TrpM4 channel in spinal cord injury

描述(申请人提供)：脊髓损伤后反应性星形胶质细胞增生症(SCI)是一种关键的防御机制，但它也可能是有害的，导致肿胀导致缺血，并形成神经胶质疤痕，抑制轴突再生。星形胶质细胞增生症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是必不可少的。因此，下调星形胶质细胞和Aqp4的表达，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，其输送量由渗透梯度决定。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。在静止的星形胶质细胞中，Aqp4与瞬时受体电位(Trp)V4物理上相互作用，这是一种非选择性的阳离子通道，组装的分子复合体是正常细胞体积控制所必需的。我们发现，当星形胶质细胞激活时，处理H2O的分子机制发生了根本性的变化。在脊髓损伤后，Aqp4共结合的伙伴从TRPV4切换到TrpM4，分子实验表明TrpM4优先与Aqp4-M23共结合，Aqp4-M23是一种异构体，可以组装成与高速率H2O运输相关的“正交颗粒阵列”。这种转换提供了一个机会，通过抑制TrpM4优先下调Aqp4-M23，从而在不直接或完全抑制Aqp4的情况下有利地影响星形胶质细胞增生。在这次竞争性更新中，我们将使用一种独特的模型，包括向脊髓内注射纤维蛋白原来研究原位反应性星形胶质细胞增生症。我们提出了3个特定的目标(SA)来扩展我们关于TrpM4在调节Aqp4和反应性星形胶质细胞增生中的作用的新的初步数据。描述：在特定目的(SA)1中，我们将检验TrpM4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SA2中，我们将检验Aqp4和TrpM4物理上相互作用形成一个完整的分子复合体的假设，以控制反应性星形胶质细胞中的H2O运输。在SA3中，我们将检验TrpM4激活通过Aqp4在反应性星形胶质细胞中渗透驱动水通量的假设。每个目标中的每个假设都得到了稳健的初步数据的支持。

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.