Lysosomal Dysfunction Promotes Autophagic Stress and NPC Death

Abstract

Autophagic pathways have been shown to regulate neural precursor cell (NPC) survival during nervous system development. To determine whether lysosomal dysfunction leads to increased autophagic vacuoles (AVs) and NPC death, we treated mouse cerebellar neural stem cells (C17.2 cell line) and FGF2‐expanded primary cerebellar NPCs with lysosomal dysfunction inducing agents chloroquine (CQ), bafilomycin A1 (Baf A1), and ammonium chloride (NH4Cl). CQ, NH4Cl, and Baf A1 treated NPCs underwent apoptosis, as evidenced by cytological features and cleaved caspase‐3 immunoreactivity. In comparison to wild‐type NPCs, Bax‐deficient NPCs exhibited significantly less caspase‐3 activation and cell death in response to lysosomal dysfunction inducing agents. These agents also produced increased levels of autophagy‐associated proteins LC3‐II and Beclin1. Cycloheximide, a protein synthesis inhibitor, attenuated AV formation in response to CQ as evidenced by decreased LC3‐II. However, cycloheximide had no effect on caspase‐3 activation or cell death indicating that AV accumulation is not necessary for CQ‐induced NPC death. p53‐deficient NPCs showed significant protection from CQ‐induced death compared to wild‐type NPCs. In total, our data demonstrate that lysosomal dysfunction leads to AV accumulation as well as p53‐ and Bax‐ dependent NPC death.