| Abstract |
The computationally intricate organization of neuronal circuits is etched within the highly polarized nature of is neuronal components which necessitates the subcellular compartmentalization specialization of their cellular architecture. In order to sustain such structural and functional polarity and effectively coordinate multivariate and rapid responses to cell intrinsic and extrinsic signals, neurons have (evolutionarily) adapted various mechanisms that enable semi-autonomous spatiotemporal regulation of protein turnover in distal parts of their oftentimes vast neurite networks. Numerous recent studies have proposed that targeted mRNA transportation and localized mRNA translation hold key roles during development, in establishing neuronal connectivity, and during maturation, in maintaining neuronal homeostasis. Both of those functions have been shown to be elegantly orchestrated by a vast array of RNA binding proteins (RPBs), which along with other regulatory proteins form ribonucleoprotein complexes (RNPs) that serve as safeguards and specialized transporters of mRNA molecules to distal sites of protein synthesis. During neuronal development, local mRNA transportation/translation, in addition to and in concert with active actin cytoskeleton remodeling, have been shown to be important for axon elongation and guidance, dendritogenesis, synaptogenesis, synaptic pruning and have also been implicated in various forms of synaptic plasticity later in life. Despite the fact that dysregulation of those processes is implicated in many neurodevelopmental and neurodegenerative disorders, there is relatively limited knowledge on their regulation and coordination at the molecular level.
During the course of evolution, the emergence of ever more complex neuronal systems was endorsed by many multifaceted protein constructs with auxiliary functional domains, with extensive interactome profiles which are involved in complex gene regulatory programs. This enabled them to participate in a variety of locally restricted and time sensitive events that regulate many of the specialized processes guiding nervous system (NS) development and function. One such protein is Mena which is the mammalian homologue of ENA(enabled) that is expressed by many cell types throughout the NS and is known to participate in the polymerization of actin through its interaction with related proteins VASP and EVL. In addition to that, previous work from our lab in developing neurons, has shown that Mena is involved in regulating the local expression of specific mRNAs in axons through the formation of an RNP complex containing the RBPs HnrnpK and PCBP1, which was shown to contribute to proper axon elongation and growth-cone guidance. Related to this, could be the fact the fact that mice deficient in Mena are known to display clear defects in NS development. This dual nature of Mena, hints its possible role as a key mediator between those two processes in neurons and prompted us to suspect that Mena could function similarly in synapses, by coordinating actin dynamics and shaping the local synaptic proteome, via regulation of local mRNA translation. However, the exact way Mena mediates those functions, as well as its potential role beyond development, to this day remain elusive. Uncovering this mechanism could be important, since the NS defects displayed by Mena knock-out mice show similarities with neurodevelopmental disorders like autism spectrum disorders.
As an initial step in addressing this puzzle, this study aims to expand our knowledge about Mena beyond the point of birth, by employing in-vivo, ex-vivo and in-vitro approaches that allow us to investigate the spatiotemporal expression profile and sub-neuronal localization of Mena protein and its RNP components across the entire NS, in relation to different neuronal and synaptic markers, whilst at the same time comparing the differences between adult and juvenile mice. We observed a highly heterogeneous intracellular and extracellular distribution of Mena protein across distinct isolated neuronal populations within the peripheral NS and central NS with strong indications for axonal, dendritic and synaptic localization, particularly in many specialized synaptic structures. More notably, we detected an interesting staining pattern in neurons belonging to the trigeminal and auditory circuits in the brain-stem, and in forebrain areas like parts of the cerebral cortex and limbic structures like the hippocampus and basolateral amygdala (BLA) and also in dorsal root ganglion (DRG) neurons and in the spinal cord. At the same time, we noted small distinct differences in the immunoreactivity of Mena in some regions between P22 and 2mo animals. By staining with different oligodendroglial markers, we were able to prove the presence of Mena in forebrain oligodendrocyte populations. Furthermore, we tested to see if Mena expression coincides with the expression of some of its main identified interactors and we observed signal overlap in some of those areas. In addition, we were able to confirm the presence of Mena protein and some of its main interactors in axonal, dendritic and pre- and post-synaptic compartments in long-term cultures from dissociated primary cortical neurons. Lastly, using an in vitro method, we manage to isolate and quantify the presence of Mena protein and some of its interactors from cortical synaptoneurosome extracts, which revealed an increased enrichment in synaptic compartments.
All these separate evidence support the presence of Mena in various subneuronal compartments and substantiate our efforts to further investigate its potential contribution in synapse formation and function. We believe that this increased subcellular and subcompartmental variability in the posttranslational expression profile of Mena could indicate its possible role in shaping the local synaptic proteome possibly by acting as a regulatory node that coordinates and balances actin polymerization and local protein synthesis. Factors that disturb this balance have been linked with a multitude of neurodevelopmental and neurodegenerative disorders.
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