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）：此飞行员和可行性赠款申请（NIDDK R21 PA-02-008公告）寻求资金来进行一系列探索性实验，以表征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是一个最近发现的过程，该过程利用了双链RNA（DSRNA）的高度保守监视机制，以引起同源mRNA的选择性，转录后基因沉默。因此，将细胞与特定mRNA一致的细胞暴露在dsRNA上，应使该mRNA和随后的肽的表达保持沉默。 RNAi应用于大脑中的神经元，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，该方法可以应用于在下丘脑（PVN）的室室核中修饰肽神经递质的特定食欲，以检查其在食物摄入调节中的作用。这种适应性神经科学方法的总体实用性可能是显着的。虽然初步数据揭示了该程序的可行性，但必须以其效用和可靠性进行评估和特征。我们建议通过使用dsRNA对神经肽甘酸蛋白（GAL）和甘丙蛋白受体1（GAL-R1）使用DSRNA来评估和表征RNAi的局部应用。我们将在PVN中选择性地确定使用DSRNA对GAL和GAL-R1的关键参数（剂量，体积，损失的时间过程和恢复）。然后，我们将使用这些最佳参数来评估PVN GAL和GAL-R1的功能效应。饮食行为将是检查的功能输出，因为GAL对食物摄入的行为的特征很好。结果完成后，将提供一个详细的程序，用于可靠地使用RNAi作为一种高度选择性，神经化学特异性的“病变”方法，并证明了其在研究大脑中特定神经元亚群的生理功能方面的实用性。