Activation of sodium-dependent glutamate transporters regulates the morphological aspects of oligodendrocyte maturation via signaling through calcium/calmodulin-dependent kinase IIβ's actin-binding/-stabilizing domain

Abstract

Signaling via the major excitatory amino acid glutamate has been implicated in the regulation of various aspects of the biology of oligodendrocytes, the myelinating cells of the central nervous system (CNS). In this respect, cells of the oligodendrocyte lineage have been described to express a variety of glutamate-responsive transmembrane proteins including sodium-dependent glutamate transporters. The latter have been well characterized to mediate glutamate clearance from the extracellular space. However, there is increasing evidence that they also mediate glutamate-induced intracellular signaling events. Our data presented here show that the activation of oligodendrocyte expressed sodium-dependent glutamate transporters, in particular GLT-1 and GLAST, promotes the morphological aspects of oligodendrocyte maturation. This effect was found to be associated with a transient increase in intracellular calcium levels and a transient phosphorylation event at the serine (S)(371) site of the calcium sensor calcium/calmodulin-dependent kinase type IIβ (CaMKIIβ). The potential regulatory S(371) site is located within CaMKIIβ's previously defined actin-binding/-stabilizing domain, and phosphorylation events within this domain were identified in our studies as a requirement for sodium-dependent glutamate transporter-mediated promotion of oligodendrocyte maturation. Furthermore, our data provide good evidence for a role of these phosphorylation events in mediating detachment of CaMKIIβ from filamentous (F)-actin, and hence allowing a remodeling of the oligodendrocyte's actin cytoskeleton. Taken together with our recent findings, which demonstrated a crucial role of CaMKIIβ in regulating CNS myelination in vivo, our data strongly suggest that a sodium-dependent glutamate transporter-CaMKIIβ-actin cytoskeleton axis plays an important role in the regulation of oligodendrocyte maturation and CNS myelination.

Keywords: actin cytoskeleton; calcium signaling; central nervous system; differentiation; myelin.

FIGURE 1

Sodium-dependent glutamate transporters are expressed in differentiating oligodendrocytes. A: Bar graph representing sodium-dependent glutamate transporter mRNA levels as determined by qRT-PCR analysis. Total glutamate transporter mRNA levels (Glas t+Glt-1+Eaac1) were set to 100% and the values for the individual gene-specific mRNA levels were adjusted accordingly. Data represent means ± SEM (n = 3 independent experiments, ***p≤0.001, ANOVA). B: Representative Western blots depicting sodium-dependent glutamate transporter protein expression. Numbers to the left indicate molecular weights in kDa. C: Representative images of differentiating oligodendrocytes double-immunostained using anti-GLAST, -GLT-1, or -EAAC1 antibodies in combination with O4 hybridoma supernatants. Scale bars: 20 μm.

FIGURE 2

Activation of sodium-dependent glutamate transporters promotes the morphological aspects of oligodendrocyte differentiation. A-F: Differentiating oligodendrocytes were treated for 6h (unless noted otherwise) as indicated: control (Ctrl), L-glutamate (Glu, 100 μM), a non-transportable inhibitor of sodium-dependent glutamate transport (TBOA, 100 μM), D-aspartate (Asp, 100 μM). A: Representative images of differentiating oligodendrocytes immunostained using O4 hybridoma supernatants. Scale bars: 20 μm. B-D: Bar graphs representing quantitative analyses of oligodendrocyte network areas (Dennis et al. 2008). Data represent means ± SEM (***p≤0.001 compared to control, ANOVA). E: Representative images of differentiating oligodendrocytes stained with an antibody specific for myelin basic protein (MBP) as well as with Hoechst 33342 (Hoechst) to visualize nuclei. Scale bars: 100 μm. F: Bar graph depicting the number of MBP immunopositive cells normalized to the number of Hoechst-positive nuclei. Data represent means ± SEM. ANOVA revealed no statistically significant difference (p≤0.05). G: Bar graph depicting oligodendrocyte network areas upon siRNA-mediated knock-down of individual sodium-dependent glutamate transporters (as indicated) and subsequent treatment with L-glutamate (Glu, 100 μM). Data represent means ± SEM (***p≤0.001 compared to siCtrl non-treated, ANOVA).

FIGURE 3

The activation of sodium-dependent glutamate transporters increases intracellular calcium levels in the processes of early stage differentiating oligodendrocytes. A: Representative pseudo-colored images of fura-2 AM fluorescence ratio measurements upon treatment with D-aspartate (Asp). The bar to the left represents a relative color scale indicating low (L) and high (H) calcium levels. B-C: Time course of changes in free intracellular calcium concentrations [Ca²⁺] upon different treatments as indicated in the inset shown in the upper right. Start of treatment is indicated by the arrow. The graphs represent means ± SEM. D: Bar graph depicting a quantitative analysis of free intracellular calcium concentrations [Ca²⁺] at the time-point of highest response to D-aspartate. The mean value for D-aspartate treated cells (+ Asp) was set to 100% and the remaining values were calculated accordingly. Treatments are indicated along the x-axis. Data represent means ± SEM (***p≤0.001 compared to control (untreated), ^###p≤0.001 and ^##p≤0.01 compared to ‘+ Asp’, ANOVA).

FIGURE 4

Activation of sodium-dependent glutamate transporters leads to a transient phosphorylation event at CaMKIIβ’s S³⁷¹ site. A: Bar graph depicting a quantitative analysis of the oligodendrocyte network area (Dennis et al. 2008). Cells were pre-treated with the pharmacological CaMKII inhibitor KN-93 or its inactive derivative KN-92 and then incubated in the absence or presence (+Glu) of 100 μM L-glutamate. The mean values for control cells (pre-treated with KN-92 and incubated in the absence of L-glutamate) were set to 100% and experimental values were calculated accordingly. Data represent means ± SEM (***p≤0.001 compared to control, ANOVA). The inset (upper right) depicts representative images of differentiating oligodendrocytes treated with KN-92 plus L-glutamate (left) or KN-93 plus L-glutamate (right) and immunostained using O4 hybridoma supernatants. Scale bars: 5 μm. B, inset in H: Representative Western blots depicting CaMKII phosphorylation (pCaMKIIβ S³⁷¹, pCaMKII T^286/7) or total CaMKIIβ protein levels. GAPDH protein levels are shown representatively for the Western blot for which anti-pCaMKII T^286/7 (B) or anti-pCaMKIIβ S³⁷¹ (inset in H) antibodies were used. C-H: Bar graphs depicting the levels of pCaMKIIβ S³⁷¹ (C,F- H), total CaMKIIβ (D) or pCaMKII T^286/7 (E) at different time-points after addition of L-glutamate (Glu) (C-F), at the time-point of 60 min after addition of L-glutamate and prior pre-treatment with or without TBOA (G) or after transfection with siRNA pools as indicated and L-glutamate treatment for 60 min (H). All CaMKII protein levels were normalized to GAPDH protein levels. The mean normalized values for control (non-treated) cells were set to 100% (horizontal line) and experimental values were calculated accordingly. Data represent means ± SEM of 3 independent experiments (***p≤0.001, *p≤0.05 compared to control, ANOVA).

FIGURE 5

Phosphorylation events within CaMKIIβ’s actin binding/stabilizing domain regulate the association of CaMKIIβ with filamentous (F)-actin. A: Representative images of CIMO cells nucleofected with plasmids encoding eGFP fusion proteins of CaMKIIβ (WT or mutant form as indicated) and stained for F-actin (phalloidin). Scale bars: 5 μm. B: Bar graph depicting the weighted colocalization coefficients for eGFP fusion proteins and phalloidin. Data represent means ± SEM (***p≤0.001 compared to eGFP, ANOVA).

FIGURE 6

Phosphorylation events within CaMKIIβ’s actin binding/stabilizing domain regulate the glutamate-mediated promotion of the morphological aspects of oligodendrocyte maturation. A: Representative images of differentiating oligodendrocytes co-nucleofected with plasmids encoding an eGFP fusion protein of CaMKIIβ (WT or mutant form as indicated) and Lifeact-mRuby. Arrows point toward F-actin-enriched regions along cellular processes as visualized via Lifeact-mRuby fluorescence. Scale bars: 20 μm. B: Bar graph depicting a quantitative analysis of the oligodendrocyte network area (Dennis et al. 2008). Cells were nucleofected as indicated and then incubated in the absence or presence (+Glu) of 100 μM L-glutamate. The mean values for control cells (nucleofected with an eGFP encoding plasmid and incubated in the absence of L-glutamate) were set to 100% and experimental values were calculated accordingly. Data represent means ± SEM (***p≤0.001 compared to control, ANOVA).

FIGURE 7

Proposed model for the role of a sodium-dependent glutamate transporter-CaMKIIβ-actin cytoskeleton axis in the regulation of oligodendrocyte maturation. A: L-glutamate (Glu) stimulates sodium-dependent glutamate transporter activity, which in turn leads to an increase in intracellular sodium (Na⁺) levels, activation of the reverse mode of the sodium/calcium exchanger and a transient increase in intracellular calcium (Ca²⁺) levels. B: The transient increase in intracellular calcium levels leads to phosphorylation events within CaMKIIβ’s actin binding/stabilizing domain (pCaMKIIβ^S371), detachment of CaMKIIβ from filamentous (F)-actin and the opening of a time window during which cytoskeletal rearrangements and morphological remodeling can occur. C: Upon de-activation (de-phosphorylation), CaMKIIβ binds to F-actin and thereby stabilizes the newly arranged cytoskeleton. Such cycles of activation and de-activation of CaMKIIβ’s actin binding activity allow re-organization of the actin cytoskeleton while at the same time preventing its uncontrolled disintegration. adapted from Okamoto et al. (2009).