Silencing Hypothalamic Galanin with RNA Interference

描述(由申请人提供)：这项试点和可行性拨款申请(NIDDK R21 PA-02-008公告)寻求资金，以进行一系列探索性实验，以表征RNA干扰(RNAi)作为一种抑制哺乳动物大脑中特定神经肽表达的方法的使用。RNAi是最近发现的一个过程，它利用对双链RNA(DsRNA)的高度保守的监控机制，导致同源mRNA的选择性转录后基因沉默。因此，细胞暴露于dsRNA与特定的信使核糖核酸相一致，应该会抑制该信使核糖核酸的表达，并随后抑制多肽的表达。应用于大脑中的神经元，RNAi可以被证明是一种选择性地消除任何神经化学特有的神经元集合的方法，这些神经元涉及神经元的信号机制中的蛋白质(例如神经肽递质、生物合成酶)。例如，该方法可以应用于抑制下丘脑室旁核(PVN)中的特定食欲调节肽神经递质，以研究它们在食物摄入调节中的作用。这种适用于神经科学的方法的普遍实用性可能会令人惊叹。虽然初步数据显示了该程序的可行性，但必须对其实用性和可靠性进行评估和表征。我们建议通过使用针对神经肽甘丙素(GAL)和甘丙素受体-1(GAL-R1)的dsRNA来评估和表征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、丢失和恢复的时间进程)。然后，我们将使用这些最佳参数来评估PVN、GAL和GAL-R1的功能效果。饮食行为将是被检查的功能输出，因为GAL对食物摄取的作用是很好的特征。结果完成后，将提供一个详细的程序，可靠地使用RNAi作为一种高度选择性的、神经化学特异性的“损伤”方法，并证明其在研究大脑中特定神经元亚群的生理功能方面的有效性。

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.