COINVOLGIMENTO DELLA NEURITINA NELLA MIGRAZIONE DEI NEURONI GNRH: EVIDENZE SPERIMENTALI IN VITRO ED IN VIVO

In this thesis we studied Neuritin (also called Nrn or cpg15), a GPI-anchored protein identified among a pool of candidate plasticity-related genes induced by kainate in rat hippocampal dentate gyrus (Nedivi, Hevroni et al. 1993). It has been shown that it is involved in neurites elongation and in promoting synaptic maturation (Putz, Harwell et al. 2005). Neuritin acts in a non-cell autonomous manner to coordinately regulate the growth of apposing dendritic and axonal arbors. Its expression is regulated by neurotrophines, NGF, calcium levels and androgens. Our previous studies demonstrated that NGF up-regulates Nrn expression during PC12 neuron differentiation, and that Nrn enhances the differentiating effects of NGF on PC12 cells favoring the extension of longer neurites (Cappelletti, Galbiati et al. 2007). Interestingly, Nrn expression peaks during neuronal development and, particularly, in the embryonic proliferative zones. During embryogenesis one of the most important aspects is the active neuronal migration in order to reach the final position and obtain the correct development of the nervous system. The migration of neurons and precursor cells involves molecular mechanisms which are often similar to those involved in neurite outgrowth. The generation of the GnRH neuron network, that plays a key role in the regulation of the reproduction system, represent a particular example of neuronal migration. These neurons generates from the olfactory epithelium and move to the hypothalamus where they exerts their regulatory function. For this reason a neuronal immortalized cell line of GnRH neurons, named Gn11 cells, is considered a good model to study neuronal migration (Radovick, Wray et al. 1991; Maggi, Pimpinelli et al. 2000; Cariboni, Maggi et al. 2007). A preliminary microarray analysis performed on Gn11 cells showed neuritin as one of the most abundant genes expressed in this cell line, leading us to hypothesize that it is involved in the neuronal migration process. To demonstrate this hypothesis, we analyzed neuritin mRNA and protein expression levels in different cell lines, characterized by a different migratory behavior. Interestingly, neuritin expression is much higher in migrating than in non-migrating cells. In Boyden microchemotaxis and wound-healing assays, the migratory ability of Gn11 cells is reduced when neuritin expression is silenced, while it is increased by neuritin over-expression. We also demonstrated through a Boyden microchemotaxis assay that the over-expression of Nrn in primary culture of ganglionic eminences (the area generating the migrating interneurons) increases interneuron migration. Furthermore, our hypothesis is strenghtened by results obtained with the selective elettroporation of ganglionic eminences of rat’s E16 brain slices indicating an increased migration of interneurons when neuritin is over-expressed. Post-translational modifications of alpha-tubulin are known to be associated with microtubule dynamics. ICC and Western blot analyses showed an enrichment of stable microtubules, that likely reflects the diminished migratory capability of the cells, in neuritin silenced cells; moreover, neuritin over-expression, that induces an increase in the migratory ability in the cells, is also able to up-regulates the tyrosinated form of a-tubulin reflecting the presence of more dynamic microtubules. Altogether these data demonstrate a novel function of neuritin in promoting migration of neuronal cells, both in vitro and in vivo, through the modulation of microtubule stability, and makes neuritin a good candidate as a therapeutic target for different diseases in which cellular migration is involved. Cappelletti, G., M. Galbiati, et al. (2007). "Neuritin (cpg15) enhances the differentiating effect of NGF on neuronal PC12 cells." J Neurosci Res 85(12): 2702-2713. Cariboni, A., R. Maggi, et al. (2007). "From nose to fertility: the long migratory journey of gonadotropin-releasing hormone neurons." Trends Neurosci 30(12): 638-644. Maggi, R., F. Pimpinelli, et al. (2000). "Immortalized luteinizing hormone-releasing hormone neurons show a different migratory activity in vitro." Endocrinology 141(6): 2105-2112. Nedivi, E., D. Hevroni, et al. (1993). "Numerous candidate plasticity-related genes revealed by differential cDNA cloning." Nature 363(6431): 718-722. Putz, U., C. Harwell, et al. (2005). "Soluble CPG15 expressed during early development rescues cortical progenitors from apoptosis." Nat Neurosci 8(3): 322-331. Radovick, S., S. Wray, et al. (1991). "Migratory arrest of gonadotropin-releasing hormone neurons in transgenic mice." Proc Natl Acad Sci U S A 88(8): 3402-3406.