Regulation of APP Pathway Gene Promoters in Alzheimer's

描述(申请人提供)：我们的目标是研究阿尔茨海默病(AD)的基因调控，基于阿尔茨海默病的“淀粉样假说”。淀粉样β-肽(Abeta)的过量产生会导致一系列神经退行性步骤，导致斑块形成和神经元丢失，这是阿尔茨海默病的特征。该领域尚未解决的关键问题是，是什么因素导致Abeta及其大Abeta前体蛋白(APP)的过度生产。Abeta产量的增加可能是由于APP表达的增加，或者是由于限制性的β-APP裂解酶(BACE)对其蛋白质分解过程的影响。这个建议的目的是研究i)APP的转录调控，因为APP(因此，AP)的生物发生始于转录水平，以及II)BACE基因，因为Aβ的过量生产可能是由于该基因上调导致BACE水平上升所致。具体目的是：1)研究APP启动子的功能结构域及不同试剂对其活性的影响。我们将对7.9kb的APP启动子进行功能鉴定，并研究内在(细胞因子)和外在(金属)因素如何调节启动子活性。启动子的研究将通过在不同细胞类型和原代神经元培养中进行系列缺失、突变和转基因实验来进行。2)确定APP和BACE基因调控所共有的特定因素和血管因子的影响。我们将研究IL-1α、TNF-α和CREB转录因子(Tf)在4.1kb BACE启动子活性中的作用。3)鉴定特定细胞类型的核因子。APP启动子的一个30bp的新区(-76-47)包含一个调控结构域，该结构域至少与两种蛋白质PUF和SKIP相互作用。我们将利用凝胶漂移分析和DNA亲和层析技术检测调控APP启动子活性的候选转录因子和调控APP启动子活性的候选转录因子在正常和AD脑组织中的表达情况。4)研究APP基因多态性对晚发性阿尔茨海默病发病风险的影响。我们在-3829和-1023处发现了两个可能与阿尔茨海默病相关的基因多态。我们将i)用启动子变体进行功能和DNA-蛋白质结合研究，以及ii)研究启动子与APP和Abeta水平的相关性。5)APP-5‘-UTR区的研究。APP的表达也通过5‘-非翻译区(UTR)进行调节。我们将在转录和转录后水平上测试APP5‘-UTR的双重作用，并研究其与细胞因子的相互作用。来自具有特征性FAD家族的细胞系将被分析APP和BACE基因的差异表达。研究APP和BACE基因调控对于理解APP产生导致AA生成至关重要。这些研究应该有助于开发治疗阿尔茨海默病的合适药物靶点。

DESCRIPTION (provided by applicant): Our goal is to study gene regulation in Alzheimer's disease (AD), based on the "amyloid hypothesis" of Alzheimer's disease. Overproduction of the amyloid beta-peptide (Abeta) causes a cascade of neurodegenerative steps resulting in plaque formation and neuronal loss that characterize Alzheimer's disease. The unresolved key question in the field is what factors cause overproduction of Abeta and its large Abeta precursor protein (APP). Increased Abeta production may result from an increase in APP expression, or in its proteolytic processing by the limiting beta-APP cleaving enzyme (BACE). The goals of this proposal are to investigate the transcriptional regulation of i) APP, because APP (and, hence, AP) biogenesis begins at the level of transcription, and ii) BACE gene, as Abeta overproduction may be due to increased BACE level as a result of upregulation in this gene. Specific Aims are: 1) To study the functional domains of the APP promoter and effects of different agents on its activity. We will functionally characterize the 7.9 kb APP promoter and study how intrinsic (cytokines) and extrinsic (metals) factors regulate promoter activity. Promoter will be studied by serial deletions, mutagenesis and transfection experiments in different cell types and primary neuronal cultures. 2) To identify the effects of specific factors and cvtokines common to both APP and BACE gene regulation. We will characterize the role of IL-1alpha, TNF-alpha and CREB transcription factor (TF) on 4.1kb BACE promoter activity. 3) To identify cell type-specific nuclear factors. A 30 bp novel region (-76-47) of the APP promoter contains a regulatory domain that interacts with at least two proteins, PuF and SkiP. We will test i) the candidate TFs that control APP promoter activity and ii) the status of such TF in normal and AD brain tissues using gel shift assay and DNA-affinity chromatography. 4) To characterize APP gene polymorphisms that influences the risk of late-onset Alzheimer's disease. We discovered two polymorphisms at -3829 and -1023 that may be associated with Alzheimer's disease. We will i) do functional and DNA-protein binding studies with promoter variants and ii) correlate promoter studies with levels of APP and Abeta. 5) To study the APP-5'-UTR region. APP expression is also regulated via the 5'-untranslated region (UTR). We will test a dual role for the APP5'-UTR at both transcriptional and post-transcriptional levels, and study its interaction with cytokines. Cell lines from families with characterized FAD will be analyzed for differential expression of the APP and BACE genes. Studying APP and BACE gene regulation is crucial to understand APP production leading to Aa generation. These studies should help developing suitable drug targets for the treatment of Alzheimer's disease.