Novel mechanisms for Alzheimer's disease prevention and/or treatment

描述(由申请人提供)： 问题：衰老是阿尔茨海默病(AD)最重要的危险因素，阿尔茨海默病是我国最常见的痴呆症原因。这种疾病目前还没有可用的治疗方法，在我们日益老龄化的退伍军人中变得越来越普遍。初步数据：我们的团队最近发现了一种新的翻译后调控形式，它同时影响BACE1的水平和活性。具体地说，我们发现新生的BACE1在内质网的管腔中被两种乙酰基转移酶瞬时乙酰化，我们称之为ATase1和ATase2。新生的BACE1的乙酰化中间产物能够完全成熟，而非乙酰化的中间产物迅速降解。一致地，ATase1和ATase2的上调会增加BACE1的水平和A？的生成，而下调则有相反的效果。ATase1和ATase2都可以在各种通常用于神经系统研究的细胞系中检测到。在大脑中，它们优先在神经元中表达。最后，这两种酶在迟发性AD患者的大脑中都上调了。鉴于BACE1在AD发病机制中的作用，BACE1是AD翻译研究的积极靶点。不幸的是，BACE1抑制剂的生物化学设计已被证明是具有挑战性的，因为酶的催化口袋相当大。因此，人们正在积极寻找影响BACE1表达水平而不是催化活性的方法。考虑到这一点，我们成功地开发了一种体外检测ATase1和ATase2活性的方法，并对14,400种化合物的文库进行了高通量筛选(HTS)。筛选的结果是发现了ATase1和ATase2的新型生化抑制剂，它们显著降低了细胞系统中BACE1的水平和A？的生成。最后，我们成功地完成了必要的物理/化学(配方前)表征和配方开发，并启动了一种成功化合物的动物试验。初步结果表明，我们的化合物：(I)能够高效地穿过脑-血屏障(BBB)并到达中枢神经系统(CNS)；(Ii)降低大脑中BACE1和A？的水平；以及(Iii)防止突触缺陷，这种缺陷是小鼠早期AD样病理的特征。假设：我们的中心假设是，ATase1和ATase2生物学作用的功能表征将有助于我们了解AD发病的分子机制，ATase1和ATase2的生化抑制剂可能有助于预防或延缓AD痴呆。研究设计：特定目标1将确定调节大脑中ATase1和ATase2生物学功能的分子机制。这一目标将采用体外、体外和体内相结合的方法。具体地说，我们将使用染色质免疫沉淀、DNA：蛋白质下拉和荧光素酶报告分析来确定在衰老和AD过程中负责激活ATase1和ATase2表达的转录机制(S)(目标1A)。相反，将使用质谱学、定点突变以及生化和生物物理方法的组合来确定调节ATase1活性的“开-关开关”的功能含义(目标1B)。体外研究的生理学和病理学意义将通过使用细胞系、原代神经元、死后AD脑组织和衰老动物模型来确定。具体目标2将评估ATase1和ATase2的生化抑制剂是否能够在该疾病的动物模型中起到预防或延缓AD痴呆的作用。动物将接受行为和认知评估。大脑的尸检切片将用于突触功能的电生理分析以及全面的组织学和生化评估(目标2A)。最后，将使用生化、生物物理和细胞方法的组合来评估新发现的化合物是否可以作为额外的抑制工具(目标2B)。

DESCRIPTION (provided by applicant): PROBLEM: Aging is the most important risk factor for Alzheimer's disease (AD), which represents the most common cause of dementia in our country. The disease, for which there is no currently available treatment, is becoming increasingly prevalent among our aging Veteran population. PRELIMINARY DATA: Our group has recently identified a novel form of post-translational regulation that affects both levels and activity of BACE1. Specifically, we discovered that nascent BACE1 is transiently acetylated in the lumen of the ER by two acetyltransferases, which we named ATase1 and ATase2. The acetylated intermediates of nascent BACE1 are able to complete maturation whereas non-acetylated intermediates are rapidly degraded. Consistently, up-regulation of ATase1 and ATase2 increases BACE1 levels and A¿ generation while down-regulation has the opposite effects. Both ATase1 and ATase2 can be detected in a variety of cell lines that are commonly used for the study of the nervous system. In the brain they are preferentially expressed in neurons. Finally, both enzymes are up-regulated in the brain of late-onset AD patients. In light of its role in pathogenesis of the diseae, BACE1 is an active target for AD translational research. Unfortunately, biochemical design of BACE1 inhibitors has proven to be challenging due to the rather large size of the catalytic pocket of the enzyme. Therefore, approaches that affect expression levels rather than catalytic activity of BACE1 are being actively sought. With this in mind, we successfully developed an in vitro assay to monitor ATase1 and ATase2 activity and conducted a High Throughput Screen (HTS) of a library of 14,400 compounds. The screen resulted in the identification of novel biochemical inhibitors of ATase1 and ATase2 that significantly reduced the levels of BACE1 and the generation of A¿ in cellular systems. Finally, we successfully completed the necessary physical/chemical (pre-formulation) characterization and formulation development and initiated animal testing of one successful compound. The initial results show that our compound: (i) is able to cross the brain-blood-barrier (BBB) and reach the Central Nervous System (CNS) with high efficiency; (ii) reduce both BACE1 and A¿ levels in the brain; and (iii) prevent the synaptic deficits that characterize the early AD-like pathology of the mice. HYPOTHESIS: Our central hypothesis is that functional characterization of the biological roles of ATase1 and ATase2 will help us understand the molecular mechanisms involved with the pathogenesis of AD, and that biochemical inhibitors of ATase1 and ATase2 can potentially serve to prevent or delay AD dementia. STUDY DESIGN: Specific Aim 1 will identify the molecular mechanisms that regulate the biological functions of ATase1 and ATase2 in the brain. This Aim will use a combination of in vitro, ex vivo and in vivo approaches. Specifically, we will use chromatin immunoprecipitation, DNA:protein pull-down and luciferase reporter assays to identify the transcriptional mechanism(s) responsible for the activation of ATase1 and ATase2 expression during aging and AD (Aim 1A). Mass spectrometry, site-directed mutagenesis, and a combination of biochemical and biophysical approaches will instead be used to determine the functional implications of the "on-off switch" that regulates ATase1 activity (Aim 1B). The physiologic and pathologic implications of the in vitro studies will be determined by using cell lines, primary neurons, post-mortem AD brain tissue and animal models of aging. Specific Aim 2 will assess whether biochemical inhibitors of ATase1 and ATase2 can serve to prevent or delay AD dementia in animal models of the disease. Animals will undergo behavioral and cognitive assessment. Post-mortem sections of the brain will be used for electrophysiological analysis of synaptic functions as well as comprehensive histological and biochemical assessment (Aim 2A). Finally, a combination of biochemical, biophysical, and cellular approaches will be used to assess whether newly identified compounds can serve as additional inhibitory tools (Aim 2B).