Autophagy is a conserved mechanism responsible for the continuous clearance of unnecessary organelles or misfolded
proteins in lysosomes. Three types of autophagy have been reported in the difference of substrate delivery to lysosome: macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA). Among these types, CMA is a unique autophagy system that selectively degrades substrates detected by heat shock cognate
protein 70 (HSC70). Recently, autophagic cell death has been reported to be involved in neuronal death following
brain ischemia; however, the contribution of CMA to neuronal death/survival after ischemic stress has not been addressed. In the present study, we determined whether quantitative alterations in LAMP-2A, which is the key molecule in CMA, would modulate neuronal cell survival under hypoxic conditions. Incubation of Neuro2A cells in a hypoxic chamber (1% O(2), 5% CO(2)) increased the level of LAMP-2A and induced accumulation of LAMP-2A-positive lysosomes in the perinuclear area, which is a hallmark of CMA activation. The activation of CMA in response to
hypoxia was also confirmed by the GAPDH-
HaloTag CMA
indicator system at the single cell level. Next, we asked whether CMA was involved in cell survival during
hypoxia. Blocking LAMP-2A expression with
siRNA increased the level of cleaved
caspase-3 and the number of
propidium iodide-positive cells after hypoxic stress regardless of whether macroautophagy could occur, whereas the administration of
mycophenolic acid, a potent CMA activator, rescued
hypoxia-mediated cell death. Finally, we asked whether CMA was activated in the neurons after
middle cerebral artery occlusion in vivo. The expression of LAMP-2A was significantly increased in the ischemic hemisphere seven days after
brain ischemia. These results indicate that CMA is activated during
hypoxia and contributes to the survival of cells under these conditions.