Identification of an Abeta fragment produced by BACE2

描述(申请人提供)：唐氏综合症(DS)是由人类21号染色体(Chr21)的三倍体(Chr21)引起的，是导致智力低下的最常见的遗传原因。据推测，DS是由位于三重chr21上的基因表达增加引发的，导致DS患者大脑发育异常，但也会导致进一步的神经并发症，特别是随着DS患者成年后出现阿尔茨海默病(AD)样痴呆。根据许多神经学和神经病理学标准，DS人群患AD样神经变性的风险大大增加--而且比普通人群早至少20-30年。在对DS三体上促进淀粉样前体蛋白(APP)诱导的神经变性的基因进行假设中性筛选时，我们发现其中一个基因，分泌酶BACE2，似乎提高了基于脑片的AD模型中神经元退化的速度/程度。根据以前的文献报道和我们的初步研究，我们假设APP的BACE2裂解产生一个截短版本的β-淀粉样蛋白(A？)，它可能不是淀粉样变性的，但即使在没有全长A？的情况下也会驱动显著的神经退变过程。为了严格检验这一假设，有必要直接鉴定和表征由BACE2产生的这个假定的A？片段。然而，目前可用的免疫反应物不适合于此目的。因此，这项研究计划的目标是开发和测试针对假定的BACE2 A？片段的新抗体；使用这些试剂来证明APP的BACE2裂解产生截短形式的A？；最后分离BACE2截短的A？片段并直接测序。因此，特定的目的是：特定的目标1：研制针对标准A？序列的C-末端一半的抗体。特定目的2：直接对BACE2裂解产生的A？片段进行MS测序。如果成功，这一研究策略将提供进一步的证据和必要的工具来检验BACE2可能是减缓/防止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BACE2也在普通人群的大脑中表达，尽管表达水平较低，这种针对BACE2的药物在散发性和家族性AD的背景下研究也可能是重要的，因为今天正在开发的高选择性BACE1抑制剂药物可以揭示由BACE2介导的神经退化过程，这可能也是在普通人群中潜伏的。 公共卫生相关性：唐氏综合症(DS)是由人类21号染色体(Chr21)三倍体(Chr21)引起的，发病率为每750名新生儿中就有一名，是导致智力低下的最常见的遗传原因。可悲的是，随着DS患者进入成年期，他们会遭受进一步的神经并发症，最明显的是阿尔茨海默病(AD)样痴呆--目前，还没有药物可以减缓或阻止DS患者早发性AD的进展。因此，迫切需要了解导致DS中AD的潜在分子遗传学机制，以支持合理设计新的治疗方法来对抗DS中的AD。在此背景下，本提案试图开发关键的免疫反应物，以验证以下假设：DS三体基因之一BACE2可能是治疗DS中的AD的重要候选药物，也可能用于治疗普通人群中的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.