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

Lead Research Organisation: University of Oxford
Department Name: Psychiatry


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.

Technical Summary

Central N-methyl-D-aspartate receptors (NMDARs) are integral to neurodevelopment, neurotoxicity and cognitive processing. The activation of the NMDAR requires the concomitant binding of glutamate and D-serine (or glycine) to the receptor complex, and in the hippocampus D-serine co-activation predominates. The catabolising enzyme, D-amino acid oxidase (DAO), has recently been suggested to be a key modulator of brain D-serine concentrations, and thus may have direct influences on NMDAR function. To begin to test this hypothesis we first propose to develop an RNA-interference (RNAi) strategy to down regulate DAO gene expression in the mouse brain. The RNAi technique is a powerful approach to examine gene function in isolated neural cells and in vivo. The appoach has the potential to provide genetically modified animals in a relatively short time, and requires only small numbers of rodents akin to pharmacological studies. The need for expensive breeding programmes such as those used in current transgenic technology is therefore precluded, making RNAi more favourable to animal welfare. Using RNAi to study brain function, however, is still in its infancy and a critical evaluation of this application is necessary to ensure its validity. We have access to small interferring RNAs (siRNAs) incorporating several lipid-based chemistries and have constructed small hairpin RNAs (shRNAs) to provide optimal RNAi in vivo. The aim of this study is therefore to test the hypothesis that DAO-siRNAs containing certain chemistries and virally delivered shRNAs produce potent DAO gene silencing in the mouse brain with minimal neurotoxic/neuropathological responses. The RNAi-trigger molecules will be injected into the hippocampus (siRNAs and shRNAs) and cerebral ventricles (siRNAs only) of the mouse, and their diffusion kinetics and toxicity will be monitored. The latter will be assessed by evaluating the expression of inflammatory and glial markers in neural tissues.


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Description We have fulfilled the major aim of our proposal in that we have successfully delivered DAO RNAi to the mouse brain, without incurring any neuro/cellular toxicity or long-term discomfort to the animals. The objectives outlined for Year 2 in our original application have been achieved. Thus, delivery of shRNA constructs with lentiviral vectors has proved to be an effective strategy for safe, long-term reduction of mouse DAO gene expression in the cerebellum and hippocampus. The preparation of this molecular tool is relatively inexpensive and simple compared to transgenic programs. It can be applied to any gene, with neuroanatomical precision and will reduce the number of animals required for molecular investigations. This system also has therapeutic implications.
Exploitation Route The sequence of DAO which initiates degradation of DAO mRNA, was identified by this project. Attenuating the activity of DAO will help understand the pathophysiological contribution of this gene to disorders such as schizophrenia, autism and athero-lateral sclerosis (ALS). Viral-mediated delivery of genetic material to the human brain has already been trialled and so, theoretically, our preparations of Lentivirus-containing DAO RNAi are ready for injection into the brain.
Sectors Pharmaceuticals and Medical Biotechnology

Description Our findings have only been used within the scientific research community, particularly neuroscience.
Sector Education
Description Prebiotic effects on the brain 
Organisation Clasado BioSciences
Country Jersey 
Sector Private 
PI Contribution We have demonstrated that dietary supplementation with prebiotics: 1) alters the chemistry and electrical responses of neurons in the rodent brain; 2) improves problem solving abilities of rats, which corresponds to the chemical and electrophysiological changes in the brain; and 3) reduces stressful behaviour in rodents and healthy human subjects. This collaboration has now extended to include investigations into the effects of early-life prebiotics and manipulation of gut bacteria, on adult brain function.
Collaborator Contribution Clasado Ltd, measured fecal gut bacteria numbers in test groups and also provided prebiotic and placebo compounds for all studies. The company also made intellectual contributions to the study (microbiology, immunity, etc)
Impact See specific research outcomes
Start Year 2010
Description The therapeutic potential of RNA interference for brain disorders 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach International
Primary Audience Participants in your research or patient groups
Results and Impact The author has been invited by Nova Publishers to submit a chapter for book entitled; RNA Interefernce: New Research, which was a direct result of the two publications resulting from this project.

The chapter reviews the approaches and RNAi molecules used to deliver RNAi to the rodent brain. The successes, failures and the factors limiting the application of RNAi to the human brain will be discussed. Novel methods of RNAi delivery, such as the use of 'endosomes' will also be highlighted. Finally, the practicalities of RNAi delivery to the human brain will be discussed with examples of recent trials.

no actual impacts realised to date
Year(s) Of Engagement Activity 2012