A modulação da via do AMPc/PKA altera a morfologia da oligodendroglia e a distribuição das proteínas CNPase e MAG in vitro

Abstract

Oligodendroglial differentiation depends on coordinated changes in the cytoskeleton and on its relationship with the plasmatic membrane, a critical site regarding the formation of the myelin sheath. 2´3´cyclic nucleotide 3´ phosphodiesterase (CNPase) is related to cytoskeleton modulation, anchoring microtubules to the plasmatic membrane. In vitro, CNPase composes, together with F-actin and microtubules, the vein-like structures or radial components in myelin sheath. Myelin associated glycoprotein (MAG) is important to membrane vellum formation and is associated with CNPase and tubulin. Besides, the AMPc/PKA pathway can regulate these three proteins. In order to evaluate the effects of the cAMP/PKA pathway modulation on oligodendroglial differentiation, cultures of cerebral hemispheres were treated for 30 min or 24 h with the adenylyl cyclase inhibitor SQ22536 SQ [1 lM] or with its activator forskolin [10 lM], or with the PKA inhibitor H-89 [1 lM]. Oligodendroglia was identified using anti-CNPase antibody as also by morphology. At 30 min, the cells treated with forskolin showed bigger processes and a shorter membrane vellum when compared to control cultures, the treatment with SQ caused an increase in the processes length, the H-89 treatment reduced the processes length and the membrane vellum. At 24 h, cultures treated with forskolin showed few processes when compared to control cultures. Cultures treated with SQ showed an increase in the processes length and after H89 treatment oligodendroglial cells presented a reduction in the membrane vellum. We also observed alterations in the distribution of CNPase, tubulin and MAG, the first showed an increase in the distribution closest to nucleus in both periods of H-89 treatment, the same pattern occurred for tubulin. The CNPase acquired a punctiform pattern after 24 hours of treatment with both inhibitors. The MAG also showed an increase closest to nucleus after 30 minutes of treatment with forskolin and SQ. The SQ treatment also caused a reduction in the protein distribution to membrane vellum. The same reduction is observed after 24 hours of treatment with H-89.These findings reinforce a role for AMPc/PKA pathway in oligodendroglial differentiation including the processes extension and arborization as also the formation of membranar vellum, with possible consequences in the formation and maintenance of myelin sheath.