Abstract
Autophagy is a catabolic mechanism facilitating degradation of cytoplasmic proteins and organelles in a lysosome-dependent manner. Autophagy flux is necessary for normal neuronal homeostasis and its dysfunction contributes to neuronal cell death in several neurodegenerative diseases. Elevated autophagy has been reported after spinal cord injury (SCI); however, its mechanism, cell type specificity and relationship to cell death are unknown. Using a rat model of contusive SCI, we observed accumulation of LC3-II-positive autophagosomes starting at posttrauma day 1. This was accompanied by a pronounced accumulation of autophagy substrate protein p62, indicating that early elevation of autophagy markers reflected disrupted autophagosome degradation. Levels of lysosomal protease cathepsin D and numbers of cathepsin-D-positive lysosomes were also decreased at this time, suggesting that lysosomal damage may contribute to the observed defect in autophagy flux. Normalization of p62 levels started by day 7 after SCI, and was associated with increased cathepsin D levels. At day 1 after SCI, accumulation of autophagosomes was pronounced in ventral horn motor neurons and dorsal column oligodendrocytes and microglia. In motor neurons, disruption of autophagy strongly correlated with evidence of endoplasmic reticulum (ER) stress. As autophagy is thought to protect against ER stress, its disruption after SCI could contribute to ER-stress-induced neuronal apoptosis. Consistently, motor neurons showing disrupted autophagy co-expressed ER-stress-associated initiator caspase 12 and cleaved executioner caspase 3. Together, these findings indicate that SCI causes lysosomal dysfunction that contributes to autophagy disruption and associated ER-stress-induced neuronal apoptosis.
Highlights
Macroautophagy is a lysosome-dependent catabolic pathway degrading cytoplasmic proteins, protein aggregates and organelles.[5,6,7] Autophagy is initiated by the formation of autophagosomes, double membrane vesicles containing cytoplasmic components that include potentially toxic protein aggregates and damaged organelles
To directly evaluate the possibility that defects in autophagy flux may contribute to ER stress observed after spinal cord injury (SCI), we examined the expression of ER stress markers, the 78 kDa glucose-regulated protein (GRP78) and transcription factors ATF4 and CHOP (DDIT3)
Disruption of autophagy flux has been reported to contribute to neuronal cell death in neurodegenerative diseases, such as Parkinson disease, Alzherimer’s disease and Huntington disease.[5,31,32]
Summary
Macroautophagy (hereafter called autophagy) is a lysosome-dependent catabolic pathway degrading cytoplasmic proteins, protein aggregates and organelles.[5,6,7] Autophagy is initiated by the formation of autophagosomes, double membrane vesicles containing cytoplasmic components that include potentially toxic protein aggregates and damaged organelles. Pathologically increased autophagy can contribute to cell death,[21,24] when autophagy flux is blocked, for example, because of lysosomal defects. Defects in autophagy flux can exacerbate ER stress and potentiate ERstress-induced apoptosis.[16,17] ER stress has long been implicated as part of the secondary injury after central nervous system trauma,[25,26] but its mechanisms remain unknown. Our data demonstrate that autophagosome accumulation after SCI is not due to increased initiation of autophagy, but rather due to inhibition of autophagy flux. This likely reflects the disruption of lysosomal function after SCI. Our findings suggest that autophagy is disrupted after SCI and may exacerbate ER stress and neuronal cell death
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