Silencing Hypothalamic Galanin with RNA Interference

通过 RNA 干扰沉默下丘脑甘丙肽

• 批准号: 6703316
• 负责人: SuJean Choi
• 金额: $ 14.87万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-03-01 至 2006-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6703316
• 关键词: RNA interference; biological signal transduction; corticotropin releasing factor; double stranded RNA; galanin; gene expression; gene induction /repression; hypothalamus; immunocytochemistry; in situ hybridization; laboratory rat; messenger RNA; neurochemistry; neuropeptide receptor; neuropeptides; nutrient intake activity; paraventricular nucleus

DESCRIPTION (provided by applicant): This pilot and feasibility grant application (NIDDK R21 PA-02-008 announcement) seeks funding to conduct a series of exploratory experiments to characterize the use of RNA interference (RNAi) as a method to suppress the expression of specific neuropeptides in the mammalian brain. RNAi is a recently discovered process that exploits a highly conserved surveillance mechanism for double stranded RNA (dsRNA), to cause a selective, post-transcription gene silencing of homologous mRNA. Thus, exposure of cells to dsRNA coinciding to a specific mRNA should silence the expression of that mRNA and subsequently the peptide. Applied to neurons in brain, RNAi could prove to be a method for the selective elimination of any neurochemically-specific set of neurons that involve a protein in the neuron's signaling mechanism (e.g. neuropeptide transmitters, biosynthetic enzymes). For example, the method could be applied to the suppression of specific appetite modifying peptide neurotransmitters in the paraventricular nucleus of the hypothalamus (PVN) to examine their roles in food intake regulation. The general utility of such an adaptable method to neuroscience would potentially be remarkable. While preliminary data reveal the feasibility of the procedure, it must be evaluated and characterized for its utility and reliability. We propose to evaluate and characterize local application of RNAi by using dsRNA against the neuropeptide galanin (GAL) and the galanin receptor-1 (GAL-R1). We will determine the key parameters (dose, volume, time course of loss and recovery) of using dsRNA against GAL and GAL-R1 selectively in the PVN. We will then use these optimal parameters to evaluate the functional effect of PVN GAL and GAL-R1. Eating behavior will be the functional output examined, because GAL's actions on food intake are well characterized. The results, when completed, will offer a detailed procedure for using RNAi reliably as a highly selective, neurochemically-specific "lesioning" method and evidence of its utility in studying physiological functions of particular neuronal subpopulations in the brain.

描述（由申请人提供）：该试点和可行性资助申请（NIDDK R21 PA-02-008公告）寻求资金进行一系列探索性实验，以表征使用RNA干扰（RNAi）作为抑制哺乳动物大脑中特定神经肽表达的方法。RNAi是近年来发现的一种利用双链RNA（dsRNA）的高度保守的监视机制来引起同源mRNA的选择性转录后基因沉默的过程。因此，将细胞暴露于与特定mRNA一致的dsRNA应沉默该mRNA的表达，随后沉默肽的表达。应用于大脑中的神经元，RNAi可以被证明是一种选择性消除任何神经化学特异性神经元的方法，这些神经元涉及神经元信号传导机制中的蛋白质（例如神经肽递质，生物合成酶）。例如，该方法可应用于抑制下丘脑室旁核（PVN）中的特定食欲调节肽神经递质，以检查它们在食物摄入调节中的作用。这种适应性强的方法在神经科学中的普遍效用可能是惊人的。虽然初步数据显示该程序的可行性，但必须对其实用性和可靠性进行评估和表征。我们建议使用dsRNA对神经肽甘丙肽（GAL）和甘丙肽受体-1（GAL-R1）进行评估和表征RNAi的局部应用。我们将确定在PVN中选择性地使用针对GAL和GAL-R1的dsRNA的关键参数（剂量、体积、损失和恢复的时间过程）。然后，我们将使用这些最佳参数来评价PVN GAL和GAL-R1的功能效应。进食行为将是功能输出检查，因为GAL的行动对食物摄入的特点。结果，完成后，将提供一个详细的程序，可靠地使用RNAi作为一个高度选择性的，神经化学特异性的“病变”的方法和证据，其效用在研究大脑中的特定神经元亚群的生理功能。