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 plasmidexpressing neuritin display a significant increase in the response to NGF: 1) in the levels of SMI312 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 NGFmediated 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 NGFinduced differentiation of PC12 cells. PC12 cells could be considered a valuable model to unravel the mechanism of action of neuritin on neurite outgrowth.