Valosin-Containing Protein (VCP)/p97 is a key mediator between autopahgic cell death and apoptosis in adult hippocampal neural stem cells followin insulin withdrawal

Programmed cell death (PCD) plays essential roles in regulation of survival and function of neural stem cells (NSCs). Abnormal regulation of this process is associated with aging and neurodegenerative diseases. However, the mechanisms underlying the PCD of NSCs remain largely unknown. Therefore, understanding the mechanism of PCD in NSCs is crucial for exploring therapeutic strategy for the treatment of neurodegenerative diseases. 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 insulin-deprived HCN cells. Valosin-containing protein (VCP)/p97 is known to be essential for autophagosome maturation in mammalian cells. In this study, we report that VCP regulates the rate of autophagic flux in HCN cells following insulin withdrawal, suggesting the novel roles of VCP at other steps of autophagy as well as maturation. Particularly, VCP is expressed abundantly in HCN cells compared to hippocampal tissue and neurons. Pharmacological and genetic inhibition of VCP significantly decreased ACD and autophagy markers, while apoptotic cell death was induced in insulin-depleted HCN cells. Taken together, these data demonstrate that VCP may play an essential role in completion of ACD and mediation of crosstalk between ACD and apoptosis in HCN cells following insulin withdrawal. Elucidating the mechanism by which VCP regulates the crosstalk of ACD and apoptosis will contribute to understanding the molecular mechanism of PCD in NSCs. ⓒ 2015 DGIST1. INTRODUCTION 1 -- 2. Material and methods 3-- 2.1 Antibodies and reagents 3 -- 2.2 Cell culture 3 -- 2.3 Plasmids, siRNA and Transfection 3 -- 2.4 Cell death assay 4 -- 2.5 Western blotting 4 -- 2.6 Quantitative reverse transcription polymerase chain reaction (qRT-PCR) 5 -- 2.7 Annexin V staining and flow cytometry analysis 5 -- 2.8 Statistic analysis 5 -- 3. Results 6-- 3.1 VCP is degraded through autophagy in HCN cells following insulin withdrawal 6 -- 3.2 Inhibition of VCP switched autophagic cell death to apoptosis in insulin depleted HCN cells 6 -- 3.3 Inhibition of VCP significantly reduced autophagic flux in HCN cells following insulin withdrawal 7 -- 3.4 VCP regulates autophagy initiation signaling in insulin-deprived HCN cells 8 -- 4. Discussion 9 -- 5. Figure legends 11 -- 6. Figures 13 -- References 22 -- Abstract in Korean 24 -- Acknowledgement 25Programmed cell death (PCD)는 신경줄기세포의 생존과 기능조절에 필수적인 역할을 한다. 따라서 신경줄기세포의 비정상적인 PCD는 노화와 퇴행성 질환에 밀접한 관련이 있다고 보고되고 있다. 그러나, 신경줄기세포의 세포사멸기작에 대한 이해는 여전히 많은 부분이 밝혀져 있지 않아신경 줄기세포를 이용한 퇴행성 치료기술개발을 위한 큰 학문적 난제로 남아 있다. 그러므로, 신경줄기 세포에서 일어나는 PCD의 기작을 이해 하는 것은 노화와 퇴행성 질환의 치료 탐색을 위해 중요하다. 우리는 선행연구를 통해 성인 쥐의 해마성체신경줄기세포가 인슐린 결핍 조건에서 apoptotic 대신 순수한 autophagic cell death (ACD)를 겪는다는 것을 입증하였다. 성체해마신경줄기 세포는 정상적인 apoptosis 능력을 가지고 있기 때문에 인슐린이 결핍된 해마성체신경줄기세포에서왜 특이적으로 ACD가 유도되는지, 그리고 ACD와 apoptosis간의 상관관계는 어떻게 조절되는지는 아직 알려지지 않았다. Valosin-containing protein (VCP)는 포유류의 세포에서 autophagosome maturation에 필수적인 단백질이라고 알려져 있다. 본 연구에서는VCP가 인슐린이 결핍된 해마성체 신경줄기세포에서 autophagic flux의 속도를 조절한다는 것을 밝혔으며, 이는 autophagosome maturation뿐만 아니라 autophagy initiation을 포함한 ACD의 다른 과정에서 VCP의 새로운 역할들을 제시한다. 특이적으로, VCP는 hippocampal tissue와 hippocampal neuron 비해 해마성체신경줄기세포에 많이 발현했다. VCP의 약리학적 유전적인 억제는 인슐린이 결핍된 해마성체신경줄기세포에서 apoptosis가 유발 되는 동안 ACD와 autophagy markers를 감소시킴을 관찰하였다. 이러한 결과들은 VCP가 인슐린이 결핍된 해마성체신경줄기세포에서 ACD 유도와 ACD와 apoptosis 사이의 상호작용 조절에 중요한 역할을 할 것이라는 것을 입증하였다. VCP가 ACD와 apoptosis간의 상호작용을 조절하는 기작을 입증하는 것은 신경줄기세포에서 PCD의 분자적 기작을 이해하는데 도움을 줄 것이라 사료된다. ⓒ 2015 DGISTMasterdCollectio

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

Outcome of neonatal palliative procedure for pulmonary atresia with ventricular septal defect or tetralogy of Fallot with severe pulmonary stenosis: experience in a single tertiary center

Purpose The present study aimed to evaluate progression and prognosis according to the palliation method used in neonates and early infants aged 3 months or younger who were diagnosed with pulmonary atresia with ventricular septal defect (PA VSD) or tetralogy of Fallot (TOF) with severe pulmonary stenosis (PS) in a single tertiary hospital over a period of 12 years. Methods Twenty with PA VSD and 9 with TOF and severe PS needed initial palliation. Reintervention after initial palliation, complete repair, and progress were reviewed retrospectively. Results Among 29 patients, 14 patients underwent right ventricle to pulmonary artery (RV-PA) connection, 11 palliative BT shunt, 2 central shunt, and 2 ductal stent insertion. Median age at the initial palliation was 13 days (1–98 days). Additional procedure for pulmonary blood flow was required in 5 patients; 4 additional BT shunt operations and 1 RV-PA connection. There were 2 early deaths among patients with RV-PA connection, one from RV failure and the other from severe infection. Finally, 25 patients (86%) had a complete repair. Median age of total correction was 12 months (range, 2–31 months). At last follow-up, 2 patients had required reintervention after total correction; 1 conduit replacement and 1 right ventricular outflow tract (RVOT) patch enlargements. Conclusion For initial palliation of patients with PA VSD or TOF with severe PS, not only shunt operation but also RV-PA connection approach can provide an acceptable outcome. To select the most proper surgical strategy, we recommend thorough evaluation of cardiac anomalies such as RVOT and PA morphologies and consideration of the patient’s condition

GSK3B induces autophagy by phosphorylating ULK1

Unc-51-like autophagy activating kinase 1 (ULK1), a mammalian homolog of the yeast kinase Atg1, has an essential role in autophagy induction. In nutrient and growth factor signaling, ULK1 activity is regulated by various posttranslational modifications, including phosphorylation, acetylation, and ubiquitination. We previously identified glycogen synthase kinase 3 beta (GSK3B) as an upstream regulator of insulin withdrawal-induced autophagy in adult hippocampal neural stem cells. Here, we report that following insulin withdrawal, GSK3B directly interacted with and activated ULK1 via phosphorylation of S405 and S415 within the GABARAP-interacting region. Phosphorylation of these residues facilitated the interaction of ULK1 with MAP1LC3B and GABARAPL1, while phosphorylation-defective mutants of ULK1 failed to do so and could not induce autophagy flux. Furthermore, high phosphorylation levels of ULK1 at S405 and S415 were observed in human pancreatic cancer cell lines, all of which are known to exhibit high levels of autophagy. Our results reveal the importance of GSK3B-mediated phosphorylation for ULK1 regulation and autophagy induction and potentially for tumorigenesis. © 2021, The Author(s).1

Valosin-containing protein (VCP)/p97 promotes autophagic cell death and prevents apoptosis in adult hippocampal neural stem cells under corticosterone treatment

Autophagic cell death; Apoptosis; Adult neural stem cells; Corticosterone; Valosin-containing protein; Calpain 2; p38DoctordCollectio

Valosin-containing protein (VCP): structure, functions, and implications in neurodegenerative diseases

Valosin-containing protein (VCP) is a hexameric protein belonging to the type II AAA+ (ATPases Associated with diverse cellular Activities) protein family. VCP governs multiple cellular processes and its diverse functions are determined by its interaction with a wide variety of partners and cofactors. Recently, mutations in VCP were suggested to cause inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia, amyotrophic lateral sclerosis, and Huntington’s disease. However, the pathogenic mechanisms of VCP mutations in these diseases are still largely unknown. In this review, we summarize the structure and cellular functions of VCP, especially focusing on apoptosis and two major cellular degradation pathways, the ubiquitin–proteasome system and autophagy. We also list the representative VCP mutations and discuss their potential association with neurodegenerative diseases. © 2016 Korean Society for Integrative Biology.FALS

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

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

FAIM2 stability controls the balance of cell death and survival in adult neural stem cells by regulating autophagy

Adult neural stem cells maintain self-renewal capacity and multipotency in the adult brain, thus regulating the output of neurogenesis. Autophagy thought to play a pro-survival role in the cells can kill cells as we have previously shown that autophagic cell death (ACD) is a dominant programmed cell death mode in adult hippocampal neural stem cells subjected to insulin withdrawal in vitro. In postnatal mouse hippocampus, Fas apoptotic inhibitory molecule 2 (FAIM2) gradually increases with age. We found that FAIM2 is expressed in adult hippocampal neural stem cells. Decrease in FAIM2 protein is accompanied by ACD following insulin withdrawal. Prevention of protein synthesis by cyclohexamide showed shorter half-life of FAIM2 in ACD in comparison to that in basal state. Indeed, FAIM2 was post-translationally degraded via autophago-lysosomal pathway. FAIM2 knockdown via siRNA augmented cell death and increased autophagy markers. In summary, our result suggests that FAIM2 protein stability is a critical determinant of neural stem cells’ susceptibility to ACD. Our results expand the role of FAIM2, which was previously established as a neuron-specific anti-apoptotic protein, to regulation of autophagy in neural stem cells