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

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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.