Project Grant R01EY036019

INVESTIGATING PHOTORECEPTOR NUCLEAR MIGRATION DEFECTS CAUSED BY LOSS OF RETINITIS PIGMENTOSA 2. - ABSTRACT RETINAL INHERITED DISEASES ARE A LEADING CAUSE OF BLINDNESS WORLDWIDE. DESPITE MAJOR PROGRESS IN THE DISCOVERY OF CAUSATIVE GENES UNDERLYING INHERITED RETINAL DISEASE, UNDERSTANDING THE NATURAL HISTORY AND PHENOTYPE OF A PARTICULAR MOLECULAR DIAGNOSIS REMAINS LIMITED. RECENTLY, WE FOUND THAT DOMINANT HUMAN MUTATIONS IN ARL3 CAUSE HIGHER LEVELS OF ARL3-GTP ACTIVITY, RESULTING IN A DEVELOPMENTAL PHENOTYPE THAT PREVENTS PHOTORECEPTOR NUCLEI FROM TRANSLOCATING PROPERLY INTO THE OUTER NUCLEAR LAYER OF THE RETINA. ARL3 IS A SMALL GTPASE THAT REGULATES THE ENRICHMENT OF LIPID PROTEINS INTO THE CILIUM. BUILDING FROM THIS DISCOVERY, WE WANTED TO DETERMINE WHETHER OTHER MUTATIONS THAT CAUSE ELEVATED LEVELS OF ARL3-GTP ACTIVITY HAD A SIMILAR PHENOTYPE. RP2 IS THE GTPASE ACTIVATING PROTEIN (GAP) THAT FACILITATES GTP HYDROLYSIS BY RETURNING ARL3 RETURNING IT TO THE INACTIVE GDP STATE. HUMAN MUTATIONS IN THE RP2 GENE CAUSE X-LINKED RETINITIS PIGMENTOSA AND LOSS OF RP2 GAP ACTIVITY WOULD BE EXPECTED TO INCREASE LEVELS OF ARL3-GTP IN THE CELL. IN OUR PRELIMINARY DATA, WE REDERIVED THE RP2NULL MOUSE AND FOUND THAT IT HAS THE SAME PHOTORECEPTOR NUCLEAR MIGRATION DEFECT AS WE OBSERVED WITH DOMINANT HUMAN MUTATIONS IN ARL3. THE MISLOCALIZATION OF PHOTORECEPTOR NUCLEI INTO THE INNER NUCLEAR LAYER WAS OVERLOOKED IN PREVIOUS PUBLICATIONS AND DEMANDS A THOROUGH EXAMINATION. THE GOALS OF OUR PROPOSAL ARE TO INVESTIGATE HOW THIS DEVELOPMENTAL PHENOTYPE AFFECTS PHOTORECEPTOR FUNCTION AND/OR HEALTH, WHETHER THIS PHENOTYPE CAN BE RESTORED BY SEQUESTERING THE OVERLY ACTIVE ARL3-GTP AND IDENTIFY THE CILIARY SIGNAL REGULATING NUCLEAR MIGRATION THAT IS PERTURBED BY EXCESS ARL3-GTP ACTIVITY. IN ADDITION TO BUILDING A COMPREHENSIVE UNDERSTANDING OF HOW MISLOCALIZED PHOTORECEPTOR NUCLEI AFFECT FUNCTION AND LEAD TO RETINAL DEGENERATION, OUR AIMS UTILIZE PROXIMITY LABELING APPROACHES TO UNCOVER THE CILIARY SIGNAL AND THE MECHANISM BY WHICH PHOTORECEPTORS GUIDE NUCLEAR MIGRATION DURING DEVELOPMENT. FURTHERMORE, WE WILL EMPLOY GENE DELIVERY TECHNIQUES IN THE RP2NULL RODS TO RAPIDLY SCREEN CANDIDATES THAT WE PREVIOUSLY FOUND RESTORED THE MIGRATION DEFECT CAUSED BY DOMINANT ARL3 MUTATIONS. COMPLETING OUR AIMS WILL PROVIDE INSIGHT INTO POST-MITOTIC NUCLEAR MIGRATION WITHIN THE DEVELOPING RETINA, HOW THESE PROCESSES FAIL DURING DISEASE, AND HOW MISLOCALIZED PHOTORECEPTOR NUCLEI IMPACT RETINAL HEALTH AND FUNCTION. THIS PROPOSAL WILL PROVIDE FUNDAMENTAL KNOWLEDGE ABOUT THE PATHOBIOLOGY UNDERLYING RP2 AND ARL3 GENE MUTATIONS AND IDENTIFY POTENTIAL THERAPEUTIC TARGETS FOR PATIENTS SUFFERING FROM INHERITED BLINDNESS.