| Abstract |
Neuronal networks constitute perhaps the most complex and intricate formations of an organism. Due to their oftentimes extensive dimensions, cellular compartments such as axons, dendrites, and synapses can be located at a considerable distance from the cellular body. However, they still contain the vast majority of the cytoplasm, and are responsible for many diverse functions. In order to maintain their neuronal homeostasis, the ability to respond rapidly to intra- and extra-cellular stimuli must be independently preserved for different sub-neuronal compartments.
Localized mRNA transport and translation are some of the mechanisms that allow for this spatiotemporal regulation. Although the exact molecular mechanism of local mRNA translation remains elusive, it appears to be based on the semi-autonomous protein translation of mRNA that has been specifically transported and stored in sub-neuronal compartments, in response to internal and external stimuli. Ribonucleoprotein complexes (RNPs) formed by RNA-binding Proteins (RBPs), mRNA molecules, and other regulatory proteins, play a crucial role in the transportation, as well as the regulation of the localized translation of these mRNA molecules. Local mRNA transportation and subsequent translation, along with the regulation of actin cytoskeleton, has been shown to have a key role in axon elongation and guidance, synaptogenesis, and post-development synaptic plasticity, among other processes. Its importance during neuronal development is highlighted by the fact that dysregulation of these two processes has been implicated in several neurodegenerative and neurodevelopmental disorders such as autism spectrum disorders (ASDs), Fragile X syndrome, spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS).
Mena (ENAH) is a protein of the Ena/Vasp protein family and is highly expressed in the nervous system. Previous work from our lab has shown that, in addition to its role in actin cytoskeleton regulation Mena also plays a regulatory role in local translation; Mena has been found to form an RNP complex with known translation regulators HnrnpK and PCBP1 and cytosolic mRNAs in developing axons. The presence of Mena is required for translation of the associated mRNAs locally. This newly discovered dual nature of Mena’s function raises several questions: does its general role in axonal local translation extend to other sub-cellular compartments, namely in synapses? Does it still interact with the same regulators, and if not, what other proteins does it interact with? The further elucidation of Mena’s role could help us shed light on different neurodevelopmental disorders.
Mena’s diverse role in the sub-cellular compartments of the NS remains for the greater part elusive, and so, in order to learn more about it, our lab has started a study on its spatiotemporal expression pattern throughout the developing mouse CNS. This project has brought this effort a step further, adding two new timepoints (P8-10 and P15 mice) to the preexisting ones (P22 and 2m.o. mice). We have continued to study Mena’s localization across the entire CNS, using different neuronal and synaptic markers, and comparing our findings with the timepoints that were previously studied. We discovered interesting similarities and differences across the different CNS structures and timepoints, and most importantly were able to acquire strong evidence of the synaptic localization of Mena in structures such as layers L2/3 and L5 of the Cortex, the Hippocampus, the Cerebellum, the Medulla, the Hindbrain, and the DRGs.
Furthermore, by testing and using a synaptoneurosome isolation method, followed by co-immunoprecipitation, we were able to test Mena’s interaction in synapses with several molecules of interest: EVL, VASP, EEF2, SAFB, HNRNPK, and PCBP1. Mena was found to interact with EVL, VASP, EEF2, and SAFB but, surprisingly, no interaction was observed for known developmental interactors HNRNPK and PCBP1. This presents a very interesting avenue for future study and leads us to believe that Mena’s interaction profile in the synapses could be quite different from its general neuronal one.
|
In vivo distribution of Mena in postnatal CNS synapses
