TrpM4 channel in spinal cord injury

描述（由申请人提供）：脊髓损伤（SCI）后的反应性星形胶质细胞增生是一种重要的防御机制，但它也可能是有害的，引起肿胀，导致缺血，并形成胶质瘢痕，抑制轴突再生。星形胶质细胞增生可以通过删除水通道蛋白（Aqp）4来预防，但这也可能是有害的，因为胶质瘢痕对于限制炎症和病变大小至关重要。因此，下调星形胶质细胞增生和Aqp 4，而不是完全抑制，是重要的治疗目标。Aqp通道被动地跨膜转运H2O，转运的H2O量由渗透梯度决定。调节渗透梯度以驱动H2O通量的分子机制知之甚少，但可能涉及离子通道。在静息星形胶质细胞中，Aqp 4与瞬时受体电位（Trp）V4（一种非选择性阳离子通道）物理共缔合，其中组装的分子复合物是正常细胞体积控制所需的。我们发现，当星形胶质细胞被激活时，处理H2O的分子机制发生了根本性的变化。SCI后，Aqp 4共缔合的伴侣从TrpV 4切换到TrpM 4，分子实验表明TrpM 4优先与Aqp 4-M23共缔合，Aqp 4-M23是组装成与H2O高速率转运相关的“正交颗粒阵列”的同种型。这种开关提供了通过抑制TrpM 4优先下调Aqp 4-M23的机会，从而有利地影响星形胶质细胞增生而不直接或完全抑制Aqp 4。在这个竞争性的更新，我们将使用一个独特的模型，涉及纤维蛋白原注射到脊髓原位研究反应性星形胶质细胞增生。我们提出了3个具体目标（SA），以扩大我们的新的初步数据的作用TrpM 4在调节Aqp 4和反应性星形胶质细胞增生。描述：在特定目标（SA）1中，我们将检验TrpM 4缺失将有利地影响星形胶质细胞增生和星形胶质细胞肿胀的假设。在SA 2中，我们将检验Aqp 4和TrpM 4物理共缔合以形成用于控制反应性星形胶质细胞中的H2O转运的完整分子复合物的假设。在SA 3中，我们将测试TrpM 4激活在反应性星形胶质细胞中通过Aqp 4逆向驱动水通量的假设。每个目标中的每个假设都得到了可靠的初步数据的支持。

DESCRIPTION (provided by applicant): Reactive astrogliosis after spinal cord injury (SCI) serves as a critical defense mechanism, but it also can be detrimental, causing swelling that leads to ischemia, and formation of a glial scar that inhibits axonal regeneration. Astrogliosis can be prevented by deleting aquaporin (Aqp) 4, but this too can be detrimental, since a glial scar is essential for restricting inflammation and lesion size. Therefore, down-modulation of astrogliosis and of Aqp4, but not total inhibition, are important therapeutic goals. Aqp channels passively transport H2O across membranes, with the amount of H2O transported determined by the osmotic gradient. The molecular mechanisms by which osmotic gradients are regulated in order to drive the flux of H2O are poorly understood, but likely involve ion channels. In resting astrocytes, Aqp4 physically co-associates with transient receptor potential (Trp) V4, a non-selective cation channel, with the assembled molecular complex being required for normal cell volume control. We discovered that the molecular mechanism by which H2O is handled undergoes a fundamental change when astrocytes become activated. Following SCI, the partner for Aqp4 co-association switches from TrpV4 to TrpM4, with molecular experiments showing that TrpM4 co-associates preferentially with Aqp4-M23, the isoform that assembles into 'orthogonal arrays of particles' that are associated with high rates of H2O transport. This switch provides an opportunity to down-modulate Aqp4-M23 preferentially by inhibiting TrpM4, and thereby favorably influence astrogliosis without directly or completely inhibiting Aqp4. In this competitive renewal, we will use a unique model involving fibrinogen injection into the spinal cord to study reactive astrogliosis in situ. We propose 3 Specific Aims (SA) to expand on our novel preliminary data on the role of TrpM4 in regulating Aqp4 and reactive astrogliosis. DESCRIPTION: In Specific Aim (SA) 1, we will test the hypothesis that TrpM4 deletion will favorably influence astrogliosis and astrocyte swelling. In SA2, we will test the hypothesis that Aqp4 and TrpM4 physically co-associate to form an integral molecular complex for the control of H2O transport in reactive astrocytes. In SA3, we will test the hypothesis that TrpM4 activation osmotically drives the flux of water via Aqp4 in reactive astrocytes. Each hypothesis in each aim is supported by robust preliminary data.