Expression of ammonia-sensitive proteins in the CNS

中枢神经系统中氨敏感蛋白的表达

• 批准号: 6720095
• 负责人: I. David Weiner
• 金额: $ 15.33万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-12-01 至 2005-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6720095
• 关键词: RNA interference; Saccharomyces cerevisiae; ammonia; biological signal transduction; central nervous system; enzyme activity; gene expression; glycoproteins; hepatic coma /encephalopathy; immunocytochemistry; inborn urea cycle disorder; laboratory mouse; neurons; polymerase chain reaction; sensory feedback; septicemia; tissue /cell culture; western blottings

DESCRIPTION (provided by applicant): Encephalopathy due to elevated ammonia levels is a common and costly clinical condition associated either with congenital deficiency of hepatic urea cycle enzymes, with acquired liver disease and with a variety of other conditions. Despite a long recognition that elevated ammonia levels can lead to impaired neurologic functioning, the exact mechanism through which this occurs remains incompletely understood. Moreover, treatment of hyperammonemic encephalopathy has been limited by a lack of knowledge of the specific CNS protein(s) with which ammonia directly interacts. The broad, long-term objective of this project is to examine the hypothesis that Rh C Glycoprotein (RhCG) is a central nervous system (CNS) ammonia 'sensor.' We hypothesize that RhCG protein is expressed in specific CNS neuron populations, that ammonia-stimulation of RhCG is coupled to specific intracellular signaling pathways, most likely including MAP kinase, and that RhCG expression is regulated by specific physiology/pathophysiologic stimuli. We will examine this hypothesis with two specific aims. In the first, we will determine whether ammonia's stimulation of RhCG, or, possibly the related proteins, Rh A Glycoprotein (RhAG) or Rh B Glycoprotein (RhBG), activates specific intracellular signaling pathways in cultured neurons. We will use primary neuronal cultures, and will use RNA interference techniques to inhibit RhCG expression to show specificity of response. In parallel, we will determine whether RhCG can function as an ammonia sensor by determining whether it can complement the pseudohyphal transformation-defect of Amep2-Amep2 S. cerevisiae. To examine the second aim, we will determine whether cecal ligation and puncture-induced sepsis increases CNS expression of either RhCG, or of RhAG or RhBG. These studies will combine immunohistochemical analyses of cellular protein expression patterns with quantitative analyses of protein and mRNA expression with immunoblot and real-time RT-PCR, respectively. These studies fit the purpose of the R21 mechanism in two different manners. These studies will provide pilot data to assess the feasibility of a novel avenue of investigation into the role of RhCG, or related proteins, as CNS ammonia 'sensors.' Second, while these studies are admittedly high risk, their results could lead to a breakthrough in the field of hyperammonemic and hepatic encephalopathy.

描述（由申请人提供）： 由于氨水平升高引起的脑病是一种常见且昂贵的临床病症，其与肝尿素循环酶的先天性缺乏、获得性肝病和各种其他病症相关。尽管长期以来人们认识到氨水平升高会导致神经功能受损，但这种情况发生的确切机制仍不完全清楚。此外，由于缺乏对氨直接相互作用的特定CNS蛋白的了解，高氨血症性脑病的治疗受到限制。 本项目的广泛、长期目标是检验Rh C糖蛋白（RhCG）是中枢神经系统（CNS）氨传感器的假设。“我们假设RhCG蛋白在特定的CNS神经元群体中表达，RhCG的氨刺激与特定的细胞内信号传导途径偶联，最有可能包括MAP激酶，并且RhCG表达受特定的生理/病理生理刺激调节。 我们将以两个具体目标来检验这一假设。首先，我们将确定氨对RhCG的刺激，或者可能的相关蛋白质，Rh A糖蛋白（RhAG）或Rh B糖蛋白（RhBG），是否激活培养神经元中特定的细胞内信号通路。我们将使用原代神经元培养物，并将使用RNA干扰技术来抑制RhCG表达以显示反应的特异性。同时，我们将通过确定RhCG是否可以补充Amep 2-Amep 2 S的假菌丝转化缺陷来确定RhCG是否可以作为氨传感器。啤酒。为了检验第二个目标，我们将确定盲肠结扎和穿孔诱导的脓毒症是否增加RhCG或RhAG或RhBG的CNS表达。这些研究将结合联合收割机的细胞蛋白质表达模式的免疫组化分析与定量分析的蛋白质和mRNA表达的免疫印迹和实时RT-PCR，分别。这些研究以两种不同的方式符合R21机制的目的。这些研究将提供试点数据，以评估一种新的途径的可行性调查的作用，RhCG，或相关蛋白质，作为中枢神经系统氨的传感器。其次，虽然这些研究被公认为高风险，但它们的结果可能会导致高氨血症和肝性脑病领域的突破。