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. Furthermore, serum-deprivation leads to depletion of intracellular zinc, while many zinc-binding proteins are induced. 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. Experiments are underway to block AMTN expression or inhibit AMTN function in cells and in transgenic mice. This could lead to new approaches to slowing the progression of AMD in humans, Retbindin is a novel protein of retinal photoreceptors. A knockout mouse model shows progressive deficits in visual response and age-related defects in the outer retina that have striking similarities to some forms of age-related macular degeneration. This includes formation of drusen, RPE dysfunction, deposition of lipids and calcium, activation of microglia and premature loss of light sensitive retinal ganglion cells. Retbindin is required for light-damage in dark-adapted mouse retina, suggesting a role in 11-cis retinal transport. Recombinant retbindin has high affinity for retinoids, particularly 11-cis-retinal. This suggests a role for retbindin in the visual cycle, probably involving retinoid supply particularly to rods. Before rods are mature, retbindin is expressed in a subset of cells of the inner retina, suggesting a role in control of light entrained diurnal rhythms.