TRPM4 channel in spinal cord injury

描述（由申请人提供）：脊髓损伤（SCI）导致“进行性出血性坏死”（PHN），这是一种30多年前描述的病理实体，其导致脊髓组织的破坏性损失和使人衰弱的神经功能障碍。我们最近发现，非选择性阳离子通道的调节亚基，NC（Ca-ATP）通道，是关键参与PHN，但通道的孔形成亚基没有分子鉴定。我们实验室的新实验提供了TRPM 4可能是通道的成孔亚基的证据。本提案的目的是通过确定TRPM 4在SCI后PHN中的作用来扩展这一发现。我们在大鼠和小鼠挫伤SCI模型中的初步数据表明，TRPM 4的药理学阻断和基因抑制显著减少了出血和进行性病变扩展，并且这些作用与神经行为功能结局的显著改善相关。在特定目标（SA）1中，我们将使用TRPM 4-KO小鼠来确定TRPM 4通道参与PHN和SCI继发性损伤的其他表现的程度。其他初步数据表明，PHN中最关键的细胞是毛细血管和毛细血管后微静脉内皮细胞。在SA 2中，使用膜片钳的新鲜分离的脊髓毛细血管后SCI和培养的CNS微血管内皮细胞暴露于TNF α，我们将确定的生理调节和TRPM 4通道在内皮细胞中的功能作用。其他初步数据表明，NF κ B是TNF α的下游效应子，已知其显著参与SCI，可能作为TRPM 4通道的重要转录调节因子。在SA 3中，使用来自大鼠SCI模型的组织和CNS微血管内皮细胞培养物，我们将确定转录因子NF κ B在TRPM 4通道表达中的作用，并且我们将检查NF κ B抑制对SCI维斯-`-维斯TRPM 4表达结果的影响。总的来说，了解TRPM 4通道在SCI中的作用将为这种毁灭性的人类疾病带来新的分子见解和新的治疗方法。使用脊髓损伤的啮齿动物模型，我们发现TRPM 4通道的药理学和反义抑制引起出血性坏死的显著减少和神经功能的显著改善。在这项提案中，我们将使用小鼠基因敲除模型，新鲜分离的脊髓毛细血管和CNS内皮细胞培养物来牢固地建立控制TRPM 4通道表达和功能的基本分子原理，这将成为未来脊髓损伤新疗法的基础。

DESCRIPTION (provided by applicant): Spinal cord injury (SCI) results in "progressive hemorrhagic necrosis" (PHN), a poorly understood pathological entity described over 30 years ago that leads to devastating loss of spinal cord tissue and debilitating neurological dysfunction. We recently discovered that the regulatory subunit of the non-selective cation channel, the NC(Ca- ATP) channel, is critically involved in PHN, but the pore-forming subunit of the channel was not molecularly identified. New experiments in our lab provide evidence that TRPM4 is likely to be the pore-forming subunit of the channel. The purpose of this proposal is to expand upon this finding by establishing the role of TRPM4 in post-SCI PHN. Our preliminary data in rat and mouse models of contusion SCI demonstrated that hemorrhage and progressive lesion expansion were dramatically reduced by pharmacological block and gene suppression of TRPM4, and that these effects were associated with a dramatic improvement in neurobehavioral functional outcome. In specific aim (SA) 1 we will use TRPM4-KO mice to determine the extent to which TRPM4 channels are involved in PHN and other manifestations of secondary injury in SCI. Other Preliminary Data indicate that the cells most critically involved in PHN are capillary and post-capillary venular endothelial cells. In SA2, using patch clamp of freshly isolated spinal cord capillaries post-SCI and cultured CNS microvascular endothelial cells exposed to TNFalpha, we will determine the physiological regulation and the functional role of TRPM4 channels in endothelial cells. Other Preliminary Data demonstrate that NFkappaB, which is the downstream effector of TNFalpha and which is known to be prominently involved in SCI, is likely to act as an important transcriptional regulator of TRPM4 channels. In SA3, using tissues from a rat SCI model and cultures of CNS microvascular endothelial cells, we will determine the role of the transcription factor, NFkappaB, in expression of TRPM4 channels, and we will examine the effect of NFkappaB suppression on outcome in SCI vis-`-vis TRPM4 expression. Overall, an understanding of the role of TRPM4 channels in SCI will lead to novel molecular insights and novel treatments for this devastating human condition. PUBLIC HEALTH RELEVANCE Using rodent models of spinal cord injury, we discovered that pharmacological and antisense inhibition of TRPM4 channels cause a striking reduction in hemorrhagic necrosis and a dramatic improvement of neurological function. In this proposal, we will use a murine gene knock out model, freshly isolated spinal cord capillaries, and cultures of CNS endothelial cells to firmly establish essential molecular principles governing TRPM4 channel expression and function that will form the basis for novel future therapies for spinal cord injury.