Novel roles for the mitotic kinase Nek7 in hippocampal neurons

Abstract

[eng] 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 kinase-dependent 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.

[spa] Los microtúbulos son una componente importante del citoesqueleto, esenciales en la división celular, migración, transporte intracelular y diferenciación. La polaridad, estabilidad y dinámica de los microtúbulos son reguladas por muchos factores, como MAPs (proteínas asociadas a microtúbulos), quinesinas, dineínas, quinasas, fosfatasas, entre otros. Muchos de estos reguladores fueron descubiertos y caracterizados por su función durante la mitosis, pero algunos también están presentes en células diferenciadas, como por ejemplo neuronas. Las neuronas dependen mucho de la organización de los microtúbulos para su función. En una neurona, el axón tiene microtúbulos de polaridad uniforme, mientras que en las dendritas la polaridad es mixta, y esto es esencial para la transmisión unidireccional de la señal nerviosa. El sistema de diferenciación de neuronas hipocampales in vitro se utiliza para estudiar la morfología neuronal y funciones del citoesqueleto. En mi trabajo de tesis doctoral, he caracterizado la función de una quinasa mitótica, Nek7, como reguladora de la diferenciación de neuronas hipocampales. He observado que Nek7, junto con Nek6, regula el crecimiento axonal en neuronas inmaduras (5/6DIV). En ausencia de Nek7 o Nek6 los axones son más largos, mientras que la depleción de Nek9, otra quinasa que funciona en conjunto con Nek6/7 en mitosis, genera axones más cortos. En neuronas maduras (14DIV), Nek7 controla la morfología de dendritas y espinas a través de la regulación de la quinesina Eg5, que también es su substrato en mitosis. Los defectos generados por la depleción de Nek7 se rescatan con un mutante fosfo-mimético de Eg5 (S1033D) pero no con un mutante no fosforilable (S1033A). Además, Nek7 controla el reclutamiento y acumulación de Eg5 en la parte distal de las dendritas, a través de esta fosforilación. En la base de estos fenotipos encontramos problemas en la estabilidad y polaridad de microtúbulos en las dendritas. Tanto la depleción de Nek7 como la inactivación de Eg5 aumentan el porcentaje de microtúbulos de polaridad reversa en la parte distal de la dendrita, y disminuyen la acetilación de microtúbulos, un indicador de estabilidad. Finalmente se presenta un modelo en lo cual Eg5 regula la estabilidad, polaridad y deslizamiento de los microtúbulos dendríticos para favorecer el crecimiento dendrítico.