Cilia Assembly and Transport in Photoreceptor Cells

感光细胞中纤毛的组装和运输

• 批准号: 10206144
• 负责人: Brian D Perkins
• 金额: $ 42.12万
• 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 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10206144
• 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 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.

项目摘要 CEP 290的突变导致许多称为纤毛虫病的遗传性疾病，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Cep 290的功能，光感受器死亡的原因主要是 未知在脊椎动物的光感受器中，Cep 290定位于连接纤毛， 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 Cep 290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，Cep 290的缺失是否影响这种睫状门还没有被证实。 在光感受器中表现出来。此外，CEP 290相关的高度可变性 疾病表型不能用传统的基因型-表型相关性来解释。两 已经提出了模型来解释这种可变性。一种可能是第二个位点的遗传 修饰剂增强了某些患者的疾病严重性。第二种可能性是， 无义突变并且也在相同的阅读框中开始和结束的突变可以被优先 跳过。在这种情况下，产生的mRNA转录物逃避无义介导的衰变，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相关- 长度和近全长的蛋白质产生。这项建议旨在解决一些根本问题， 与感光细胞生物学和Cep 290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cep 290突变体来确定睫状门控缺陷是否发挥作用 在退化。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arl 13 b、ahi 1或cc 2d 2a作为cep 290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CEP 290突变患者中提取成纤维细胞， 产生人类诱导多能干细胞（hiPSC），随后分化成3D 视网膜杯这些hiPSC衍生的视网膜杯（hiPSC-DRC）将用于确定基础外显子 携带CEP 290突变的人中发生跳跃，以及总蛋白水平是否与 疾病的严重程度。此外，致病突变如何导致纤毛改变 结构和蛋白质运输将被调查。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。 了解表型变异的基础将提供急需的澄清， 发病机制，并导致更好的治疗睫状体疾病。

项目成果

Early defects in photoreceptor outer segment morphogenesis in zebrafish ift57, ift88 and ift172 Intraflagellar Transport mutants.

• DOI: 10.1016/j.visres.2008.12.009
• 发表时间: 2009-02
• 期刊: VISION RESEARCH
• 影响因子: 1.8
• 作者: Sukumaran, Sujita;Perkins, Brian D.
• 通讯作者: Perkins, Brian D.

The adult zebrafish retina: In vivo optical sectioning with Confocal Scanning Laser Ophthalmoscopy and Spectral-Domain Optical Coherence Tomography.

• DOI: 10.1016/j.exer.2016.10.001
• 发表时间: 2016-12
• 期刊: EXPERIMENTAL EYE RESEARCH
• 影响因子: 3.4
• 作者: Bell, Brent A.;Yuan, Alex;Dicicco, Rose M.;Fogerty, Joseph;Lessieur, Emma M.;Perkins, Brian D.
• 通讯作者: Perkins, Brian D.

Retrograde intraflagellar transport by cytoplasmic dynein-2 is required for outer segment extension in vertebrate photoreceptors but not arrestin translocation.

• DOI: 10.1167/iovs.09-3828
• 发表时间: 2009-11
• 期刊: Investigative ophthalmology & visual science
• 影响因子: 4.4
• 作者: Krock BL;Mills-Henry I;Perkins BD
• 通讯作者: Perkins BD

myosin 7aa(-/-) mutant zebrafish show mild photoreceptor degeneration and reduced electroretinographic responses.

• DOI: 10.1016/j.exer.2014.03.007
• 发表时间: 2014-05
• 期刊: EXPERIMENTAL EYE RESEARCH
• 影响因子: 3.4
• 作者: Wasfy, Meagan M.;Matsui, Jonathan I.;Miller, Jessica;Dowling, John E.;Perkins, Brian D.
• 通讯作者: Perkins, Brian D.

Intraflagellar transport proteins are involved in thrombocyte filopodia formation and secretion.

• DOI: 10.1080/09537104.2017.1361524
• 发表时间: 2018-12
• 期刊: Platelets
• 影响因子: 3.3
• 作者: Radhakrishnan U;Alsrhani A;Sundaramoorthi H;Khandekar G;Kashyap M;Fuchs JL;Perkins BD;Omori Y;Jagadeeswaran P
• 通讯作者: Jagadeeswaran P