MOLECULAR BIOLOGY OF OUTER RETINA-SPECIFIC PROTEINS

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The retinal pigment epithelium (RPE) plays a pivotal role in the development and function of the outer retina. We are interested in RPE-specific mechanisms, at both the regulatory and functional levels. This will help us gain a better understanding of the RPE in normal and disease states and provide us with tools for further analysis of the RPE. To this end we have been studying the function and regulation of RPE65, a gene whose expression is restricted to the RPE. We studied the possiblity that RPE65 is the retinol isomerase, an enzyme essential to vision and (like RPE65) restricted to the RPE. Firstly, we found that isomerase activity co-purifies with RPE65 and is separable from retinyl ester synthase activity. We found that isomerase activity was inhibited by 2 antibodies to RPE65, suggesting that RPE65 plays a role in the isomerase reaction or may be the isomerase itself. To better understand the function of RPE65 and its role in RPE/photoreceptor interactions we are making an RPE65 knockout mouse. Mouse embryonic stem cells containing a targeted disruption of the RPE65 locus were used to generate chimeric mice. Genotyping of progeny from these chimeras confirmed germline disruption of RPE65. Verified heterozygous males and females are being mated to generate homozygous knockout mice. These will be tested to establish a phenotype for this knockout. RPE65 promoter function was studied with transgenic mice containing RPE65 promoter/reporter constructs. In all constructs, activity was restricted to the posterior segment of the eye/RPE. From our data we conclude that: i) the RPE65 promoter is RPE-specific; ii) the minimal length for promoter activity is found from between -700 and -300 bp upstream of the start site; iii) there may be positive and negative control elements upstream of this region. Given the implication of RPE65 in retinoid metabolism, mutations in the RPE65 gene might cause an early dysfunction of photoreceptors. Thus, patients with Leber's congenital amaurosis (LCA), a heterogeneous disorder characterized by blindness at birth, were screened for mutations in the RPE65 gene. Compound heterozygous mutations resulting in premature termination of RPE65 were found in 2 siblings. Heterozygous carriers of either mutation were asymptomatic, strongly supporting that these two mutations together are responsible for this form of LCA, assigned as LCA2.