Translating Pathomechanisms into Treatment for Spinal Muscular Atrophies

项目摘要 脊髓性肌萎缩（SMA）是单基因运动神经元（MN）疾病， 肌肉无力和过早死亡我的研究项目集中在两种先进的治疗方法 SMA的形式：由运动神经元存活1的隐性、功能丧失突变引起的近端SMA 基因（SMN 1）和远端SMA（dSMA）由瞬时受体电位的显性突变引起 香草素4基因（TRPV 4）。我们的总体方法是将人类患者的发现与 在动物和iPSC衍生模型中进行实验，以阐明与以下相关的病理机制途径： 人类疾病和确定有希望的治疗机会。在这里，我们将利用独特的资源， 最先进的技术，以确定限制当前SMA治疗有效性的因素，表征细胞 以及驱动SMA病理的分子机制，并确定和验证新的治疗策略。 近端SMA处于快速发展的基因靶向治疗的最前沿， SMN诱导治疗和第三个在FDA审查。虽然这是一个变革性的成功， 这些治疗是高度可变的，从早期运动里程碑的正常实现到没有 改善运动功能。在过去的5年中，我们的研究表明，近端SMA病理 开始于子宫内，在患者目前开始治疗之前。在人类和小鼠中，SMA MN 在妊娠期间表现出成熟受损和新生儿急剧变性， SMN表达下降。在这里，我们将建立在这些观察1）解剖的具体机制 在发育和治疗过程中调节SMN表达，2）确定引起SMN表达的分子机制， SMA MN的成熟和变性受损，以及3）使用这些见解来开发新的和在子宫内 SMA治疗策略。在对dSMA的平行研究中，我们最近证明了神经致病性 细胞表面阳离子通道TRPV 4的突变破坏了调节蛋白-蛋白相互作用，并导致细胞凋亡。 通道功能的增益。现有的TRPV 4拮抗剂在人体中具有良好的耐受性，使该通道成为一种新的药物。 有希望的治疗靶点。引人注目的是，突变TRPV 4基因敲入小鼠模型发生了严重的神经系统疾病， 表型由于局部破坏血液神经屏障（BNB），这是拯救选择性遗传 从内皮细胞（EC）中缺失TRPV 4或用TRPV 4拮抗剂治疗有症状的小鼠。 这些研究表明，TRPV 4激活可以通过以下方式以非细胞自主方式驱动神经病理学： 监管BNB。在这里，我们将1）表征调节TRPV 4通道活性的蛋白质相互作用，2） 评估TRPV 4在调节EC屏障功能中的作用，以及3）评估TRPV 4拮抗剂作为一种抗肿瘤药物的作用。 在dSMA小鼠和最终以BNB破坏为特征的其他病症中的治疗策略。在一起， 我们的研究将进一步加深我们对SMA病理机制的理解，描绘新的治疗靶点， 和策略，以及SMA和相关神经肌肉疾病患者的高级护理。

PROJECT SUMMARY Spinal muscular atrophies (SMAs) are monogenetic motor neuron (MN) diseases that cause debilitating muscle weakness and often early mortality. My research program focuses on advancing therapeutics for two forms of SMA: proximal SMA caused by recessive, loss-of-function mutations of the survival motor neuron 1 gene (SMN1) and distal SMA (dSMA) caused by dominant mutations of the transient receptor potential vanilloid 4 gene (TRPV4). Our overarching approach is to integrate findings from human patients with experimentation in animal and iPSC-derived models to elucidate pathomechanistic pathways relevant to human disease and identify promising therapeutic opportunities. Here, we will leverage unique resources and state-of-the-art technologies to define factors limiting efficacy of current SMA therapeutics, characterize cellular and molecular mechanisms driving SMA pathology, and identify and validate novel therapeutic strategies. Proximal SMA is at the forefront of rapidly evolving gene-targeting therapeutics, with two recently approved SMN-inducing treatments and a third under FDA review. While a transformative success, the clinical efficacy of these treatments is highly variable, ranging from normal attainment of early motor milestones to no improvement in motor function. In the last 5 years, our studies have revealed that proximal SMA pathology begins in utero, before treatments are currently initiated in patients. In both humans and mice, SMA MNs exhibit impaired maturation during gestation and precipitous neonatal degeneration, paralleled by a marked decline in SMN expression. Here, we will build on these observations to 1) dissect the specific mechanisms regulating SMN expression during development and treatment, 2) identify the molecular mechanisms causing impaired maturation and degeneration of SMA MNs, and 3) use these insights to develop novel and in utero SMA therapeutic strategies. In parallel studies on dSMA, we have recently demonstrated that neuropathogenic mutations in TRPV4, a cell surface cation channel, disrupt regulatory protein-protein interactions and cause a gain of channel function. Existing TRPV4 antagonists have good tolerability in humans, making the channel a promising therapeutic target. Strikingly, mutant TRPV4 knock-in mouse models develop severe neurological phenotypes due to focal breakdown of blood-neural barriers (BNBs), which are rescued by selective genetic deletion of TRPV4 from endothelial cells (ECs) or treatment of symptomatic mice with TRPV4 antagonists. These studies suggest that TRPV4 activation can drive neuropathology in a non-cell autonomous manner by regulating BNBs. Here, we will 1) characterize protein interactions regulating TRPV4 channel activity, 2) evaluate the role of TRPV4 in modulating EC barrier function, and 3) assess TRPV4 antagonists as a therapeutic strategy in dSMA mice and ultimately other disorders characterized by BNB disruption. Together, our studies will further our mechanistic understanding of SMA pathology, delineate novel therapeutic targets and strategies, and advance care of patients with SMAs and related neuromuscular diseases.