Searching for a role of protein tyrosine nitration during neuronal differentiation

Among the myriad of cellular functions played by nitric oxide (NO) in the brain, there is increasing evidence that NO might be a primary player in the programme of neurogenesis and neuronal differentiation. The signalling pathway triggered by NO in physiological processes involves the activation of soluble guanylate cyclase, S-nitrosylation of proteins, and, as recently proposed, nitration of tyrosine residues in proteins. We have previously showed that the cytoskeleton becomes the main cellular fraction containing nitrotyrosinated proteins during nerve growth factor (NGF)-induced differentiation of cultured cells. On the ground that cytoskeleton is crucial in neuronal morphogenesis to determine axonal guidance and branching, here we have investigated the possible role of nitration in controlling cytoskeleton remodeling, in particular microtubule rearrangement and dynamics, that underlines neuronal differentiation. We have initially studied the association of nitrated proteins with the cytoskeletal fraction in differentiating neuronal cells following exposure to microtubule depolymerising treatments. Through morphological and biochemical approaches we found that nitration correlates with the increased microtubule stability underlying the progression of neuronal differentiation. Given thatalfa-tubulin is one of the major target of nitration, we have undertaken an in vitro study to selectively nitrate purified tubulin and to asses the role of this post-translational modification in modulating tubulin assembly and stability. We found that modified tubulin remains competent to assembly and that nitrated microtubules show an increased stability to depolymerising agents. Finally, we are trying to modulate the level of nitrated proteins in differentiating cells and study the effects on microtubule stability and dynamics by live cell imaging. We conclude that nitration of cytoskeletal proteins could play a novel functional role in the complex and dynamic organisation of the cytoskeleton underlying neuritogenesis and differentiation.