Identifying genes in Beta-Amyloid Production

描述（由申请人提供）：大脑中β -淀粉样蛋白（Abeta）肽的过量产生和积累被认为是阿尔茨海默病（AD）的病理级联反应的开始。常染色体显性形式的阿尔茨海默病似乎是通过促进β的产生，特别是Abeta42而引起疾病的。β来源于β和γ分泌酶活性对β -淀粉样蛋白前体蛋白（betaAPP）的蛋白水解裂解。抑制这两种分泌酶是阿尔茨海默病治疗的主要目标。β -分泌酶最近被发现，负责Abeta42生成的γ -分泌酶尚未被发现，尽管早老素1被证明是γ -分泌酶的核心成分，可能由蛋白质复合物组成。因此，进一步阐明γ -分泌酶复合物，以及发现影响β -或γ -分泌酶活性的蛋白质（基因），是阿尔茨海默病研究的主要目标。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方法，已成功用于鉴定参与肿瘤发生、泛素介导的蛋白质降解和对感染因子的抗性的基因。具体来说，RHKO通过使用含有调控反义启动子的基因搜索（逆转录病毒）载体盒式实现染色体基因的两个等位基因的随机失活。因此，一个失活的基因随后可以被激活来验证基因的敲除。此外，载体中还包含一个标签，因此可以很容易地分离出基因。RHKO的最新版本使转染的细胞产生含有目标基因的环状质粒，可用于转化细菌以进行快速DNA克隆。在几个新开发的分析系统中，RHKO的改良版本将用于鉴定其失活改变β和γ分泌酶活性的基因。这些基因对β产生的影响将在四环素调节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。该结果有望阐明Abeta产生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。特异性目标1提出鉴定γ分泌酶基因或影响γ或β分泌酶活性的基因。具体目标2是验证和表征所鉴定的基因，并了解这些基因产物如何影响betaAPP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.