Silencing Hypothalamic Galanin with RNA Interference

描述（由申请人提供）：本试点和可行性资助申请（NIDDK R21 PA-02-008公告）寻求资助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是最近发现的一个过程，利用双链RNA （dsRNA）的高度保守的监视机制，引起同源mRNA的选择性转录后基因沉默。因此，将细胞暴露于与特定mRNA一致的dsRNA应使该mRNA和随后的肽的表达沉默。应用于大脑神经元，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，该方法可以应用于下丘脑室旁核（PVN）中特定食欲调节肽神经递质的抑制，以研究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领域的普遍应用可能是显著的。虽然初步数据显示了该程序的可行性，但必须对其效用和可靠性进行评估和表征。我们建议通过dsRNA对神经肽甘丙肽（GAL）和甘丙肽受体-1 （GAL- r1）的靶向来评估和表征RNAi的局部应用。我们将确定在PVN中选择性使用dsRNA对抗GAL和GAL- r1的关键参数（剂量、体积、损失和恢复的时间过程）。然后，我们将使用这些最优参数来评估PVN GAL和GAL- r1的功能效果。饮食行为将是功能性输出检查，因为GAL对食物摄入的作用是很好的特征。研究结果完成后，将为RNAi作为一种高度选择性的、神经化学特异性的“损伤”方法提供详细的程序，并为其在研究大脑中特定神经元亚群的生理功能方面的应用提供证据。

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.