Cell-type Specific Genomics in the Aging Human Retina

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DESCRIPTION (provided by applicant): Our goal for this exploratory proposal is to understand how neuronal aging depends upon morphological phenotype in the human retina. We will generate a comprehensive genetic profile of aging photoreceptors by combining immunocytochemical identification of rods and different spectral types of cone with serial analysis of gene expression (SAGE). This information will allow us to compare cell-type specific patterns that could either contribute to or impede age-related photoreceptor degeneration. Age-related deficits in vision are a costly, debilitating and psychologically onerous consequence of overall senescence of the brain. These deficits are associated with many physiological changes in the neural retina, including most prominently the bss of rods and cones. Photoreceptors are also primarily targeted in the leading age-related retinal disease, macular degeneration. The susceptibility of photoreceptors to aging and disease depends upon both neuronal phenotype and individual variation. For example, rods are far more vulnerable than cones, while psychophysical studies indicate that blue-light sensitive "S" cones may decline more rapidly than green- and red-sensitive M and L cones. These losses are not uniform across different individuals. While extrinsic factors in the eye must contribute to photoreceptor decline, we hypothesize that the differential susceptibility of distinct photoreceptor phenotypes corresponds to an intrinsic pattern of cell-type specific gene expression. We have developed a unique and highly sensitive technology to construct, probe and compare complete gene libraries (cDNA) from the RNA harvested from select, labeled neurons in aldehyde-preserved retina. This "micro-harvesting" allows us to compare with great precision expressed sequences across different neuronal cell types. We will construct cDNA libraries from rods, S and MIL cones in young and aged human central retina. From these cDNAs, we propose to use a SAGE protocol modified for small amounts of RNA to (1) determine how gene expression within each photoreceptor cell type varies between human donors of the same age, (2) test whether aging correlates with patterned changes in gene expression for photoreceptors and whether expression correlates with photoreceptor survival, and (3) determine how different photoreceptor cell types respond genetically to aging. The information generated will provide a basis for more extensive investigations of the mechanisms that mediate neuronal aging in the retina and brain.