Gransee, Heather M.

Neuromuscular dysfunction increases with age and mobility restrictions decrease quality of life for the elderly. A hallmark of aging is the aggregation of proteins and damaged components within a cell, which is especially detrimental to post‐mitotic cells such as motor neurons. Autophagy, a multistep catabolic process, is responsible for the disposal of damaged cellular components. Accumulation of autophagy markers may differ across motor neuron pools consistent with differences in aging effects across muscles. Autophagy is characterized by the elongation of a double‐membrane vacuole around the tagged cargo which fuses with a lysosome and is subsequently degraded. In the present study, expression levels of factors for autophagy initiation (Beclin1), elongation (LC3I, LC3II, the LC3II/I ratio, ATG7, and the ATG5/12 complex), and degradation (p62 clearance) were determined in the cervical (CSC) and lumbar spinal cord (LSC) of male and female C57BL/6 mice at 6, 18, and 24 months of age (n=8/age) using Western blot, spanning a period where aging differences manifest in forelimb vs. hindlimb muscles. A mixed linear model with animal as a random effect was used to compare the effect of age, tissue, sex, and their interaction on the relative expression of each autophagy factor. There was an age*tissue interaction for Beclin1 at 24 mo, with greater Beclin1 expression in LSC than CSC. There was an overall effect of age on LC3I expression, with 24 mo greater than 6 mo, and no changes in LC3II expression. The LC3II/I ratio was significantly different by tissue, whereby CSC had a greater LC3II/I ratio than that of LSC. Expression of ATG7 had a tissue*sex interaction, with ATG7 in females greater in LSC than CSC. There were no changes in ATG5/12 complex. Finally, there was an age and age*tissue interaction in p62 expression. p62 was significantly greater at 24 mo than at 18 mo, and at 18mo the LSC had greater p62 expression than CSC. These results suggest greater impairment of autophagy flux in LSC compared to CSC at comparable ages, consistent with greater susceptibility to aging effects in hindlimb muscles. Future studies will evaluate motor neuron‐specific changes in markers of autophagy elongation (i.e., LC3) and p62 clearance.

Autophagy is a cellular digestion process that contributes to cellular homeostasis by the elimination of proteins and damaged organelles. Impaired autophagy has been implicated in aging‐related disorders and in neurodegenerative diseases, including motor neuron disorders. Therefore, upregulation of autophagy may serve as a promising therapeutic approach. Lanthionine ketenamine (LK), an amino acid metabolite found in mammalian brain tissue at low concentrations, activates autophagy in rat glioma and human neuroblastoma cells in vitro, as well as in hippocampus and cortex after in vivo administration. With the overall goal of using LK to induce autophagy flux to prevent age‐related motor neuron dysfunction, we recently tested the effects of two lanthionine ketenamine phosphonates (LK‐Ps) and one lanthionine ketenamine ethyl (ester) phosphonate (LKE‐P) on autophagy modulation in a mouse motor neuron‐like hybrid cell line (NSC‐34). For fluorescence visualization of autophagy flux, NSC‐34 cells were transfected with mCherry‐GFP‐LC3 plasmid. Motor neurons treated with LK analogs displayed increased autophagy flux measured by the ratio of mCherry area (lysosomes) over GFP area (autophagosomes that have not fused with lysosomes) compared to control cells. There results were corroborated by increased LC3‐II/LC3‐I ratio as measured by protein expression using Western blot; and presence of autophagic vacuoles identified using transmission electron microscopy. In conclusion, LK analogs constitute a promising tool to induce autophagy flux in motor neurons, which may serve to prevent age‐related motor axon withdrawal and muscle denervation.

Brain‐derived neurotrophic factor (BDNF) binds with high affinity to tropomyosin‐related kinase receptor subtype B (TrkB), and plays an important role in neuromuscular junction (NMJ) function (regulating neuromuscular transmission and muscle innervation). We previously showed impaired neuromuscular transmission following acute inhibition of TrkB kinase activity in TrkB F616A mice harboring a point mutation that renders the kinase domain susceptible to inhibition by 1NMPP1 treatment. Neuromuscular transmission failure is expected to predominantly affect the ability of more fatigable motor units to generate force and thus primarily impair higher force motor behaviors. Accordingly, we hypothesized that acute administration of 1NMPP1 will impair the ability of the diaphragm muscle to accomplish higher force behaviors in adult, 6‐month old TrkB F616A mice. Transdiaphragmatic pressure (Pdi) measurements were conducted across a range of motor behaviors in lightly anesthetized mice during eupnea, exposure to hypoxia (10% O 2 )/hypercapnia (5% CO 2 ), tracheal occlusion, spontaneous deep breaths (“sighs”) and during maximal activation elicited by bilateral phrenic nerve stimulation. Adult male and female TrkB F616A mice at 6 months were injected with 1NMPP1 or vehicle (DMSO) one hour prior to Pdi measurements. The Pdi generated during eupnea and hypoxia/hypercapnia was ~8 cm H 2 0 compared to ~70 cm H 2 0 during maximal stimulation. There was a significant reduction in Pdi during maximal force with bilateral phrenic nerve stimulation in the 1NMPP1 group compared to the vehicle control (p=0.01). There was no effect of 1NMPP1 on Pdi during eupnea, hypoxia/hypercapnia, tracheal occlusion, or sigh behaviors. The reduction in Pdi is generally similar to the impairment in neuromuscular transmission consistent with a predominant effect on motor units innervating fast‐twitch fatigable muscle fibers in the diaphragm muscle. Thus, impaired trophic BDNF/TrkB signaling can be expected to limit the range of motor behaviors accomplished by the diaphragm muscle and may be an important contributor to neuromuscular dysfunction. Support or Funding Information This study was supported by NIH grant R01 AG057052. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Brain derived neurotrophic factor (BDNF) acting through the high affinity tropomyosin‐related kinase receptor B (TrkB) plays an important role in synaptic plasticity and function at the neuromuscular junction (NMJ) as well as maintenance of neuromuscular transmission. Our previous studies showed that susceptibility to age‐related changes in diaphragm muscle (DIAm) NMJ morphology and denervation depends on trophic influences exerted by BDNF/TrkB signaling. Indeed, TrkB kinase activity is necessary to stabilize DIAm NMJ pre‐synaptic terminals in 18 month old mice (representing early old age; ~90% survival). However, by 24 months of age (~75% survival), BDNF/TrkB signaling has no effect on DIAm neuromuscular transmission, suggesting reduced TrkB kinase activity in the older age groups. We hypothesized that the NMJs that are resilient to old age do not depend on TrkB kinase activity for stabilization of pre‐synaptic terminals. Male and female 24 month old TrkB F616A mice were used to examine the structural properties of NMJs across various muscles with varying susceptibility to age‐related effects (DIAm, EDLm, and SOLm). Confocal microscopy was used to compare pre‐ and post‐synaptic morphology and denervation following a 7 day treatment with the phosphoprotein phosphatase‐1 derivative 1NMPP1, which inhibits TrkB kinase activity in TrkB F616A mice vs. vehicle treatment. Inhibition of TrkB kinase activity had no effect on pre‐synaptic or post‐synaptic terminal volume in DIAm, EDLm, and SOLm. Similarly, the overlap of pre‐ and post‐synaptic structures and the proportion of denervated NMJs did not change with 1NMPP1 treatment in all muscle types. The average percent of denervated NMJs was 8, 22, and 7% in DIAm, EDLm, and SOLm, respectively. Collectively these results suggest that the requirement of TrkB kinase activity for stabilization of NMJs in DIAm, EDLm and SOLm wanes in old age. Support or Funding Information Supported by R01‐AG057052. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Following spinal cord hemisection at C2 (SH), removal of descending excitatory drive to phrenic motoneurons results in ipsilateral diaphragm muscle paralysis with partial spontaneous recovery of rhythmic phrenic activity over time. Brain‐derived neurotrophic factor (BDNF) plays an important role in neuroplasticity following SH. Intraspinal transplantation of mesenchymal stem cells engineered to produce BDNF‐GFP (BDNF‐MSCs) at the time of injury promotes functional recovery of all animals, despite low levels of MSC survival at 14 days post‐SH. The goal of the present study was to determine the survival and localization of BDNF‐MSCs after intraspinal transplantation. BDNF‐MSCs were injected intraspinally at C4 (2 sites, total 2 x 10 5 cells in 4 µl) in adult male Sprague‐Dawley rats. After one and three days, the cervical spinal cord was fixed, excised, and cut longitudinally. Survival of transplanted BDNF‐MSCs was confirmed in all animals at 1 day post‐transplantation (n=5) and in five of 6 animals at 3 days post‐transplantation. BDNF‐MSCs remained localized at the site of injection in the white matter at 1 and 3 days, with an average volume of 1.1 ± 0.3 µl. There was no localization of MSCs in the vicinity of phrenic motoneurons identified by retrograde labeling with cholera toxin subunit B. Understanding the time course of stem cell survival after transplantation is important in defining windows of opportunity during which the beneficial effects of BDNF on respiratory function after spinal cord injury are executed. Supported by HL96750 and Mayo Clinic Center for Regenerative Medicine.

The population at very old age is increasing dramatically throughout the world. Aging adults experience increased health and medical needs associated with debility and sarcopenia (i.e., muscle atrophy and force loss). Respiratory complications and diseases are common causes of morbidity and mortality in the elderly. Sarcopenia of respiratory muscles such as the diaphragm muscle (DIAm) may contribute to respiratory complications since the DIAm, the main inspiratory muscle in mammals, plays an important role in ventilation and generating the forces necessary to maintain airway clearance. Presently, there is limited information about aging effects into very old age. Specifically, whether DIAm sarcopenia is exacerbated in old age and if it will effect ventilation into very old age are presently unknown. We hypothesized that DIAm sarcopenia in very old mice (>30 mo; ~30% survival) will result in reduced DIAm force generation across motor behaviors, exhibit impaired ventilation, decreased isometric force, and reduced fiber cross‐sectional area compared to old (24 mo; ~80% survival), and young (6 mo, 100% survival). C57BL/6 × 129 female and male mice (n = 30) at 6 mo, 24 mo, and >30 mo were used to examine transdiaphragmatic pressure (Pdi) generation during eupnea, exposure to hypoxia‐hypercapnia, as well as higher force behaviors in airway occlusion, and/or bilateral phrenic stimulation. Ventilation was assessed using whole body plethysmography and venous blood gases. Isometric force was examined via physiological muscle force testing, and fiber cross‐sectional area was determined in immunohistochemically labeled sections across fiber types. There was a significant reduction in Pdi between young and old mice for higher force behaviors – tracheal occlusion and bilateral phrenic nerve stimulation, (p < 0.001); however, there was no further decline in Pdi for all behaviors between old and very old mice. There was no evidence of impaired ventilation (ventilatory parameters – respiratory rate, minute ventilation, or tidal volume) across age groups, and no evidence of differences between sex in any of the measurements. A loss of ability to generate higher levels of force necessary to maintain airway clearance may predispose older individuals to recurrent infection. Understanding the mechanisms of sarcopenia in old age will be important in mitigating the effects of aging on quality of life and maintaining the health span. Support or Funding Information This work was supported by grants R25‐GM075148, R01‐AG044615, and R01‐AG057052 from the National Institutes of Health. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Unilateral C2 spinal cord hemisection (C2SH) removes premotor drive to phrenic motoneurons, paralyzing the ipsilateral diaphragm muscle (DIAm). In previous studies, we found that intrathecal brain‐derived neurotrophic factor (BDNF) increases the proportion of rats displaying recovery of ipsilateral DIAm activity over time. We hypothesized that enhancing expression of the tropomyosin‐related kinase receptor subtype B (TrkB) in phrenic motoneurons will promote functional recovery ipsilateral to C2SH. Adult rats were injected intrapleurally with AAV7 expressing TrkB.FL‐FLAG, and 3 weeks later C2SH was performed. A separate group of C2SH animals served as controls. Bilateral DIAm EMG activity was chronically monitored to assess recovery up to 2 weeks after C2SH. In AAV‐treated rats, delivery of TrkB.FL‐FLAG to phrenic motoneurons was evident by the presence of FLAG protein in the phrenic motor nucleus. At 7‐days post‐C2SH, the proportion of rats displaying ipsilateral DIAm EMG activity was 13% and 40% in the untreated control and AAV‐treated groups, respectively. At 14‐days post‐C2SH, all AAV‐treated rats displayed functional recovery compared to 40% of C2SH rats. We conclude that targeted delivery of TrkB.FL to phrenic motoneurons enhances functional recovery of DIAm respiratory activity following upper cervical spinal cord injury. Supported by NIH grant HL96750 and Paralyzed Veterans of America.

Brain‐derived neurotrophic factor (BDNF) acting through full‐length tropomyosin related kinase receptor subtype B (TrkB.FL) plays an important role in neuroplasticity following spinal cord injury. BDNF/TrkB.FL signaling in phrenic motoneurons is both necessary and sufficient to enhance spontaneous recovery of ipsilateral rhythmic phrenic activity after C2 spinal cord hemisection (SH), where premotor drive to phrenic motoneurons is removed. Phrenic motoneuron expression of the excitatory glutamatergic NMDA receptor correlates with the time course of spontaneous functional recovery post‐SH. We hypothesized that increased BDNF/TrkB.FL signaling in phrenic motoneurons enhances recovery of rhythmic phrenic activity after SH via regulation of glutamatergic receptor expression. Rats were injected intrapleurally with AAV7 expressing human TrkB.FL‐FLAG at 3 weeks prior to SH. FLAG was present in the cervical ventral horn region, and AAV7‐GFP expression was specifically in phrenic motoneurons. At 14 days post‐SH, quantitative real‐time RT‐PCR verified human TrkB.FL expression in microdissected phrenic motoneurons. Phrenic motoneuron expression of NMDA increased ~10 fold compared to control, suggesting that increased BDNF/TrkB.FL signaling magnified SH‐induced changes in phrenic motoneuron NMDA expression. Supported by HL96750 and T32‐HL105355, and Paralyzed Veterans of America.

Cervical spinal hemisection at C2 (SH) paralyzes the diaphragm muscle by interrupting descending bulbospinal pathways to phrenic motoneurons. Signaling via the neurotrophin receptor TrkB in phrenic motoneurons enhances functional recovery after SH. However, it is not clear if the delay in gene expression resulting from adeno‐associated virus (AAV)‐mediated delivery would blunt the therapeutic efficacy of AAV delivered after SH. The goal of the present study was to examine the efficacy of intrapleurally‐delivered AAV7 encoding the full‐length TrkB receptor at 3 days post‐SH. Chronic diaphragm EMG recordings were conducted in adult male Sprague‐Dawley rats confirming absence of ipsilateral activity 3 days after SH. The proportion of animals displaying recovery of ipsilateral diaphragm EMG activity increased over time post‐SH and was greater in those treated with AAV‐TrkB (by 14 days post‐SH: AAV‐TrkB: 10/15 vs. AAV‐GFP: 2/11; p=0.01). Furthermore, peak root‐mean‐square amplitude of the diaphragm EMG activity increased progressively over time post‐SH. During eupnea, animals displaying recovery of ipsilateral diaphragm EMG activity that were treated with AAV‐TrkB achieved 40% of pre‐injury values (vs. 10% in those treated with AAV‐GFP). Targeting phrenic motoneurons via intrapleural delivery of AAV7‐TrkB promotes recovery of diaphragm activity when administered 3 days post‐injury. Grant Funding Source : Supported by R01 HL96750, T32 HL105355 and Mayo Clinic Center for Regenerative Medicine

Ventilatory behaviors across a range of species including humans require less than 50% of maximal force‐generating capacity by the diaphragm muscle, the main inspiratory muscle in mammals. Near maximal forces are only generated during expulsive behaviors usually associated with airway clearance. There is ongoing controversy regarding the impact of unilateral lesions (e.g., spinal cord injury, denervation) on ventilatory outcomes. We hypothesized that unilateral denervation of the diaphragm muscle in rats and mice would minimally impact ventilation. Transdiaphragmatic pressure (Pdi) measurements were used to estimate diaphragm muscle force in adult male rats and mice. Following acute, unilateral phrenic nerve transection in the neck, there was no change in Pdi during eupnea or hypoxia‐hypercapnia in rats or mice. Reduced Pdi was evident during deep breaths and tracheal occlusion (behaviors associated with ~70% of maximal Pdi elicited by bilateral phrenic nerve stimulation) in both species. These results are also consistent with the lack of changes in tidal volume or respiratory pattern during eupnea measured by whole body plethysmography in unrestrained, spontaneously breathing rats after mid‐cervical contusion or hemisection injuries. These results highlight the large reserve capacity for force generation by the diaphragm muscle in rodents and the importance of measuring multiple motor behaviors. Grant Funding Source : Supported by HL096750, AG044615, HL105355 and Mayo Center for Regenerative Medicine