Sunday, May 18, 2025
5/18/2025
IRBP in the visual cycles and retinal diseases - PROJECT SUMMARY/ABSTRACT: The interphotoreceptor retinoid-binding protein (IRBP) secreted by photoreceptors (PRs) is large (135-kD) and the most abundant soluble protein in the inter-PR matrix (IPM). Mutations and reduced expression of IRBP cause retinitis pigmentosa (RP), childhood-onset retinal dystrophy with high-myopia (CRDHM), cornea curvature, or increased susceptibility to diabetic retinopathy with unclear pathogenic mechanisms. Our newly generated knock-in (KI) model of RP demonstrated that the equivalent mouse mutation (D1078N) abolished secretion of IRBP from the PRs of the KI mouse. The D1078N IRBP was exclusively localized to the PR inner segments (IS) including the endoplasmic reticulum (ER), and formed insoluble high molecular complexes (HMC) via the disulfide bonds. The abundance of IRBP were dramatically reduced in the KI retina, possibly due to the ER- associated degradation (ERAD) of the D1078N IRBP in the PRs. The KI mouse recapitulated many disease phenotypes of the D1080N-caused RP patients, including progressive degeneration of rods & cones, aberrant visual cycle, delayed rod dark adaptation, and significantly diminished visual function of rods, M- & S-cones. Importantly, we were able to rescue the function of the RP (D1080N)- & CRDHM (E1152x)-associated IRBP mutants in the preliminary studies. To capitalize on these findings, we will accomplish three specific aims in this proposal. In Aim-1, we will elucidate the mechanisms by which D1080N mutation causes severe defect in secretion & stability of IRBP. Through computational analysis, we have identified several amino acid (AA) residues that may contribute to the secretory defect of D1080N IRBP. An AA substitution in one of these identified AAs rescued the secretion & function of the mutant IRBP. We will test whether AA substitutions in the other identified AAs can also rescue secretion, stability & function of the D1080N IRBP. We will also define the mechanisms causing ERAD of IRBP and death of PRs in the KI mice. In Aim-2, we will further characterize and expand our preliminary findings that can rescue the function of E1152x IRBP in cultured cells. Using a mouse model, we will then analyze the pathological mechanisms of the truncated IRBP that causes early loss of cone- & rod-mediated vision in children with the E1152x mutation. In Aim-3, we will test pharmacological interventions that may alleviate retinal degeneration & vision loss in the D1078N KI mouse model of RP. Through preliminary studies, we have identified small compounds that either rescue secretion & stability of D1080N IRBP in vitro or improve survival & function of rods & cones in Irbp-/- mice. We will test if these compounds improve secretion & function of D1078N IRBP as well as survival & function of PRs in the KI mice. To elucidate the in vivo action mechanisms of the compounds, we will also analyze the visual cycle, bis-retinoid formation, unfolded protein responses (UPR), oxidative stress, inflammation and cell death pathways in the KI retina. The results of these studies will provide a knowledge basis for the development of safe & effective therapeutic strategy for patients with IRBP mutations, aberrant visual cycle & oxidative or ER stress implicated in many forms of retinal diseases.
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