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During peripheral nervous system development, Schwann cells (SCs) surrounding single large axons differentiate into myelinating SCs. Previous studies implicate RhoGTPases in SC myelination, but the mechanisms involved in RhoGTPase regulation of SC myelination are unknown. Here, we show that SC myelination is arrested in Rac1 conditional knockout (Rac1-CKO) mice. Rac1 knockout abrogated phosphorylation of the effector p21-activated kinase (PAK) and decreased NF2/merlin phosphorylation. Mutation of NF2/merlin rescued the myelin deficit in Rac1-CKO mice in vivo, and the shortened processes in cultured Rac1-CKO SCs in vitro. Mechanistically, cyclic adenosine monophosphate (cAMP) levels and E-cadherin expression were decreased in the absence of Rac1, and both were restored by mutation of NF2/merlin. Reduced cAMP is a cause of the myelin deficiency in Rac1-CKO mice, as elevation of cAMP by rolipram in Rac1-CKO mice in vivo allowed myelin formation. Thus NF2/merlin and cAMP function downstream of Rac1 signaling in SC myelination, and cAMP levels control Rac1-regulated SC myelination.
SCs ensheath single large diameter axons to form myelin sheaths in the peripheral nervous system (Jessen and Mirsky 2005). These myelinating SCs electrically insulate axons for rapid impulse conduction (Bhatheja and Field 2006; Jessen and Mirsky 2005). Transcription factors necessary for myelination have been identified, and intracellular signaling pathways controlling SC myelination are being intensively analyzed. Several studies implicate the Rho family GTPase Rac1 in SC functions. Specifically, Rac1 is important for neurotrophin regulated SC migration (Yamauchi et al. 2005) and Rac1 mediates initial SC-axon interaction (Nakai et al. 2006). Rac1 has also been implicated in proper radial sorting and myelination (Benninger et al. 2007; Nodari et al. 2007). Downstream effectors of Rac1 in SC differentiation are unknown.
One protein implicated both upstream and downstream of Rac1 signaling is NF2/merlin, the product of the neurofibromatosis type 2 gene. NF2/merlin is a tumor suppressor and NF2/merlin mutation or loss predisposes to SC tumors (Curto and McClatchey 2008; Roche et al. 2008; Scoles 2008). NF2/merlin is phosphorylated on serine 518 by PAK, a downstream effector of Rac1. This phosphorylation alters NF2/merlin conformation and interactions with binding partners (Rong et al. 2004; Ye 2007). NF2/merlin also regulates Rac1 signaling, so that NF2/merlin null fibroblasts and NF2/merlin mutant schwannoma cells exhibit membrane ruffling, characteristic of elevated active Rac1 (Flaiz et al. 2009; Kaempchen et al. 2003; Yi et al. 2008). On a cellular level, NF2/merlin stabilizes the bipolar morphology of SCs through inhibition of Rac1 (Thaxton et al. 2011). Specific pathways in which NF2/merlin acts related to SC biology and pathology remain undefined.
NF2/merlin can stabilize cadherin-containing adherens junctions (Lallemand et al. 2003). Expression of cadherins in SCs is under developmental control (Fannon et al. 1995; Menichella et al. 2001; Wanner et al. 2006). E-cadherin maintains the structural integrity of non-compact myelin domains in vivo (Perrin-Tricaud et al. 2007; Young et al. 2002). cAMP regulates E-cadherin in postnatal SCs (Crawford et al. 2008). Notably, cAMP can act downstream of Rac1 in mouse embryonic fibroblast cells (Chen et al. 2008), and cAMP is critical for SC myelination (Arthur-Farraj et al. 2011; Monk et al. 2009).
Here we studied SC myelination in Rac1-CKO mice to identify Rac1-related targets in SC differentiation. First, we showed SC myelination was arrested in Rac1-CKO mice and that Rac1 knockout abrogated phosphorylation of p21-activated kinase and decreased NF2/merlin phosphorylation. We further generated Rac-CKO&NF2-del double mutant mice and showed that mutation of NF2/merlin rescued the myelin deficit in Rac1-CKO mice in vivo and shortened processes in Rac1-CKO SCs in vitro. Second, we showed that cAMP levels and E-cadherin expression decreased in Rac1-CKO mice, and each was restored by mutation of NF2/merlin. We further confirmed that reduced cAMP is one cause of the myelin deficits in Rac1-CKO mice, as these defects were rescued via rolipram elevation of cAMP in vivo. Thus, our observations define a novel pathway through which Rac1 regulates SC differentiation via NF2/merlin and cAMP signaling. The results establish a functional link between NF2/merlin, cAMP and Rac1 in SC myelination.
Rac1flox/flox (Guo et al. 2008) were bred to DhhCre mice (Jaegle et al. 2003) to obtain DhhCre+; Rac1flox/flox (Rac1-CKO) mice. Littermate DhhCre-; Rac1flox/WT mice were used as controls. The genotypes of Dhh and Rac1 alleles for all of the mice in our experiments were analyzed by PCR as previously described (Guo et al. 2008; Williams et al. 2008; Wu et al. 2008; Yang et al. 2007; Yang et al. 2006). The primers for Dhh were sense: 5′-ACCCTGTTACGTATAGCCGA-3′ and anti-sense: 5′-CTCGGTATTAAACTCCAG-3′. The primers for Rac1 were sense: 5′-TCCAATCTGTGCTGCCCATC-3′ and anti-sense: 5′-GATGCTTCTAGGGGTGAGCC-3′. Primer 5'- CAG AGC TCG AAT CCA GAA ACT AGT A -3' was used to identify the Rac1 knockout band. NF2-del mice, are transgenic mice (P0-SCH-Δ39-121) expressing a mutant NF2/merlin, in which exons 2 and 3 (amino acids 39–121) are deleted from the genomic sequence, mimicking a human mutation (Giovannini et al. 1999). This NF2/merlin mutant allele is expressed under the control of a SC-specific P0 promoter. These mice were crossbred with Rac1 knockout mice to generate DhhCre+;Rac1flox/flox;NF2-del (Rac1-CKO&NF2-del) crosses. Littermate Dhh-Cre-; Rac1flox/WT; NF2-wt, or Dhh-Cre-; NF2-wt genotype mice were used as controls. Dhh-Cre mice were maintained on the C57BL/6 background. Rac1flox/flox mice were maintained on a mixed 129Sv and C57BL/6J background. NF2-del mice were maintained on the FVB/N background. All animal experiments were conducted in mixed gender mice.
Mice were anaesthetized, then perfused and fixed with electron microscopy fixation solution (3% paraformaldehyde and 3% glutaraldehyde in phosphate buffered saline, pH 7.4 to 7.6). Sciatic nerves of wild type and mutant mice at ages designated in the text were dissected, post-fixed, osmicated, embedded and sectioned. High magnification pictures of ultrathin sections were taken by a Hitachi H-7600 transmission electron microscope following staining with lead citrate and uranyl acetate.
Sciatic nerve tissue was homogenized using a TissueRuptor (Qiagen) and lysed in lysis buffer (20 mM NaPO4, 150 mM NaCl, 2 mM MgCl2, 0.1% Nonidet P-40, 10% glycerol, 10 mM sodium fluoride, 0.1 mM sodium orthovanadate, 10 mM sodium pyrophosphate, 10 nM okadaic acid, 1 mM dithiothreitol, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 10 µg/ml pepstatin, 10 µg/ml tosyl-L-phenylalanine chloromethyl ketone, and 10 µg/ml N α-tosyl-L-lysine chloromethyl ketone). Homogenates were centrifuged at 10,000g for 10 mins and protein extract supernatants were collected. Protein concentration was measured on a spectrophotometer using Bio-Rad DC Protein Assay Kit (Bio-Rad). Equal amounts of protein were fractionated by 4–20% SDS-PAGE (NuStep Inc., Ann Arbor, MI) and transferred to PVDF membrane. Membranes were incubated with primary antibodies followed by appropriate secondary antibodies, and developed by Amersham ECL Detection Reagents (GE Healthcare Biosciences). The following primary antibodies were used: Rac1 (BD Transduction Laboratories, 1:800), P-Erk1/2 (Cell Signaling, 1:2000), P-merlin (Abcam, 1:500), merlin (Abcam, 1:800), P-PAK1/2 (Cell Signaling 1: 1000), PAK (Cell Signaling 1:1000), Erk1/2 (Cell Signaling, 1:1000), E-cadherin (Cell Signaling 1:1000), neurofilament (Abcam, 1:1000), Pmp22 (Abcam, 1:5000) and beta-actin (Cell Signaling, 1:10000). Anti-rabbit and anti-mouse HRP conjugated secondary antibodies (1:5000) were purchased from Bio-Rad.
Mice were sacrificed and perfused with 4% paraformaldehyde (Wieser et al.) and sciatic nerves were dissected. Sciatic nerves were fixed in 4% PFA overnight, incubated in 20% sucrose buffer and then frozen in OCT compound (Sakura, Finetek, Inc., USA). Frozen blocks were cut into 6–8 µm frozen sections using a Leica Cryostat. For staining, frozen sciatic nerve sections were incubated with 4% PFA for 20 min at room temperature then washed in PBS and permeabilized with 0.3% Triton X-100 in PBS when necessary. Sections were blocked for one hour with blocking buffer (10% serum in PBS) and incubated with primary antibody at 4°C overnight. The next day, sections were washed with PBS and incubated in Alexa Fluor® 488 or Alexa Fluor® 594 conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, Inc. 1:400) for one hour at room temperature. To visualize nuclei, sections were stained with DAPI for 10 mins then washed with PBS and mounted in FluoromountG (Electron Microscopy Sciences, Hatfield, PA). Cultured SCs were immunostained with P75NTR antibody (Chemicon, Temecula, CA) and Alexa 549 Fluor-phalloidin antibody (Invitrogen) to visualize SC protrusions and lamellipodia in vitro. A Rac-GTP antibody was used at 1:500 (NewEast Biosciences) to detect active Rac1 in cultures. All the images were acquired using a fluorescence microscope with 10×/0.4 or 40×/0.6 objectives (Carl Zeiss, Inc.). Acquisition software ImageJ was used.
Primary mouse SC cultures were obtained from P30 sciatic nerves. In brief, sciatic nerves were sterilely dissected from euthanized P30 mice and incubated at 37°C, 7.5% CO2 in DMEM medium (Invitrogen) supplemented with 10% fetal bovine serum (Gemini Bio-Products, Inc.), 1% penicillin-streptomycin (Fisher Scientific), forskolin (2 µM, Calbiochem), and beta-heregulin (HRG, 10 ng/ml, R&D Systems). Pretreatment medium was replaced every 2 days. After 6–9 days nerves were incubated in dissociation medium [Leibovitz medium (Invitrogen) containing collagenase type I (130 U/ml, Worthington Biochemical Corporation,Lakewood, NJ), dispase II (2.5 mg/ml, Roche Diagnostics, Indianapolis, IN), gentamycin (50 µg/ml, Lonza walkersville), fungizone (2.5 µg/ml, Invitrogen)] for 3 hours at 37°C. Cells were dissociated using a narrowed Pasteur pipette then were centrifuged for 5 min at 1000 rpm. Cells were resuspended in DMEM/ F-12 medium (Invitrogen) supplemented with N2 supplement solution (Invitrogen), forskolin (2 µM, Calbiochem), beta-heregulin (HRG) (10 ng/ml), gentamycin (50 µg/ml, Lonza walkersville) and fungizone (2.5 µg/ml, Invitrogen). Cells were plated on poly-L-lysine (Sigma) and laminin (BD biosciences) coated plates and incubated at 37°C, 7.5% CO2. Medium was changed every 3 days and the cells were passaged when confluent. Cells were used at passage 0–1.
Levels of cAMP were measured and quantified using a cAMP assay kit (Enzo Life Sciences). Homogenates of sciatic nerves or lysates from cell cultures were used for the cAMP assay according to manufacturer’s instructions. To elevate cAMP levels in cultured SCs, forskolin (5µM, Sigma) was added into serum free cell culture medium for 30 mins. The Rac1 specific inhibitor NSC 23766 was used to treat cells for 30 mins before adding forskolin (Gao et al., 2004). To elevate cAMP levels in mice in vivo, Rolipram (5mg/kg/daily, Calbiochem) was administered daily via i.p. injection for 6–8 weeks.
Morphometric measurements of myelination were performed in electron micrographs. Myelin sheath thickness was quantified by g-ratio analysis (the ratio of the axon diameter to the myelinated fibre diameter). The diameter of the axon and the myelinated fibre were measured in the electron microscopy images of the nerve cross sections.
A cell counter plugin from ImageJ software was used to analyze the number of one-to-one myelinated axons, non-myelinated axons, and the axon number in each axonal bundle. The quantification of SC process number, length and SC lamellipodia number in primary cultures was performed using ImageJ software. Statistical significance was determined between two individual samples with the Student’s t test. For multiple comparisions, one-way ANOVA followed by Tukey’s post-hoc test was used. Significance was denoted as *P < 0.05, **P < 0.01, or ***P < 0.001.
To analyze the role of the small GTPase Rac1 in SCs in vivo, Rac1 conditional knockout mice (Rac1-CKO) were generated by using the Cre recombinase-LoxP recombination system under the control of desert hedgehog (Dhh) gene. In these mice, Dhh activates Cre recombinase expression in SCs at embryonic day 12.5 (Jaegle et al. 2003). Exon1 of the Rac1 alleles were excised upon Dhh-Cre recombination (Fig. 1A). The Rac1 knockout alleles were confirmed by PCR (Fig. 1B).
Conditional knockout of Rac1 in SCs led to hind limb dysfunction in mutant mice (Fig. 1C), which is consistent with a previous study using a different set of conditional knockout mice (Benninger et al. 2007). Rac1-CKO mice showed tremors by 20-days old and progressive paralysis of their hind limbs as the mice aged. Rac1-CKO mice displayed complete paralysis of hind limbs by two month of age. Rac1-CKO mice lived for over six months provided that food was easily accessible. At postnatal day (P) 30, sciatic nerves from Rac1-CKO were thinner than the sciatic nerves of wild type mice (Fig. 1D). Reduction of Rac1 protein expression in the sciatic nerves of these mutant mice was verified by Western blot (Fig. 1E and F). There remained residual Rac1 protein in mutant nerves. We hypothesized that this was generated from axons in the nerve. Consistent with this hypothesis, Rac1 protein was barely detectable in Rac1-CKO sciatic nerve from which axons were removed by in vitro degeneration for six days (Fig. 1E and F). GTP-bound active Rac1 was also eliminated in Rac1-CKO SCs (Fig. 1G, arrows) as shown by immunostaining, using an antibody that detects Rac-GTP.
To investigate the pathological changes underlying these abnormalities in Rac1-CKO mice, nerve ultrastructure in Rac1-CKO sciatic nerves was compared to control sciatic nerves by electron microscopy (EM) at different developmental stages. At P1, some axons were sorted and already thinly myelinated in control sciatic nerves, however, no myelinated axons were observed in Rac1-CKO sciatic nerves (Fig. 2A). At P30, P60 and P120, large axons in control sciatic nerves were well myelinated. However, fewer than 5% of axons in Rac1-CKO sciatic nerves were myelinated (Fig. 2A and B), even though most SCs had differentiated to one-to-one interactions with large axons in Rac1-CKO sciatic nerves (Fig. 2A and C). The vast majority of large axons in Rac1-CKO mice were sorted but unmyelinated even at P15. These results indicate that Rac1 plays a critical role in SC myelination.
A previous study reported SC axonal sorting defects in E17.5 to P24 nerves in the absence of Rac1 (Benninger et al. 2007). Consistent with that study we observed irregular membrane-delineated cytoplasmic protrusions from SCs in adult Rac1-CKO mouse sciatic nerves. These protrusions from Rac1-CKO SCs were largely from SCs in 1:1 relationship with axons (Fig. 2A and E, arrowheads). Remak bundles in adult Rac1-CKO sciatic nerves contain similar numbers of small axons as control mice (Fig. 2 D). The numbers of Remak bundles also did not change in the absence of Rac1. We quantified aspects of axons and SCs within Remak bundles in sections from five mice. Most (96±3%) of unmyelinated small axons in adult Rac1-CKO sciatic nerves were normally segregated by SC processes in Remak bundles (Fig. 2E). However, some Remak bundles abnormalities were observed in Rac1-CKO nerves. For example, 2±0.8% of Remak bundles contained one or two axons with redundant SC wraps (Fig. 2Ee. black arrow). In addition, in 49±6% of Remak bundles contained one to three large diameter axons (>1µM) which remained unsorted (Fig. 2Ed), so that 6±2% of axons within Remak bundles were large diameter axons. These data confirm an axonal sorting defect in Rac1-CKO nerves. Taken together, our data indicate that Rac1 is necessary for SC myelin sheath formation and plays role in axonal sorting in SC.
Next, we used western blots to define the possible downstream effectors that mediate the myelination deficiency in Rac1-CKO sciatic nerves. Phosphorylation and activity of NF2/merlin is regulated by a Rac1 effector, PAK (Rong et al. 2004; Xiao et al. 2002; Ye 2007). Phosphorylation of PAK at Thr423(PAK1)/Thr402(PAK2) and phosphorylation of merlin at Ser518 were dramatically downregulated in Rac1-CKO sciatic nerves (Fig. 3A and B). Expression of P-Erk1/2, total Erk1/2, total PAK and NF2/merlin were not changed in Rac1-CKO sciatic nerves (Fig. 3A and B). These results indicate that Rac1 regulates Pak and NF2/merlin phosphorylation in SCs in vivo. NF2/merlin phosphorylation at Ser518 changes merlin conformation and activity (Sher et al. 2012) Our data support the idea that NF2/merlin is de-phosphorylated in the absence of Rac1 and the change in NF2/merlin phosphorylation may lead to the myelin deficits found in Rac1-CKO mice.
To test if NF2/merlin function is critical for the absence of SC myelination in Rac1-CKO mice, Rac1 knockout mice were intercrossed with NF2-del transgenic mice in which exons 2 and 3 (amino acids 39–121) of NF2/merlin are deleted (Giovannini et al. 1999). This NF2/merlin mutant allele is expressed under the control of the SC-specific P0 promoter. We confirmed SC hyperplasia in NF2-del mouse nerves and observed no defects in myelin internodes at P30 to P180. NF2-del transgenic mice and homozygous NF2/merlin knockout mice (P0Cre; NF2 flox2/flox2) mice have similar paranodal defects, SC hyperplasia, and both types of NF2/merlin mutants develop tumors with similar incidence and timing (Giovannini et al., 1999; 2000; Denisenko et al., 2008). Analysis of SC cultures from these two mouse strains reveals identical growth after confluence, and identical gene expression (Macro Giovannini, personal communication), indicating that the NF2-del acts as a functional null allele. Strikingly, paralysis of hind limbs in adult Rac1-CKO mice was improved in Rac1-CKO&NF2-del double mutant mice. In contrast to the thinner sciatic nerves in Rac1-CKO mice, sciatic nerves in Rac1-CKO&NF2-del double mutant mice were close to the normal size of control sciatic nerves in wild type mice (Fig. 4A). Next, we used electron microscopy to test whether SC myelination defects in Rac1-CKO mice were rescued in Rac1-CKO&NF2-del mice. At four months old, a dramatic increase in myelinated axons was observed in Rac1-CKO&NF2-del mice compared to the profound absence of myelinated axons in Rac1-CKO mice (Fig. 4B and C), indicating that NF2/merlin mutant rescues the myelination deficiency in Rac1-CKO mice. Consistent with this finding, decreased expression of the peripheral myelin protein, Pmp22 in Rac1-CKO nerves was rescued in Rac1-CKO&NF2-del nerves (Fig. 4E). The myelin sheath thickness (g-ratio; ratio of axon diameter to axon + myelin sheath diameter) was calculated for myelinated axons in wild type control and Rac1-CKO&NF2-del mice. The g-ratio for Rac1-CKO&NF2-del mice was 0.771 (n=613) compared to 0.646 (n=618) for control mice. Thus, myelin sheaths are present in the Rac1-CKO&NF2-del mice but myelin is thinner than normal.
The Remak bundle defects including one or two large axons within some Remak bundles in Rac1-CKO nerves were not rescued in Rac1-CKO&NF2-del nerves. The numbers of axons within Remak bundles also did not change in Rac1-CKO&NF2-del nerves (data not show). Irregular SC protrusions in Rac1-CKO sciatic nerves in vivo remained in Rac1-CKO&NF2-del sciatic nerves (Fig. 4B and F, arrowheads). Effector(s) apart from NF2/merlin may mediate those abnormalities.
A well-known role of Rho GTPases is regulation of the cellular cytoskeleton, and previous evidence supports a role of Rac1 activity for SC process elongation and lamellipodia formation (Nodari et al. 2007). To test whether NF2/merlin is involved in Rac1 mediated SC process elongation, we cultured primary SCs from sciatic nerves of P30 control, Rac1-CKO, NF2-del and Rac1-CKO&NF2-del mice. Compared to control SCs, SC process length was shorter in Rac1-CKO cultures (Fig. 5A, B and D) and lamellipodia (including both radial lamellipodia and axial lamellipodia) were largely lost in the absence of Rac1 (Fig. 5A, B and E). Shortened SC processes in Rac1-CKO SCs were rescued by NF2/merlin mutation (Fig. 5A, B and D), while the lamellipodia defects in Rac1-CKO SCs were not affected by NF2/merlin function (Fig. 5A, B and E). These results confirm that Rac1 promotes SC process elongation and lamellipodia formation under physiological conditions. These data suggest that NF2/merlin plays a role in Rac1 regulated SC process elongation, which correlates with our observation that NF2/merlin functions in Rac1 mediated myelination in vivo.
NF2/merlin is thought to stabilize cadherin-containing adherens junctions (Lallemand et al. 2003). E-cadherin is an important constituent of adherens junctions and plays a key role at cell-cell junctions, although it is not itself necessary for myelination (Young et al. 2002). We used western blot to test E-cadherin expression in sciatic nerves. E-cadherin expression was significantly decreased in the absence of Rac1 (Fig. 6A and B). Decreased E-cadherin expression in Rac1-CKO sciatic nerves was rescued in Rac1-CKO&NF2-del sciatic nerves (Fig. 6A and B). As cAMP is critical for E-cadherin expression in postnatal SCs (Crawford et al. 2008), we tested if elevation of cAMP levels could restore E-cadherin expression in Rac1-CKO nerves. Forskolin was used to elevate cAMP levels in wild type or Rac1-CKO nerve segments in vitro. Forskolin treatment increased both E-cadherin expression and phosphorylation of merlin (Fig. 6C and D). These results suggest that Rac1 regulates E-cadherin expression in SCs through NF2/merlin and/or cAMP signaling.
To better define relationships among Rac1, cAMP and NF2/merlin, we measured cAMP levels in sciatic nerves of control, Rac1-CKO, NF2-del and Rac1-CKO&NF2-del mice. cAMP levels in Rac1-CKO nerves were significantly lower than those in control nerves, while cAMP levels in NF2-del nerves were significantly higher than those in control nerves (Fig. 6E). Importantly, compared to Rac1-CKO nerves, cAMP levels were increased in Rac1-CKO&NF2-del nerves (Fig. 6E). The difference in cAMP levels between Rac1-CKO vs. Rac1-CKO&NF2-del was not significant by ANOVA followed by Tukey analysis using multiple group comparison with wild type and NF2-del nerves. However, t-test in six independent samples shows that the difference in cAMP between Rac1 and Rac1-CKO&NF2-del nerves is significant (p<0.05). These results suggest that Rac1 regulates cAMP at least in part through NF2/merlin signaling.
To confirm a link between Rac1, cAMP and NF2/merlin we used a Rac1 specific inhibitor, NSC23766 (Gao et al. 2004), in cultured wild type or NF2-del SCs in vitro. Both basal and stimulated levels of cAMP were increased in NF2-del SCs compared to wild type SCs (Fig. 6F). Rac1 inhibition by NSC23766 reduced forskolin-stimulated cAMP levels in cultured wild type and NF2-del SCs. These results confirm that Rac1 and NF2/merlin regulate cAMP in SCs.
To test whether low cAMP prevents myelination in Rac1 mutants in vivo, we used the phosphodiesterase inhibitor, rolipram, to elevate cAMP levels in Rac1-CKO mice in vivo. A significant increase in myelinated axons was observed in Rac1-CKO mice after 6–8 weeks daily treatment with rolipram (Fig. 7A and B). These results demonstrate that elevating cAMP in Rac1 mutants is sufficient to enable SC myelination in vivo. However, SCs protrusions remained in Rac1-CKO nerves after rolipram treatment (Fig. 7C, arrow). In addition, abnormal folding of SC myelin sheath (Fig. 7C, arrow heads) was occasionally observed in Rac1-CKO nerves after rolipram treatment. Thus, the SC myelination defects in Rac1-CKO mice are partially rescued by NF2-del mutant or rolipram treatment, both of which increase cAMP in vivo.
We identified a novel pathway in which Rac1 promotes SC myelination through cAMP signaling and NF2/merlin. Our data in Rac1-CKO mice showed that Rac1 plays an important role in SC myelination, as SCs did not form myelin sheaths in the absence of Rac1. Rac1-CKO SCs had decreased NF2/merlin phosphorylation. Rac1 regulates SC myelination through NF2/merlin, as the myelin deficits in Rac1-CKO SCs were rescued by loss of NF2/merlin function. Decreased NF2/merlin phosphorylation in Rac1-CKO nerves also correlated with decreased cAMP and NF2/merlin mutant nerves had increased cAMP levels. Acute elevation of cAMP in vitro using forskolin increased NF2/merlin phosphorylation. In addition, using rolipram to elevate cAMP in vivo rescued the myelin deficiency in Rac1-CKO mice. The results of this study are summarized in Fig. 8.
In contrast to a previous study that described delay in radial sorting at developmental stages (E17.5 to P24) after Rac1 knockout (Benninger et al. 2007; Nodari et al. 2007), our study at later time points (P0 to P120) support a major role for Rac1 in SC myelination. We found that myelin sheaths do not form in the Rac1 mutant mice at P15, P30, P60 and P120, even though most SCs establish one-to-one relationship with large axons. Thus, we conclude that Rac1 is required at the initiation of SC myelination, after axonal sorting is complete. These differences in phenotypes may be due to strain background differences, and/or be related to the difference in gene targeting strategies (Benitah et al. 2005; Cappello et al. 2006; Castilho et al. 2007; Chen et al. 2006; Chrostek et al. 2006). Consistent with our results that Rac1 is critical for SC myelination, a recent study reported that active Rac1 is localized to the axon-glial interface in SCs by Par3 and that polarization of Rac1 activation is critical for myelination (Tep et al. 2012).
Unmyelinated large axons in sciatic nerves of Rac1-CKO mice became myelinated in Rac1-CKO&NF2-del double mutants. The role of NF2/merlin in cellular differentiation is poorly studied. Indeed, a minor role for NF2/Merlin in SC differentiation was proposed, yet contacts between SCs and axons at paranodes regions were altered in NF2-del mice (Denisenko et al. 2008). We confirmed the absence of significant defects in myelin internodes at P30 to P180 in NF2-del mutant mice. Our data show that NF2/merlin plays a critical role downstream of Rac1 in SC differentiation in vivo, which is revealed by the absence of Rac1.
Our in vitro analyses using SCs with genetic loss of Rac1 confirm data of Benninger et al. (2007). Both studies show that Rac1 null cells have short processes, indicating that Rac1 normally promotes SC processes elongation. In contrast, SCs expressing dominant negative Rac1 extended abnormally long bipolar processes (Thaxton et al. 2011). The difference between using the dominant negative Rac1 and the genetic loss of Rac1 in SC process elongation remains to be analyzed. Comparison of SC morphology in primary cultures provided compelling additional evidence that Rac1 function is NF2/merlin-dependent, as short processes in Rac1-CKO SCs were rescued in Rac1-CKO&NF2-del SCs.
Myelin sheath thickness in the Rac1-CKO&NF2-del mice was not completely rescued to the myelin thickness characteristic of control mice and resembles the thin myelin seen after remyelination. It is also likely that NF2/merlin is not the only Rac1 downstream effector that contributes to SC function, especially as Rac1-CKO&NF2-del cultured SCs were partially rescued in process length. The irregular SC protrusions in Rac1-CKO sciatic nerves and the lamellipodia defects in Rac1-CKO SCs were unaffected by NF2/merlin function.
SC myelination requires cAMP. For example, addition of cAMP to SC cultures increases the expression of myelin genes and proteins (Sobue et al. 1986), the G protein-coupled receptor GPR126 is essential for SC myelination in vivo (Monk et al. 2009), and cAMP dependent phosphorylation of the transcription factor nuclear factor-kappaB is required for myelin formation (Yoon et al. 2008). Our results provide in vitro and in vivo evidence for a novel pathway in which Rac1 promotes myelination through cAMP signaling and NF2/merlin. Upstream activators of Rac1/cAMP signaling in this myelination pathway remain to be identified. Rac1 has been linked to laminins and β1 integrins in SC myelination (Nodari et al. 2007; Yu et al. 2009). Intriguingly, integrins activate cAMP signaling through Gαs in endothelial cells (Alenghat et al. 2009). It is possible that integrin, signaling through Gαs and Rac1, activates cAMP to control SC myelination.
We found that PAK is a major effector of Rac1 in SCs, as when Rac1 is lost P-PAK at Thr423 (PAK1)/Thr402 (PAK2) is absent. Consistent with absence of P-PAK in Rac1-CKO SCs, we observed decreased NF2/merlin phosphorylation at Ser518, a site known to be a substrate of PAK. Protein kinase A (PKA) can also phosphorylate NF2/merlin on Ser518 (Laulajainen et al. 2008) and the reduction in cAMP in Rac1-CKO Schwann cells is predicted to lead to decreased PKA activity, which may contribute to reduced merlin phosphorylation. Consistent with PKA acting upstream of merlin, forskolin increased NF2/merlin phosphorylation. However, cAMP levels also increased in NF2-del mutants in vitro and in vivo, suggesting a feedback mechanism. In this sense, NF2/merlin may be upstream of cAMP. It is notable that a similar feedback loop has been described between Rac, PAK, and merlin (Kissil et al., 2003).
In normal nerve, Rac-GTP is predicted to phosphorylate PAK; PAK would phosphorylate merlin, possibly increasing cAMP and promoting myelination. In the absence of Rac1, the de-phosphorylated NF2/merlin correlates with decreased cAMP, preventing myelination. Non-phosphorylated NF2/merlin is increasingly believed to have critical functions, for example in cell growth (Li et al. 2010; Sher et al. 2012). Intriguingly, both mutation in PKA and mutation in NF2, correlate with SC tumorigenesis (Jones et al. 2008). It will be of interest to study whether cAMP levels are changed in NF2/merlin deficient SC tumors.
In summary, our study provided in vitro and in vivo evidence supporting a novel pathway in which Rac1 regulates SC myelination through NF2/merlin and cAMP. We have also established a functional link between NF2/merlin, cAMP and Rac1 in SC myelination. Myelin defects in the PNS are associated with demyelinating diseases as well as tumors. Therefore, these observations may be relevant to SC development, nerve pathology, and tumorigenesis.
We thank Georgianne Ciraolo for assistance with electron microscopy, Dr. Marco Giovannini for providing NF2-delta2,3 (NF2-del) mice and Dr. Robert Hennigan for helpful discussions. This study was supported by NIHR01CA118032 to N.R. and Y.Z.