Identification of an Abeta fragment produced by BACE2

描述（由申请人提供）：唐氏综合征（DS）是由人类21号染色体（chr 21）的三倍引起的，是智力迟钝的最常见遗传原因。据推测，DS是由位于三重chr 21上的基因表达增加引起的，导致DS中的异常脑发育，但也导致进一步的神经系统并发症，尤其是阿尔茨海默病（AD）样痴呆，随着DS患者长大成人。根据一些神经学和神经病理学标准，DS人群AD样神经退行性变的风险显著升高，并且比普通人群早至少20-30年。在对位于DS三体上的基因进行中性假设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BACE 2似乎增强了神经元退行性变的速率/程度在基于脑切片的AD模型中。基于文献中先前的报道和我们的初步研究，我们假设BACE 2裂解APP产生截短形式的淀粉样蛋白（A <$），其可能不是淀粉样蛋白，但即使在缺乏全长A <$的情况下，仍然驱动显著的神经退行性过程。为了严格检验这一假设，有必要直接鉴定和表征由BACE 2产生的假定A？片段。然而，目前可用的免疫试剂不适合此目的。因此，本研究提案的目标是开发和测试针对推定的BACE 2 A ²片段的新型抗体;使用这些试剂来证明APP的BACE 2切割产生了截短形式的A ²;最后分离并直接测序BACE 2截短的A ²片段。因此，具体目标是：具体目标1：开发针对典型A <$序列C-末端一半的抗体。具体目标2：直接MS测序由BACE 2切割产生的A？片段。如果成功，该研究策略将提供进一步的证据和必要的工具来检验BACE 2可能是减缓/预防DS中AD样神经变性进展的有利潜在药物靶点的假设。此外，鉴于BACE 2也在一般人群的大脑中表达，尽管水平较低，这种BACE 2靶向药物在散发性和家族性AD的背景下也可能是重要的，因为今天正在开发的高选择性BACE 1抑制剂药物可以“揭示”由BACE 2介导的神经退行性过程，这些过程也可能潜伏在一般人群中。 公共卫生相关性：唐氏综合征（DS）是由人类21号染色体（chr 21）的三重化引起的，其发病率为1/750，是智力低下的最常见遗传原因。可悲的是，随着DS患者长大成人，他们遭受进一步的神经系统并发症，最明显的是阿尔茨海默病（AD）样痴呆-目前，没有药物可用于患者，可以减缓或停止DS中早发性AD的进展。因此，迫切需要了解导致DS中AD的潜在分子遗传机制，以支持新疗法的合理设计来对抗DS中的AD。在这种情况下，本提案旨在开发关键的免疫试剂，以验证以下假设：DS三体基因之一BACE 2可能是治疗DS中AD的重要候选药物靶标，也可能用于治疗一般人群中的AD。

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.