PHOSPHOPROTEOMIC ANALYSIS OF SYNAPTIC PROTEINS IN ALZHEIMER DISEASE MOUSE MODELS

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 阿尔茨海默病(AD)和相关小鼠模型的认知缺陷与突触缺陷密切相关。我们最近发现，tau的减少有效地防止了人类淀粉样前体蛋白(HAPP)转基因小鼠的认知和突触缺陷，这些小鼠在大脑中有高水平的A？，并发展成几个AD样异常。由于突触的功能关键依赖于突触蛋白的翻译后修饰(PTM)，A？可能以tau依赖的方式改变这一过程。为了了解AD的发病机制以及tau降低如何预防AD样异常，特别是突触连接处信号转导通路的变化，将从行为测试的Happ/tau+/+、Happ/tau/、tau+/+和tau/小鼠的脑中制备突触小体和突触后密度(PSD)蛋白。蛋白质样品将用蔗糖密度分级法制备，然后进行处理，并用同位素编码的标记试剂进行差示标记。样品中的磷酸化肽将用金属亲和层析进行浓缩，样品中的O-GlcNacylated多肽将用WGA亲和层析进行浓缩，样品中富含和不含PTM的部分将用其他液相色谱方法进一步分离。差异标记的分离样本将在具有ETD能力的LTQ Orbitrap平台上进行分析，并将比较四种基因型之间不同突触体蛋白及其PTM的相对丰度。整个项目将使用加州大学旧金山分校质谱学设施开发的磷酸和O-GlcNAc蛋白质组分析技术平台以协作方式进行。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Cognitive deficits in Alzheimer's disease (AD) and related mouse models correlate closely with synaptic deficits. We recently discovered that tau reduction effectively prevents cognitive and synaptic deficits in human amyloid precursor protein (hAPP) transgenic mice, which have high levels of A¿ in the brain and develop several AD-like abnormalities. Because synaptic functions critically depend on the posttranslational modification (PTM) of synaptic proteins, A¿ may alter this process in a tau-dependent manner. To understand AD pathogenesis and how tau reduction prevents AD-like abnormalities, particularly changes of signal transduction pathways at the synaptic junction, synaptosomal and post-synaptic density (PSD) proteins will be prepared from brains of behaviorally tested hAPP/tau+/+, hAPP/tau/, tau+/+ and tau/ mice. Protein samples will be prepared using sucrose density fractionation and then processed and differentially labeled with isotope-coded labeling reagents. The phosphorylated peptides in the sample will be enriched using metal affinity chromatography, the O-GlcNAcylated peptides in the sample will be enriched using WGA affinity chromatography, and both the PTM-enriched and the PTM-depleted portions of the sample will be further fractionated using other liquid chromatography approaches. The differentially labeled, fractionated samples will be analyzed on the LTQ Orbitrap platform with ETD capabilities and the relative abundance of the different synaptosomal proteins and their PTMs will be compared between the four genotypes. The whole project will be conducted in a collaborative manner using the phospho- and O-GlcNAc-proteomics analysis technical platform developed in the mass spectrometry facility at UCSF.