DAPK1 Interaction with NMDA Receptor NR2B Subunits Mediates Brain Damage in Stroke

“CaMK-II is one of the most abundant Ca/CaM-dependent enzymes in the central neurons and participates in synaptic plasticity (Lisman et al., 2002) as well as in the cellular events responsible for ischemic neuronal death (Waxham et al.”

“Ca overload triggers multiple intracellular catabolic processes, and thus induces an irreversible death of neuronal cells in the brain (for review, see Lipton, 2006; Lo et al., 2003).”

“DISCUSSION NMDA receptor NR2B subunits have recently become the major focus of studies in the treatment of stroke because of their unique features (Hardingham et al., 2002; Simpkins et al., 2003; Liu et al., 2007 ).”

“These three groups of neurons were examined because they are known to be vulnerable to transient global ischemic insult (Simon et al., 1984; Pellegrini-Giampietro et al., 1997; Wang et al., 2003a; Liu et al., 2004; Peng et al., 2006).”

“This feature enables the receptor to detect extrasynaptic glutamate spillover in the brain during ischemic insults (Drejer et al., 1985; Rossi et al., 2000 ).”

“Alternatively, recent studies have focused on NMDA receptor NR2B subunit downstream cell death signaling via interaction with PSD-95 (Aarts et al., 2002).”

“Ca 2+ overload triggers multiple intracellular catabolic processes, and thus induces an irreversible death of neuronal cells in the brain (for review, see Lipton, 2006; Lo et al., 2003).”

“Ca 2+ overload triggers several downstream lethal reactions including nitrosative stress (Aarts et al., 2003; Uehara et al., 2006), oxidative stress (Kinouchi et al., 1991) and mitochondrial dysfunction (Krajewski et al., 1999; Fiskum et al., 1999).”

“Glutamate is the major excitatory transmitter in the mammalian central nervous system (CNS) and plays an essential role in neural development, excitatory synaptic transmission, and plasticity (Greengard, 2001).”

“Ca 2+ overload triggers several downstream lethal reactions including nitrosative stress (Aarts et al., 2003; Uehara et al., 2006), oxidative stress (Kinouchi et al., 1991) and mitochondrial dysfunction (Krajewski et al., 1999; Fiskum et al., 1999).”

“…DAPK1 enhances the channel conductance of the recombinant NR1/NR2B receptors, which are known to be expressed predominantly in the extrasynaptic sites (Tovar and Westbrook, 1999; Brickley et al., 2003), we reasoned that an activated DAPK1 may functionally interact with extrasynaptic NMDA receptors.”

“…5 (Cdk5) (Wang et al., 2003a), tyrosine kinases (Khanna et al., 2007), and nNOS (Eliasson et al., 1999), however, also control the physiological processes of gene expression, as well as synaptic plasticity (Ghosh, 2002; Salter and Kalia, 2004), and thus are not promising targets for stroke therapy.”

“D) and possibly the more recently identified NR3 subunit (Chatterton et al., 2002).”

“Ca 2+ overload triggers several downstream lethal reactions including nitrosative stress (Aarts et al., 2003; Uehara et al., 2006), oxidative stress (Kinouchi et al., 1991) and mitochondrial dysfunction (Krajewski et al., 1999; Fiskum et al., 1999).”

“However, this strategy has been challenged by its parallel inhibition of synaptic plasticity (El-Husseini et al., 2002).”

“These three groups of neurons were examined because they are known to be vulnerable to transient global ischemic insult (Simon et al., 1984; Pellegrini-Giampietro et al., 1997; Wang et al., 2003a; Liu et al., 2004; Peng et al., 2006).”

“Ca 2+ overload triggers several downstream lethal reactions including nitrosative stress (Aarts et al., 2003; Uehara et al., 2006), oxidative stress (Kinouchi et al., 1991) and mitochondrial dysfunction (Krajewski et al., 1999; Fiskum et al., 1999).”

“, 2004), and possibly via a cleavage of Na/Ca exchangers (Bano et al., 2005).”

“Immediately following ischemia, however, glutamate accumulates at synapses (Drejer et al., 1985; Rossi et al., 2000), resulting in extensive stimulation of its receptors that can eventually be toxic to neurons (Lipton, 2006; Lo et al., 2003).”

“Since the NR2C(and possibly NR2D-) containing receptors have very low probability for their channel opening (Sprengel et al., 1998), both NR2A- and NR2B-containing NMDA receptors are considered as the main types of functional NMDA receptor channels in CNS neurons (Madden, 2002).”

“Second, extrasynaptic NMDA receptors are reported to link the specific signals that induce neuronal death in ischemia (Finkbeiner, 2000; Hardingham et al., 2002, Takasu et al., 2002; Liu et al., 2007).”

“DAPK1 belongs to a Ca 2+ /CaM-dependent protein kinase family, and comprises a kinase domain, a CaM-binding motif, eight ankyrin repeats, and a death domain (for reviews, see Bialik and Kimchi, 2006; Van Eldik, 2002; Velentza et al., 2001).”

“, 2007), and nNOS (Eliasson et al., 1999), however, also control the physiological processes of gene expression, as well as synaptic plasticity (Ghosh, 2002; Salter and Kalia, 2004), and thus are not promising targets for stroke therapy.”

“Ca 2+ overload triggers several downstream lethal reactions including nitrosative stress (Aarts et al., 2003; Uehara et al., 2006), oxidative stress (Kinouchi et al., 1991) and mitochondrial dysfunction (Krajewski et al., 1999; Fiskum et al., 1999).”

“Interestingly, the NR2B at Ser-1303 is the same residue as that catalyzed by CaMK-II (Omkumar et al., 1996).”

“However, it is now believed that NMDA receptor NR2B subunits may be also combined with NR1/NR2A subunits to form NR1/NR2A/NR2B complex receptors at synapses (Thomas et al., 2006).”

“…the most parsimonious explanation for our findings is that under physiological conditions, an initial Ca2+ influx through NMDA receptor channels produces partial inhibition of NMDA receptors via Ca2+-dependent inactivation, thereby preventing the intracellular Ca2+ overloading (Krupp et al., 1999).”

“Local blood flow was monitored using BLF21C Laser Doppler Flow-meter with LL-M1043 fiber flow-probe (Transonic System Inc, New York), as described before (Wang et al., 2003a; Liu et al., 2004; Peng et al., 2006, see also Extended Experimental Procedures).”

“, 1999), Ca/CaM-activated protein kinase II (CaMK-II) (Mabuchi et al., 2001), protein kinase C (Sun et al.”

“However, CaMK-II was 230 Cell 140, 222–234, January 22, 2010 ª2010 Elsevier Inc. found to become inactivated in the brain in response to ischemic insults (Hanson et al., 1994; Waxham et al., 1996; Petersen et al., 2003).”

“However, CaMK-II was found to become inactivated in the brain in response to ischemic insults (Hanson et al., 1994; Waxham et al., 1996; Petersen et al., 2003).”

“…DAPK1 enhances the channel conductance of the recombinant NR1/NR2B receptors, which are known to be expressed predominantly in the extrasynaptic sites (Tovar and Westbrook, 1999; Brickley et al., 2003), we reasoned that an activated DAPK1 may functionally interact with extrasynaptic NMDA receptors.”

“Catalytic activity of DAPK1 is controlled by its autophosphorylation at serine-308 (pS308); dephosphorylation of pS308 allows DAPK1 to bind with CaM, thereby activating DAPK1 (Shohat et al., 2001).”

“DISCUSSION NMDA receptor NR2B subunits have recently become the major focus of studies in the treatment of stroke because of their unique features (Hardingham et al., 2002; Simpkins et al., 2003; Liu et al., 2007 ).”

“, 2001), protein kinase C (Sun et al., 2008), cycline-dependetn kinase 5 (Cdk5) (Wang et al.”

“It has been known that enzymatic activity of CaN depends on Ca2+ influx through NMDA receptor channels (Marshall et al., 2003).”

“, 2003a), tyrosine kinases (Khanna et al., 2007), and nNOS (Eliasson et al.”

“DAPK1 belongs to a Ca 2+ /CaM-dependent protein kinase family, and comprises a kinase domain, a CaM-binding motif, eight ankyrin repeats, and a death domain (for reviews, see Bialik and Kimchi, 2006; Van Eldik, 2002; Velentza et al., 2001).”

“Also, some other studies indicated that inhibition of PSD-95 increased brain damage in stroke (Gar- doni et al., 2002).”

“However, CaMK-II was 230 Cell 140, 222–234, January 22, 2010 ª2010 Elsevier Inc. found to become inactivated in the brain in response to ischemic insults (Hanson et al., 1994; Waxham et al., 1996; Petersen et al., 2003).”

“We next examined whether an activated DAPK1 phosphorylates NR2B subunit at Ser-1303, (Figure 3E), a residue corresponding to a phosphorylation site (KXXRXXSX) of DAPK1 (Schumacher and Schavocky, 2004).”

“Multiple intracellular signals are able to regulate NMDA receptor channel activity (Ghosh, 2002) and induce NMDA receptor-dependent cell death (Simon et al., 1984; Wang et al., 1999 ), protein kinase C (Sun et al., 2008), cycline-dependetn kinase 5 (Cdk5) (Wang et al., 2003a), tyrosine kinases…”

“DAPK1 belongs to a Ca 2+ /CaM-dependent protein kinase family, and comprises a kinase domain, a CaM-binding motif, eight ankyrin repeats, and a death domain (for reviews, see Bialik and Kimchi, 2006; Van Eldik, 2002; Velentza et al., 2001).”

“In particular, deleterious effects can be mediated by extrasynaptic receptors containing GluN2B subunits (Tu et al., 2010).”

“A recent study has demonstrated that extrasynaptic NMDAR activity may be selectively enhanced in ischaemia through an intracellular signalling cascad”

“excitotoxic neuronal death following ischemia in vitro and in vivo, without affecting NMDAR channel activity or other downstream signaling proteins (Aarts et al., 2002; Sattler et al., 1999; Tu et al., 2010; Wang et al., 2003).”