GABAB receptor agonist baclofen promotes central nervous system remyelination

Abstract

Promoting remyelination is considered as a potential neurorepair strategy to prevent/limit the development of permanent neurological disability in patients with multiple sclerosis (MS). To this end, a number of clinical trials are investigating the potential of existing drugs to enhance oligodendrocyte progenitor cell (OPC) differentiation, a process that fails in chronic MS lesions. We previously reported that oligodendroglia express GABA_B receptors (GABA_B Rs) both in vitro and in vivo, and that GABA_B R-mediated signaling enhances OPC differentiation and myelin protein expression in vitro. Our goal here was to evaluate the pro-remyelinating potential of GABA_B R agonist baclofen (Bac), a clinically approved drug to treat spasticity in patients with MS. We first demonstrated that Bac increases myelin protein production in lysolecithin (LPC)-treated cerebellar slices. Importantly, Bac administration to adult mice following induction of demyelination by LPC injection in the spinal cord resulted in enhanced OPC differentiation and remyelination. Thus, our results suggest that Bac repurposing should be considered as a potential therapeutic strategy to stimulate remyelination in patients with MS.

Keywords: GABAB receptor; baclofen; multiple sclerosis; myelin; oligodendrocyte; remyelination.

FIGURE 1

Baclofen increases myelin‐related protein synthesis in organotypic cultures without altering the proliferation ratio of oligodendroglial lineage. (a) Oligodendroglial cells, distinguished as Olig2⁺ cells (red), are positive for GABA_B1 and GABA_B2 subunits (green) of GABA_BRs in cerebellar slices of P5‐P7 rats. (b) Mature oligodendrocytes (OLs), identified as APC⁺ cells (red), express GABA_B1, and GABA_B2 subunits (green) of GABA_BRs in the same preparations. Arrows indicate double‐stained cells and arrowheads show the cell magnified in the corresponding inset. Scale bars = 20 μm. (c) Representative western blot image‐showing expression of myelin‐associated glycoprotein (MAG), CNPase, and MBP proteins in control and baclofen‐treated cerebellar slices. Quantification of MAG (d), CNPase (e), and myelin basic protein (MBP) (f) expression normalized to GAPDH values. *p < .05 and ** p < .01 versus control; paired Student's t‐test. (g) Representative images showing immunofluorescence of mature OLs (APC⁺, red) and total oligodendroglial cells (Olig2⁺, green) co‐labeled with EdU (cyan) to identify mature OLs (APC⁺) and Olig2⁺ cells in cerebellar slices in the indicated condition. Arrows indicate mature OLs (APC⁺Olig2⁺) or newly generated oligodendroglial cells (Olig2⁺EdU⁺). Scale bar = 50 μm. Quantification of (h) percentage of mature OL from total oligodendroglial cell pool (APC⁺Olig2⁺/Olig2⁺) and (i) density of newly formed oligodendroglial cells (Olig2⁺EdU⁺), in the indicated conditions. One‐way ANOVA followed by Tukey's post‐test. (j) Optic nerve‐retina unit from P11 PLP‐DsRed transgenic mice. Scale bar = 500 μm. (k) Optic nerves explants cultured in control conditions (left) or in presence of baclofen (right) showing DsRed fluorescent signal. Scale bars = 100 μm; higher magnification scale bar = 315 μm. (l) Quantification of DsRed fluorescent signal in control and treated optic nerve explants. *p < .05 versus control; unpaired Student's t‐test. (a–f): Control and bac 6 slices from different animals. (g–i): Control 15, GABA 18, and bac 18 images from cerebellar slices. (j–l): Control 27, bac 24 images from optic nerve explants

FIGURE 2

GABA_BRs modulate remyelination in lysolecithin (LPC)‐treated cerebellar organotypic slices. (a) Time course showing the experimental design in LPC‐induced demyelination in cerebellar organotypic cultures obtained from P11 rats. (b) Representative western blot image showing in duplicates the effect of GABA and baclofen in modulating myelin‐related protein restoration in LPC‐treated organotypic cultures following the paradigm shown in a. (c, d) Quantification of myelin‐associated glycoprotein (MAG) (c) and myelin basic protein (MBP) (d) levels in indicated conditions. **p < .01 and ****p < .0001 versus control, ^## p < .01 and ^### p < .001 versus LPC; one‐way ANOVA followed by Tukey's post‐test. (e) Representative images of cerebellar slices from P11 PLP‐DsRed transgenic mice showing DsRed fluorescence in indicated conditions. Scale bar = 100 μm. (f) Treatments were added to the slices in conjunction with LPC for 16 h and maintained thereafter for 4 days after. GABA_BRs were selectively activated with baclofen or with GABA plus the GABA_AR antagonist gabazine. (g) Quantification of DsRed fluorescent signal in indicated conditions. ****p < .0001 versus control, ^# p < .05 and ^#### p < .001 versus LPC; one‐way ANOVA followed by Tukey's post‐test. (b–d): Control 9, LPC 8, GABA 9, bac 8 slices from different animals. (e–g): Control 27, LPC 25, GABA 8, bac 19, Gbz 10, GABA + Gbz 10 images from optic nerve explants

FIGURE 3

GABA_B receptors are expressed by oligodendrocyte progenitor cell (OPCs) and mature oligodendrocytes from the mouse spinal cord. (a) Confocal images showing OPCs (Nkx2.2⁺, gray) expressing GABA_B1 (red) and GABA_B2 (green) subunits of GABA_BRs in the dorsal funiculus of the spinal cord of unlesioned mice. (B, C) Confocal images showing OPCs (Nkx2.2⁺, red) (b) and mature OLs (APC⁺, red) (c) expressing GABA_B1 (green, top) and GABA_B2 (green, bottom) subunits of GABA_BRs in the dorsal funiculus of the spinal cord of control lysolecithin (LPC)‐injected mice. White dash line indicates lesion border. Arrowheads point at cells shown at higher magnification in each photograph. Scale bars = 50 μm. Higher magnification = 10 μm

FIGURE 4

GABA_B receptor activation by baclofen promotes oligodendrocyte differentiation but does not impact on oligodendrocyte progenitor cell (OPC), microglia/macrophague population or astrocytes in lysolecithin (LPC)‐induced demyelinated spinal cords. (a) Diagram representing the time course of the studies in the LPC‐induced demyelination model. (b) Spinal cord sections of LPC‐injected control (top) and baclofen‐treated (bottom) mice immunostained with anti‐MBP (gray), anti‐PDGFRα (green) and anti‐Iba1 (red) antibodies. Dapi was used to identify cell nuclei. White dash line indicates lesion border. Quantification of number of PDGFRα⁺ cells per mm² (c) and percentage of Iba1⁺ occupied area (d) in LPC‐injected mice. (e) Spinal cord sections of LPC‐injected control (top) and baclofen‐treated (bottom) mice immunostained with anti‐MBP (gray), anti‐Nkx2.2 (green) and anti‐GFAP (red) antibodies. Dapi was used to identify cell nuclei. White dash line indicates lesion border. Quantification of number of Nkx2.2⁺ cells per mm² (f) and percentage of GFAP stained area (g) in LPC‐injected mice. (h) Spinal cord sections of LPC‐injected control (top) and baclofen‐treated mice immunostained with anti‐MBP (gray), anti‐APC/CC1 (green) and anti‐Olig2 (red) antibodies. Dapi was used to identify cell nuclei. White dash line indicates lesion border. Histograms showing percentage of APC⁺Olig2⁺ cells from total Olig2⁺ cells (i), and number of Olig2⁺ cells per mm² (j) in LPC‐injected mice. At least 3 lesion areas from 3 different animals were analyzed. **p < .01 versus control; unpaired Student's t‐test, control 3 animals, bac 3 animals. Scale bars: B, E, H = 50 μm. Higher magnification = 20 μm. Arrows show positive staining

FIGURE 5

Baclofen treatment promotes remyelination following spinal cord lysolecithin (LPC)‐induced demyelination. (a) Representative images of semithin sections of spinal cord control (left) and baclofen‐treated (right) mice showing LPC‐induced lesions, stained with Richardson's blue. (b) Electron micrographs of ultrathin spinal cord sections showing remyelinated (green arrows) and unmyelinated (red arrows) axons. Histograms showing percentage of total remyelinated axons within the lessions (c), axons remyelinated by oligodendrocytes (OLs) (d) and axons remyelinated by Schwann cells (SCs) (e). *p < .05 and ***p < .001 versus control; unpaired Student's t‐test, control 5 animals, bac 6 animals. Scale bars: A = 100 μm; B = 2 μm