Visualization and manipulation of microRNA in neural cells

Abstract: microRNA (miRNA) are small non-coding RNA, 21-23 nucleotides long. miRNA provide a new layer of regulatory control over gene expression programs. It is increasingly evident that miRNA are cell type and tissue specific. Many miRNAs have been identified to be highly abundant in certain regions of the brain. It has also been shown that alterations in levels of specific miRNAs have implications in various neurodegenerative disorders. However, the role of individual miRNA remains poorly understood. There are various methods that are used to visualize miRNA, depending on type of sample, resolution and throughput. There is currently no gold standard for transcriptional profiling of miRNA and the use of independent techniques to verify results is preferable. This thesis takes a look at the various techniques available to look at miRNA expression profile, providing insight into advantages, disadvantages, complexities and challenges with each technique. To study the function of miRNA it is also essential to regulate its expression. The thesis highlights various methods used by researchers to downregulate or overexpress miRNA. This thesis comprises of three papers where I have tried to evolve tools to visualize distinct miRNA profile to differentiate between cells, and manipulate miRNA expression in vitro and in vivo. In paper 1, I have used miRNA-regulated vectors to differentiate between embryonic stem cells and brain specific cells. This enabled me to sort out neural cells and transplant them in a Parkinson disease mouse model. Using this method I was able to reduce the frequency of tumor formation post transplantation. [1] It has been shown that miRNA-124 (miR-124) plays a crucial role in establishing and maintaining a neuronal transcription network. In paper 2, I used a similar miRNA-regulated vector to look specifically at miR-124 expression in the brain. Here I also used tools to manipulate the expression of miR-124. I was able to demonstrate that miR-124 is a neuronal fate determinant in the subventricular zone [2]. Finally in paper 3, I showed, using miR-9 regulated vectors that miR-9 is not expressed in microglia. Thereby I developed a novel system by which one can specifically target microglia. This system could be used to target genetic modifications to resident microglia in the rodent brain [3]. Overall, further understanding of biogenesis and functionality of this exceptional gene regulator will in turn enhance techniques used to study them. Hopefully, in the future this leads to opportunities to safely pursue miRNA as therapeutic strategies.