Novel roles for the mitotic kinase Nek7 in hippocampal neurons

Abstract/Resumen The microtubule cytoskeleton plays essential roles during cell division, migration, differentiation, defining cell morphology and organizing intracellular transport. The properties of microtubules, such as their stability, polarity and dynamics, are spatially and temporally regulated by several factors, including post-translational modifications, stabilizing/destabilizing MAPs, motors, kinases, phosphatases, etc. Many of these factors were identified in cycling cells and particularly during mitosis. Nevertheless, some bona-fide mitotic microtubule regulators are also expressed in differentiated cells such as neurons. In a neuron, the microtubule cytoskeleton is organized differently in axons and dendrites, to guarantee a unidirectional transmission of the signal in a neuronal network. In axons, microtubules are generally more stable and are oriented with their plus-ends growing towards the axon cone, while in dendrites microtubules have mixed polarity. In the work described in this thesis, we performed an RNAi screen in neurons with a short list of mitotic/microtubule – related genes that were found upregulated or constantly expressed in a microarray during hippocampal neuron differentiation in vitro. In this screen, we found that the mitotic kinase Nek7 regulated axon length in immature neurons (5/6DIV). Nek7 depletion generates longer axons, and interestingly depletion/absence of Nek6, a kinase that works together with Nek7 in mitosis to phosphorylate the kinesin Eg5, generated the same phenotype. Eg5 pharmacological inhibition also increased axon length, as described by others, suggesting that these kinases are regulating axon length through Eg5. However, depletion of Nek9, another kinase form the same mitotic module, gave rise to shorter axons, indicating that the whole module is not conserved in neurons. In mature neurons (14DIV) Nek7 depletion decreased the total length and branching of dendrites and affected dendritic spines, in a kinasedependent way. Nek6 and Nek9 had no effect on these morphological parameters, but Eg5 inhibition also decreased dendrite length and branching, and spine density. Indeed, co-expression of an Eg5 S1033D phosphomimetic but not of a S1033A phosphor-null mutant, rescued the effects of Nek7 depletion. Furthermore, Nek7 controls Eg5 accumulation in dendrites, in a S1033 phosphorylation-dependent way. To explore the mechanisms behind these dendritic phenotypes, we analyzed microtubule polarity and stability in these dendrites, and observed that Nek7 depletion/Eg5 inhibition increases the percentage of retrograde microtubule EB3 comets in the distal parts of the dendrite. Additionally, Eg5 inhibition with STLC also increased EB3 comet density and decreased tubulin acetylation in dendrites. Ectopic generation of excess of microtubules and of minus-end distal microtubules in the distal regions of the dendrites by expression of the CM1 domain of CDK5Rap2 also gave rise to similar dendritic phenotypes, suggesting that these observations are correlated. I also observed that Eg5 inhibition with STLC can counteract the effects of KIF23 depletion in terms of dendritic microtubule polarity, a motor kinesin that is involved in establishing the mixed polarity microtubule array in dendrites. Furthermore, this depends on Eg5 binding to microtubules and on its motor function, since FCPT treatment did not rescue KIF23 –depletion phenotypes. We suggest a model where Nek7 phosphorylates Eg5 S1033 in dendrites, thus mediating Eg5 transport by dynein and accumulation in dendritic microtubules via TPX2, by analogy with mechanisms existent during mitosis. As expected, I observed that depletion of TPX2 also decreased total dendrite length. In dendrites, immobile Eg5 likely crosslinks and stabilizes microtubules in parallel bundles, and mobile Eg5 may also help to guide and sort microtubules into parallel bundles, and to mediates sliding of antiparallel microtubules. It is also possible that Eg5 can regulate the rate of short microtubule transport in dendrites, as demonstrated by others in axons. Altogether, these functions would promote dendritic growth and branching and correct spine formation.