An evaluation of modified RNA-interference strategies to study the function of D-amino acid oxidase (DAO) in vivo.

现在有强有力的证据表明，NMDA谷氨酸受体对记忆和解决问题等大脑功能很重要。一种自然产生的化学物质，一种名为D-丝氨酸的氨基酸，有助于激活这种受体，因此可以控制我们的思维方式。然而，关于大脑中D-丝氨酸水平是如何控制的，人们知之甚少。有一种叫做DAO的分子负责代谢(分解)D-丝氨酸，我们已经在小鼠大脑中与思维和记忆有关的部分(额叶皮质和海马体)中发现了大量的DAO。因此，我们想知道大脑中DAO水平的降低是否会增加D-丝氨酸的浓度，进而导致NMDA受体的激活。然而，在我们验证这一假设之前，我们需要找到一种方法来降低小鼠大脑中的DAO水平。我们将开发一种名为RNA干扰(RNAi)的新技术来减少海马区和整个大脑中的DAO。这种方法使用被称为小干扰RNA(SiRNAs)的分子，它干扰脑细胞中的机制，这些细胞根据保存在每个细胞DNA中的蓝图或“基因”制造DAO。尽管这种方法开始被广泛用于其他基因的研究，但人们对它的安全性以及这种方法在多大程度上能够降低大脑分子水平知之甚少。因此，我们将把不同类型的siRNA注射到小鼠大脑中，看看它们降低DAO水平的效果如何，并将使用其他现有的方法来检查这些分子是否对脑细胞有毒。然后，我们将重复对小发夹RNA(ShRNAs)的研究，shRNAs是另一种产生RNAi的分子。在以后的工作中，我们将使用最安全和最有效的RNAi方法来降低大脑中的DAO水平，并观察D-丝氨酸浓度和NMDA受体激活是否增加。总体而言，这项研究将提供有关RNAi方法在研究对不同大脑功能重要的分子的有用性的信息。

There is now strong evidence showing that the NMDA glutamate receptor is important for brain functions such as memory and problem solving. A naturally occurring chemical, an amino acid called D-serine, helps activate this receptor and therefore can control the way we think. However, not much is known about how D-serine levels of the brain are controlled. One molecule, called DAO, is responsible for metabolising (breaking down) D-serine, and we have found large amounts of DAO made in parts of the mouse brain (the frontal cortex and hippocampus) which are involved in thinking and memory. So, we wonder if a decrease in DAO levels in the brain increases concentrations of D-serine, which in turn leads to the activation of the NMDA receptor. Before we can test this hypothesis, however, we need to find a way of decreasing DAO levels in the mouse brain. We will develop a new technique called RNA-interference (RNAi) to decrease DAO in the hippocamus and whole brain. This approach uses molecules called small interferring RNAs (siRNAs) which interfere with the machinery in brain cells that manufacture DAO from the blue-prints or 'genes'which are kept in the DNA of each cell. Although this method is beginning to be widely used to study other genes, not a lot is known about its safety or how well the approach can decrease the levels of brain molecules. We will therefore inject different types of siRNAs into the mouse brain to see how well they decrease DAO levels, and will also check whether these molecules are poisonous or toxic to brain cells by using other established methods. We will then repeat the study on small hairpin RNAs (shRNAs) which are another type of molecule that produces RNAi. In later work we will use the safest and most powerful RNAi approach to decrease DAO levels in the brain and see if D-serine concentrations and NMDA receptor activation is increased. Overall, the study will provide information on the usefullness of the RNAi approach to study molecules important to different brain functions.