REGULATION AND PROCESSING OF AMYLOID PRECURSOR PROTEIN GENES AND GENE PRODUCTS

淀粉样前体蛋白基因和基因产物的调控和加工

• 批准号: 5200317
• 负责人: J W KUSIAK
• 金额: --
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/5200317
• 关键词: Alzheimer's disease; PC12 cells; acidity /alkalinity; aging; amyloid proteins; antioxidants; apoptosis; calcium; cell adhesion; cellular pathology; cysteine; flow cytometry; gene expression; gene mutation; mitochondria; molecular pathology; mutant; oxidative stress; posttranslational modifications; scanning electron microscopy; tissue /cell culture; transmission electron microscopy

Alzheimer's Disease (AD) is a major neurological disorder of the elderly affecting as much as 50% of the population over 80. No cure for this disease is known and its etiology is obscure. However, several genetic loci have been identified in subsets of AD patients causing early onset forms of the disorder or increasing the risk of a late onset form. One of these mutations is in the gene coding for the amyloid precursor protein (APP). Normal processing of this protein generates a peptide fragment, termed Ab, which is a major component of the senile plaques in brains of AD patients. These observations suggest an important role of APP in the causation of this disorder. Our work has focused on establishing a model system in which mutant APP genes are over-expressed in several types of cultured cells thought to be important in AD. We previously showed that neuronal-like PC-12 cells and endothelial cells over-expressing mutant APPs undergo morphological changes and shift the processing of APP towards production of larger carboxyl terminal amyloidogenic fragments within the cells. We have now determined by a combination of TUNEL staining and FACS analysis that as many as 20% of PC-12 cells expressing these mutant APPs undergo an apoptotic cell death. Conditioned media from these cells, containing slightly increased amounts of Ab peptide did not induce apoptosis in untransfected cells, suggesting an intracellular site for this effect. Scanning electron microscopic analysis of these cells revealed cell body compaction and membrane blebbing while transmission EM showed nuclear chromatin condensation at the periphery of the nucleus, both phenomenon characteristic of apoptosis. Over-expression of mutant APPs may cause apoptosis by inducing oxidative damage to cells since an anti-oxidant compound, L-cysteine prevents DNA laddering and cell death. We are currently examining other possible mechanisms of apoptosis in these cells due to mutant APP over-expression. Our emphasis is on examining intracellular calcium levels, pH changes, cell adhesion properties, and mitochondrial oxidative damage.

阿尔茨海默氏病（AD）是老年人的主要神经系统疾病 影响80岁以上人口的50％。无法治愈 疾病是已知的，其病因是晦涩的。但是，几个遗传 在引起早发病的AD患者子集中已经确定了基因座 疾病的形式或增加发病形式的风险。一 这些突变是编码淀粉样前体的基因 蛋白质（APP）。该蛋白质的正常加工会产生肽 碎片，称为AB，它是老年斑块的主要组成部分 广告患者的大脑。这些观察表明 应用于这种疾病的因果。我们的工作重点是 建立一个模型系统，其中突变应用程序基因过表达 在几种类型的培养细胞中，人们认为在AD中很重要。我们 以前表明神经元样PC-12细胞和内皮细胞 过表达的突变应用程序会经历形态变化并改变 处理应用程序以生产较大的羧基终端 细胞内的淀粉样生成片段。我们现在由 TUNEL染色和FACS分析的组合，多达20％ 表达这些突变应用程序的PC-12细胞会经历凋亡细胞死亡。 来自这些单元的条件培养基，含量略有增加 AB肽的肽不会诱导未转染的细胞凋亡，这表明 用于这种效果的细胞内部位。扫描电子显微镜 对这些细胞的分析揭示了细胞体压实和膜 传播EM时吹散的核染色质冷凝 核的外围，这都是现象的特征 凋亡。突变应用程序的过表达可能会通过诱导引起凋亡 由于抗氧化剂化合物L-半胱氨酸，对细胞的氧化损伤 防止DNA梯子和细胞死亡。我们目前正在研究其他 由于突变APP，这些细胞中凋亡的可能机制 过表达。我们的重点是检查细胞内钙 水平，pH变化，细胞粘附特性和线粒体氧化 损害。