Aging‐Related Changes in Motoneuron Autophagy

Autophagy is a series of catabolic processes that involves degradation of cytoplasmic components through lysosomal pathways. There is an important balance that must be met, with impaired autophagy contributing to cell dysfunction and excessive autophagy leading to cell death likely via apoptosis. Indeed autophagy is a dynamic multi‐step process that is modulated positively and negatively at several steps. The role of autophagy in aging‐related motoneuron dysfunction or death is not known. Therefore, it is important to differentiate between the accumulation of autophagosomes due to increased induction of autophagy during conditions of cell stress common across aging‐related conditions, and the accumulation of autophagosomes due to inefficient clearance of damaged organelles and other sequestered cargoes, which reflects impairments in autophagy that would lead to cell dysfunction. We hypothesized that with aging, there is autophagy imbalance causing accumulation of autophagosomes and ultimately neuromuscular dysfunction. Male and female mice at 6 and 24 months of age were used to assess markers of autophagy in the spinal cord. Protein expression of autophagy‐related genes LC3A and LC3B increased in the cervical and lumbar spinal cord of old compared to young mice, whereas protein expression of Atg5, Atg7, and Bcn1 did not change with age. Quantitative real‐time RT‐PCR of microdissected phrenic motoneurons showed an increase in LC3 mRNA expression with age. Overall autophagic flux, which assesses inclusion (or exclusion) of cargo within autophagosomes, delivery of cargo to lysosomes, and subsequent breakdown and release of macromolecules back into the cytosol was evaluated using an mCherry‐GFP‐LC3 sensor. This genetic sensor is designed to monitor LC3 flux through autophagy pathways since the GFP signal is sensitive to the acidic/proteolytic conditions of the lysosome lumen, whereas mCherry fluorescence persists under these environments. We used motoneuron‐like NSC‐34 cells transfected with mCherry‐GFP‐LC3 plasmid to detect changes in the autophagic degradation rate in cells treated with tunicamycin (which leads to cell stress via induction of the unfolded protein response). We observed an increase in yellow vesicles indicating an accumulation of autophagosomes due to inefficient clearance of sequestered cargoes. This genetic sensor indicates impaired autophagy flux in motoneurons in conditions of elevated stress likely present in old age. Taken together, these results suggest impaired autophagy in motoneurons, including phrenic motoneurons, with an associated increase in markers of early autophagosome formation (LC3). Support or Funding Information Supported by R01‐AG044615.