Drusen and AMD: sub-type isolation and characterization

描述(申请人提供)：本申请的广泛、长期目标是用分子术语确定黄斑中视网膜色素上皮(RPE)下方软性玻璃体积聚与发生老年性黄斑变性(AMD)风险增加之间的联系。黄斑部软性玻璃体的存在是发生AMD的显著危险因素。令人惊讶的是，人们对黄褐斑的成分或来源知之甚少。为此，已经开发了一种新的玻璃体分离方法，该方法允许从供体眼组织中收集微克量的玻璃体。在分离时，可以识别和分离不同的酒渣子亚型，以用于表征其分子组成的研究。肌萎缩侧索硬化症的诊断作用可以与动脉粥样硬化的血胆固醇水平相提并论。玻璃体的存在和丰富程度，就像血液中的胆固醇水平一样，表明了患者患上这种疾病的风险程度。由于玻璃疣与老年性黄斑变性的关系，了解不同玻璃疣亚型的组成将提供与玻璃疣形成相关的可能途径的重要信息。新的蛋白质或存在于玻璃疣中的蛋白质的常见修饰，应该为治疗AMD的治疗剂的潜在药物靶点提供洞察力。目前的应用集中在利用这种玻璃膜分离程序来确定正常和AMD组织中玻璃膜亚型的分子组成、分布和细胞来源。这三个具体目标是：(1)检验可以从供体眼组织中分离出不同亚群的玻璃体的假设。(硬与软、凹与外周、旧与旧、球形与扁平、琥珀色与透明、不透明与颗粒状，等等)。(2)验证不同的结构特征反映出不同的分子组成的假说(光镜、组织化学、电子显微镜、SDS/PAGE、Western blotting、质谱仪)。(3)检验存在于玻璃膜中的某些分子是新的，并且没有在Bruch膜中发现的假设。

DESCRIPTION (provided by applicant): The broad, long-term objective of this application is to define in molecular terms the linkage between the accumulation of soft drusen below the retinal pigment epithelium (RPE) in the macula and the increased risk of developing age-related macular degeneration (AMD). The presence of soft drusen in the macula is the hallmark risk factor for developing AMD. Surprisingly little is known of the composition or origin of drusen. To this end a novel method for drusen isolation has been developed that allows the collection of microgram quantities of drusen from donor eye tissue. At the time of isolation, different drusen sub-types can be identified and separated for use in studies that will characterize their molecular composition. The diagnostic utility of drusen in AMD can be likened to that of blood levels of cholesterol in atherosclerosis. The presence and abundance of drusen, like the level of cholesterol in the blood, indicates the degree to which a patient is at risk for developing the disease. Because of the relationship of drusen and AMD, understanding the composition of different drusen sub-types will provide important information on possible pathways that are causally involved in drusen development. Novel proteins or common modifications of proteins present in drusen, should provide insight as to potential drug targets of therapeutic agents to treat AMD. The current application is focused on exploiting this drusen isolation procedure to define the molecular composition, distribution and cellular origin of drusen sub-types in normal and AMD tissues. The three specific aims are: (1) To test the hypothesis that different sub-populations of drusen can be isolated from donor eye tissue. (hard vs. soft, foveal vs. peripheral, old vs. older, spherical vs. flat, amber vs. clear, opaque vs. granular, etc.). (2) To test the hypothesis that different structural features of drusen sub-populations reflect different molecular composition (light microscopy, histochemistry, electron microscopy, SDS/PAGE, Western blotting, mass spectrometry). (3) To test the hypothesis that some of the molecules present in drusen are novel and are not found in Bruch's membrane.