Analisi del ruolo neuronale di Morgana (Chordc1) in colture primarie ippocampali

Cell polarization is a crucial aspect of the neuronal differentiation program, responsible for the establishment of the axonal and dendritic compartments. The centrosome is a very important organizing center for the membrane trafficking and cytoskeletal events required for neuronal polarization. Morgana, also called Chp-1 and Chordc1, is a strongly conserved CHORD (cysteine and histidine-rich domains) containing protein that plays a very important role in centrosomes biogenesis. Indeed, it has been recently shown that Morgana regulates centrosome duplication in proliferating cells of both Drosophila and mice, by inhibiting the activity of ROCKII through NPM-dependent mechanism. ROCK (RhoA kinase) is another important cue in the signaling pathway of neuronal polarity. The identification of high expression levels of this protein in the mature brain raised the interesting possibility that it may play an important role in neuronal differentiation and/or physiology. To directly address this possibility we studied the expression and function of Morgana in rat hippocampal neurons, a well-known primary culture system widely used to recapitulate in vitro all the milestones of the in vivo neuronal differentiation program. We found that Morgana is expressed throughout brain embryogenesis, with highest levels during neurogenesis. Furthermore, Morgana is expressed at constant levels during the in vitro differentiation of rat hippocampal neurons. At the subcellular level, Morgana accumulates specifically in a polarized cytoplasmic area near the nucleus at early stages of differentiation, and is evenly redistributed to the cytoplasm after axon specification. The polarized distribution of Morgana corresponds to the main structures implicated in neuronal polarization, such as the Golgi, the ER and the early endosomes, all of which are tightly associated to the centrosome, even though Morgana do not colocalize precisely with them. These results suggest a potential role of Morgana in early stages of neuronal polarization. To test this possibility, we tried to perform both knock-down and overexpression studies. Interestingly, we found that the upregulation of Morgana in hippocampal neurons does not affect axon specification but inhibits axonal elongation. Moreover, we demonstrated that ROCKII physically interacts with Morgana also in neuronal cells and that its activity is required for the Morgana-induced differentiation delay. Importantly, the effects elicited by the inhibition of ROCK in Morgana-overexpressing neurons are completely opposite to the effects elicited under basal conditions. In conclusion, our results strongly suggest that Morgana forms a specific functional complex with ROCKII that plays an important role in controlling axonal growth.