Autophagy markers LC3 and p62 in aging lumbar motor neurons.

Autophagy is a ubiquitous process through which damaged cytoplasmic structures are recycled and degraded within cells. Aging can affect autophagy regulation in different steps leading to the accumulation of damaged organelles and proteins, which can contribute to cell dysfunction and death. Motor neuron (MN) loss and sarcopenia are prominent features of neuromuscular aging. Previous studies on phrenic MNs showed increased levels of the autophagy proteins LC3 and p62 in 24 month compared to 6 month old mice, consistent with the onset of diaphragm muscle sarcopenia. In the present study, we hypothesized that aging leads to increased expression of the autophagy markers LC3 and p62 in single lumbar MNs. Expression of LC3 and p62 in lumbar MNs (spinal levels L1-L6) was assessed using immunofluorescence and confocal imaging of male and female mice at 6, 18 and 24 months of age, reflecting 100 %, 90 % and 75 % survival, respectively. A mixed linear model with animal as a random effect was used to compare relative LC3 and p62 expression in choline acetyl transferase-positive MNs across age groups. Expression of LC3 and p62 decreased in the white matter of the lumbar spinal cord with aging, with ~29 % decrease in LC3 and ~ 7 % decrease in p62 expression at 24 months of age compared to 6 months of age. There was no change in LC3 or p62 expression in the gray matter with age. LC3 expression in MNs relative to white matter increased significantly with age, with 150 % increase at 24 months of age compared to 6 months of age. Similarly, p62 expression in MNs relative to white matter increased significantly with age, with ~14 % increase at 24 months of age compared to 6 months of age. No effect of sex or MN pool was observed in LC3 and p62 expression in MNs. Overall, these data suggest autophagy impairment during elongation (increased LC3) and degradation (increased p62) phases with aging in lumbar MNs.

TrkB signaling contributes to transdiaphragmatic pressure generation in aged mice.

Ventilatory deficits are common in old age and may result from neuromuscular dysfunction. Signaling via the tropomyosin-related kinase receptor B (TrkB) regulates neuromuscular transmission and, in young mice, is important for the generation of transdiaphragmatic pressure (Pdi). Loss of TrkB signaling worsened neuromuscular transmission failure and reduced maximal Pdi, and these effects are similar to those observed in old age. Administration of TrkB agonists such as 7,8-dihydroxyflavone (7,8-DHF) improves neuromuscular transmission in young and old mice (18 mo; 75% survival). We hypothesized that TrkB signaling contributes to Pdi generation in old mice, particularly during maximal force behaviors. Old male and female TrkBF616A mice, with a mutation that induces 1NMPP1-mediated TrkB kinase inhibition, were randomly assigned to systemic treatment with vehicle, 7,8-DHF, or 1NMPP1 1 h before experiments. Pdi was measured during eupneic breathing (room air), hypoxia-hypercapnia (10% O2/5% CO2), tracheal occlusion, spontaneous deep breaths ("sighs"), and bilateral phrenic nerve stimulation (Pdimax). There were no differences in the Pdi amplitude across treatments during ventilatory behaviors (eupnea, hypoxia-hypercapnia, occlusion, or sigh). As expected, Pdi increased from eupnea and hypoxia-hypercapnia (∼7 cm H2O) to occlusion and sighs (∼25 cm H2O), with no differences across treatments. Pdimax was ∼50 cm H2O in the vehicle and 7,8-DHF groups and ∼40 cm H2O in the 1NMPP1 group (F8,74 = 2; P = 0.02). Our results indicate that TrkB signaling is necessary for generating maximal forces by the diaphragm muscle in old mice and are consistent with aging effects of TrkB signaling on neuromuscular transmission.NEW & NOTEWORTHY TrkB signaling is necessary for generating maximal forces by the diaphragm muscle. In 19- to 21-mo-old TrkBF616A mice susceptible to 1NMPP1-induced inhibition of TrkB kinase activity, maximal Pdi generated by bilateral phrenic nerve stimulation was ∼20% lower after 1NMPP1 compared with vehicle-treated mice. Treatment with the TrkB agonist 7,8-dihydroxyflavone did not affect Pdi generation when compared with age-matched mice. Inhibition of TrkB kinase activity did not affect the forces generated during lower force behaviors in old age.

Age-related impairment of autophagy in cervical motor neurons.

Neuromuscular dysfunction is common in old age. Damaged cytoplasmic structures aggregate with aging, especially in post-mitotic cells like motor neurons. Autophagy is a ubiquitous cell process that aids in the clearance of damaged aggregates. Accordingly, we hypothesized that autophagy is impaired in old age, contributing to neuromuscular dysfunction via an effect in motor neurons. Autophagy flux may be impaired as a result of deficits in the initiation, elongation or degradation phases. Changes in the expression levels of core proteins necessary for each of the autophagy phases were evaluated by Western blotting in the cervical spinal cord (segments C2-C6 corresponding to the phrenic motor pool) of adult male and female mice at 6-, 18-, and 24-months of age (reflecting 100%, 90% and 75% survival, respectively). There was no evidence of an effect of age on the expression of the autophagy markers Beclin-1 (Becn-1; initiation), ATG7 and ATG5/12 complex (elongation) or LC3 (elongation/degradation). Reduced p62 expression (a marker of degradation) was evident in the cervical spinal cord of adult mice at 18-months compared to 24-months. Accordingly, expression of LC3 and p62 in motor neurons was analyzed using immunofluorescence and confocal microscopy in separate animals. LC3 and p62 immunoreactivity was evident in the gray matter with minimal expression in the white matter across all age groups. A mixed linear model with animal as a random effect was used to compare relative LC3 and p62 expression in motor neurons to gray matter across age groups. Expression of both LC3 and p62 was higher in choline acetyl transferase (ChAT)-positive motor neurons (~2-3 fold vs. gray matter). Across age groups, there were differences in the relative expression of LC3 (F2,12 = 7.59, p < 0.01) and p62 (F2,12 = 8.00, p < 0.01) in cervical motor neurons. LC3 expression in motor neurons increased ~20% by 24-months of age in both male and female mice. p62 expression in motor neurons increased ~70% by 18-months compared to 6-months with no further changes by 24-months of age in male mice. p62 expression did not change across age groups in female mice, and was ~20% higher than in males. Our findings highlight important changes in autophagy pathways that likely contribute to the development of aging-related neuromuscular dysfunction in mice. At 18-months of age, increased autophagosome clearance (reduced p62 expression) appears to be a global effect not restricted to motor neurons. By 24-months of age, increased expression of LC3 and p62 indicates impaired autophagy with autophagosome accumulation in cervical motor neurons.

Inhibition of TrkB kinase activity impairs transdiaphragmatic pressure generation.

Signaling via the tropomyosin-related kinase receptor subtype B (TrkB) regulates neuromuscular transmission, and inhibition of TrkB kinase activity by 1NMPP1 in TrkBF616A mice worsens neuromuscular transmission failure (NMTF). We hypothesized that acute inhibition of TrkB kinase activity will impair the ability of the diaphragm muscle to produce maximal transdiaphragmatic pressure (Pdi) without impacting the ability to generate forces associated with ventilation, consistent with the greater susceptibility to NMTF in motor units responsible for higher-force nonventilatory behaviors. Adult male and female TrkBF616A mice were injected with 1NMPP1 (n = 8) or vehicle (DMSO; n = 8) 1 h before Pdi measurements during eupneic breathing, hypoxia/hypercapnia (10% O2/5% CO2), tracheal occlusion, spontaneous deep breaths ("sighs") and during maximal activation elicited by bilateral phrenic nerve stimulation. In the vehicle-treated group, Pdi increased from ~10 cmH2O during eupnea and hypoxia/hypercapnia, to ~35 cmH2O during sighs and tracheal occlusion, and to ~65 cm H2O during maximal stimulation. There was no effect of acute 1NMPP1 treatment on Pdi generated during most behaviors, except during maximal stimulation (~30% reduction; P < 0.05). This reduction in maximal Pdi is generally similar to the worsening of NMTF previously reported with TrkB kinase inhibition in rodents. Accordingly, impaired TrkB signaling limits the range of motor behaviors accomplished by the diaphragm muscle and may contribute to neuromuscular dysfunction, primarily by impacting fatigable, higher force-generating motor units.NEW & NOTEWORTHY TrkB signaling plays an important role in maintaining neuromuscular function in the diaphragm muscle and may be necessary to accomplish the various motor behaviors ranging from ventilation to expulsive, behaviors requiring near-maximal forces. This study shows that inhibition of TrkB kinase activity impairs maximal pressure generation by the diaphragm muscle, but the ability to generate the lower pressures required for ventilatory behaviors is not impacted.