Role of post-transcriptional gene regulation in differentiating retinal ganglion cells

Neurons are specialized cells which communicate with each other and other cells via processes called dendrites and axons. During their growth and differentiation, neuronal dendrites and axons protrude and elongate from the cell body in response to trophic factor cues until they reach their target. This process, called neuritogenesis, is essential for the proper establishment of functional and viable connections between neurons and their targets. Neurons that fail to establish such contact undergo cell death. Neurites need a constant supply of proteins, lipids and other molecules in order to elongate and function properly. These molecules are generally produced in the cell body and then transported along the neurite to the synapse by an elaborate network of molecules. Alongside this shuttling mechanism, it has been recently shown that many proteins are actually produced locally in the dendrite when and where they are needed. This process allows neurons to have a faster and more precise response to specific stimuli. Proteins are produced from molecules called messenger RNA or mRNA which are normally found close to the cellular core, or nucleus. Very recently mRNAs have been found in distal parts of neurites where they are translated into proteins. The regulation of mRNAs stability and half-life is one of the mechanisms used by cells to modulate protein synthesis. Amongst the several means used to regulate this process, microRNAs, very short RNA molecules, have been found to play a very important part. By binding to a specific part of the mRNA they trigger its degradation and thus indirectly reduce the production of specific proteins. In our experimental proposal we would thus like to study the sub-cellular localization and the role played by microRNAs in the elongation of neurites. This will be complemented by analyzing the localization and expression of Dicer, one of the key enzymes involved in the production of mature microRNAs. In particular we want to understand whether Dicer and microRNAs are relocated in elongating neurites and whether they play a causative role in this process. This will allow us to better understand how protein synthesis is regulated in growing neurons and by experimentally modulating Dicer's activity, whether any impairment of this mechanism can cause any damage to nerve cells. Furthermore, following our recent results on the regulation of the Brn-3b transcription factor mRNA by microRNAs, we would like to analyze the occurrence of a similar mechanism in retinal ganglion cells (RGC) induced to elongate neurites. Brn-3b is expressed during RGC development, and is essential for their survival as evidenced by the loss of up to 70% of RGC in mice devoid of Brn-3b. The analysis of Brn-3b post-transcriptional regulation in explanted RGC will be thus be a paradigm of the overall role of post-transcriptional regulation in RGC.

The aim of this project is to understand whether post-transcriptional gene regulation has a significant role in elongating neurites of retinal ganglion cells and whether microRNAs are involved in this process. Our initial approach will be to analyse the localization, regulation and role of Dicer, one of the enzymes involved in the production of microRNAs. Dicer expression and localization will be measured by western blot, realtime RT-PCR and immunohystochemistry in primary retinal ganglion cells (RGC) induced to elongate axonal projections when exposed to neurotrophic factor. These experiments will be complemented by analysing the sub-cellular localization of microRNAs in similar experimental conditions. Furthermore, we would like to understand whether Dicer has a direct role in neuronal differentiation. We will thus over-express or conversely silence Dicer expression and analyse whether RGCs are still able to elongate neurites in response to the application of growth stimuli. Subsequently, via a microarray assay we will proceed to analyse the expression profile of microRNAs in neurons induced to elongate neurites. Due to the role played by the Brn-3b transcription factor in RGC development in mice and following our previous findings concerning the role of microRNAs in its post-transcriptional regulation in the ND7 neuroblastoma cell line, we would like to analyse whether such events also occur in primary RGCs. The levels of Brn-3b will be assessed via realtime RT-PCR and western blot in RGCs induced to elongate axons as described above. Furthermore, the data obtained from the microRNA array will be cross-analysed by examining whether any of the up-regulated microRNAs has a potential binding site on the 3'UTR of Brn-3b. These ultimately will be mutated and their effect evaluated on the stability of a GFP-Brn-3b-3'UTR reporter construct as described previously (Calissano et al, 2007, FEBS in press).