Cilia Assembly and Transport in Photoreceptor Cells

感光细胞中纤毛的组装和运输

• 批准号: 9762111
• 负责人: Brian D Perkins
• 金额: $ 43.23万
• 依托单位: CLEVELAND CLINIC LERNER COM-CWRU
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-01 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9762111
• 关键词: 3-Dimensional; Address; Affect; Alleles; Alternative Splicing; Architecture; Bardet-Biedl Syndrome; Binding; Blindness; Cell Culture Techniques; Cellular biology; Cessation of life; Childhood; Cilia; Clinic; Clinical; Consensus; Data; Defect; Diffusion; Disease; Disease Progression; Exhibits; Exons; Eye; Fibroblasts; Frameshift Mutation; Gatekeeping; Genes; Genetic; Genetic Diseases; Genotype; Human; Immunohistochemistry; In Vitro; Institutes; Invertebrates; Joubert syndrome; Knowledge; Lead; Leber' s amaurosis; Length; Mediating; Messenger RNA; Microtubules; Modeling; Molecular Genetics; Mutation; Nature; Nonsense Mutation; Nonsense-Mediated Decay; Organoids; Outcome; Pathogenesis; Patients; Penetrance; Phenotype; Photoreceptors; Play; Point Mutation; Process; Proteins; Reading Frames; Retina; Retinal; Retinal Degeneration; Retinal Diseases; Retinal Dystrophy; Role; Severities; Severity of illness; Site; Symptoms; Technology; Testing; Transcript; Transgenic Organisms; Variant; Vertebrate Photoreceptors; Work; Zebrafish; ciliopathy; disease phenotype; disease-causing mutation; exon skipping; experience; experimental study; induced pluripotent stem cell; kinetosome; mutant; photoreceptor degeneration; protein complex; protein protein interaction; protein transport; therapeutic development; trafficking

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.

项目概要 CEP290 突变会导致许多称为纤毛病的遗传性疾病，其表现为 各种临床症状，包括视网膜变性。虽然众所周知 光感受器的生存需要Cep290功能，光感受器死亡的原因仍然很大 未知。在脊椎动物的光感受器中，Cep290 定位于连接纤毛，即 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 组织蛋白质复合物的组装，在细胞内形成“纤毛门” 过渡区。然而，Cep290 的丢失是否会影响这样的睫状门尚未得到证实。 在光感受器中得到证明。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型-表型相关性来解释。二 已经提出模型来解释这种变化。一种可能性是第二位点遗传 修饰剂会加重某些患者的疾病严重程度。第二种可能性是外显子携带 无义突变并且也在同一阅读框中开始和结束可以优先 跳过了。在这种情况下，产生的 mRNA 转录本可以避开无义介导的衰变，并且可以 产生接近全长的蛋白质。因此，疾病的严重程度与全量的总量相关。 产生的长度和接近全长的蛋白质。该提案旨在解决基本问题 与感光细胞生物学和 Cep290 在感光细胞变性中的作用有关。在目标 1 中， 我们将利用两种不同的斑马鱼 cep290 突变体来确定纤毛门控缺陷是否起作用 处于退化状态。在目标 2 中，我们将确定是否有其他基因与 Joubert 综合征相关， 即 arl13b、ahi1 或 cc2d2a 作为 cep290 相关视网膜变性的遗传修饰剂 斑马鱼。最后，在目标 3 中，我们将从具有 CEP290 突变的患者身上获取成纤维细胞， 产生人类诱导多能干细胞 (hiPSC)，并随后分化为 3D 视网膜杯。这些 hiPSC 衍生的视网膜杯 (hiPSC-DRC) 将用于确定基底外显子是否 携带 CEP290 突变的人类中发生的跳跃与总蛋白水平是否相关 与疾病的严重程度有关。此外，致病突变如何导致纤毛改变 将研究结构和蛋白质运输。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。 了解表型变异的基础将为我们提供急需的澄清。 发病机制并导致更好地治疗纤毛疾病。