INDUCTION OF CHOLINERGIC DIFFERENTIATION WITH NEURITE SPROUTING BY DE-NOVO BIOSYNTHESIS AND EXPRESSION OF GD3 AND B-SERIES GANGLIOSIDES IN NEURO2A CELLS

The expression of a single glycosyltransferase, GD3 synthase, caused c holinergic differentiation with neu rite sprouting. The cells that exp ressed GD3 were established from Neuro2a cells by transfection of a ma mmalian expression vector into which were carried a cDNA encoding GD3 synthase and the blasticidin-S-deaminase gene with a SV40 promoter, fo llowed by selection with blasticidin-S-hydrochloride. The blasticidin- S-hydrochloride-resistant colonies derived from the cells transfected with the cDNA encoding GD3 synthase and the clonal cells (N2a-GD3) wer e spontaneously sprouting neurites but not those derived from cells tr ansfected with only the vector without the cDNA encoding GD3 synthase (N2a-bsr). GD3 expression by N2a-GD3 was confirmed by immunostaining o f the cells using the anti-GD3 monoclonal antibody, KM643. N2a-GD3 exp ressed not only GD3 but also GQ1b, one of the b-series ganglio sides, whereas N2a-bsr did not express these ganglio sides. Cell proliferatio n of N2a-GD3 was greatly reduced, as compared with that of N2a-bsr, an d, after several passages, it completely stopped. In addition, N2a-GD3 expressed acetylcholine esterase, indicating that the differentiation of Neuro2a cells was induced by expression of GD3 synthase and subseq uent modification of the biosynthesis and expression of gangliosides. These results strongly suggest that the de novo synthesis and expressi on of GD3 and/or b-series gangliosides induce neurite outgrowth and di fferentiation of Neuro2a cells. Exogenous GM1 stimulated the neuritoge nesis of N2a-bsr but not differentiated N2a-GD3, indicating that the m echanism of neurite sprouting in this system may be overlapped en rout e with that of exogenous GM1.