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

总结 这个合作的R 01应用程序之间的神经科学实验室（由斯图尔特利普顿在Scintllon Inst./ UC San Diego）和化学实验室（由MIT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蛋白质修饰称为S-亚硝基化 该平台用于筛选S-亚硝基蛋白质组，并导致蛋白质功能的改变， 阿尔茨海默病（AD）的发病机制。我们的假设是，整个生化途径对 神经元功能受到多种蛋白质的异常S-亚硝基化的影响，这些异常的氧化还原反应 （其至少部分地位于AD损伤的下游）有助于AD的发病机制， 在散发性和家族性病例中均发生反应。S-的化学和功能分析 亚硝基化蛋白将通过生物化学测定以及通过细胞和组织成像进行评估，包括 人AD脑和各种体外和体内AD模型，从转基因小鼠到基于hiPSC的 模型系统我们还将使用定点诱变和CRISPR/Cas9技术来产生DNA 编码不能被S-亚硝基化（从而形成不可亚硝基化）的蛋白质的构建体或基因 蛋白质）。因此，我们的具体目标如下： 目标1。目的：研究人AD脑组织和转基因小鼠模型中S-亚硝基蛋白质组的变化。我们将 验证了我们最近在CK-p25 AD小鼠模型中的S-亚硝基蛋白质组研究结果（发表于PNAS，2016） 并确定它是否推广到人类AD脑和其他AD转基因小鼠模型，例如，hAPP-J20 Tg2576 目标#2。为了使用从暴露于以下的人AD患者或WT产生的hiPSC衍生的皮质神经元， 寡聚腺苷酸（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， 它是如何影响生化途径的这种方法将使我们能够研究SNO蛋白的功能效应 in AD AD in a human人context上下文. 目标#3。在基于hiPSC的模型中筛选各种S-亚硝基蛋白对潜在的 生物功能，例如，对突触损失或神经元细胞死亡的影响。这将通过 产生不可亚硝基化的蛋白质构建体（例如，用Ala代替Cys）， CRISPR/Cas9技术。对于选定的基因产品，表现出深刻的影响S- 在基于hiPSC的模型中，非亚硝基化的亚硝酰化对突触功能和神经元细胞存活的影响 也可以使用CRISPR/Cas9在小鼠中创建该基因的版本，以在体内机械地测试其效果。

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.