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30多年前描述的一种鲜为人知的病理实体，导致脊髓组织的毁灭性损失和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控亚基，即NC（Ca- ATP）通道，在PHN中起关键作用，但该通道的成孔亚基尚未被分子鉴定。我们实验室的新实验提供了证据，证明TRPM4可能是通道的成孔亚基。本提案的目的是通过确定TRPM4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性脊髓损伤模型中的初步数据表明，药物阻断和基因抑制TRPM4可显著减少出血和进行性病变扩展，并且这些作用与神经行为功能结果的显着改善有关。在特异性目的（SA） 1中，我们将使用TRPM4- ko小鼠来确定TRPM4通道在多大程度上参与了脊髓损伤的PHN和其他继发性损伤的表现。其他初步数据表明，参与PHN最关键的细胞是毛细血管内皮细胞和毛细血管后静脉内皮细胞。在SA2实验中，我们将利用膜片钳固定脊髓损伤后新鲜分离的脊髓毛细血管和体外培养的中枢神经系统微血管内皮细胞暴露于TNFalpha，确定TRPM4通道在内皮细胞中的生理调控和功能作用。其他初步数据表明，NFkappaB可能是TRPM4通道的重要转录调节因子，它是TNFalpha的下游效应因子，已知在SCI中起重要作用。在SA3中，我们将利用大鼠脊髓损伤模型组织和CNS微血管内皮细胞培养物，确定转录因子NFkappaB在TRPM4通道表达中的作用，并研究NFkappaB抑制对脊髓损伤与TRPM4表达结果的影响。总的来说，了解TRPM4通道在脊髓损伤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。通过使用啮齿类动物脊髓损伤模型，我们发现TRPM4通道的药理学和反义抑制可显著减少出血性坏死并显著改善神经功能。在这项提议中，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内皮细胞培养来牢固地建立控制TRPM4通道表达和功能的基本分子原理，这将为未来新的脊髓损伤治疗奠定基础。

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.