Ducaji, Emily C.

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 maintains cellular homeostasis by the elimination of damaged proteins or organelles. Our overarching hypothesis is that autophagy contributes to aging‐related neuromuscular dysfunction. The mechanisms regulating autophagy flux are promising therapeutic targets to mitigate the effects of neuromuscular dysfunction across conditions such as aging. In previous studies, we found that autophagy flux is reduced in early old age (18‐month old mice; 90% survival), with progressive autophagosome accumulation in old age evident by increased LC3 and p62 protein expression in cervical motor neurons (24‐month old mice; 75% survival). Neurotrophins acting through the tropomyosin‐related kinase receptor subtype B (TrkB) are critical to the maintenance of neuromuscular function including neuromuscular transmission in the diaphragm muscle and generation of maximal transdiaphragmatic pressure (Pdi), both in young and aged mice. Specifically, inhibition of TrkB signaling impairs maximal Pdi generation whereas activation of TrkB signaling blunts failure in neuromuscular transmission. We hypothesize that inhibiting TrkB kinase activity reduces autophagy flux resulting in increased LC3 and p62 expression, like the aging effects at phrenic motor neurons. TrkB F616A mice at 18‐ and 24‐ months of age, harboring a mutation that induces susceptibility to 1NMPP1‐mediated TrkB kinase inhibition, were randomized to receive 1‐week treatment with vehicle or 1NMPP1 (n=6 per age and treatment group). Protein expression of the autophagy markers LC3 and p62 in cervical spinal cord (CSC) segments C2‐C6 was analyzed by Western blotting. There was a significant effect on LC3 and p62 expression of age, but not of 1NMPP1 treatment, and no age*treatment interaction. LC3B expression increased by ~50% and p62 expression increased by ~30% in 24 month‐old compared to 18 month‐old mice. These results are consistent with previous reports that there is an aging‐related impairment of autophagy and accumulation of autophagosomes; however, inhibiting TrkB kinase activity does not modulate autophagy in these age groups. Understanding the role of TrkB signaling in mediating changes in autophagy and age‐related neuromuscular dysfunction will be important in advancing potential therapeutic applications of TrkB agonists.

Autophagy is a homeostatic process for the elimination of damaged, unnecessary proteins or organelles within cells. Our overarching hypothesis is that autophagy contributes to neuromuscular dysfunction, specifically increased NMTF seen with aging. Understanding the mechanisms regulating autophagy flux is essential to discovering therapeutic agents which may mitigate the effects of neuromuscular dysfunction across conditions including old age. In recent studies, we found that in 18 month old mice autophagy in the cervical spinal cord is accelerated and neuromuscular transmission failure (NMTF) in diaphragm muscle‐phrenic nerve preparations is increased. Neurotrophin activity, specifically brain‐derived neurotrophic factor (BDNF) signaling through its high affinity receptor tropomyosin‐related kinase receptor subtype B (TrkB) decreases NMTF. Thus, we predict BDNF/TrkB signaling suppresses autophagy in phrenic motor neurons located in the cervical spinal cord. We hypothesize that inhibiting TrkB kinase activity accelerates autophagy in phrenic motor neurons. TrkB F616A mice at 18 months of age were randomized to receive 1 week treatment with vehicle (n=4) or 1NMPP1 (TrkB kinase inhibitor; n=6). Additionally, 18 month old wild type C57BL/6 mice (n=2) were studied. Whole cervical spinal cord (CSC) was harvested for measurements of autophagy marker protein expression analyzed by Western blotting. Data for the vehicle‐treated TrkB F616A and wild type mice was combined given lack of differences for any measurement. p62, a marker associated with the initiation of autophagy, was significantly decreased in the 1NMPP1‐treated group compared to vehicle‐treated or wild type mice. LC3, a protein marking the autophagosome membrane elongation phase, was not significantly changed across groups indicating that LC3‐tagged autophagosomes proceed to lysosomal degradation and do not accumulate. These results support our hypothesis that inhibiting BDNF/TrkB signaling accelerates autophagy flux in the CSC and reveal trophic effects of BDNF/TrkB signaling in the neuromuscular system. Accelerated autophagy may underlie age‐related neuromuscular dysfunction in a BDNF/TrkB signaling dependent manner. Support or Funding Information Support: NIH R01 AG057052