Amyloidogenic processing of APP

描述（由申请人提供）：阿尔茨海默病是一种破坏性的神经退行性疾病，其特征是脑内沉积？淀粉样肽（A？），通过BACE 1和β-淀粉样前体蛋白（APP）的连续蛋白水解产生分泌酶胆固醇和阿尔茨海默病易感性之间的关系引起了流行病学的极大兴趣。来自各种体外和体内研究的证据表明，富含胆固醇和鞘脂的膜微区（称为脂筏）可能是细胞胆固醇水平和APP淀粉样蛋白形成过程之间的关键联系。分泌酶亚基和APP C-末端片段（CTF）富含从脑中分离的脂筏;全长APP和BACE 1也存在于脂筏中，尽管水平较低。特别是，BACE 1和2？分泌酶亚基（nicastrin和APH 1）经历S-棕榈酰化，这是一种通常在筏相关蛋白中发现的翻译后脂质修饰。除了脂筏靶向，S-棕榈酰化是动态调节膜运输和调节蛋白质如神经元跨膜（AMPA和NMDA受体）和胞质蛋白（PSD-95）的功能的关键修饰。 神经元中APP分泌酶的S-棕榈酰化的体内生理意义仍然未知。我们已经开始使用转基因小鼠并通过在海马神经元中应用新的活细胞成像策略来解决这个重要问题。在未发表的初步研究中，我们观察到表达S-棕榈酰化缺陷？分泌酶亚单位。此外，在海马神经元中，我们发现了S-棕榈酰化依赖的差异调节BACE 1贩运到神经元细胞表面，在树突棘的定位，和轴突运输。这些后来的发现与阿尔茨海默病的发病机制高度相关，因为在神经元中，APP沿着外周和中央轴突顺行运输，并在运输过程中进行蛋白水解处理。因此，在体内以生理表达水平对神经元中S-棕榈酰化缺陷的BACE 1进行功能分析以明确确定BACE 1的S-棕榈酰化如何调节APP加工和脑中淀粉样蛋白沉积是极其重要的。以下是这次调查的具体目的。 目的1：进行功能分析？-转基因小鼠中的分泌酶S-棕榈酰化。 目的2：对BACE 1的S-棕榈酰化进行体内功能鉴定。 目的3：研究S-棕榈酰化对BACE 1转运的动态调控。 我们的研究将揭示新的和重要的见解差异调节AP分泌酶的定位和功能的S-棕榈酰化在培养的海马神经元和在体内。 公共卫生相关性：阿尔茨海默病（AD）是老年痴呆症的主要原因，影响超过50%的80岁以上的人口;目前有510万美国人患有这种毁灭性的疾病。AD患者以及老年人在大脑中积累β-淀粉样肽作为沉积物，称为老年斑。使用培养的细胞，转基因小鼠和基因敲入小鼠作为实验模型，我们研究APP分泌酶的翻译后修饰及其在细胞中的动态运动如何调节β-淀粉样蛋白的产生和沉积。我们的研究对于开发旨在减少大脑中β-淀粉样蛋白负担的新型合理AD治疗方法至关重要。

DESCRIPTION (provided by applicant): Alzheimer's disease is a devastating neurodegenerative disorder characterized by the cerebral deposition of ?-amyloid peptides (A?), generated by sequential proteolysis of amyloid precursor protein (APP) by BACE1 and ?-secretase. There has been considerable epidemiological interest in the relationship between cholesterol and susceptibility to Alzheimer's disease. Evidence from a variety of in vitro and in vivo studies indicates that cholesterol- and sphingolipid-rich membrane microdomains, termed lipid rafts, might be a critical link between cellular cholesterol levels and amyloidogenic processing of APP. Indeed, each of the ?-secretase subunits and APP C-terminal fragments (CTF) are enriched in lipid rafts isolated from brain; full-length APP and BACE1 are also found in lipid rafts, albeit at lower levels. Notably, BACE1 and two ?-secretase subunits (nicastrin and APH1) undergo S-palmitoylation, a post-translational lipid modification commonly found in raft-associated proteins. In addition to lipid raft targeting, S-palmitoylation is a critical modification that dynamically regulates membrane trafficking and modulates the function of proteins such as neuronal transmembrane (AMPA and NMDA receptors) and cytosolic proteins (PSD-95). The in vivo physiological significance of S-palmitoylation of APP secretases in neurons remains unknown. We have begun to address this important issue using transgenic mice and by applying novel live-cell imaging strategies in hippocampal neurons. In unpublished preliminary studies we observe a significant decrease in amyloid burden in the brains of transgenic mice expressing S-palmitoylation-deficient ?-secretase subunits. Moreover, in hippocampal neurons we have discovered S-palmitoylation-dependent differential regulation of BACE1 trafficking to the neuronal cell surface, localization in dendritic spines, and axonal transport. These later findings are highly relevant to Alzheimer's disease pathogenesis because in neurons APP is trafficked anterogradely along peripheral and central axons, and proteolytically processed during transit. Therefore, it is extremely important to perform functional analysis of S-palmitoylation-deficient BACE1 in neurons at physiological expression levels in vivo to unequivocally determine how S-palmitoylation of BACE1 regulates APP processing and amyloid deposition in the brain. The following are the specific aims of this investigation. Aim 1: To perform functional analysis of ?-secretase S-palmitoylation in transgenic mice. Aim 2: To perform functional characterization of BACE1 S-palmitoylation in vivo. Aim 3: To investigate the dynamic regulation of BACE1 trafficking by S-palmitoylation. Our studies will uncover novel and significant insights on differential regulation of AP secretase localization and function by S-palmitoylation in cultured hippocampal neurons and in vivo. PUBLIC HEALTH RELEVANCE: Alzheimer's disease (AD) is the major cause of dementia in the elderly, afflicting more than 50% of the population over 80 years of age; presently 5.1 million Americans suffer from this devastating disorder. AD patients as well as aged individuals accumulate beta-amyloid peptides as deposits in brain, called senile plaques. Using cultured cells, transgenic mice, and knock-in mice as experimental models we investigate how post-translational modification of APP secretases and their dynamic movement in cells regulate beta-amyloid production and deposition. Our studies will be critical to develop novel rational AD therapeutics aimed at reducing beta-amyloid burden in the brain.