Liver X receptors alpha and beta promote myelination and remyelination in the cerebellum

Significance

Liver X receptors (LXRs) α and β are the two major receptors of oxysterols, oxygenated derivatives of cholesterol. They control the homeostasis of cholesterol, an important lipid constituent of myelin sheaths. In the central nervous system, these insulating structures are generated by oligodendrocytes and are stabilized by myelin proteins. Here, we provide evidence of a new role of LXRs in the myelin physiology of the cerebellum. Mice invalidated for both LXRs exhibit alteration in motor coordination and spatial learning linked with myelination deficits. We demonstrated that LXRs intervene both in oligodendroglial cell maturation and in the transcriptional control of myelin gene expression during (re)myelination processes.

Abstract

The identification of new pathways governing myelination provides innovative avenues for remyelination. Liver X receptors (LXRs) α and β are nuclear receptors activated by oxysterols that originated from the oxidation of cholesterol. They are crucial for cholesterol homeostasis, a major lipid constituent of myelin sheaths that are formed by oligodendrocytes. However, the role of LXRs in myelin generation and maintenance is poorly understood. Here, we show that LXRs are involved in myelination and remyelination processes. LXRs and their ligands are present in oligodendrocytes. We found that mice invalidated for LXRs exhibit altered motor coordination and spatial learning, thinner myelin sheaths, and reduced myelin gene expression. Conversely, activation of LXRs by either 25-hydroxycholesterol or synthetic TO901317 stimulates myelin gene expression at the promoter, mRNA, and protein levels, directly implicating LXRα/β in the transcriptional control of myelin gene expression. Interestingly, activation of LXRs also promotes oligodendroglial cell maturation and remyelination after lysolecithin-induced demyelination of organotypic cerebellar slice cultures. Together, our findings represent a conceptual advance in the transcriptional control of myelin gene expression and strongly support a new role of LXRs as positive modulators in central (re)myelination processes.

Myelin sheaths, synthesized by oligodendrocytes in the central nervous system (CNS), form an insulating layer around axons. Specific myelin proteins, such as Proteolipid Protein (PLP) and Myelin Basic Protein (MBP), stabilize this structure, which is particularly rich in cholesterol. Myelination is a fine-tuned process that involves complex crosstalk between several signaling pathways, regulating oligodendroglial cell proliferation, migration, and differentiation.

Demyelination in the CNS can occur as a result of oligodendrocyte cell death or injury, which is usually accompanied or followed by axonal degeneration, functional impairments, and clinical disability. Currently available treatments for demyelinating diseases (i.e., multiple sclerosis) mostly target their inflammatory components and have a modest impact on the regenerative process of remyelination. Therefore, the development of therapeutic strategies enhancing remyelination is crucial for preventing axonal loss and consequent disability.

Experimental animal models of CNS demyelination indicate that remyelination is mediated by newly generated oligodendrocytes derived from oligodendrocyte progenitor cells (OPCs) (1). The eventual failure of remyelination may result from a nonpermissive environment or intrinsic deficits in the remyelinating cells. Thus, pharmacological agents that promote remyelination are predicted to act on OPC maturation/differentiation and myelin maintenance. They are expected to be important determinants of long-term functional outcome in demyelinating diseases.

Liver X receptors (LXRs) are members of the nuclear receptor superfamily (2). There are two isoforms of LXRs: LXRα and LXRβ. The natural ligands of LXRs are oxysterols originating from the oxidation of cholesterol [e.g., 24(S)-hydroxycholesterol (OH), 25-OH, and 27-OH]. The synthetic agonist TO901317 (TO9) also potently activates those receptors. Oxysterol levels are altered in several psychiatric or neurodegenerative diseases (3).

Studies with transgenic mice revealed important roles of LXRs within the nervous system. LXRα/β double knockout (LXR dKO) mice show several defects like axonal atrophy, neuronal loss, reduction in ventral midbrain neurogenesis (4), astrogliosis, and lipid accumulation in specific brain regions (5). We previously showed that, in the peripheral nervous system (PNS), LXRs inhibit myelin gene expression by mechanisms involving the canonical Wnt/β-catenin pathway (6, 7).

The aim of this study was to understand the role of LXRs in cerebellar myelination and remyelination. Here, we show that LXR dKO mice have both motor coordination and spatial learning alterations linked to cerebellar deficits. They exhibit thinner myelin sheaths and decreased myelin gene expression in the cerebellum. We also demonstrated that LXRs intervene both in oligodendrocyte maturation and in the transcriptional control of PLP and MBP expression. Finally, using organotypic cultures of cerebellar slices, we found that LXR activation enhances remyelination after lysolecithin-induced demyelination. Our data strongly support an unravelled role of LXRs as positive modulators of myelination and give original insight in the role of oxysterol/LXR signaling in myelination and remyelination.

Results

Invalidation of LXRs Impairs Motor Coordination and Spatial Learning.

First, we observed that the main actors of LXR signaling pathways are present in the cerebellum and in the spinal cord. We detected in these structures 24(S)-OH, 25-OH, and 27-OH (Fig. S1A); their biosynthetic enzymes (Fig. S1B); and the nuclear receptors LXRα and β (Fig. S2 A and B).

To assess the functional role of LXRs in the cerebellum, we evaluated LXR dKO mice for motor coordination and spatial learning ability, both of which are associated with cerebellar integrity. Global motor ability was overall preserved in LXR dKO mice. There was no difference in ambulation measured in an actimeter between LXR dKO and wild-type (WT) mice (Fig. 1A). Moreover, all of the WT and LXR dKO mice successfully completed the fixed rotarod paradigm (Fig. 1B, trial 0). However, on an accelerating rotarod test, LXR dKO mice showed little improvement in comparison with control mice, which managed to stay on the turning rod for a significantly longer time over repeated training sessions (Fig. 1B; margin for progression from 17 to 29 rpm for WT mice versus 7.5–12 rpm for LXR dKO). Although the LXR dKO mice achieved the inverted screen and the rod-crossing tests, they showed impaired motor coordination through a detailed analysis of motor patterns in the rod-crossing test. Indeed, the LXR dKO mice exhibited a higher hind paw slip score while crossing the 70-cm-long rod (LXR dKO, 6.35 ± 1.58 faults vs. WT, 1.22 ± 0.62 faults; Fig. 1C) and also displayed a higher stop number (Fig. S3A).

Fig. 1.

Invalidation of LXRs impairs motor coordination and spatial learning linked to cerebellar deficit. (A) Number of ambulation over 30 min for WT and LXR dKO mice in the actimeter. Data represent mean ± SEM (n = 5–7 animals per group; Student’s t test). (B) Performance of WT and LXR dKO mice in the rotarod. Trial 0 refers to a fixed (4 rpm) rotarod schedule. Trials 1–5 refer to an accelerating rotarod schedule. Results represent the rotarod speed at which the mice fall across five trial sessions (mean ± SEM of n = 5–6 animals per group; *P < 0.05 by ANOVA between genotype and session). (C) Number of faults (hind paw slips) counted during rod-crossing test (mean ± SEM of n = 5–7 animals per group; *P < 0.05 by Student’s t test). (D) Latency to reach the platform during acquisition trials of the place learning in the spatial version of the MWM (mean ± SEM of n = 5–7 animals per group; *P < 0.05 by ANOVA between genotype and session). (E) Distance traveled and (F) percentage of time spent in the relevant quadrant during probe test (relevant quadrant, NE; irrelevant quadrant, SW; mean ± SEM of n = 5–7 mice per group; *P < 0.05 compared with WT by ANOVA and Duncan’s post hoc).

Spatial learning was also severely impaired in LXR dKO mice, as evidenced in the hidden-spatial version of the Morris water maze (MWM) (Fig. 1 D–F and Fig. S3B). For the WT mice, 43.8% of the overall traveled distance was done in the relevant quadrant, whereas only 9.14% was observed for LXR dKO (Fig. 1E). WT mice stayed twice as long in the relevant quadrant than LXR dKO mice (Fig. 1F). However, there was no difference between LXR dKO and WT littermate performances in the visible-cued version of the MWM (Fig. S3C), suggesting a specific deficit in spatial learning, which has been related to cerebellar impairment (8) rather than to a generalized learning problem in LXR dKO mice.

Effect of LXR Modulation on Myelin in Vivo.

The motor coordination and spatial learning defects observed in LXR dKO mice prompted us to study the structure of myelin sheaths in the cerebellum of adult WT and LXR dKO mice by using electron microscopy. Myelin was altered in the cerebellum of LXR dKO animals (Fig. 2 A and B). We detected thinner myelin sheaths around the axons, as reflected by the significant higher g-ratio (axonal perimeter/outer perimeter) in LXR dKO animals (0.8292 ± 0.0055) compared with WT (0.8069 ± 0.0049) (Fig. 2C).

Fig. 2.

Invalidation or activation of LXRs alters myelin structure and myelin gene expression in the cerebellum. (A) Low magnification of cerebellar WT and LXR dKO electron micrographs. (Scale bar, 2 µm.) (B) High-magnification electron micrographs of a representative axon and its myelin layers. (Scale bar, 50 nm.) (C) Myelin thickness estimated by g-ratio (mean ± SEM, 3 animals per group; **P < 0.01 by t test). (D) Quantitative RT-PCR of WT and LXR dKO cerebellar RNA with primers recognizing PLP, MBP, or ABCA1 (positive control) normalized with GAPDH (glyceraldehyde 3-phosphate dehydrogenase) (mean ± SEM of n = 5 animals per group; **P < 0.01 and ***P < 0.001 by t test). (E) Western blots from WT and LXR dKO cerebellar proteins using anti-PLP and anti-MBP antibodies normalized with β-actin (mean ± SEM of n = 4 animals per group; **P < 0.01 and ***P < 0.001 by t test). Pictures represent a typical experiment. (F) Quantitative RT-PCR of cerebellar RNA from WT mice force-fed with TO9 or vehicle (veh) with primers recognizing PLP, MBP, or ABCA1 (mean ± SEM of n = 5 animals per group; *P < 0.05 and **P < 0.01 by t test). (G) Western blots of cerebellar proteins from WT force-fed with TO9 using anti-PLP and anti-MBP antibodies normalized with β-actin (mean ± SEM of n = 5 animals per group; *P < 0.05 by t test). (H) Cerebellar RNA from LXR dKO force-fed with TO9 as analyzed in F.

We analyzed the effect of the invalidation of both LXRs on the expression of the two major CNS myelin proteins, PLP and MBP, in the cerebellum. As depicted in Fig. 2D, the cerebellum of LXR dKO mice showed a significant 80% decrease in the expression of ABCA1, a LXR target gene used as a positive control. Concomitantly, we observed a 50% decrease in the amount of PLP and MBP transcripts (Fig. 2D) and proteins (–21.7 ± 6.8% and –31.6 ± 7.9%, respectively; Fig. 2E). Altogether, these results suggest that endogenous LXR signaling is an important actor of cerebellar myelin gene expression and myelination.

We next tested the influence of LXR activation on the expression of CNS myelin genes in vivo by force-feeding WT mice with the LXR agonist TO9. LXR activation was effective, according to the 15-fold increase in ABCA1 mRNA expression (Fig. 2F). MBP and PLP mRNA expression was increased by four- and fivefold, respectively. At the protein levels, TO9 increased the amount of PLP (1.8-fold) and MBP (2.2-fold) (Fig. 2G). We confirmed that the activation of myelin genes was mediated by LXRs, as the TO9 effect on PLP, MBP, and ABCA1 expression levels was greatly reduced in LXR dKO-treated mice (Fig. 2H). These results show that LXRs positively regulate myelin gene expression in the cerebellum.

LXR Activation Enhances Myelin Gene Expression and Oligodendroglial Maturation.

In primary mixed glial cells, TO9 and 25-OH enhanced by four- and twofold the mRNA amount of ABCA1, respectively, whereas PLP and MBP were increased by 150% and 120% (Fig. 3A). In isolated cells, LXR activation was less potent than in vivo but was significant. We confirmed the stimulation of the PLP transcript by TO9 and 25-OH in oligodendroglial cell line 158N (Fig. S4). The effects of oxysterols are exerted on myelin gene promoters, as we observed a 1.5-fold stimulation of PLP promoter activity by both TO9 and 25-OH (Fig. 3B). These results show that LXRs positively regulate myelin gene expression through promoter activation.

Fig. 3.

LXR agonists enhance myelin gene expression and oligodendrocyte maturation. (A) Quantitative RT-PCR of RNA from 7 DIV primary mixed glial cells treated with EtOH (control), TO9, or 25-OH using primers recognizing PLP, MBP, or ABCA1 and normalized using 26S (mean ± SEM of at least five independent experiments; *P < 0.05 by ANOVA one-way and Tukey’s post hoc test). (B) PLP promoter transactivation in 158N oligodendrocyte cells transiently transfected with PLP-luc and incubated with EtOH (control), TO9, or 25-OH (mean ± SEM of at least six independent experiments performed in duplicate; *P < 0.05 and ***P < 0.001 by t test). (C and D) Primary mixed glial cells from PLP-EGFP mice (7, 9 and 14 DIV) treated with EtOH or TO9. (C) After immunostaining for Olig2, positive cells for either Olig2 alone (Olig2⁺/PLP-EGFP^–) or Olig2 and PLP-EGFP (Olig2⁺/PLP-EGFP⁺) were counted using ImageJ software (mean ± SEM; **P < 0.01 by t test). (D) Proportion of three classes of PLP-EGFP oligodendrocytes (at 7, 9, or 14 DIV) determined by their ramified morphologies: simple, mild, or complex process outgrowth (mean ± SEM; *P < 0.05 and **P < 0.01 by t test).

We analyzed the effect of TO9 on oligodendroglial cell maturation using primary mixed glial cell cultures from PLP-EGFP transgenic mice. We counted the number of Olig2⁺/PLP-EGFP⁻ cells and Olig2⁺/PLP-EGFP⁺ cells to underline oligodendroglial lineage progression. Our data showed that LXR activation by TO9 led to a shift in the differentiation status of the cultured cells (Fig. 3C). Indeed, after 7 d in vitro (DIV), TO9 treatment significantly reduced by half the Olig2⁺/PLP-EGFP⁻ cell population and concomitantly increased the Olig2⁺/EGFP-PLP⁺ population rate (from 52% to 75%). This effect of TO9 was still present in 9 DIV cultures but greatly attenuated in 14 DIV cultures (Fig. 3C).

It is noteworthy that the cell differentiation program is concomitant to morphological changes with the emergence of multiple branched processes. Therefore, we analyzed the effect of a 24-h TO9 treatment on branching processes of PLP-EGFP⁺ cells cultured for 7, 9, and 14 DIV. We determined three classes of cell shape on the basis of their ramified morphologies (9), as defined by simple or mild process outgrowth (harboring zero to two and three to four processes emerging from the cell body, respectively) or complex process outgrowth (with more than four processes emerging from the cell body). Between 7 and 14 DIV, PLP-EGFP⁺ cell processes became more complex, leading to notable changes in the proportion of cell shape categories (Fig. 3D). TO9 stimulated branching processes in 7 DIV cultured oligodendrocytes (Fig. 3D, first panel, and Fig. S5) by increasing the proportion of complex branching cells (from 30% to 39%). This effect is more obvious after 14 DIV, where TO9 led to a drastic increase of the complex branching population from 53% to more than 70% (Fig. 3D, last panel, and Fig. S5). This increase was concomitant with a reduction in the simple branching population after 7 DIV (24% to 15%) and 14 DIV (14% to 8%). Interestingly, at 14 DIV, TO9 treatment also induced a significant reduction of the mild branching population (33% to 22%) to the benefit of the complex branching category.

Altogether these results suggest that oxysterols/LXRs are implicated in both oligodendroglial cell differentiation and maturation.

LXR Activation Increases Remyelination in Cerebellar Organotypic Cultures.

The results described above prompted us to address the question of a potential therapeutic use of LXR ligands in the treatment of demyelinating events. For that purpose, we treated lysolecithin-demyelinated cerebellar organotypic cultures with TO9 and 25-OH to assess remyelination status.

We showed that Purkinje cell axons, stained with the Calbindin Protein (CaBP) antibody, were not affected by lysolecithin treatment (Fig. 4A, red), as confirmed by axon number counting (Fig. 4B). In contrast, lysolecithin induced massive demyelination, leading to a sparse MBP staining (Fig. 4A, green) and to a 60% loss of myelinated Purkinje cell axons (Fig. 4C). Interestingly, the treatment of lysolecithin-demyelinated slices with TO9 reverted the demyelinated phenotype (Fig. 4A) by restoring the percentage of myelinated axons to normal levels (75%; Fig. 4C). The 25-OH exerted similar but less potent effects (54%; Fig. 4C). In LXR dKO organotypic cultures, lysolecithin treatment led to equivalent demyelination, whereas TO9 and 25-OH were unable to restore myelination status (Fig. S6 A–C), demonstrating that the beneficial effect of TO9 and 25-OH depends on LXRs presence.

Fig. 4.

LXR activation accelerates remyelination in organotypic cerebellar cultures. Control (vehicle) and lysolecithin-demyelinated cerebellar slices were treated for 3 DIV with EtOH, TO9, or 25-OH. After (A) double immunostaining for MBP and CaBP and confocal acquisition, (B) axon number and (C) percentage of myelinated fibers were quantified (***P < 0.001 by ANOVA and Tukey’s post hoc test; n = 18 animals per group). (Scale bar, 20 µm.) (D and E) Quantitative RT-PCR with primers recognizing PLP, MBP, and ABCA1 normalized using 26S (mean ± SEM of at least three independent experiments performed in duplicate; *P < 0.05 by t test). (F) Western blots for PLP and MBP normalized with β-actin and reproduced at least in three independent experiments (mean ± SEM; *P < 0.05 and **P < 0.01 by t test). (G) Control (vehicle) and lysolecithin-demyelinated organotypic slices treated with EtOH, TO9, or 25-OH. After immunostaining for Nkx2.2, numbers of immature oligodendrocytes were quantified with ImageJ (n = 12 animals per group; *P < 0.05, **P < 0.01, and ***P < 0.001 by t test). Representative micrographs are shown. (Scale bar, 50 μm.) (H) Control (vehicle) and demyelinated organotypic slices treated for 3 DIV with EtOH, TO9, or 25-OH and doubled immunostained for Caspr and MAG. A 5× magnification of a single Caspr cluster (pointed by the open arrowhead) is presented in each picture. (I) Number of Caspr clusters, (J) internode lengths, and (K) node lengths measured using ImageJ (means ± SEM; *P < 0.05, **P < 0.01, and ***P < 0.001 by t test).

Concomitantly, we studied the effect of TO9 after demyelination on myelin gene expression. PLP and MBP transcripts were decreased after lysolecithin treatment by 45% and 50%, respectively, whereas LXR signaling was not affected according to stable ABCA1 expression (Fig. 4D). Fig. 4E shows that in demyelinated slices, TO9 or 25-OH activated LXRs, as assessed by the fourfold increase in ABCA1 expression, without affecting LXRα and β expression. They potently stimulated PLP and MBP mRNA expression (by 2.5- and fivefold for TO9 and 25-OH, respectively). These observations were confirmed at the protein level, where TO9 and 25-OH increased the expression of both proteins after demyelination by two- and 1.5-fold, respectively (Fig. 4F and Fig. S7).

Because the results so far support that LXRs act on terminal myelination, we next investigated the effect of TO9 and 25-OH on immature (Nkx2.2⁺) oligodendrocytes. We highlighted a differential effect between the natural oxysterol and the synthetic LXR ligand, depending on pathophysiological conditions. Indeed, in normal slices, TO9 or 25-OH increased by threefold the number of Nkx2.2⁺ cells (Fig. 4G). Interestingly, after demyelination, TO9 but not 25-OH enhanced the number of immature oligodendrocytes. These results point out the influence of oxysterols on the immature oligodendrocyte population.

We investigated remyelination status by staining cerebellar slices with the Contactin-associated protein (Caspr) antibody to highlight paranodal junctions. In contrast to the demyelinated slices treated with vehicle alone, slices treated with TO9 or 25-OH were characterized by the detection of aggregates of Caspr (Fig. 4H). Counts of Caspr-positive clusters (Fig. 4I) showed a severe decrease of the number of nodes of Ranvier (dispersion of Caspr immunostaining) after lysolecithin demyelination in vehicle-treated slices, which was counteracted by TO9 or 25-OH treatment (35 ± 2.8 for control vehicle, 0.5 ± 0.4 for control + lysolecithin, 17.33 ± 1.76 for TO9 + lysolecithin, and 12 ± 1.15/0.02 mm² for 25-OH + lysolecithin). It is noteworthy that remyelination is accompanied by reorganization of Caspr aggregates and modifications in node and internode lengths (10). We highlighted that following LXR activation, aggregates differed by internode (Fig. 4J) and node lengths (Fig. 4K). Indeed, demyelinated slices treated with TO9 or 25-OH exhibited a significantly higher number of <30 μm internodes (21.2 ± 12.82% for control vehicle vs. 55.1 ± 32.3% for TO9 + lysolecithin and 73.8 ± 4.2% for 25-OH + lysolecithin) and larger nodes with respect to controls (0.85 ± 0.32 μm for control vehicle vs. 1.71 ± 0.14 μm for TO9 + lysolecithin and 2.08 ± 0.29 μm for 25-OH + lysolecithin), suggesting that these organotypic slices underwent remyelination. Conversely, LXRα/β invalidation abolished the remyelination properties of TO9 and 25-OH, demonstrating that oxysterols promote ex vivo remyelination in a LXR-dependent manner (Fig. S6D).

Collectively, these data indicate that oxysterols, via the LXR pathway, enhanced oligodendroglial differentiation and myelin gene expression and therefore the remyelination process under pathological conditions. Given that the modulation of the LXR pathway is already beneficial for the treatment of other pathologies of the nervous system (11, 12), our observations open new perspectives for the development of therapies targeting LXRs in demyelinating diseases.

Discussion

LXRs are crucial for homeostasis of cholesterol (13), a major lipid constituent of myelin sheath, required for both oligodendrocyte maturation and myelination (14). The cerebellum is a CNS region known to be involved in motor coordination and spatial learning but also cognition and emotion (15). Here, we show that this highly myelinated structure has large amounts of oxysterols and expresses their biosynthetic enzymes and their cognate receptors LXRα and β to exert their biological effects. We also demonstrate that both the natural (25-OH) and the synthetic (TO9) ligands of LXRs are able to stimulate myelin gene expression within oligodendrocytes. We propose that the endogenous oxysterols are able to enhance the expression of myelin genes through the LXR signaling pathway, contributing to the basal expression of those proteins.

The first study on LXRs in the CNS described variations in the expression level of LXR isoforms during development (16). Kainu et al. showed that LXRβ expression increased postnatally in the cerebellum to reach its maximum after 21 postnatal days. During this period, the cerebellum is known to undergo a myelination process, so we could speculate the involvement of LXR signaling during cerebellar development. Data collected so far appeared, however, inconclusive, as Andersson et al. did not notice any morphological structural alterations in the cerebellum of adult LXRβ KO mice nor neurodegeneration through Nissl staining (5). Conversely, LXRβ invalidation led to abnormal cerebellar layer development, and LXR activation was shown to promote granule neuron migration during postnatal development of the cerebellum (17, 18). We focused our study on the impact of both LXRα and β invalidation on cerebellar myelination status and revealed an alteration of myelin gene expression and a deregulation of the myelination process in LXR dKO mice. These mice also displayed impaired performances in motor and spatial learning behavioral paradigms, suggesting a functional role of LXRs in the cerebellum-related functions (8).

In line with the prominent regulatory roles of LXRs in CNS myelination that we demonstrate in the cerebellum, Xu et al. showed that LXRβ, by acting on OPC specification in another brain region, the dorsal cortex, is critical for white matter development and CNS myelination, especially in the corpus callosum (19). We provide evidence that LXR isoforms differentially affect CNS myelination. In fact, we also observed down-regulation of PLP and MBP mRNA expression in the corpus callosum of LXRβ KO mice in accordance with Xu et al. (Fig. S8A), but we lost this effect in the LXR dKO (Fig. S8B). Hence, it appears that part of the phenotype seen in the corpus callosum of LXRβ KO mice would be due to the presence of LXRα. By using dKO animals, we eliminated the effect of LXRα, unraveling a cerebellar implication of both LXRs in CNS myelination.

We also observed an opposite effect of LXR invalidation toward myelin gene expression in the CNS and PNS. Although LXRs increase central myelin gene expression in the cerebellum, we previously showed that LXRs repress peripheral ones in the sciatic nerve (6). We assume that this differential effect of LXRs depends in part on Wnt signaling modulation (6, 7). As a matter of fact, invalidation of LXRs enhances β-catenin expression in the sciatic nerve but represses it in the cerebellum (Fig. S9A). Interestingly, the treatment of demyelinated cerebellar slices by TO9 or 25-OH enhanced the amount of β-catenin (Fig. S9B). Therefore, Wnt and LXR pathways would be tightly associated in the myelination process.

The modulation of the LXR pathway is beneficial for the treatment of several pathologies of the nervous system either by modulating nervous cell functions or through its ability to modulate the inflammation process. LXR activation promotes synaptic plasticity and axonal regeneration in stroke patients (11) and is required for dopaminergic neuron differentiation, which is useful for Parkinson’s disease treatment (4, 20). In Alzheimer’s disease, LXR activation increases the number of cholinergic neurons and attenuates cognitive impairment by blocking the inflammatory process (12). In Experimental Autoimmune Encephalitis, a mouse model of multiple sclerosis, TO9 (via LXR) attenuates the inflammatory process to reduce the severity of the pathology (21, 22).

Besides the implication of LXRs in the modulation of inflammation, we provide here evidence that LXR ligands also act as remyelinating agents. We demonstrated both in vitro and ex vivo that LXR activation stimulates morphological cell maturation of oligodendroglial cells. Interestingly, Hendriks’ group showed that a 72-h treatment with TO9 of late immature rat oligodendrocytes reduced cell branching (23). These data and ours suggest that LXR activation exerts a differential effect regarding the differentiation status of oligodendrocytes. Indeed, as the culture grows and oligodendrocytes undergo differentiation, we noticed that the stimulation of both the branching process and myelin gene expression by TO9 observed at 7 DIV declines afterward.

Although we cannot exclude that part of the beneficial effect of LXRs could be due to reduced demyelination severity, we bring evidence that remyelination is stimulated, as suggested by enhanced oligodendrocyte differentiation status and Caspr staining analyses. We state that both LXRα and β are implicated in the remyelination potency of both natural oxysterol and synthetic LXR ligands (25-OH and TO9) as remyelination is abolished in LXR dKO demyelinated cerebellar slices. Accordingly, Magalon et al. demonstrated the beneficial use of another small cholesterol-like compound, olesoxime, for the treatment of demyelinating pathologies (24). Targeting nuclear receptors to stimulate remyelination is a challenging opportunity. It is noteworthy that the activation of LXRs by TO9 enhances the production of neuroprogesterone, which exerts a neuroprotective and promyelinating effect (25). We have previously shown that the modulation of steroid receptors (progesterone and glucocorticoid receptors) stimulates remyelination (26). Moreover, Retinoic X Receptor (RXR) has been identified as a good candidate for promoting remyelination. Franklin’s group showed that administration of 9-cis-retinoic acid to either demyelinated cerebellar slices or rats after demyelination caused an increase of remyelinated axons (27). Because LXRs have to form a heterodimer with RXR to induce gene expression, our findings, together with those of Franklin’s group, point out the LXR/RXR pathway as a promising avenue for remyelination strategies.

From a physiological and fundamental point of view, our results underline the complexity of the regulation of myelin genes and unravel the implication of LXRs in myelin gene expression. From a pathophysiological point of view, our observations open new perspectives for the development of therapies targeting LXRs for the treatment of demyelinating diseases.

Materials and Methods

In Vitro and ex Vivo Culture.

The immortalized mouse oligodendrocyte cell line 158N was transiently transfected by using Effectene reagent (Qiagen), as previously described (28). This cell line expresses PLP but not MBP. Primary glial cells were prepared as described in ref. 29 from the P1–3 mouse brain from c57/bl6 or PLP-EGFP mice provided by W. B. Macklin, University of Colorado School of Medicine, Aurora, CO (30), and were grown during 7–14 DIV. P10 organotypic slices of cerebellum (350 μm) were prepared as described in refs. 26 and 31. After 7 DIV, they were demyelinated with lysolecithin overnight (0.5 mg/mL). All cultures were treated with EtOH (0.05%), 25-OH, or TO9 (10 μM).

Animals and in Vivo Experiments.

Eight-week-old male LXR dKO mice and their WT controls were provided by J. M. Lobaccaro, UMR 6547, CNRS, Clermont-Ferrand, France, and Herve Guilloux, Institut National de la Recherche Agronomique, UMR 1331, Toulouse, France. All aspects of animal care were approved by the Regional Ethics Committee (authorization CEEA34.MJT.048.12). Each experiment was made to minimize the number of animals used and the suffering of animals. Mice were tested for motor function and in the MWM as previously described (32, 33) (SI Materials and Methods). Independent groups of mice were force-fed with 45 mg/kg TO9 or vehicle (methylcellulose, vehicle) 16 h before the sacrifice (34). Cerebella were collected and frozen in liquid nitrogen.

Plasmids and Chemicals.

PLP-luc was provided by W. B. Macklin, University of Colorado School of Medicine, Aurora, CO. Lysolecithin and 25-OH were purchased from Sigma-Aldrich and TO9 from Bertin Pharma (France).

Oxysterol Quantification by GC/MS.

Cerebella and spinal cords from either WT or LXR dKO animals (n = 4 per group) were collected, and oxysterols were extracted and assayed as described in ref. 28 (SI Materials and Methods).

Western Blot, Immunochemistry, and Semi- and Quantitative RT-PCR.

Western blot, semiquantitative RT-PCR, and quantitative RT-PCR were performed as previously described (6, 29), and immunochemistry was as described in ref. 26 (SI Materials and Methods).

Methylene Blue/Azur II Staining and Transmission Electron Microscopy.

Sample preparation and EM were performed as described in ref. 6.

Statistical Analysis.

Unless otherwise specified, two group comparisons were performed by Student’s t test. Means of multiple treated groups were compared by one-way analysis of variance (ANOVA). When the ANOVA showed that there were significant differences between the groups, Tuckey’s post hoc tests were used to identify the sources of these differences. A P value ≤ 0.05 was considered statistically significant. Analyses were performed with GraphPad Prism 4 Software, and we indicated *P < 0.05, **P < 0.01, and ***P < 0.001. For the accelerating rotarod and the MWM paradigms, data were analyzed with one-way, repeated-measures (between factors, genotype; within, session) ANOVA followed by Duncan’s test. Analysis was performed with STATISTICA software. Significance was set at P < 0.05.