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的时间长度可能是蛋白质在疾病中作用的重要决定因素。随着时间的流逝，蛋白质可能会受到损坏或修饰，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营业额来从细胞中取出。因此，蛋白质的寿命和维持在神经元等有线后细胞中尤为重要。传统上，使用放射性同位素的脉冲追踪技术是一种测量蛋白质表达和周转的方法。我们使用15N标记的动物开发了一种体内脉冲练习方法来测量蛋白质的寿命，并使用这些方法，我们出人意料地观察到内核核孔蛋白是意外的长寿命蛋白。这些研究使用了从3个月开始延伸至12个月的大鼠中的线性衰减分析模型。在这项研究建议中，我们将用15N标记硼北阿尔茨海默氏病小鼠模型，并用14N食物追逐被标记的动物。 14N与15N比率的测量将确定APP，ABETA1-42和其他蛋白质中存在的寿命。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的正常过程失败了。该R03提案的第二个目标是使用L-氮杂类丙氨酸（AHA）开发一种方法，以测量小鼠新翻译的蛋白质的表达。作为原则的证明，我们成功地将标记为L-氮杂丙氨酸的小鼠标记，以确定安全水平和掺入水平。在拟议的研究中，我们将在阿尔茨海默氏病小鼠小鼠模型中以固定时间点的固定时间点脉动AHA。我们将富集并测量在前额叶皮层和不溶性脑聚集体中掺入AHA的蛋白质。只有新翻译的蛋白质才能掺入AHA，并将使用“点击”化学富集。我们将确定被翻译和招募到前额叶皮层和聚集的新蛋白质。我们的假设是，正在表达并招募新的蛋白质以帮助消除它们。