There is fundamental gap in understanding the function of the ciliary protein RPGR (Retinitis Pigmentosa GTPase Regulator) and how mutations result in severe retinal degeneration in a majority of XLRP patients. Such a gap has hampered the progress to generate therapeutic modalities for this blinding disorder. The long- term goal of this application is to better understand the function of RPGR and the mechanism of RPGR- associated photoreceptor degeneration so that effective therapeutic interventions can be designed. The objective in this application is to identify how RPGR, as part of multiprotein ciliary complexes, regulates protein trafficking at the connecting cilium (CC or transition zone; TZ) of photoreceptors. The central hypothesis is that RPGR facilitates the assembly of distinct TZ protein complexes to regulate sorting and trafficking of cargo proteins. Hence, the primary goals of this project are to elucidate the mode of action of RPGR and the mechanism by which mutations in RPGR or its interacting proteins alter ciliary transport in photoreceptors and result in retinal degeneration. The rationale for the proposed research is that understanding the function of RPGR in ciliary transport has the potential to translate into better understanding of the disease pathogenesis. The current application proposes to delineate the role of RPGR in modulating the integrity and formation of TZ protein complexes (Aim 1), test the role of activity of RPGR over small GTPases in regulating ciliary transport in photoreceptors (Aim 2), and examine the mechanism of associated pathogenesis by testing the function of RPGR disease mutations. All reagents, assays, model systems and equipments that are already on hand will be utilized. Under the first aim, the role of RPGR and its selected interacting proteins in maintaining the ultrastructure and protein content of the OS will be evaluated. Studies proposed in the second aim focus on delineating the role of RPGR as an activator of small GTPases and on identifying additional small GTPases as substrates of RPGR in the retina. Under the third aim, effect of human disease mutations in RPGR on its interaction with different TZ proteins and on the localization of RPGR and other ciliary membrane proteins using zebrafish and mouse as model systems will be tested. We will also determine the pathogenic potential of various RPGR mutations in zebrafish. The approach is innovative, because it focuses on (i) ascertaining the precise function of RPGR as a regulator of small GTPases at the TZ; (i) using zebrafish to understand the pathogenic potential of human disease mutations in RPGR, and (iii) analyzing expression and localization of disease-associated RPGR mutants in photoreceptors of mice. The proposed research is significant, because it will not only be relevant to the understanding of the fundamental aspects of maintenance of ciliary function but will also provide novel mechanistic insights into the pathogenesis of photoreceptor degeneration in a common cause of human RP. Ultimately, such knowledge has the potential to inform the development of treatment modalities that will help to reduce the growing problem of retinal degenerative diseases in human beings.
The proposed research is relevant to public health because these studies are expected to increase the understanding of the retinal pathogenesis RPGR-associated disease. In addition, we will gain new knowledge on the pathogenic mechanisms of many other retinal degenerative disorders that are part of pleiotropic disorders due to dysfunction of ciliary transport mechanisms, collectively termed 'ciliopathies'. Thus, the proposed research is relevant to the part of NIH's mission that pertains to developing fundamental knowledge that will help reduce the burdens of human disability.
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