Functional Analysis of Novel Genes in Eye Development and Vision

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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. Analysis of gene expression in the cell model has led to the discovery of 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 morphological changes in RPE and Bruch's membrane similar to those seen in AMD. We have now found an experimental wound system that induces HAP/AMTN deposits in RPE in mice. This is being used to test the effects of siRNA inhibition of AMTN for potential therapeutic use. We gave identified several FDA approved drugs that inhibit AMTN function and will be tested as potential therapies to slow AMD progression. We are also collaborating to identify any effects of these drugs on AMD progression in patient populations. We have initiated collaborative structural studies on AMTM. The protein is intrinsically disordered and presents opportunities for new methodological approaches. Our collaborators have already provided new insights into the structure and behavior of AMTN with important implications for work on similar proteins. Combined with molecular modelling, this is giving hints of possible functional mechanisms,