Abstract
Cytoplasmic Ca2+ actively engages in diverse intracellular processes from protein synthesis, folding and trafficking to cell survival and death. Dysregulation of intracellular Ca2+ levels is observed in various neuropathological states including Alzheimer’s and Parkinson’s diseases. Ryanodine receptors (RyRs) and inositol 1,4,5-triphosphate receptors (IP3Rs), the main Ca2+ release channels located in endoplasmic reticulum (ER) membranes, are known to direct various cellular events such as autophagy and apoptosis. Here we investigated the intracellular Ca2+-mediated regulation of survival and death of adult hippocampal neural stem (HCN) cells utilizing an insulin withdrawal model of autophagic cell death (ACD). Despite comparable expression levels of RyR and IP3R transcripts in HCN cells at normal state, the expression levels of RyRs—especially RyR3—were markedly upregulated upon insulin withdrawal. While treatment with the RyR agonist caffeine significantly promoted the autophagic death of insulin-deficient HCN cells, treatment with its inhibitor dantrolene prevented the induction of autophagy following insulin withdrawal. Furthermore, CRISPR/Cas9-mediated knockout of the RyR3 gene abolished ACD of HCN cells. This study delineates a distinct, RyR3-mediated ER Ca2+ regulation of autophagy and programmed cell death in neural stem cells. Our findings provide novel insights into the critical, yet understudied mechanisms underlying the regulatory function of ER Ca2+ in neural stem cell biology.
Highlights
A subtle balance between cell survival and death is maintained through intricate networks of molecular signaling machinery
Through live-imaging and Western blot analyses we found that absence of insulin gradually induces autophagic response in hippocampal neural stem (HCN) cells as early as 2 h after insulin withdrawal, since live cell imaging with Lysotracker Green and RFP-LC3 at 30’, 60’, 90’, and 120’ showed gradual appearance of LC3 puncta co-localized with the lysosomes (Figure 1A)
While the intracellular Ca2+ level in insulindeprived HCN cells was two-fold greater than control cells, the fluorescence intensity of CEPIA1er revealed that endoplasmic reticulum (ER) Ca2+ levels were halved following insulin withdrawal (Figures 1E,F)
Summary
A subtle balance between cell survival and death is maintained through intricate networks of molecular signaling machinery. Ca2+ is a critical intracellular signal that regulates many cellular processes in development stages of embryo, neuronal proliferation and cognitive ability in the brain (Webb and Miller, 2003; Rosenberg and Spitzer, 2011; Bading, 2013). Due to its versatile nature, molecular mechanisms of Ca2+-dependent signaling exist in all types of mammalian cells, and its malfunction fails networks of intracellular signals that maintain cellular homeostasis and cell survival. The importance of Ca2+ regulation in neuronal cell death has led to the Ca2+ hypothesis in the pathogenesis of Alzheimer’s disease (AD), first proposed by Khachaturian in 1980s (Khachaturian, 1987, 1994). The Ca2+ hypothesis states that the persistent upregulation of Ca2+ signaling in the hippocampus is responsible for neuronal death and progressive decline in cognition and memory (Guo et al, 1996; Palop et al, 2003; Du et al, 2008) commonly observed in AD
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