Neuritin (cpg15) enhances the differentiating effect of NGF on neuronal PC12 cells

Neuritin is a small, highly conserved GPI-anchored protein involved in neurite outgrowth. We have analyzed the involvement of neuritin in NGF-induced differentiation of PC12 cells by investigating the time-course of neuritin expression, the effects of its overexpression or silencing, and the possible mechanisms of its regulation and action. Real-time PCR analysis has shown that neuritin gene is upregulated by NGF in PC12 cells hours before neurite outgrowth becomes appreciable. PC12 cells transfected with a plasmid expressing neuritin display a significant increase in the response to NGF: 1) in the levels of SM1312 positive phosphorylated neurofilament proteins (markers for axonal processes) and tyrosine hydroxylase; 2) in the percentage of cells bearing neurites; as well as 3) in the average length of neurites when compared to control cells. On the contrary, neuritin silencing significantly reduces neurite outgrowth. These data suggest that neuritin is a modulator of NGF-induced neurite extension in PC12 cells. We also showed that neuritin potentiated the NGF-induced differentiation of PC12 cells without affecting TrkA or EGF receptor mRNAs expression. Moreover, the S-methylisothiourea (MIU), a potent inhibitor of inducible nitric oxide synthases, partially counteracts the NGF-mediated neuritin induction. These data suggest that NGF regulates neuritin expression in PC12 cells via the signaling pathway triggered by NO. This study reports the first evidence that neuritin plays a role in modulating neurite outgrowth during the progression of NGF-induced differentiation of PC12 cells. PC12 cells could be considered a valuable model to unravel the mechanism of action of neuritin on neurite outgrowth.