Local mRNA translation in the regulation of neurite outgrowth

Local mRNA translation allows to synthesize proteins in discrete subcellular locations upon induction by various stimuli, therefore contributing to the control of gene expression in space and in time. The possibility to rapidly produce big amounts of proteins from few molecules of localized transcripts makes this mechanism extremely cost-efficient, since it avoids the long-distance transport of proteins. This is important especially in neurons, where local translation has been shown to be involved in the control of synaptic plasticity and axonal guidance. Nevertheless, it has never been studied during the early phases of neuronal polarization, before the axon/dendrite specification step.
In N1E-115 cells, a neuron-like cell line that mimics the early stages of differentiation, we identified 80 mRNAs that are enriched in neurites compared to cell bodies by a genome-wide gene CHIP analysis. This suggests that also at these stages, targeting of transcripts to specific subcellular regions can play a role in cell morphogenesis. One of the detected messengers encodes MKK7, a MAP kinase kinase that directly activates the c-JUN NH2-terminal kinases (JNKs). We showed that the 3’UTRs of MKK7 mRNA target the transcript specifically to the growth cone. Here local synthesis of the protein allows the formation of a zone of activated, phosphorylated MKK7 that is confined to the neurite shaft. Depletion of MKK7 by siRNA leads to instable neurite extension, due to defects in microtubule bundling at the base of the neurites.
With a bioinformatic analysis of the published proteome of the N1E-115 cell line we built an MKK7-centered interactome, which includes MAPKKKs (the upstream kinase of MKK7), MKKs, JNKs, microtubule associated proteins (the effectors of JNKs), scaffold proteins and phosphatases. Immunofluorescence analysis for the localization of the components of the network, combined with knock down experiments allowed us to identify a specific signaling module consisting of DLK, MKK7, JNK1 and MAP1B that regulates microtubule bundling in the neurite shaft and promotes neurite extension. FRET experiments using an activity probe for JNK further confirmed the involvement of JNK in the neurite shaft. Moreover, with immunofluorescence experiments we demonstrated the localization of the JNK signaling module also in mice E15 hippocampal primary neurons.
This thesis proposes a mechanism by which local translation of MKK7 mRNA in the growth cone enables the activation of a specific branch of the JNK signaling pathway to regulate neurite extension. Therefore, local protein synthesis allows the spatio-temporal control of gene expression during early stages of neuronal differentiation.