DAPK1 regulation of NMDA receptors in ischemic neuronal death

描述（由申请人提供）：n -甲基- d -天冬氨酸（NMDA）受体是谷氨酸受体的主要亚型，通常参与整个中枢神经系统的快速兴奋性突触传递。迄今为止，已经克隆了多种NMDA受体亚基蛋白（NR1, NR2A-D）。天然NMDA受体似乎是由一个必需的NR1亚基和一个或多个调控的NR2亚基（NR2A-D）以及最近发现的NR3亚基组成的异聚复合物。NR2A和NR2B受体通道的激活可渗透到Na+和K+以及Ca2+，从而触发多种细胞内分解代谢过程，导致神经元细胞的不可逆死亡。最近，我们利用反相纳米lc -MS/MS质谱法分析了假性或局灶性脑缺血小鼠前脑NR2B受体复合物的蛋白质成分。我们已经证明脑缺血将死亡相关蛋白激酶（DAPK1）招募到NR2B受体复合体中。DAPK1是Ca2+/calmodulin （CaM）依赖性丝氨酸/苏氨酸激酶家族的成员之一，是细胞死亡的关键介质。全细胞膜片钳记录显示，DAPK1的激活增加了NR1/NR2B受体介导的电流。随后，我们产生了基因修饰小鼠（cdDAPK1），其中DAPK1的催化结构域被选择性地删除。我们发现cdDAPK1突变小鼠的前脑神经元对缺血性损伤具有抵抗性。因此，我们假设DAPK1在物理和功能上与NR2B受体相互作用，这种相互作用有助于缺血性卒中的神经元损伤。拟议的研究将解决这个问题。迄今为止，所有针对谷氨酸受体（AMPA或NMDA）的中风临床试验都失败了，可能是因为受体拮抗剂也阻断了谷氨酸的生理作用。本文首次提出了一种靶向DAPK1选择性阻断NR2B受体病理作用的分子方法。因此，这种方法不应该影响大脑中谷氨酸受体的生理作用，从而为中风治疗确定了一个有希望的靶点。公共卫生相关性：中风是美国第三大死亡原因。该疾病的一个关键特征是通过激活谷氨酸受体通道使大脑神经元选择性变性。然而，迄今为止，所有针对谷氨酸受体的中风临床试验都失败了，因为受体拮抗剂也阻断了谷氨酸的生理作用。本文首次提出了一种靶向DAPK1酶选择性阻断谷氨酸受体病理作用的分子方法。因此，这种方法不应该影响大脑中谷氨酸受体的生理作用，从而为中风治疗确定了一个有希望的靶点。

DESCRIPTION (provided by applicant): N-methyl-D-aspartate (NMDA) receptors constitute the major subtype of glutamate receptors and normally participate in rapid excitatory synaptic transmission throughout the CNS. To date, a variety of NMDA receptor subunit proteins (NR1, NR2A-D) have been cloned. Native NMDA receptors appear to be heteroligomeric complexes consisting of an essential NR1 subunit and one or more regulatory NR2 subunits (NR2A-D) and possibly the more recently identified NR3 subunit. Activation of NR2A and NR2B receptor channels are permeable to Na+ and K+ and also to Ca2+, which triggers multiple intracellular catabolic processes, leading to the irreversible death of neuronal cells. Recently, we have used reverse phase nano-LC-MS/MS mass spectrometry to analyze protein components in the NR2B receptor complex from forebrain of mice that had been subjected to sham or focal cerebral ischemia. We have shown that cerebral ischemia recruits death-associated protein kinase (DAPK1) into the NR2B receptor complex. DAPK1 is one member of Ca2+/calmodulin (CaM)-dependent serine/threonine kinase family and functions as a critical mediator of cell death. Whole-cell patch clamp recordings have demonstrated that activation of DAPK1 increases the NR1/NR2B receptor-mediated currents. Subsequently, we have generated genetically modified mice (cdDAPK1), in which catalytic domain of DAPK1 is selectively deleted. We have found that neurons in the forebrain of the cdDAPK1 mutant mice are resistant to ischemic insults. Thus, we hypothesize that DAPK1 physically and functionally interacts with NR2B receptors and this interaction contributes to neuronal injury in ischemic stroke. Proposed studies will address this question. To date, all clinical stroke trials targeting glutamate receptors (AMPA or NMDA) have failed, possibly because receptor antagonists block the physiological actions of glutamate as well. This proposal describes, for the first time, a molecular approach to selectively block the pathological effects of NR2B receptors by targeting DAPK1. Thus, this approach should not affect the physiological actions of glutamate receptors in the brain, thereby defining a promising target for stroke therapy. PUBLIC HEALTH RELEVANCE: Stroke is a third leading cause of death in the United States. A critical feature of the disease is selective degeneration of neurons in the brain by activation of glutamate receptor channels. To date, all clinical stroke trials targeting glutamate receptors have, however, failed, because receptor antagonists block the physiological actions of glutamate as well. This proposal describes, for the first time, a molecular approach to selectively block the pathological effects of glutamate receptors by targeting DAPK1 enzyme. Thus, this approach should not affect the physiological actions of glutamate receptors in the brain, thereby defining a promising target for stroke therapy.