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: 6823260
• 负责人: I. David Weiner
• 金额: $ 15.35万
• 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 2006-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6823260
• 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）： 氨水平升高引起的脑病是一种常见且昂贵的临床病症，与先天性肝尿素循环酶缺乏、后天性肝病和各种其他病症有关。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，但发生这种情况的确切机制仍不完全清楚。此外，由于缺乏对与氨直接相互作用的特定中枢神经系统蛋白的了解，高氨血症性脑病的治疗受到限制。 该项目的广泛、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通路（最有可能包括 MAP 激酶）耦合，并且 RhCG 的表达受到特定的生理/病理生理刺激的调节。 我们将通过两个具体目标来检验这一假设。首先，我们将确定氨对 RhCG 或可能相关的蛋白质 Rh A 糖蛋白 (RhAG) 或 Rh B 糖蛋白 (RhBG) 的刺激是否会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Amep2-Amep2酿酒酵母的假菌丝转化缺陷来确定它是否可以作为氨传感器发挥作用。为了检验第二个目标，我们将确定盲肠结扎和穿刺引起的脓毒症是否会增加中枢神经系统中 RhCG、RhAG 或 RhBG 的表达。这些研究将分别通过免疫印迹和实时 RT-PCR 对细胞蛋白表达模式进行免疫组织化学分析，对蛋白和 mRNA 表达进行定量分析。这些研究以两种不同的方式满足了 R21 机制的目的。这些研究将提供试点数据，以评估研究 RhCG 或相关蛋白质作为中枢神经系统氨“传感器”作用的新途径的可行性。其次，虽然这些研究无疑具有高风险，但其结果可能会在高氨血症和肝性脑病领域带来突破。