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Myosin II has distinct functions in PNS and CNS myelin sheath formation.

Wang H, Tewari A, Einheber S, Salzer JL, Melendez-Vasquez CV - J. Cell Biol. (2008)

Bottom Line: We have found that inhibition of myosin II, a key regulator of actin cytoskeleton dynamics, has remarkably opposite effects on myelin formation by Schwann cells (SC) and oligodendrocytes (OL).In contrast, OL branching, differentiation, and myelin formation are potentiated by inhibition of myosin II.Our data indicate that the mechanisms regulating myelination in the peripheral and central nervous systems are distinct.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Hunter College, City University of New York, New York, NY 10065, USA.

ABSTRACT
The myelin sheath forms by the spiral wrapping of a glial membrane around the axon. The mechanisms responsible for this process are unknown but are likely to involve coordinated changes in the glial cell cytoskeleton. We have found that inhibition of myosin II, a key regulator of actin cytoskeleton dynamics, has remarkably opposite effects on myelin formation by Schwann cells (SC) and oligodendrocytes (OL). Myosin II is necessary for initial interactions between SC and axons, and its inhibition or down-regulation impairs their ability to segregate axons and elongate along them, preventing the formation of a 1:1 relationship, which is critical for peripheral nervous system myelination. In contrast, OL branching, differentiation, and myelin formation are potentiated by inhibition of myosin II. Thus, by controlling the spatial and localized activation of actin polymerization, myosin II regulates SC polarization and OL branching, and by extension their ability to form myelin. Our data indicate that the mechanisms regulating myelination in the peripheral and central nervous systems are distinct.

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Myosin II inhibition in oligodendrocyte-DRG cocultures enhances myelin formation. (A) Purified OL progenitors (OPC) were seeded onto 3-wk-old DRG neuronal cultures. After 2 wk in media containing 1 μg/ml TrkA-Fc, cultures were stained for Olig2, MBP, and neurofilament. Treatment with blebbistatin resulted in a prominent dose-dependant increase in the amount of myelin segments formed in cultures. (B) Quantitation of myelin segments (left), OL cell number (middle), and MBP expression (right) in control and blebbistatin-treated cultures. A significant dose-dependent increase in the amount of MBP+ segments was observed in cultures treated with blebbistatin (P < 0.001, ANOVA). The percentage of MBP+ cells is comparable in all conditions, and at the highest blebbistatin concentration (25 μM), a significant reduction in the number of Olig2+ cells was observed. Data represent mean ± SEM from two independent experiments (two cultures per condition per experiment). (C, left) Quantitation of the number of myelin segments formed per single OL in cultures. Low-density 16-d-old myelinating cocultures were stained for Olig2, MBP, and neurofilament to identify individual OL nuclei, OL expressing MBP, and OL actively myelinating axons. A significant increase in the mean number of myelin segments made by a single OL in treated cultures was observed (P < 0.001, ANOVA). Data represent mean ± SEM from two independent experiments (two cultures per condition per experiment). (right) Representative examples of myelinating OL in control and treated cultures are shown. Bars: (A) 50 μm; (C) 20 μm.
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fig4: Myosin II inhibition in oligodendrocyte-DRG cocultures enhances myelin formation. (A) Purified OL progenitors (OPC) were seeded onto 3-wk-old DRG neuronal cultures. After 2 wk in media containing 1 μg/ml TrkA-Fc, cultures were stained for Olig2, MBP, and neurofilament. Treatment with blebbistatin resulted in a prominent dose-dependant increase in the amount of myelin segments formed in cultures. (B) Quantitation of myelin segments (left), OL cell number (middle), and MBP expression (right) in control and blebbistatin-treated cultures. A significant dose-dependent increase in the amount of MBP+ segments was observed in cultures treated with blebbistatin (P < 0.001, ANOVA). The percentage of MBP+ cells is comparable in all conditions, and at the highest blebbistatin concentration (25 μM), a significant reduction in the number of Olig2+ cells was observed. Data represent mean ± SEM from two independent experiments (two cultures per condition per experiment). (C, left) Quantitation of the number of myelin segments formed per single OL in cultures. Low-density 16-d-old myelinating cocultures were stained for Olig2, MBP, and neurofilament to identify individual OL nuclei, OL expressing MBP, and OL actively myelinating axons. A significant increase in the mean number of myelin segments made by a single OL in treated cultures was observed (P < 0.001, ANOVA). Data represent mean ± SEM from two independent experiments (two cultures per condition per experiment). (right) Representative examples of myelinating OL in control and treated cultures are shown. Bars: (A) 50 μm; (C) 20 μm.

Mentions: We next examined whether myosin II was also required for central myelination. To this end, cocultures of DRG neurons and purified rat cortical OL precursor cells (OPC) were grown for 2 wk in myelination-promoting conditions with or without blebbistatin. Cultures were then stained for Olig2, MBP, and neurofilament to identify individual OL nuclei, OL expressing MBP, and those actively myelinating axons. In striking contrast to SC myelination, inhibition of myosin II activity by blebbistatin resulted in a prominent increase in the amount of myelin formed in DRG-OL cocultures (Fig. 4 A). Quantitation of the number of MBP+ segments showed a significant dose-dependent increase (P < 0.001, analysis of variance [ANOVA]) in the amount of MBP+ segments in cultures treated with blebbistatin (Fig. 4 B). Interestingly, despite the difference in the total number of myelin segments, we found that differentiation of OL as measured by MBP expression (Fig. 4 B) was comparable between control and treated cultures (∼70–80% MBP+ cells). Furthermore, at the highest blebbistatin concentration tested (25 μM), a significant decrease in proliferation (Table S1) and in the total number of Olig2+ cells was consistently observed, yet these cultures had, on average, more myelinated segments per field than control cultures (P < 0.001). These results strongly suggest that in blebbistatin-treated cultures, individual myelinating OL give rise to more internodes than OL in control cultures. To facilitate the quantitation of the number of internodes made by a single myelinating OL, we established cocultures with a lower number of OPC (50,000 instead of 100,000). As anticipated, the mean number of myelin segments made by a single OL in blebbistatin-treated cultures was 3–4 times more than OL in control cultures (control, 4.8 ± 3.5; 10 μM blebbistatin, 14.6 ± 5.5; 25 μm blebbistatin, 18.5 ± 9.3; mean ± SEM; P < 0.001; Fig. 4 C). Collectively, these results indicate that myosin II activity is not required for OL differentiation and myelination, and that in the presence of blebbistatin, individual OL are more branched and make more myelin.


Myosin II has distinct functions in PNS and CNS myelin sheath formation.

Wang H, Tewari A, Einheber S, Salzer JL, Melendez-Vasquez CV - J. Cell Biol. (2008)

Myosin II inhibition in oligodendrocyte-DRG cocultures enhances myelin formation. (A) Purified OL progenitors (OPC) were seeded onto 3-wk-old DRG neuronal cultures. After 2 wk in media containing 1 μg/ml TrkA-Fc, cultures were stained for Olig2, MBP, and neurofilament. Treatment with blebbistatin resulted in a prominent dose-dependant increase in the amount of myelin segments formed in cultures. (B) Quantitation of myelin segments (left), OL cell number (middle), and MBP expression (right) in control and blebbistatin-treated cultures. A significant dose-dependent increase in the amount of MBP+ segments was observed in cultures treated with blebbistatin (P < 0.001, ANOVA). The percentage of MBP+ cells is comparable in all conditions, and at the highest blebbistatin concentration (25 μM), a significant reduction in the number of Olig2+ cells was observed. Data represent mean ± SEM from two independent experiments (two cultures per condition per experiment). (C, left) Quantitation of the number of myelin segments formed per single OL in cultures. Low-density 16-d-old myelinating cocultures were stained for Olig2, MBP, and neurofilament to identify individual OL nuclei, OL expressing MBP, and OL actively myelinating axons. A significant increase in the mean number of myelin segments made by a single OL in treated cultures was observed (P < 0.001, ANOVA). Data represent mean ± SEM from two independent experiments (two cultures per condition per experiment). (right) Representative examples of myelinating OL in control and treated cultures are shown. Bars: (A) 50 μm; (C) 20 μm.
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fig4: Myosin II inhibition in oligodendrocyte-DRG cocultures enhances myelin formation. (A) Purified OL progenitors (OPC) were seeded onto 3-wk-old DRG neuronal cultures. After 2 wk in media containing 1 μg/ml TrkA-Fc, cultures were stained for Olig2, MBP, and neurofilament. Treatment with blebbistatin resulted in a prominent dose-dependant increase in the amount of myelin segments formed in cultures. (B) Quantitation of myelin segments (left), OL cell number (middle), and MBP expression (right) in control and blebbistatin-treated cultures. A significant dose-dependent increase in the amount of MBP+ segments was observed in cultures treated with blebbistatin (P < 0.001, ANOVA). The percentage of MBP+ cells is comparable in all conditions, and at the highest blebbistatin concentration (25 μM), a significant reduction in the number of Olig2+ cells was observed. Data represent mean ± SEM from two independent experiments (two cultures per condition per experiment). (C, left) Quantitation of the number of myelin segments formed per single OL in cultures. Low-density 16-d-old myelinating cocultures were stained for Olig2, MBP, and neurofilament to identify individual OL nuclei, OL expressing MBP, and OL actively myelinating axons. A significant increase in the mean number of myelin segments made by a single OL in treated cultures was observed (P < 0.001, ANOVA). Data represent mean ± SEM from two independent experiments (two cultures per condition per experiment). (right) Representative examples of myelinating OL in control and treated cultures are shown. Bars: (A) 50 μm; (C) 20 μm.
Mentions: We next examined whether myosin II was also required for central myelination. To this end, cocultures of DRG neurons and purified rat cortical OL precursor cells (OPC) were grown for 2 wk in myelination-promoting conditions with or without blebbistatin. Cultures were then stained for Olig2, MBP, and neurofilament to identify individual OL nuclei, OL expressing MBP, and those actively myelinating axons. In striking contrast to SC myelination, inhibition of myosin II activity by blebbistatin resulted in a prominent increase in the amount of myelin formed in DRG-OL cocultures (Fig. 4 A). Quantitation of the number of MBP+ segments showed a significant dose-dependent increase (P < 0.001, analysis of variance [ANOVA]) in the amount of MBP+ segments in cultures treated with blebbistatin (Fig. 4 B). Interestingly, despite the difference in the total number of myelin segments, we found that differentiation of OL as measured by MBP expression (Fig. 4 B) was comparable between control and treated cultures (∼70–80% MBP+ cells). Furthermore, at the highest blebbistatin concentration tested (25 μM), a significant decrease in proliferation (Table S1) and in the total number of Olig2+ cells was consistently observed, yet these cultures had, on average, more myelinated segments per field than control cultures (P < 0.001). These results strongly suggest that in blebbistatin-treated cultures, individual myelinating OL give rise to more internodes than OL in control cultures. To facilitate the quantitation of the number of internodes made by a single myelinating OL, we established cocultures with a lower number of OPC (50,000 instead of 100,000). As anticipated, the mean number of myelin segments made by a single OL in blebbistatin-treated cultures was 3–4 times more than OL in control cultures (control, 4.8 ± 3.5; 10 μM blebbistatin, 14.6 ± 5.5; 25 μm blebbistatin, 18.5 ± 9.3; mean ± SEM; P < 0.001; Fig. 4 C). Collectively, these results indicate that myosin II activity is not required for OL differentiation and myelination, and that in the presence of blebbistatin, individual OL are more branched and make more myelin.

Bottom Line: We have found that inhibition of myosin II, a key regulator of actin cytoskeleton dynamics, has remarkably opposite effects on myelin formation by Schwann cells (SC) and oligodendrocytes (OL).In contrast, OL branching, differentiation, and myelin formation are potentiated by inhibition of myosin II.Our data indicate that the mechanisms regulating myelination in the peripheral and central nervous systems are distinct.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Hunter College, City University of New York, New York, NY 10065, USA.

ABSTRACT
The myelin sheath forms by the spiral wrapping of a glial membrane around the axon. The mechanisms responsible for this process are unknown but are likely to involve coordinated changes in the glial cell cytoskeleton. We have found that inhibition of myosin II, a key regulator of actin cytoskeleton dynamics, has remarkably opposite effects on myelin formation by Schwann cells (SC) and oligodendrocytes (OL). Myosin II is necessary for initial interactions between SC and axons, and its inhibition or down-regulation impairs their ability to segregate axons and elongate along them, preventing the formation of a 1:1 relationship, which is critical for peripheral nervous system myelination. In contrast, OL branching, differentiation, and myelin formation are potentiated by inhibition of myosin II. Thus, by controlling the spatial and localized activation of actin polymerization, myosin II regulates SC polarization and OL branching, and by extension their ability to form myelin. Our data indicate that the mechanisms regulating myelination in the peripheral and central nervous systems are distinct.

Show MeSH
Related in: MedlinePlus