Davis, Anne S.

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.

Neural control of the diaphragm muscle (DIAm) is essential to sustain breathing. During breathing, fatigue resistant DIAm motor units are recruited comprising smaller phrenic motor neurons (PhMNs) and type I and IIa muscle fibers. Larger PhMNs comprise more fatigable DIAm motor units that are infrequently active during expulsive behaviors. The difference in activation history is reflected in the higher mitochondrial volume density in smaller PhMNs. In PhMNs, brain-derived neurotrophic factor (BDNF) signals through its high-affnity receptor, tropomyosin receptor kinase B (TrkB.FL). Previously, we found that intrathecal BDNF treatment induces CREB phosphorylation at Serine 133 (pCREBs133). We hypothesized that pCREBs133 binds to the PGC1a promoter, transcriptionally targeting PGC1a expression, mediating downstream increased expression of NRF1, NRF2, and TFAM and mitochondrial biogenesis. To test this hypothesis, we used a chemosensitive TrkBF616 rat model with a knock-in allele sensitive to 1NMPP1 in that TrkB kinase is inhibited following 1NMPP1, a small molecule derivative of the protein kinase inhibitor protein phosphatase 1 that plays a significant role inhibiting the TrkB phosphorylation and activation. Systemic treatment of 1NMPP1 in TrkBF616 rats inhibits TrkB kinase activity. A bioinformatic analysis was performed to identify a putative binding site for pCREBs133 in the PGC1a promoter sequence in rats. Using a chromatin immunoprecipitation (ChIP) assay we found that pCREBS133 binds to the PGC1a promoter in rat cervical ventral horn. We showed that intrathecal BDNF treatment increased pCREBs133 phosphorylation, which was blocked by 1NMPP1 treatment in TrkBF616 rats. PGC1a, NRF1/2, and TFAM protein was compared in cervical ventral horn lysates from BDNF- and BDNF+1NMPP1-treated TrkBF616 rats by qRT-PCR and Western Blot. Relative mtDNA copy number (normalized to nuclear DNA) was quantified by qPCR to assess the mitochondrial biogenesis. All results were analyzed by paired t-test. Following 1NMPP1-induced inhibition of TrkB kinase, PGC1a, NRF1/2, and TFAM mRNA and protein expression was reduced. Consistent with these findings, mtDNA copy number was decreased following TrkB kinase inhibition by 1NMPP1. We conclude that inhibition of TrkB kinase by 1NMPP1 reduces PGC1a-mediated mitochondrial biogenesis in rat cervical ventral horn. Supported by NIH grants AG44615 and HL146114. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.