REGULATION AND PROCESSING OF AMYLOID PRECURSOR PROTEIN GENES AND GENE PRODUCTS

工作总结：本项目的一个主要重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。APP是重要的研究，因为APP的加工和早老素（PS）突变对APP加工的影响直接影响AD中A-β肽的产生和细胞外沉积的增加。APP的加工还产生两种分泌形式的蛋白质，其可能具有神经营养或神经毒性特性。AD患者的大脑表现出突触连接性降低和选择性和大量神经元损失。我们有兴趣研究这种细胞死亡的机制。以前，我们表明，在稳定转染的PC 12细胞中APP的FAD突变体形式的过度表达导致了几天内增加的凋亡性细胞死亡，并且通过腺病毒介导的基因转移过度表达这些相同的APP导致了原代皮层神经元的细胞死亡。最近，我们一直在研究几种A-β肽对人神经母细胞瘤和星形细胞瘤细胞的影响。低浓度的A-β 1-42通过细胞凋亡杀死SH-SY 5 Y细胞，如通过ELISA方法所测量的，而较高浓度的A-β 1 - 42通过坏死杀死。A-β 1-40的效力要小得多。通过α-和γ-分泌酶切割APP衍生自APP的A-β 17-42，剂量依赖性地杀死这些细胞。通过cDNA阵列技术监测的这些细胞A-β肽处理后基因表达的早期变化包括骨化相关和氧化应激相关基因的增加以及神经营养因子和生长相关原癌基因的减少。用A-β肽处理星形细胞瘤细胞导致其活化并诱导MCP-1基因转录和蛋白质合成。初步研究表明，A-β 1-42和1-40通过不同的信号转导途径激活这些细胞。这些结果为靶向细胞凋亡和炎症通路的特定元件以及APP加工用于AD的治疗干预提供了理论基础。

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 Alzheimer's 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 production and extracellular deposition of A-beta peptides in AD. The processing of APP also generates two secreted forms of the protein, which may have neurotrophic or neurotoxic properties. Brains of AD patients exhibit decreased synaptic connectivity and selective and massive neuronal loss. We are interested in examining the mechanisms involved in this cell death. Previously, we showed that over-expression of FAD mutant forms of APP in stably transfected PC12 cells led to increased apoptotic cell death over several days and that over-expression of these same APPs by adenovirus-mediated gene transfer led to cell death of primary cortical neurons. More recently, we have been studying the effects of several A-beta peptides on human neuroblastoma and astrocytoma cells. Low concentrations of A-beta 1-42 kill SH-SY5Y cells by apoptosis as measured by an ELISA method while higher concentrations kill by necrosis. A-beta 1-40 is much less potent. A-beta 17-42, derived from APP by alpha- and gamma-secretase cutting of APP, dose-dependently kills these cells apoptotically. Early changes in gene expression after A-beta peptide treatment of these cells as monitored by cDNA array techniques include increases in apoptosis-related and oxidative stress-related genes and decreases in neurotrophic factor and growth-related proto-oncogenes. Treatment of astrocytoma cells with A-beta peptides leads to their activation and induction of MCP-1 gene transcription and protein synthesis. Initial studies suggest that A-beta 1-42 and 1-40 are activating these cells by different signal transduction pathways. These results provide a rationale for targeting particular elements of apoptotic and inflammatory pathways as well as APP processing for therapeutic intervention in AD.