title: Pharmacological Amelioration of Cone Survival and Vision in a Mouse Model for Leber Congenital Amaurosis.
abstract: RPE65, an abundant membrane-associate protein in the retinal pigment epithelium (RPE), is a key retinoid isomerase of the visual cycle necessary for generating 11-cis-retinal that functions not only as a molecular switch for activating cone and rod visual pigments in response to light stimulation, but also as a chaperone for normal trafficking of cone opsins to the outer segments. Many mutations in RPE65 are associated with Leber congenital amaurosis (LCA). A R91W substitution, the most frequent LCA-associated mutation, results in a severe decrease in protein level and enzymatic activity of RPE65, causing cone opsin mislocalization and early cone degeneration in the mutation knock-in mouse model of LCA. Here we show that R91W RPE65 undergoes ubiquitination-dependent proteasomal degradation in the knock-in mouse RPE due to misfolding. The 26S proteasome non-ATPase regulatory subunit 13 mediated degradation specifically of misfolded R91W RPE65. The mutation disrupted membrane-association and colocalization of RPE65 with lecithin:retinol acyltransferase (LRAT) that provides the hydrophobic substrate for RPE65. Systemic administration of sodium 4-phenylbutyrate (PBA), a chemical chaperone, increased protein stability, enzymatic activity, membrane-association, and colocalization of R91W RPE65 with LRAT. This rescue effect increased synthesis of 11-cis-retinal and 9-cis-retinal, a functional iso-chromophore of the visual pigments, led to alleviation of S-opsin mislocalization and cone degeneration in the knock-in mice. Importantly, PBA-treatment also improved cone-mediated vision in the mutant mice. These results indicate that PBA, a U.S. Food and Drug Administration-approved safe oral medication, may provide a noninvasive therapeutic intervention that delays daylight vision loss in patients with RPE65 mutations.
  
  
  
  LCA is a severe early onset retinal dystrophy. Recent clinical trials of gene therapy have implicated the need of an alternative or combination therapy to improve cone survival and function in patients with LCA caused by RPE65 mutations. Using a mouse model carrying the most frequent LCA-associated mutation (R91W), we found that the mutant RPE65 underwent ubiquitination-dependent proteasomal degradation due to misfolding. Treatment of the mice with a chemical chaperone partially corrected stability, enzymatic activity, and subcellular localization of R91W RPE65, which was also accompanied by improvement of cone survival and vision. These findings identify an in vivo molecular pathogenic mechanism for R91W mutation and provide a feasible pharmacological approach that can delay vision loss in patients with RPE65 mutations.
authors.affiliation: Department of Ophthalmology and Neuroscience Center, Louisiana State University School of Medicine, New Orleans, Louisiana 70112, and.
authors.forename: Songhua
authors.initials: S
authors.lastname: Li
authors.affiliation: Department of Ophthalmology, University of Zurich, CH-8952 Schlieren, Switzerland.
authors.forename: Marijana
authors.initials: M
authors.lastname: Samardzija
authors.affiliation: Department of Ophthalmology and Neuroscience Center, Louisiana State University School of Medicine, New Orleans, Louisiana 70112, and Department of Ophthalmology, University of Zurich, CH-8952 Schlieren, Switzerland.
authors.forename: Zhihui
authors.initials: Z
authors.lastname: Yang
authors.affiliation: Department of Ophthalmology, University of Zurich, CH-8952 Schlieren, Switzerland.
authors.forename: Christian
authors.identifier: 0000-0001-9318-4352
authors.initials: C
authors.lastname: Grimm
authors.affiliation: Department of Ophthalmology and Neuroscience Center, Louisiana State University School of Medicine, New Orleans, Louisiana 70112, and mjin@lsuhsc.edu.
authors.forename: Minghao
authors.initials: M
authors.lastname: Jin
mesh_terms: D000818:Animals; D001766:Blindness / Q000378:metabolism / Q000473:pathology / Q000517:prevention & control*; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D005260:Female; D057130:Leber Congenital Amaurosis / Q000188:drug therapy* / Q000378:metabolism* / Q000473:pathology; D008297:Male; D051379:Mice; D010654:Phenylbutyrates / Q000008:administration & dosage*; D017949:Retinal Cone Photoreceptor Cells / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D016896:Treatment Outcome; D014792:Visual Acuity / Q000187:drug effects; D019745:cis-trans-Isomerases / Q000235:genetics / Q000378:metabolism*; D000099178:Retinoid Isomerohydrolase
chemical_list: D010654:Phenylbutyrates; D019745:cis-trans-Isomerases; D000099178:Retinoid Isomerohydrolase; C075773:4-phenylbutyric acid
keywords: Leber congenital amaurosis; Rpe65; chemical chaperone; cone photoreceptor; retina; retinoid visual cycle