Involvement of the Na+/Ca2+exchanger isoform 1 (NCX1) in Neuronal Growth Factor (NGF)-induced neuronal differentiation through Ca2+-dependent Akt phosphorylation∗

Abstract

NGF induces neuronal differentiation by modulating [Ca2+]i. However, the role of the three isoforms of the main Ca2+-extruding system, the Na+/Ca2+exchanger (NCX), in NGF-induced differentiation remains unexplored. We investigated whether NCX1, NCX2, andNCX3isoforms could play a relevant role in neuronal differentiation through the modulation of [Ca2+]iand the Akt pathway. NGF caused progressive neurite elongation; a significant increase of the well known marker of growth cones, GAP-43; and an enhancement of endoplasmic reticulum (ER) Ca2+content and of Akt phosphorylation through an early activation of ERK1/2. Interestingly, during NGF-induced differentiation, theNCX1protein level increased, NCX3 decreased, and NCX2 remained unaffected. At the same time, NCX total activity increased. Moreover, NCX1 colocalized and coimmunoprecipitated with GAP-43, and NCX1 silencing prevented NGF-induced effects on GAP-43 expression, Akt phosphorylation, and neurite outgrowth.Onthe other hand, the overexpression of its neuronal splicing isoform, NCX1.4, even in the absence of NGF, induced an increase in Akt phosphorylation and GAP-43 protein expression. Interestingly, tetrodotoxin-sensitive Na+currents and 1,3-benzenedicarboxylic acid, 4,4′-[1,4,10-trioxa-7,13-diazacyclopentadecane-7,13-diylbis(5-methoxy-6,12-benzofurandiyl)]bis-, tetrakis[(acetyloxy)methyl] ester-detected [Na+]isignificantly increased in cells overexpressing NCX1.4 as well as ER Ca2+content. This latter effect was prevented by tetrodotoxin. Furthermore, either the [Ca2+]ichelator (1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid) (BAPTA-AM) or the PI3K inhibitor LY 294002 prevented Akt phosphorylation and GAP-43 protein expression rise in NCX1.4 overexpressing cells. Moreover, in primary cortical neurons, NCX1 silencing prevented Akt phosphorylation, GAP-43 and MAP2 overexpression, and neurite elongation. Collectively, these data show that NCX1 participates in neuronal differentiation through the modulation of ER Ca2+content and PI3K signaling.