Neurotrophin-3 (NT-3) modulates early differentiation of oligodendrocytes in rat brain cortical cultures

Multiple extracellular signals are required for oligodendroglia survival, p roliferation and differentiation, and increasing evidence has accumulated t hat also neurotrophins regulate glial cell development in the central nervo us system (CNS). In the present study we have investigated the influence of neurotrophin-3 (NT-3) on the in vitro differentiation and proliferation of oligodendrocytes prepared from the brains of newborn rats. Cells were grow n in chemically defined growth medium, in the absence of fetal calf serum ( FCS). RT-PCR analysis confirmed the expression of mRNA encoding the NT-3 re ceptor trkC in oligodendrocytes throughout in vitro development. Cell morph ology was observed by phase contrast microscopy and indirect immunofluoresc ence staining using anti-galactocerebroside (GalC) antibodies. An increase in process formation and arborization was observed 8-24 h after the treatme nt with NT-3 (5-50 ng/ml). Concomitantly, NT-3 caused an increase in the ap pearance of GalC-positive cells. Long-term treatment with NT-3 (up to seven days) did not yield any further improvement of process formation. To eluci date the molecular mechanisms and signal transduction pathways underlying t he effect of NT-3 in oligodendrocytes, the time- and concentration-dependen t effect of NT-3 on c-Fos protein expression was studied by Western blot an alysis. The data show that NT-3 stimulated the appearance of two c-Fos immu noreactive polypeptides with apparent molecular weights of 62 and 55 kDa, r espectively. This effect was maximal at a concentration of 50 ng/ml of NT-3 after 8-24 h. NT-3-modulated morphological differentiation and c-Fos prote in expression was regulated by protein kinases. Whereas the protein kinase C (PKC) inhibitors staurosporine and chelerythrine chloride had a stimulato ry effect on NT-3-promoted process formation, the tyrosine kinase inhibitor genistein had an inhibitory effect and mainly cells with a bipolar and imm ature morphology were observable. The inhibition of tyrosine kinase activit y prevented NT-S-promoted induction of c-Fos protein. Thus, in addition to its mitogenic effects, NT-3 during early time points influences the in vitr o differentiation of oligodendrocytes. This process involves the induction of c-Fos protein and is mediated by PKC and trosine kinase activities. (C) 1999 Wiley-Liss, Inc.