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.

The Impact of Midcervical Contusion Injury on Diaphragm Muscle Function.

Midcervical contusion injuries disrupt descending ipsilateral excitatory bulbospinal projections to phrenic motoneurons, compromising ventilation. We hypothesized that a unilateral contusion injury at C3 versus C5 would differentially impact phrenic activity reflecting more prominent disruption of ipsilateral descending excitatory drive to more caudal segments of the phrenic motor pool with more cranial injuries. Phrenic motoneuron counts and evidence of diaphragm muscle denervation at individual neuromuscular junctions (NMJ) were evaluated at 14 days post-injury after unilateral contusion injury (100 kDynes). Whole body plethysmography and chronic diaphragm EMG were measured before the injury and at 3, 7, and 14 days post-injury. Contusion injuries at either level resulted in a similarly sized cavity. C3 contusion resulted in loss of 39 ± 13% of ipsilateral phrenic motoneurons compared with 13 ± 21% after C5 contusion (p = 0.003). Cervical contusion injuries resulted in diaphragm muscle denervation (C3 contusion: 17 ± 4%; C5 contusion: 7 ± 4%; p = 0.047). The pattern of denervation revealed segmental innervation of the diaphragm muscle, with greater denervation ventrally after C3 contusion and dorsally after C5 contusion. Overall, diaphragm root mean square electromyography activity did not change ipsilaterally after C3 or C5 contusion, but increased contralaterally (∼ 11%) after C3 contusion only on the first day post-injury (p = 0.026). Similarly, there were no significant changes in breathing parameters during eupnea or exposure to hypoxia (10% O2) - hypercapnia (5% CO2) at any time post-injury. Unilateral midcervical contusions minimally impair ventilatory behaviors despite phrenic motoneuron loss and diaphragm muscle denervation.

Role of TrkB kinase activity in aging diaphragm neuromuscular junctions.

Brain derived neurotrophic factor (BDNF) acting through the tropomyosin-related kinase receptor B (TrkB) enhances neuromuscular transmission in the diaphragm muscle of adult mice, reflecting presynaptic effects. With aging, BDNF enhancement of neuromuscular transmission is lost. We hypothesize that disrupting BDNF/TrkB signaling in early old age will reveal a period of susceptibility evident by morphological changes at neuromuscular junctions (NMJ). Adult, male TrkB(F616A) mice (n=25) at 6 and 18 months of age, were used to examine the structural properties of diaphragm muscle NMJs (n=1097). 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. In early old age (18 months), presynaptic terminal volume decreased compared to 6 month old diaphragm NMJs (~20%). Inhibition of TrkB kinase activity significantly decreased the presynaptic terminal volume (~20%) and motor end-plate 2D planar area (~10%), independent of age group. Inhibition of TrkB kinase activity in early old age significantly reduced overlap of pre- and post-synaptic structures and increased the proportion of denervated NMJs (to ~20%). Collectively these results support a period of susceptibility in early old age when BDNF/TrkB signaling at diaphragm NMJs supports the maintenance of NMJs structure and muscle innervation.

Functional impact of diaphragm muscle sarcopenia in both male and female mice.

To perform a range of ventilatory and nonventilatory behaviors, the diaphragm muscle (DIAm) must be able to generate sufficient forces throughout the lifespan. We hypothesized that sarcopenia impacts DIAm force generation and thus limits performance of expulsive, higher force, nonventilatory behaviors. Male and female mice (n = 79) at 6 and 24 mo of age (100 vs. 70-75% survival, respectively) were used to examine transdiaphragmatic pressure (Pdi) generation across motor behaviors in vivo and in vitro DIAm specific force. We found a significant effect of age on maximum Pdi (20-41% decline during tracheal occlusion and bilateral phrenic nerve stimulation), maximum DIAm specific force (30% decline), and DIAm fatigue resistance (15% increase). There were no differences between sexes in these age effects on DIAm performance. These results support our hypothesis that sarcopenia primarily impacts higher force, nonventilatory motor behaviors of the DIAm. Such functional limitations may have negative implications in the ability of the DIAm to generate forces needed for airway clearance in old age and thereby contribute to age-related respiratory complications.

TrkB kinase activity maintains synaptic function and structural integrity at adult neuromuscular junctions.

Activation of the tropomyosin-related kinase receptor B (TrkB) by brain-derived neurotrophic factor acutely regulates synaptic transmission at adult neuromuscular junctions (NMJs). The role of TrkB kinase activity in the maintenance of NMJ function and structure at diaphragm muscle NMJs was explored using a chemical-genetic approach that permits reversible inactivation of TrkB kinase activity in TrkB(F616A) mice by 1NMPP1. Inhibiting TrkB kinase activity for 7 days resulted in significant, yet reversible, impairments in neuromuscular transmission at diaphragm NMJs. Neuromuscular transmission failure following 2 min of repetitive phrenic nerve stimulation increased from 42% in control to 59% in 1NMPP1-treated TrkB(F616A) mice (P = 0.010). Recovery of TrkB kinase activity following withdrawal of 1NMPP1 treatment improved neuromuscular transmission (P = 0.006). Electrophysiological measurements at individual diaphragm NMJs documented lack of differences in quantal content in control and 1NMPP1-treated mice (P = 0.845). Morphological changes at diaphragm NMJs were modest following inhibition and recovery of TrkB kinase activity. Three-dimensional reconstructions of diaphragm NMJs revealed no differences in volume at motor end plates (labeled by α-bungarotoxin; P = 0.982) or presynaptic terminals (labeled by synaptophysin; P = 0.515). Inhibition of TrkB kinase activity by 1NMPP1 resulted in more compact NMJs, with increased apposition of presynaptic terminals and motor end plates (P = 0.017) and reduced fragmentation of motor end plates (P = 0.005). Recovery of TrkB kinase activity following withdrawal of 1NMPP1 treatment resulted in postsynaptic remodeling likely reflecting increased gutter depth (P = 0.007), without significant presynaptic changes. These results support an essential role for TrkB kinase activity in maintaining synaptic function and structural integrity at NMJs in the adult mouse diaphragm muscle.

TrkB kinase activity is critical for recovery of respiratory function after cervical spinal cord hemisection.

Neuroplasticity following spinal cord injury contributes to spontaneous recovery over time. Recent studies highlight the important role of brain-derived neurotrophic factor (BDNF) signaling via the high-affinity tropomyosin-related kinase (Trk) receptor subtype B (TrkB) in recovery of rhythmic diaphragm activity following unilateral spinal hemisection at C2 (C2SH). We hypothesized that TrkB kinase activity is necessary for spontaneous recovery of diaphragm activity post-C2SH. A chemical-genetic approach employing adult male TrkB(F616A) mice (n=49) was used to determine the impact of inhibiting TrkB kinase activity by the phosphoprotein phosphatase 1 inhibitor derivative 1NMPP1 on recovery of ipsilateral hemidiaphragm EMG activity. In mice, C2SH was localized primarily to white matter tracts comprising the lateral funiculus. The extent of damaged spinal cord (~27%) was similar regardless of the presence of functional recovery, consistent with spontaneous recovery reflecting neuroplasticity primarily of contralateral spared descending pathways to the phrenic motor pools. Ipsilateral hemidiaphragm EMG activity was verified as absent in all mice at 3days post-C2SH. By 2weeks after C2SH, ipsilateral hemidiaphragm EMG activity was present in 39% of vehicle-treated mice compared to 7% of 1NMPP1-treated mice (P=0.03). These data support the hypothesis that BDNF/TrkB signaling involving TrkB kinase activity plays a critical role in spontaneous recovery of diaphragm activity following cervical spinal cord injury.