Sinalização da MAPK/ERK na diferenciaçãao da oligodendroglia: efeitos de inibidores da MEK sobre a morfologia e distribuição de proteínas de oligodendrócitos/mielina in vitro

Abstract

The mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway is important for both long-term survival and timing of the progression of oligodendrocyte differentiation. In this work, the MAPK/ERK signaling in oligodendroglia was studied in vitro by using MEK inhibitors. Cell morphology and distribution of proteins were analyzed in different stages of maturation. Primary cultures of oligodendroglia were treated with the MEK inhibitors PD98059 or U0126, at 5 or 11div for 30min, 24 or 48h. Oligodendroglial cells were distinguished by using stage specific markers: NG2 proteoglycan, A2B5, 2´3´nucleotide-cyclic 3´phosphodiesterase (CNPase) and myelin basic protein (MBP), and classified according to their morphology into different developmental stages. Treatment significantly increased the number of cells with more immature morphologies and decreased the number of mature cells. Furthermore, it increased the number of rounded cells that could not be classified into any of the oligodendroglial developmental stages. The strongest effects were usually observed shortly after treatment. Rounded cells were CNPase/MBP positive and they were not stained by anti-NG2 or A2B5, indicating that they were mature cells unable either to extend and/or to maintain their processes. In fact, these changes were accompanied by alterations in the distribution of the oligodendroglial proteins MBP and CNPase, and alterations in cytoskeleton proteins, as actin, tubulin and the focal adhesion kinase (FAK). MBP was observed in a continuous distribution in cell body and processes in control cultures. Furthermore, in treated cultures a disorganized pattern of distribution of CNPase, actin and tubulin was observed. In control cultures, these proteins compose the cytoskeleton vein-like structures. By the other side, after a short time of MEK inhibition (30min), actin and tubulin showed the same punctual pattern observed in CNPase distribution. Treatment also caused a reduction of focal adhesion sites showed by FAK. As treatment progressed, after 24 and 48h, actin and tubulin seemed to be rearranged into a filament-like pattern. These data showed an effect of the MAPK/ERK pathway on oligodendroglial branching, with possible consequences for the formation of the myelin sheath.