LINGO-1, a transmembrane signaling protein, inhibits oligodendrocyte differentiation and myelination through intercellular self-interactions

Abstract

Overcoming remyelination failure is a major goal of new therapies for demyelinating diseases like multiple sclerosis. LINGO-1, a key negative regulator of myelination, is a transmembrane signaling protein expressed in both neurons and oligodendrocytes. In neurons, LINGO-1 is an integral component of the Nogo receptor complex, which inhibits axonal growth via RhoA. Because the only ligand-binding subunit of this complex, the Nogo receptor, is absent in oligodendrocytes, the extracellular signals that inhibit myelination through a LINGO-1-mediated mechanism are unknown. Here we show that LINGO-1 inhibits oligodendrocyte terminal differentiation through intercellular interactions and is capable of a self-association in trans. Consistent with previous reports, overexpression of full-length LINGO-1 inhibited differentiation of oligodendrocyte precursor cells (OPCs). Unexpectedly, treatment with a soluble recombinant LINGO-1 ectodomain also had an inhibitory effect on OPCs and decreased myelinated axonal segments in cocultures with neurons from dorsal root ganglia. We demonstrated LINGO-1-mediated inhibition of OPCs through intercellular signaling by using a surface-bound LINGO-1 construct expressed ectopically in astrocytes. Further investigation showed that the soluble LINGO-1 ectodomain can interact with itself in trans by binding to CHO cells expressing full-length LINGO-1. Finally, we observed that soluble LINGO-1 could activate RhoA in OPCs. We propose that LINGO-1 acts as both a ligand and a receptor and that the mechanism by which it negatively regulates OPC differentiation and myelination is mediated by a homophilic intercellular interaction. Disruption of this protein-protein interaction could lead to a decrease of LINGO-1 inhibition and an increase in myelination.

FIGURE 1.

LINGO-1 expression decreases during OPC maturation in vitro. Newborn rat OPCs were grown for 3 days in PDGF/FGF as described under “Experimental Procedures.” PDGF/FGF was removed and replaced for 10 ng/ml CNTF and 15 nm T3 for an additional 4 days to promote oligodendrocyte differentiation. A, OPC differentiation in culture was assessed by immunocytochemistry and fluorescence microscopy for NG2 and MBP. B, representative Western blot analyses of cultured rat OPCs show an increase in MBP protein expression that correlated with a decrease in LINGO-1 expression as cells differentiated for 4 days in the absence or presence of CNTF/T3. C, densitometric quantitation of four independent experiments for changes in MBP and LINGO-1 expression, normalized to GAPDH. Expression is represented as percent of levels in PDGF/FGF. HMW, LINGO-1 high molecular weight band; LMW, LINGO-1 low molecular weight band; MBP, *, p < 0.05 one-way ANOVA with Bonferroni post hoc test; LINGO-1, **, p < 0.01; ***, p < 0.001 two-way ANOVA with Bonferroni post hoc test.

FIGURE 2.

Overexpression of LINGO-1 prevents OPC maturation. Rat and mouse OPC cultures were infected with lentiviral constructs expressing full-length LINGO-1 (FL-LINGO), a truncated form containing the extracellular and transmembrane domains of LINGO-1 (ECTO-LINGO), or a control virus (CTRL VIRUS), as described under “Experimental Procedures.” After PDGF/FGF removal, cells were treated for an additional 4 days for rat and 6 days for mouse in the absence or presence of 1 ng/ml CNTF and 1.5 nm T3. A, representative Western blot analysis of eight independent experiments in rat OPCs infected at 2.5 m.o.i. shows that overexpression of FL-LINGO-1 or ECTO-LINGO-1 prevented expression of MBP. Each panel contains samples run on the same gel. B, representative Western blot analysis of mouse OPCs infected at 0.5 and 2.5 m.o.i., as indicated, also shows decreased MBP expression after overexpression of these two LINGO-1 constructs. GAPDH was used as a loading control for Western blot analyses. C, decreased differentiation of mouse OPCs by FL-LINGO and ECTO-LINGO confirmed by quantitative immunofluorescence microscopy for MBP expression. Data are representative of three experiments. Values are mean ± S.E. of four replicate cultures/condition. *, p < 0.05, one-way ANOVA with Bonferroni post hoc test. D, representative images of oligodendrocyte differentiation in infected mouse cultures stained for MBP show reduced numbers of mature cells.

FIGURE 3.

The soluble LINGO-1 ectodomain prevents oligodendrocyte maturation. Newborn rat OPC cultures were grown for 4 days in medium containing PDGF/FGF and subsequently treated for 4 days with 50 μg/ml recombinant soluble LINGO-1 ectodomain (B–D) or at the doses indicated (A) after removal of PDGF/FGF and addition of 1 ng/ml CNTF and 1.5 nm T3. A, representative Western blot analysis of five independent experiments showing a dose-responsive decrease in the amount of MBP protein expression after exposure to soluble LINGO-1. The graph in the right panel represents the densitometric quantitation of the MBP bands normalized to GAPDH. Values are expressed as percent of levels in CNTF/T3. B, quantitative immunofluorescence microscopy analysis for MBP showing that treatment with soluble LINGO-1 extracellular domain prevented OPC differentiation. Data are representative of three experiments. *, p < 0.05 one-way ANOVA with Bonferroni post-hoc test. C and D, rat OPC cultures were double-immunostained for O4 and MBP. Visual (C) and quantitative (D) immunofluorescence show that the effect of soluble LINGO-1 was restricted to reducing MBP expression (green cells) and did not affect the expression of O4 (red cells). The arrows in C indicate O4⁺/MBP⁺ double-labeled cells. Data are representative of two experiments. Quantitative immunofluorescence microscopy values were obtained as in Fig. 2. *, p < 0.05, Student's t test.

FIGURE 4.

Soluble LINGO-1 ectodomain prevents myelination. Two-week-old DRG cultures were cocultured with newborn rat OPCs for an additional 2 weeks in the presence of DAPT or soluble LINGO-1 ectodomain, as indicated. A, representative pictures of myelination assessed by fluorescence microscopy show bundles of MBP+ linear segments aligning with axons identified with Neurofilament-H in control cultures (left panel). Addition of 2 μm DAPT increased myelination (right panel), whereas incubation with 50 μg/ml soluble LINGO-1 ectodomain prevented the appearance of myelinated axons in these cultures (center panel). Scale bar = 40 μm. B, myelination was assessed blindly by two trained independent observers using fluorescence microscopy, and MBP+ myelin segments were counted in high-power fields (field area 65 μm × 65 μm). Values represent the mean ± S.E. of at least 18 fields from duplicate wells in two independent experiments. ***, p < 0.001 one-way ANOVA with Bonferroni post hoc test.

FIGURE 5.

LINGO-1 inhibits OPC differentiation through intercellular interactions. Confluent astrocyte cultures were infected with control, FL-LINGO-1, and ECTO-LINGO-1 lentiviruses at 2 m.o.i. After 3 days in vitro, freshly isolated OPCs were seeded on the infected astrocytes and examined 4 days later to assess oligodendrocyte differentiation by immunocytochemistry for MBP and O4. A, MBP⁺ cell counts show that exposure to astrocytes expressing LINGO-1 decreased OPC differentiation. The effects of FL-LINGO-1 or ECTO-LINGO-1 were similar. Values represent the mean ± S.E. of three replicate cultures/condition. ***, p < 0.001 one-way ANOVA with Bonferroni post hoc test. B, representative images of OPCs cocultured with infected astrocytes and double-stained for MBP and O4 show that exposure to astrocytes infected with LINGO-1 inhibited expression of MBP (top panels, red cells) without a noticeable change in the expression of O4 (center panels, grayscale). Successful lentiviral infection was confirmed by expression of the eGFP fluorescent reporter downstream of the IRES (see “Experimental Procedures” and supplemental Fig. 4).

FIGURE 6.

The soluble LINGO-1 ectodomain binds to itself in trans. A, CHO cells stably expressing FL-LINGO-1 and control cells were incubated with increasing doses of soluble LINGO-1 extracellular domain, and bound protein was measured by alkaline phosphatase activity as described under “Experimental Procedures.” Specific binding (solid line) was calculated by subtracting the signal obtained with control CHO cells from the signal obtained with CHO cells stably transfected with FL-LINGO-1 (dashed lines). The soluble LINGO-1 ectodomain displayed specific, saturable binding to CHO cells stably transfected with FL-LINGO-1 compared with control CHO cells. Each point represents the mean ± S.E. of four replicate wells. Binding experiments were performed independently at least eight times with three separate batches of purified recombinant sLINGO-1 protein. B, newborn rat OPCs were exposed to increasing doses of soluble LINGO-1 ectodomain, rinsed, and processed for Western blotting. A representative Western blot analysis shows the bound exogenous LINGO-1 ectodomain detected with an antibody against the Fc tag. The graph below represents a densitometric quantitation of the soluble LINGO-1 bands normalized to GAPDH. The curve was fit by non-linear regression using GraphPad Prism. C, competitive binding displacement of sLINGO-1-Fc with untagged sLINGO-1. OPCs were incubated as described above for CHO cells, with 50 μg/ml tagged soluble LINGO-1-Fc and variable concentrations of untagged soluble LINGO-1. The value for half maximal binding inhibition was ∼1 μm, determined by non-linear regression with the competitive binding, one site-fit logIC50 model, using GraphPad Prism. Each point represents mean ± S.E. of three replicate wells.

FIGURE 7.

LINGO-1 activates RhoA in trans. OPCs grown for 3 days in PDGF/FGF were exposed to 5 ng/ml LPA, 100 μg/ml soluble LINGO-1 ectodomain, or Fc control for the times indicated in the figure (see “Experimental Procedures”). Soluble LINGO-1 and LPA activated RhoA, as assessed by immunoprecipitation of GTP-bound RhoA, followed by Western blotting of the immunoprecipitates and whole cell lysates with a total RhoA antibody. The graph below shows the densitometric quantitation of the RhoA bands. Values represent the ratio of RhoA-GTP/Total RhoA expressed as percent levels with respect to Control-Fc at 15 min.

FIGURE 8.

Model for LINGO-1 inhibition of myelination. LINGO-1, expressed by axons and NG2⁺ OPCs, binds to itself in trans to prevent oligodendrocyte differentiation and myelination during axo-glial interactions (left). A developmental decrease in LINGO-1 protein in O4+/MBP− premyelinating oligodendrocytes (center), and possibly in the axon as well, leads to a condition permissive for myelination (upper arrow path). Sustained LINGO-1 expression that maintains the self-interaction in trans leads to a reduced capacity of O4+/MBP− oligodendrocytes to mature to the MBP+ stage and to myelinate (lower arrow path). Neurons, red; OPC/Oligodendrocytes, green; LINGO-1, blue lines. Stage-specific oligodendrocyte markers used in this study are indicated. Gray objects represent additional axons myelinated by the same oligodendrocyte and their myelinated internodes originating from other oligodendrocytes (not pictured).