Hypoxic stress activates chaperone-mediated autophagy and modulates neuronal cell survival

Abstract

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% O₂, 5% CO₂) 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.

Introduction

Three different forms of autophagic mechanism have been proposed so far: macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA). Of these three, CMA is a unique autophagic pathway in that it provides a selective pathway for the degradation of cytosolic proteins in lysosomes. During CMA, cytoplasmic substrates that have a specific pentapeptide amino acid motif are specifically recognized by heat shock cognate protein 70 (HSC70). The substrates are transferred to the lysosomal membrane through an interaction with lysosomal-associated membrane protein type 2A (LAMP-2A), translocated into the lysosome with the assistance of several co-chaperones (HSP40, HSP90, Hip, Hop, and Bag-1), and subsequently degraded by lysosomal proteases (Agarraberes and Dice, 2001, Cuervo and Dice, 1996, Salvador et al., 2000). CMA is activated by various stimuli, such as long-term serum deprivation (Dice, 1982) and oxidative stress (Kiffin et al., 2004), suggesting that CMA contributes to the removal of damaged protein in these conditions. When neurons suffer from injuries, the removal of damaged and misfolded proteins by the protein degradation system is critical for neuronal survival because of the non-proliferative nature of neurons. Recent reports have revealed that CMA is related to the pathogenesis of Parkinson’s disease (Cuervo et al., 2004, Yang et al., 2009) and Alzheimer disease (Liu et al., 2009, Wang et al., 2009). However, the role of CMA in neuronal cell survival under ischemic conditions has not been elucidated.

Ischemic cerebral stroke is one of the leading causes of death and morbidity in humans. During focal brain ischemia, necrotic cell death occurs at the ischemic core, followed by apoptotic cell death at the ischemic border zone, called the “penumbra area” (Astrup et al., 1977, Ferrer, 2006). However, accumulating evidence has revealed that autophagy may also mediate neuronal death during cerebral ischemia (Aggoun-Zouaoui et al., 1998, Koike et al., 2008, Nitatori et al., 1995, Puyal et al., 2009, Rami et al., 2008). Transient forebrain ischemia induces selective neuronal death in vulnerable areas (Kirino, 1982). However, when sub-lethal ischemia is initiated prior to lethal ischemia, neuronal death is spared even in the vulnerable area (Kirino et al., 1991, Kitagawa et al., 1990). We previously reported that HSC70 and HSP40 are synergistically expressed in the neurons of the vulnerable area in response to sub-lethal ischemia (Tanaka et al., 2002). Furthermore, the combination of HSC70 and HSP40 may suppress aggregate formation and apoptosis in neurons (Kobayashi et al., 2000). These results suggested that CMA may be activated during ischemia and may facilitate the survival of neurons.

In the present study, we examined whether hypoxic stress would induce CMA activity in neuronal cells. Further, we evaluated the contribution of CMA to cell survival under hypoxic conditions.