ALTERNATIVE DEATH PATHWAYS IN NEURAL STEM CELLS

Identifying the molecular pathways that regulate neural stem cell (NSC) death is important for understanding both normal nervous system development and neurodegenerative diseases. Autophagy is a normally occurring cellular process that is responsible for degrading long lived proteins and damaged organelles. Autophagic death is morphologically defined by the accumulation of autophagic vacuoles in the cytoplasm of degenerating cells. Chloroquine (CQ), a weak base, causes lysosomal dysfunction by increasing there pH resulting in decreased protease activity. In our current studies we utilized C17.2 cells, a mouse cerebellar neural stem cell line, and FGF2 expanded primary cerebellar neural precursor cells (NPCs) to dissect the molecular pathways associated with CQ‐induced NSC death. CQ treated C17.2 cells underwent apoptosis, as evidenced by nuclear morphology and cleaved caspase‐3 immunoreactivity (IR). Pharmacological inhibition of caspases with BAF, a broad spectrum caspase inhibitor, attenuated CQ‐induced death in both C17.2 cells and FGF2 expanded NPCs. LC3 IR appears diffuse under normal conditions and becomes clumped in appearance when associated with autophagic vacuoles. Robust clumped LC3 IR was observed in CQ treated C17.2 cells but not in untreated cells. p53 is relatively highly expressed in NSCs where it critically regulates NSC proliferation and death. Immunocytochemical analysis showed an increase in both cytosolic and nuclear p53 IR after CQ treatment. In addition, western blot analysis revealed increased phosphorylation and total levels of p53 in CQ treated C17.2 whole cell lysates. Our data suggests that p53 activation may regulate both autophagic and apoptotic death pathways in NSCs. This work was supported by NIH Grant NS35107 and NS41962 (KAR), and the UAB Civitan International Research Center (KCW).