Mutations in the gene BEST1 are causally associated with 4 clinically distinct human retinal degenerative diseases characterized by accumulation of lipofuscin in the retinal pigment epithelium (RPE). Best vitelliform macular dystrophy (BVMD), the most common. is characterized by diminished central vision resulting from an """"""""egg-yolk"""""""" like lesion in the macula. Eventually the lesion can become disrupted leading to an atrophic form of macular degeneration. All individuals with BVMD exhibit a depressed electrooculogram light peak (LP) with a normal clinical electroretinogram (ERG). The LP is generated by a Ca2+ dependent Cl- conductance across the RPE, where Best1 is localized. This led to the hypothesis that Best1 is a Ca2+ activated Cl- channel (CaCC) that generates the LP, and that BVMD results from loss of Best1 CaCC activity. While this hypothesis is supported by heterologous expression studies, our past goal was to test the """"""""CaCC hypothesis"""""""" in animal models and physiologically relevant RPE cell cultures. We found that Best1 is not required to generate the LP. Thus, our data do not support the """"""""CaCC hypothesis"""""""", and neither do the clinical presentations of autosomal dominant vitreoretinochoroidopathy (ADVIRC), and autosomal recessive bestrophinopathy (ARB), diseases more severe than BVMD that also result from mutations in BEST1. Over the past 5 years, we demonstrated that the LP requires voltage dependent Ca2+ channels (VDCC), and Best1 interacts with them. Analysis of Best1W93C knock-in mice, and human RPE cultures, revealed that mutations in Best1 suppress Ca2+ dependent ion transport and alter the intracellular pH (pHi) of RPE cells. Thus, we propose to test a new hypothesis;that Best1 dysfunction causes aberrant regulation of intracellular pH, Ca2+ dependent ion transport, and photoreceptor outer segment phagocytosis resulting in the pathogenic accumulation of lipofusin and defective RPE fluid transport. This will be accomplished via 3 specific aims: 1. To Determine How Different Mutations in Best1 Cause 4 Clinically Distinct Diseases.
Specific Aim 2. To Dissect the Events Leading to Lipofuscin Accumulation in Bestrophinopathies Specific Aim 3. To Determine the Physiological Consequences of Bestrophin Dysfunction on ion homeostasis and fluid transport.
These aims will be accomplished by the production of mouse models of ARB and ADVIRC. How Best1 mutations cause lipofuscin accumulation will be assessd by determining the impact of Best1 mutations on the kinetics of photoreceptor outer segment (OS) phagocytosis. Finally we will seek to determine how bestrophin dysfunction leads to the fluid filled retinal detachments that occur in bestrophinopathies by examining the effects of Best1 mutations on, Ca2+ signaling, pHi, and fluid transport. At its conclusion this work will provide a comprehensive model of the pathogenesis of bestrophinopathies that should focus the search for therapies for these incurable blinding eye diseases.
Mutations in the gene BEST1, encoding the protein bestrophin-1 (Best1), cause 4 clinically distinct retinal degenerative eye diseases in man21, 26-29. Understanding the function of Best1 and determining how mutations in Best1 cause disease, is a critical public health objective. Success in this application will set the stage for identification of therapies to prevent or cure vision loss in carriers of BEST1 mutations, and potentially other blinding eye diseases.
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