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

预防和/或治疗阿尔茨海默病的新机制

• 批准号: 8536999
• 负责人: Luigi Puglielli
• 金额: --
• 依托单位: WM S. MIDDLETON MEMORIAL VETERANS HOSP
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-01 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8536999
• 关键词: Acetyltransferase; Affect; Aging; Alzheimer disease prevention; Alzheimer' s Disease; American; Amyloid; Amyloid beta-Protein Precursor; Animal Disease Models; Animal Model; Animal Testing; Animals; Area; Asses; Behavioral; Binding; Binding Proteins; Biochemical; Biological; Biological Assay; Biological Process; Biology; Blood - brain barrier anatomy; Brain; Cell Line; Cell model; Chemicals; Cleaved cell; Clinical; Cognitive; Cognitive deficits; Country; DNA; Dementia; Deposition; Development; Diffuse; Disease; Down-Regulation; Drug Formulations; Education; Elderly; Enzymes; Event; Extracellular Protein; Generations; Glutamate-ammonia-ligase adenylyltransferase; Goals; Healthcare; Hospitals; Impaired cognition; In Vitro; Individual; Knock-out; Laboratories; Late Onset Alzheimer Disease; Lead; Libraries; Light; Link; Luciferases; Mass Spectrum Analysis; Membrane Proteins; Memory; Memory impairment; Microtubule Stabilization; Microtubules; Mind; Mission; Molecular; Monitor; Mus; Names; Nervous system structure; Neuraxis; Neurofibrillary Tangles; Neurons; Pathogenesis; Pathologic; Pathology; Patients; Peptides; Personality; Physiological; Play; Population; Post-Translational Regulation; Prevention; Process; Property; Proteins; Quality of Care; Reporter; Reporting; Research; Risk Factors; Role; Screening Result; Senile Plaques; Site; Site-Directed Mutagenesis; Synapses; System; Testing; Tissue Model; Toxic effect; Translational Research; Up-Regulation; Veterans; age related; aged; beta-site APP cleaving enzyme 1; brain tissue; chromatin immunoprecipitation; design; disorder prevention; high throughput screening; immortalized cell; improved; in vitro Assay; in vivo; inhibitor/antagonist; neuron loss; neuropathology; novel; novel therapeutic intervention; prevent; protein aggregate; public health relevance; sequential proteolysis; synaptic function; tau Proteins; tau phosphorylation; tau-1; tool

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).

描述（由申请人提供）： 问题：老龄化是阿尔茨海默病（AD）最重要的危险因素，这是我国痴呆症最常见的原因。这种疾病，目前还没有可用的治疗方法，在我们的老年退伍军人中越来越普遍。 初步数据：我们的研究小组最近发现了一种新的翻译后调节形式，它影响BACE 1的水平和活性。具体来说，我们发现新生BACE 1在ER的内腔中被两种乙酰转移酶瞬时乙酰化，我们将其命名为ATase 1和ATase 2。新生BACE 1的乙酰化中间体能够完全成熟，而非乙酰化中间体迅速降解。因此，ATase 1和ATase 2的上调增加BACE 1水平和A？生成，而下调具有相反的效果。ATase 1和ATase 2都可以在神经系统研究中常用的多种细胞系中检测到。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患者的大脑中上调。鉴于其在疾病发病机制中的作用，BACE 1是AD转化研究的活性靶标。不幸的是，BACE 1抑制剂的生物化学设计已被证明是具有挑战性的，因为该酶的催化口袋相当大。因此，正在积极寻求影响BACE 1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方法来监测ATase 1和ATase 2的活性，并对14，400种化合物的文库进行了高通量筛选（HTS）。该筛选导致鉴定了ATase 1和ATase 2的新型生化抑制剂，其显著降低了细胞系统中BACE 1的水平和A？的产生。最后，我们成功地完成了必要的物理/化学（预制剂）表征和制剂开发，并开始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）能够穿过脑血屏障（BBB）并高效到达中枢神经系统（CNS）;（ii）降低大脑中的BACE 1和A？水平;（iii）预防小鼠早期AD样病理特征的突触缺陷。 假设：我们的中心假设是，ATase 1和ATase 2的生物学作用的功能表征将帮助我们了解与AD的发病机制，和ATase 1和ATase 2的生化抑制剂可以潜在地用于预防或延迟AD痴呆的分子机制。 研究设计：特定目标1将确定调节脑中ATase 1和ATase 2生物学功能的分子机制。该目标将使用体外、离体和体内方法的组合。具体而言，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基因测定，以确定负责在衰老和AD（Aim 1A）过程中激活ATase 1和ATase 2表达的转录机制。质谱法，定点诱变，以及生物化学和生物物理方法的组合将被用来确定调节ATase 1活性的“开关”的功能意义（目的1B）。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体外研究的生理和病理意义。具体目标2将评估ATase 1和ATase 2的生化抑制剂是否可以在疾病的动物模型中预防或延迟AD痴呆。动物将接受行为和认知评估。脑的死后切片将用于突触功能的电生理学分析以及全面的组织学和生物化学评估（目的2A）。最后，生物化学，生物物理和细胞方法的组合将用于评估新鉴定的化合物是否可以作为额外的抑制工具（目的2B）。