[1] 7w Role of DAPK in neuronal cell death

[1] 164w Neuronal cell death happens as a result of the normal physiological process that occurs during development, or as part of the pathological process that occurs during disease. Death-associated protein kinase (DAPK) is an intracellular protein that mediates cell death by its serine/threonine kinase activity, and transmits apoptotic cell death signals in various cells, including neurons. DAPK is elevated in injured neurons in acute models of injury such as ischemia and seizure. The absence of DAPK has been shown to protect neurons from a wide variety of acute toxic insults. Moreover, DAPK also regulates neuronal cell death during central nervous system development. Neurons are initially overproduced in the developing nervous system, following which approximately one-half of the original cell population dies. This ''naturally-occurring'' or ''programmed'' cell death is essential for the construction of the developing nervous system. In this review, we focus on the role of DAPK in neuronal cell death after neuronal injury. The participation of DAPK in developmental neuronal death is also explained.

[1] 61w Neuronal cell death is a major phenomenon in the injury and development of the nervous system. Death-associated protein kinase (DAPK) is a serine/threonine protein kinase that mediates cell death. DAPK is abundantly expressed in the developing and adult central nervous system. It has been shown that an increase in DAPK expression is involved in neuronal damage in vivo and in vitro.

[2] 203w In the developing nervous system, the elimination of excess cells through programmed cell death (PCD) is essential for the establishment and maintenance of the nervous system. PCD has several major functions in embryonic development: it limits the progenitor pool size during neurogenesis, corrects errors, and quantitatively matches neurons with efferent targets and afferent inputs for optimal neuronal connections during synaptogenesis [1,2]. Thus, as a result of PCD, the developing nervous system is refined. Neuronal death is regulated by typical signal transduction, which is initiated by ligand-receptor associations and alternative signal transduction, which, in turn, is initiated by the withdrawal of ligands from specific receptors called ''dependence receptors'' [3]. These receptors mediate diverse biological functions. In the presence of the ligand, these receptors transduce a positive signal, such as differentiation and migration. However, in the absence of ligand, the receptors trigger PCD. It has been shown that some dependence receptors regulate cell death through DAPK in neurons [4,5]. The absence of a ligand induces self-activation of the receptor with its subsequent proteolytic processing, thereby initiating cell death through interaction with DAPK. Here, we review current knowledge concerning the mechanisms of neuronal cell death through DAPK in the injury and development of the nervous system.

[3] 144w Many ligand-receptor systems were found to depend on particular ligands for cell survival, such as the NGF-Trk signaling pathway in PCD [3]. These receptors have also been called ''dependence receptors'' because they depend on their respective ligands to create the cellular state, including apoptosis [44]; i.e., in the presence of a ligand, these receptors transduce positive signals, such as survival conversely, in the absence of a ligand, these receptors induce apoptosis. Various dependence receptors are identified as follows: p75 neurotrophin receptor [45], deleted in colorectal cancer (DCC) [46], uncoordinated gene-5 homologs (UNC5H) [47], RET [48], androgen receptor (AR) [49], integrins (a v b 3 and a 5 b 1 ) [50,51], and patched (Ptc) [52]. All of these receptors are structurally unrelated and have been shown to work as membrane sensors for cell fate decisions in neural development and in mammalian nervous systems [53][54][55][56].

[1] 48w The evidence obtained from acute injury models and cultured neurons implicate DAPK protein in the control of neuronal cell death. However, many unrevealed questions remain regarding the role of DAPK in neurons. For example, how does Neogenin or UNC5H2 induce DAPK dephosphorylation in the absence of its ligand?

[2] 64w The control of apoptosis by DAPK has clear significance in normal processes during CNS development. Furthermore, these proteins may play a role in certain neurodegenerative diseases that occur as a result of increased apoptosis. The ablation of DAPK appears to be important in protecting neurons from apoptosis. Therefore, agents that inhibit the ability of DAPK to control apoptosis are likely to have clinical relevance.

[1] 59w DAPK, a calcium/calmodulin (CaM)-regulated serine/ threonine protein kinase, was originally identified as a factor that could protect against interferon-gamma (INF-c)induced apoptosis [6,7]. DAPK consists of several functional domains, including a kinase domain, a Ca 2? /CaMbinding motif, eight ankyrin repeats, a cytoskeleton-binding region, and a death domain; the kinase and death domains are both important for proapoptotic activity [8,9].

[2] 72w It has been shown that DAPK is negatively regulated by autophosphorylation in a Ca 2? /CaM-dependent manner [9]. Binding of Ca 2? -activated CaM to the Ca 2? /CaM-binding motif leads to the dephosphorylation of Ser308 in the CaMregulatory domain and consequently promotes catalytic activity of DAPK [10]. DAPK autophosphorylation occurs in the absence of Ca 2? /CaM. Thus, the proapoptotic function of DAPK is inhibited by autophosphorylation in the basal state.

[3] 91w Catalytic activity is also essential for the proapoptotic function of DAPK. DAPK catalytic activity is abolished by the substitution of a conserved lysine in the ATP-binding loop in the kinase domain with an alanine residue (K42A). Overexpression of wild-type DAPK reduces the number of viable cells, whereas the catalytically inactive mutant of DAPK (K42A) completely loses its death-inducing activity [9]. These results show that the induction of cell death depends on the status of the intrinsic kinase activity of DAPK, and that catalytic activity is clearly required for DAPK proapoptotic function.

[4] 79w Furthermore, the death domain is important for the death-promoting function of DAPK. Deletion of the CaMregulatory domain generates a constitutively active mutant of DAPK that induces cell death. Deletion of the death domain from this mutant reduces the number of apoptotic cells [8]. In addition, co-transfection of the death domaincoding fragment of DAPK reduces the cell death induced by DAPK overexpression [8]. Thus, the death domain of DAPK also plays an important role in the apoptotic effect of DAPK.

[5] 109w DAPK stimulates caspase-linked apoptosis [11] and p53-mediated cell death [12]. It also enhances autophagyassociated cell death via interaction with Beclin-1, a BH3domain-only protein [13,14]. Although the regulatory pathways of DAPK-induced apoptosis have been largely defined for non-neuronal cells, DAPK is also critically involved in neuronal cell death. DAPK activity is increased in response to various neurotoxic insults, such as hypoxic ischemia [15,16], and a specific peptide inhibitor of DAPK is known to significantly attenuate brain damage after ischemic stroke [16,17]. DAPK also regulates neuronal death in the developmental nervous system. Dependence receptors, which induce apoptosis when their ligand is absent, have been shown to regulate apoptosis through DAPK [4,5].

[6] 83w DAPK mRNA is abundantly expressed in the developing and adult CNS of rats, and is observed in both proliferative and postmitotic cells within the cerebral cortex, hippocampus, and cerebellum from embryonic day 13 [18,19]. DAPK mRNA expression in the brain is markedly decreased postnatally, but remains high in several neuronal populations, such as the olfactory bulb, hippocampal formation, and cerebellar purkinje and granule cells. This temporal and spatial regulation of DAPK expression suggests that it may be involved in developmental neuronal cell death.

[7] 48w The DAPK knockout mouse is viable and fertile [12]; however, it has abnormal renal tubule morphology, possibly because of decreased renal tubule apoptosis [20]. Although an abnormality in the CNS has not been reported in DAPK knockout mice, deletion of DAPK shows neuroprotective effects against various toxic assaults.

[8] 149w In the CNS, cell death is normally observed during development, in response to injury and in neurodegenerative disorders [21][22][23][24]. An increase in DAPK expression has been associated with neuronal damage in both in vivo and in vitro models. The in vivo models include those of acute injury, such as seizure induction produced by administration of excitatory amino acids, and ischemic injury resulting from middle cerebral artery occlusion (MCAO). Administration of kainic acid into the rat amygdala nucleus induces seizures, which results in the death of CA3 neurons. This is accompanied by increased levels of DAPK protein and an increase in the interaction of DAPK and p53 [25,26]. Elevated expression of DAPK has also been observed following ischemia. Global cerebral ischemia causes cell death, which is associated with an increase in the expression of DAPK mRNA. In addition DAPK is dephosphorylated and activated after focal cerebral ischemia by MCAO [15,16].

[9] 108w The results obtained with in vitro models of neuronal injury are consistent with the aforementioned data for in vivo models. Oxygen-glucose deprivation of cultured primary cortical neurons mimics the conditions of ischemia in vitro and causes cell death. DAPK is dephosphorylated and activated under oxygen-glucose deprivation. This activation can be reduced by an inhibitor of calcineurin, FK506, or by MK-801, a selective N-methyl-d-aspartate (NMDA) receptor antagonist [16]. Increased DAPK activity has also been detected in an in vitro model of neurodegenerative attack. Low molecular weight betaamyloid peptide (Ab), which contains Ab species from the monomeric to the tetrameric form, induces apoptosis accompanied by the activation of DAPK [27].

[10] 56w These observations demonstrate that DAPK mRNA or protein can be increased in neurons, and that DAPK is activated in response to diverse conditions of cellular insults, including seizure and ischemia. Taken together, these studies suggest that DAPK is widely involved in the neuronal cell death response to different forms of acute neuronal damage and neurological disorders.

[11] 279w The relationship between DAPK expression and neuronal cell death has been evaluated in various injury and disease models. DAPK-deficient mice or DAPK inhibitors have been used to assess its role in apoptotic cell death. The genetic deletion of DAPK in vivo has been demonstrated to protect neurons from various neurotoxic insults including glutamate toxicity [28] and a mouse model of ischemia [29]. Glutamate is the excitatory neurotransmitter in the CNS. In mammalian retinal ganglion cells, as well as in other CNS neurons, an abnormal increase in glutamate concentration causes cell death. NMDA receptors, which are a glutamate receptor subtype activated by NMDA, are widely thought to be responsible for the neurotoxic effect of glutamate. Knockout of DAPK protects retinal ganglion cells from glutamate toxicity, but not from the toxicity of NMDA [28]. Genetic deletion of DAPK1 protects against ischemic neuronal death, and is consistent with the results obtained from treatment with a DAPK inhibitor. NMDA receptor NR2B subunits are considered to be the main types of functional NMDA receptor channels in CNS neurons and are known to contribute to ischemic neuronal death. Activation of DAPK increases Ca 2? influx through extrasynaptic NMDA receptor channels, leading to cell death. Knockout of DAPK1 suppresses NR2B receptor channel activity, and reduces the brain infarction volume [29]. In addition, inhibition of DAPK activity using pharmacological interventions protects neurons from apoptosis. Administration of an aminopyridazine-based small molecule, which specifically inhibits DAPK activity, reduces brain infarction volume in the mouse model of MCAO [16,17]. Suppression of DAPK dephosphorylation contributes to neuroprotective effects in a mouse model of ischemia [30]. Thus, the inhibition of DAPK expression or function protects neurons in the context of neuronal injury.

[12] 111w Cultured cells deficient in both DAPK alleles also exhibit protection from death responses. DAPK knockout fibroblasts are resistant to endoplasmic reticulum (ER) stress [20]. A high concentration of ceramide induces apoptotic neuronal cell death in a manner that is dependent on the stage of development [31]. In cultured hippocampal neurons, deficiency of DAPK inhibits apoptosis induced by a high concentration of ceramide [32]. In contrast, mouse embryonic fibroblasts lacking DAPK show normal death responses to UV light, adriamycin and staurosporine [20]. These observations show that DAPK plays an important role in apoptosis both in vivo and in vitro although its deficiency does not protect cells against all forms of toxic insults.

[13] 173w Furthermore, DAPK has a critical role in neurodegenerative diseases, such as Alzheimer's disease (AD). DAPK is highly expressed in the adult rat brain, and its expression in parts of the brain including the hippocampus and cortex, is particularly affected in AD [18]. Deletion of DAPK kinase activity promotes learning and memory [33]. A large-scale genetic study revealed that the association of two single nucleotide polymorphisms (SNPs) in DAPK occurs with late onset Alzheimer's disease (LOAD) [34]. A recent study has also demonstrated possible molecular mechanisms for the effect of DAPK on LOAD. Tau phosphorylation at microtubule-affinity regulating kinase (MARK)-directed sites is increased in the brains of AD patients and AD transgenic mouse models [35][36][37]. DAPK endogenously interacts with MARK2, and inhibition of DAPK expression by siRNA reduces MARK2-induced phosphorylation on endogenous tau. Reduction of tau phosphorylation is also observed in brain extracts prepared from DAPK-deficient mice. These results indicate that DAPK enhances MARK-induced tau phosphorylation and tau toxicity, and provide a molecular linkage between DAPK and tauopathy-related neurodegenerative disorders such as AD [38].

[14] 169w A role for DAPK in apoptosis has also been demonstrated during development. The development of individual neurons is characterized by progressive cellular events involving proliferation, migration, differentiation, pathway formation, and synaptogenesis. Death, along with these progressive events, is essential for the establishment and maintenance of the nervous system. In the developing nervous system, neurons are initially overproduced after which approximately one-half of the original cell population dies. This naturally occurring cell death during normal development has been called PCD. PCD is defined as the reproducible, spatiotemporally specific occurrence of individual cell loss during development [39]. It is less clear whether the cells that will die perform some developmental function before their death, and whether the process of PCD is important for the developmental nervous system. However, possible functions of PCD are thought to include removing overproduced cells, limiting the progenitor pool size during neurogenesis, or eliminating aberrant connections during synaptogenesis [1,2]. Therefore, regressive cellular events, such as PCD, may be essential for the establishment of a functional neural network.

[15] 157w Both progressive and regressive events during development are regulated by intercellular signaling pathways. PCD has been suggested to occur by default signals from other cells [40]. From this point of view, neurons may escape PCD only by receiving the appropriate survival signals. Some intercellular signals that contribute to the regulation of progressive events during nervous system development also regulate regressive events. It is well established that the survival of many mammalian cell types, including developing immature neurons, depends on functionally diverse growth factors such as nerve growth factor (NGF). These growth factors suppress cell death by binding to and initiating survival signaling through their cognate cell surface receptors [41][42][43]. For example, NGF maintains cellular survival through tropomyosin receptor kinases (Trks) and downstream signaling via the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway. While these positive survival signals are extremely important, removal of NGF leads to neuronal cell death, usually involving a major class of death proteases known as caspases [41][42][43].

[16] 155w DAPK is a crucial protein as the proapoptotic partner of the dependence receptor (Fig. 1). Llambi et al. [4] showed that DAPK was required for UNC5H2-mediated apoptosis. The receptors in the Unc5 family (UNC5H1-4; also named Unc5 A, B, C, and D in human) function as netrin-1 receptors, and were originally proposed to mediate the chemorepulsive effects of netrin-1 [57,58]. It has been demonstrated that netrin-1 mediates chemoattractive effects in axons that express only DCC, whereas it mediates chemorepulsive effects when axons express both DCC and UNC5H. In the absence of netrin-1, expression of UNC5H induces apoptosis [59]. Therefore, UNC5H proteins have been described as dependence receptors. The death domain of UNC5H2 interacts with DAPK, and this interaction is required for UNC5H2induced cell death. Binding of netrin-1 to UNC5H2 inhibits DAPK activity by reducing DAPK autophosphorylation. These results indicate that UNC5H2 induces cell death by activating DAPK in the absence of its ligand, netrin-1 [4].

[17] 176w DAPK has also been shown to be involved in the apoptosis induced by another dependence receptor, Neogenin [5]. Neogenin was first isolated from embryonic chicken cerebellum as a DCC homologue [60]. Both Neogenin and DCC are transmembrane proteins of the immunoglobulin superfamily, and comprise four immunoglobulins and six fibronectin III domains in their extracellular domains. Neogenin mediates axon guidance by binding its ligands, either repulsive guidance molecule (RGM) or netrin-1 [61], and controls neuronal differentiation and survival during CNS development [62,63]. In the ligand-free state, Neogenin induces apoptosis [64]. Therefore, Neogenin is also described as a dependence receptor. Neogenin interacts with DAPK, triggering cell death through DAPK activity. Neogenin-induced cell death is inhibited in the presence of its ligand, RGMa. The ligandreceptor complex, RGMa/Neogenin, enhances DAPK autophosphorylation. Further, knockdown of RGMa by siRNA induces apoptosis in the chick neural tube. This effect is inhibited by coelectroporation of DAPK siRNA, or by a plasmid encoding DAPK dominant negative. These observations indicate that Neogenin induces cell death by activating DAPK in the absence of its ligand, RGMa [5].

[18] 157w Neogenin and DCC are the vertebrate orthologs of the Caenorhabditis elegans protein, UNC-40 [65]. Neogenin lacks a death domain and is not structurally related to the UNC5H2 receptor. While it contains domains with a high homology to DCC in its extracellular portion, the cytoplasmic domains of both proteins are less conserved [66,67]. The P3 domain in the intracellular domain of DCC is necessary for the axonal projection of corticospinal tract fibers into the spinal cord in mice [68]. Although the amino acid sequence of the P3 domain in Neogenin shows high similarity to DCC, DAPK does not interact with DCC. These observations suggest that even if DAPK regulates death signaling induced by several dependence receptors, the mechanisms by which the receptors recruit DAPK are different from one another. Consistently, the functional role of DAPK in dependence receptor-induced cell death can be similar in UNC5H2 and Neogenin. Indeed, DAPK is activated only in the absence of their ligands.