The Role of Cilia and Cerebrospinal Fluid Flow in Spine Development and Human Disease

项目摘要/摘要 青少年特发性脊柱侧凸(AIS)是一种流行的疾病，影响全球3%的儿童。的确有 目前还不知道AIS中出现的三维脊柱曲线的潜在基础，因此， 治疗仅限于侵入性手术干预或发病后的支撑。当前的一个主要障碍 对AIS的理解和治疗是我们对这种疾病的潜在病因缺乏了解， 这种情况由于我们缺乏动物模型而进一步恶化。这项建议的目的是 通过阐明AIS的原因，通过生成和表征AIS来解决这些障碍 特殊的斑马鱼模型的条件，并通过了解疾病的机制基础。 在我的初步数据中，我证明了具有异常纤毛运动和脑脊液流动的斑马鱼突变体 展示了迟发性脊柱曲线，它模拟了人类AIS的定义特征。此外，我还证明了 斑马鱼ptk7突变体是现存的唯一的AIS模型，其脊椎弯曲的原因可能起源于 纤毛功能失调。这让我对AIS的原因提出了一个新的假说：脑脊液异常 部分由纤毛产生的液体(脑脊液)流动导致AIS脊柱曲线异常。为了测试这一点 中心假说，我将通过定位和蛋白质组学研究来检验C21ORF59的功能，这是一种蛋白质I 已经发现纤毛运动和纤毛极性对纤毛介导流的产生是至关重要的。自.以来 纤毛生物学的这两个方面之间的联系很差，这两个方面都对生产流的产生至关重要。 理解，我的新蛋白质组学方法将导致知识的重要增长。以及 研究纤毛产生流动的分子基础，我将产生并表征几种斑马鱼 AIS模型，以评估这些患者是否存在脑脊液流动缺陷，并测试基因突变是否与 人类人工免疫系统影响斑马鱼脑脊液流量的产生。这将消除在这一领域取得进展的主要障碍。我 还将调查纤毛运动基因的人类变异是否会导致动物模型的AIS。这些 实验将使我能够破译异常的纤毛运动和功能障碍的脑脊液流动是如何与 人类人工智能系统。最后，从指导阶段开始，延伸到独立阶段，我将执行 确定斑马鱼AIS发病的空间和时间基础的机械实验，实验 需要使用我们独特的温度敏感型纤毛运动突变体。这将使我能够开始解释 功能失调的脑脊液流动如何导致脊柱弯曲。我的建议将测试脊柱弯曲是否可以解决 通过恢复脑脊液流量和恢复的程度，潜在地提供了一种原则证明， 脊柱弯曲可以非侵入性治疗，并为未来的治疗干预铺平道路。 流行且知之甚少的疾病。因为我的研究有力地表明，脑脊液流量对维持 脊椎直立，我以一系列有针对性的实验结束，目的是了解脑脊液流动是如何 在发育中的椎管中感觉到。

Project Summary/Abstract Adolescent Idiopathic Scoliosis (AIS) is a prevalent condition that impacts 3% of children worldwide. There is currently no known underlying basis for the three-dimensional spinal curves that occur in AIS and, as such, treatment is restricted to invasive surgical intervention or bracing post onset. A major obstacle to current understanding and treatment of AIS is our poor knowledge of the underlying etiology of the condition, a situation which is further compounded by our lack of animal models. The objective of this proposal is to address these barriers to progress by elucidating the cause of AIS, by generating and characterizing exceptional zebrafish models of the condition, and by understanding the mechanistic basis of the disease. In my preliminary data, I demonstrate that zebrafish mutants with abnormal cilia motility and CSF flow exhibit late-onset spinal curves that model the defining features of human AIS. Moreover, I demonstrate that the cause of spinal curves in zebrafish ptk7 mutants, the only existing AIS model, is likely to originate with dysfunctional cilia. This led me to propose a novel hypothesis for the cause of AIS: abnormal cerebrospinal fluid (CSF) flow, which is generated in part by cilia, causes the abnormal spinal curves in AIS. To test this central hypothesis, I will examine by localization and proteomic studies the function of C21ORF59, a protein I have found to be critical for cilia-mediated flow generation by linking cilia motility and cilia polarity. Since the links between these two facets of cilia biology, both critical for productive flow generation, are poorly understood, my novel proteomic approaches will lead to an important increase in knowledge. As well as investigating the molecular basis of flow generation by cilia, I will generate and characterize several zebrafish AIS models to assess whether these have defective CSF flow and to test whether mutations in genes linked to human AIS affect CSF flow generation in zebrafish. This will remove the major barrier to progress in this field. I will also investigate whether human variants in cilia motility genes cause AIS in animal models. These experiments will allow me to decipher how abnormal cilia motility and dysfunctional CSF flow are linked to human AIS. Lastly, beginning in the mentored phase but extending into the independent phase, I will perform mechanistic experiments to define the spatial and temporal basis for AIS onset in zebrafish, experiments that require the use of our unique temperature sensitive cilia motility mutant. This will allow me to begin to explain how dysfunctional CSF flow causes spinal curves. My proposal will test whether spinal curves can be resolved post-onset by restoring CSF flow and the extent of that recovery, potentially providing a proof-of-principle that spinal curves can be treated non-invasively and paving the way for future therapeutic interventions for this prevalent and poorly understood disease. Since my work strongly suggests that CSF flow is critical to maintain spine straightness, I end with a series of targeted experiments aimed at understanding how CSF flow is sensed in the developing spinal canal.