Mantilla, Carlos B

This study found that inhibiting TrkB kinase activity decreases the extent of neuromuscular transmission failure during repetitive stimulation at low frequency while increasing failure with high-frequency stimulation. The frequency-dependent effect of 1NMPP1 treatment suggests that the role of TrkB signaling in maintaining neuromuscular transmission during repetitive stimulation may differ between motor unit types (slow-twitch, fatigue-resistant vs. fast-twitch, fatigable) and likely reflect effects at the presynaptic terminal without major effects on the muscle itself.

Aging selectively impacts the function of type IIx/IIb fibers in the diaphragm muscle. We show that sarcopenia reduces maximum specific force, maximum shortening velocity, and peak power output, whereas endurance during repetitive contractions is preserved. These findings explain why higher force airway clearance behaviors decline with age, whereas fatigue-resistant breathing behavior remains resilient.

The present study provides novel physiological insights into aging by measuring airway compliance and residual volume via forced oscillation perturbations using a programmable mechanical ventilator (flexiVent—currently considered a gold standard for in vivo lung function measurements). In addition, dynamic measurements of lung inflation reported herein may serve as early indicators of altered elastic properties. Taken together, the findings offer a comprehensive assessment of age-related changes in lung structure and function.

ABSTRACT Motor neuron plasticity and survival depend on several trophic factors, with signaling via the tropomyosin receptor kinase B (TrkB) receptor being particularly important beyond development and across the lifespan. We hypothesized that differences in motor neuron properties may reflect differential trophic influences across motor units within a single motor unit pool. Based on extensive previous characterization of rat phrenic motor neurons (PhMN), somal surface area was used as an indicator of motor unit type (smaller neurons innervating slow‐twitch, fatigue‐resistant units versus larger neurons innervating fast‐twitch, fatigable units). To examine whether TrkB expression reflects these size‐related properties, TrkB mRNA transcripts were studied in individual PhMNs labeled retrogradely with Alexa Fluor 488‐conjugated cholera toxin subunit β from five adult male Sprague–Dawley rats. Longitudinal sections of the cervical C3–C5 spinal cord (10 μm thick) were processed using fluorescence in situ hybridization (RNAscope) and imaged using confocal microscopy to measure individual PhMN size and full‐length TrkB mRNA transcripts in nuclear and cytoplasmic compartments. Across 287 PhMNs, significant size variation existed both within and between animals. Across all animals, somal surface area averaged 4385 ± 1627 μm 2 with mean values per animal ranging from 3544 to 5461 μm 2 . PhMNs were classified into tertiles within each animal based on somal surface area. Individual PhMNs averaged 1070 ± 685 TrkB mRNA transcripts per motor neuron. Larger PhMNs in the upper tertile by size expressed a greater total number of TrkB mRNA transcripts per motor neuron, with values on average threefold higher compared to smaller motor neurons in the lower tertile ( p < 0.001). Cytoplasmic and nuclear TrkB mRNA density (transcripts/volume) displayed no significant effect of PhMN somal surface area tertile. Overall, the greater TrkB mRNA levels in larger motor units responsible for higher force, expulsive motor behaviors may underlie their functional adaptations and resilience to injury or disease. image

Neuromotor control of the diaphragm muscle (DIAm) after C 2 spinal hemisection (C 2 SH) is impacted differentially between ipsilateral and contralateral sides. We show neural drive to ipsilateral phrenic motor neurons decreases after C 2 SH, leading to a reduction in the number of DIAm motor units (MUs) recruited. Contralateral neural drive increases after C 2 SH, leading to an increase in the number of MU recruited. These results provide novel information about the impact of C 2 SH on DIAm neuromotor control.

Autophagy impairments have been implicated in various aging conditions. Previous studies in cervical motor neurons show an age-dependent increase in the key autophagy proteins LC3 and p62, reflecting autophagy impairment and autophagosome accumulation. Chloroquine is commonly used to inhibit autophagy by preventing autophagosome–lysosome fusion and may thus emulate the effects of aging on the neuromuscular system. Indeed, acute chloroquine administration in old mice decreases maximal transdiaphragmatic pressure generation, consistent with aging effects. We hypothesized that chloroquine alters diaphragm muscle neuromuscular junction (NMJ) morphology and increases denervation. Adult male and female C57BL/6 × 129J mice between 5 and 8 months of age were used to examine diaphragm muscle NMJ morphology and denervation following daily intraperitoneal injections of chloroquine (10 mg/kg/d) or vehicle for 7 days. The motor end-plates and pre-synaptic terminals were fluorescently labeled with α-bungarotoxin and anti-synaptophysin, respectively. Confocal microscopy was used to assess pre- and post-synaptic morphology and denervation. At diaphragm NMJs, chloroquine treatment decreased pre-synaptic volume by 12% compared to the vehicle (p < 0.05), with no change in post-synaptic volume. Chloroquine treatment increased the proportion of partially denervated NMJs by 2.7-fold compared to vehicle treatment (p < 0.05). The morphological changes observed were similar to those previously reported in the diaphragm muscles of 18-month-old mice. These findings highlight the importance of autophagy in the maintenance of the structural properties at adult NMJs in vivo.

Aging disrupts multiple homeostatic processes, including autophagy, a cellular process for the recycling and degradation of defective cytoplasmic structures. Acute treatment with the autophagy inhibitor chloroquine blunts the maximal forces generated by the diaphragm muscle, but the mechanisms underlying neuromuscular dysfunction in old age remain poorly understood. We hypothesized that chloroquine treatment increases the presynaptic retention of the styryl dye FM 4-64 following high-frequency nerve stimulation, consistent with the accumulation of unprocessed bulk endosomes. Diaphragm-phrenic nerve preparations from 24-month-old male and female C57BL/6 × 129 J mice were incubated with FM 4-64 (5 µM) and either chloroquine (50 µM) or vehicle during 80 Hz phrenic nerve stimulation. Acute chloroquine treatment significantly decreased FM 4-64 intensity at diaphragm neuromuscular junctions following 80 Hz phrenic nerve stimulation, consistent with disrupted synaptic vesicle recycling. A similar reduction was evident in regions with the greatest FM 4-64 fluorescence intensity, which most likely surround synaptic vesicle release sites. In the absence of nerve stimulation, chloroquine treatment significantly increased FM 4-64 intensity at diaphragm neuromuscular junctions. These findings highlight the importance of autophagy in regulating presynaptic vesicle retrieval (including vesicle recycling and endosomal processing) and support the role of autophagy impairments in age-related neuromuscular dysfunction.

Abstract Ageing‐related neuromuscular dysfunction is associated with reduced tropomyosin‐related kinase receptor subtype B (TrkB) signalling and accumulation of damaged cytoplasmic aggregates in motor neurons. Autophagy functions to remove these damaged aggregates, and we previously reported increased cervical motor neuron expression of LC3 and p62 in old age. We hypothesized that inhibition of TrkB kinase activity results in an increase in the relative expression of both LC3 and p62 in cervical motor neurons, consistent with impaired progression of autophagy. TrkB F616A mice, which possess a mutation that renders TrkB kinase activity susceptible to rapid inhibition by 1NMPP1, were treated at 6, 18 or 24 months of age with vehicle or 1NMPP1 for 7 days. Immunofluorescence intensity was measured to determine LC3 and p62 expression in choline acetyltransferase‐positive motor neurons in the cervical spinal cord. The effect of inhibiting TrkB kinase activity on progression of autophagy was age dependent. In 6‐month‐old mice, inhibiting TrkB kinase activity increased cervical motor neuron expression of LC3 by 11% ( P < 0.001) and p62 by 8% ( P = 0.019) compared with vehicle treatment. In 18‐ and 24‐month‐old mice, there was no effect of inhibiting TrkB kinase activity on motor neuron LC3 or p62 expression. We provide evidence that inhibition of TrkB signalling impairs progression of autophagy in motor neurons of young mice, similar to the response to ageing. Accordingly, a reduction of TrkB signalling in old age might contribute to neuromuscular dysfunction by impairing progression of autophagy in motor neurons.