Pulse-Chase Labeling with 15N and AHA in an Alzheimer's Mouse Model

在阿尔茨海默病小鼠模型中使用 15N 和 AHA 进行脉冲追踪标记

• 批准号: 8919211
• 负责人: John R Yates III
• 金额: $ 9.19万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8919211
• 关键词: Alzheimer' s Disease; Animals; Brain; Cells; Chemistry; Disease; Extracellular Matrix; Food; Goals; Health; Label; Length; Life; Longevity; Maintenance; Measurement; Measures; Methods; Mitotic; Modeling; Mus; Neurons; Nuclear Pore; Physiologic pulse; Pore Proteins; Prefrontal Cortex; Process; Protein Fragment; Proteins; Proteolytic Processing; Proteomics; Radioisotopes; Rattus; Recruitment Activity; Research; Research Proposals; Role; Safety; System; Techniques; Time; Translating; expectation; in vivo; mouse model; multicatalytic endopeptidase complex; protein degradation; protein misfolding; response

DESCRIPTION (provided by applicant): The length of time a protein persists in a cell or the extracellular matrix can be an important determinant of the protein's role in disease. Over time proteins can be damaged or modified and then must be removed from the cell by proteolytic processes involving the proteasome or autophagic systems within the cell or by turnover of the whole cell. Accordingly, protein lifetimes and maintenance are particularly important in post-mitotic cells such as neurons. Traditionally, pulse-chase techniques using radioactive isotopes have been a method to measure the expression and turnover of proteins. We have developed an in vivo pulse-chase method using 15N labeled animals to measure the lifetimes of proteins and using these methods we observed surprisingly that inner core nuclear pore proteins are unexpectedly long- lived proteins. These studies used a linear rate decay analysis model in rats starting at 3 months and extending through 12 months. In this research proposal, we will label the Borchelt Alzheimer's disease mouse model with 15N and chase the labeled animal with 14N food. Measurement of the 14N to 15N ratios will determine the lifetime of APP, abeta1-42 and other proteins present in aggregates. Our expectation is that protein found in the aggregates will be long lived because the normal processes that remove misfolded proteins have failed. A second goal of this R03 proposal is to develop a method using L-azidohomoalanine (AHA) to measure the expression of newly translated proteins in mice. As proof of principle we have successfully pulse labeled a mouse with L-azidohomoalanine to determine safety and incorporation levels. In the proposed research we will pulse AHA in the mouse's food at set timepoints in a Borchelt Alzheimer's mouse model of Alzheimer's disease. We will enrich and measure proteins incorporating AHA in the prefrontal cortex and in insoluble brain aggregates. Only newly translated proteins should have incorporated AHA and will be enriched using "click" chemistry. We will identify new proteins being translated and recruited to the prefrontal cortex and to aggregates. Our hypothesis is that new proteins are being expressed and recruited to aggregates to help remove them.

描述（由申请人提供）：蛋白质在细胞或细胞外基质中持续存在的时间长度可能是蛋白质在疾病中的作用的重要决定因素。随着时间的推移，蛋白质可能会被损坏或修饰，然后必须通过涉及细胞内蛋白酶体或自噬系统的蛋白水解过程或通过整个细胞的周转从细胞中去除。因此，蛋白质寿命和维持对于有丝分裂后细胞（例如神经元）尤其重要。传统上，使用放射性同位素的脉冲追踪技术是测量蛋白质表达和更新的一种方法。我们开发了一种体内脉冲追踪方法，使用 15N 标记的动物来测量蛋白质的寿命，并且使用这些方法，我们令人惊讶地观察到内核核孔蛋白质是出乎意料的长寿蛋白质。这些研究在大鼠中使用了线性速率衰减分析模型，从 3 个月开始一直持续到 12 个月。在这项研究计划中，我们将用 15N 标记 Borchelt 阿尔茨海默病小鼠模型，并用 14N 食物追逐标记的动物。 14N 与 15N 比率的测量将确定 APP、abeta1-42 和聚集体中存在的其他蛋白质的寿命。我们的期望是，在聚集体中发现的蛋白质将长期存在，因为去除错误折叠蛋白质的正常过程已经失败。 R03 提案的第二个目标是开发一种使用 L-叠氮高丙氨酸 (AHA) 测量小鼠中新翻译蛋白表达的方法。作为原理证明，我们成功地用 L-叠氮高丙氨酸对小鼠进行脉冲标记，以确定安全性和掺入水平。在拟议的研究中，我们将在 Borchelt 阿尔茨海默病小鼠模型的设定时间点向小鼠食物中添加 AHA。我们将丰富并测量前额皮质和不溶性大脑聚集体中含有 AHA 的蛋白质。只有新翻译的蛋白质才应掺入 AHA，并使用“点击”化学进行富集。我们将识别被翻译并招募到前额皮质和聚集体的新蛋白质。我们的假设是，新的蛋白质正在表达并招募到聚集体中，以帮助去除它们。