MOLECULAR PROBES TO STUDY ALZHEIMER'S DISEASE

研究阿尔茨海默病的分子探针

• 批准号: 6098117
• 负责人: BRIDGET SHAFIT-ZAGARDO
• 金额: $ 18.08万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-05-15 至 2000-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6098117
• 关键词: Alzheimer's disease; Lewy body; antisense nucleic acid; cell population study; cellular pathology; electron microscopy; gene expression; human fetus tissue; human tissue; immunocytochemistry; in situ hybridization; laboratory rat; microtubule associated protein; microtubules; molecular pathology; neurons; nucleic acid sequence; protein structure function; western blottings

The development and maintenance of cell shape depends on the assembly of microtubules from tubulin monomers. This process is facilitated by microtubule-associated protein-2 (MAP-2) which is expressed throughout neuronal development. MAP-2 binds to tubulin and reduces the critical concentration of tubulin required to polymerize into microtubules. Multiple MAP-2 isoforms regulate process outgrowth and the spacing of microtubules. The goal of this proposal is to understand the regulation of MAP-2 function and to determine the temporal and spatial expression of newly isolated MAP-2 isoforms in brain and spinal cord. We have cloned and characterized six novel MAP-2 exons. By in situ hybridization and antisense strategies, we will test the hypothesis that unique regions of the 5/UTR and/or CA repeats located in the 3/ UTR play a role in intracellular localization of MAP-2 transcripts or targeting to subpopulations of neurons. Using the rat as a model system, we will test the hypothesis that MAP_2 isoforms containing newly identified exons 8 and/or 13 are developmentally regulated in subpopulations of rat spinal cord and brain neurons. Preliminary data support this hypothesis. RT- PCR, and Northern and Western blot analyses will determine the developmental expression of the transcripts and the respective proteins. The neuronal subpopulations will be examined by immunocytochemistry, and the association of these isoforms with microtubules will be examined by electron microscopy. Western blots and immunocytochemistry will be carried out using MAP-2-specific polyclonal antibodies generated in our laboratory from exons 8 and 13 translated sequences. In human brain and spinal cord, we will determine the sub-populations of neurons expressing these MAP-2 isoforms. Also, brain sections from normal, Alzheimer disease and Lewy body disease will be compared. This study will permit us to assess for the first time the developmental expression of novel MAP-2 isoforms expressing exons 8 and 13 and to understand their role in the development and maintenance of the differentiated state. Further, the ability to distinguish between the multiple MAP-2 isoforms will permit analysis of neurodegenerative diseases as assessed by aberrant MAP-2 expression.

细胞形态的形成和维持依赖于细胞内 微管蛋白单体。 这一过程由以下方面推动： 微管相关蛋白-2（MAP-2），其在整个 神经元发育 MAP-2与微管蛋白结合并降低了关键的 微管蛋白的浓度是进入微管所需的。 多种MAP-2亚型调节过程生长和细胞间隔， 微管 本提案的目的是了解该法规 MAP-2函数的时间和空间表达 脑和脊髓中新分离的MAP-2亚型。 我们有 克隆并鉴定了六个新的MAP-2外显子。 通过原位杂交 和反义策略，我们将测试的假设，独特的区域， 位于3/ UTR中的5/UTR和/或CA重复序列在以下方面起作用： MAP-2转录物的细胞内定位或靶向 神经元的亚群。 使用大鼠作为模型系统，我们将测试 MAP_2亚型含有新鉴定的外显子8的假设 和/或13在大鼠脊髓神经元亚群中受到发育调节 脊髓和脑神经元。 初步数据支持这一假设。 RT- PCR、北方和西方印迹分析将确定 转录物和相应蛋白质的发育表达。 将通过免疫细胞化学检查神经元亚群， 这些同种型与微管的结合将通过 电镜 免疫印迹和免疫细胞化学将 使用MAP-2特异性多克隆抗体进行， 实验室从外显子8和13翻译序列。 在人脑中， 脊髓，我们将确定表达神经元的亚群， 这些MAP-2亚型。 同时，正常人，老年痴呆症患者 疾病和路易体病进行比较。 这项研究将使 我们第一次评估了小说的发展表达 MAP-2亚型表达外显子8和13，并了解其在 分化状态的发展和维持。 此外，本发明还 区分多种MAP-2同种型的能力将 允许分析神经退行性疾病， MAP-2表达。