Cell-type Specific Genomics in the Aging Human Retina

描述（由申请人提供）：我们这个探索性提案的目标是了解人类视网膜中神经元老化如何依赖于形态表型。我们将通过结合视杆细胞和视锥细胞不同光谱类型的免疫细胞化学鉴定和基因表达序列分析（SAGE），生成衰老光感受器的全面遗传图谱。这些信息将使我们能够比较细胞类型的特定模式，这些模式可能有助于或阻碍与年龄相关的光感受器变性。与年龄相关的视力缺陷是大脑全面衰老的代价高昂、使人衰弱和心理负担沉重的后果。这些缺陷与神经视网膜的许多生理变化有关，其中最显著的是杆状和锥状细胞的缺失。光感受器也主要针对与年龄相关的视网膜疾病黄斑变性。光感受器对衰老和疾病的易感性取决于神经元表型和个体变异。例如，视杆细胞远比视锥细胞脆弱，而心理物理学研究表明，对蓝光敏感的“S”视锥细胞可能比对绿色和红色敏感的M和L视锥细胞衰退得更快。这些损失在不同的个体中并不一致。虽然眼睛中的外在因素一定会导致光感受器的衰退，但我们假设不同光感受器表型的不同易感性与细胞类型特异性基因表达的内在模式相对应。我们开发了一种独特的、高度敏感的技术来构建、探测和比较完整的基因文库（cDNA），这些基因文库来自醛保存视网膜中精选的标记神经元的RNA。这种“微收获”使我们能够在不同的神经细胞类型中比较非常精确的表达序列。我们将从年轻和年老的人中央视网膜的视杆细胞、S细胞和MIL视锥细胞中构建cDNA文库。从这些cdna中，我们建议使用经过少量RNA修饰的SAGE方案来(1)确定相同年龄的人类供体中每种光感受器细胞类型的基因表达如何变化，(2)测试衰老是否与光感受器基因表达的模式变化相关，以及表达是否与光感受器存活相关，以及(3)确定不同光感受器细胞类型对衰老的遗传反应。所产生的信息将为更广泛的研究视网膜和大脑神经元老化的机制提供基础。

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.