. Bardet-Biedl syndrome (BBS) is a genetically heterogeneous disorder characterized by retinal degeneration (RD), obesity, polydactyly, renal defects, and genital defects. This proposal focuses on one BBS-associated gene, Centrosomal protein of 290 kDa (CEP290), with the goal of understanding the role of CEP290 in the normal structure and function of the rod sensory cilium, and the mechanisms of pathophysiology in RD caused by CEP290 deficiency. CEP290 was chosen because depending on the allele and background, CEP290 defects cause an array of diverse defects, all involving RD. In contrast to most other BBS gene products, CEP290 protein does not form a stable part of the BBSome membrane coat complex, but it interacts functionally and physically with other BBS proteins. While CEP290 has been proposed to be a ciliary gate-keeper or a structural scaffold within the connecting cilium (CC), the field is currently divided on the localization and the structural and functional roles of CEP290 within the CC, and the mechanism for CEP290 regulation of ciliary trafficking is not well understood.
The Specific Aims are: 1. Chronology of CEP290 (BBS14) Subcellular Localization and BBS Protein Localization in CEP290- or BBS4-Mediated Retinal Degeneration To test the hypothesis that CEP290 localizes peripherally to microtubule doublets and elucidate the structural role of CEP290, the superresolution imaging techniques, Structured Illumination Microscopy (SIM) and Stochastic Optical Reconstruction Microscopy (STORM) will be used. To assess the hypothesis that mislocalization of interacting proteins such as BBS4 and BBS8,in CEP290-mediated RD is the direct result of the absence of CEP290, the localization of BBS4 over the course of RD will be tracked as well as CEP290 localization in BBS4-mediated RD. To correlate mislocalization with disease progression over time, histology, Optical Coherence Tomography (OCT), Transmission Electron Microscopy (TEM), and electroretinography (ERG) will be used. 2. Identification of the BBSome Interacting Domains of CEP290. BBS4 and BBS8 are BBSome subunits that both depend on the presence of CEP290 for proper localization. To test the hypotheses that CEP290 interacts with BBS4 and BBS8 through its N-terminal SMC homology domain, and that BBS4 and BBS8 interact with CEP290 through their tetratricopeptide repeats (TPR) domains, the proposed domains, or full length proteins with and without mutations within the proposed interaction domains, will be expressed in hRPE1 cells and tests for both proper co-localization and co-immunoprecipitation will be performed. 3. Functional Rescue of Retinal Degeneration with In Vivo CEP290 domain Expression. To determine the in vivo role of CEP290 and its domains in protein mislocalization, CEP290 mutant mice will receive sub-retinal injections and electroporation of expression constructs for rescue domains and full-length CEP290 protein. Understanding the functions and disease mechanisms of CEP290 is essential for understanding the pathophysiological mechanisms of BBS and other retinal ciliopathies, and designing new therapies.
Mutations in a gene called CEP290 result in ciliopathies, an extensive list of diseases with diverse symptoms, all including retinal degeneration and blindness. To better understand these diseases and how the mutations cause blindness, this research will use the latest techniques to determine where the protein product of the CEP290 gene is located in rod cells of the retina, and which parts of the protein are important for its normal functions.
