Identification of an Abeta fragment produced by BACE2

描述（由申请人提供）：唐氏综合症（DS）是由人类21号染色体（chr21）的三倍引起的，是导致智力迟钝的最常见遗传原因。据推测，DS是由位于三倍chr21上的基因表达增加引发的，导致DS的大脑发育异常，但也导致进一步的神经系统并发症，特别是阿尔茨海默病（AD）样痴呆，随着DS患者长大成人。根据许多神经学和神经病理学标准，退行性痴呆人群发生ad样神经退行性变的风险明显升高，而且比一般人群至少早20-30年。在对位于DS三体的基因进行假设中性筛选以增强淀粉样前体蛋白（APP）诱导的神经退行性变时，我们发现其中一个基因，分泌酶BACE2，似乎在基于脑片的AD模型中增强了神经元退行性变的速度/程度。基于先前的文献报道和我们的初步研究，我们假设BACE2切割APP会产生一个截断的¿-淀粉样蛋白（a¿），它可能不是淀粉样蛋白，但即使在没有全长a¿的情况下，也会驱动重要的神经退行性过程。为了严格验证这一假设，有必要直接识别和表征由BACE2产生的假定A¿片段。然而，目前可用的免疫试剂不适合用于此目的。因此，本研究计划的目标是开发和测试针对假定的BACE2 A¿片段的新型抗体；利用这些试剂证明BACE2切割APP会产生a¿的截短形式；最后分离并直接测序bace2截断的A¿片段。因此，具体目标是：具体目标1：开发针对典型A¿序列c端一半的抗体。特异性目的2：直接对BACE2切割产生的A¿片段进行质谱测序。如果成功，该研究策略将为验证BACE2可能是减缓/预防退行性痴呆样神经退行性变进展的有利潜在药物靶点这一假设提供进一步的证据和必要的工具。此外，鉴于BACE2也在普通人群的大脑中表达，尽管表达水平较低，因此针对BACE2的药物在散发性和家族性AD的研究中可能也很重要，因为目前正在开发的高选择性BACE1抑制剂药物可以“揭示”由BACE2介导的神经退行性过程，这些过程可能在普通人群中也可能是潜伏的。

DESCRIPTION (provided by applicant): Down syndrome (DS) results from triplication of human chromosome 21 (chr21) and is the most common genetic cause of mental retardation. It is hypothesized that DS is initiated by increased expression of genes located on the triplicated chr21, causing abnormal brain development in DS, but also resulting in further neurological complications, prominently Alzheimer's disease (AD)-like dementia, as DS patients grow into adulthood. By a number of neurological and neuropathological criteria, the DS population is at substantially elevated risk for AD-like neurodegeneration-and by at least 20-30 years earlier than the general population. In conducting a hypothesis-neutral screen of genes located on the DS trisomy for enhancement of amyloid precursor protein (APP)-induced neurodegeneration, we found that one of these genes, the - secretase enzyme BACE2, appears to enhance the rate/extent of neuronal degeneration in a brain slice-based model of AD. Based on previous reports in the literature and our preliminary studies, we hypothesize that BACE2 cleavage of APP generates a truncated version of ¿-amyloid (A¿) that may not be amyloidogenic but nevertheless drives significant neurodegenerative processes even in the absence of full-length A¿. In order to rigorously test this hypothesis, it will be necessary to directly identify and characterize this presumptive A¿ fragment generated by BACE2. However, currently available immunoreagents are not suitable for this purpose. Thus, the goal of this research proposal is to develop and test novel antibodies directed at the presumptive BACE2 A¿ fragment; to use these reagents to demonstrate that a truncated form of A¿ is produced by BACE2 cleavage of APP; and finally to isolate and directly sequence the BACE2-truncated A¿ fragment. The specific aims are thus to: Specific Aim 1: Develop an antibody to the C-terminal half of the canonical A¿ sequence. Specific Aim 2: Directly MS sequence the A¿ fragment produced by BACE2 cleavage. If successful, this research strategy will provide further evidence and the tools necessary for testing the hypothesis that BACE2 could be a favorable potential drug target for slowing/preventing the progression of AD-like neurodegeneration in DS. Moreover, given that BACE2 is also expressed in the brains of the general population, albeit at lower levels, such BACE2-targeted drugs may also be important to investigate in the context of sporadic and familial AD, as the highly-selective BACE1 inhibitor drugs being developed today could "unmask" neurodegenerative processes mediated by BACE2 that may be latent in the general population as well. PUBLIC HEALTH RELEVANCE: Down syndrome (DS) results from the triplication of human chromosome 21 (chr21) and, with an incidence of 1 in ~750 births, is the most common genetic cause of mental retardation. Tragically, as DS patients grow into adulthood, they suffer from further neurological complications, most notably Alzheimer's disease (AD)-like dementia - at present, there are no drugs available to patients that can slow or halt the progression of early-onset AD in DS. There is thus an urgent need to understand the underlying molecular genetic mechanisms that lead to AD in DS in order to support the rational design of new therapeutics to combat AD in DS. In this context, the present proposal seeks to develop key immunoreagents to enable testing the hypothesis that one of the DS trisomy genes, BACE2, may be an important drug target candidate for the treatment of AD in DS, and potentially for treating AD in the general population as well.