Abstract
In the adult brain, programmed death of neural stem cells is considered to be critical for tissue homeostasis and cognitive function and is dysregulated in neurodegeneration. Previously, we have reported that adult rat hippocampal neural (HCN) stem cells undergo autophagic cell death (ACD) following insulin withdrawal. Because the apoptotic capability of the HCN cells was intact, our findings suggested activation of unique molecular mechanisms linking insulin withdrawal to ACD rather than apoptosis. Here, we report that phosphorylation of autophagy-associated protein p62 by AMP-activated protein kinase (AMPK) drives ACD and mitophagy in HCN cells. Pharmacological inhibition of AMPK or genetic ablation of the AMPK α2 subunit by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing suppressed ACD, whereas AMPK activation promoted ACD in insulin-deprived HCN cells. We found that following insulin withdrawal AMPK phosphorylated p62 at a novel site, Ser-293/Ser-294 (in rat and human p62, respectively). Phosphorylated p62 translocated to mitochondria and induced mitophagy and ACD. Interestingly, p62 phosphorylation at Ser-293 was not required for staurosporine-induced apoptosis in HCN cells. To the best of our knowledge, this is the first report on the direct phosphorylation of p62 by AMPK. Our data suggest that AMPK-mediated p62 phosphorylation is an ACD-specific signaling event and provide novel mechanistic insight into the molecular mechanisms in ACD.
Highlights
In the adult brain, programmed death of neural stem cells is considered to be critical for tissue homeostasis and cognitive function and is dysregulated in neurodegeneration
To corroborate these results derived from the pharmacological inhibition of AMPK, we knocked out AMPK ␣1 or ␣2 subunit using the clustered regularly interspaced short palindromic repeats (CRISPR)/ Cas9 gene editing approach
Our previous observations that insulin withdrawal drives the mode of cell death toward autophagic cell death (ACD) rather than apoptosis suggest the existence of uniquely programmed cell death mechanisms in hippocampal neural (HCN) cells [17,18,19,20]
Summary
In the adult brain, programmed death of neural stem cells is considered to be critical for tissue homeostasis and cognitive function and is dysregulated in neurodegeneration. We have reported that adult rat hippocampal neural (HCN) stem cells undergo autophagic cell death (ACD) following insulin withdrawal. Because the apoptotic capability of the HCN cells was intact, our findings suggested activation of unique molecular mechanisms linking insulin withdrawal to ACD rather than apoptosis. According to the criteria suggested by Shen and Codogno [16], ACD is distinguished from other modes of PCD by the lack of apoptotic features and ineffectiveness of caspase inhibition, increased autophagic flux, and dependence of cell death on autophagy-related genes (Atgs) or other key autophagy genes. We further report that AMPK-mediated phosphorylation of p62 is required for mitophagy and ACD following insulin withdrawal but not for apoptotic death in HCN cells. Our study suggests that novel AMPK-mediated p62 phosphorylation can be an ACD-specific signaling event and that this AMPK–p62 axis drives ACD and mitophagy in HCN cells. Our findings warrant further studies on the molecular mechanisms that link autophagy to cell death; understanding these mechanisms is required to distinguish the role of autophagy in facilitation of cell death from its general protective role
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