CXCR4 promotes differentiation of oligodendrocyte progenitors and remyelination

Abstract

Multiple sclerosis is a neurodegenerative disease characterized by episodes of autoimmune attack of oligodendrocytes leading to demyelination and progressive functional deficits. Because many patients exhibit functional recovery in between demyelinating episodes, understanding mechanisms responsible for repair of damaged myelin is critical for developing therapies that promote remyelination and prevent disease progression. The chemokine CXCL12 is a developmental molecule known to orchestrate the migration, proliferation, and differentiation of neuronal precursor cells within the developing CNS. Although studies suggest a role for CXCL12 in oligodendroglia ontogeny in vitro, no studies have investigated the role of CXCL12 in remyelination in vivo in the adult CNS. Using an experimental murine model of demyelination mediated by the copper chelator cuprizone, we evaluated the expression of CXCL12 and its receptor, CXCR4, within the demyelinating and remyelinating corpus callosum (CC). CXCL12 was significantly up-regulated within activated astrocytes and microglia in the CC during demyelination, as were numbers of CXCR4+NG2+ oligodendrocyte precursor cells (OPCs). Loss of CXCR4 signaling via either pharmacological blockade or in vivo RNA silencing led to decreased OPCs maturation and failure to remyelinate. These data indicate that CXCR4 activation, by promoting the differentiation of OPCs into oligodendrocytes, is critical for remyelination of the injured adult CNS.

Fig. 1.

CXCL12 expression in the CC after CPZ exposure. (A) qRT-PCR analysis of CXCL12 expression in dissected whole CC derived from naive mice and from mice after 6 and 12 wk of CPZ ingestion. Each group represents three to five individual animals, *, P < 0.05. (B) Confocal IHC analysis of CC from naive mice and after 6 and 12 wk of CPZ ingestion. Images were stained for GFAP or CD11b (green, Upper and Lower, respectively), CXCL12 (red), and nuclei (blue). Higher power images of boxed areas in images from 6-wk time-point are shown. Representative images shown for three sections from three mice in two separate experiments. IC = isotype control. (Scale bars, 25 μm.) (C and D) Quantitation of GFAP+ (C) and CD11b+ (D) cells that express CXCL12 within rostral, central, and caudal sections of the CC in naive mice (white bars) and after 6 (gray bars) and 12 (black bars) wk of CPZ exposure; n = 6 images taken from three to five mice/group. *, P < 0.05 and **, P < 0.005.

Fig. 2.

CXCR4 expression in the CC after CPZ exposure. (A) QRT-PCR analysis of CXCR4 expression in dissected whole CC derived from naive mice and from mice after 6 and 12 wk of CPZ ingestion. Each group represents three to five individual animals, *, P < 0.05. (B) Confocal IHC analysis of CC from naive mice and after 6 and 12 wk of CPZ ingestion. Images were stained for NG2 (green), CXCL12 (red), and nuclei (blue). Higher power images of boxed area in image from 6-wk time-point are shown. Representative images shown for three sections from three mice in two separate experiments. IC = isotype control. (Scale bars, 25 μm.) (C) Quantitation of NG2+ cells that express CXCL12 within rostral, central, and caudal sections of the CC in naive mice (white bars) and after 6 (gray bars) and 12 (black bars) wk of CPZ exposure; n = 6 images taken from three to five mice/group. *, P < 0.05 and **, P < 0.005.

Fig. 3.

CXCR4 antagonism prevents maturation of OPCs and remyelination. (A) Confocal IHC analysis of CC from mice after 6 wk of CPZ ingestion then 2 wk of treatment with PBS (Left) or AMD3100 (Right, AMD). Sections were stained for CXCR4 (green), NG2 (red), and nuclei (blue). Note linear organization of NG2-negative, oligodendrocyte nuclei in PBS-treated specimen (arrowheads). (Inset) Isotype control. (Scale bar, 25 μm.) (B) Quantitation of CXCR4+NG2+ cells within rostral, central and caudal sections of the CC in naive mice (white bars) and after 6 (gray bars) and 12 (black bars) wk of CPZ exposure; n = 6 images taken from four mice/group. *, P < 0.05. (C) Sections from CC of PBS- (Left) or AMD3100- (Right, AMD) treated mice were stained for CNPase (green) and nuclei (blue). (Inset) Isotype control. (Scale bar, 25 μm.) (D) Quantitation of CNPase+ cells within rostral, central, and caudal sections of the CC in naive mice (white bars) and after 6 (gray bars) and 12 (black bars) wk of CPZ exposure; n = 6 images taken from four mice/group. *, P < 0.05. (E) LFB myelin staining of CC in mice at 0 and 6 wk of CPZ treatment and after 6 wk of CPZ treatment plus 2 wk of treatment with PBS or AMD3100 (AMD). (F) IHC analysis of MBP expression (red) within CC of naive mice (Upper Left) and in mice treated with 4 wk of PBS (Lower Left) or AMD3100 (Lower Right, AMD) after 6 wk of CPZ ingestion. Nuclei counterstained with DAPI (blue). (Scale bar, 50 μm.) (G) Quantitation of MBP expression within all regions of the CC in mice treated with 4 wk of PBS or AMD3100 (AMD) after 6 wk of CPZ ingestion; n = 6 images taken from four mice/group. *, P < 0.05.

Fig. 4.

CXCR4 activation differentially affects OPC proliferation within the SVZ and CC. (A) Confocal IHC analysis of rostral subventricular zones (SVZ) and CC from mice after 6 wk of CPZ ingestion plus 2 wk of treatment with PBS (Left) or AMD3100 (Right) with daily i.p. injection of BrDU. Images were stained for BrDU (green), NG2 (red), and nuclei (blue). Representative images shown from three sections from three individual mice per group. (Scale bars, 25 μm.) (B) Quantitation of BrDU+NG2+ cells within rostral, central, and caudal sections of the CC and SVZ of mice after 6 wk of CPZ ingestion and 2 wk of treatment with PBS (gray bars) or AMD3100 (black bars); n = 6 images taken from three mice/group, *, P < 0.05.

Fig. 5.

CXCR4 RNA silencing in glial cells in vitro. (A) Schematic of lentiviral construct used to silence CXCR4 expression. pLKO.1 plasmids carrying shRNAs (CXCR4 and nonsense) driven by the human U6 promoter and HIV packaging signal were reengineered to contain sequence encoding GFP with a polyA tail driven by the CMV promoter. CXCR4 shRNA and nonsense sequences are shown. (B) qPCR analysis of CXCR4 mRNA levels in primary astrocytes infected with CXCR4 shRNA lentivirus at multiplicity of infection (MOI) of 5, 10, and 20. Data are presented as the fold-change in CXCR4 mRNA levels normalized to those observed in astrocytes infected with nonsense shRNA lentivirus. (C) Confocal IHC analysis of GFP (green), CXCR4 (red), and NG2 (blue) expression by primary oligodendroglia infected with nonsense (Left) versus CXCR4 (Middle) shRNA lentiviruses at 20 MOIs reveals significant knock-down of CXCR4 as assessed by confocal IHC (Right); n = 5–6 images taken from three replicates/group, *, P < 0.05. (D) Low power images depicting stereotactic infection of CC with GFP-expressing lentiviruses expressing nonsense (Left) and CXCR4 (Right) shRNAs. Arrow = site of injection. (Scale bar, 50 μm.)

Fig. 6.

In vivo CXCR4 silencing prevents OPC maturation and remyelination. (A and B) Confocal IHC analysis of CC from mice after 6 wk of CPZ ingestion followed by stereotactic injection of lentivirus bearing shRNAs (Nonsense, Left, versus CXCR4, Right). Images depict GFP expression (green) with staining for CXCR4 (A), NG2 (B), and CNPase (C). (Scale bar, 20 μm.) Quantitation of CXCR4+ (A), NG2+ (B), and CNPase+ (C) cells within CC of mice injected with lentivirus expressing Nonsense and CXCR4 shRNAs after 6 wk of CPZ exposure (Far Right); n = 6 images taken from three to five mice/group. *, P < 0.05. (D) LFB-stained sections of CC from mice injected with lentivirus expressing Nonsense (Upper) and CXCR4 (Lower) shRNAs after 6 wk of CPZ exposure. (A–D) Representative images shown for three sections from three to five mice in two separate experiments. Note linear area without remyelination in mouse injected with CXCR4 shRNA lentivirus (arrowheads). (E) IHC analysis of MBP expression (red) within CC of mice injected with lentivirus expressing GFP (green) plus Nonsense (Left) and CXCR4 (Right) shRNAs after 6 wk of CPZ exposure. Note area of demyelination in CC of mouse with CXCR4 RNA silencing that correspond to sites of GFP staining (arrowheads). Nuclei counterstained with DAPI (blue). Quantitation of MBP staining in areas of GFP expression was performed on two to three sections/mouse in three to five mice/group in two separate experiments, *, P < 0.05 (Far Right).