Nitric oxide stabilizes microtubules during neuronal differentiation

Nitric oxide (NO) is a signalling molecule in the nervous system playing a role in neurotransmitter release, synaptic plasticity, excitability, learning, differentiation and development. The signalling pathway triggered by NO in physiological processes involves the activation of soluble guanylate cyclase, S-nitrosylation and nitration of proteins. Focusing on neuronal differentiation and development, NO-induced axonal retraction is involved in the refinement of neuronal projections during brain development and modulated by a 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 the positive effects of NO on neuritogenesis. Our previous results showed that nitrated proteins accumulate during neuronal differentiation and the cytoskeleton becomes the main cellular fraction containing nitrated proteins, being alpha-tubulin and tau two of the main targets, and that nitration correlates with increased microtubule stability. Here we have addressed the question of the possible role played by protein tyrosine nitration and microtubules during neuronal differentiation and neuritogenesis. We modulated the level of intracellular NO by donors and investigated the effects on nitration of proteins, neuritogenesis, arrangement and dynamics of microtubules. Our results show that low-dose NO exposure inducing an increase in nitrated proteins stimulates neurite elongation, microtubule growth and stabilization as shown by indirect immunofluorescence and live cell imaging. On the contrary, high-dose NO exposure induces axonal retraction, causes the accumulation of nitrated proteins at growth cone and destabilizes microtubules. We suggest that protein nitration plays a dual role during neuronal differentiation and modulates NO signalling to the microtubular cytoskeleton.