Generation of a mouse model to monitor ERAD in neurons

生成监测神经元 ERAD 的小鼠模型

• 批准号: 9251591
• 负责人: Mervyn J Monteiro
• 金额: $ 23.11万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9251591
• 关键词: Animals; Biochemical; Biological Process; Biology; Brain; Caenorhabditis elegans; Cell Death; Cell physiology; Cells; Cognitive deficits; Communities; DNA; DNA cassette; Defect; Disease; Endoplasmic Reticulum; Ensure; Equilibrium; Generations; Homeostasis; Human; Imagery; Immunoblotting; In Vitro; Lead; Length; Life; Link; Longevity; Measures; Membrane; Membrane Proteins; Methods; Molecular Chaperones; Monitor; Motor Neuron Disease; Mus; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Organism; Population; Process; Proteins; Reporter; Research; Signal Transduction; Site; Spinal Cord; Staging; Symptoms; System; Testing; Therapeutic; Time; Tissues; Transgenic Animals; Transgenic Mice; Transgenic Organisms; Validation; Wild Type Mouse; base; cell type; early onset; endoplasmic reticulum stress; environmental change; in vivo; link protein; mouse model; mutant; neuron loss; overexpression; promoter; protein degradation; protein folding; protein function; protein misfolding; secretory protein; tool; transgene expression

Summary Proper protein folding is vital for biological function. Unfortunately, many proteins, especially membrane proteins, tend to misfold either because of errors during synthesis, mutations, changes in the balance of protein folding/chaperone systems, or because of environmental changes. Misfolded proteins must be eliminated otherwise they could interfere with cellular function, eventually causing disease. Furthermore, accumulation of misfolded proteins in organisms can shorten lifespan. Therefore, understanding how misfolded proteins are removed from cells has important implications both in terms of our basic understanding of the biology of the process as well as for devising therapeutic methods to treat diseases linked to protein misfolding. A particular challenge is to understand how misfolded proteins are eliminated from the endoplasmic reticulum (ER), the site where most membrane and secretory proteins are made. An appreciation of the importance of protein degradation from the ER, called ER-associated degradation (ERAD), is underscored by defects that sometimes occur in the system, which can lead to activation of ER stress, prolonged activation of which leads to cell death and tissue malfunction. Unfortunately, at present there are few, if any, reporters that can be used to directly monitor and/or visualize ERAD in living organisms. In this application we propose to fill this void by generating transgenic mice that could be used for studying ERAD in vivo. Accordingly, in Aim 1 we propose to generate a transgenic mouse model that expresses a fluorescent-based reporter that can be used to biochemically and visually measure ERAD activity in neurons (driven by the Thy1.2 promoter). The ERAD reporter we have chosen is CD3δ tagged with the photoswitchable fluorescent protein Dendra2. We enumerate the many advantages why this reporter is particularly well suited for measuring ERAD activity in neurons. Once generated the mice will be characterized to ensure that the reporter can be reliably used to measure defects in ERAD. Upon validation we will cross the reporter mice in Aim 2 with wild type (WT) and a P497S UBQLN2 transgenic mouse model of ALS, which we recently generated. The P497S line develops cognitive deficits and motor neuron disease, whereas UBQLN2 WT mice do not show similar disease. Immunoblots indicate a build- up of ubiquitinated proteins in the brain and spinal cord of the P497S animals compared to WT and non- transgenic animals. In addition, ER stress markers (PDI and phosphorylated eIF2α) are elevated in spinal cord of end-stage P497S animals. The increase in ER stress is consistent with expression of the mutant P497S protein interfering with ERAD, which is a known function of UBQLN2 protein. The animal cross will allow us to evaluate if this is the case, and the time course of any interference. The generation and validation Thy1.2 expressing CD3δ-Dendra2 mice as proposed here should provide an extremely powerful tool for evaluating interference in ERAD caused by UBQLN2 and other mutant proteins linked to neurodegenerative diseases.

概括 正确的蛋白质折叠对于生物功能至关重要。不幸的是，许多蛋白质，尤其是膜 由于合成过程中的错误、突变、平衡的变化，蛋白质往往会错误折叠 蛋白质折叠/伴侣系统，或由于环境变化。错误折叠的蛋白质一定是 消除它们，否则它们可能会干扰细胞功能，最终导致疾病。此外， 生物体中错误折叠蛋白质的积累会缩短寿命。因此，了解如何错误折叠 从细胞中去除蛋白质对于我们对蛋白质的基本理解具有重要意义 该过程的生物学以及设计治疗与蛋白质相关疾病的治疗方法 错误折叠。一个特殊的挑战是了解如何从内质中消除错误折叠的蛋白质 网状结构 (ER)，大多数膜蛋白和分泌蛋白的生成场所。欣赏 内质网蛋白质降解（称为内质网相关降解（ERAD））的重要性被强调 系统中有时会出现缺陷，这可能导致内质网应激的激活、内质网应激的长时间激活 这会导致细胞死亡和组织功能障碍。不幸的是，目前很少有记者（如果有的话） 可用于直接监测和/或可视化活生物体中的 ERAD。在此申请中，我们建议填写 通过产生可用于体内研究 ERAD 的转基因小鼠，可以解决这一问题。因此，在目标 1 中，我们 提议生成表达可使用的基于荧光的报告基因的转基因小鼠模型 以生化和视觉方式测量神经元中的 ERAD 活性（由 Thy1.2 启动子驱动）。 ERAD 我们选择的记者是带有光开关荧光蛋白 Dendra2 标记的 CD3δ。我们列举 该报告器具有众多优点，特别适合测量神经元中的 ERAD 活性。一次 生成的小鼠将被表征，以确保报告器可以可靠地用于测量缺陷 ERAD。验证后，我们将 Aim 2 中的报告小鼠与野生型 (WT) 和 P497S UBQLN2 杂交 我们最近生成的 ALS 转基因小鼠模型。 P497S 系会出现认知缺陷 运动神经元疾病，而 UBQLN2 WT 小鼠没有表现出类似的疾病。免疫印迹表明构建 与 WT 和非 P497S 动物相比，P497S 动物大脑和脊髓中泛素化蛋白的增加 转基因动物。此外，脊髓中的 ER 应激标记物（PDI 和磷酸化 eIF2α）升高 末期 P497S 动物。 ER 应激的增加与突变体 P497S 的表达一致 蛋白质干扰 ERAD，这是 UBQLN2 蛋白质的已知功能。动物杂交将使我们能够 评估是否属于这种情况，以及任何干扰的时间过程。 Thy1.2的生成和验证 这里提出的表达 CD3δ-Dendra2 小鼠应该为评估提供极其强大的工具 UBQLN2 和其他与神经退行性疾病相关的突变蛋白引起的 ERAD 干扰。