Role of TRP channels in secondary injury after brain trauma

描述（由申请人提供）：创伤性脑损伤（TBI）不可避免地并发微血管功能障碍和/或衰竭，导致水肿形成，甚至更糟的是，“进行性继发性出血”（PSH）。这些异常导致缺血、血液和炎性细胞外渗、CNS组织的自身破坏和神经学结果的恶化。我们最近发现NCCa-ATP通道在PSH中起关键作用，强有力的证据显示通道的调节亚基磺酰脲受体1（SUR 1）的关键参与。新的初步数据强烈表明，一个不寻常的成员的瞬时受体电位（TRP）家庭，TRPM 4，作为孔形成亚基的通道，和该通道是转录上调微血管内皮细胞通过机械激活NF-？B，其在力传递到体内组织和体外培养的内皮细胞的几分钟内发生。我们的首要假设是，创伤相关的机械力激活机械敏感的转录因子，NF-？B，导致微血管内皮中TRPM 4通道的转录上调，并且这些通道在TBI后进行性继发性出血（PSH）的病理生理学中起关键作用。这一假说的许多分支将使用分子和细胞生物学，膜片钳电生理学进行测试。在特定目标（SA）1中，我们将使用TRPM 4基因敲除小鼠来确定TRPM 4通道对PSH的整体病理生理学的贡献程度。出于同样的目的，我们还将评价2种具有较强翻译潜力的治疗：使用IV反义寡核苷酸（AS-ODN）进行短暂（24小时）TRPM 4基因抑制，以及利鲁唑（一种FDA批准的多效性药物，是TRPM 4通道的强效阻断剂）。在SA 2中，我们将研究生理调制的通道在新鲜分离的CNS毛细血管从TBI小鼠，并在培养的内皮细胞暴露于“创伤样”的机械刺激（拉伸），我们将确定的具体作用的通道功能障碍和死亡的毛细血管内皮细胞。在SA 3中，我们将研究机械活化转录因子NF-？B，在通道的转录调节中，我们将评估在TBI中靶向该转录因子是否由于干扰TRPM 4通道的表达而有益。TBI仍然是死亡和残疾的主要原因，目前还没有治疗方法来减少继发性损伤。我们期望发现TRPM 4在TBI后导致PSH的毛细血管破坏中起着关键作用，并且通过基因抑制或转录水平阻断TRPM 4将显著减少PSH和出血引起的继发性损伤的多个级联反应。这些发现将在TBI中建立一个新的和强大的治疗靶点，导致AS-ODN和/或利鲁唑的进一步临床前和临床试验，我们预测这将对人类TBI的急性治疗产生重大影响。

DESCRIPTION (provided by applicant): Traumatic brain injury (TBI) is inevitably complicated by microvascular dysfunction and/or failure, resulting in formation of edema and, even worse, "progressive secondary hemorrhage" (PSH). These abnormalities lead to ischemia, extravasation of blood and inflammatory cells, autodestruction of CNS tissues, and worsening of neurological outcome. We recently discovered that the NCCa-ATP channel is critically involved in PSH, with strong evidence showing critical involvement of sulfonylurea receptor 1 (SUR1), the regulatory subunit of channel. New preliminary data strongly suggest that an unusual member of the transient receptor potential (TRP) family, TRPM4, acts as the pore-forming subunit of the channel, and that the channel is transcriptionally upregulated in microvascular endothelial cells via mechano-activation of NF-?B, which occurs within minutes of force delivery to tissues in vivo and to cultured endothelial cells in vitro. Our overarching hypothesis is that trauma-associated mechanical forces activate the mechano-sensitive transcription factor, NF-?B, resulting in transcriptional upregulation of TRPM4 channels in microvascular endothelium, and that these channels play a critical role in the pathophysiology of progressive secondary hemorrhage (PSH) following TBI. Numerous ramifications of this hypothesis will be tested using molecular and cell biology, and patch clamp electrophysiology. In Specific Aim (SA) 1, we will use TRPM4 gene knock-out mice to determine the extent to which TRPM4 channels contribute to the overall pathophysiology of PSH. In the same Aim, we will also evaluate 2 treatments with strong translational potential: brief (24-hr) TRPM4 gene suppression using IV antisense oligodeoxynucleotide (AS-ODN), and riluzole, a pleiotropic FDA-approved drug that is a potent blocker of TRPM4 channels. In SA2, we will examine physiological modulation of the channel in freshly isolated CNS capillaries from TBI-mice, and in cultured endothelial cells exposed to a "trauma-like" mechanical stimulus (stretch), and we will determine the specific role of the channel in dysfunction and death of capillary endothelial cells. In SA3, we will examine the role of the mechano-activated transcription factor, NF-?B, in transcriptional regulation of the channel, and we will assess whether targeting this transcription factor in TBI is beneficial due to interference with expression of TRPM4 channels. TBI continues to be a leading cause of death and disability, with no treatments yet available to reduce secondary injury. We expect to find that TRPM4 plays a critical role as end-executioner in capillary destruction leading to PSH following TBI, and that block of TRPM4 by gene suppression, at the transcriptional level, or pharmacologically will yield significant reductions in the multiple cascades of secondary injury attributable to PSH and hemorrhage. These findings would establish a novel and powerful therapeutic target in TBI, leading to further pre-clinical as well as clinical trials with AS-ODN and/or riluzole, which we predict will have a major impact on acute treatment of TBI in humans.