Influence of Na+, K+-ATPase and Na+/Ca2+ exchanger on developmental ion signaling and MBP synthesis in murine oligodendrocyte precursor cells
Loading...
Files
Date issued
Authors
Editors
Journal Title
Journal ISSN
Volume Title
Publisher
Reuse License
Abstract
In the present thesis it was hypothesized that the expression of MBP could be altered by local [Na+]i and [Ca2+]i transients occurring in murine OPCs. In OPC monocultures MBP synthesis starts at around DIV4. Therefore, experiments in OPC monocultures at day in vitro (DIV) 2-6 were performed. Also transient elevations of resting [Ca2+]i and [Na+]i in OPCs were observed at DIV4-5. A similar, but slower increase of resting [Ca2+]i and [Na+]i was also observed in acute callosal brain slices. Chronic treatment of OPCs for 24 hours with culture medium containing elevated [K+] (+5 mM) decreased resting [Na+]i and enhanced MBP synthesis. At the same time, blocking the reverse mode of the Na+, Ca2+ exchanger (NCX) for 12 hours with KB-R7943 (1 µM) elevated resting [Na+]i and decreased MBP synthesis. OPC depolarization after 12 hours of chronic ouabain application (500 nM), a Na+, K+-ATPase (NKA) blocker, stimulated MBP synthesis as well. At single cell level NCX blockade, the elevation of extracellular [K+] and partial NKA inhibition led to [Na+]i transients in OPCs. The latter two, [K+]e elevations and NKA blockade resulted in [Ca2+]i oscillations. Those [Ca2+]i oscillations were blocked by application of KB-R7943 (1 µM), but not with Cd2+ (100 µM), indicating an involvement of the NCX reverse mode. Already small concentrations of ouabain (10 nM) successfully induced [Ca2+]i oscillations as well. Moreover it was demonstrated that cultured OPCs express the alpha2 isoform of NKA (α2-NKA) which has a high affinity for ouabain. Therefore it was hypothesized that the [Ca2+]i oscillations are mediated by α2-NKA. Similar to its blocking, knocking down the α2-NKA with small interfering (si)RNA (α2-siRNA) significantly potentiated MBP synthesis at DIV4 and 5. This potentiation was completely abolished by a chronic application of KB-R7943 (1 μM) for 24 hours. Additionally to the MBP potentiation, α2-NKA knockdown also increased the frequency of NCX-mediated spontaneous Ca2+ transients ([Ca2+]t) at DIV4, while in control cultures comparable frequency of [Ca2+]t was observed at DIV5. The [Ca2+]t were only observed in a narrow time window (DIV4-5), disappeared nearly completely at DIV6, in control as well as in α2 siRNA-treated cultures. However, knockdown of MBP did not show any influence on the α2-NKA expression, but the [Ca2+]t remained even at DIV6. A homogeneous distribution of α2-NKA was detected via immunocytochemical analyses and showed co-localization with MBP in proximal processes of immature OPCs. Later in cell development, the co-localization was only weakly present in MBP-enriched membrane sheets. In cortical organotypic slice cultures the knockdown of α2-NKA did not alter the levels of MBP but reduced co-localization of neurofilament- and MBP-positive compartments.
Summarizing the results, it is suggested that α2-NKA keeps the local membrane potential of the OPCs close to the reversal potential of NCX. A depolarization, caused by a neuronal activity dependent elevation of [K+]e in the vicinity, leads to a flip of the NCX into reverse mode, intruding Ca2+ into the cell. This Ca2+ influx initiates [Ca2+]t in OPC processes which seems to positively influence the local MBP expression.