TRPV4 links the blood-neural barrier to motor neuron dysfunction

项目总结 血-神经屏障(BNB)通透性增加与多发性硬化的发病机制有关。 急性和慢性神经疾病，包括神经退行性疾病，但 BNB损伤对神经元功能障碍和变性的影响一直难以准确定位。在.期间 我们和其他人之前发现的遗传性运动神经元病患者的特征 常染色体显性突变的细胞表面表达的阳离子通道瞬时受体电位 香草素样蛋白4(TRPV4)可引起亚型脊髓性肌萎缩症和夏科-玛丽-牙病。而我们的 在培养细胞中的研究表明，TRPV4突变导致通道功能增强，但几乎没有证据 TRPV4在运动神经元中有功能表达。为了进一步剖析TRPV4的细胞学基础 通道病，我们最近产生了新的突变的TRPV4敲入小鼠模型，该模型发展为严重的 与BNB局灶性崩溃相关的神经学表型，特别是在脑角腹角 颈髓和脑干。令人惊讶的是，内皮细胞中TRPV4的细胞类型特异性基因缺失 细胞(ECs)或用TRPV4小分子拮抗剂治疗有症状的小鼠明显逆转 这些表型。总之，这些研究表明，TRPV4的激活在 提示TRPV4拮抗剂可能是一种促进BNB功能的新的治疗方法。 在这里，在特定的目标1中，我们将表征TRPV4在 并测定TRPV4突变对两种细胞TRPV4通道活性的影响 原代小鼠神经血管内皮细胞和人IPSC来源的神经血管内皮细胞。在具体目标2中，我们将 确定TRPV4活性如何在体外改变BNB的通透性和结构，包括在2D融合中 在单层和3D工程微血管中，以及在体内突变的TRPV4小鼠模型中。最后，在 具体目标3，利用脊髓切片的膜片钳电生理学，我们将确定BNB是如何泄漏的 影响运动神经元的功能和结构，并决定TRPV4小分子拮抗剂是否可以 逆转突变的TRPV4小鼠的疾病表现。总而言之，这些研究将定义以前的 TRPV4在神经血管内皮细胞中调节BNB完整性的未知作用，决定BNB的作用 运动神经元功能的崩溃，并研究TRPV4小分子拮抗剂是否可能是 针对TRPV4突变患者以及其他神经系统疾病患者的新疗法 以BNBs受损为特征的。

PROJECT SUMMARY Increased permeability of blood-neural barriers (BNBs) has been implicated in the pathogenesis of multiple acute and chronic neurological disorders, including neurodegenerative disease, but the specific contributions of BNB impairments to neuronal dysfunction and degeneration have been difficult to pinpoint. During characterization of patients with inherited forms of motor neuron disease, we and others previously discovered that autosomal dominant mutations of the cell surface-expressed cation channel transient receptor potential vanilloid 4 (TRPV4) cause subtypes of spinal muscular atrophy and Charcot-Marie-Tooth disease. While our studies in cultured cells suggest that TRPV4 mutations cause a gain of channel function, there is little evidence that TRPV4 is functionally expressed in motor neurons. In order to further dissect the cellular basis of TRPV4 channelopathy, we recently generated novel mutant TRPV4 knock-in mouse models that develop severe neurological phenotypes associated with focal breakdown of BNBs, particularly in the ventral horn of the cervical spinal cord and brainstem. Strikingly, cell type-specific genetic deletion of TRPV4 from endothelial cells (ECs) or treatment of symptomatic mice with a TRPV4 small molecule antagonist markedly reverses these phenotypes. Together, these studies suggest that TRPV4 activation plays a fundamental role in regulating BNB integrity and that TRPV4 antagonists could be a novel therapeutic promoting BNB function. Here, in Specific Aim 1, we will characterize the topographical and temporal expression patterns of TRPV4 in neural vascular ECs and determine the effects of TRPV4 mutations on TRPV4 channel activity in both cultured primary mouse neural vascular ECs and human iPSC-derived neural vascular ECs. In Specific Aim 2, we will determine how TRPV4 activity alters BNB permeability and structure in vitro, including in both 2D confluent monolayers and in 3D engineered microvessels, as well as in mutant TRPV4 mouse models in vivo. Finally, in Specific Aim 3, using patch clamp electrophysiology in spinal cord slices, we will determine how BNB leak affects motor neuron function and structure, and determine whether TRPV4 small molecule antagonists can reverse disease manifestations in mutant TRPV4 mice. Together, these studies will define a previously uncharacterized role for TRPV4 in neural vascular ECs in regulating BNB integrity, determine effects of BNB breakdown on motor neuron function, and investigate whether a TRPV4 small molecule antagonist could be a novel treatment for patients with TRPV4 mutations, as well as for patients with other neurological diseases characterized by impaired BNBs.