TRPM4 channel in spinal cord injury

TRPM4通道在脊髓损伤中的作用

• 批准号: 7662256
• 负责人: Vladimir Gerzanich
• 金额: $ 32.81万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7662256
• 关键词: Accounting; Blood capillaries; Capillary Endothelial Cell; Cations; Cell Culture Techniques; Cell Death; Cell Line; Cells; Cessation of life; Contusions; Data; Dose; Edema; Electrophoretic Mobility Shift Assay; Employee Strikes; Endothelial Cells; Estrogens; Flufenamic Acid; Future; Gene Knock-Out Model; Genes; Hemorrhage; Human; In Vitro; Inflammation; Injury; Knock-out; Knockout Mice; Lead; Lesion; Mediating; Modeling; Molecular; Mus; Necrosis; Necrotic Lesion; Nervous System Physiology; Neurologic Dysfunctions; Neurophysiology - biologic function; Nuclear; Outcome; Phosphatidylinositol 4,5-Diphosphate; Phospholipase C; Physiological; Preparation; Property; Proteins; Rattus; Regulation; Reporter Genes; Reporting; Rodent Model; Role; Series; Spinal Cord; Spinal cord injury; Time; Tissues; Transcriptional Regulation; Wild Type Mouse; base; capillary; chromatin immunoprecipitation; cytotoxic; functional outcomes; in vivo; insight; knock-down; mouse model; neurobehavioral; novel; patch clamp; public health relevance; research study; response; transcription factor

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.

描述(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节亚基NC(Ca-ATP)通道与PHN密切相关，但该通道的致孔亚基尚未从分子上鉴定。我们实验室的新实验提供了证据，证明TRPM4很可能是通道的成孔亚单位。这项建议的目的是通过确定TRPM4在SCI后PHN中的作用来扩大这一发现。我们在大鼠和小鼠脊髓挫伤模型上的初步数据表明，通过药物阻断和TRPM4的基因抑制，出血和进行性病变扩展显著减少，并且这些效应与神经行为功能结果的显著改善有关。在特定目的(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PHN和其他SCI继发性损伤的表现。其他初步数据表明，在PHN中最关键的细胞是毛细血管和毛细血管后静脉内皮细胞。在SA2中，我们利用脊髓损伤后新鲜分离的脊髓毛细血管和培养的CNS微血管内皮细胞的膜片钳技术，研究了TRPM4通道在内皮细胞中的生理调节和功能作用。其他初步数据表明，NFkappaB可能是TRPM4通道的重要转录调节因子。NFkappaB是TNFpha的下游效应因子，已知与脊髓损伤密切相关。在SA3中，利用大鼠脊髓损伤模型的组织和CNS微血管内皮细胞的培养，我们将确定转录因子NFkappaB在TRPM4通道表达中的作用，并将检测NFkappaB抑制对SCI预后的影响。总体而言，了解TRPM4通道在脊髓损伤中的作用将导致对这种毁灭性的人类疾病的新的分子见解和新的治疗方法。公共卫生相关性利用啮齿动物脊髓损伤模型，我们发现，药物和反义抑制TRPM4通道可显著减少出血性坏死，并显著改善神经功能。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中枢神经系统内皮细胞培养来牢固地建立调控TRPM4通道表达和功能的基本分子原理，这将为未来脊髓损伤的新疗法奠定基础。