α2 isoform of Na+,K+-ATPase via Na+,Ca2+ exchanger modulates myelin basic protein synthesis in oligodendrocyte lineage cells in vitro

Oligodendrocytes in the CNS myelinate neuronal axons, facilitating rapid propagation of action potentials. Myelin basic protein (MBP) is an essential component of myelin and its absence results in severe hypomyelination. In oligodendrocyte lineage cell (OLC) monocultures MBP synthesis starts at DIV4. Ouabain (10 nM), a Na+,K+-ATPase (NKA) blocker, stimulates MBP synthesis. As OLCs express the alpha 2 isoform of NKA (alpha 2-NKA) that has a high affinity for ouabain, we hypothesized that alpha 2-NKA mediates this effect. Knockdown of alpha 2-NKA with small interfering (si)RNA (alpha 2-siRNA) significantly potentiated MBP synthesis at DIV4 and 5. This effect was completely blocked by KB-R7943 (1 mu M), a Na+,Ca2+ exchanger (NCX) antagonist. alpha 2-NKA ablation increased the frequency of NCX-mediated spontaneous Ca2+ transients ([Ca2+]t) at DIV4, whereas in control OLC cultures comparable frequency of [Ca2+]t was observed at DIV5. At DIV6 almost no [Ca2+]t were observed either in control or in alpha 2-siRNA-treated cultures. Immunocytochemical analyses showed that alpha 2-NKA co-localizes with MBP in proximal processes of immature OLCs but is only weakly present in MBP-enriched membrane sheets. Knockdown of alpha 2-NKA in cortical slice cultures did not change MBP levels but reduced co-localization of neurofilament- and MBP-positive compartments. We conclude that alpha 2-NKA activity in OLCs affects NCXmediated [Ca2+]t and the onset of MBP synthesis. We suggest therefore that neuronal activity, presumably in form of local extracellular [K+] changes, might locally influence NCX-mediated [Ca2+]t in OLC processes thus triggering local MBP synthesis in the vicinity of an active axon.