REGULATION AND PROCESSING OF AMYLOID PRECURSOR PROTEIN GENES AND GENE PRODUCTS

工作总结：该项目的一个主要焦点是发现淀粉样前体蛋白(APP)在阿尔茨海默病(AD)的病因和病理中的作用。这种蛋白质的正常生理作用也在研究中。由于APP的加工过程以及早老素(PS)突变对APP加工的影响直接关系到AD衰老斑块中A-β多肽的细胞内分泌和细胞外沉积，因此APP的研究具有重要意义。APP的加工过程还会产生可能具有神经营养特性的分泌形式的蛋白质。阿尔茨海默病患者的大脑表现出选择性和大量的神经元丢失。鉴于我们实验室最近的证据表明，在细胞培养中，家族性AD突变形式的APP和PS的过度表达会导致细胞凋亡，我们有兴趣研究这种细胞死亡的机制。我们实验室的目的之一是发现APP或PS突变是如何导致AD的特定神经细胞丢失的。以前我们发现，在稳定转染的PC12细胞中，突变形式的APP过表达导致APP细胞内淀粉样蛋白C末端片段的产生增加。伴随而来的是几天内细胞凋亡性增加。我们最近发现，通过腺病毒介导的基因转移，FAD突变APP的过度表达导致原代皮质神经元的细胞死亡。细胞内可检测到大量的羧基末端APP片段和A-β多肽。根据一种检测与寡核小体结合的片段DNA的酶联免疫吸附试验方法，过度表达导致72小时内凋亡细胞死亡增加约四倍。此外，野生型APP的过度表达导致细胞死亡增加，这表明正常APP水平的升高可能具有神经毒性特征。在初级皮质神经元中，这种细胞死亡，进而在AD中，可能是由于APP的神经毒性和神经营养加工产物的产生不平衡造成的。这些产物可能作用于细胞内，或者可能具有旁分泌效应，释放到介质中影响邻近的细胞。这些结果为AD的治疗干预提供了靶向凋亡途径和APP处理的特定元件的理论基础。-淀粉样蛋白、A-β肽、衰老、神经退行性变、细胞凋亡

Summary of Work:A major focus of this project is to discover the role of the Amyloid Precursor Protein (APP) in the etiology and pathology of Alzheimers Disease (AD). The normal physiological role of this protein is also under investigation. APP is important to study since the processing of APP and the effect of Presenilin (PS) mutations on APP processing bear directly on the increased intracellular production and extracellular deposition of A-beta peptides in senile plaques of AD. The processing of APP also generates secreted forms of the protein which may have neurotrophic properties. Brains of AD patients exhibit selective and massive neuronal loss. In light of recent evidence from our laboratory showing that, in cell culture, over-expression of familial AD mutated forms of APP and PS causes apoptotic cell death, we are interested in examining the mechanisms involved in this cell death. One of the aims of our laboratory is to discover how APP or PS mutations lead to specific neuronal cell loss in AD.Previously we showed that over-expression of mutated forms of APP in stably transfected PC12 cells led to an increased production of intracellular, amyloidogenic C-terminal fragments of APP. This was accompanied by increased apoptotic cell death over several days. We recently showed that over-expression of FAD mutant APP by adenovirus-mediated gene transfer leads to cell death of primary cortical neurons. Increased amounts of carboxyl terminal APP fragments and A-beta peptide were detected intracellularly. The overexpression led to about a four-fold increase in apoptotic cell death over 72 hours as measured by an ELISA method detecting fragmented DNA bound to oligonucleosomes. In addition wild-type APP over-expression caused an increase in cell death suggesting that elevated levels of normal APP may have neurotoxic characteristics. This cell death in primary cortical neurons and by extension, in AD, may result from an imbalance in the generation of neurotoxic and neurotrophic processing products of APP. These products may act within the cell or may have a paracrine effect, being released into the media to affect neighboring cells. These results provide a rationale for targeting particular elements of apoptotic pathways and APP processing for therapeutic intervention in AD. - amyloid, A-beta peptide, aging, neurodegeneration, apoptosis