Myelin-mediated inhibition of oligodendrocyte precursor differentiation can be overcome by pharmacological modulation of Fyn-RhoA and protein kinase C signalling

Abstract

Failure of oligodendrocyte precursor cell (OPC) differentiation contributes significantly to failed myelin sheath regeneration (remyelination) in chronic demyelinating diseases. Although the reasons for this failure are not completely understood, several lines of evidence point to factors present following demyelination that specifically inhibit differentiation of cells capable of generating remyelinating oligodendrocytes. We have previously demonstrated that myelin debris generated by demyelination inhibits remyelination by inhibiting OPC differentiation and that the inhibitory effects are associated with myelin proteins. In the present study, we narrow down the spectrum of potential protein candidates by proteomic analysis of inhibitory protein fractions prepared by CM and HighQ column chromatography followed by BN/SDS/SDS-PAGE gel separation using Nano-HPLC-ESI-Q-TOF mass spectrometry. We show that the inhibitory effects on OPC differentiation mediated by myelin are regulated by Fyn-RhoA-ROCK signalling as well as by modulation of protein kinase C (PKC) signalling. We demonstrate that pharmacological or siRNA-mediated inhibition of RhoA-ROCK-II and/or PKC signalling can induce OPC differentiation in the presence of myelin. Our results, which provide a mechanistic link between myelin, a mediator of OPC differentiation inhibition associated with demyelinating pathologies and specific signalling pathways amenable to pharmacological manipulation, are therefore of significant potential value for future strategies aimed at enhancing CNS remyelination.

Fig. 1

OPCs plated on MPEs (protein/cm²) display a concentration dependent down-regulation of relative MBP mRNA expression as assessed by qPCR after 3d culture in differentiation medium (for this and subsequent figures: MPE: myelin protein extract; error bars: SEM).

Fig. 2

(A) Myelin inhibitors impair Fyn-1/Y-418 phosphorylation in OPCs, (B) immunoblot following anti-Fyn-1-immunoprecipitation and loading control. (C) Myelin inhibitors also induce activation of RhoA. (D) Autoradiograph of Rho assay (MPE: 40 µg/cm²; PLL: poly-l-lysine; incubation in differentiation medium for 24 h).

Fig. 3

(A) Immunoblot of OPC lysates demonstrates that differentiating OPCs express ROCK-II. (B) Inhibition of ROCK-II by culturing OPCs on MPE with Fasudil (HA-1077) results in a more than 160% increase of differentiating cells after 48 h culture in differentiation medium. (C) Whereas OPCs on MPE down-regulate O4 immunoreactivity. (D) O4 expression is largely restored by treatment with HA-1077 (scale bar = 30 µM).

Fig. 4

(A–F) MARCKS is a down-stream effector of PKC. While in control cells MARCKS expression is associated with the cell membrane, myelin inhibitors lead to a cytoplasmatic MARCKS presence. (G–N) Inhibition of PKC signaling in OPCs on MPE with BIM (G–J; max. mean increase 169%) or Gö6976 (K–N; max. mean increase 269%) strongly induces OPC differentiation after 48 h incubation in differentiation medium. (Scalebar: in A–C: 15.9 µm, in D–F: 14.6 µm in H–J, L–N: 30 µm).

Fig. 5

(A and B) Transfection efficiency of OPCs was monitored by transfecting cells with RNAi (cy-3) and assessing the proportion of cy-3-labelled cells after 48 h in differentiation medium (n = 3); in all experiments >95% of the cells were cy-3 positive. Immunoblots of OPC lysates after transfection with (C) RNAi for RhoA as well as (D) RNAi for PKC-α demonstrate downregulation of RhoA or PKC-α on protein level after 48 h. (D) Silencing of RhoA or PKC-α results in a significant increase of O4 positive cells when cultured for 48 h on MPE. (F) Whereas transfection with control RNAi (RNAi(scrambled, scr)) was not able to restore O4-immuroreactivity of OPCs on MPE (G and H) gene silencing induced a strong increase in the number of differentiating cells (scalebar = 30 µM).

Fig. 6

(A–C) Blocking PKC and ROCK-II simultaneously in OPCs plated on myelin by co-incubation with HA-1077 and Gö6976 induces an additional increase in the percentage of O4-positive cells as compared to single treatment after incubation in differentiation medium for 48 h. (B) While MPE induces down-regulation of O4 immunoreactivity in OPCs (C: the same cells stained for A2B5), (D) O4 expression is restored after treatment with the pharmacological inhibitors. (E–G) The presence of MPE is associated with a reduction of the complexity of OPC processes and earlier morphological stages as compared to OPCs plated on control substrate. (H) Treatment with HA-1077 and Gö6976 restores the presence of more mature phenotypes (scalebar = 30 µM).