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. © 2016 Yeo et al.1

Parkin Promotes Mitophagic Cell Death in Adult Hippocampal Neural Stem Cells Following Insulin Withdrawal

Regulated cell death (RCD) plays a fundamental role in human health and disease. Apoptosis is the best-studied mode of RCD, but the importance of other modes has recently been gaining attention. We have previously demonstrated that adult rat hippocampal neural stem (HCN) cells undergo autophagy-dependent cell death (ADCD) following insulin withdrawal. Here, we show that Parkin mediates mitophagy and ADCD in insulin-deprived HCN cells. Insulin withdrawal increased the amount of depolarized mitochondria and their colocalization with autophagosomes. Insulin withdrawal also upregulated both mRNA and protein levels of Parkin, gene knockout of which prevented mitophagy and ADCD. c-Jun is a transcriptional repressor of Parkin and is degraded by the proteasome following insulin withdrawal. In insulin-deprived HCN cells, Parkin is required for Ca2+ accumulation and depolarization of mitochondria at the early stages of mitophagy as well as for recognition and removal of depolarized mitochondria at later stages. In contrast to the pro-death role of Parkin during mitophagy, Parkin deletion rendered HCN cells susceptible to apoptosis, revealing distinct roles of Parkin depending on different modes of RCD. Taken together, these results indicate that Parkin is required for the induction of ADCD accompanying mitochondrial dysfunction in HCN cells following insulin withdrawal. Since impaired insulin signaling is implicated in hippocampal deficits in various neurodegenerative diseases and psychological disorders, these findings may help to understand the mechanisms underlying death of neural stem cells and develop novel therapeutic strategies aiming to improve neurogenesis and survival of neural stem cells

Dasatinib regulates LPS-induced microglial and astrocytic neuroinflammatory responses by inhibiting AKT/STAT3 signaling

Background: The FDA-approved small-molecule drug dasatinib is currently used as a treatment for chronic myeloid leukemia (CML). However, the effects of dasatinib on microglial and/or astrocytic neuroinflammatory responses and its mechanism of action have not been studied in detail. Methods: BV2 microglial cells, primary astrocytes, or primary microglial cells were treated with dasatinib (100 or 250 nM) or vehicle (1% DMSO) for 30 min or 2 h followed by lipopolysaccharide (LPS; 200 ng/ml or 1 μg/ml) or PBS for 5.5 h. RT-PCR, real-time PCR; immunocytochemistry; subcellular fractionation; and immunohistochemistry were subsequently conducted to determine the effects of dasatinib on LPS-induced neuroinflammation. In addition, wild-type mice were injected with dasatinib (20 mg/kg, intraperitoneally (i.p.) daily for 4 days or 20 mg/kg, orally administered (p.o.) daily for 4 days or 2 weeks) or vehicle (4% DMSO + 30% polyethylene glycol (PEG) + 5% Tween 80), followed by injection with LPS (10 mg/kg, i.p.) or PBS. Then, immunohistochemistry was performed, and plasma IL-6, IL-1β, and TNF-α levels were analyzed by ELISA. Results: Dasatinib regulates LPS-induced proinflammatory cytokine and anti-inflammatory cytokine levels in BV2 microglial cells, primary microglial cells, and primary astrocytes. In BV2 microglial cells, dasatinib regulates LPS-induced proinflammatory cytokine levels by regulating TLR4/AKT and/or TLR4/ERK signaling. In addition, intraperitoneal injection and oral administration of dasatinib suppress LPS-induced microglial/astrocyte activation, proinflammatory cytokine levels (including brain and plasma levels), and neutrophil rolling in the brains of wild-type mice. Conclusions: Our results suggest that dasatinib modulates LPS-induced microglial and astrocytic activation, proinflammatory cytokine levels, and neutrophil rolling in the brain. © 2019 The Author(s).1

Chronic Restraint Stress Induces Hippocampal Memory Deficits by Impairing Insulin Signaling

세포의 항상성은 세포의 생존과 사멸 간의 균형있는 조절을 통해 유지되며, 이를 잘 조절하여 다양한 신경퇴행성 질환들을 개선하기위한 보다 나은 치료를 제시할 수 있다. 많은 신경퇴행성 질환을 가진 환자들은 인슐린의 감소와 학습 및 기억력의 감퇴를 보인다. 이를 관장하는 해마는 인슐린 저항성이나 감소에 가장 민감하게 반응하는 기관이다. 스트레스는 학습 및 기억에 영향을 미칠 수 있는 주요한 원인 중 하나이다. 이러한 학습 및 기억은 해마에 의해 통제 받고 있으며, 해마의 발달 및 기능은 인슐린 신호전달 경로에 의해 주로 조절을 받는다. 하지만, 해마에 영향을 미칠 수 있는 스트레스와 인슐린 신호전달 경로 사이에 연관된 분자적 수준의 메커니즘은 잘 알려져 있지않다. 이번 연구는 만성적인 스트레스와 해마의 기능 간에 인슐린 신호전달 경로가 중요하게 작용하는지를 밝혀냈다. 스트레스는 세포수준에서 인슐린 신호전달 경로를 손상시키고 자가포식현상을 일으켰다. 또한, 만성적인 스트레스를 받은 마우스에서 해마 내 인슐린 신호전달 경로가 손상되어 해마의 기능이 떨어진다는 결과를 인슐린 신호전달 경로에 관여하는 물질들의 변화와 인지 및 기억 능력의 저하를 확인하는 행동실험을 통해 밝혀냈다. 또한 코를 통해 인슐린을 흡입시키는 방법을 통해 인슐린 신호전달 경로의 회복 및 해마의 기능 회복을 확인함으로써, 마우스에서 만성적 스트레스가 인슐린 신호전달 경로에 손상을 일으켜 해마의 기능을 떨어뜨린다는 연구결과를 다시 한번 뒷받침하였다. 배양한 해마 신경줄기세포와 신경세포에 스트레스 호르몬인 코티코스테론을 처리하였을 때, 이 세포들에서 인슐린 신호전달 경로의 활성을 나타내는 대표 물질들(IRS, Akt, mTOR)이 비활성화 및 억제되어 인슐린 신호전달 경로가 저해되는 것을 확인하였고, 이때 이 세포들은 자가포식현상을 보이며 죽었다. 마우스에 만성적 스트레스를 주었을 때, 높아진 혈액 내 코티코스테론 농도 및 체중 감소를 통해 마우스가 충분히 스트레스를 받고있는 상황임을 확인하였다. 또한 해마의 기능 이상이 있을 때 나타나는 보금자리를 만들지 않는 결과를 보였다. 만성적 스트레스는 해마 내 인슐린 신호전달 경로에 관여하는 주요 물질들(IRS, Akt, mTOR)의 활성을 억제하여 인슐린 신호전달 경로를 저해했으며, 마우스 해마 신경세포 배양에서 코티코스테론을 처리했을 때와 마찬가지로 만성적 스트레스 또한 해마에서의 자가포식현상을 보였다. 해마의 공간 인지능력 및 기억력을 확인할 수 있는 Y형 미로를 이용한 실험을 통하여 만성적 스트레스를 받은 마우스들이 인지 및 기억과 관계된 해마의 기능 장애를 보임을 확인하였다. 만성적 스트레스가 해마 내 인슐린 신호전달 경로와 인지 및 기억 능력을 저하시킴을 확인 후, 인슐린 신호전달 경로가 주된 메커니즘임을 확인하기위해 코를 통해 외부에서 인슐린을 다시 흡입시키는 회복실험을 진행하였다. 우선 형광물질이 달린 인슐린을 이용해 코로의 흡입을 통해 해마에 인슐린에 달린 형광물질이 해마에 잘 도달함을 확인하였다. 이후 코를 통해 인슐린이 주입된 마우스들은 만성적 스트레스를 받았음에도 불구하고 체중 감소 및 보금자리를 만들지 않는 등의 결과를 보이지 않았다. 또한, 해마 내 인슐린 신호전달 물질들의 억제되었던 활성도 회복되었으며, Y형 미로를 이용한 실험에서도 회복된 공간 인지능력 및 기억력을 보였다. 즉, 만성적 스트레스는 혈중 코티코스테론 농도가 증가시키고, 인슐린 신호전달 경로를 손상시켜 해마 내 자가포식현상에 의해 해마의 기능이 떨어뜨리는데, 코를 통한 인슐린의 흡입은 이런 스트레스에 의한 인슐린 신호전달 경로의 손상을 회복시켜 해마의 기능 또한 회복시킴을 확인할 수 있다. 이번 연구를 통해 만성적 스트레스와 해마의 기능 간에 인슐린 신호전달 경로가 중요하게 작용을 하며, 코를 통해 인슐린을 흡입하였을 때 해마 기능이 회복됨을 보임으로써 신경정신적 질병 및 신경퇴행성 질환들에 대한 쉽고 새로운 치료 방법을 제시하였다.|Normal brain development and tissue homeostasis are achieved by balancing cell survival and death, and it has been recognized that the proper regulation of programmed cell death is an important cellular process needed to take into account for better therapeutic design to treat various neurodegenerative diseases and neurological conditions. Hippocampus is responsible for certain functions of learning and memory, and previous studies have shown an association of reduction in insulin levels and decline in learning and memory functions in hippocampus during neurodegeneration. Hippocampus is one of the most vulnerable targets of insulin signaling impairment or insulin resistance. Insulin signaling plays a key role for the memory and cognitive function of the hippocampus, and chronic stress is a psychologically significant factor that impairs learning and memory in the hippocampus. However, the relation between insulin signaling and chronic stress is poorly understood. Our group previously established that insulin withdrawal induces autophagic cell death in adult hippocampal neural stem (HCN) cells. We also recently observed an induction of autophagic cell death by psychological stress. So we set to explore the relation between chronic stress and insulin signaling with the hypothesis that insulin signaling impairment may be involved in stress-induced hippocampal memory and cognitive function deficits. In this thesis, we report that chronic stress impairs insulin signaling and induces autophagy in vitro and in vivo, and thereby causes deficits in hippocampal spatial working memory and neurobehavior. Corticosterone (CORT) is a major stress hormone in rodent, and CORT treatment in HCN cells and mouse hippocampal neurons in vitro caused neurotoxicity with the features of the autophagy but not apoptosis. CORT impaired insulin signaling from early time points in HCN cells and mouse hippocampal neurons in vitro, as shown by reduction in the phosphorylation levels of IRS-1 on Y608, Akt on S473, and mTOR on S2448, and increase in the expression of IRβ. As an in vivo model of stress, mice were subjected to chronic restraint stress (CRS) for 14 days with immobilization of 6 h daily. CRS downregulated insulin signaling in a similar manner as CORT. CRS decreased the phosphorylation levels of IRS-1 on Y608, Akt on S473, and mTOR on S2448. Moreover, CRS caused the deficits in hippocampal spatial working memory and nesting behavior. However, intranasal insulin delivery during the CRS restored insulin signaling and recovered hippocampal function deficits. These data suggested that psychological stress impaired insulin signaling and caused deficits in hippocampal function, and these disorders could be rescued by intranasal insulin delivery. Interestingly, intranasal administration of insulin did not alter the level of CORT increased by CRS, which suggested that the recovery by intranasal insulin delivery was not caused by the alteration of hypothalamic-pituitary-adrenal axis. Finally, the type of hippocampal cell death in vivo was examined. The level of cleaved caspase-3 was not significantly different between control and CRS mouse hippocampus. However, the level of LC3-II, a well-known autophagy marker, was increased by CRS, which was suppressed by intranasal insulin delivery. This data suggested that the neuroprotective activity of insulin might be by suppressing autophagy in the hippocampus against the detrimental effects of CORT released by CRS. Our findings that CRS induces hippocampal memory deficits by impairing insulin signaling pathway and inducing autophagy will be the basis of the future studies in psychological stress-induced neuropathology and the effectiveness of insulin for neuroprotection and cure of hippocampal cognitive dysfunctions.openList of Contents Abstract · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · i Lists of Contents · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·iii Lists of Figures · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·vii Lists of Abbreviations · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·viii I. Introduction 1.1. Mode of Cell Death · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 1 1.1.1. Apoptosis 1.1.2. Autophagy 1.2. Insulin Signaling · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·10 1.2.1. Insulin Signaling Pathway in the Cell 1.2.2. The Role of Insulin Signaling in Hippocampal Functions and Neurodegenerative Diseases 1.3. Stress · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·14 1.3.1. Hypothalamic-Pituitary-Adrenal Axis (HPA Axis) and Stress Hormones 1.3.2. Psychological Stresses II. Materials and Methods 2.1. Materials · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·18 2.1.1. Experimental Animals 2.1.1. Reagents and Antibodies 2.2. Methods · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 19 2.2.1. Animal acclimation 2.2.2. Body weight measurement 2.2.3. Cell death assay 2.2.4. Cell viability assay 2.2.5. Chronic restraint stress (CRS) procedure 2.2.6. Confirmation of intranasal delivery of insulin into the hippocampus using insulin-FITC 2.2.7. Corticosterone (CORT) level measurement 2.2.8. Hippocampal neural stem (HCN) cell culture 2.2.9. Hippocampal neuron culture 2.2.10. Immunocytochemistry 2.2.11. Tissue histology 2.2.12. Intranasal delivery of insulin 2.2.13. Nest building behavior assay 2.2.14. Y-maze assay 2.2.15. Preparation of hippocampal lysates 2.2.16. Western blotting analysis 2.2.17. Statistical analysis III. The Effect of CORT in Insulin Signaling and Cell Death in HCN Cells and Primary Mouse Hippocampal Neurons in vitro. 3.1. Introduction & Hypothesis · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·26 3.2. Results · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·27 3.2.1. CORT is neurotoxic and induces autophagy feature in HCN cells and primary mouse hippocampal neurons in vitro. 3.2.2. CORT impairs insulin signaling from early time points in HCN cells and primary mouse hippocampal neurons in vitro. 3.3. Discussion · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·34 IV. The Effect of CRS in Insulin Signaling in the Hippocampus and Hippocampus Functions in Mice. 4.1. Introduction & Hypothesis · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·37 4.2. Results · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·37 4.2.1. CRS causes stress responses in mice. 4.2.2. CRS impairs insulin signaling in the mouse hippocampus in vivo. 4.2.3. CRS causes the deficits in hippocampal spatial working memory and nesting behavior. 4.3. Discussion · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·43 V. The Effect of Intranasal Insulin Delivery in Insulin Signaling and Hippocampus Functions in CRS Mice. 5.1. Introduction & Hypothesis · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·44 5.2. Results · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·44 5.2.1. Intranasal insulin delivery is successfully confirmed by the detection of insulin-FITC. 5.2.2. Intranasal insulin delivery mitigates the reduced body weight gain in CRS mice. 5.2.3. Intranasal insulin delivery recovers the impairment of insulin signaling in CRS mice. 5.2.4. Intranasal insulin delivery prevents the hippocampal function deficits in CRS mice. 5.3. Discussion · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·51 VI. Conclusion · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 56 References · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 58 Summary (국문요약) · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 67DOCTORdCollectio

Chronic restraint stress induces hippocampal memory deficits by impairing insulin signaling

Abstract 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

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. © 2019 Federation of American Societies for Experimental Biology.1

Electroceutical approach ameliorates intracellular PMP22 aggregation and promotes pro-myelinating pathways in a CMT1A in vitro model

Charcot-Marie-Tooth disease subtype 1A (CMT1A) is one of the most prevalent demyelinating peripheral neuropathies worldwide, caused by duplication of the peripheral myelin protein 22 (PMP22) gene, which is expressed primarily in Schwann cells (SCs). PMP22 overexpression in SCs leads to intracellular aggregation of the protein, which eventually results in demyelination. Unfortunately, previous biochemical approaches have not resulted in an approved treatment for CMT1A disease, compelling the pursuit for a biophysical approach such as electrical stimulation (ES). However, the effects of ES on CMT1A SCs have remained unexplored. In this study, we established PMP22-overexpressed Schwannoma cells as a CMT1A in vitro model, and investigated the biomolecular changes upon applying ES via a custom-made high-throughput ES platform, screening for the condition that delivers optimal therapeutic effects. While PMP22-overexpressed Schwannoma exhibited intracellular PMP22 aggregation, ES at 20 Hz for 1 h improved this phenomenon, bringing PMP22 distribution closer to healthy condition. ES at this condition also enhanced the expression of the genes encoding myelin basic protein (MBP) and myelin-associated glycoprotein (MAG), which are essential for assembling myelin sheath. Furthermore, ES altered the gene expression for myelination-regulating transcription factors Krox-20, Oct-6, c-Jun and Sox10, inducing pro-myelinating effects in PMP22-overexpressed Schwannoma. While electroceuticals has previously been applied in the peripheral nervous system towards acquired peripheral neuropathies such as pain and nerve injury, this study demonstrates its effectiveness towards ameliorating biomolecular abnormalities in an in vitro model of CMT1A, an inherited peripheral neuropathy. These findings will facilitate the clinical translation of an electroceutical treatment for CMT1A. © 2023 Elsevier B.V.FALS

Autophagic death of neural stem cells mediates chronic stress-induced decline of adult hippocampal neurogenesis and cognitive deficits

Macroautophagy/autophagy is generally regarded as a cytoprotective mechanism, and it remains a matter of controversy whether autophagy can cause cell death in mammals. Here, we show that chronic restraint stress suppresses adult hippocampal neurogenesis in mice by inducing autophagic cell death (ACD) of hippocampal neural stem cells (NSCs). We generated NSC-specific, inducible Atg7 conditional knockout mice and found that they had an intact number of NSCs and neurogenesis level under chronic restraint stress and were resilient to stress- or corticosterone-induced cognitive and mood deficits. Corticosterone treatment of adult hippocampal NSC cultures induced ACD via SGK3 (serum/glucocorticoid regulated kinase 3) without signs of apoptosis. Our results demonstrate that ACD is biologically important in a mammalian system in vivo and would be an attractive target for therapeutic intervention for psychological stress-induced disorders. Abbreviations: AAV: adeno-associated virus; ACD: autophagic cell death; ACTB: actin, beta; Atg: autophagy-related; ASCL1/MASH1: achaete-scute family bHLH transcription factor 1; BafA1: bafilomycin A1; BrdU: Bromodeoxyuridine/5-bromo-2ʹ-deoxyuridine; CASP3: caspase 3; cKO: conditional knockout; CLEM: correlative light and electron microscopy; CORT: corticosterone; CRS: chronic restraint stress; DAB: 3,3ʹ–diaminobenzidine; DCX: doublecortin; DG: dentate gyrus; GC: glucocorticoid; GFAP: glial fibrillary acidic protein; HCN: hippocampal neural stem; i.p.: intraperitoneal; MAP1LC3B: microtubule-associated protein 1 light chain 3 beta; MKI67/Ki67: antigen identified by monoclonal antibody Ki 67; MWM: Morris water maze; Nec-1: necrostatin-1; NES: nestin; NR3C1/GR: nuclear receptor subfamily 3, group C, member 1; NSC: neural stem cell; PCD: programmed cell death; PFA: paraformaldehyde; PX: Phox homology; PtdIns3P: phosphatidylinositol-3-phosphate; RBFOX3/NeuN: RNA binding protein, fox-1 homolog (C. elegans) 3; SGK: serum/glucocorticoid-regulated kinases; SGZ: subgranular zone; SOX2: SRY (sex determining region Y)-box 2; SQSTM1: sequestosome 1; STS: staurosporine; TAM: tamoxifen; Ulk1: unc-51 like kinase 1; TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling; VIM: vimentin; WT: wild type; ZFYVE1: zinc finger, FYVE domain containing 1; Z-VAD/Z-VAD-FMK: pan-caspase inhibitor. © 2019, © 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.1

Calpain Determines the Propensity of Adult Hippocampal Neural Stem Cells to Autophagic Cell Death Following Insulin Withdrawal

Programmed cell death (PCD) has significant effects on the function of neural stem cells (NSCs) during brain development and degeneration. We have previously reported that adult rat hippocampal neural stem (HCN) cells underwent autophagic cell death (ACD) rather than apoptosis following insulin withdrawal despite their intact apoptotic capabilities. Here, we report a switch in the mode of cell death in HCN cells with calpain as a critical determinant. In HCN cells, calpain 1 expression was barely detectable while calpain 2 was predominant. Inhibition of calpain in insulin-deprived HCN cells further augmented ACD. In contrast, expression of calpain 1 switched ACD to apoptosis. The proteasome inhibitor lactacystin blocked calpain 2 degradation and elevated the intracellular Ca2+ concentration. In combination, these effects potentiated calpain activity and converted the mode of cell death to apoptosis. Our results indicate that low calpain activity, due to absence of calpain 1 and degradation of calpain 2, results in a preference for ACD over apoptosis in insulin-deprived HCN cells. On the other hand, conditions leading to high calpain activity completely switch the mode of cell death to apoptosis. This is the first report on the PCD mode switching mechanism in NSCs. The dynamic change in calpain activity through the proteasome-mediated modulation of the calpain and intracellular Ca2+ levels may be the critical contributor to the demise of NSCs. Our findings provide a novel insight into the complex mechanisms interconnecting autophagy and apoptosis and their roles in the regulation of NSC death. © 2015 AlphaMed Press.