S-Nitrosylation-Induced Posttranslational Modification and Aberrant Cell Signaling in Sporadic Alzheimer's Disease

散发性阿尔茨海默病中 S-亚硝基化诱导的翻译后修饰和异常细胞信号转导

基本信息

批准号：
9355868
负责人：
STUART A LIPTON
金额：
$ 67.72万
依托单位：
SCRIPPS RESEARCH INSTITUTE, THE
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-09-30 至 2022-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9355868
关键词：
Affect Alzheimer&apos s Disease Alzheimer&apos s disease model Amyloid beta-Protein Biochemical Biochemical Pathway Biological Assay Biological Markers Biological Models Biological Process Brain Diseases CRISPR/Cas technology Cell Survival Chemicals Chemistry Critical Pathways DNA Disease Genes Human In Vitro Mass Spectrum Analysis Modeling Mus Neurodegenerative Disorders Neurons Neurosciences Oxidation-Reduction Pathogenesis Pathogenicity Pathway interactions Patients Post-Translational Protein Processing Process Production Proteins Publishing Reaction SKIL gene Signal Transduction Site-Directed Mutagenesis Synapses Techniques Technology Testing Tg2576 Therapeutic Tissue imaging Transgenic Mice base cellular imaging gene product in vitro Model in vivo in vivo Model innovation mouse model neuron loss protein E protein function synaptic function

项目摘要

SUMMARY This collaborative R01 application between a neuroscience lab (led by Stuart Lipton at Scintllon Inst./UC San Diego) and a chemistry lab (led by Steve Tannenbaum at MIT) will identify the redox posttranslational modification of proteins called S-nitrosylation by developing a more effective and integrated Mass Spec-based platform to screen for the S-nitrosoproteome and resulting alterations in protein function that contribute to the pathogenesis of Alzheimer’s disease (AD). Our hypothesis is that entire biochemical pathways critical to neuronal function are affected by aberrant S-nitrosylation of multiple proteins, these aberrant redox reactions (which are located, at least in part, downstream of Aß insult) contribute to the pathogenesis of AD, and the reactions occur in both sporadic and familial cases of the disease. Chemical and functional analysis of S- nitrosylated proteins will be assessed by biochemical assays, and by imaging of cells and tissues, including human AD brain and various in vitro and in vivo models of AD, ranging from transgenic mice to hiPSC-based model systems. We will also use site-directed mutagenesis and CRISPR/Cas9 techniques to generate DNA constructs or genes encoding proteins that that cannot be S-nitrosylated (thus forming non-nitrosylatable proteins). Accordingly, our Specific Aims are as follows: AIM #1. To determine the S-nitrosoproteome in human AD brain and transgenic mouse models. We will validate our recent S-nitrosoproteome findings in the CK-p25 mouse model of AD (published in PNAS, 2016) and determine if it generalizes to human AD brain and other transgenic mouse models of AD, e.g., hAPP-J20 and Tg2576. AIM #2. To use hiPSC-derived cerebrocortical neurons generated from human AD patients or WT exposed to oligomeric Aß (as a model of sporadic AD) as an in vitro model system to study the S-nitrosoproteome and how it affects biochemical pathways. This approach will allow us to study the functional effect of SNO-proteins in AD in a human context. AIM #3. To screen the effects of various S-nitrosoproteins in hiPSC-based models for impact on potential biological functions, e.g., effect on synaptic loss or neuronal cell death. This will be accomplished by generating non-nitrosylatable constructs of proteins (e.g., substituting Ala for Cys) by replacing the underling gene by CRISPR/Cas9 technology. For selected gene products that manifest profound effects of S- nitrosylation on synaptic functions and neuronal cell survival in hiPSC-based models, the non-nitrosylatable version of the gene can also be created in mice using CRISPR/Cas9 to mechanistically test its effect in vivo.

概括神经科学实验室（由 Scintllon Inst./UC San 的 Stuart Lipton 领导）之间的协作 R01 应用程序 Diego）和化学实验室（由麻省理工学院的 Steve Tannenbaum 领导）将鉴定氧化还原翻译后通过开发更有效和集成的基于质谱的方法来修饰称为 S-亚硝基化的蛋白质筛选 S-亚硝基蛋白质组和由此产生的蛋白质功能改变的平台，这些改变有助于我们的假设是整个生化途径对于阿尔茨海默病 (AD) 的发病机制至关重要。神经元功能受到多种蛋白质异常 S-亚硝基化的影响，这些异常的氧化还原反应（至少部分位于 Aß 损伤的下游）有助于 AD 的发病机制，并且该反应发生在散发性和家族性病例中。S-的化学和功能分析。亚硝基化蛋白质将通过生化测定以及细胞和组织成像进行评估，包括人类 AD 大脑以及各种 AD 体外和体内模型，从转基因小鼠到基于 hiPSC 的模型我们还将使用定点诱变和 CRISPR/Cas9 技术来生成 DNA。编码不能被S-亚硝基化的蛋白质的构建体或基因（从而形成不可亚硝基化的因此，我们的具体目标如下：目标#1：确定人类 AD 大脑和转基因小鼠模型中的 S-亚硝基蛋白质组。验证我们最近在 AD CK-p25 小鼠模型中的 S-亚硝基蛋白质组发现（发表于 PNAS，2016）并确定它是否适用于人类 AD 大脑和其他 AD 转基因小鼠模型，例如 hAPP-J20 和Tg2576。目标#2。使用来自人类 AD 患者或暴露于寡聚 Aß（作为散发性 AD 的模型）作为体外模型系统来研究 S-亚硝基蛋白质组和它如何影响生化途径，使我们能够研究 SNO 蛋白的功能效应。在人类背景下的AD。目标#3。筛选各种 S-亚硝基蛋白在基于 hiPSC 的模型中的潜在影响。生物学功能，例如对突触丧失或神经细胞死亡的影响，这将通过以下方式实现。通过替换下层生成不可亚硝基化的蛋白质构建体（例如，用 Ala 代替 Cys）通过 CRISPR/Cas9 技术筛选出具有 S- 深远影响的基因产物。在基于 hiPSC 的模型中，亚硝基化对突触功能和神经元细胞存活的影响还可以使用 CRISPR/Cas9 在小鼠体内创建该基因的版本，以机械方式测试其体内效果。