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First published online 29 March 2007
doi: 10.1242/dev.02819

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Identification of dystroglycan as a second laminin receptor in oligodendrocytes, with a role in myelination

¹ Department of Pharmacology, State University of New York, Stony Brook, NY 11794, USA.
² Department of Pathology and Centre for Brain Repair, University of Cambridge, Cambridge CB2 1QP, UK.
³ Department of Pathology, University of Medicine and Dentistry of New Jersey, Piscataway, NJ 08854, USA.

View larger version (30K): [in this window] [in a new window]   Fig. 1. Recombinant laminin that contains dystroglycan-binding, but not integrin-binding, sites binds to the surface of differentiated oligodendrocytes. (A) rE3 is a recombinant laminin protein that comprises the last two LG domains of the laminin -subunit. rE3 has been shown previously to contain dystroglycan-binding, but not integrin-binding, sites. Oligodendrocytes were differentiated for the indicated times in the presence or absence of 10 µg/ml rE3, washed to remove unbound protein and lysed. Western blots of lysates were performed to detect the presence of rE3. Blots were reprobed with actin antibodies as a loading control. (B) Densitometry to determine the average relative intensity of the captured rE3 signal (n=3, **P<0.01, error bars represent s.d.). (C) FLAG antibodies followed by FITC-conjugated secondary antibodies were used to detect cell-surface binding of rE3 to differentiated oligodendrocytes. Control (ctrl) image depicts FLAG immunofluorescence on cells without added rE3. Representative merged phase and fluorescent micrographs are shown.

View larger version (52K): [in this window] [in a new window]   Fig. 2. Oligodendrocytes express dystroglycan. (A) -dystroglycan (-DG) expression in primary oligodendrocytes. Secondary antibody alone is shown in control image (ctrl). Scale bar: 50 µm. (B) -DG and MBP co-expression in oligodendrocytes. Two different fields are shown. Scale bar: 100 µm. (C) Co-expression of oligodendrocyte marker (CC1) and -DG in corpus callosum from a postnatal-day 8 mouse brain.

View larger version (22K): [in this window] [in a new window]   Fig. 3. Oligodendrocytes differentiated on laminin show increased levels of dystroglycan. (A) Relative dystroglycan expression in oligodendrocytes differentiated for 1 or 4 days on poly-D-lysine (PDL) or laminin (Lm). Western blots were probed with antibodies specific for -dystroglycan (-DG) or ß-dystroglycan (ß-DG). Blots were reprobed with antibodies against actin as a loading control. A representative western blot is shown. (B) Densitometry to determine relative -DG expression. The average fold increase relative to expression at day 1 on PDL is shown (n=3, *P<0.05 and **P<0.01, error bars represent s.d.). (C) Densitometry to determine relative ß-DG expression. The average fold increase relative to expression at day 1 on PDL is shown (n=3, *P<0.05, error bars represent s.d.). ND, no significant difference.

View larger version (41K): [in this window] [in a new window]   Fig. 4. Myelin membrane sheet formation is reduced by dystroglycan-blocking antibodies. (A) Oligodendrocytes were differentiated for 2 or 4 days on PDL or laminin (Lm) in the presence of dystroglycan-blocking antibodies (anti-DG) or control antibodies. Indirect immunofluorescence was used to detect myelin basic protein (MBP) expression. The percentage of MBP-positive cells was determined using DAPI nuclear stain to obtain total cell counts per field. In the presence of blocking antibodies, no change in the percentage of MBP-positive cells was observed at either time point or on either substrate. (B) The average percentage of cells differentiated on laminin that express galactocerebroside C (GalC) following treatment with dystroglycan-blocking antibodies (anti-DG) or control antibodies. No significant change was found. (C) Typical morphology at day 2. Oligodendrocytes were differentiated for 2 days on laminin in the presence of dystroglycan-blocking or control antibodies. MBP immunofluorescence was used to visualize differentiated cells. Nuclei were detected using DAPI nuclear stain. (D) Typical morphology at day 4. Oligodendrocytes were differentiated for 4 days in the presence of dystroglycan (DG) or control antibodies. MBP immunofluorescence was used to visualize differentiated cells. Nuclei were detected using DAPI nuclear stain. (E) Myelin membrane sheet complexity is reduced by DG-blocking antibodies. A morphology classification scheme (bottom) was used to evaluate the degree of myelin membrane in MBP-positive cells. Examples of MBP-expressing cells: stages 1, 2 and 3 show increasing levels of process outgrowth and branching, without myelin membrane, whereas stages 4, 5 and 6 show increasing levels of complexity and myelin membrane. The percentage of cells within each category is shown for oligodendrocytes differentiated for 2 or 4 days in the presence or absence of DG-blocking antibodies (control, black squares; anti-DG, gray triangles). In the presence of DG-blocking antibodies, no difference in morphology profile is observed at day 2; however, at day 4, a significant shift away from more-complex myelin membrane structures is observed (*P<0.05, **P<0.01, error bars represent s.d.). Scale bars: 100 µm.

View larger version (48K): [in this window] [in a new window]   Fig. 5. Dystroglycan siRNA causes a decrease in oligodendrocyte differentiation and myelin protein production. (A) Dystroglycan expression is reduced in oligodendrocytes differentiated following transfection with siRNA designed to target dystroglycan mRNA (DG siRNA) compared with control siRNA (ctrl siRNA). Representative fields of cells visualized with -dystroglycan antibodies (anti-DG) are shown. (B) Immunoblots using lysates obtained from cells transfected with control (ctrl) or dystroglycan (DG) siRNA following 2 or 4 days differentiation. Blots were probed with antibodies to detect -dystroglycan, ß-dystroglycan, MBP (late-stage oligodendrocyte marker; MBP' shows a longer exposure to visualize the MBP at day 2), CNP (2',3'-cyclic nucleotide 3'-phosphodiesterase, mid-stage oligodendrocyte marker), NG2 (early stage oligodendrocyte marker) or actin (loading control). (C,D) Average expression, relative to control siRNA at 100%, for MBP or CNP at day 2 (C; n=3; *P<0.05 and **P<0.01) or day 4 (D; n=4; **P<0.01) (error bars represent s.d.). Black bars depict control siRNA and gray bars depict dystroglycan siRNA. (E) Average percentage of cells, treated with either control (ctrl) or dystroglycan siRNA, that express MBP, as determined by immunocytochemistry (n=4, error bars represent s.d.). (F) Representative fields of MBP immunocytochemistry as described in E.

View larger version (43K): [in this window] [in a new window]   Fig. 6. Enhancement of survival by laminin requires ß1-integrins, but not dystroglycan. (A) Indirect immunofluorescent TUNEL to detect cell death in oligodendrocytes transfected with control (ctrl) or dystroglycan (DG) siRNA. Bar graph depicts the average percentage of TUNEL-positive cells relative to total cell population (no significant difference, n=3, error bars represent s.d.). (B) Oligodendrocyte progenitors were differentiated on poly-D-lysine (PDL) or laminin for 4 days with increasing amounts of soluble growth factor PDGF. Integrin- and dystroglycan-blocking antibodies, or control antibodies, at 10 µg/ml were added as indicated. Survival was evaluated using TUNEL on the GalC-positive cell population (newly formed oligodendrocytes). Laminin caused a significant shift in the PDGF dose-response such that survival was increased in response to 0.1 and 1.0 ng/ml PDGF compared with cells grown on PDL (**P<0.01, error bars represent s.d.). Antibodies against ß1-integrin, but not dystroglycan, blocked the ability of laminin to amplify PDGF-mediated survival at all concentrations (**P<0.01, error bars represent s.d.).

View larger version (56K): [in this window] [in a new window]   Fig. 7. Myelination in oligodendrocyte-neuron co-cultures is perturbed by dystroglycan-blocking antibodies. (A) Rat dorsal root ganglion (DRG) neuron cultures, which contain laminin 2. Immunocytochemistry was used to detect laminin 2 (green) and nuclei (DAPI; blue). (B) Rat DRG neurons co-cultured with oligodendrocytes. Myelinating oligodendrocytes in rat DRG neuron co-cultures are found in association with laminin 2. Double immunocytochemistry was used to detect laminin 2 (green) and myelin basic protein (MBP; red). Nuclei were visualized using DAPI (blue). (C) Western blots to detect laminin proteins. Lysates obtained from oligodendrocytes (OL), astrocytes (A) or DRG neuron cultures were immunoblotted with a monoclonal antibody against the laminin 2 subunit (lm 2) or a polyclonal antibody against laminin-1 (lm-1). Note that laminin-1 antibodies detect the laminin 1, ß1 and 1 subunits. (D) Rat DRG neurons were seeded with oligodendrocyte progenitors in the presence of dystroglycan-blocking or control antibodies. At 2 weeks, differentiated oligodendrocytes were visualized using MBP immunofluorescence (green) in conjunction with neurofilament (NF) immunofluorescence (red) to visualize the underlying neurite network. (E) The average number of MBP-positive cells per field was scored in cultures treated with control and dystroglycan-blocking antibodies. No significant difference was observed (n=4, error bars represent s.d.). (F) The average percentage of myelinating MBP-positive oligodendrocytes (OL) was scored in dystroglycan-blocking- and control-antibody conditions. Dystroglycan-blocking antibodies significantly reduced the percentage of myelinating oligodendrocytes compared with control antibodies (n=4, **P<0.01, error bars represent s.d.). (G) The average number of myelinating segments per field was scored in cultures treated with control and dystroglycan-blocking antibodies. Dystroglycan-blocking antibodies significantly reduced the number of segments per field compared with control antibodies (n=4, **P<0.01, error bars represent s.d.). (H) Representative plots depicting the correlation between the myelinating oligodendrocyte (OL) to total oligodendrocyte (OL) ratio and neurite density (neurite area fraction, %). (I) Average slope of plots depicted in H (n=4, ***P<0.001, error bars represent s.d.). Scale bars: 100 µm.

View larger version (13K): [in this window] [in a new window]   Fig. 8. Model of potential integrin and dystroglycan hierarchies. (A) In a sequential model, laminins interact preferentially with integrins in newly formed oligodendrocytes (1) during the regulation of survival and process outgrowth, and preferentially with dystroglycan during the later stages (2), such as myelin membrane formation. (B) In a parallel model, laminins interact with both integrins and dystroglycan to promote myelin membrane formation, but receptor crosstalk or signaling changes can alter the relative contribution of each receptor during distinct oligodendrocyte developmental stages.

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