Identifying genes in Beta-Amyloid Production

描述(由申请人提供)：大脑中β-淀粉样蛋白(Abeta)多肽的过度产生和积累被认为启动了最终导致阿尔茨海默病(AD)的病理性级联反应。常染色体显性形式的AD似乎通过促进Abeta的产生而致病，尤其是Abeta42。β-淀粉样前体蛋白(β-APP)是由β-和γ-分泌酶活性分解而来的。抑制任何一种分泌酶都是AD治疗的主要目标。β-分泌酶是新近发现的，但导致Abeta42产生的γ-分泌酶尚未被鉴定，尽管早老素1被证明是γ-分泌酶的中心成分，它可能由一种蛋白质复合体组成。因此，进一步阐明伽玛分泌酶复合体，以及发现影响β或伽马分泌酶活性的蛋白质(基因)，是AD研究的主要目标。随机纯合子敲除(RHKO)是一种基于生物学功能发现基因的遗传学方法，已成功地用于识别与肿瘤发生、泛素介导的蛋白质降解和对感染性物质的抗性有关的基因。具体地说，RHKO通过使用包含受调控的反义启动子的基因搜索(逆转录病毒)载体盒来实现染色体基因的两个等位基因的随机灭活。因此，失活的基因稍后可以被打开以验证基因敲除。此外，载体中还包括一个标签，这样就可以很容易地分离基因。最近版本的RHKO使转基因细胞产生含有目标基因的环状质粒，可用于转化细菌进行快速DNA克隆。在几个新开发的检测系统中，RHKO的一个改进版本将被用来识别其失活会改变β和伽马分泌酶活性的基因。这些基因对Abeta生成的影响将在四环素调控的细胞培养系统中得到验证。基因产物的细胞生物学特性将在细胞培养系统和转基因小鼠模型中进行研究。这些结果有望阐明Abeta生成及其调控的遗传途径，并允许评估新发现的基因的诊断和治疗潜力。特定目标1建议识别影响伽马或β分泌酶活性的一个或多个基因。具体目标2是验证和鉴定已鉴定的基因，并了解这些基因产物可能如何影响β-APP代谢。

DESCRIPTION (provided by applicant): The excessive production and accumulation of beta-amyloid (Abeta) peptides within the brain is believed to initiate the pathological cascade culminating in Alzheimer Disease (AD). Autosomal dominant forms of AD appear to cause disease by promoting Abeta production, especially Abeta42. Abeta is derived from the proteolytic cleavage of beta-amyloid precursor protein (betaAPP) by beta- and gamma-secretase activities. Inhibiting either secretase is a major goal in AD therapeutics. Beta-secretase was recently identified, gamma-secretase, which is responsible for Abeta42 generation, has not been identified, though presenilin 1 was shown to be a central component of gamma-secretase which probably consists of a complex of proteins. Thus, the further elucidation of a gamma-secretase complex, as well as the discovery of proteins (genes) that influence either beta- or gamma-secretase activities, are major goals in AD research. Random Homozygous Knockout (RHKO) is a genetic approach designed to discover genes based on their biological function and has been successfully used to identify genes involved in tumorigenesis, ubiquitin-mediated protein degradation and resistance to infectious agents. Specifically, RHKO accomplishes random inactivation of both alleles of chromosomal genes by using gene search (retroviral) vector cassettes that contain a regulated antisense promoter. Hence, an inactivated gene can later be turned on to validate the knockout. Also, a tag is included in the vector so the gene can be easily isolated. A recent version of RHKO causes transfected cells to produce circularized plasmids containing the targeted gene, which can be used to transform bacteria for rapid DNA cloning. A modified version of RHKO will be used to identify genes whose inactivation alters beta- and gamma-secretase activities in several newly developed assay systems. The effects of such genes on Abeta generation will be validated in a tetracycline regulated cell culture systems. Cell biological characteristics of the gene products will be studied in cell culture systems and in transgenic mouse models. The results are expected to illuminate genetic pathways of Abeta generation and its regulation, and to allow evaluation of the diagnostic and therapeutic potential of the newly identified genes. Specific Aim 1 proposes to identify gamma-secretase gene or genes affecting gamma- or beta-secretase activities. Specific Aim 2 is to validate and characterize the genes identified and to understand how these gene products may affect betaAPP metabolism.