Validation of RNA interferences as technique to study CNS anti-obesity drug targets

Abstract

RNA interference offers the opportunity to unravel the roles of genes in a specific area of the brain. Genes implicated in energy balance are perfect targets for the application of RNA interference as their phenotypes can be easily monitored and the neural circuits involved in energy homeostasis are well described. Energy homeostasis is here studied in a rat model, since recording of feeding in rats is more accurate than in mice and the size of the rat’s brain allows more precise injection into brain areas. The overall aim of this thesis was to optimize AAV-mediated knockdown of obesity-associated genes. In order to achieve this goal different AAV serotypes commonly used to transfect the brain were injected into the VMH to determine transduction efficiency. To evaluate biosafety, half-life was calculated after injection of the AAV serotypes in the blood. These experiments are described in chapter two. Unfortunately, the AAV-mediated delivery of shRNAs to the central nervous system resulted in toxicity. To elucidate the cause of the observed toxicity, rats received a unilateral injection with an AAV carrying an shRNA and a control AAV on the other side. In situ hybridization on microRNA-124 was performed to investigate the consequences of shRNA delivery to a neuron for microRNA expression. This study shows an shRNA-mediated oversaturation of the microRNA pathway and the results are described in chapter three. As AAV-mediated knockdown has become a common method to determine gene function, it is important to determine whether the observed toxicity can result in a false phenotype. Chapter four describes how an AAV encoding an shRNA targeting the LepR results in a false phenotype. A solution to the shRNA-mediated oversaturation of the microRNA pathway is to place the shRNA in a microRNA background. Using this technique, the role of FTO in the VMH in the regulation of energy balance was studied. Results from this study are described in chapter five. Finally, chapter six summarizes and discusses the main findings of these studies.