Re-expression of a developmentally restricted potassium channel in autoimmune demyelination: Kv1.4 is implicated in oligodendroglial proliferation

Abstract

Mechanisms of lesion repair in multiple sclerosis are incompletely understood. To some degree, remyelination can occur, associated with an increase of proliferating oligodendroglial cells. Recently, the expression of potassium channels has been implicated in the control of oligodendrocyte precursor cell proliferation in vitro. We investigated the expression of Kv1.4 potassium channels in myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis, a model of multiple sclerosis. Confocal microscopy revealed expression of Kv1.4 in AN2-positive oligodendrocyte precursor cells and premyelinating oligodendrocytes in vitro but neither in mature oligodendrocytes nor in the spinal cords of healthy adult mice. After induction of myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis, Kv1.4 immunoreactivity was detected in or around lesions already during disease onset with a peak early and a subsequent decrease in the late phase of the disease. Kv1.4 expression was confined to 2',3'-cyclic nucleotide 3'-phosphodiesterase-positive oligodendroglial cells, which were actively proliferating and ensheathed naked axons. After a demyelinating episode, the number of Kv1.4 and 2',3'-cyclic nucleotide 3'-phosphodiesterase double-positive cells was greatly reduced in ciliary neurotrophic factor knockout mice, a model with impaired lesion repair. In summary, the re-expression of an oligodendroglial potassium channel may have a functional implication on oligodendroglial cell cycle progression, thus influencing tissue repair in experimental autoimmune encephalomyelitis and multiple sclerosis.

Figure 1

Decrease of Kv1.4 potassium channels during OPC differentiation in vitro. A: Undifferentiated OPCs are positive for the progenitor marker AN2 (red). These OPCs express high levels of Kv1.4 (green) localized to the soma and short cellular processes. B: Two days after induction of morphological differentiation, Kv1.4 is present in cells simultaneously expressing AN2 and CNPase. C: Five days later, mature OLs display a complex branched structure expressing markers for mature oligodendrocytes and myelin proteins like myelin basic protein (red). At that time point, Kv1.4 immunoreactivity is reduced to a residual staining (green).

Figure 2

Re-expression of Kv1.4 during autoimmune demyelination. Representative images of longitudinal lumbar spinal cord sections are shown. Nuclei are stained in blue. A: No Kv1.4 expression is detected in the spinal cord of a healthy adult control mouse. B: During the early phase of MOG-EAE at day 13 p.i., a strong immunoreactivity for Kv1.4 (green) is seen in and around demyelinated lesions.

Figure 3

Kv1.4 mRNA expression is not regulated by proinflammatory stimuli. RT-PCR analysis of Kv1.4 expression (A) and transferrin receptor expression (B) as control. In comparison with naïve OPCs (○), Kv1.4 mRNA is not up-regulated after treatment with 4% supernatant of concanavalin A-stimulated spleen cells for 4 (•) or 8 hours (▪). Likewise, there is no difference in Kv1.4 mRNA expression between unstimulated but partly differentiated OPC (▵) and partly differentiated OPC after treatment with cytokine supernatant for 4 hours (▴).

Figure 4

CNPase-positive oligodendroglial cells express Kv1.4 over the course of MOG-EAE. Representative images of longitudinal lumbar spinal cord sections are shown. A: Just after disease onset (day 9 p.i.), some cells with a strong signal in the soma for CNPase and Kv1.4 are already present. B: The number of CNPase and Kv1.4 double-positive premyelinating oligodendroglial cells increases 13 days p.i. C and D: The number subsequently decreases in the later phases of the disease (days 26 and 40 p.i.).

Figure 5

Quantification of Kv1.4- and CNPase-positive cells in the lumbar spinal cord of EAE-diseased mice. Each bar represents the mean density of double-positive cells. CNPase and Kv1.4 double-positive cells are already present at the beginning (day 9 p.i.), peak at the maximum (day 13 p.i.), and subsequently decrease in the later phases of MOG-EAE (days 26 to 40 p.i.).

Figure 6

CNPase-positive oligodendroglial cells are actively proliferating. Double staining of MOG-EAE lesions (day 13 p.i.) for CNPase (B) and the proliferation marker Ki67 (A). C: Most of the cells that are Ki67-positive are also immunoreactive for CNPase (double-labeled cells marked with arrows).

Figure 7

Kv1.4-expressing cells in MOG-EAE lesions are actively proliferating (day 13 p.i.). Representative images from lumbar spinal cord are shown. A: High-resolution imaging after immunohistochemistry for Kv1.4 and Ki67 reveals colocalization of both markers (see Merged). B: Similar results are obtained after in vivo labeling of proliferating cells with 5-bromo-2′-deoxyuridine. Kv1.4-expressing cells are also 5-bromo-2′-deoxyuridine-positive (see Merged).

Figure 8

CNPase-positive oligodendroglial cells are associated with remyelination of naked axons expressing Nav1.2 sodium channels (A–D, red). A: At the onset of the disease (day 9 p.i), CNPase-expressing oligodendroglial cells (green, arrowheads) start to appear in the lesion. B: At the maximum of disease (day 13 p.i) contact Nav1.2-positive naked axons appear (arrowheads). C: In the later phase of MOG-EAE, CNPase-positive oligodendroglial cells surround and ensheathed demyelinated axons (arrowheads). D: Finally (day 40 p.i), irregular and thin myelin sheaths suggestive of remyelination can be seen (arrowheads). Representative sections from lumbar spinal cord are shown. D: Projection of 24 optical sections.

Figure 9

Investigation of Kv1.4 in CNTF^−/− mice. A: Quantification of Kv1.4 and CNPase double-positive cells during MOG-EAE in CNTF^−/− mice at the maximum of disease (day 14 p.i.). CNTF^−/− mice display a significantly lower number of double-positive cells (***P < 0.001). B: Quantification of apoptotic CNPase-positive oligodendroglial cells at the maximum of disease. At that time point, numbers of apoptotic OLs are not increased in CNTF^−/− mice.