Article Figures & Data
Figures
- Figure 1.
LINGO-1 and NGF inhibit oligodendrocyte differentiation and myelination of TrkA positive DRG neurons. A, Anti-MBP immunostaining (red) of OPC/DRG cocultures after 14 d in culture in the presence of a control IgG, an anti-LINGO-1 antibody (1A7) or in the absence of NGF with the addition of the NGF scavenger TrkA-Fc. B, Anti-MBP immunostaining (red) of OPC/DRG cocultures after lentiviral transduction of DN- and FL-LINGO-1 in DRGs, OPCs, or both cell types. C, The number of myelinating MBP+ oligodendrocytes/field were quantified for the expression of FL-LINGO-1, DN-LINGO-1, and the control in each of the conditions. *p < 0.05; **p < 0.0001, unpaired t test. D, Western blot analysis for MAG, MBP, and β-actin, of OPC/DRG cocultures infected with FL and DN forms of LINGO-1, NgR, TROY/Taj, p75NTR, and TrkA after 14 d in culture. E, Electron micrographs of spinal cord sections were analyzed for extent of myelination in cross sections from a transgenic mouse overexpressing axonal LINGO-1 at postnatal day 8 and a wild-type littermate control. F, The number of myelinated axons in the spinal cords were counted and normalized per field. Error bars indicate SD. *p < 0.001, unpaired t test. Scale bars, 100 μm.
- Figure 2.
NGF/TrkA regulates the axonal expression of LINGO-1 on NGF-dependent DRG neurons. A, Expression of LINGO-1, TrkA, MBP, MAG, and β-actin were examined in the developing mouse spinal cord. Tissue from postnatal day 0, 2, 4, 6, 8, 10, 20, and adult were isolated and prepared for Western blot analysis. B, OPC/DRG cocultures were subjected to NGF treatment for 2 d (0–100 ng/ml) and immunoprecipitated for TrkA. Samples were probed for phosphorylated (P)-tyrosine, LINGO-1, and β-actin. C, Purified DRGs and OPCs were subjected to NGF treatment (0–100 ng/ml) in similar manner, and cells were extracted and prepared for Western blot analysis for LINGO-1 and β-actin. D, Purified DRG neuronal cultures were either treated with or without NGF (100 ng/ml) and immunostained for LINGO-1. Scale bar, 100 μm. E, RT-PCR TrkA, LINGO-1, and glyceraldehyde phosphate dyhydrogenase (GAPDH; control) was performed on purified OPCs, SCs, and DRGs in the presence or absence of NGF or BDNF. F, Combined immunostaining for TrkA and in situ hybridization for LINGO-1 mRNA were performed on frozen sections of rat postnatal day 6 dorsal root ganglia. Sections were cut and probed with a digoxigenin-labeled RNA for LINGO-1 (red) and then with an antibody to TrkA (green). A sense probe for LINGO-1 was used as a control for the in situ hybridization. G, LINGO-1 inhibits oligodendrocyte differentiation and myelination downstream of NGF/TrkA signaling. DRG cultures were infected for 2 d with DN-LINGO-1 (1), control (2), DN-TrkA (3), control (4), or a combination of DN-TrkA and FL-LINGO-1 (5) or DN-LINGO-1 and FL-TrkA (6). Purified OPCs were seeded onto the DRG cultures after infection and allowed to grow for 14 d. Cocultures were then extracted and prepared for Western blot analysis. Samples were probed for MAG, MBP, LINGO-1, and β-actin.
- Figure 3.
LINGO-1 does not influence Schwann cell myelination. A, B, Lentivirus was used to transduce FL- and DN-LINGO-1 in the SC/DRG cocultures and myelination was analyzed by immunostaining for MBP and Western blot analysis for MAG, P0, and β-actin. The expression of the myelin proteins and the formation of myelin internodes were unaltered by the expression of the FL- or DN-LINGO-1. Scale bar, 100 μm.
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