Lithium enhances remyelination of peripheral nerves - PNAS

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Feb 13, 2012 - (C) Eight days after the facial nerve crush (DPI8), the facial nerve was isolated. Ultrathin .... (C) Pri- mary Schwann cells were treated with LiCl (10 mM) for 24 h. (D) MSC80 cells were treated ..... Noble W, et al. (2005) Inhibition ...
Lithium enhances remyelination of peripheral nerves Joelle Makoukjia,1, Martin Bellea,1, Delphine Meffrea, Ruth Stassartb, Julien Greniera, Ghjuvan’Ghjacumu Shackleforda, Robert Fledrichb, Cosima Fontec, Julien Branchua, Marie Goularda, Catherine de Waelea, Frédéric Charbonniera, Michael W. Seredab,d, Etienne-Emile Baulieuc,2, Michael Schumacherc, Sophie Bernarda, and Charbel Massaada,2 a Unité Mixte de Recherche 8194, Centre National de la Recherche Scientifique, Centre d’Etude de la Sensori Motricité, University Paris Descartes, 75270 Paris Cedex 06, France; bDepartment of Neurogenetics, Max Planck Institute of Experimental Medicine, 37075 Goettingen, Germany; cUnité Mixte de Recherche 788, Institut National de la Santé et de la Recherche Médicale, Institut Fédératif de Recherche de Bicêtre 93, University Paris-Sud 11, 94276 Le Kremlin-Bicêtre Cedex, France; and dDepartment of Clinical Neurophysiology, University of Goettingen, 37075 Goettingen, Germany

Glycogen synthase kinase 3β (GSK3β) inhibitors, especially the mood stabilizer lithium chloride, are also used as neuroprotective or anti-inflammatory agents. We studied the influence of LiCl on the remyelination of peripheral nerves. We showed that the treatment of adult mice with LiCl after facial nerve crush injury stimulated the expression of myelin genes, restored the myelin structure, and accelerated the recovery of whisker movements. LiCl treatment also promoted remyelination of the sciatic nerve after crush. We also demonstrated that peripheral myelin gene MPZ and PMP22 promoter activities, transcripts, and protein levels are stimulated by GSK3β inhibitors (LiCl and SB216763) in Schwann cells as well as in sciatic and facial nerves. LiCl exerts its action in Schwann cells by increasing the amount of β-catenin and provoking its nuclear localization. We showed by ChIP experiments that LiCl treatment drives β-catenin to bind to T-cell factor/lymphoid-enhancer factor response elements identified in myelin genes. Taken together, our findings open perspectives in the treatment of nerve demyelination by administering GSK3β inhibitors such as lithium. Wnt/β-catenin

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yelin is essential for the rapid conduction of axonal impulses. Myelination, elicited in peripheral nerves by Schwann cells, is a complex and fine-tuned process. Peripheral myelin gene expression [Myelin Protein Zero (MPZ) and Peripheral Myelin Protein 22 (PMP22)] is tightly regulated (1). Myelin gene expression is stimulated during the myelination process, which occurs in rodents between postnatal days 1 and 21 (2). Afterward, those genes are expressed at lower levels, sufficient to maintain myelin turnover. They are restimulated after nerve injuries to initiate remyelination (3). Peripheral nerve injuries are frequent, and the functional outcomes are often not satisfactory. Demyelination can occur as a result of neuronal or Schwann cell injury and is usually accompanied or followed by axonal degeneration. Acquired neuropathies may be caused by aberrant immune responses, local injury, ischemia, metabolic disorders, toxic agents, or viral infections. To date, few therapeutic treatments are available. They are mostly based on anti-inflammatory agents. Few cellular signals are known to directly regulate the expression of myelin genes. For instance, we have shown that the Wnt/ β-catenin signaling pathway is a direct driver of myelin gene expression (4). Wnt activation inhibits glycogen synthase kinase 3β (GSK3β) enzyme and prevents β-catenin degradation. The stabilized β-catenin is translocated into the nucleus, where it regulates gene transcription by interacting with transcription factors called T-cell factor/lymphoid-enhancer factors (TCF/LEF or TCFs). Lithium is an enzymatic inhibitor of GSK3β, and mimics the Wnt/ β-catenin signaling pathway. This drug is widely used as a long-term mood stabilizer in the treatment of bipolar and depressive disorders (5). Growing evidence demonstrates that lithium has neuroprotective (6) and antiapoptotic (7) properties, and can therefore be used as a treatment in several neurodegenerative diseases [e.g., Huntington disease (8), Alzheimer’s disease (9), Parkinson disease (10)] and in experimental autoimmune encephalomyelitis (11). www.pnas.org/cgi/doi/10.1073/pnas.1121367109

In this study, we report several lines of evidence that the inhibitors of GSK3β (LiCl and SB216763) enhance peripheral myelin gene expression in vitro and in vivo via the binding of β-catenin to TCF/LEF transcription factors present in PMP22 and MPZ promoters. Most strikingly, LiCl administration to mice after a crush of the facial nerve, which provokes paralysis of the whiskers, elicited a rapid recovery of their movements. This recovery is concomitant with the stimulation of myelin gene expression and the restoration of the myelin sheath structure around the axons. LiCl also provoked the remyelination of axons after sciatic nerve crush. Consequently, our findings open perspectives in the treatment of demyelinating diseases by administering GSK3β inhibitors such as lithium. Results LiCl Administration Accelerates Functional Recovery and Ameliorates Phenotype of Facial Nerve After Nerve Injury. Our aim is to assess

the outcome of the administration of LiCl, a widely used inhibitor of GSK3β (IC50, 10 mM), after motor nerve injury. Therefore, we developed in mice a model of facial nerve crush that provokes a local demyelination and complete subsequent palsy of the ipsilateral (crushed) whiskers, as well as absence of ipsilateral blink reflex. The left facial nerve was crushed whereas the right one was left intact to serve as a control (Fig. 1A). We then analyzed by means of WhiskerWatcher device the angular velocity of the ipsilateral whisker movements before and after the crush at different postlesion time points [day postinjury 3 (DPI3), DPI8, and DPI14] in two groups of animals: a group treated with LiCl (i.p. injection, 50 mg/kg/d during 4 d after nerve crush) and a group treated with PBS solution (i.e., placebo) during a similar period (Fig. 1A). The mean angular velocity of the whiskers was 56.44°/s in normal animals, and it decreased to 0.2°/s at DPI3 in nervecrushed animals, indicating a dramatic alteration of whisker movements after facial nerve injury (Fig. 1B). The placebo-treated animals recovered slowly: at DPI8, the velocity was 7.96°/s, and at 14 d, the whisker movements were still below normal levels (44.99°/s vs. 56.44°/s for normal animals). Interestingly, the i.p. LiCl administration after the facial nerve crush accelerated the functional recovery during the first 8 d after lesion creation. The angular velocity was significantly improved after 3 d of treatment with LiCl: 3.48°/s with LiCl vs. 0.2°/s with placebo. At 8 d, we also observed an improvement in the movements of the whiskers, which became almost normal (43.08°/s with LiCl vs. 7.96°/s with placebo). At 14 d, we can consider that the animal recovered

Author contributions: R.S., C.d.W., F.C., M.W.S., S.B., and C.M. designed research; J.M., M.B., D.M., R.S., J.G., G.S., R.F., C.F., J.B., M.G., C.d.W., and S.B. performed research; J.M., C.d.W., F.C., M.W.S., M.S., S.B., and C.M. analyzed data; and E.-E.B., M.S., and C.M. wrote the paper. The authors declare no conflict of interest. 1

J.M. and M.B. contributed equally to this work.

2

To whom correspondence may be addressed. E-mail: [email protected] or charbel. [email protected].

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1121367109/-/DCSupplemental.

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Contributed by Etienne-Emile Baulieu, December 27, 2011 (sent for review November 24, 2011)

Fig. 1. Effect of LiCl administration on functional recovery of whiskers and phenotype of the facial nerve after nerve crush. (A) Nerve crush of the left facial nerve was performed in 8-wk-old mice. One group received four i.p. injections of LiCl (50 mg/kg/d) directly after the nerve crush during 4 d. The other group received PBS solution injection as a placebo (Pl). Normal animals (control) did not undergo nerve crush. (B) The mean angular velocity of the whisker movements of all animals was analyzed by means of WhiskerWatcher software at DPI3, DPI8, and DPI14. Results are expressed in degrees per second and represent the mean of six animals per group; the angular velocity of the whiskers of each animal was analyzed three times (***P < 0.005, Kruskal–Wallis test). (C) Eight days after the facial nerve crush (DPI8), the facial nerve was isolated. Ultrathin cross-sections were prepared from epon-embedded adult facial nerves and stained for myelin. 1 and 2, Low-magnification EM images of a section of facial nerve. (Scale bar: 2 μM.) 3 and 4, High-magnification EM images of a representative axon. (Scale bar: 1 μM.) 5 and 6, High-magnification EM images of myelin layers. (Scale bar: 100 nm.) (D) Myelin thickness was estimated by gratio determination by using EM images obtained from facial nerves (12 images per animal, three animals per condition). Data are means ± SEM. (E) The percentage of myelinated axons per square micrometer was calculated by using EM images. Three animals per condition were used. Data are means ± SEM. (F) Facial nerves were dissected at DPI3. Quantitative RT-PCR experiments were performed by using primers recognizing PMP22 and MPZ genes. 26S and GAPDH RNA were used to normalize quantitative RT-PCR (*P < 0.05 and **P < 0.01 by Mann–Whitney test vs. control).

completely because we did not have any significant mean angular velocity difference between the normal animals (56.44°/s) and LiCl-treated animals (54.78°/s). Afterward, we analyzed the structure of facial nerve at DPI8 by using EM. The crushed nerves of LiCl-treated mice exhibited a thicker myelin sheath (Fig. 1C), suggesting nerve remyelination. This assumption was reinforced by the calculation of the gratio (i.e., axon diameter/myelin sheath diameter). The g-ratio of placebo-treated animals (0.84 ± 0.01) was significantly higher than that of LiCl-treated animals (0.75 ± 0.01; Fig. 1D). Furthermore, the percentage of myelinated axons doubled after LiCl treatment (23.31 ± 3.05 with placebo vs. 47.64 ± 3.69 with LiCl; P < 0.001; Fig. 1E). We analyzed by quantitative RT-PCR the expression of myelin genes. The administration of lithium enhanced by twofold the transcripts of MPZ and PMP22 at DPI3 (Fig. 1F). At DPI8, LiCl did not enhance the expression of myelin genes. In conclusion, our results indicate that LiCl accelerates the functional recovery of whisker movements after facial nerve crush injury and improves the nerve phenotype. LiCl Administration Enhances Remyelination of Sciatic Nerve After Nerve Injury. To confirm the remyelinating effect of LiCl on an-

other peripheral nerve, we performed sciatic nerve crush on 8-wkold mice. Two weeks after the nerve crush, we treated the animals with LiCl (0.2%) in drinking water during 7 d (Fig. 2A). The administration of LiCl in drinking water is a more relevant clinical setting, as patients receive lithium orally. One week later, the animals were killed, and we performed real-time PCR to analyze the effect of LiCl on myelin gene expression, as well as histological study to observe the effect of LiCl administration on nerve shape. As expected, LiCl treatment was shown to enhance the 2 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.1121367109

mRNA expression of MPZ (threefold) and PMP22 (sixfold; Fig. 2B) compared with placebo-treated mice. Furthermore, the crushed nerves of LiCl-treated mice exhibited a thicker myelin sheath (Fig. 2C), suggesting nerve remyelination. This observation was confirmed by the fact that the g ratio of LiCl-treated mice was lower than that of placebo-treated ones (Fig. 2D). LiCltreated mice did not show any alteration in the axon diameter compared with controls. Taken together, our observations demonstrate an increase of myelin sheath diameter around the axons of sciatic nerve in LiCl-treated mice vs. placebo-treated ones. Pharmacological Inhibition of GSK3β Activates Myelin Gene Expression. We incubated Schwann cell line (MSC80) with LiCl

(10 mM) to examine whether LiCl has a direct impact on Schwann cells and on myelin genes. LiCl did not elicit any significant effect on MPZ or PMP22 transcripts after 1–8 h of treatment, but this drug stimulated MPZ transcript by threefold after 24 h of incubation (Fig. 3A). Interestingly, LiCl stimulation lasted longer. When MSC80 cells were incubated with LiCl for 48 h and 72 h, we observed a 20-fold and 54-fold stimulation of MPZ transcript, respectively (Fig. 3A). Stimulation of PMP22 transcript (2.5-fold) was also observed, but to a lesser extent than for MPZ (Fig. 3A). These results were confirmed at the protein levels. As shown in Fig. 3B, MPZ protein level was enhanced by 14 fold after 24 h and by 13 fold after 48 h of LiCl treatment. This observation is in accordance with the increase of MPZ transcript. Nevertheless, MPZ protein stimulation decreased after 72 h of treatment with LiCl (sevenfold stimulation). Such a discrepancy between MPZ transcript overexpression and protein decrease has already been described (12, 13). We also observed that the increase in PMP22 Makoukji et al.

protein expression was time-dependent: LiCl slightly enhanced PMP22 protein expression at 24 h (1.4 fold), but a clear induction of PMP22 protein was observed after 48 h (3.3 fold) and 72 h (2.3 fold) of treatment with LiCl. Similarly, LiCl enhanced myelin gene expression in Schwann cell primary cultures. As depicted in Fig. 3C, LiCl at 24 h augmented protein expression of MPZ (1.5 fold) and PMP22 (3.3 fold). LiCl did not further increase MPZ and PMP22 expression after 48 h or 72 h of treatment. To confirm the implication of GSK3β in the regulation of myelin genes, we used SB216763, a more potent and selective inhibitor of GSK3β. MSC80 cells were incubated with SB216763 (10 μM) during 3 h, 6 h, or 24 h. The expression of MPZ and PMP22 proteins was enhanced after 3 h of SB216763 treatment (1.8 fold and 2.5 fold, respectively) and lasted as long as 24 h (Fig. 3D). LiCl Enhances Expression and Nuclear Localization of β-Catenin Without Affecting Akt Signaling. LiCl is an inhibitor of GSK3β,

which is at the crossroads of several signaling pathways, including Wnt/β-catenin and Akt pathways. For that reason, we looked for the signaling pathway affected in a Schwann cell line after incubation with LiCl. LiCl treatment did not alter the phosphorylation status of Akt in MSC80 cells, ruling out the intervention of Akt (Fig. 4A). However, LiCl stimulated β-catenin protein expression by fourfold in MSC80 cells (from 24 h until 72 h of treatment; Fig. 4B) and in primary Schwann cells (24 h; Fig. 4C). SB216763 also increased the expression of β-catenin after 3 h of treatment until 24 h (Fig. 4D). The mimicking of Wnt/β-catenin pathway by LiCl was also confirmed by the 15-fold stimulation of a TOP-FLASH luciferase (luc) construct by LiCl (Fig. 4E). This Makoukji et al.

Fig. 3. Effect of LiCl on the expression of MPZ and PMP22 in MSC80 cells and in primary Schwann cells. MSC80 cells were incubated with LiCl (10 mM) from 1 h to 72 h. (A) Total RNA was extracted, and quantitative real-time PCR experiments were performed by using primers recognizing MPZ or PMP22. RT-PCR was normalized by using 26S RNA. Results represent the mean ± SEM of four independent experiments (*P < 0.05 and **P < 0.01 by Mann–Whitney test vs. control.) (B) Proteins were extracted and Western blots were performed by using anti-MPZ or anti-PMP22 antibodies. (C) Primary Schwann cells were treated with LiCl (10 mM) for 24 h. (D) MSC80 cells were treated with SB216763 (10 μM) for 3, 6, or 24 h. Proteins were extracted and Western blots were performed by using anti-MPZ or anti-PMP22 antibodies. GAPDH was used to normalize the Western blots. Results were reproduced at least in three independent experiments.

promoter contains several binding sites for TCF/LEF transcription factors that are downstream of Wnt/β-catenin signaling pathway. LiCl also enhances the expression of cyclin D1, which is a Wnt/β-catenin target gene (Fig. S1). Confocal microscopy analysis revealed that, in control MSC80, the majority of β-catenin was localized in the plasma membrane as well as in the cytoplasm. Only a small fraction was nuclear (Fig. 4 F and G). After a 24-h treatment with LiCl, a part of β-catenin was transferred into the nucleus. After 48 h and 72 h of LiCl treatment, β-catenin was localized in the cytoplasm, the cell membrane, and the nucleus (Fig. 4G). Thus, incubation of MSC80 cells with LiCl triggers the relocalization of a fraction of β-catenin into the nucleus. Taken together, these results demonstrate that, in Schwann cells, LiCl is able to mimic the Wnt/β-catenin signaling pathway (14) without affecting Akt phosphorylation. LiCl Promotes β-Catenin Recruitment on TCF/LEF Binding Sites on Myelin Gene Promoters. We investigated the incidence of LiCl

treatment on the regulation of myelin gene promoters by transfecting MSC80 cells with MPZ-luc or PMP22-luc constructs and incubating them with LiCl (10 mM) during 24 h. As shown in Fig. 5A, LiCl enhanced by fourfold and threefold the transactivation of MPZ-luc and PMP22-luc constructs, respectively. LiCl did not further increase MPZ-Luc or PMP22-Luc promoter activities after 48 h or 72 h of incubation. We performed ChIP experiments to identify the molecular targets of LiCl at the level of MPZ and PMP22 promoters. We have already shown that MPZ and PMP22 promoters contain PNAS Early Edition | 3 of 6

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Fig. 2. Effect of LiCl on the remyelination of sciatic nerve after nerve crush. (A) Nerve crush of the right sciatic nerve was performed in 8-wk-old mice. Two weeks after nerve crush, one group received 0.2% LiCl for 7 d. All animals were killed 4 wk after nerve crush, and their sciatic nerves were analyzed. (B) Sciatic nerves distal to the crush were dissected. Quantitative RT-PCR experiments were performed by using primers recognizing MPZ and PMP22. The quantitative RT-PCR was normalized by using 26S RNA (*P < 0.05 and **P < 0.01 by Mann–Whitney test vs. control.) (C) EM images of sciatic nerves 4 wk after crush from placebo- (Pl) and LiCl-treated mice. 1 and 2, Low-magnification EM images of section of sciatic nerve. (Scale bar: 5 μM.) 3 and 4, High-magnification EM images of myelin layers. (Scale bar: 0.5 μM.) (D) G-ratio analysis of sciatic nerves from placebo-treated animals (n = 8) and LiCl-treated animals (n = 8) 4 wk after crush. At least 100 axons per animal were quantified on an EM level.

Fig. 4. Effect of LiCl and SB 216763 on Akt and canonical Wnt pathway in MSC80 cells and in primary Schwann cells. (A) MSC80 cells were treated with LiCl (10 mM) during 24 h, 48 h, and 72 h. Western blots were performed by using anti-AktP or anti-Akt total antibodies. GAPDH was used to normalize the Western blots. These results were reproduced in three independent experiments. Protein extracts were prepared from MSC80 cells treated with LiCl (10 mM) during 0 h, 24 h, 48 h, and 72 h (B), primary Schwann cells treated with LiCl (10 mM) for 24 h (C), or MSC80 cells treated with SB216763 (10 μM) for 3, 6, or 24 h (D). Western blots were performed by using anti–β-catenin antibody. GAPDH was used to normalize the Western blots. Those experiments were reproduced at least three times. (E) MSC80 cells were transiently transfected with TOP-FLASH-luc plasmid, then incubated with LiCl (10 mM) for 24 h. β-Gal and luciferase activities were analyzed. Results are expressed as the induction over the basal activity, and they represent the mean ± SEM of at least 11 independent assays performed in duplicate (***P < 0.001 by Mann–Whitney test vs. control). (F) MSC80 cells were incubated in the absence (control) or in the presence of LiCl (10 mM) during 24 h, 48 h, and/or 72 h, and then monitored by confocal microscopy to localize β-catenin (Alexa 488, green) or nuclei (Topro3, red). This experiment was repeated three times, and a typical experiment is presented here. [Scale bars: Upper (low magnification, ×40), 20 μM; Lower (high magnification, ×63), 10 μM.]

active TCF/LEF-binding sites (Fig. 5 B and C) (4). ChIP analysis demonstrated that β-catenin recruitment by TCF/LEF binding domain (localized −8,487/−8,471 bp on the level of the MPZ promoter) was enhanced by 15 fold after 1 h of LiCl treatment. Concerning the PMP22 promoter, we identified a potential binding site of TCF/LEF in position +107/+124. ChIP assays showed that β-catenin recruitment increased by 30 fold after 1 h of incubation with LiCl. Discussion GSK3β is emerging as a prominent drug target in the central nervous system (15), in anticancer therapy (16), and in diabetes treatment. Lithium, a well known inhibitor of GSK3β, is commonly used as a mood stabilizer in the treatment of bipolar disorder (17, 18). Recent in vitro and in vivo studies described the effect of lithium in the treatment of acute brain injuries (e.g., ischemia, brain trauma) and in neurodegenerative diseases (e.g, Parkinson disease, Alzheimer’s disease, multiple sclerosis, and Huntington disease) (11, 19). Indeed, lithium has a neuroprotective effect by preventing neuronal death and stimulating neurogenesis (7). For example, in primary neuronal cultures, lithium protected neurons from apoptosis induced by excessive glutamate (20). Low doses of lithium were also shown to decrease striatal lesion induced by an NMDA receptor agonist in a rat excitotoxic model of Huntington disease (8) and also to reduce tau hyperphosphorylation (i.e., in Alzheimer disease) (9) through inhibiting GSK3β, Akt (21), and protein kinases A (22) and C pathways (23). Lithium is not only known to decrease brain damage, but also to promote survival, proliferation, and neuronal differentiation of neural progenitor cells in vitro and after transplantation for the treatment of spinal cord injury (24). Lithium treatment enhances the expression of cell survival genes like Bcl-2, BDNF, Hsp70, β-catenin, and CREB (20, 25, 26). This 4 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.1121367109

drug also attenuates the expression or activity of proapopotitic genes like Bax, caspase, cytochrome c, and β-amyloid peptide. In addition, lithium has an anti-inflammatory potential in the brain (27). Lithium reduces microglia activation and leukocyte infiltration in the spinal cord in EAE, suggesting its possible use in multiple sclerosis (11). Nevertheless, the effect of LiCl or other GSK3β inhibitors on remyelination of the peripheral nervous system is still unexplored. Therefore, we performed nerve crush on the facial nerve and sciatic nerve to study the effect of LiCl on remyelination after myelin injuries. We have tested two different peripheral nerves to demonstrate that the beneficial effects of lithium on remyelination are not limited a specific type of nerve. We have also used two different protocols. For the facial nerve, mice were directly treated after the lesion with i.p. injections of LiCl. For the sciatic nerve, we administered LiCl 14 d after lesion to verify its effects on myelination. Indeed, at this stage of regeneration, the remyelination of the regrowing axons has started and in addition, myelin debris have been removed by macrophages and Schwann cells, allowing to precisely evaluate the formation of new myelin. In this experiment, we also tested the efficacy of orally administered LiCl (added to drinking water) because patients receive lithium orally. In the model of facial nerve crush, LiCl administration accelerated the recovery of whisker movement in injured animals. Eight days after the crush, lithium-treated animals had practically totally recovered, whereas placebo-treated ones still had a palsy of the whiskers. LiCl greatly ameliorated the phenotype of the lesioned nerve: it enhanced the expression of myelin genes and increased the thickness of the myelin sheaths, as indicated by the decrease of the g-ratio. Consequently, after LiCl administration, the number of myelinated axons was doubled. We obtained comparable results in another peripheral nerve, the sciatic nerve. LiCl administration Makoukji et al.

promoters, as suggested by ChIP assays. Those observations are in accordance with our previous results that showed that the Wnt/ β-catenin pathway is essential for myelin gene expression (4). In conclusion, lithium is known to exert several beneficial effects on axonal regeneration and also has remyelinating effects. Therefore, this molecule could constitute a potential treatment of nerve injuries in which axonal lesions and demyelination coexist. Our results provide evidence that the GSK3β could be considered as an important actor in the expression of myelin genes, and they open approaches in the treatment of nerve injuries that use inhibitors of GSK3β such as lithium. Materials and Methods Cell Culture. Mouse Schwann cells (MSC80 cells) were transiently transfected by using Effecten reagent (Qiagen) as described by Makoukji et al. (13). Sixteen hours after transfection, the medium was replaced by DMEM with or without LiCl or SB216763. Primary Schwann cells were isolated from the sciatic nerves as described by Tawk et al. (4).

Fig. 5. Effect of LiCl on MPZ and PMP22 promoters. (A) MSC80 cells were transiently transfected with MPZ-Luc or PMP22-Luc plasmids and then incubated with LiCl (10 mM) for 24 h. Luciferase and β-gal activities were analyzed. Results are expressed as induction vs. basal activity, and they represent the mean ± SEM of at least 11 independent assays performed in duplicate (***P < 0.001 by Mann–Whitney test vs. control). Recruitment of β-catenin on the levels of MPZ (B) and PMP22 (C) promoters. LEF/TCF binding sites were localized in MPZ and PMP22 promoters (4). Cells were treated with LiCl (10 mM) for 1 h and then subjected to ChIP analysis by using control or anti–β-catenin antibody. Quantitative real-time PCR was performed to detect MPZ and PMP22 promoter regions including LEF/TCF binding elements (***P < 0.001 by Tukey post-hoc test after one-way ANOVA vs. control).

in drinking water ameliorated sciatic nerve phenotype, increased myelin sheaths thickness, and enhanced myelin gene expression. These encouraging results demonstrate that the administration of lithium either by injection or orally has beneficial effects in the treatment of myelin injuries. Does LiCl exert its positive effects on Schwann cells? To address this question, we performed in vitro experiments in primary cultures of Schwann cells and in the MSC80 cell line. LiCl increased cyclin D1 expression (Wnt/β-catenin target gene; Fig. S1) but did not augment the proliferation of MSC80 (Fig. S2). After 24 h, LiCl stimulated MPZ and PMP22 promoters, transcripts, and proteins. Interestingly, in MSC80 cells, LiCl enhanced myelin gene expression after 48 h and 72 h of treatment. We obtained a robust stimulation of MPZ mRNA (20 fold at 48 h and 50 fold at 72 h). The stimulation was lesser at the level of the PMP22 protein, probably because of posttranscriptional regulation of PMP22 mRNA. The kinetic of stimulation of MPZ and PMP22 by two distinct GSK3β inhibitors (SB216763 and LiCl) was different. Whereas SB216763 rapidly stimulated MPZ (i.e., after 1 h of treatment), LiCl activated MPZ expression only after 24 h. This discrepancy may result from the fact that these drugs vary in their potency to inhibit GSK3β. Actually, LiCl has an IC50 of 10 mM, whereas SB216763 is a more potent and selective inhibitor (IC50 of 34 nM). The treatment of cells with LiCl did not trigger any modification in the phosphorylation state of Akt, but enhanced the expression and nuclear localization of β-catenin and stimulated a TCF/LEF-containing promoter (TOPFLASH). Therefore, the stimulation of MPZ and PMP22 is not a result of the activation of the Akt pathway but rather of β-catenin/ TCF signaling. The nuclear stabilized β-catenin binds to TCF/LEF transcription factors present in the vicinity of PMP22 and MPZ Makoukji et al.

Semiquantitative RT-PCR Experiments, Western Blot, Immunocytochemistry, and ChIP. Semiquantitative RT-PCR experiments, Western blot, immunocytochemistry, and ChIP were performed as described by Tawk et al. (4), by Makoukji et al. (13), and in SI Materials and Methods. Facial Nerve Crush. Animals. Facial nerve crush experiments were performed on 8-wk-old male C57bl6 mice (Janvier). They were done in accordance with the requirements of the European 7 Communities Council Directive of November 24, 1986 (86/609/EEC). Each experiment was made to minimize the number of animals used and the suffering of animals. Nerve crush and drug treatment. Mice were anesthetized using a mixture of ketamine (0.5 mL) and xylazine (1 mL), and the left facial nerve near its exit from the stylomastoid foramen was exposed and crushed by applying pressure for 5 s by using a forceps on the trunk of the facial nerve. After surgery, the wound site was closed and animals were allowed to recover. Nonoperated mice were used as controls. All of the crushes were made by the same surgeon to minimize the variation of the crush among different animals. Following injury, the animals displayed a unilateral facial palsy with return of some eyelid and whisker movements after 14 d. Mice received a treatment after the nerve lesion: a 50 mg/kg/d i.p. injection of LiCl (n = 6 per group) during 4 d. The control or placebo received an injection of 0.1 M PBS solution. Movements of the whiskers were quantified at the three recovery times (i.e., DPI3, DPI8, and DPI14). Procedures used with WhiskerWatcher. The quantification of whisker movements was performed one day after the facial nerve crush. For that purpose, a device was fixed under general anesthesia (ketamine/xylazine) to the mouse’s skull by using dental cement (SuperBond; Sun Medical). This device restrained head movements and avoided recording parasite movements. The stand is composed of a cylinder in which the mouse is placed and of a rod enabling the fixation of the animal. A silver adhesive foil was then placed on vibrissae of the third rank on the left side. A sensitive camera placed above the vibrissae allowed the detection of all vibrissae movements (WhiskerWatcher; Arrington Research). The mice were stimulated with food or air breeze in the eyes. Every mouse was recorded at least six times each day by WhiskerWatcher software. The data collected corresponded to position Y and position X (providing 2D coordinates of the marker in millimeters), the angle variation of the vibrissae (in degrees), and the angular velocity (in degrees pe second). Sciatic Nerve Crush Injury. Adult 8-wk-old male mice were anesthetized and the right sciatic nerve was crushed by using fine forceps (13-cm smooth needle holder; FST) for 40 s just distal to the sciatic notch. As a control, the contralateral side was exposed but remained uninjured. Following the specified length of time, the mice were killed for immediate collection of tissues for histologic examination or RNA isolation. Methylene Blue/Azur II Staining and Transmission EM. Sample preparation and EM were peformed as described by Makoukji et al. (13) and in SI Materials and Methods.

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Plasmids and Chemicals. MPZ-luc was provided by G. Lemke (Salk Institute for Biological Studies, San Diego, CA), PMP22-luc by P. Patel (Baylor College of Medicine, Houston, TX), and TOP-FLASH-luc plasmid by R. T. Moon (University of Washington School of Medicine, Seattle, WA). LiCl and SB216763 were purchased from Sigma-Aldrich.

Statistical Analysis. Unless otherwise specified, means of treatment groups were compared with one-way ANOVA. When the ANOVA showed that there were significant differences between the groups, the Tukey test was used to identify the sources of these differences. A P value of 0.05 or lower was considered statistically significant. The variations of the angular speed were submitted to statistical analysis by the Kruskal–Wallis test, with results significant at P < 0.05.

ACKNOWLEDGMENTS. The authors acknowledge Dr. Alain Schmitt of the Cochin Imaging Facility. This work was funded by Institut National de la Santé et de la Recherche Médicale, Centre National de la Recherche Scientifique, University Paris Descartes, and the Association Française contre les Myopathies. J.M., M.B., G.S., and J.B. received doctoral fellowships from the French Ministry of Research. D.M. received a postdoctoral fellowship from Neuropole de Recherche Francilien.

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