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

描述(申请人提供)：每年有数以千计的生命被脊髓损伤摧毁。如果设计出阻止创伤后存活组织退化的治疗方法，这些患者经历的运动障碍和疼痛的很大一部分是可以预防的。脊髓损伤会引发一连串的生化事件，从而加剧损伤。这种持续的损伤会导致运动功能的进一步下降，并导致慢性疼痛等感觉障碍。作为这些过程的一部分，阿片肽强啡肽的浓度增加，并在创伤后期间在脊髓中释放。强啡肽此前被证明可以增强N-甲基-D-天冬氨酸(NMDA)受体的功能，从而导致脊髓神经元损伤。因此，强啡肽很可能是侮辱后退行性事件的重要参与者。本研究将检验强啡肽与不具有蛋白质外显子-5编码区(NR1a)的NR1剪接变体结合的假设，以暴露以前不活跃或低活性的NMDA受体。将被检验的第二个假说是强啡肽诱导NMDA受体的内部存储迁移到质膜。为了验证这些假说，我们提出了三个目标。在第一个目标中，将32对可能的NR1剪接变异体和NR2亚单位瞬时导入Hek-293细胞。不同亚基组合对强啡肽和NMDA诱导的兴奋性毒性的敏感性将通过高通量96孔法测量乳酸脱氢酶释放到培养上清液中来评估。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来检测对强啡肽最敏感的NR1和NR2亚基的组合，以确定强啡肽如何影响受体的功能。最终目的是利用免疫共沉淀法、双向凝胶电泳法、免疫组织化学法和RT-PCR法确定脊髓中表达的NR1剪接变异体和NR2亚单位对。这些实验将提供强啡肽增强的NMDA受体在脊髓中的亚单位特性以及强啡肽如何增强NMDA受体功能的机制。希望这一信息将为阻断强啡肽参与脊髓创伤后神经变性提供一个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.