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 .
A model for Rac1, NF2/merlin and cAMP regulate SC myelination
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.