MLCK regulates Schwann cell cytoskeletal organization, differentiation and myelination

Abstract

Signaling through cyclic AMP (cAMP) has been implicated in the regulation of Schwann cell (SC) proliferation and differentiation. In quiescent SCs, elevation of cAMP promotes the expression of proteins associated with myelination such as Krox-20 and P0, and downregulation of markers associated with the non-myelinating SC phenotype. We have previously shown that the motor protein myosin II is required for the establishment of normal SC-axon interactions, differentiation and myelination, however, the mechanisms behind these effects are unknown. Here we report that the levels and activity of myosin light chain kinase (MLCK), an enzyme that regulates MLC phosphorylation in non-muscle cells, are dramatically downregulated in SCs after cAMP treatment, in a similar pattern to that of c-Jun, a known inhibitor of myelination. Knockdown of MLCK in SCs mimics the effect of cAMP elevation, inducing plasma membrane expansion and expression of Krox-20 and myelin proteins. Despite activation of myelin gene transcription these cells fail to make compact myelin when placed in contact with axons. Our data indicate that myosin II activity is differentially regulated at various stages during myelination and that in the absence of MLCK the processes of SC differentiation and compact myelin assembly are uncoupled.

Fig. 1.

Expression of MLCK in SCs in vitro and in vivo. (A) Primary cultures of rat SCs demonstrate strong expression of MLCK (green) in the nucleus and perinuclear cytoplasm, and weaker staining in the membrane and stress fibers where it colocalizes with actin (red). Scale bars: 15 μm. (B) Western blots of SC lysates after subcellular fractionation, confirming enrichment of MLCK in the nuclear fraction (Nu). A weak MLCK signal was also detected in the cytoplasmic fraction (Cyt), but none was detected in either the membrane (Mb) or cytoskeletal fraction (Csk). N-cadherin, and Krox-20 and c-Jun were used as markers for the membrane and nuclear fractions, respectively. (C) SC–neuron cocultures maintained in either pre-myelinating or myelinating conditions, stained with antibodies to MLCK (red), neurofilament (blue) and MBP (green). Strong MLCK staining is seen in the nucleus of pre-myelinating SCs. The intensity of the staining decreases considerably in myelinating cultures. Weak axonal staining for MLCK is also apparent in these cultures. Scale bars: 25 μm. (D) Western blot of SCs, DRG and 14-day myelinating coculture (CC) showing strong MLCK expression in SCs and weaker expression of a lower molecular mass isoform in DRG (overexposed lower panel). In myelinating cocultures, MLCK expression is downregulated. (E) Frozen section of P7 rat sciatic nerve stained with antibodies to MLCK (red), Krox-20 (green) and MBP (blue). MLCK is present in axons and in the nucleus of non-myelinating SCs (arrowheads) but not in Krox-20⁺ myelinating SCs. Scale bar: 10 μm.

Fig. 2.

Elevation of cAMP downregulates MLCK expression and activates the myelination program. (A) Western blots of SC cultures maintained in defined medium (t=0) and stimulated with 1 mM db-cAMP for 0.5–72 hours. Treatment with cAMP upregulates the expression of pro-myelinating transcription factors Oct-6 and Krox-20 and myelin proteins such as MAG and P0, and downregulates the expression of c-Jun, MLCK and phosphorylated MLC. There is also an increase in MLCK phosphorylation. (B) SC cultures were maintained in defined medium and stimulated for 48 hours with 1 mM db-cAMP. A striking decrease in MLCK nuclear staining (red) is observed after cAMP treatment, concomitant with an increase in Krox-20 expression (green). (C) Detail of the changes in the actin cytoskeleton organization after a 30-minute treatment with 1 mM db-cAMP. Scale bars: 10 μm. (D) SC cultures stained with antibodies to P0 (green), phalloidin-TRITC (red) and c-Jun (blue) before and after treatment with 1 mM db-cAMP for 72 hours. Loss of stress fibers and considerable cell expansion occurs in stimulated SCs together with upregulation of P0 expression and downregulation of c-Jun. Scale bars: 25 μm.

Fig. 3.

Knockdown of MLCK in SCs activates the myelination program. (A) Primary cultures of rat SCs were infected with lentiviral constructs expressing non-targeting shRNA (shCTRL) or shRNA against MLCK (shMLCK). Upper panels: phase-contrast images showing striking morphological differences between these cultures. Lower panels: a dramatic upregulation of P0 expression (green) was observed in SC cultures treated with shMLCK compared with controls (shCTRL). Scale bars: 25 μm. (B) Western blots of SC cultures showing reduction of MLCK and MLC-P (pMLC) levels as well as activation of myelination program in SCs treated with shMLCK compared with shCTRL. (C) Western blots of SC cultures infected with non-targeting shRNA (shCTRL) or shRNA against myosin phosphatase 1 (shMYPT1). Increased levels of MLC-P are seen in shMYPT1-treated SCs, confirming knockdown of the phosphatase. No changes in P0 levels are observed whereas levels of Krox-20, c-Jun and p27^Kip are decreased compared with control cultures. (D) SCs cultures showing increased level of MLC-P (green) and robust stress fiber formation in cultures treated with shMYPT1 compared with controls (shCTRL). Scale bars: 25 μm. (E) Western blots of control- and shMLCK-treated SC cultures before (−) and after (+) stimulation with 1 mM db-cAMP for 24 hours. In MYPT1-knockdown cultures, MLC-P levels remain high, although downregulation of MLCK and c-Jun and upregulation of Oct-6 after cAMP treatment appear unaffected; upregulation of Krox-20 was reduced compared with control cultures. (F) Immunofluorescence of SCs after 24 hours of 1 mM db-cAMP stimulation, showing a reverse correlation between the expression of P0 and the presence of high levels of MLC-P. Cells with high levels of MLC-P exhibit abundant stress fibers and lack P0 expression (upper panel); whereas cells expressing high levels of P0 lack stress fibers and have reduced expression of MLC-P (lower panel). Scale bars: 20 μm.

Fig. 4.

Knockdown of MLCK in SCs does not prevent initial SCs–axon association. SCs infected with lentiviral constructs expressing non-targeting shRNA (shCTRL) or shRNA against MLCK (shMLCK) were seeded onto purified DRG neurons and allowed to proliferate for 3 days. Cultures were stained for laminin (red), c-Jun (green) and neurofilament (NF; blue). SCs (red) infected with shMLCK elaborate very thin and long processes, but they appear to contact and extend along the axons (blue). Expression of c-Jun is downregulated in shMLCK cultures compared with controls. Scale bars: 50 μm.

Fig. 5.

Knockdown of MLCK in SCs inhibits myelination. (A) SCs infected with lentivirus constructs expressing non-targeting shRNA (shCTRL) or shRNA against MLCK (shMLCK) were seeded onto purified DRG neurons and allowed to myelinate for 18 days. Cultures were stained for MBP (blue), laminin (red) and MAG (green). A considerable reduction in the amount of MBP⁺ segments is observed in cultures established with shMLCK SCs compared with controls. Despite the lack of MBP, staining for laminin shows the presence of basal lamina and in some cases expression of MAG. Scale bars: 50 μm. (B) Quantification of MBP⁺ segments per field. Values are means ± s.e.m. of three independent experiments (three cultures per condition per experiment). (C) Western blots of myelinating cocultures showing a marked reduction on the levels of myelin proteins in shMLCK-treated cocultures. Levels of P0 and Krox-20 were reduced to a lesser extent whereas the expression of laminin chains was non-affected by shMLCK-treatment. (D) Detail of a myelinating coculture (14 days) showing an example of P0 expression (green) in the absence of MBP (blue) in shMLCK cultures. Basal lamina assembly (red) appears normal. (E) Myelinating cocultures (14 days) stained for Krox-20 and MBP. Despite the absence of myelin segments in shMLCK cultures Krox-20 upregulation can be detected in the nucleus of many MBP-negative SCs. Scale bars: 50 μm.

Fig. 6.

Real-time PCR of myelinating cocultures. SCs infected with non-targeting shRNA (shCTRL) or shRNA against MLCK (shMLCK), were used to establish myelinating cocultures. RNA was isolated from a total of 12 cultures at the indicated time points and the relative increase of myelin protein mRNAs (P0, MBP and MAG), as well as Krox-20 and laminin mRNAs was measured by quantitative RT-PCR. Relative mRNA levels for each protein, normalized to actin (or L19), were calculated as indicated in Material and Methods. A significant increase in the relative amount of mRNA is observed for all proteins after 14 days (d) in coculture, reaching a plateau by 21 days. In shMLCK cultures (filled circles) the relative increase of mRNA is significantly higher (***P<0.001; **P<0.01, two-way ANOVA, Bonferroni post-test) than that of control cultures (open squares). The only exception was for the relative levels of MBP mRNA at 14 days, which was higher in control cultures.

Fig. 7.

Increased protein degradation and accumulation in MLCK-treated cocultures. (A) Lysates from shCTRL (C) and shMLCK-treated (T) cocultures were prepared at different days after the induction of myelination (4, 14 and 21 days; d) and probed with antibodies to LCB3 and LAMP1. The expression of both markers was increased in shMLCK cultures compared with controls. (B) SCs infected with lentivirus constructs expressing non-targeting shRNA (shCTRL) or shRNA against MLCK (shMLCK) were seeded onto purified DRG neurons and allowed to myelinate for 14 days. Cocultures were fixed and stained for Krox-20 (red), transferring receptor (green) and neurofilament (blue). Scale bars: 25 μm. (C) Quantification of transferrin staining area in cocultures. Values are means ± s.e.m. from two cultures. A total of 234 cells (128 shCTRL) and (106 shMLCK) were evaluated.

Fig. 8.

Ultrastructural analysis of myelinating cocultures. (A) Longitudinal section of a myelinated axon in an 18-day-old control coculture. (B) Unevenly myelinated axon in an18-day-old shMLCK culture. Arrowheads indicate aberrant compaction on one side of the internode. Asterisks mark two unmyelinated axons. (C) Detail of an abnormally enlarged rough endoplasmic reticulum (rER) in a non-myelinating shMLCK SC. The swollen and prominent lumen (L) of the rER and attached ribosomes (arrowheads) are visible. N denotes the nucleus of the SC. A non-myelinated axon (asterisk) is indicated. (D) Cross section of two axons segregated in a 1:1 association in control cultures. (E) Example of incomplete sorting by SCs in shMLCK cultures. Many small (black asterisks) and large (red asterisks) caliber axons are contained within the cytoplasm of this cell and are not properly segregated. Prominent ER (arrowheads) and SC nucleus (N) are also visible. Scale bars: 500 nm. (F) Percentage of myelinated, segregated or non-segregated axons in shCTRL and shMLCK cultures. Values are means ± s.e.m. from four cultures (two per condition). A total of 645 axons were counted (318 from shCTRL; 327 from shMLCK).

Fig. 9.

A model of changes in non-muscle myosin II activity during SC development. In immature SCs, the activity of myosin II is higher than in mature differentiated cells (Melendez-Vasquez et al., 2004; Wang et al., 2008). Myosin II activity is required for radial sorting of axons and this process is impaired by knockdown of shMLCK (Wang et al., 2008) (this study). Once the SC has achieved a 1:1 association with the axon (pro-myelinating stage), the activation of myelin gene transcription and membrane expansion is correlated with a marked downregulation of myosin II activity, a process that can be recapitulated in SCs cultures by knockdown of MLCK and/or treatment with cAMP (this study). Later morphogenetic events, such as the wrapping of compact myelin around the axons, requires coordination between the synthesis of myelin proteins and their trafficking to the plasma membrane, processes in which myosin II and MLCK also appear to play a role (Melendez-Vasquez et al., 2004) (this study). Orange ovals, SC nuclei; blue circles, axons; red lines, inhibition; blue arrow, activation.