Retinoid X receptor gamma signaling accelerates CNS remyelination

Abstract

The molecular basis of CNS myelin regeneration (remyelination) is poorly understood. We generated a comprehensive transcriptional profile of the separate stages of spontaneous remyelination that follow focal demyelination in the rat CNS and found that transcripts that encode the retinoid acid receptor RXR-γ were differentially expressed during remyelination. Cells of the oligodendrocyte lineage expressed RXR-γ in rat tissues that were undergoing remyelination and in active and remyelinated multiple sclerosis lesions. Knockdown of RXR-γ by RNA interference or RXR-specific antagonists severely inhibited oligodendrocyte differentiation in culture. In mice that lacked RXR-γ, adult oligodendrocyte precursor cells efficiently repopulated lesions after demyelination, but showed delayed differentiation into mature oligodendrocytes. Administration of the RXR agonist 9-cis-retinoic acid to demyelinated cerebellar slice cultures and to aged rats after demyelination caused an increase in remyelinated axons. Our results indicate that RXR-γ is a positive regulator of endogenous oligodendrocyte precursor cell differentiation and remyelination and might be a pharmacological target for regenerative therapy in the CNS.

Figure 1. Differential expression of Rxrg in CNS remyelination transcriptome

(a) Hierarchical clustering and graphical analysis of differentially expressed genes at 5, 14 and 28 dpl (P < 0.05). (b) Ten most upregulated genes at each time point relative to the other time points. (c) Graphical analysis showing the differential expressions of known genes associated with myelination (P < 0.05). (d) Top five overall physiological functions in lesions at 5, 14 and 28 dpl using Ingenuity pathway analysis of upregulated genes from each time point. (e) Volcano plot (x axis = log₂ FC at 14 dpl compared to 5 dpl; y axis = log₂ P) showing highly differentially expressed genes associated with myelination genes. Rxrg (green triangle; x, y = 3.3752, 2.7084) is shown as a highly expressed transcript at 14 dpl compared to 5 dpl. (f) Real-time qPCR detection of Rxra, Rxrb and Rxrg expression from laser-captured lesions during remyelination (n = 3). Rxrg is barely detectable in non-lesioned CCPs and at 5 dpl, and highly expressed at 14 and 28 dpl. (g) In situ hybridization shows significant increase of Rxrg⁺ cells in the CCP at 14 dpl and 28 dpl compared to non-lesioned and 5 dpl CCP. Scale bar, 50 μm. (h) Quantification of Rxrg⁺ cells in lesioned CCPs at 5, 14 and 28 dpl (n = 3 per time point). Mean ± s.e.m. are shown. *P < 0.05, ***P < 0.001, one-way ANOVA.

Figure 2. RXR-γ expression by oligodendrocyte lineage cells

Figure 3. Expression of RXR-γ in multiple sclerosis lesions

(a–f) Co-immunolabeling for RXR-γ (green) and (in red) MOG (a), Sox10 (b), Olig1 (c), NOG0-A (d), MHCII (e) or GFAP (f) in active multiple sclerosis lesion areas. Nuclei were visualized with Hoechst (blue). (g) Luxol fast blue staining followed by anti-MHCII immunoperoxidase labeling showing a typical chronic active multiple sclerosis lesion with active border (A) and chronic inactive core (C), as well as peri-plaque white matter (PPWM). (h) Quantification of nuclear and cytoplasmic RXR-γ+ cells in multiple sclerosis lesions reveals significantly more nuclear RXR-γ+ cells in active lesions, PPWM and remyelinated shadow plaques (RM) compared to chronic inactive lesions and normal appearing white matter (WM) from non-neurological cases. Scale bars: a–f, 50 μm; g, 2 mm. Mean ± s.e.m. are shown. *P < 0.05, ***P < 0.001; one-way ANOVA.

Figure 4. Loss of RXR-γ function impairs oligodendrocyte differentiation

(a–c) Purified OPCs transfected with non-targeting siRNAs (a), RXRα siRNAs (b) and RXR-γ siRNAs (c) and visualized with O4 (red) and antibodies to MBP (green) after 72 h in differentiation medium. Scale bar, 25 μm. (d) Morphological criteria for the maturation state of differentiating oligodendrocyte defined as simple, complex or membrane morphologies. Cells transfected with RXR-γ siRNAs resulted in increased percentage of O4⁺ oligodendrocytes with simple morphologies and decreased percentage of complex membrane morphologies compared to mock-treated and non-targeting siRNA–transfected cells. (e) Western blot shows the specificity of RXR-α or RXR-γknockdowns. The position of molecular weight standards (in kilodaltons) is shown on the left. Full-length blot presented in Supplementary Figure 4. C, untransfected control; M, mock-transfected; NT, non-targeting siRNA; RXRα, β, γ, RXRα, β or γ siRNA. (f,g) Ventral spinal cord lesions of Rxrg^+/− (f) and Rxrg^−/− (g) mice stained with antibodies to CC1 (green) and Olig2 (red) 15 d after demyelination. Nuclei visualized with Hoechst (blue). (h) Quantification of oligodendrocyte lineage cells at 15 and 30 dpl shows no difference in the density of total Olig2⁺ cells between homozygous and heterozygous mutant mice, but a reduction of CC1⁺ cells and increased Nkx2.2⁺ cells in lesions of homozygous compared to heterozygous mutant mice. Scale bar, 50 μm. Mean ± s.e.m. are shown. *P < 0.05, **P < 0.01; Student’s t test.

Figure 5. Rexinoids influence oligodendrocyte differentiation and myelination

(a–d) OPC cultures immunolabeled with antibodies to O4 (red) and MBP (green) after RXR antagonist treatment for 72 h. Compared to non-treated cells (b), treatment with HX531 (c; 2 μM) or PA452 (d; 5 μM) resulted in fewer mature oligodendrocytes. Scale bar, 25 μm. (e) Increasing antagonist concentration resulted in decreasing number of membrane sheet-bearing oligodendrocytes. (f–j) Oligodendrocyte-DRG co-cultures maintained for 10 d after addition of OPCs immunolabeled with anti-MBP (green), anti-Caspr (red) and anti-NFH (blue). (g) Control co-culture; (h) HX531 (2 μM); (i) PA452 (5 μM); (j) 9cRA (50 nM). Scale bar, 100 μm. (k,l) Increasing antagonist concentration resulted in decreased MBP⁺ oligodendrocytes (k) and less myelination (l). (m–p) OPC cultures labeled with O4 (red) and anti-MBP (green). (m) Untreated; (n) 9cRA alone; (o) 9cRA and HX531; (p) 9cRA and PA452. Scale bar, 25 μm. (q) Quantification showing that 50 nM 9cRA increased mature oligodendrocyte membranes, and low concentrations of HX531 and PA452 were sufficient to abrogate 9cRA-mediated differentiation. (r) Treatment of cultured OPCs with 9cRA, HX630 or PA024 resulted in increased oligodendrocyte membrane sheets. Mean ± s.e.m. are shown. *P < 0.05 versus control,**P < 0.005 versus control, ^†P < 0.05 versus 50 nM 9cRA, ^††P < 0.005 versus 50 nM 9cRA; Student’s t test.

Figure 6. CNS remyelination is enhanced by 9 cis-retinoic acid

(a) Control cerebellar slices fixed 10 d after demyelination with lysolecithin and immunolabeled with antibodies to NFH (red) and MBP (green). (b–d) Remyelination was increased by 9cRA (b), and decreased by HX531 (c) or PA452 (d). Scale bar, 20 μm. (e,f) Quantification of NG2⁺ and MBP⁺ cells, 48 h (e) or 14 d (f) after treatment. *P < 0.05; Student’s t test. (g) Quantification of remyelination after addition of 9cRA, or HX531 or PA452 at high (H, 2 or 5 μM, respectively) or low (L, 0.2 or 0.5 μM) concentrations. *P< 0.05, **P < 0.001; one-way ANOVA. (h) Treatment with 9cRA increased CC1⁺ cells in lesions in rats. Student’s t test. (i) Real-time qPCR analysis shows increased Mbpexpression in 9cRA-treated mice. Student’s t test. (j) Semi-thin section of a lesioned CCP 27 dpl after 9cRA treatment. Upper left corner shows normal myelinated axons. To the right is a large area of lesion showing axons outlined by thinly remyelinating membranes and dark macrophages. Scale bar, 50 μm. (k) Ultrastructural microscopy (1,500×) shows many remyelinated axons (pink) compared to axons that remained demyelinated. (l,m) Control animal (images as in j and k, respectively) shows few visible remyelinated axons. (n) Ranking analysis. Highest rank represents most remyelination. Mann-Whitney U test. (o) G-ratio is lower in 9cRA-treated mice compared to control mice. Student’s t test. (p) Representative images of myelinated, demyelinated, control remyelinated and 9cRA remyelinated axons. Mean ± s.e.m. are shown. ***P < 0.001.