Cilia Assembly and Transport in Photoreceptor Cells

感光细胞中纤毛的组装和运输

基本信息

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
项目类别：
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&apos 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

项目摘要

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 CEP290中的突变导致许多称为纤毛病的遗传疾病，这表现为各种临床症状，包括残留变性。虽然公平的光感受器的存活需要CEP290功能，感光器死亡的原因在很大程度上仍然存在未知。在脊椎动物感光器中，CEP290定位于连接纤毛，这是类似于原发性纤毛的过渡区。细胞培养和无脊椎动物的工作表明 CEP290组织了蛋白质复合物的组装，该蛋白质复合物在过渡区。但是，CEP290的损失是否影响这种睫毛闸在感光器中证明。此外，CEP290相关的高度可变性质疾病表型不能用传统的基因型 - 表型相关性来解释。二已经提出了模型来解释这种可变性。一种可能性是第二站点遗传修饰符增加了某些患者的疾病严重程度。第二可能是藏有的外显子胡说八道的突变，也可以优先以同一阅读框架开始和结束跳过。在这种情况下，由此产生的mRNA转录本避免了废话介导的衰减，可以产生近乎满的长度蛋白质。因此，疾病的严重程度与全部总量相关长度和接近长度的蛋白质产生。该建议旨在解决基本问题与感光细胞生物学以及CEP290在光感受器变性中的作用有关。在AIM 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中的缺陷是否起作用在AIM 2中，我们将确定其他基因是否与Joubert综合征相关，即ARL13B，AHI1或CC2D2A作为CEP290相关的残留变性的遗传修饰剂斑马鱼。最后，在AIM 3中，我们将从CEP290突变患者和产生人类诱导的多能干细胞（HIPSC），然后分化为3D 这些HIPSC衍生的视网膜杯（HIPSC-DRC）将被确定是否基本外显子跳过来自携带CEP290突变的人类，以及总蛋白水平是否相关疾病严重程度。另外，引起疾病的突变如何导致纤毛改变将研究建筑和蛋白质运输。这些实验将确定决定疾病进展严重程度的分子和遗传机制。了解表型变异性的基础将为急需的澄清发病机理的机制，导致更好地治疗睫状性疾病。

项目成果

期刊论文数量（22）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

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

Comparative analysis of transcriptional changes in zebrafish cep290 and bbs2 mutants by RNA-seq reveals upregulation of inflammatory and stress-related pathways.