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

• 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
• 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' s Disease; Alzheimer' 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

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 神经科学实验室之间的合作R01应用程序（由Scintllon Inst./uc San的Stuart Lipton领导 迭戈）和化学实验室（由麻省理工学院的史蒂夫·坦嫩鲍姆（Steve Tannenbaum）领导）将确定氧化还原后翻译后 通过开发更有效和基于质量规格的蛋白质修饰称为S-亚硝基化的蛋白质 筛选S-亚硝基蛋白质组的平台和蛋白质功能的改变，这有助于 阿尔茨海默氏病（AD）的发病机理。我们的假设是整个生化途径至关重要 神经元功能受多种蛋白质异常S-硝基化的影响，这些异常氧化还原反应 （至少部分位于Aß侮辱的下游）有助于AD的发病机理，并有助于 反应发生在零星和家庭病例中。 S-的化学和功能分析 亚硝基化蛋白将通过生化测定和细胞和组织的成像来评估 人类广告大脑以及各种体外和体内AD模型，从转基因小鼠到基于HIPSC 模型系统。我们还将使用位置定向的诱变和CRISPR/CAS9技术生成DNA 编码不能被S-亚硝基化的蛋白质的构建体或基因（从而形成了不可硝基的 蛋白质）。根据所有人，我们的具体目标如下： 目标＃1。确定人AD脑和转基因小鼠模型中的S-硝化蛋白酶。我们将 在AD的CK-P25小鼠模型中验证我们最近的S-亚硝化蛋白质组发现（PNAS，2016年出版） 并确定它是否普遍为人类AD大脑和其他AD的转基因小鼠模型，例如Happ-J20 和TG2576。 目标＃2。使用由人类AD患者产生的HIPSC衍生的脑皮层神经元或暴露于WT 寡聚Aß（作为零星AD的模型）作为一种体外模型系统，用于研究S-硝化蛋白质组和 它如何影响生化途径。这种方法将使我们能够研究SNO蛋白的功能效应 在人类背景下的广告中。 目标＃3。筛选基于HIPSC的模型中各种S-硝基蛋白的影响，以影响潜在 生物学功能，例如对合成损失或神经元细胞死亡的影响。这将通过 通过替换下层的蛋白质（例如，代替ALA代替ALA）的非亚硝基基构建体 CRISPR/CAS9技术的基因。对于某些表现出S-深远影响的基因产物 基于HIPSC的模型，非亚硝基化的突触功能和神经元细胞存活的硝基化 也可以使用CRISPR/CAS9在小鼠中创建该基因的版本，以机械测试其在体内的作用。