Liver X receptor β is essential for the differentiation of radial glial cells to oligodendrocytes in the dorsal cortex

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Subjects

Abstract

Several psychiatric disorders are associated with aberrant white matter development, suggesting oligodendrocyte and myelin dysfunction in these diseases. There are indications that radial glial cells (RGCs) are involved in initiating myelination, and may contribute to the production of oligodendrocyte progenitor cells (OPCs) in the dorsal cortex. Liver X receptors (LXRs) are involved in maintaining normal myelin in the central nervous system (CNS), however, their function in oligodendrogenesis and myelination is not well understood. Here, we demonstrate that loss of LXRβ function leads to abnormality in locomotor activity and exploratory behavior, signs of anxiety and hypomyelination in the corpus callosum and optic nerve, providing in vivo evidence that LXRβ deletion delays both oligodendrocyte differentiation and maturation. Remarkably, along the germinal ventricular zone-subventricular zone and corpus callosum there is reduced OPC production from RGCs in LXRβ−/− mice. Conversely, in cultured RGC an LXR agonist led to increased differentiation into OPCs. Collectively, these results suggest that LXRβ, by driving RGCs to become OPCs in the dorsal cortex, is critical for white matter development and CNS myelination, and point to the involvement of LXRβ in psychiatric disorders.

Introduction

Deterioration of white matter structures including axons, myelin sheaths and oligodendrocytes (OLs) contribute to the pathophysiology of central nervous system (CNS) diseases.1,2

Mature OLs derived from oligodendrocyte precursor cells (OPCs) are the myelin-forming cells of the CNS, which surround axons to form myelin and mediate the fast conduction of neuronal information.3,4 The earliest differentiated OPCs come from the ventral cortex of the embryo and are gradually degraded and disappear after birth,5,6 whereas the OPCs produced from the dorsal cortex continue to differentiate into OLs and are involved in myelination after birth.7,8 However, the pathway of generation of OPCs in the dorsal cortex has not yet been clarified. In the developing CNS, radial glial cells (RGCs) have two functions: (1) they act as neural precursor cells to form new neurons in the cortex; and (2) they are migrational guides for later-born neurons, and thus are critical for morphogenesis of the cerebral cortex.9, 10, 11 During late neurogenesis, RGCs primarily transform into glia cells.10 Recently, it has been suggested that RGCs may also contribute to the production of OPCs in the dorsal cortex,12 although the underlying mechanism remains unclear.

Liver X receptors (LXRs) α and β are ligand-activated nuclear receptors, active in the regulation of lipid metabolism.13,14 Analysis of the expression patterns of these receptor subtypes has shown that although LXRα is mainly expressed in tissues with high lipid metabolism such as liver, intestine, adipose tissue and macrophages, LXRβ is expressed ubiquitously.15 Furthermore, LXRβ has an important role in lamination of the cerebral cortex by guiding migration of later-born neurons during corticogenesis. Loss of LXRβ in mice causes significant reduction of RGC long fibers in the cerebral cortex during later embryonic stages.16 In the cerebellum, LXR agonist treatment of neonatal mice promotes the migration of granular neurons by inhibition of RGC transformation into astrocytes during development.17 Consistent with these findings, LXRβ deletion induced astrogliosis in the spinal cord and substantia nigra.18,19 Early studies with LXRβ−/− mice first revealed the importance of LXRs in maintaining the intregrity of myelin sheaths.20 Later loss of LXRβ was found to exacerbate demyelination in mouse experimental autoimmune encephalomyelitis.21 Accordingly, as an important regulatory factor involved in the maintenance of RGCs, LXRβ may determine the choice of differentiation of RGCs to astrocytes or OPCs, respectively, and thereby affect myelination in the adult CNS.

In the present study, we examined the role of LXRβ in the myelination and oligodendrogenesis in the corpus callosum. We report here that LXRβ−/− animals exhibit behavioral abnormalities, delayed myelination and abnormal myelin thickness. Furthermore, we show that ablation of LXRβ negatively regulates OL production and typically inhibits RGC differentiation into OPCs. Thus, our study reveals a previously uncharacterized role for LXRβ in myelination.

Materials and methods

Animals

Generation of LXRβ−/− mice was described previously.22 LXRβ+/ female mice were mated overnight with LXRβ+/− males and inspected at 0900 hours on the following day for the presence of vaginal plugs. Noon of this day was assumed to correspond to E0.5. All animals were housed in the animal facility of the Third Military Medical University in a controlled environment on a 12-h light/12-h dark illumination schedule and were fed a standard pellet diet with water provided ad libitum. All experimental procedures were performed in accordance with approved principles of laboratory animal care and ethical approval by the Third Military Medical University.

Immunohistochemistry and Immunofluorescence

Brains were dissected and fixed in 4% paraformaldehyde for 24 h at 4 °C. For paraffin sections, tissues were processed for paraffin embedding, and coronal sections (5 μm thick) were collected. For cryostat sections, brain was postfixed in 30% sucrose solution with 4% paraformaldehyde at 4 °C, and coronal cryosections (40 or 15 μm thick) were collected. Paraffin sections were used for LXRβ immunohistochemistry according to Fan et al.6 In brief, brain sections were deparaffinized in xylene and rehydrated through graded ethanol. Antigens were retrieved by boiling in 10 mM citrate buffer (pH 6.0). Endogenous peroxidase was quenched with 0.3% hydrogen peroxide in 50% methanol and nonspecific binding blocked with 1% bovine serum albumin. For LXRβ staining, slides were incubated with 0.15 units ml−1 of β-galactosidase for 2 h at room temperature. Thereafter, sections were incubated with goat anti-LXRβ antibody (1:1000; made in the Jan-Ake Gustafsson's laboratory, Karolinska Institute, Novum, Sweden) at 4 °C overnight. After thorough washing, sections were incubated for 2 h with biotinylated secondary antibody (Vector Laboratories, Burlingame, CA, USA) at a 1:200 dilution in phosphate-buffered saline. The sections were rinsed and incubated with avidin-biotinylated horseradish peroxidase complex (Vectastain Elite ABC kit; Vector Laboratories) for 1 h. The peroxidase–substrate reaction was visualized by 3,3′-diaminobenzidine (DAKO, Carpenteria, CA, USA). The sections were slightly counterstained with hematoxylin.

Coronal cryosections, 40 μm thick, were incubated overnight at 4 °C with the primary antibodies in 1% bovine serum albumin: goat polyclonal anti-MBP (1:200) and rabbit anti-PDGFRα (1:200) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-Olig2 (1:1000) (Millipore, Temecula, CA, USA), and mouse anti-APC/CC-1 (1:100, Calbiochem, San Diego, CA, USA). Sections were then incubated with the avidin–biotin complex, followed by the biotinylated secondary antibodies (1:200; 2 h, 37 °C) and finally staining was visualized using the 3,3′-diaminobenzidine substrate kit. For immunofluorescence, sections (15 μm thick) were incubated with rabbit anti-PDGFRα 1:200 and mouse anti-Olig2 (1:1000, Millipore), mouse anti-APC/CC-1 (1:100) and rabbit anti-Olig2 (1:1000), rabbit anti-BLBP (1:500, Millipore) and rat anti-PDGFRα (1:300, Millipore) or mouse anti-Olig2 (1:1000) overnight at 4 °C. Bovine serum albumin alone served as the negative control. The secondary antibodies, Cy3- or 488-conjugated (both at 1:500, 3 h; Jackson ImmunoResearch, West Grove, PA, USA) were then added (3 h, at room temperature), respectively. Sections were counterstained with 4',6-diamidino-2-phenylindole (Sigma-Aldrich, St. Louis, MO, USA) and then viewed using the Leica TCS SP2 spectral confocal laser-scanning microscope (Leica, Wetzlar, Germany).

Western blot analysis

The cerebral cortex was harvested from wild-type (WT) and LXRβ−/− mice at P2, P7, P10, P14 and P6W. The protein content was measured using a bicinchoninic acid protein assay with bovine serum albumin as a standard. Protein was mixed with 5 × loading buffer and then submitted for electrophoresis in a 12% SDS-polyacrylamide gel electrophoresis gel and electrically transferred onto a polyvinylidene difluoride transfer membrane. Membranes were blocked in 5% fat-free milk and then incubated overnight at 4 °C with the primary antibodies followed by peroxidase-conjugated secondary antibody labeling for 1 h at room temperature. The following primary antibodies were used: anti-β-actin (1:1000, Santa Cruz Biotechnology), anti-MBP (1:1000, Santa Cruz Biotechnology), anti-PDGFRα (1:1000, Santa Cruz Biotechnology), anti-Olig2 (1:1000, Chemicon, Temecula, CA, USA) and anti-β-catenin (1:1000, Sigma, St Louis, MO, USA). Final visualization was achieved using an enhanced chemiluminescence western blotting analysis system (Pierce, Rockford, IL, USA), and the signals were exposed to X-ray films (Kodak, Rochester, NY, USA) and analyzed by the Gel-Pro analyzer (Quantity One 4.0; Bio-Rad Laboratories, Hercules, CA, USA). Western blots were obtained from the cerebral cortex from three animals of each genotype and age. Data were averaged and represented as means ±s.e.m.

Electron microscopy and morphometric analysis

Six-week- and three-month-old littermate mice were processed for electron microscopy (three LXRβ−/− and three WT. Male mice were perfused intracardially with 4% paraformaldehyde/2.5% glutaraldehyde in 0.1 M phosphate-buffered saline. Optic nerves and corpus callosum were cut into small cubes (1 mm) under a dissecting microscope, then postfixed overnight at 4 °C. Tissues were rinsed in phosphate-buffered saline, postfixed in 1% osmium tetroxide for 2 h, dehydrated in a graded series of ethyl alcohol, infiltrated with propylene oxide and embedded in Epon. Ultrathin sections (~60 nm thick) were generated by an ultramicrotome (LKB-V, LKB Produkter AB, Bromma, Sweden) and counterstained with uranyl acetate and lead citrate. Sections were viewed with a transmission electron microscope (TECNAI10, Philips, Eindhoven, The Netherlands). Digital images were acquired with an AMT XR-60 CCD Digital Camera System (Advanced Microscopy Techniques, Danvers, MA, USA) and compiled and analyzed using Adobe Photoshop and ImageJ (NIH). The g-ratio of myelinated axons was determined by dividing the axon diameter by the myelin diameter. A minimum of 344 axons was measured for the corpus callosum of each genotype at each age.

Behavioral tests

Male LXRβ−/− mice and their WT littermates (6 weeks old, 3, 7 and 10 months old) were housed in a controlled environment (20–23 °C) with free access to food and water and maintained on a 12 h/12 h day/night cycle, light on at 0600 hours. Behavioral experiments were performed between 1000 and 1400 hours in an arrangement of 24 h in between behavioral tests. The following assays were performed: open-field and plus-maze tests to measure motor exploration and anxiety, respectively. For all behavioral experiments, investigators were blinded for LXRβ−/− or WT mice. After each experiment all the apparatuses were wiped clean to remove traces of the previous assay.

Open field was measured using an open-field activity system (Biowill, Shanghai, China) and activity software (Biowill). Mice were placed in the center of the open-field box, and activity was recorded for a period of 10 min. The total and center-area distances were measured and the time in the central area was recorded.

Elevated plus-maze test was used to measure anxiety levels of mice as they avoid the open arms of the plus maze. Briefly, a mouse was initially placed in the center area facing an open arm. The mouse’s entry into any of the four arms was counted when all four paws crossed from the central region into an arm. The number of total arm entries and the amount of time spent in the open arms during a 10-min testing period were recorded.

Quantification of cholesterol in the cerebral cortex

Cholesterol in the cerebral cortex was extracted by homogenization with chloroform–Triton X-100 (1% Triton X-100 in pure chloroform) in a microhomogenizer. The mixture was vortexed and centrifuged at 16,000 g for 10 min at 4 °C. The lower organic phase was collected and air-dried at 50 °C, and chloroform traces were removed with N2 flow. The cholesterol content was then determined with a cholesterol quantification kit (BioVision, Milpitas, CA, USA) according to the manufacture's instructions.

Cell culture, differentiation and factor treatments

Cultures of radial glial clone L2.3 (a gift from Professor HD Li) have been described previously.23 Briefly, culture medium contained DMEM/F12 (Invitrogen, Carlsbad, CA, USA) supplemented with 25 mM glucose (Sigma), 2 mM glutamine (Invitrogen), penicillin/streptomycin (Invitrogen), 10 ng ml−1 FGF2 (BD Biosciences, San Jose, CA, USA), 2 μg ml−1 heparin (Sigma), and 1 × B27 (Invitrogen). Cells were propagated as neurospheres and passaged by mild trypsinization (0.025% for 5 min) every 3 days. For differentiation,24 cells were cultured on laminin-coated coverslips in FGF2 containing serum-free medium for 1 day, then the medium was replaced with DMEM/F12 plus N2 in the presence of FGF2, PDGF and forskolin for 4 days, and the growth factors withdrawn in the presence of 3,3,5-tri-iodothyronine hormone (T3) and ascorbic acid for another 4 days. Treatments with or without the LXR agonist T0901317 (0.1 μM or 1 μM) started on day 1 until the end of differentiation.

Cultured cells were fixed with 4% paraformaldehyde for 15 min at room temperature, washed three times with phosphate-buffered saline, followed by incubation for 1 h at room temperature with the primary mouse anti-NG2 antibody (1/200; Chemicon), and rabbit anti-BLBP antibody (1/400). Next, the cells were washed and incubated for 1 h with Cy3-conjugated goat anti-mouse or 488-conjugated goat anti-rabbit antibody. Cells were counterstained with 4',6-diamidino-2-phenylindole, and coverslips were viewed with the Carl Zeiss Axioplan microscope (Oberkochen, Germany).

Statistical analysis

All data are expressed as the mean±s.e. of the mean, and were analyzed by the Student’s t-test, or a one-way analysis of variance followed by Fisher’s protected least-significant difference post hoc test or a least-significant difference multiple-comparison t-test. Significance was reached at values of P<0.05. Statistical analysis was performed using the Statistical Product and Service Solutions software V13.0 (SPSS, Chicago, IL, USA).

Results

LXRβ expression in the corpus callosum and ventricular zone (VZ) /subventricular zone (SVZ) of mice from P2 to P6W

Immunohistochemistry with an anti-LXRβ antibody was used to map the spatiotemporal expression pattern of LXRβ protein in the corpus callosum and SVZ in postnatal mouse brain, at P2, P7, P14 and P6W. LXRβ expression was detected in both corpus callosum (Figures 1a, c, e, g and i) and the SVZ (Figures 1b, d, f, h and j) from P2 to adulthood. LXRβ expression was higher in the SVZ than that in the corpus callosum from P2 to P10 (Figures 1a–f). There was a similar level of LXRβ expression in the corpus callosum and SVZ at P14 (Figures 1g and h) and P6W (Figures 1i and j), of which LXRβ expression was less at 6W compared with P14. This expression pattern indicates that LXRβ might be related to postnatal white matter development and myelination.

Figure 1
figure1

Liver X receptor (LXR)β expression in the corpus callosum and subventricular zone (SVZ) of mice from P2 to P 6W. LXRβ expression was detected in both corpus callosum and the SVZ from P2 to P 6W. LXRβ expression was higher in the SVZ from P2 to P10 (af), which was of similar level in the corpus callosum and SVZ at P14 (g and h) and P6W (i and j). Scale bar: aj, 50 μm. CC, corpus callosum; LV, lateral ventricle; P, postnatal day; SVZ, subventricular zone; VZ, ventricular zone; W, week.

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Abnormality in the total locomotor activity of LXRβ KO mice

We examined the pattern of free movement of mice at 6 weeks and 3 months of age using open-field equipment. In the open-field test (Figure 2), the LXRβ knockout (KO) mice at 6 weeks of age showed significantly decreased total locomotor activity (P<0.01), and a similar trend was confirmed for mice aged 3 months (P<0.05). However, at 6 weeks and 3 months of age loss of LXRβ in mice did not significantly affect the time or distance in the central area, indicating that the KO mice had no altered anxiety level. Abnormal exploratory behavior remained in the KO mice at 7 and 10 months of age (Supplementary Figure S1).

Figure 2
figure2

An illustrative example of travel pathway in the open-field test of a control and an liver X receptor (LXR)β knockout (KO) mouse at 6 weeks of age (a and b) and 3 months of age (f and g). LXRβ KO mice at 6 weeks of age traveled less distance overall (c) compared with wild-type (WT) controls (n=11, **P<0.01), whereas there was no change either in the time (d) or distance traveled (e) in the center. Similarly, traveled distance overall (h) was decreased by loss of LXRβ in the mice of 3 months of age (n=16, *P<0.05). Meanwhile, LXRβ KO mice and WT controls spent similar time (i) and traveled similar distances (j) in the center. NS, not significant.

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In the elevated plus-maze assay (Figure 3), a two-way analysis of variance revealed no difference between WT and LXRβ KO mice at 6 weeks of age for either percentage open-arm entries or percentage open-arm time. By 3 months of age, the percentage of open-arm entries for LXRβ KO mice was less than that in WT mice (P<0.05), although no alteration was recorded for the percentage of open-arm time. But, in mice 7 and 10 months of age, the percentage open-arm entries or percentage open-arm time was not different from those of WT mice (Supplementary Figure S1). These results suggest that the anxiety in LXRβ KO mice at 3 months is temporary.

Figure 3
figure3

An illustrative example of the travel pathway on the elevated plus-maze assay of a control and an liver X receptor (LXR)β knockout (KO) mouse at 6 weeks of age (a and b) and 3 months of age (e and f). There is no alteration in the percentages of open-arm entries (c) or open-arm time (d) between wild-type (WT) and LXRβ KO mice at 6 weeks of age. By 3 months of age, percentage of open-arm entries of the LXRβ KO mice was decreased compared with WT mice (g, *P<0.05), although no alteration was observed in the percentage open-arm time (h). NS, not significant.

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Electron micrographs reveal extensive hypomyelination in the corpus callosum and optic nerve in LXRβ KO mice at ages of 6 weeks and 3 months

To examine myelin sheaths in LXRβ KO mice, we analyzed the corpus callosum and optic nerve by electron microscopy. At 6 weeks of age, LXRβ KO (Figures 4c and d) compared with WT mice (Figures 4a and b) had thinner myelin sheaths with higher g ratios (numerical ratio between the diameter of the axon proper and the outer diameter of the myelinated fiber) in the corpus callosum. At 3 months of age, the demyelination of LXRβ KO mice (Figures 4g and h) in the corpus callosum was more severe and the thickness of myelin surrounding axons was reduced compared with WT (Figures 4e and f) resulting from a decrease in the number of wraps (Figures 4i and j). We also observed slight axonal swelling or degeneration in the LXRβ KO mouse (Figures 4d and h). These changes were not limited to the corpus callosum, as hypomyelination was also detected in the optic nerve (Supplementary Figure S2). Hypomyelination in the optic nerve (Supplementary Figure S3) was confirmed by the prolonged latency of the P2 component of the Flash visual evoked potentials in the LXRβ KO mice.

Figure 4
figure4

Myelinated axons in 6-week-old and 3-month-old liver X receptor (LXR)β-deficient corpus callosum. (a–d) Corpus callosum prepared from 6-week-old wild-type (WT) (a and b) and LXRβ-deficient (c and d) mice were examined by electron microscopy. (eh) Corpus callosum prepared from 3-month-old WT (e and f) and LXRβ-deficient (g and h) mice were examined by electron microscopy. Scale bar: a, c, e and g, 1 μm; b, d, f and h, 1 μm. Higher magnification of images demonstrate proper axonal myelination (b and f), and loosely wrapped or lack of myelin ensheathment (d and h) (examples denoted by asterisk). (i) The number of myelinated axons is significantly decreased in LXRβ knockout (KO) mice compared with WT controls (n=15 fields from at least 3 animals per genotype at each age were analyzed). *P<0.05, **P<0.01 by analysis of variance statistical analysis, followed by Tukey’s test. Error bars indicate +/−s.e.m. (j) Graph shows that loss of LXRβ induced changes in G ratios compared with WT littermates at P6W and P3M (n=3 brains for each age and each genotype; 344 axons analyzed (**P<0.01). M, month; W, week.

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Delay in the initiation of myelination and hypomyelination in LXRβ KO mice

The level of the major CNS myelin component, myelin basic protein (MBP), was examined in LXRβ KO mice. In 10-day-old (Figures 5a–d) and 14–day-old (Figures 5e–h) animals there was a reduction in MBP level of LXRβ KO mice. Immunostaining of the brain from 6-week-old (Figures 5i–l) mice with anti-MBP antibody revealed widespread loss of fine MBP reactive fibers in all layers at this late stage of myelination. Western blots confirmed lower levels of MBP in LXRβ KO than in WT mice (Figures 5m and n).

Figure 5
figure5

The expression of myelin basic protein (MBP) is reduced during postnatal development in liver X receptor (LXR)β knockout (KO) mice compared with littermate controls. At P10 (a–d), P14 (eh) and P6W (il), MBP immunohistochemistry reveals a reduction in MBP expression in the corpus callosum and overlying cortex of LXRβ KO mice compared with their WT littermates. Scale bar: a–h, 100 μm; il, 200 μm. CC, corpus callosum; CTX, cortex; P, postnatal day; W, week. (m) Western blots reveal reductions in MBP in LXRβ KO mice compared with controls. (n) Graph represents changes in expressions of MBP in the cerebral cortical lysates indicating a reduction induced by loss of LXRβ (n=3; brains for each age and each genotype; **P<0.01).

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Loss of LXRβ affects postnatal myelination in the corpus callosum

Myelin is generated by mature OLs that express adenomatous polyposis coli known as CC1 marker of postmitotic OLs. Quantification of CC1-positive cells at P6W showed that, compared with WT mice, there was a 41% (Figure 6e) decrease in CC1-labeled OLs in corpus callosum from LXRβ-KO mice (Figures 6a–e). The number of CC1+/Olig2+ cells was reduced by 31% (Figure 6l) in multiple regions, including corpus callosum of LXRβ KO mice at P14 (Figures 6f–l), suggesting that lack of mature OLs could account for the hypomyelination in the LXRβ KO brain.

Figure 6
figure6

Maturation of oligodendrocytes is delayed during postnatal development in liver X receptor (LXR)β knockout (KO) mice compared with littermate controls. (a–d) At P6W, CC1 immunohistochemistry reveals a reduction of CC1-positive cells in the corpus callosum and overlying cortex of LXRβ KO mice (b and d) compared with their wild-typw (WT) littermates (a and c). (e) The mean cell density of CC1 per volume (mm3) of corpus callosum was determined for WT (white bar) and LXRβ KO (black bar) animals at P6W. (f–k) At P14, Olig2 and CC1 double-positive cells were decreased by loss of LXRβ. (l) The mean cell densities co-expressing CC1 and Olig2 per volume (mm3) of corpus callosum were determined for WT (white bar) and LXRβ KO (black bar) animals at P14. *P<0.05, **P<0.01 compared with WT. Scale bar: 100 μm (a and b); 50 μm (c, d and fk). CC, corpus callosum; P, postnatal day; W, week.

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LXRβ regulates the number of OL progenitors in developing white matter

Significantly fewer PDGFRα-labeled OPCs were found in the developing corpus callosum of LXRβ−/− mice (Figures 7b, d and f) compared with WT mice (Figures 7a, c and e) at P7 (Figures 7a and b), P10 (Figures 7c and d) and P14 (Figures 7e and f). Western blots revealed that PDGFRα protein levels were consistently lower in cerebral cortical lysates from LXRβ−/− than in WT mice (Figures 7o and q). These findings indicate that LXRβ is essential for maintaining the correct number of OPCs to populate the developing corpus callosum.

Figure 7
figure7

The expressions of PDGFRα and Olig2 were reduced during postnatal development in liver X receptor (LXR)β knockout (KO) mice compared with littermate controls. (al) Immunohistochemistry on corpus callosum sections from P7, P10 and P14, with antibodies against PDGFRα (af) and Olig2 (gl) as indicated. Oligodendrocyte progenitor cells (PDGFRα+) (m) and (Olig2+) (n) per mm3 in the corpus callosum in wild-type (WT) (white bars) and LXRβ KO (black bars) littermates at ages of P7, P10 and P14. Graphs are mean (±s.e.m.) counts obtained from four different areas of the corpus callosum (n=3; brains for each age and each genotype; *P<0.05, **P<0.01). Western blot analysis showed a marked decrease in PDGFRα (o) and Olig2 (p) expression in the cerebral cortical lysates from LXRβ KO mice compared with WT from P7 to P14. Graphs represent changes in PDGFRα (q) and Olig2 (r) expression from P7 to P14 indicating a reduction in their level by loss of LXRβ in brains for each age and each genotype; *P<0.05, **P<0.01). Scale bar: 50 μm (a–f and g–l). CC, corpus callosum; P, postnatal day.

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The Olig2 transcription factor is a primary determinant of OL fate specification. Analysis of Olig2-positive cells reflected the summation of changes in CC1-positive mature OLs and PDGFRα-positive OPCs. Loss of LXRβ was accompanied by a significant reduction of Olig2-positive cells compared with WT mice at P7, P10 and P14 (Figures 7g–l). Western blots revealed that Olig2 protein levels were consistently lower in cerebral cortical lysates from LXRβ KO than in WT mice (Figures 7p and r). These results indicate that LXRβ is involved in the differentiation of OPCs to mature OLs.

Co-expression of Olig2 and PDGFRα is an indication of OPCs specification from Pre-OPCs or neural stem cells. We examined the densities of cells positive for both Olig2 and PDGFRα at P2, early postnatal development (Figure 8). Compared with WT mice (Figures 8a, c, e and g), there were fewer double-labeled cells immunopositive for both Olig2 and PDGFRα in the VZ/SVZ (Figures 8e and f), medial (Figures 8a and c) and lateral (Figures 8b and d) corpus callosum in the LXRβ KO mice (Figures 8b, d, f and g). β-catenin, which is involved in the specification of OPCs from neural stem cells,25,26 was upregulated in the cerebral cortical lysates of LXRβ KO mice (Figure 8h). These results constitute strong evidence that LXRβ determines OPCs specification through inhibition of β-catenin.

Figure 8
figure8

Effects of liver X receptor (LXR)β ablation on PDGFRα+/Olig2+ cell densities in the corpus callosum at P2. (a–d) PDGFRα (green) and Olig2 (red) expression in the corpus callosum (a–d) and ventricular zone/subventricular zone (VZ/SVZ) (e and f) of mice at P2 was examined by immunohistochemistry. Arrowheads indicate PDGFRα+/Olig2+ double-staining cells. The mean cell densities co-expressing PDGFRα and Olig2 per volume (mm3) of corpus callosum and SVZ were determined for wild-type (WT) (black bars) and LXRβ KO (white bars) animals at P2 (g). LXRβ KO mice had significantly fewer PDGFRα+/Olig2+ double-staining cells in the corpus callosum compared with their WT littermates (n=3, **P<0.01). Graphs depict mean (±s.e.m.) cell densities measured from four different areas of the corpus callosum and VZ/SVZ. Scale bar: af, 50 μm. (h) Western blot analysis showed that loss of LXRβ induced a decrease in PDGFRα and Olig2 expression, but increase in β-catenin expression in the cerebral cortical lysates compared with WT at P2.

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LXRβ contributes to differentiation of RGCs into OPCs

RGCs, which have long been thought of as glia or glial progenitors, can also give rise to neurons. Brain lipid-binding protein (BLBP), which is a reliable marker for RGC during development, combined with OPC markers such as PDGFRα or Olig2, was used to study the effect of LXRβ on the differentiation of RGCs into OPCs. At P2 (Figures 9a–d) and P7 (Figures 9e–h), there was a large number of BLBP-labeled RGCs robustly co-expressing OPC surface marker PDGFRα in the VZ/SVZ (Figures 9c and d) and corpus callosum (Figures 9a, b, e–h) of WT mice (Figures 9a, c, e and g), whereas the double-labeled BLBP+/PDGFRα+ cells were significantly reduced in LXRβ KO mice (Figures 9b, d, f and h).

Figure 9
figure9

Effects of liver X receptor (LXR)β ablation on PDGFRα+/BLBP+ cell densities in the ventricular zone/subventricular zone (VZ/SVZ) and corpus callosum. PDGFRα (red) and BLBP (green) expressions in the corpus callosum (a,b and eh) and VZ/SVZ (c and d) of mice at P2 (a–d) and P7 (eh) were examined by immunohistochemistry. LXRβ knockout (KO) mice had significantly fewer PDGFRα+/BLBP+ double-staining cells in the corpus callosum and VZ/SVZ compared with their wild-type (WT) littermates. Arrowheads indicate PDGFRα+/BLBP+ double-staining cells. Scale bar: 50 μm (a, b, cf). P, postnatal day.

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Early expression of Olig2 has a role in the initial specification of the OL lineage, both in human27 and in animal models.28 We confirmed that there was a large number of BLBP-labeled RGCs co-expressed with Olig2 in the VZ/SVZ (Figure 10e) and corpus callosum (Figures 10a, c and k) of WT mice at P2, whereas the double-labeled cells were significantly reduced in LXRβ KO mice (Figures 10b, d, f and k). At P7, there were fewer double-stained BLBP +/Olig2+ cells in the corpus callosum of WT (Figures 10g, i and k) and LXRβ KO mice (Figures 10h, j and k).

Figure 10
figure10

Effects of liver X receptor (LXR)β ablation on the BLBP+/Olig2+ cell densities in the corpus callosum of mice at P2 and P7. BLBP (green) and Olig2 (red) co-expression in the corpus callosum (a–d) and ventricular zone/subventricular zone (VZ/SVZ) (e and f) of mice at P2 and in the corpus callosum (gj) at P7 was examined by immunohistochemistry. Arrowheads indicate BLBP+/Olig2+ double-staining cells. (k) The mean cell densities co-expressing BLBP and Olig2 per volume (mm3) of corpus callosum were determined for wild-type (WT) (white bars) and LXRβ knockout (KO) (black bars) animals at P2 and P7. LXRβ KO mice had significantly fewer BLBP+/Olig2+ double-staining cells in the white matter compared with their WT littermates (n=3, **P<0.01). Graphs depict mean (±s.e.m.) cell densities measured from four different areas of the corpus callosum. Scale bar: ah, 50 μm. P, postnatal day.

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Loss of LXRβ did not alter cholesterol contents in the cerebral cortex

There was no difference in cholesterol levels between WT and LXRβ mice in the cerebral cortex from P10 to P6W, at the peak of OL myelination/maturation (Figure 11).

Figure 11
figure11

Measurement of cholesterol in the cerebral cortex of mice from P10 to P6W. The cholesterol content of the brain was determined with a cholesterol quantification kit (BioVision). (a) Data are expressed as micrograms of cholesterol per milligram of brain tissue. n=4 per group; Student's t-test. P, postnatal day; W, week.

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LXR agonist promotes the differentiation of RGCs to OPCs

When radial glial clone L2.3 cells were maintained in differentiation medium with LXR agonist T0901317 (1 μM) for 8 days, the percentage of NG2+ (OPC surface marker) cells increased by 67% from 44.3 to 74% (Figures 12a, c and j; P<0.01), and percentage of NG2+ cells arising from BLBP+cells increased by 36% from 61.6 yo 83.6% (Figures 12d, f, g, i and k; P<0.05) in comparison with non-treated cultures. With 0.1 μM T0901317 treatment, no significant upregulation in the proportion of NG2+ cells (51.6%) or NG2+-positive cells from BLBP+cells (67.6%) was detected.

Figure 12
figure12

T0901317 promotes radial glial cell differentiation into oligodendrocyte progenitor cells (OPCs). (ac) OPCs are labeled with anti-NG2 antibody 8 days after being cultured in differentiation medium. (di) NG2-positive cells are co-labeled with BLBP antibody 8 days after being cultured in differentiation medium; (d and g) without T0901317; (e and h) with T0901317 (0.1 μM); (f and i) with T0901317 (1 μM). (j) Quantification of percentage of NG2+ cells (**P<0.01). (k) Quantification of percentage of NG2+ cells in the BLBP+ cells (*P<0.05). Scale bar: a–i, 50 μm.

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Discussion

Our previous studies have shown that male mice with deleted LXRβ exhibit impaired performance on the rota-rod and that this phenotype is associated with lipid accumulation and loss of motor neurons in the spinal cord, together with axonal atrophy and astrogliosis.18,19 Here, we further confirm that LXRβ−/− male mice display significant abnormal locomotor activity in the open-field test. Demyelination and hypomyelination in the corpus callosum of LXRβ KO mice were confirmed by electron microscopy and MBP staining. Meanwhile, there was hypomyelination in the corpus callosum of LXRβ KO mice accompanied by an obvious defect in production and maturation of OLs.

LXRs are known to have a key role in the homeostasis of cholesterol,14 a major lipid constituent of the myelin sheaths and required for OL maturation and myelination.29,30 LXRβ is expressed in a mouse OL cell line (158N) and in primary OLs isolated from neonatal and adult rats. It has a key role in the regulation of cholesterol metabolism in OLs.31,32 Deficiency in cholesterol synthesis by OLs leads to impaired myelination.32 Surprisingly, LXRβ KO mice exhibited defects in OL myelination/maturation by P14 without alteration of cholesterol content in the cerebral cortex. This may infer that LXRβ affects early postnatal OL differentiation independent of cholesterol homeostasis. Indeed, generating or maintaining the correct number of OPCs is critical to populate OLs in the developing corpus callosum. PDGFRα-labeled OPCs were decreased in the corpus callosum of LXRβ KO mice from P7 to P14, strongly indicating that LXRβ is involved in the production of OPCs.

It is important to note that postnatal regulation is critical for establishment of the number of OLs in the mature CNS. Spatially restricted Olig2 ablation leads to a nearly complete absence of myelination in the cortex at early postnatal stages and severe dysmyelination even at adulthood,33,34 suggesting that dorsal progenitor cells are a critical source for OL myelination in the developing cortex. It has also been demonstrated by the anatomical fate-mapping strategy in postnatal mice that the progeny of dorsal RGCs include OLs.35 In rodents, dorsal RGCs have been implicated in generation of OPCs and OLs in the postnatal cortex and subcortical white matter.36, 37, 38 In the fetal brain, RGCs localized in the cortical VZ/SVZ are also involved in generation of OLs at a specific developmental stage, further confirmed by generation of OPCs from cultured isolated RGCs under Shh influence.39 We have previously shown that LXRβ contributes to cerebral cortex lamination by maintenance of RGCs.16 We noted that LXRβ was expressed in the cortical VZ/SVZ and corpus callosum with high level from P2 to P10, implying its possible role in oligodendrogenesis of the emerging white matter. This is supported by our finding of decreased densities of cells positive for BLBP+/PDGFRα+ and BLBP+/Olig2+ along the VZ and emerging corpus callosum at P2 and P7. Together with the promotion of RGC differentiation into OPCs by LXR agonist, these data suggest that LXRβ specifies development of RGCs to OPCs at early postnatal development. Furthermore, in support of the finding that an LXR agonist decreased β-catenin level in the developing cerebellum,40 we confirmed upregulation of β-catenin in the cortex at P2 by loss of LXRβ. These data suggest that the inhibited oligodendrogenesis observed in LXRβ KO mice may result from a dysregulation of Wnt/β-catenin signaling, which has previously been shown to affect early postnatal OL differentiation.41,42 Thus LXRβ may interplay with β-catenin and Olig2 to control differentiation of OPCs from RGCs.

Together, our findings indicate that LXRβ regulates oligodendrogenesis in the dorsal cerebral cortex, and may therefore regulate the differentiation potential of newly born OPCs derived from RGCs, in addition to OL maturation/myelination (Figure 13). Therefore, activation of LXRβ provides a new strategy to promote OPC production, maturation and myelination. Moreover, similar to our previous finding of an anxiogenic phenotype in female LXRβ KO mice,43 signs of anxiety were also observed in the male LXRβ KO mice at 3 months of age. From this we may infer that impairment of OPC development in the emerging white matter postnatally could be involved in the pathology of psychiatric diseases.

Figure 13
figure13

Schematic model of liver X receptor (LXR)β modulation of the differentiation of radial glial cells (RGCs) to oligodendrocyte progenitor cells (OPCs) during dorsal cortex oligodendrogenesis through interplay with β-catenin and Olig2. A subpopulation of RGCs in the ventricular zone/subventricular zone (VZ/SVZ), which are differentiated from the neuroepithelial cells, retain the capacity of neural stem cells in the neonate and divide asymmetrically to generate OPC and astrocyte precursor cells. (LXR)β has a key role in the transformation of RGCs to OPCs by repression of the expression of β-catenin and enhancing the expression of Olig2 transcription factors simultaneously. BLBP, brain lipid-binding protein; IOs, immature oligodendrocyte cells; MA, mantle; MBP, myelin basic protein; MOs, mature oligodendrocyte cells; MZ, marginal zone; NCs, neuroepithelial cells.

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Acknowledgements

This study was supported by the National Nature Science Foundation of China (No. 31271051 and 81371197), Natural Science Foundation Project of CQ CSTC 2013jjB10028, the Swedish Research Council and a grant from the Robert A Welch Foundation (E-0004, J-ÅG).

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Correspondence to J-A Gustafsson or X Fan.

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Supplementary Information accompanies the paper on the Molecular Psychiatry website

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