rat) in which insertion of a knock-in allele sensitive to 1NMPP1 (a selective kinase inhibitor) allows rapid inhibition of TrkB.FL kinase activity. Using this TrkB F616 rat, we found that in younger animals, a 14-day period of 1NMPP1-induced TrkB.FL kinase inhibition resulted in an ~20% loss of larger PhMNs similar to that found with aging. Previous studies from our laboratory demonstrated that BDNF/TrkB.FL signaling induces pCREB S133 phosphorylation at serine residue 133 in PhMNs. 1NMPP1 induced inhibition of TrkB.FL kinase activity in TrkB F616 rats acutely blunts pCREB S133 phosphorylation induced by intrathecal BDNF treatment. In another cell type, we showed that pCREB S133 transcriptionally targets PGC1α expression with downstream upregulation of expression of mitochondrial transcription factor A (TFAM) and mitochondrial biogenesis. Based on a bioinformatic analysis, we identified a putative binding site for pCREB S133 in the rat PGC1α promoter. In the present study, we hypothesized that BDNF/TrkB.FL signaling mediates pCREB S133 phosphorylation and downstream activation of PGC1α thereby promoting mitochondrial biogenesis. Adult female and male TrkB F616 rats (~ 3 months of age; 300 g) were anesthetized (ketamine/xylazine) and either 25 μM 1NMPP1 or DMSO (vehicle control) was injected ip. After 2 h, the rats were treated intrathecally (infused at C 4 ) with 100 ng/ml BDNF for 40 min. Thereafter, the animals were euthanized and the ventral cervical spinal cord containing PhMNs was dissected, and lysates were prepared for subsequent analyses. Western blot was performed to quantify pCREB S133 phosphorylation, and PGC1α mRNA and protein expression were measured by qPCR and Western blot, respectively. A chromatin immunoprecipitation (ChIP) assay confirmed that pCREB S133 binds to the PGC1α promoter and that compared to vehicle treated controls, 1NMPP1induced TrkB.FL kinase inhibition reduced the binding affinity of pCREB S133 . Consistent with these results, we found that pCREB S133 phosphorylation and PGC1α mRNA and protein expression were reduced following 1NMPP1-induced TrkB.FL kinase inhibition. We conclude that in the rat cervical ventral horn, BDNF/TrkB.FL signaling induces pCREB S133 phosphorylation which transcriptionally activates PGC1α expression and downstream mitochondrial biogenesis. The BDNF/TrkB.FL signaling pathway acting in concert with activity dependent signaling may converge on pCREB S133 phosphorylation to maintain the survival of PhMNs by promoting mitochondrial biogenesis. The loss of the contribution of BDNF/TrkB.FL signaling in old age and with neurodegeneration may lead to PhMN death. Supported by NIH grants AG44615 and HL146114 This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

In the elderly, age-associated muscle weakness and atrophy (sarcopenia) is prevalent in both limb and respiratory muscles. In the tongue muscles, dyscoordination and sarcopenia predisposes elderly individuals to dysphagia, dysphonia and a greater risk for aspiration pneumonias and obstructive sleep apnea. In humans and in the Fischer 344 rat model of aging, motor neuron (MN) death occurs in old age, and the selective vulnerability of fast-fatigable motor units to loss is consistent with behavioral deficits in airway defense (e.g., cough). However, whether the timing of MN loss is gradual across the lifespan or precipitous from late-middle-age is unknown. Recently, the concept of an age -related increase of systemic inflammatory cytokines has been implicated in the development of sarcopenia, although the mechanisms by which this occurs remain obscure. In other cell types, inflammation triggers an unfolded protein response and mitochondrial fragmentation – processes implicated in neuronal death and neurodegeneration. In this study of female and male Fischer 344 rats at 6-, 18- and 24-months-old, we evaluated serum inflammatory cytokines (TNFα, IL1β & IL6) via ELISA, medullary unfolded protein response (pIRE1a S724 & CDK5) and mitochondrial fragmentation (pDRP1 S616 ) via Western blot, hypoglossal MN mitochondrial volume density and mitochondrial fragmentation via serial blockface electron microscopy (SBFEM), hypoglossal MN survival via Nissl histology and stereological counting and tongue muscle (longitudinal and genioglossus) sarcopenia via uniaxial specific force assessment and cross-sectional muscle fiber areas. All data were analyzed by 1-way ANOVAs or Kruskal-Wallis nonparametric tests, depending on data distributions. Here, we show that a systemic inflammation-induced unfolded protein response in the medulla and hypoglossal MNs mitochondrial fragmentation is present at 18-months-old. This mitochondrial fragmentation is consistent with increased medullary expression of pDRP1 S616 . At this age, we did not observe any evidence of tongue muscle weakness or atrophy, nor any evidence of hypoglossal MN death. At 24-months old, hypoglossal MN mitochondrial deficits remained readily observable using SBFEM, with hypoglossal MN death (specific to larger MNs) and tongue muscle sarcopenia (selective to fast-fatigable type IIx fibers) present at this age. Overall, we conclude that denervation-related sarcopenia is precipitous from late-middle-age to old age in Fischer 344 rats. This MN death is triggered (or at least preceded), by an unfolded protein response and mitochondrial fragmentation. Interventions ameliorating inflammation, the unfolded protein response and/or mitochondrial breakdown may preserve MNs and prevent or reduce age-associated sarcopenia. This work was supported by NIH grants: HL166204 (MJF), AG086136 (MJF), HL157984 (GCS) & AG44615 (GCS) This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Airway inflammation occurs as a defense mechanism in response to pathogens, allergens, toxins, or pollutants. The physiological responses of airway inflammation are mediated by proinflammatory cytokines such as tumor necrosis factor alpha (TNFα), that causes airway remodeling, characterized by airway smooth muscle (ASM) hyperreactivity and proliferation. The molecular mechanism underlying TNFα-induced ASM cell proliferation is not fully understood. Previous studies from our lab demonstrated that TNFα exposure induces accumulation of unfolded proteins in the endoplasmic reticulum (ER) triggering an ER stress response that involves autophosphorylation of inositol requiring enzyme 1α at Serine 724 (pIRE1α S724 ). pIRE1α S724 promotes alternative splicing of X-box binding protein 1 (XBP1s) mRNA. XBP1s acts as a ubiquitous transcription factor in ASM cells, targets a number of genes associated with different cellular responses. In the present study, we hypothesized that TNFα promotes XBP1s mediated transcriptional activation of the Cyclin B1, a protein essential for the control of the cell cycle, thus promoting ASM cell proliferation. Human ASM (hASM) cells were dissociated from bronchiolar tissue samples collected during lung surgery from female and male patients (49 to 61 years of age) with no history of chronic respiratory diseases or current smoking. Isolated hASM cells from the same patient were serum-deprived for 48 h and separated into two groups: 1) untreated controls, and 2) treated with TNFα (20 ng/ ml) for 6 h. To confirm the effect of TNFα on hASM cell proliferation, loss of function experiments were employed by: 1) transfecting hASM cells with a non-spliceable XBP1 mutant, and 2) treating the cells with 4μ8C, a pharmacological inhibitor for IRE1α mediated XBP1 splicing. hASM cell proliferation was measured using CyQuant cell proliferation assay. The binding site sequence of XBP1s to the Cyclin B1 promoter was identified by bioinformatic analysis and confirmed by chromatin immunoprecipitation (ChIP) assay. mRNA and protein expressions of Cyclin B1 were determined by qRT-PCR and Western blot respectively. Nuclear localization of Cyclin B1 was measured by nuclear fractionation, followed by western blot of nuclear and cytosolic fraction. Results were analyzed by paired t-tests. TNFα treatment for 6 h induced pIRE1α S724 phosphorylation and XBP1s splicing. Bioinformatic analysis revealed the presence of an XBP1s binding site on the Cyclin B1 promoter. The ChIP assay confirmed that XBP1s binds to the promoter region of the Cyclin B1 genes, and the binding affinity was increased in TNFα-treated hASM cells. Consistent with these results, we found that TNFα treatment increased both mRNA and protein expressions of Cyclin B1 in hASM cells. Nuclear localization of Cyclin B1 increased significantly in TNFα treated cells consistent with an increase in hASM cell proliferation. Inhibition of XBP1 splicing reduced the expression of Cyclin B1 in hASM cells and hASM cell proliferation. In summary, TNFα induces an ER stress response involving pIRE1α S724 phosphorylation and XBP1s splicing. In turn, XBP1s transcriptionally targets expression of Cyclin B1 that mediates hASM cell proliferation. Supported by NIH grant HL157984 This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.