Fas-apoptotic inhibitory molecule 2 localizes to the lysosome and facilitates autophagosome-lysosome fusion through the LC3 interaction region motif-dependent interaction with LC3.

Fas-apoptotic inhibitory molecule 2 (FAIM2) is a member of the transmembrane BAX inhibitor motif-containing (TMBIM) family. TMBIM family is comprised of six anti-apoptotic proteins that suppress cell death by regulating endoplasmic reticulum Ca2+ homeostasis. Recent studies have implicated two TMBIM proteins, GRINA and BAX Inhibitor-1, in mediating cytoprotection via autophagy. However, whether FAIM2 plays a role in autophagy has been unknown. Here we show that FAIM2 localizes to the lysosomes at basal state and facilitates autophagy through interaction with microtubule-associated protein 1 light chain 3 proteins in human neuroblastoma SH-SY5Y cells. FAIM2 overexpression increased autophagy flux, while autophagy flux was impaired in shRNA-mediated knockdown (shFAIM2) cells, and the impairment was more evident in the presence of rapamycin. In shFAIM2 cells, autophagosome maturation through fusion with lysosomes was impaired, leading to accumulation of autophagosomes. A functional LC3-interacting region motif within FAIM2 was essential for the interaction with LC3 and rescue of autophagy flux in shFAIM2 cells while LC3-binding property of FAIM2 was dispensable for the anti-apoptotic function in response to Fas receptor-mediated apoptosis. Suppression of autophagosome maturation was also observed in a null mutant of Caenorhabditis elegans lacking xbx-6, the ortholog of FAIM2. Our study suggests that FAIM2 is a novel regulator of autophagy mediating autophagosome maturation through the interaction with LC3.

Chronic restraint stress induces hippocampal memory deficits by impairing insulin signaling.

Chronic stress is a psychologically significant factor that impairs learning and memory in the hippocampus. Insulin signaling is important for the development and cognitive function of the hippocampus. However, the relation between chronic stress and insulin signaling at the molecular level is poorly understood. Here, we show that chronic stress impairs insulin signaling in vitro and in vivo, and thereby induces deficits in hippocampal spatial working memory and neurobehavior. Corticosterone treatment of mouse hippocampal neurons in vitro caused neurotoxicity with an increase in the markers of autophagy but not apoptosis. Corticosterone treatment impaired insulin signaling from early time points. As an in vivo model of stress, mice were subjected to chronic restraint stress. The chronic restraint stress group showed downregulated insulin signaling and suffered deficits in spatial working memory and nesting behavior. Intranasal insulin delivery restored insulin signaling and rescued hippocampal deficits. Our data suggest that psychological stress impairs insulin signaling and results in hippocampal deficits, and these effects can be prevented by intranasal insulin delivery.

Phosphorylation of p62 by AMP-activated protein kinase mediates autophagic cell death in adult hippocampal neural stem cells.

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.

Control of adult neurogenesis by programmed cell death in the mammalian brain.

The presence of neural stem cells (NSCs) and the production of new neurons in the adult brain have received great attention from scientists and the public because of implications to brain plasticity and their potential use for treating currently incurable brain diseases. Adult neurogenesis is controlled at multiple levels, including proliferation, differentiation, migration, and programmed cell death (PCD). Among these, PCD is the last and most prominent process for regulating the final number of mature neurons integrated into neural circuits. PCD can be classified into apoptosis, necrosis, and autophagic cell death and emerging evidence suggests that all three may be important modes of cell death in neural stem/progenitor cells. However, the molecular mechanisms that regulate PCD and thereby impact the intricate balance between self-renewal, proliferation, and differentiation during adult neurogenesis are not well understood. In this comprehensive review, we focus on the extent, mechanism, and biological significance of PCD for the control of adult neurogenesis in the mammalian brain. The role of intrinsic and extrinsic factors in the regulation of PCD at the molecular and systems levels is also discussed. Adult neurogenesis is a dynamic process, and the signals for differentiation, proliferation, and death of neural progenitor/stem cells are closely interrelated. A better understanding of how adult neurogenesis is influenced by PCD will help lead to important insights relevant to brain health and diseases.

Autophagy for the quality control of adult hippocampal neural stem cells.

Autophagy plays an important role in neurodegeneration, as well as in normal brain development and function. Recent studies have also implicated autophagy in the regulation of stemness and neurogenesis in neural stem cells (NSCs). However, little is known regarding the roles of autophagy in NSC biology. It has been shown that in addition to cytoprotective roles of autophagy, pro-death autophagy, or ׳autophagic cell death (ACD),' regulates the quantity of adult NSCs. A tight regulation of survival and death of NSCs residing in the neurogenic niches through programmed cell death (PCD) is critical for maintenance of adult neurogenesis. ACD plays a primary role in the death of adult hippocampal neural stem (HCN) cells following insulin withdrawal. Despite the normal apoptotic capability of HCN cells, they are committed to death by autophagy following insulin withdrawal, suggesting the existence of a unique regulatory program that controls the mode of cell death. We propose that dual roles of autophagy for maintenance of NSC pluripotency, as well as for elimination of defective NSCs, may serve as a combined NSC quality control mechanism. This article is part of a Special Issue entitled SI:Autophagy.

Valosin-containing protein is a key mediator between autophagic cell death and apoptosis in adult hippocampal neural stem cells following insulin withdrawal.

BACKGROUND: Programmed cell death (PCD) plays essential roles in the regulation of survival and function of neural stem cells (NSCs). Abnormal regulation of this process is associated with developmental and degenerative neuronal disorders. However, the mechanisms underlying the PCD of NSCs remain largely unknown. Understanding the mechanisms of PCD in NSCs is crucial for exploring therapeutic strategies for the treatment of neurodegenerative diseases. RESULT: We have previously reported that adult rat hippocampal neural stem (HCN) cells undergo autophagic cell death (ACD) following insulin withdrawal without apoptotic signs despite their normal apoptotic capabilities. It is unknown how interconnection between ACD and apoptosis is mediated in HCN cells. Valosin-containing protein (VCP) is known to be essential for autophagosome maturation in mammalian cells. VCP is abundantly expressed in HCN cells compared to hippocampal tissue and neurons. Pharmacological and genetic inhibition of VCP at basal state in the presence of insulin modestly impaired autophagic flux, consistent with its known role in autophagosome maturation. Of note, VCP inaction in insulin-deprived HCN cells significantly decreased ACD and down-regulated autophagy initiation signals with robust induction of apoptosis. Overall autophagy level was also substantially reduced, suggesting the novel roles of VCP at initial step of autophagy. CONCLUSION: Taken together, these data demonstrate that VCP may play an essential role in the initiation of autophagy and mediation of crosstalk between ACD and apoptosis in HCN cells when autophagy level is high upon insulin withdrawal. This is the first report on the role of VCP in regulation of NSC cell death. Elucidating the mechanism by which VCP regulates the crosstalk of ACD and apoptosis will contribute to understanding the molecular mechanism of PCD in NSCs.