Cilia Assembly and Transport in Photoreceptor Cells

项目摘要 CEP290的突变导致了许多称为纤毛病的遗传性疾病，这些疾病表现为 各种临床症状，包括视网膜变性。虽然众所周知， 光感受器的存活需要CEP290的功能，光感受器死亡的原因在很大程度上仍然存在 未知。在脊椎动物的光感受器中，CEP290定位于连接纤毛，这是 类似于初级纤毛的过渡区。细胞培养和无脊椎动物的研究表明 CEP290组织蛋白质复合体的组装，这些复合体在 过渡区。然而，CEP290的缺失是否影响了这样的睫状门 在光感受器中表现出来。此外，CEP290相关的高度可变的性质 疾病表型不能用传统的基因-表型相关性来解释。二 已经提出了一些模型来解释这种变异性。一种可能性是第二位基因 修饰剂可提高某些患者的病情严重程度。第二种可能性是外显子含有 无义突变和也在同一阅读框中开始和结束的突变可以优先 已跳过。在这种情况下，由此产生的mrna转录本避免了无意义介导的衰退，并可以 产生一种近乎全长的蛋白质。因此，疾病的严重程度与全血中的 生产出长度和接近全长的蛋白质。这项提议旨在解决根本问题 与光感受器细胞生物学和CEP290在光感受器退变中的作用有关。在目标1中， 我们将利用两个不同的斑马鱼CEP290突变体来确定睫状门的缺陷是否起作用 在退化中。在目标2中，我们将确定其他基因是否与Joubert综合征有关， 即Arl13b、ahi1或cc2d2a作为CEP290相关性视网膜变性的遗传修饰物。 斑马鱼。最后，在目标3中，我们将从CEP290突变患者和 产生人诱导多能干细胞(HiPSCs)并随后分化为3D 视网膜杯子。这些HiPSC来源的视网膜杯子(hiPSC-DRCs)将被用来确定基础外显子 携带CEP290突变的人会发生跳过，总蛋白水平是否与 与疾病的严重性有关。此外，致病突变是如何导致纤毛变化的 将对建筑和蛋白质贩运进行调查。这些实验将建立 决定疾病进展严重程度的分子和遗传机制。 理解表型变异的基础将提供急需的澄清 探讨其发病机制，有助于更好地治疗睫状体病。

Project Summary Mutations in CEP290 result a number of genetic diseases termed ciliopathies, which manifest with a variety of clinical symptoms, including retinal degeneration. While it is well-established that photoreceptor survival requires Cep290 function, the causes of photoreceptor death remain largely unknown. In vertebrate photoreceptors, Cep290 localizes to the connecting cilium, which is analogous to the transition zone of primary cilia. Work from cell culture and invertebrates suggest that Cep290 organizes the assembly of protein complexes that form a “ciliary gate” within the transition zone. However, whether loss of Cep290 impacts such a ciliary gate have not been demonstrated in photoreceptors. Furthermore, the highly variable nature of CEP290-associated disease phenotypes cannot be explained by traditional genotype-phenotype correlations. Two models have been proposed to explain this variability. One possibility is that second-site genetic modifiers enhance disease severity in some patients. The second possibility is that exons harboring nonsense mutations and that also begin and end in the same reading frame can be preferentially skipped. In such a case the resulting mRNA transcript eludes nonsense-mediated decay and can produce a near-full-length protein. Disease severity therefore correlates with the total amount of full- length and near-full-length protein produced. This proposal seeks to address fundamental questions related to photoreceptor cell biology and the role of Cep290 in photoreceptor degeneration. In Aim 1, we will utilize two distinct zebrafish cep290 mutants to determine if defects in ciliary gating play a role in degeneration. In Aim 2, we will determine if other genes associated with Joubert Syndrome, namely arl13b, ahi1 or cc2d2a act as genetic modifiers to cep290-associated retinal degeneration in zebrafish. Finally, in Aim 3, we will take fibroblasts from patients with CEP290 mutations and generate human induced pluripotent stem cells (hiPSCs) and subsequently differentiated into 3D retinal cups. These hiPSC-derived retinal cups (hiPSC-DRCs) will be used determine if basal exon skipping occurs in from humans carrying CEP290 mutations and whether total protein levels correlate with disease severity. In addition, how disease-causing mutations lead to alterations in cilia architecture and protein trafficking will be investigated. These experiments will establish the molecular and genetic mechanisms that determine the severity of disease progression. Understanding the basis for phenotypic variability will provide much-needed clarification on the mechanisms of pathogenesis and lead to better treatment of ciliary disease.