Protein tyrosine nitration: a beneficial or detrimental cue during neuronal differentiation?

Nitric oxide (NO) is well established as an intracellular and transcellular signalling molecule in the nervous system playing a role in neurotransmitter release, synaptic plasticity, excitability, learning, differentiation and development. The majority of NO actions under physiological conditions occur through the activation of soluble guanylate cyclase, leading to the intracellular increase of cGMP. However, NO can also produce cGMP-independent effects in living cells through protein modification including S-nitrosylation and tyrosine nitration. Focusing on neuronal differentiation and development, NO-induced axonal retraction is involved in the refinement of neuronal projections during brain development and modulated by an S-nitrosylation-dependent signal –transduction pathway leading to the reconfiguration of axonal microtubules. On the other hand, NO donors have been reported to enhance neurite outgrowth suggesting a positive effects of NO on neuritogenesis. We have addressed the question of the possible role played by protein tyrosine nitration in the signalling pathway triggered by NO during neuronal differentiation and neuritogenesis. Our previous results showed that nitrated proteins accumulate during NGF-induced differentiation of PC12 cells, the cytoskeleton becomes the main cellular fraction containing nitrated proteins, and that nitration correlates with increased microtubule stability. Here we have modulated the level of intracellular NO by donors and investigated the effects on nitration of proteins, neuritogenesis, arrangement and dynamics of the microtubular cytoskeleton. Our results show that NO donor (Glyco-SNAP-2) significantly affects neuritogenesis in NGF-differentiating PC12 cells. Low-dose NO exposure inducing an increase in nitrated protein stimulates neurite elongation and causes microtubule stabilization as shown by indirect immunofluorescence and live cell imaging analyses. In conclusion, our results reveal a dual role of protein nitration during neuronal differentiation and neuritogenesis, with a beneficial or cytotoxic actions depending on the NO concentration, and suggest the involvement of this posttranslational modification in modulation of NO signalling to the cytoskeleton.