Dynorphin Modulation of N-Methyl-D-Aspartate (NMDA) Receptor Function

描述（由申请人提供）：每年有成千上万的生命因脊髓损伤而毁灭。如果设计出能够阻止创伤后残存组织退化的治疗方法，这些患者所经历的运动缺陷和疼痛的很大一部分是可以预防的。脊髓创伤会引发一系列生化反应，从而加重损伤。延长的损伤导致运动功能的进一步下降和感觉障碍，如慢性疼痛。作为这些过程的一部分，阿片肽啡肽浓度增加，并在创伤后的脊髓中释放。Dynorphin先前被证明可以增强n -甲基- d -天冬氨酸（NMDA）受体功能，从而在脊髓中产生神经元损伤。因此，肌啡可能是侮辱后退行性事件的重要参与者。本研究将验证一种假设，即dynorphin与不具有NRla外显子5编码区域的NR1剪接变异体结合，以暴露先前失活或低活性的NMDA受体。第二个有待验证的假设是，肌啡诱导NMDA受体的内部储存向质膜迁移。为了验证这些假设，提出了三个目标。在第一个目的中，将32对可能的NR1剪接变异体和NR2亚基瞬时转染Hek-293细胞。各种亚基组合对dynorphin和NMDA诱导的兴奋性毒性的敏感性将通过使用高通量96孔试验测量乳酸脱氢酶释放到培养基中来评估。在第二个目标中，将使用全细胞膜片钳和细胞表面标记技术检查对肌啡肽最敏感的NR1和NR2亚基的组合，以确定肌啡肽如何影响受体的功能。在最终目的中，NR1剪接变异体和NR2亚基在脊髓中的表达将通过免疫共沉淀、2d电泳、免疫组织化学和RT-PCR来确定。这些实验将提供dynorphin增强脊髓NMDA受体的亚基身份以及dynorphin增强NMDA受体功能的机制。希望这一信息将为阻断dynorphin参与脊髓损伤后神经退行性变提供有价值的药理学靶点。

DESCRIPTION (provided by applicant): Thousands of lives are devastated each year by spinal cord injury. A significant portion of the motor deficits and pain experienced by these patients could be prevented if therapies were devised to block posttrauma degradation of the surviving tissue. Trauma to the spinal cord initiates a cascade of biochemical events that exacerbate the injury. The extended damage leads to an additional decline in motor function and to sensory disturbances such as chronic pain. As part of these processes the opioid peptide dynorphin increases in concentration and is released in the spinal cord during the post-trauma period. Dynorphin was previously demonstrated to enhance N-methyl-D-aspartate (NMDA) receptor function to produce neuronal damage in the spinal cord. Thus, dynorphin is likely to be a significant participant in the post-insult degenerative events. This study will test the hypothesis that dynorphin binds to NR1 splice variants that do not possess the exon-5 coded region of the protein (NRla) to expose previously inactive or low activity NMDA receptors. A second hypothesis that will be tested is that dynorphin induces migration of internal stores of NMDA receptors to the plasma membrane. To test these hypotheses three aims are proposed. In the first aim Hek-293 cells will be transiently transfected with the 32 possible pairs of NR1 splice variants and NR2 subunits. The sensitivity of the various subunit combinations to dynorphin and NMDA induced excitotoxicity will be evaluated by measuring the release of lactate dehydrogenase into the culture media using a high throughput 96 well assay. In the second aim the combinations of NR1 and NR2 subunits that are most sensitive to dynorphin (determined from aim 1) will be examined using whole cell patch clamp and cell surface labeling techniques to determine how dynorphin influences the receptor's function. In the final aim the pairs of NR1 splice variants and NR2 subunits expressed in the spinal cord will be determined using co-immunoprecipitation, 2D-electrophoresis, immunohistochemistry and RT-PCR. These experiments will provide the subunit identity of the dynorphin enhanced NMDA receptors in the spinal cord and the mechanism for how dynorphin enhances NMDA receptor function. It is hoped that this information will provide a valuable pharmacological target for interrupting dynorphin's participation in post spinal trauma neurodegeneration.