Autophagic Death of Adult Hippocampal Neural Stem Cells Following Insulin Withdrawal

Abstract

Novel therapeutic approaches using stem cell transplantation to treat neurodegenerative diseases have yielded promising results. However, survival of stem cells after transplantation has been very poor in animal models, and considerable efforts have been directed at increasing the viability of engrafted stem cells. Therefore, understanding the mechanisms that regulate survival and death of neural stem cells is critical to the development of stem cell-based therapies. Hippocampal neural (HCN) stem cells derived from the adult rat brain undergo cell death following insulin withdrawal, which is associated with downregulation of antiapoptotic Bcl-2 family members. To understand the type of cell death in HCN cells following insulin withdrawal, apoptosis markers were assessed. Of note, DNA fragmentation or caspase-3 activation was not observed, but rather dying cells displayed features of autophagy, including increased expression of Beclin 1 and the type II form of light chain 3. Electron micrographs showed the dramatically increased formation of autophagic vacuoles with cytoplasmic contents. Staurosporine induced robust activation of caspase-3 and nucleosomal DNA fragmentation, suggesting that the machinery of apoptosis is intact in HCN cells despite the apparent absence of apoptosis following insulin withdrawal. Autophagic cell death was suppressed by knockdown of autophagy-related gene 7, whereas promotion of autophagy by rapamycin increased cell death. Taken together, these data demonstrate that HCN cells undergo a caspase-independent, autophagic cell death following insulin withdrawal. Understanding the mechanisms governing autophagy of adult neural stem cells may provide novel strategies to improve the survival rate of transplanted stem cells for treatment of neurodegenerative diseases.

Insulin withdrawal-induced cell death in adult hippocampal neural stem cells as a model of autophagic cell death

“…13 Insulin withdrawal-induced HCN cell death was characterized by multiple complementary assays, e.g., cell morphology, propidium iodide (PI) staining, cell viability assay dependent on mitochondrial metabolic activity, and cell proliferation assay employing BrdU incorporation. 13 Insulin withdrawal typically induces more than 50% of cell death at day 3, when assessed by PI staining. Interestingly, even after 3 days of insulin withdrawal caspases-3 activation or chromosomal DNA fragmentation is not observed.…”

Autophagy Provides Nutrients but Can Lead to Chop-dependent Induction of Bim to Sensitize Growth Factor–deprived Cells to Apoptosis

“…Autophagy may also promote survival by clearance of damaged organelles or protein aggregates in stressed cells (Abedin et al, 2007;Colell et al, 2007). Alternately, autophagy can induce cell death and autophagic death has been observed in cells in which caspases have been inhibited (Yu et al, 2004) and has also been implicated in the death of Drosophila larval salivary glands (Lee and Baehrecke, 2001), the elimination of cells in the inner cell mass of blastocysts (Qu et al, 2007), and the death of insulin-deprived neural stem cells (Yu et al, 2008).The Bcl-2 family of proteins regulate cell death upon growth factor withdrawal and is critical in the regulation and function of both apoptosis and autophagy. Anti-apoptotic Bcl-2 family proteins such as Bcl-2 and Bcl-xL are up-regulated by growth factors and are frequently overexpressed in cancer (Walensky, 2006).…”

Increased leptin by hypoxic-preconditioning promotes autophagy of mesenchymal stem cells and protects them from apoptosis

“…However, as previous studies reported, the expression of beclin1 is augmented significantly when autophagy is induced [22,27]. This discrepancy may be due to the difference in cell types, stimuli, and duration of treatment applied to individual studies.…”