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，其特征是大脑沉积？-淀粉样肽（A?），由BACE1和-分泌酶对淀粉样前体蛋白（APP）进行顺序蛋白水解而产生。流行病学对胆固醇与阿尔茨海默病易感性之间的关系有相当大的兴趣。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域（称为脂筏）可能是细胞胆固醇水平和APP淀粉样变性过程之间的关键联系。-分泌酶亚基和APP c -末端片段（CTF）在脑脂筏中富集；全长APP和BACE1也存在于脂筏中，尽管水平较低。值得注意的是，BACE1和2 ？-分泌酶亚基（nicastrin和APH1）经历s -棕榈酰化，这是一种翻译后脂质修饰，常见于筏子相关蛋白。除了脂质筏靶向，s -棕榈酰化是一种关键的修饰，可以动态调节膜运输和调节蛋白质的功能，如神经元跨膜（AMPA和NMDA受体）和细胞质蛋白（PSD-95）。神经元中APP分泌酶s -棕榈酰化的体内生理意义尚不清楚。我们已经开始使用转基因小鼠和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 -棕榈酰化缺陷的转基因小鼠大脑中淀粉样蛋白负荷显著减少。分泌酶亚基。此外，在海马神经元中，我们发现了s -棕榈酰化依赖性的BACE1转运到神经元细胞表面、树突棘定位和轴突运输的差异调节。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，在体内生理表达水平上对神经元中s -棕榈酰化缺失的BACE1进行功能分析，以明确确定BACE1的s -棕榈酰化如何调节APP加工和脑内淀粉样蛋白沉积，是非常重要的。以下是本次调查的具体目的。目标1：对？-分泌酶s -棕榈酰化转基因小鼠。目的2：在体内对BACE1 s -棕榈酰化进行功能表征。目的3：研究s -棕榈酰化对BACE1转运的动态调控。我们的研究将揭示培养海马神经元和体内s -棕榈酰化对AP分泌酶定位和功能的差异调节的新颖和重要见解。

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 In unpublished preliminary studies we observe a significant decrease in amyloid burden in the brains of transgenic mice expressing S-palmitoylation-deficient ?-secretase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.