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• W2724886670 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. 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. Autophagy is an evolutionarily conserved cellular degradation and recycling process characterized by an increased generation of autophagic vesicles (1.Klionsky D.J. Emr S.D. Cell biology—autophagy as a regulated pathway of cellular degradation.Science. 2000; 290: 1717-1721Crossref PubMed Scopus (2969) Google Scholar). Autophagy plays various cytoprotective roles, including degradation of toxic proteins and damaged organelles, and provision of biochemical intermediates for the synthesis of macromolecules and metabolism (2.Lum J.J. DeBerardinis R.J. Thompson C.B. Autophagy in metazoans: cell survival in the land of plenty.Nat. Rev. Mol. Cell Biol. 2005; 6: 439-448Crossref PubMed Scopus (645) Google Scholar). A growing body of evidence also suggests the involvement of autophagy in programmed cell death (PCD), 2The abbreviations used are: PCD, programmed cell death; ACC, acetyl-CoA carboxylase; ACD, autophagic cell death; AMPK, AMP-activated protein kinase; Atg, autophagy-related gene; BafA1, bafilomycin A1; CA, constitutively active; CC, compound C; CCCP, carbonyl cyanide m-chlorophenylhydrazone; CRISPR, clustered regularly interspaced short palindromic repeats; HCN, hippocampal neural stem cells; LC3, microtubule-associated protein light chain 3; STS, staurosporine; ULK1, Unc-51-like kinase 1; Z-VAD, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone; I, insulin; CaMKK, Ca2+/calmodulin-dependent protein kinase kinase; mRFP, monomeric red fluorescent protein; rp62, rat p62; mp62, mouse p62; hp62, human p62; SR-SIM, super-resolution microscopy for structured illumination; mt-mKeima, mitochondrially targeted version of monomeric Keima. 2The abbreviations used are: PCD, programmed cell death; ACC, acetyl-CoA carboxylase; ACD, autophagic cell death; AMPK, AMP-activated protein kinase; Atg, autophagy-related gene; BafA1, bafilomycin A1; CA, constitutively active; CC, compound C; CCCP, carbonyl cyanide m-chlorophenylhydrazone; CRISPR, clustered regularly interspaced short palindromic repeats; HCN, hippocampal neural stem cells; LC3, microtubule-associated protein light chain 3; STS, staurosporine; ULK1, Unc-51-like kinase 1; Z-VAD, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone; I, insulin; CaMKK, Ca2+/calmodulin-dependent protein kinase kinase; mRFP, monomeric red fluorescent protein; rp62, rat p62; mp62, mouse p62; hp62, human p62; SR-SIM, super-resolution microscopy for structured illumination; mt-mKeima, mitochondrially targeted version of monomeric Keima. although its role in PCD remains controversial, and the detailed mechanisms of the mediation of PCD by autophagy remain to be elucidated (3.Codogno P. Meijer A. Autophagy and signaling: their role in cell survival and cell death.Cell Death Differ. 2005; 12: 1509-1518Crossref PubMed Scopus (946) Google Scholar). In terms of the prodeath role of autophagy, autophagy may be a bona fide mechanism for induction and execution of cell death (autophagic cell death (ACD)) or be a prerequisite for apoptotic or necrotic cell death (autophagy-mediated cell death) (4.Puyal J. Ginet V. Clarke P.G. Multiple interacting cell death mechanisms in the mediation of excitotoxicity and ischemic brain damage: a challenge for neuroprotection.Prog. Neurobiol. 2013; 105: 24-48Crossref PubMed Scopus (173) Google Scholar). Autophagosome biogenesis can be assessed by monitoring the autophagy markers microtubule-associated protein light chain 3 (LC3) and p62. LC3 is a ubiquitin-like protein; pro-LC3 is cleaved by Atg4B, resulting in the cytosolic form LC3-I (5.Kabeya Y. Mizushima N. Ueno T. Yamamoto A. Kirisako T. Noda T. Kominami E. Ohsumi Y. Yoshimori T. LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing.EMBO J. 2000; 19: 5720-5728Crossref PubMed Scopus (5433) Google Scholar). When autophagy is activated, LC3-I is enriched in autophagosomes and converted to LC3-II through conjugation with phosphatidylethanolamine (6.Ichimura Y. Kirisako T. Takao T. Satomi Y. Shimonishi Y. Ishihara N. Mizushima N. Tanida I. Kominami E. Ohsumi M. Noda T. Ohsumi Y. A ubiquitin-like system mediates protein lipidation.Nature. 2000; 408: 488-492Crossref PubMed Scopus (1504) Google Scholar). Therefore, LC3-II is a reliable biochemical marker of autophagosome formation (5.Kabeya Y. Mizushima N. Ueno T. Yamamoto A. Kirisako T. Noda T. Kominami E. Ohsumi Y. Yoshimori T. LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing.EMBO J. 2000; 19: 5720-5728Crossref PubMed Scopus (5433) Google Scholar, 7.Suzuki K. Kirisako T. Kamada Y. Mizushima N. Noda T. Ohsumi Y. The pre-autophagosomal structure organized by concerted functions of APG genes is essential for autophagosome formation.EMBO J. 2001; 20: 5971-5981Crossref PubMed Scopus (799) Google Scholar, 8.Mizushima N. Yamamoto A. Hatano M. Kobayashi Y. Kabeya Y. Suzuki K. Tokuhisa T. Ohsumi Y. Yoshimori T. Dissection of autophagosome formation using Apg5-deficient mouse embryonic stem cells.J. Cell Biol. 2001; 152: 657-668Crossref PubMed Scopus (1158) Google Scholar). In a similar manner, a punctate pattern of fluorescently tagged LC3 can be indicative of autophagy induction (9.Mizushima N. Yamamoto A. Matsui M. Yoshimori T. Ohsumi Y. In vivo analysis of autophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker.Mol. Biol. Cell. 2004; 15: 1101-1111Crossref PubMed Scopus (1923) Google Scholar). The ubiquitin-binding protein p62 interacts with LC3-II and thereby delivers cargo molecules to autophagosomes. During this process, p62 is also degraded by autophagy (10.Bjørkøy G. Lamark T. Brech A. Outzen H. Perander M. Overvatn A. Stenmark H. Johansen T. p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death.J. Cell Biol. 2005; 171: 603-614Crossref PubMed Scopus (2506) Google Scholar, 11.Pankiv S. Clausen T.H. Lamark T. Brech A. Bruun J.-A. Outzen H. Øvervatn A. Bjørkøy G. Johansen T. p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy.J. Biol. Chem. 2007; 282: 24131-24145Abstract Full Text Full Text PDF PubMed Scopus (3307) Google Scholar). Hence, a decrease in p62 is another measure of autophagic flux (12.Ichimura Y. Kominami E. Tanaka K. Komatsu M. Selective turnover of p62/A170/SQSTM1 by autophagy.Autophagy. 2008; 4: 1063-1066Crossref PubMed Scopus (191) Google Scholar). However, accumulation of autophagic vesicles at one static time point can reflect either an increase (on-rate) or decrease (off-rate) of autophagic flux with diametrically opposite implication for the role of autophagy in cell death. Therefore, description of autophagy morphology in dying cells without data to clarify the functional role of autophagy in the cell death process has led to confusion about the concept of ACD. In this regard, observation of ACD in apoptosis-defective cells or cells with suppressed apoptosis, the lack of the specific pharmacological reagents for modulation of autophagy, and other difficulties and technical concerns in addressing the exact role of autophagy in relation to cell death were well discussed by Kroemer and Levine (13.Kroemer G. Levine B. Autophagic cell death: the story of a misnomer.Nat. Rev. Mol. Cell Biol. 2008; 9: 1004-1010Crossref PubMed Scopus (1162) Google Scholar). Despite the controversy, an increasing number of studies support a causative role of autophagy in cell death, especially in insects and other model organisms (14.Kourtis N. Tavernarakis N. Autophagy and cell death in model organisms.Cell Death Differ. 2009; 16: 21-30Crossref PubMed Scopus (207) Google Scholar). Examples of ACD in mammals are much fewer (15.Kim N.-Y. Lee M. The pro-death role of autophagy and apoptosis in cell death induced by the BH3 mimetic gossypol.Animal Cells Syst. 2014; 18: 183-189Crossref Scopus (3) Google Scholar). According to the criteria suggested by Shen and Codogno (16.Shen H.M. Codogno P. Autophagic cell death: Loch Ness monster or endangered species?.Autophagy. 2011; 7: 457-465Crossref PubMed Scopus (267) Google Scholar), 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 have previously demonstrated that insulin withdrawal induces ACD in adult hippocampal neural (HCN) stem cells despite their normal apoptotic capabilities. Insulin-deprived HCN cells show enhanced autophagic flux but do not display signs of apoptosis, such as caspase activation, chromosomal DNA fragmentation, and exposure of phosphatidylserine on the outer leaflet of the plasma membrane (17.Yu S.W. Baek S.H. Brennan R.T. Bradley C.J. Park S.K. Lee Y.S. Jun E.J. Lookingland K.J. Kim E.K. Lee H. Goudreau J.L. Kim S.W. Autophagic death of adult hippocampal neural stem cells following insulin withdrawal.Stem Cells. 2008; 26: 2602-2610Crossref PubMed Scopus (98) Google Scholar, 18.Baek S.-H. Kim E.-K. Goudreau J.L. Lookingland K.J. Kim S.W. Yu S.-W. Insulin withdrawal-induced cell death in adult hippocampal neural stem cells as a model of autophagic cell death.Autophagy. 2009; 5: 277-279Crossref PubMed Scopus (32) Google Scholar, 19.Ha S. Ryu H.Y. Chung K.M. Baek S.-H. Kim E.-K. Yu S.-W. Regulation of autophagic cell death by glycogen synthase kinase-3β in adult hippocampal neural stem cells following insulin withdrawal.Mol. Brain. 2015; 8: 30-41Crossref PubMed Scopus (32) Google Scholar, 20.Chung K.M. Park H. Jung S. Ha S. Yoo S.-J. Woo H. Lee H.J. Kim S.W. Kim E.-K. Moon C. Yu S.-W. Calpain determines the propensity of adult hippocampal neural stem cells to autophagic cell death following insulin withdrawal.Stem Cells. 2015; 33: 3052-3064Crossref PubMed Scopus (23) Google Scholar). In addition, the pan-caspase inhibitor Z-VAD failed to protect HCN cells from insulin withdrawal (17.Yu S.W. Baek S.H. Brennan R.T. Bradley C.J. Park S.K. Lee Y.S. Jun E.J. Lookingland K.J. Kim E.K. Lee H. Goudreau J.L. Kim S.W. Autophagic death of adult hippocampal neural stem cells following insulin withdrawal.Stem Cells. 2008; 26: 2602-2610Crossref PubMed Scopus (98) Google Scholar, 19.Ha S. Ryu H.Y. Chung K.M. Baek S.-H. Kim E.-K. Yu S.-W. Regulation of autophagic cell death by glycogen synthase kinase-3β in adult hippocampal neural stem cells following insulin withdrawal.Mol. Brain. 2015; 8: 30-41Crossref PubMed Scopus (32) Google Scholar, 20.Chung K.M. Park H. Jung S. Ha S. Yoo S.-J. Woo H. Lee H.J. Kim S.W. Kim E.-K. Moon C. Yu S.-W. Calpain determines the propensity of adult hippocampal neural stem cells to autophagic cell death following insulin withdrawal.Stem Cells. 2015; 33: 3052-3064Crossref PubMed Scopus (23) Google Scholar). In contrast, Atg7 knockdown efficiently suppresses ACD in insulin-deprived HCN cells (17.Yu S.W. Baek S.H. Brennan R.T. Bradley C.J. Park S.K. Lee Y.S. Jun E.J. Lookingland K.J. Kim E.K. Lee H. Goudreau J.L. Kim S.W. Autophagic death of adult hippocampal neural stem cells following insulin withdrawal.Stem Cells. 2008; 26: 2602-2610Crossref PubMed Scopus (98) Google Scholar, 19.Ha S. Ryu H.Y. Chung K.M. Baek S.-H. Kim E.-K. Yu S.-W. Regulation of autophagic cell death by glycogen synthase kinase-3β in adult hippocampal neural stem cells following insulin withdrawal.Mol. Brain. 2015; 8: 30-41Crossref PubMed Scopus (32) Google Scholar, 20.Chung K.M. Park H. Jung S. Ha S. Yoo S.-J. Woo H. Lee H.J. Kim S.W. Kim E.-K. Moon C. Yu S.-W. Calpain determines the propensity of adult hippocampal neural stem cells to autophagic cell death following insulin withdrawal.Stem Cells. 2015; 33: 3052-3064Crossref PubMed Scopus (23) Google Scholar, 21.Yeo B.K. Hong C.J. Chung K.M. Woo H. Kim K. Jung S. Kim E.-K. Yu S.-W. Valosin-containing protein is a key mediator between autophagic cell death and apoptosis in adult hippocampal neural stem cells following insulin withdrawal.Mol. Brain. 2016; 9: 31-44Crossref PubMed Scopus (22) Google Scholar). Therefore, insulin withdrawal-induced death of HCN cells fulfills all the criteria for ACD (16.Shen H.M. Codogno P. Autophagic cell death: Loch Ness monster or endangered species?.Autophagy. 2011; 7: 457-465Crossref PubMed Scopus (267) Google Scholar, 22.Hong C.J. Park H. Yu S.-W. Autophagy for the quality control of adult hippocampal neural stem cells.Brain Res. 2016; 1649: 166-172Crossref PubMed Scopus (22) Google Scholar, 23.Denton D. Nicolson S. Kumar S. Cell death by autophagy: facts and apparent artefacts.Cell Death Differ. 2012; 19: 87-95Crossref PubMed Scopus (314) Google Scholar, 24.Chung K.M. Yu S.-W. Interplay between autophagy and programmed cell death in mammalian neural stem cells.BMB Rep. 2013; 46: 383-390Crossref PubMed Scopus (26) Google Scholar, 25.Ryu J.R. Hong C.J. Kim J.Y. Kim E.-K. Sun W. Yu S.-W. Control of adult neurogenesis by programmed cell death in the mammalian brain.Mol. Brain. 2016; 9: 43-62Crossref PubMed Scopus (85) Google Scholar, 26.Clarke P.G. Puyal J. Autophagic cell death exists.Autophagy. 2012; 8: 867-869Crossref PubMed Scopus (95) Google Scholar). Furthermore, we also identified several key regulators of ACD and mediators for the cross-talk between ACD and apoptosis, including glycogen synthase kinase-3β, calpain, type 3 ryanodine receptor, and p97/valosin-containing protein (19.Ha S. Ryu H.Y. Chung K.M. Baek S.-H. Kim E.-K. Yu S.-W. Regulation of autophagic cell death by glycogen synthase kinase-3β in adult hippocampal neural stem cells following insulin withdrawal.Mol. Brain. 2015; 8: 30-41Crossref PubMed Scopus (32) Google Scholar, 20.Chung K.M. Park H. Jung S. Ha S. Yoo S.-J. Woo H. Lee H.J. Kim S.W. Kim E.-K. Moon C. Yu S.-W. Calpain determines the propensity of adult hippocampal neural stem cells to autophagic cell death following insulin withdrawal.Stem Cells. 2015; 33: 3052-3064Crossref PubMed Scopus (23) Google Scholar, 21.Yeo B.K. Hong C.J. Chung K.M. Woo H. Kim K. Jung S. Kim E.-K. Yu S.-W. Valosin-containing protein is a key mediator between autophagic cell death and apoptosis in adult hippocampal neural stem cells following insulin withdrawal.Mol. Brain. 2016; 9: 31-44Crossref PubMed Scopus (22) Google Scholar, 27.Chung K.M. Jeong E.J. Park H. An H.K. Yu S.W. Mediation of autophagic cell death by type 3 ryanodine receptor (RyR3) in adult hippocampal neural stem cells.Front. Cell. Neurosci. 2016; 10: 116-130Crossref PubMed Scopus (34) Google Scholar). AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase composed of a catalytic subunit (α1 or α2) and regulatory β and γ subunits (28.Stapleton D. Mitchelhill K.I. Gao G. Widmer J. Michell B.J. Teh T. House C.M. Fernandez C.S. Cox T. Witters L.A. Kemp B.E. Mammalian AMP-activated protein kinase subfamily.J. Biol. Chem. 1996; 271: 611-614Abstract Full Text Full Text PDF PubMed Scopus (562) Google Scholar). AMPK regulates various cellular processes, including metabolic homeostasis, cell proliferation, and cell death (29.Mihaylova M.M. Shaw R.J. The AMPK signalling pathway coordinates cell growth, autophagy and metabolism.Nat. Cell Biol. 2011; 13: 1016-1023Crossref PubMed Scopus (1980) Google Scholar, 30.Motoshima H. Goldstein B.J. Igata M. Araki E. AMPK and cell proliferation—AMPK as a therapeutic target for atherosclerosis and cancer.J. Physiol. 2006; 574: 63-71Crossref PubMed Scopus (441) Google Scholar). AMPK can also induce autophagy (31.Han D. Yang B. Olson L.K. Greenstein A. Baek S.H. Claycombe K.J. Goudreau J.L. Yu S.W. Kim E.K. Activation of autophagy through modulation of 5′-AMP-activated protein kinase protects pancreatic β-cells from high glucose.Biochem. J. 2010; 425: 541-551Crossref PubMed Scopus (52) Google Scholar, 32.Liang J. Shao S.H. Xu Z.-X. Hennessy B. Ding Z. Larrea M. Kondo S. Dumont D.J. Gutterman J.U. Walker C.L. Slingerland J.M. Mills G.B. The energy sensing LKB1–AMPK pathway regulates p27kip1 phosphorylation mediating the decision to enter autophagy or apoptosis.Nat. Cell Biol. 2007; 9: 218-224Crossref PubMed Scopus (707) Google Scholar, 33.Matsui Y. Takagi H. Qu X. Abdellatif M. Sakoda H. Asano T. Levine B. Sadoshima J. Distinct roles of autophagy in the heart during ischemia and reperfusion roles of AMP-activated protein kinase and beclin 1 in mediating autophagy.Circ. Res. 2007; 100: 914-922Crossref PubMed Scopus (1243) Google Scholar). Given the close connection of insulin signaling with AMPK in the regulation of cell metabolism, proliferation, survival, and autophagy, we postulated that AMPK might be intimately involved in ACD of HCN cells upon insulin withdrawal. In the present study, we found that AMPK was readily activated in insulin-deprived HCN cells and served as a critical trigger of ACD. Looking further into the molecular mechanism, we identified p62 as a new molecular target of AMPK. Although many substrates connecting AMPK to metabolism regulation are known, only a few AMPK targets relevant to autophagy have been identified so far (32.Liang J. Shao S.H. Xu Z.-X. Hennessy B. Ding Z. Larrea M. Kondo S. Dumont D.J. Gutterman J.U. Walker C.L. Slingerland J.M. Mills G.B. The energy sensing LKB1–AMPK pathway regulates p27kip1 phosphorylation mediating the decision to enter autophagy or apoptosis.Nat. Cell Biol. 2007; 9: 218-224Crossref PubMed Scopus (707) Google Scholar, 34.Kim J. Kundu M. Viollet B. Guan K.-L. AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1.Nat. Cell Biol. 2011; 13: 132-141Crossref PubMed Scopus (4513) Google Scholar, 35.Gwinn D.M. Shackelford D.B. Egan D.F. Mihaylova M.M. Mery A. Vasquez D.S. Turk B.E. Shaw R.J. AMPK phosphorylation of raptor mediates a metabolic checkpoint.Mol. Cell. 2008; 30: 214-226Abstract Full Text Full Text PDF PubMed Scopus (2749) Google Scholar, 36.Egan D.F. Shackelford D.B. Mihaylova M.M. Gelino S. Kohnz R.A. Mair W. Vasquez D.S. Joshi A. Gwinn D.M. Taylor R. Asara J.M. Fitzpatrick J. Dillin A. Viollet B. Kundu M. et al.Phosphorylation of ULK1 (hATG1) by AMP-activated protein kinase connects energy sensing to mitophagy.Science. 2011; 331: 456-461Crossref PubMed Scopus (1815) Google Scholar, 37.Kim J. Kim Y.C. Fang C. Russell R.C. Kim J.H. Fan W. Liu R. Zhong Q. Guan K.-L. Differential regulation of distinct Vps34 complexes by AMPK in nutrient stress and autophagy.Cell. 2013; 152: 290-303Abstract Full Text Full Text PDF PubMed Scopus (555) Google Scholar). 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. To address the role of AMPK in HCN cell death, we initially assessed the activation status of AMPK in insulin-deprived HCN cells. Hereafter, we will refer to HCN cells cultured in insulin-containing and insulin-deficient media as I(+) and I(−), respectively, as in our prior reports (19.Ha S. Ryu H.Y. Chung K.M. Baek S.-H. Kim E.-K. Yu S.-W. Regulation of autophagic cell death by glycogen synthase kinase-3β in adult hippocampal neural stem cells following insulin withdrawal.Mol. Brain. 2015; 8: 30-41Crossref PubMed Scopus (32) Google Scholar, 20.Chung K.M. Park H. Jung S. Ha S. Yoo S.-J. Woo H. Lee H.J. Kim S.W. Kim E.-K. Moon C. Yu S.-W. Calpain determines the propensity of adult hippocampal neural stem cells to autophagic cell death following insulin withdrawal.Stem Cells. 2015; 33: 3052-3064Crossref PubMed Scopus (23) Google Scholar). We assessed Thr-172 phosphorylation of the AMPK α subunit because this post-translational modification in the kinase activation loop is associated with increased AMPK activity (38.Hawley S.A. Davison M. Woods A. Davies S.P. Beri R.K. Carling D. Hardie D.G. Characterization of the AMP-activated protein kinase kinase from rat liver and identification of threonine 172 as the major site at which it phosphorylates AMP-activated protein kinase.J. Biol. Chem. 1996; 271: 27879-27887Abstract Full Text Full Text PDF PubMed Scopus (1005) Google Scholar, 39.Stein S.C. Woods A. Jones N.A. Davison M.D. Carling D. The regulation of AMP-activated protein kinase by phosphorylation.Biochem. J. 2000; 345: 437-443Crossref PubMed Scopus (490) Google Scholar). Following insulin withdrawal, AMPK was rapidly activated, and the time course of its activation was well correlated with the up-regulation of autophagic flux as revealed by an increase in LC3-II and decrease in p62 (Fig. 1A). The AMPK inhibitor compound C (CC; 0.5 μm) efficiently blocked AMPK activation and substantially reduced the autophagy level and cell death in I(−) HCN cells (Fig. 1, B–D). CC also reduced the number of GFP-LC3 puncta in these cells (Fig. 1, E and F). To corroborate these results derived from the pharmacological inhibition of AMPK, we knocked out AMPK α1 or α2 subunit using the CRISPR/Cas9 gene editing approach. The relative levels of α1 and α2 mRNAs were similar and were not changed by insulin withdrawal (supplemental Fig. S1A). Single guide RNA sequences targeting each subunit efficiently ablated the AMPK α1 or α2 gene (supplemental Fig. S1B). Of note, the deletion of the α2 but not α1 gene markedly reduced cell death (Fig. 1, G and supplemental Fig. S1C) and autophagic flux in I(−) HCN cells (Fig. 1, H and I). These data indicated that pharmacological or genetic inactivation of AMPK substantially diminished ACD in I(−) HCN cells, suggesting a critical role of AMPK in HCN cell death following insulin withdrawal. With regard to the mechanism of AMPK activation, two upstream kinases, LKB1 and Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ), are well documented to phosphorylate Thr-172 of the AMPK α subunit (40.Kim J. Yang G. Kim Y. Kim J. Ha J. AMPK activators: mechanisms of action and physiological activities.Exp. Mol. Med. 2016; 48: e224Crossref PubMed Scopus (400) Google Scholar). AMPK is activated in response to metabolic stresses that deplete the cellular ATP level, and binding of AMP to the γ subunit allosterically activates the AMPK complex by enhancing LKB1-dependent Thr-172 phosphorylation. AMPK can also be directly phosphorylated by CaMKKβ in response to increases in intracellular Ca2+ without significant change in ATP/ADP/AMP levels (40.Kim J. Yang G. Kim Y. Kim J. Ha J. AMPK activators: mechanisms of action and physiological activities.Exp. Mol. Med. 2016; 48: e224Crossref PubMed Scopus (400) Google Scholar). To elucidate how insulin withdrawal activated AMPK, we first examined the cellular ATP level. Interestingly, insulin withdrawal did not induce depletion of cellular ATP compared with the substantial depletion of cellular ATP by glucose deprivation for 5 h (supplemental Fig. S2A). In contrast, a specific CaMKK inhibitor, STO-609, decreased AMPK Thr-174 phosphorylation and attenuated cell death (supplemental Fig. S2, B and C). Our previous report also demonstrated an increase in the intracellular Ca2+ level that was due to ryanodine receptor 3-mediated endoplasmic reticulum Ca2+ efflux following insulin withdrawal (27.Chung K.M. Jeong E.J. Park H. An H.K. Yu S.W. Mediation of autophagic cell death by type 3 ryanodine receptor (RyR3) in adult hippocampal neural stem cells.Front. Cell. Neurosci. 2016; 10: 116-130Crossref PubMed Scopus (34) Google Scholar). These results suggest that insulin withdrawal activated AMPK through Ca2+–CaMKK pathway rather than ATP depletion in HCN cells. If AMPK is critical for ACD in I(−) HCN cells, its forced activation may enhance the effects of insulin withdrawal and boost ACD. To test this assumption, we generated an AMPK construct encoding the constitutively active (CA) mutant form of the α2 subunit by introducing a point mutation of Thr-172 to Asp in combination with the deletion of C-terminal amino acid residues 313–548 containing the autoinhibitory sequence and the binding sites to the regulatory β/γ subunits (41.Crute B.E. Seefeld K. Gamble J. Kemp B.E. Witters L.A. Functional domains of the α1 catalytic subunit of the AMP-activated protein kinase.J. Biol. Chem. 1998; 273: 35347-35354Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar, 42.Woods A. Azzout-Marniche D. Foretz M. Stein S.C. Lemarchand P. Ferré P. Foufelle F. Carling D. Characterization of the role of AMP-activated protein kinase in the regulation of glucose-activated gene expression using constitutively active and dominant negative forms of the kinase.Mol. Cell. Biol. 2000; 20: 6704-6711Crossref PubMed Scopus (358) Google Scholar). Persistent activation of AMPK was confirmed by phosphorylation of its well known substrate, acetyl-CoA carboxylase (ACC) (supplemental Fig. S2D). As expected, constitutive activation of AMPK in I(−) HCN cells led to a more marked increase in LC3-II and decrease in p62 levels than insulin withdrawal alone but did not activate caspase-3 (Fig. 2, A and B). AMPK α2-CA also induced a higher level of cell death than insulin withdrawal (Fig. 2C). In line with the ineffectiveness of Z-VAD against insulin withdrawal-induced ACD (19.Ha S. Ryu H.Y. Chung K.M. Baek S.-H. Kim E.-K. Yu S.-W. Regulation of autophagic cell death by glycogen synthase kinase-3β in adult hippocampal neural stem cells following insulin withdrawal.Mol. Brain. 2015; 8: 30-41Crossref PubMed Scopus (32) Google Scholar, 20.Chung K.M. Park H. Jung S. Ha S. Yoo S.-J. Woo H. Lee H.J. Kim S.W. Kim E.-K. Moon C. Yu S.-W. Calpain determines the propensity of adult hippocampal neural stem cells to autophagic cell death following insulin withdrawal.Stem Cells. 2015; 33: 3052-3064Crossref PubMed Scopus (23) Google Scholar), Z-VAD (20 μm) failed to diminish the AMPK α2-CA-induced boost of cell death but efficiently decreased staurosporine (STS)-induced apoptosis in HCN cells (Fig. 2C). Autophagic flux, as assessed by Western blotting of LC3-II in combination with bafilomycin A1 (BafA1), was further increased by the expression of AMPK α2-CA in I(−) HCN cells (Fig. 2, D and E). Lentiviral transduction with tandem labeled monomeric mRFP-GFP-LC3 also indicated a higher level of autophagic flux in I(−) HCN cells co-expressing AMPK α2-CA than I(−) HCN cells alone (Fig. 2, F and G). The quenching of the GFP fluorescence but not mRFP fluorescence at the acidic pH yields red puncta upon fusion of autophagosomes and lysosomes (43.Kimura S. Noda T. Yoshimori T. Dissection of the autophagosome maturation process by a novel reporter protein, tandem fluorescent-tagged LC3.Autophagy. 2007; 3: 452-460Crossref PubMed Scopus (1662) Google Scholar). Co" @default.
• W2724886670 created "2017-07-14" @default.
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• W2724886670 cites W1488940119 @default.
• W2724886670 cites W1550290005 @default.
• W2724886670 cites W1555054913 @default.
• W2724886670 cites W1563676220 @default.
• W2724886670 cites W1569812687 @default.
• W2724886670 cites W1839260472 @default.
• W2724886670 cites W1853192652 @default.
• W2724886670 cites W1968016394 @default.
• W2724886670 cites W1970297447 @default.
• W2724886670 cites W1970637701 @default.
• W2724886670 cites W1972762474 @default.
• W2724886670 cites W1973472942 @default.
• W2724886670 cites W1977767269 @default.
• W2724886670 cites W1978763812 @default.
• W2724886670 cites W1979566598 @default.
• W2724886670 cites W1980621373 @default.
• W2724886670 cites W1981358316 @default.
• W2724886670 cites W1988848089 @default.
• W2724886670 cites W1989051519 @default.
• W2724886670 cites W1989202029 @default.
• W2724886670 cites W1990753965 @default.
• W2724886670 cites W1995642346 @default.
• W2724886670 cites W2009233209 @default.
• W2724886670 cites W2010008722 @default.
• W2724886670 cites W2014918593 @default.
• W2724886670 cites W2022675211 @default.
• W2724886670 cites W2025872915 @default.
• W2724886670 cites W2030430693 @default.
• W2724886670 cites W2035019166 @default.
• W2724886670 cites W2038976751 @default.
• W2724886670 cites W2040605398 @default.
• W2724886670 cites W2063761428 @default.
• W2724886670 cites W2070224555 @default.
• W2724886670 cites W2071221374 @default.
• W2724886670 cites W2072948635 @default.
• W2724886670 cites W2073704223 @default.
• W2724886670 cites W2075410089 @default.
• W2724886670 cites W2077247026 @default.
• W2724886670 cites W2079580613 @default.
• W2724886670 cites W2080693065 @default.
• W2724886670 cites W2081920342 @default.
• W2724886670 cites W2083796374 @default.
• W2724886670 cites W2086299467 @default.
• W2724886670 cites W2096045103 @default.
• W2724886670 cites W2099686283 @default.
• W2724886670 cites W2103230459 @default.
• W2724886670 cites W2103659405 @default.
• W2724886670 cites W2105793503 @default.
• W2724886670 cites W2106684995 @default.
• W2724886670 cites W2108086395 @default.
• W2724886670 cites W2115742671 @default.
• W2724886670 cites W2131007152 @default.
• W2724886670 cites W2134535786 @default.
• W2724886670 cites W2136994247 @default.
• W2724886670 cites W2144687501 @default.
• W2724886670 cites W2145265436 @default.
• W2724886670 cites W2145572250 @default.
• W2724886670 cites W2146124002 @default.
• W2724886670 cites W2160411684 @default.
• W2724886670 cites W2161234475 @default.
• W2724886670 cites W2170880090 @default.
• W2724886670 cites W2233205187 @default.
• W2724886670 cites W2294662276 @default.
• W2724886670 cites W2306350261 @default.
• W2724886670 cites W2317113145 @default.
• W2724886670 cites W2338879794 @default.
• W2724886670 cites W2345823565 @default.
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