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

Abstract

Multiple extracellular signals are required for oligodendroglia survival, proliferation and differentiation, and increasing evidence has accumulated that also neurotrophins regulate glial cell development in the central nervous 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 grown 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 receptor trkC in oligodendrocytes throughout in vitro development. Cell morphology was observed by phase contrast microscopy and indirect immunofluorescence staining using anti-galactocerebroside (GalC) antibodies. An increase in process formation and arborization was observed 8-24 h after the treatment with NT-3 (5-50 ng/ml). Concomitantly, NT-3 caused an increase in the appearance of GalC-positive cells. Long-term treatment with NT-3 (up to seven days) did not yield any further improvement of process formation. To elucidate the molecular mechanisms and signal transduction pathways underlying the effect of NT-3 in oligodendrocytes, the time- and concentration-dependent effect of NT-3 on c-Fos protein expression was studied by Western blot analysis. The data show that NT-3 stimulated the appearance of two c-Fos immunoreactive polypeptides with apparent molecular weights of 62 and 55 kDa, respectively. 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 protein expression was regulated by protein kinases. Whereas the protein kinase C (PKC) inhibitors staurosporine and chelerythrine chloride had a stimulatory effect on NT-3-promoted process formation, the tyrosine kinase inhibitor genistein had an inhibitory effect and mainly cells with a bipolar and immature morphology were observable. The inhibition of tyrosine kinase activity prevented NT-3-promoted induction of c-Fos protein. Thus, in addition to its mitogenic effects, NT-3 during early time points influences the in vitro differentiation of oligodendrocytes. This process involves the induction of c-Fos protein and is mediated by PKC and trosine kinase activities.