Phosphorylation of Neuronal Survival Factor MEF2D by Glycogen Synthase Kinase 3β in Neuronal Apoptosis
2009; Elsevier BV; Volume: 284; Issue: 47 Linguagem: Inglês
10.1074/jbc.m109.067785
ISSN1083-351X
AutoresXuemin Wang, Hua She, Zixu Mao,
Tópico(s)Nuclear Receptors and Signaling
ResumoGlycogen synthase kinase 3β (GSK3β) has been identified to play important roles in neuronal death. Evidence from both in vitro and in vivo studies indicates that increased GSK3β activity contributes to neurodegeneration and to the pathogenesis of Alzheimer disease. But the molecular mechanisms that underlie GSK3β-mediated neurotoxicity remain poorly understood. We reported here that myocyte enhancer factor 2D (MEF2D), a nuclear transcription factor known to promote neuronal survival, is directly phosphorylated by GSK3β. Our data showed that phosphorylation of MEF2D by GSK3β at three specific residues in its transactivation domain inhibits MEF2D transcriptional activity. Withdrawal of neuronal activity in cerebellar granule neurons activated GSK3β in the nucleus, leading to GSK3β-dependent inhibition of MEF2 function. This inhibition contributed to GSK3β-mediated neuronal toxicity. Overexpression of MEF2D mutant that is resistant to GSK3β inhibition protected cerebellar granule neurons from either GSK3β activation- or neuronal activity deprivation-induced toxicity. These results identify survival factor MEF2D as a novel downstream effector targeted by GSK3β and define a molecular link between activation of GSK3β and neuronal survival machinery which may underlie in part GSK3β-mediated neurotoxicity. Glycogen synthase kinase 3β (GSK3β) has been identified to play important roles in neuronal death. Evidence from both in vitro and in vivo studies indicates that increased GSK3β activity contributes to neurodegeneration and to the pathogenesis of Alzheimer disease. But the molecular mechanisms that underlie GSK3β-mediated neurotoxicity remain poorly understood. We reported here that myocyte enhancer factor 2D (MEF2D), a nuclear transcription factor known to promote neuronal survival, is directly phosphorylated by GSK3β. Our data showed that phosphorylation of MEF2D by GSK3β at three specific residues in its transactivation domain inhibits MEF2D transcriptional activity. Withdrawal of neuronal activity in cerebellar granule neurons activated GSK3β in the nucleus, leading to GSK3β-dependent inhibition of MEF2 function. This inhibition contributed to GSK3β-mediated neuronal toxicity. Overexpression of MEF2D mutant that is resistant to GSK3β inhibition protected cerebellar granule neurons from either GSK3β activation- or neuronal activity deprivation-induced toxicity. These results identify survival factor MEF2D as a novel downstream effector targeted by GSK3β and define a molecular link between activation of GSK3β and neuronal survival machinery which may underlie in part GSK3β-mediated neurotoxicity. IntroductionNeuronal survival plays an important role in both immature neurons during development and mature neurons under stress. Although the molecular mechanisms that underlie neuronal death are complex, some key regulators have been identified. One such regulator is glycogen synthase kinase 3β (GSK3β). 3The abbreviations used are: GSK3βglycogen synthase kinase 3βMEF2myocyte enhancer factor 2ERK5extracellular-regulated kinase 5CGNcerebellar granule neuronsPIpropidium iodideGFPgreen fluorescent proteinDIVday(s) in vitroPBSphosphate-buffered salineCdk5cyclin-dependent kinase 5. Indeed, in vitro and in vivo evidence suggests that the GSK3β signaling pathway plays a prominent role in neurodegeneration and in the formation of plaque and neurofibrillary tangle in Alzheimer disease (1.Kaytor M.D. Orr H.T. Curr. Opin. Neurobiol. 2002; 12: 275-278Crossref PubMed Scopus (183) Google Scholar, 2.Mora A. Sabio G. González-Polo R.A. Cuenda A. Alessi D.R. Alonso J.C. Fuentes J.M. Soler G. Centeno F. J. Neurochem. 2001; 78: 199-206Crossref PubMed Scopus (94) Google Scholar). In vitro, withdrawal of trophic support from cultured neurons increases GSK3β activity, which is accompanied with neuronal death (3.Hetman M. Cavanaugh J.E. Kimelman D. Xia Z. J. Neurosci. 2000; 20: 2567-2574Crossref PubMed Google Scholar), whereas overexpression of it is sufficient to induce apoptosis (4.Pap M. Cooper G.M. J. Biol. Chem. 1998; 273: 19929-19932Abstract Full Text Full Text PDF PubMed Scopus (954) Google Scholar). In vivo, overexpression of GSK3β also leads to activation of caspase, neuronal loss, and gliosis (5.Lucas J.J. Hernández F. Gómez-Ramos P. Morán M.A. Hen R. Avila J. EMBO J. 2001; 20: 27-39Crossref PubMed Scopus (804) Google Scholar). Consistent with its role in neuronal death, GSK3β activity is inhibited by the insulin signaling pathway. But how GSK3β acts to induce neuronal death and promote neurodegeneration is not entirely clear and remains an area of active investigation.As a kinase, one of the most likely modes of action by which GSK3β modulates neuronal viability is to phosphorylate downstream effectors. Indeed, although identified first to regulate glycogen synthesis (6.Embi N. Rylatt D.B. Cohen P. Eur. J. Biochem. 1980; 107: 519-527Crossref PubMed Scopus (803) Google Scholar), GSK3β is now known to target many protein substrates to modulate many processes including apoptosis (7.Jope R.S. Johnson G.V. Trends Biochem. Sci. 2004; 29: 95-102Abstract Full Text Full Text PDF PubMed Scopus (1318) Google Scholar, 8.Beurel E. Jope R.S. Prog. Neurobiol. 2006; 79: 173-189Crossref PubMed Scopus (458) Google Scholar). An interesting feature of GSK3β is how it recognizes its substrates. Some GSK3β substrates require prior phosphorylation by another kinase to prime the substrates for GSK3β phosphorylation. A second category of GSK3β substrates can be phosphorylated directly by GSK3β without the priming event. Although GSK3β has been shown to regulate several substrates implicated in the pathogenesis of Alzheimer disease, including microtubule-associated protein Tau (9.Takashima A. Honda T. Yasutake K. Michel G. Murayama O. Murayama M. Ishiguro K. Yamaguchi H. Neurosci. Res. 1998; 31: 317-323Crossref PubMed Scopus (263) Google Scholar), much remains to be learned on whether GSK3β directly regulates factors required for neuronal survival.Members of myocyte enhancer factor 2 (MEF2A-D) belong to the MADS (MCM1, agamous, deficiens, serum-response factor) family of transcription factor. They have been shown to play critical roles in neuronal development and survival. Studies have shown that MEF2s are required for the survival of various types of neurons in several model systems. For example, recent studies have shown that various isoforms of MEF2s are required for neuronal activity-dependent survival of cerebellar granule neurons (10.Mao Z. Bonni A. Xia F. Nadal-Vicens M. Greenberg M.E. Science. 1999; 286: 785-790Crossref PubMed Scopus (441) Google Scholar, 11.Gong X. Tang X. Wiedmann M. Wang X. Peng J. Zheng D. Blair L.A. Marshall J. Mao Z. Neuron. 2003; 38: 33-46Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar, 12.Tang X. Wang X. Gong X. Tong M. Park D. Xia Z. Mao Z. J. Neurosci. 2005; 25: 4823-4834Crossref PubMed Scopus (105) Google Scholar) and for trophic factor-induced survival of developing cortical neurons (13.Liu L. Cavanaugh J.E. Wang Y. Sakagami H. Mao Z. Xia Z. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 8532-8537Crossref PubMed Scopus (141) Google Scholar). MEF2s are the targets for several key intracellular signaling pathways that regulate cell survival and apoptosis. For example, the phosphorylation of MEF2 by p38 mitogen-activated protein kinase (10.Mao Z. Bonni A. Xia F. Nadal-Vicens M. Greenberg M.E. Science. 1999; 286: 785-790Crossref PubMed Scopus (441) Google Scholar), extracellular-regulated kinase 5 (ERK5), or protein kinase A (14.Kato Y. Kravchenko V.V. Tapping R.I. Han J. Ulevitch R.J. Lee J.D. EMBO J. 1997; 16: 7054-7066Crossref PubMed Scopus (491) Google Scholar, 15.Wang X. Tang X. Li M. Marshall J. Mao Z. J. Biol. Chem. 2005; 280: 16705-16713Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) promotes MEF2 function, enhancing neuronal survival. In contrast, the phosphorylation of MEF2 by cyclin-dependent kinase 5 (Cdk5) in response to overt neurotoxic insults inhibits MEF2 function (11.Gong X. Tang X. Wiedmann M. Wang X. Peng J. Zheng D. Blair L.A. Marshall J. Mao Z. Neuron. 2003; 38: 33-46Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar). However, the kinases that mediate inhibitory effects on MEF2 upon neuronal activity withdrawal have not been identified.In this study we examined the regulating of MEF2D in cerebellar granule neuron death induced by potassium withdrawal. We found that potassium withdrawal triggered activation of GSK3β. GSK3β directly phosphorylated MEF2D at multiple sites, which inhibits MEF2 transactivation activity. MEF2 mutants that are resistant to GSK3β phosphorylation rescued neurons from potassium withdrawal-induced apoptosis.DISCUSSIONStrong evidences indicate that transcription factor MEF2s play a critical role in the survival of different types of neurons (10.Mao Z. Bonni A. Xia F. Nadal-Vicens M. Greenberg M.E. Science. 1999; 286: 785-790Crossref PubMed Scopus (441) Google Scholar, 11.Gong X. Tang X. Wiedmann M. Wang X. Peng J. Zheng D. Blair L.A. Marshall J. Mao Z. Neuron. 2003; 38: 33-46Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar, 12.Tang X. Wang X. Gong X. Tong M. Park D. Xia Z. Mao Z. J. Neurosci. 2005; 25: 4823-4834Crossref PubMed Scopus (105) Google Scholar, 13.Liu L. Cavanaugh J.E. Wang Y. Sakagami H. Mao Z. Xia Z. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 8532-8537Crossref PubMed Scopus (141) Google Scholar, 15.Wang X. Tang X. Li M. Marshall J. Mao Z. J. Biol. Chem. 2005; 280: 16705-16713Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). MEF2 activity is tightly controlled by both survival and death signals. Multiple key pathways converge on MEF2 to regulate its activity and neuronal survival. For example, membrane depolarization promotes the survival of CGNs via p38MAPK-mediated phosphorylation and activation of MEF2s (10.Mao Z. Bonni A. Xia F. Nadal-Vicens M. Greenberg M.E. Science. 1999; 286: 785-790Crossref PubMed Scopus (441) Google Scholar), whereas brain-derived neurotrophic factor seems to activate MEF2 through a ERK5-mediated mechanism (13.Liu L. Cavanaugh J.E. Wang Y. Sakagami H. Mao Z. Xia Z. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 8532-8537Crossref PubMed Scopus (141) Google Scholar). Protein kinase A has also been shown to modulate MEF2 activity (15.Wang X. Tang X. Li M. Marshall J. Mao Z. J. Biol. Chem. 2005; 280: 16705-16713Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). In contrast to our knowledge on the positive regulation of MEF2 by survival signals, how death signals regulate MEF2 is not well understood. It is known that Cdk5 phosphorylates the transactivation domain of MEF2s, which facilitates caspase-dependent degradation (11.Gong X. Tang X. Wiedmann M. Wang X. Peng J. Zheng D. Blair L.A. Marshall J. Mao Z. Neuron. 2003; 38: 33-46Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar, 12.Tang X. Wang X. Gong X. Tong M. Park D. Xia Z. Mao Z. J. Neurosci. 2005; 25: 4823-4834Crossref PubMed Scopus (105) Google Scholar). This mechanism appears to function predominantly in response to overt excitotoxic and oxidative stress as KCl withdrawal does not appear to induce significant activation of Cdk5 in the nucleus (25.Tian B. Yang Q. Mao Z. Nat Cell Biol. 2009; 11: 211-218Crossref PubMed Scopus (132) Google Scholar). In this study we identified GSK3β-mediated phosphorylation of MEF2D as a major mode of inhibitory regulation in response to KCl withdrawal. Using potassium withdrawal-induced apoptosis in CGNs as a model, we found that KCl withdrawal increases GSK3β activity in the nucleus. GSK3β directly phosphorylates the transactivation domain of MEF2D, which leads to inhibition of MEF2 activity. MEF2D mutants that are resistant to GSK3β phosphorylation can effectively rescue cerebellar granule neurons from potassium withdrawal-induced apoptosis. Taken together, our data define GSK3β as a novel negative regulator of MEF2D in mediating apoptotic signal in cerebellar granule neurons.In addition to the positive regulators that directly phosphorylate MEF2 and stimulate its activity, MEF2 function is also enhanced upon the activation of phosphatidylinositol 3-kinase-Akt pathway (26.Xu Q. Wu Z. J. Biol. Chem. 2000; 275: 36750-36757Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 27.Wiedmann M. Wang X. Tang X. Han M. Li M. Mao Z. J. Neurosci. Res. 2005; 81: 226-234Crossref PubMed Scopus (18) Google Scholar). However, Akt does not seem to phosphorylate MEF2D efficiently (27.Wiedmann M. Wang X. Tang X. Han M. Li M. Mao Z. J. Neurosci. Res. 2005; 81: 226-234Crossref PubMed Scopus (18) Google Scholar). Because Akt is a potent inhibitor of GSK3β, our finding that MEF2 is phosphorylated and inhibited by GSK3β provides a link between MEF2 and Akt.GSK3β has been implicated in the induction of apoptosis in response to many forms of apoptotic signals, including trophic signal withdrawal and neurotoxicity (28.Nair V.D. Olanow C.W. J. Biol. Chem. 2008; 283: 15469-15478Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 29.Mishra R. Barthwal M.K. Sondarva G. Rana B. Wong L. Chatterjee M. Woodgett J.R. Rana A. J. Biol. Chem. 2007; 282: 30393-30405Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Activation of GSK3β has been proposed to underlie the pathogenic process in neurodegenerative disorders including Alzheimer disease. Despite the important role for GSK3β in controlling neuronal apoptosis, the molecular mechanisms by which GSK3β regulates this process were not well illustrated. The identification of survival factor MEF2D as a direct substrate of GSK3β during neuronal apoptotic process expands the targets by which GSK3β transmits apoptotic signal. It also provides a mechanism that underlies GSK3β-mediated neurotoxicity. Because both GSK3β and MEF2D are known to play important roles in non-neuronal tissues such as mediating insulin signal and regulating glucose metabolism (30.Liu M.L. Olson A.L. Edgington N.P. Moye-Rowley W.S. Pessin J.E. J. Biol. Chem. 1994; 269: 28514-28521Abstract Full Text PDF PubMed Google Scholar), it would be interesting to investigate whether GSK3β-dependent regulation of MEF2 also plays a role in this and other processes.An interesting feature of GSK3β is its substrate specificity. Many GSK3β substrates require prior phosphorylation by different kinases at a downstream S/T site to generate a motif (S/T)XXX(S/T)P, where the upstream S/T can serve as the phosphorylation site for GSK3 (31.Liu C. Li Y. Semenov M. Han C. Baeg G.H. Tan Y. Zhang Z. Lin X. He X. Cell. 2002; 108: 837-847Abstract Full Text Full Text PDF PubMed Scopus (1636) Google Scholar). Other GSK3β substrates seem not to require prior phosphorylation for their recognition by the kinase (32.Cho J.H. Johnson G.V. J. Biol. Chem. 2003; 278: 187-193Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). Our data suggest that GSK3β can directly phosphorylate MEF2D at 145TXXXSXXXS153. Residues Thr-145, Ser-149, and Ser-153 are required for efficient phosphorylation (Fig. 4). Because robust phosphorylation of MEF2D at these sites by GSK3β can be easily achieved in vitro without prior incubation of MEF2D with another kinase, it is consistent with the notion that MEF2D is efficiently recognized as an unprimed substrate by GSK3β. Hyperphosphorylation of microtubule associate protein Tau is the pathological hallmark of Alzheimer disease (33.Engel T. Goñi-Oliver P. Gómez de Barreda E. Lucas J.J. Hernández F. Avila J. Neurodegener. Dis. 2008; 5: 247-249Crossref PubMed Scopus (42) Google Scholar). GSK3β phosphorylates Tau at both primed and unprimed sites. Moreover, the GSK3β mediated phosphorylation of Tau at the primed sites, which are prephosphorylated by Cdk5/p25 (32.Cho J.H. Johnson G.V. J. Biol. Chem. 2003; 278: 187-193Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). Given that MEF2 is phosphorylated by Cdk5 in response to neurotoxic insults and given the involvement of Cdk5 in Alzheimer disease (11.Gong X. Tang X. Wiedmann M. Wang X. Peng J. Zheng D. Blair L.A. Marshall J. Mao Z. Neuron. 2003; 38: 33-46Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar), it would be important to investigate if MEF2 may be synergistically regulated by GSK3β and Cdk5 under the pathological conditions associated with the development of Alzheimer disease. IntroductionNeuronal survival plays an important role in both immature neurons during development and mature neurons under stress. Although the molecular mechanisms that underlie neuronal death are complex, some key regulators have been identified. One such regulator is glycogen synthase kinase 3β (GSK3β). 3The abbreviations used are: GSK3βglycogen synthase kinase 3βMEF2myocyte enhancer factor 2ERK5extracellular-regulated kinase 5CGNcerebellar granule neuronsPIpropidium iodideGFPgreen fluorescent proteinDIVday(s) in vitroPBSphosphate-buffered salineCdk5cyclin-dependent kinase 5. Indeed, in vitro and in vivo evidence suggests that the GSK3β signaling pathway plays a prominent role in neurodegeneration and in the formation of plaque and neurofibrillary tangle in Alzheimer disease (1.Kaytor M.D. Orr H.T. Curr. Opin. Neurobiol. 2002; 12: 275-278Crossref PubMed Scopus (183) Google Scholar, 2.Mora A. Sabio G. González-Polo R.A. Cuenda A. Alessi D.R. Alonso J.C. Fuentes J.M. Soler G. Centeno F. J. Neurochem. 2001; 78: 199-206Crossref PubMed Scopus (94) Google Scholar). In vitro, withdrawal of trophic support from cultured neurons increases GSK3β activity, which is accompanied with neuronal death (3.Hetman M. Cavanaugh J.E. Kimelman D. Xia Z. J. Neurosci. 2000; 20: 2567-2574Crossref PubMed Google Scholar), whereas overexpression of it is sufficient to induce apoptosis (4.Pap M. Cooper G.M. J. Biol. Chem. 1998; 273: 19929-19932Abstract Full Text Full Text PDF PubMed Scopus (954) Google Scholar). In vivo, overexpression of GSK3β also leads to activation of caspase, neuronal loss, and gliosis (5.Lucas J.J. Hernández F. Gómez-Ramos P. Morán M.A. Hen R. Avila J. EMBO J. 2001; 20: 27-39Crossref PubMed Scopus (804) Google Scholar). Consistent with its role in neuronal death, GSK3β activity is inhibited by the insulin signaling pathway. But how GSK3β acts to induce neuronal death and promote neurodegeneration is not entirely clear and remains an area of active investigation.As a kinase, one of the most likely modes of action by which GSK3β modulates neuronal viability is to phosphorylate downstream effectors. Indeed, although identified first to regulate glycogen synthesis (6.Embi N. Rylatt D.B. Cohen P. Eur. J. Biochem. 1980; 107: 519-527Crossref PubMed Scopus (803) Google Scholar), GSK3β is now known to target many protein substrates to modulate many processes including apoptosis (7.Jope R.S. Johnson G.V. Trends Biochem. Sci. 2004; 29: 95-102Abstract Full Text Full Text PDF PubMed Scopus (1318) Google Scholar, 8.Beurel E. Jope R.S. Prog. Neurobiol. 2006; 79: 173-189Crossref PubMed Scopus (458) Google Scholar). An interesting feature of GSK3β is how it recognizes its substrates. Some GSK3β substrates require prior phosphorylation by another kinase to prime the substrates for GSK3β phosphorylation. A second category of GSK3β substrates can be phosphorylated directly by GSK3β without the priming event. Although GSK3β has been shown to regulate several substrates implicated in the pathogenesis of Alzheimer disease, including microtubule-associated protein Tau (9.Takashima A. Honda T. Yasutake K. Michel G. Murayama O. Murayama M. Ishiguro K. Yamaguchi H. Neurosci. Res. 1998; 31: 317-323Crossref PubMed Scopus (263) Google Scholar), much remains to be learned on whether GSK3β directly regulates factors required for neuronal survival.Members of myocyte enhancer factor 2 (MEF2A-D) belong to the MADS (MCM1, agamous, deficiens, serum-response factor) family of transcription factor. They have been shown to play critical roles in neuronal development and survival. Studies have shown that MEF2s are required for the survival of various types of neurons in several model systems. For example, recent studies have shown that various isoforms of MEF2s are required for neuronal activity-dependent survival of cerebellar granule neurons (10.Mao Z. Bonni A. Xia F. Nadal-Vicens M. Greenberg M.E. Science. 1999; 286: 785-790Crossref PubMed Scopus (441) Google Scholar, 11.Gong X. Tang X. Wiedmann M. Wang X. Peng J. Zheng D. Blair L.A. Marshall J. Mao Z. Neuron. 2003; 38: 33-46Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar, 12.Tang X. Wang X. Gong X. Tong M. Park D. Xia Z. Mao Z. J. Neurosci. 2005; 25: 4823-4834Crossref PubMed Scopus (105) Google Scholar) and for trophic factor-induced survival of developing cortical neurons (13.Liu L. Cavanaugh J.E. Wang Y. Sakagami H. Mao Z. Xia Z. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 8532-8537Crossref PubMed Scopus (141) Google Scholar). MEF2s are the targets for several key intracellular signaling pathways that regulate cell survival and apoptosis. For example, the phosphorylation of MEF2 by p38 mitogen-activated protein kinase (10.Mao Z. Bonni A. Xia F. Nadal-Vicens M. Greenberg M.E. Science. 1999; 286: 785-790Crossref PubMed Scopus (441) Google Scholar), extracellular-regulated kinase 5 (ERK5), or protein kinase A (14.Kato Y. Kravchenko V.V. Tapping R.I. Han J. Ulevitch R.J. Lee J.D. EMBO J. 1997; 16: 7054-7066Crossref PubMed Scopus (491) Google Scholar, 15.Wang X. Tang X. Li M. Marshall J. Mao Z. J. Biol. Chem. 2005; 280: 16705-16713Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) promotes MEF2 function, enhancing neuronal survival. In contrast, the phosphorylation of MEF2 by cyclin-dependent kinase 5 (Cdk5) in response to overt neurotoxic insults inhibits MEF2 function (11.Gong X. Tang X. Wiedmann M. Wang X. Peng J. Zheng D. Blair L.A. Marshall J. Mao Z. Neuron. 2003; 38: 33-46Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar). However, the kinases that mediate inhibitory effects on MEF2 upon neuronal activity withdrawal have not been identified.In this study we examined the regulating of MEF2D in cerebellar granule neuron death induced by potassium withdrawal. We found that potassium withdrawal triggered activation of GSK3β. GSK3β directly phosphorylated MEF2D at multiple sites, which inhibits MEF2 transactivation activity. MEF2 mutants that are resistant to GSK3β phosphorylation rescued neurons from potassium withdrawal-induced apoptosis.
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