Project Grant R01EY035884

MOLECULAR MECHANISM OF THE VISUAL DISORDERS CAUSED BY G90D AND G90V RHODOPSIN MUTATIONS - PROJECT SUMMARY/ABSTRACT VISION BEGINS IN PHOTORECEPTOR CELLS WITH LIGHT ACTIVATION OF THE VISUAL PIGMENT, RHODOPSIN, WHICH TRIGGERS A TRANSDUCTION CASCADE TO PRODUCE THE CELLULAR ELECTRICAL RESPONSE. A WIDE RANGE OF BLINDING DISORDERS HAVE BEEN ASSOCIATED WITH MUTATIONS IN RHODOPSIN. FROM THESE, MUTATIONS OF GLYCINE 90 TO ASPARTATE (G90D) AND TO VALINE (G90V) HAVE BEEN REPORTED TO CAUSE CONGENITAL STATIONARY NIGHT BLINDNESS (CSNB) AND RETINITIS PIGMENTOSA (RP), RESPECTIVELY. STRUCTURAL AND BIOCHEMICAL IN VITRO STUDIES HAVE SHOWN THAT THE G90D/G90V MUTATIONS CAUSE RHODOPSIN DESTABILIZATION THAT COULD INTERFERE WITH NORMAL LIGHT DETECTION. HOWEVER, DESPITE DECADES OF RESEARCH, THE MECHANISMS BY WHICH THESE MUTATIONS CAUSE VISION LOSS REMAIN UNCLEAR AND EFFECTIVE TREATMENTS FOR PEOPLE WITH THESE MUTATIONS ARE NOT AVAILABLE. TO ADDRESS THESE QUESTIONS, WE HAVE CREATED G90D AND G90V RHODOPSIN KNOCKIN MICE. WE WILL PERFORM COMPREHENSIVE ANALYSIS OF THESE MICE TO DETERMINE THE PHENOTYPE OF THEIR ROD PHOTORECEPTORS. THESE EXPERIMENTS WILL INCLUDE MORPHOLOGICAL ANALYSIS AND IN VIVO ELETRORETINOGRAPHY AND SINGLE- CELL SUCTION ELECTRODE RECORDINGS TO DETERMINE THE PHYSIOLOGICAL PROPERTIES OF THE MUTANT RODS, COMBINED WITH MICROSPECTROPHOTOMETRIC AND BIOCHEMICAL ANALYSIS TO DETERMINE THE MOLECULAR PROPERTIES OF THE MUTANT MOUSE RHODOPSINS (AIM 1). WE WILL PERFORM EXPERIMENTS TO DETERMINE THE MOLECULAR MECHANISMS BY WHICH THE G90D AND G90V RHODOPSINS CAUSE BLINDNESS BY EVALUATING THE STABILITY OF THEIR COVALENT BONDS BETWEEN OPSIN AND CHROMOPHORE, THE BINDING AND RELEASE OF CHROMOPHORE, AND THE EQUILIBRIUM BETWEEN CHROMOPHORE-FREE AND CHROMOPHORE-BOUND MUTANT OPSINS (AIM 2). FINALLY, WE WILL ALSO TEST THE EFFICIENCY OF GENOME EDITING FOR RESCUING THE FUNCTION OF RODS WITH THE G90D OR G90V RHODOPSIN MUTATIONS (AIM 3). TOGETHER, THESE EXPERIMENTS WILL ESTABLISH THE UNIQUE DISEASE MECHANISMS OF TWO DISTINCT HUMAN VISUAL DISORDERS CAUSED BY MUTATIONS IN THE SAME RESIDUE OF RHODOPSIN AND WILL DEVELOP THERAPEUTIC APPROACHES FOR REVERSING THEIR EFFECTS THAT COULD EVENTUALLY BE USED IN HUMAN CLINICAL STUDIES. ANALYZING OUR G90D AND G90V MUTANT MICE SIDE BY SIDE WILL ALSO HELP UNDERSTAND WHY THESE TWO SIMILAR MUTATIONS PRODUCE DISTINCT CLINICAL PHENOTYPES.