Functional Analysis of Novel Genes in Eye Development and Vision

National Eye Institute

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Abstract

Age-related macular degeneration (AMD) is a major cause of vision loss. We have developed a cell-culture model for serum-deprivation AMD using RPE-derived cells. In AMD, changes at Bruch's membrane and in the capillary bed are likely to restrict access of serum components to the RPE. We have shown that serum deprivation of RPE cells leads to a marked upregulation of cholesterol synthesis and transport and the accumulation of cholesterol in the RPE. This is strongly reminiscent of the accumulation of cholesterol RPE that we and others have seen in human AMD. A major discovery of the analysis of gene expression in the cell model is the induction of amelotin (AMTN), a protein of calcium/hydroxyapatite (HAP) mineralization. In cell culture AMTN is responsible for HAP deposition and siRNA knockdown of AMTN inhibits calcification. Furthermore, we showed that AMTN is expressed in human donor eyes with dry AMD and is associated with HAP spherules. We have also developed a transgenic mouse model which specifically targets expression of human AMTN to retinal pigment epithelium using a novel cassette based on the RPE65 gene. This efficiently and specifically expresses human AMTN in mouse RPE and causes several morphological changes in RPE and Bruch's membrane similar to those seen in AMD. However, the transgene alone does not produce HAP deposits, presumably because of insufficient availability of calcium. Since tissue wounding is associated with increased calcium flux, we used the well established technique of retinal laser injury in the TG mice. This approach led to formation of lesions resembling the large "soft" drusen associated with Geographic Atrophy(GA)/dry AMD. These lesions contain AMTN associated with HAP spherules along with cholesterol and Fibulin3. This is the first animal model to include these important features of GA. Initial experiments have been performed to test suppression of human AMTN expression in transgenic mice using siRNA. Protocols are being optimized. We have also developed lines of AMTN KO mice to study the function role of AMTN in retinal wound repair. First experiments are under evaluation and suggest that AMTN is required for normal healing. We have developed a custom antibody for mouse AMTN We have identified several FDA approved drugs that inhibit AMTN function and will be tested as potential therapies to slow AMD progression. We have collaborated with a major health care provider to investigate any association of these drugs with AMD onset and progression in patient populations. This has been challenging in terms of data extraction and cost and is currently on hold.