The receptor for advanced glycation end products (RAGE) is a multiligand receptor protein thought to play an important role in neuronal differentiation. RAGE can bind a number of ligands and activate a variety of signalling pathways that lead to diverse downstream effects. Amphoterin and S100B are endogenous ligands, the interaction of which with RAGE is known to be involved in defined physiological processes. The present study investigated the spatiotemporal pattern of the expression for RAGE and its ligands, amphoterin and S100B, during neuronal differentiation of NT2/D1 cells. In this study, all three proteins were shown to increase with progression of neuronal differentiation as determined by Western blotting, raising the possibility that both amphoterin and S100B may interact with RAGE and have important functions during the process of cell differentiation. Moreover, blocking the activation of RAGE with neutralizing antibody in the presence of retinoic acid disrupted the progression of normal neuronal differentiation. Immunocytochemistry (ICC) studies showed that amphoterin partially colocalized with RAGE within differentiating NT2 cells, whereas S100B showed a high degree of colocalization. This result suggests that S100B is more likely to be the principal ligand for RAGE during the differentiation process and that RAGE and amphoterin might have both independent and combined roles. Moreover, RAGE was expressed only in cells that were committed to a neuronal phenotype, suggesting direct involvement of RAGE in mediating cellular changes within differentiating neuronal cells. Further detailed studies are now required to characterize fully the role of RAGE during the neuronal differentiation period.

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While connexins (Cxs) are thought to be involved in differentiation, their expression and role has yet to be fully elucidated. We investigated the temporal expression of Cx30, Cx36 and Cx43 in two in vitro models of neuronal differentiation: human NT2/D1 and murine P19 cells, and the spatial localisation of Cx30 and Cx43 in these models.A temporal Cx43 downregulation was confirmed in both cell lines during RA-induced neuronal differentiation using RT-PCR (P < 0.05) preceding an increase in neuronal doublecortin protein. RT-PCR showed Cx36 was upregulated twofold in NT2/D1 cells (P < 0.05) and sixfold in P19 cells (P < 0.001) during neuronal differentiation. Cx30 exhibited a transient peak in expression midway through the timecourse of differentiation increasing threefold in NT2/D1 cells (P < 0.001) and eightfold in P19 cells (P < 0.01).Qualitative immunocytochemistry was used to examine spatiotemporal patterns of Cx protein distribution alongside neuronal differentiation markers. The temporal immunolabelling pattern was similar to that seen using RT-PCR. Cx43 was observed intracellularly and on cell surfaces, while Cx30 was seen as puncta. Spatially Cx43 was seen on doublecortin-negative cells, which may indicate Cx43 downregulation is requisite for differentiation in these models. Conversely, Cx30 puncta were observed on doublecortin-positive and -negative cells in NT2/D1 cells and examination of the Cx30 peak showed puncta also localized to nestin-positive cells, with few puncta on MAP2-positive cells. In P19 cells Cx30 was localized on clusters of cells surrounded by MAP2- and doublecortin-positive processes. The expression pattern of Cx30 indicates a role in neuronal differentiation; the nature of that role warrants future investigation.