CILIA are unique cellular organelles that extend from the surface of the cell in the form of an antenna. They are generated and maintained by an elaborate process of vesicular protein trafficking, microtubule extension and resorption and stringently regulated activity of microtubule motor proteins. This process is called Intraflagellar Transport (IFT). Cilia are involved in regulating diverse developmental and adulthood signaling cascades, including sonic hedgehog signaling and Wnt signaling. Being nearly ubiquitous, dysfunction of cilia results in severe developmental disorders, including neurodegenerative diseases of the eye and brain.
Our lab investigates the Molecular and Cell Biological basis of Neurodegenerative Diseases of the Eye, with special focus on those caused due to defects of ciliary function in the photoreceptors, the light-sensing neurons of the Retina. These diseases include Retinitis Pigmentosa (RP) and Leber congenital amaurosis (LCA) and are characterized by progressive loss of night and day vision in patients. Photoreceptors are polarized sensory neurons with a distinct inner segment (IS) and the photosensory outer segment (OS). The OS is a sensory (or primary) cilium, which contains membranous discs arranged in a coin-stack like fashion. These discs are periodically shed at the tip and are renewed at the base. Such phenomena require a high level of stringently regulated trafficking of membrane and protein components from IS to OS via a narrow bridge-like structure, called the transition zone (TZ). The unique and distinct protein composition of the ciliary OS membrane of photoreceptors is essential to maintain the polar nature of the photoreceptors. Consistently, any defects in the trafficking machinery of photoreceptors due to dysfunction of the cilia results in degeneration and blindness.
The TZ of photoreceptor cilium is implicated in regulating the sorting and trafficking of specific protein and other cargo components to the OS. We are interested in understanding the development, composition and function of the photoreceptor ciliary TZ in order to obtain mechanistic insights into the mode of regulated protein trafficking and maintenance of photoreceptor polarity. Our studies have identified three key ciliary proteins that are involved in photoreceptor ciliary protein: RPGR (retinitis pigmentosa GTPase regulator), RP2 (retinitis pigmentosa 2), and CEP290 (centrosomal protein of 290 kDa). These proteins not only modulate ciliary transport but are also mutated in various forms of human retinal degenerative diseases. Following three major projects are currently being carried out in our laboratory.
PROJECT I: Functional Analysis of RPGR: RPGR is a ciliary protein mutated in a majority of X-linked RP cases (>75%) and is one of the most common cause of RP in humans. This project focuses on investigating the role of the TZ-associated protein RPGR in photoreceptor cilia. Our recent studies have revealed that RPGR exists in multiple protein complexes in mammalian retina, such as with Nephronophthisis (NPHP)-associated proteins and IFT proteins. Moreover, RPGR acts as a guanine exchange factor (GEF) for the small GTPase RAB8A, which is involved in cilia formation and photoreceptor protein trafficking. Our studies are specifically aimed at (i) delineating the role of RPGR as a GEF in photoreceptors, (ii) the cargo that is specifically delivered by RPGR to the outer segment, and (iii) the effect of human disease mutations on the function of RPGR. We have developed zebrafish and mouse models of RPGR dysfunction, which represent the human disease condition. Our investigations have also led to a successful gene therapy study to ameliorate RPGR-associated disease in two canine models.
PROJECT II: RP2 in X-linked RP: The RP2 gene is mutated in 10-15% of X-linked RP cases. Previous studies indicate a role of RP2 in ciliary trafficking and maintenance. However, its precise role in photoreceptors is still unclear. Our lab utilizes cell culture and animal models to investigate the function of RP2 and pathology of associated disease.
PROJECT III: LCA due to CEP290 mutations: CEP290 is another ciliary protein involved in regulating cilia formation. Mutations in CEP290 are a frequent cause of LCA, a childhood blindness disorder. Our lab has identified a naturally occurring mouse mutant of Cep290. Additionally, we have shown that CEP290 interacts with several distinct ciliary proteins in the retina and is involved in regulating protein trafficking as well as protein degradation. This project focuses on delineating the role of CEP290 in regulating photoreceptor cilia formation and maintenance and understanding the pathogenesis of CEP290-associated retinal degenerative diseases.
We employ following strategies to evaluate the function of these proteins in mediating the development and function of the TZ of photoreceptors as well as to develop treatment strategies for associated disorders:
Protein-Protein interactions: We utilize large-scale co-immunoprecipitation followed by mass spectrometry analyses to identify the macromolecular protein complexes involved in mediating the sorting and transport of proteins.
In vivo animal models: We utilize mouse and zebrafish models to assess the role of ciliary proteins in photoreceptor development and maintenance. We also utilize these assays to evaluate the pathogenic potential of human disease mutations in ciliary proteins.
Develop gene-based and small molecule based therapeutic approaches: Our recent studies have revealed new knowledge about the function of some ciliary proteins, which have prompted us to develop assays to assess the potential of gene augmentation or identification of small molecules in ameliorating ciliary defects associated with RPGR, RP2 and CEP290 mutations.