DAPK1 regulation of NMDA receptors in ischemic neuronal death

DAPK1 对 NMDA 受体在缺血性神经元死亡中的调节

• 批准号: 7522367
• 负责人: YOUMING LU
• 金额: $ 21.93万
• 依托单位: UNIVERSITY OF CENTRAL FLORIDA
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-01 至 2008-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7522367
• 关键词: AMPA Receptors; Address; Affect; Binding; Brain; C-terminal; Calcineurin; Calmodulin; Catabolic Process; Catalytic Domain; Cause of Death; Cell Death; Cell Death Signaling Process; Cell Nucleus; Cells; Cerebral Ischemia; Cerebrum; Cessation of life; Clinical; Complex; Consensus; Cytoplasm; DAP kinase; Disease; Enzymes; Extracellular Signal Regulated Kinases; Family; Genetic; Glutamate Receptor; Glutamates; Injury; Ischemia; Ischemic Neuronal Injury; Ischemic Stroke; Link; Mass Spectrum Analysis; Mediating; Mediator of activation protein; Mitogen-Activated Protein Kinases; Molecular; Mus; Mutant Strains Mice; N-Methyl-D-Aspartate Receptors; N-Methylaspartate; NR1 gene; NR2B NMDA receptor; Nerve Degeneration; Neuronal Injury; Neurons; Nuclear Translocation; Phase; Phosphotransferases; Physiological; Prosencephalon; Protein Dephosphorylation; Protein Subunits; Protein phosphatase; Protein-Serine-Threonine Kinases; Proteins; Public Health; Recruitment Activity; Regulation; Resistance; Stroke; Synaptic Transmission; Tail; Testing; Time; United States; human NR1 protein; member; nano; neuron loss; neuronal survival; patch clamp; protein protein interaction; receptor; receptor function; stroke therapy

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.

描述（由申请人提供）：N-甲基-D-天冬氨酸（NMDA）受体构成谷氨酸受体的主要亚型，并且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，多种NMDA受体亚基蛋白（NR1、NR2A-D）已被克隆。天然 NMDA 受体似乎是异寡聚复合物，由一个必需的 NR1 亚基和一个或多个调节性 NR2 亚基 (NR2A-D) 以及可能是最近发现的 NR3 亚基组成。 NR2A 和 NR2B 受体通道的激活可渗透 Na+ 和 K+ 以及 Ca2+，从而触发多种细胞内分解代谢过程，导致神经元细胞不可逆死亡。最近，我们使用反相纳米LC-MS/MS质谱分析了来自假手术或局灶性脑缺血的小鼠前脑的NR2B受体复合物中的蛋白质成分。我们已经证明，脑缺血会将死亡相关蛋白激酶 (DAPK1) 招募到 NR2B 受体复合物中。 DAPK1 是 Ca2+/钙调蛋白 (CaM) 依赖性丝氨酸/苏氨酸激酶家族的成员之一，作为细胞死亡的关键介质发挥作用。全细胞膜片钳记录表明，DAPK1 的激活会增加 NR1/NR2B 受体介导的电流。随后，我们培育了转基因小鼠（cdDAPK1），其中 DAPK1 的催化结构域被选择性删除。我们发现 cdDAPK1 突变小鼠前脑中的神经元能够抵抗缺血性损伤。因此，我们假设 DAPK1 在物理和功能上与 NR2B 受体相互作用，并且这种相互作用导致缺血性中风中的神经元损伤。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）的临床中风试验都失败了，可能是因为受体拮抗剂也阻断了谷氨酸的生理作用。该提案首次描述了一种通过靶向 DAPK1 选择性阻断 NR2B 受体病理效应的分子方法。因此，这种方法不应影响大脑中谷氨酸受体的生理作用，从而确定中风治疗的一个有希望的目标。公共卫生相关性：中风是美国第三大死因。该疾病的一个关键特征是谷氨酸受体通道的激活导致大脑中神经元的选择性变性。然而，迄今为止，所有针对谷氨酸受体的临床中风试验都失败了，因为受体拮抗剂也会阻断谷氨酸的生理作用。该提案首次描述了一种通过靶向 DAPK1 酶来选择性阻断谷氨酸受体病理效应的分子方法。因此，这种方法不应影响大脑中谷氨酸受体的生理作用，从而确定中风治疗的一个有希望的目标。