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. 2020 Feb 5;21(3):1062.
doi: 10.3390/ijms21031062.

A Role of Microtubules in Oligodendrocyte Differentiation

Bo Yoon Lee  1   2 , Eun-Mi Hur  2
Affiliations

A Role of Microtubules in Oligodendrocyte Differentiation

Bo Yoon Lee et al. Int J Mol Sci. .

Abstract

Oligodendrocytes are specialized cells that myelinate axons in the central nervous system. Defects in oligodendrocyte function and failure to form or maintain myelin sheaths can cause a number of neurological disorders. Oligodendrocytes are differentiated from oligodendrocyte progenitor cells (OPCs), which extend several processes that contact, elaborate, and eventually wrap axonal segments to form multilayered myelin sheaths. These processes require extensive changes in the cytoarchitecture and must be regulated by reorganization of the cytoskeleton. Here, we established a simple protocol to isolate and differentiate mouse OPCs, and by using this method, we investigated a role of microtubules (MTs) in oligodendrocyte differentiation. Oligodendrocytes developed a complex network of MTs during differentiation, and treatment of differentiating oligodendrocytes with nanomolar concentrations of MT-targeting agents (MTAs) markedly affected oligodendrocyte survival and differentiation. We found that acute exposure to vincristine and nocodazole at early stages of oligodendrocyte differentiation markedly increased MT arborization and enhanced differentiation, whereas taxol and epothilone B treatment produced opposing outcomes. Furthermore, treatment of myelinating co-cultures of oligodendrocytes and neurons with nanomolar concentrations of MTAs at late stages of oligodendrocyte differentiation induced dysmyelination. Together, these results suggest that MTs play an important role in the survival, differentiation, and myelination of oligodendrocytes.

Keywords: differentiation; microtubule; mouse oligodendrocyte; survival.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Isolation and differentiation of mouse OPCs. (A) Schematics and timeline for isolation and differentiation of oligodendrocytes from mouse OPCs. (B) Cell population at DIV 1. Cells were fixed and stained with anti-Olig2, anti-GFAP, and anti-TuJ1 antibodies, and percentage of cells was quantified. Graph shows mean ± SEM. n = 4. (C) Representative images of oligodendrocyte cultures. Cells were fixed at DIV 1, 3, 8, and 10 and stained for anti-α-tubulin and anti-MBP antibodies. Scale bar, 50 µm. (D) Representative images of oligodendrocytes stained with anti-α-tubulin, anti-MBP, and anti-Olig2 antibodies. Scale bar, 25 µm. (E) Representative immunoblots of oligodendrocyte lysates probed with antibodies against α-tubulin (α -tub), Olig2, MBP, and GAPDH. Quantification of MBP, α-tubulin, and GAPDH is shown. Graph shows mean ± SEM. n = 7. (F) mRNA levels of myelin-associated genes, plp and mbp (normalized to gapdh). n = 8. * p < 0.05, ** p < 0.01, and *** p < 0.001, Mann–Whitney test.
Figure 2
Figure 2
Effects of MTAs in oligodendrocytes and neurons. (A,B) Cell viability of oligodendrocytes (A) or cortical neurons (B) cultured in the absence or presence of varying concentrations of MTAs. In oligodendrocytes, MTAs were treated at DIV 3 until fixation at DIV 10. In neurons, MTAs were treated at 5–6 h after plating until fixation at DIV 3. Cell viability was quantified as described in Materials and Methods. Graph shows mean ± SEM. n = 12 in (A), n = 5 in (B). (C,D) Representative images of oligodendrocytes (C) and cortical neurons (D) treated with MTAs (2 nM) or vehicle control. Scale bar, 25 µm. (E) Percentage of differentiated oligodendrocytes (stages 3 and 4) cultured in the absence or presence of varying concentrations of MTAs. Inset, morphological classification of differentiating oligodendrocytes. Detailed criteria for each stage are described in the text. Graph shows mean ± SEM. Total numbers of cells pooled from at least four independent experiments are depicted in each bar. (F) Quantification of axon length from cortical neurons cultured in the absence or presence of varying concentrations of MTAs. Graph shows mean ± SEM. n = 5. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001; ns, statistically not significant. Student’s t-test in (A,B,E), and Mann–Whitney test in (F).
Figure 3
Figure 3
Effect of MTAs on oligodendrocyte morphology. (A,B) Oligodendrocytes were treated with MTA (2 nM) and immunostained with anti-α-tubulin and anti-MBP antibodies. (A) Number of branches of individual oligodendrocytes (left) and total branch length (right) were quantified. Graphs show mean ± SEM. Total numbers of cells pooled from at least seven independent experiments are depicted in each bar. (B) Representative images (left) and quantification of process complexity (right) of control and MTA-treated oligodendrocytes. Complexity was defined as the actual length of the process divided by the distance from the starting point of main branch to the distal tip. Graph shows mean complexity ± SEM of 42–75 cells pooled from at least four independent experiments. * p < 0.05, ** p < 0.01, and **** p < 0.0001, Student’s t-test.
Figure 4
Figure 4
MTAs affect MT arborization, MBP expression, and oligodendrocyte differentiation. (A–C) Oligodendrocytes were treated with 2 nM of each MTA at DIV 3. Cells were fixed at DIV 5 and immunostained with anti-α-tubulin and anti-MBP antibodies. (A) Representative images of control and MTA-treated oligodendrocytes. White boxes are enlarged at right. Scale bar, 25 µm. (B) Quantification of area occupied by MBP immunostaining in each oligodendrocyte. Graph shows mean ± SEM. Total number of cells pooled from at least three independent experiments are depicted in each bar. (C) Quantification of differentiation stages. Morphological criteria of differentiation stage are identical to Figure 1A. Graph shows mean ± SEM. n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. Mann–Whitney test in (B,C).
Figure 5
Figure 5
MT dynamics is essential for myelination. (A) Experimental timeline of myelinating co-cultures of OPCs and dorsal root ganglion (DRG) explants (left). Illustrations of myelinating oligodendrocytes are shown on right. DRGs were cultured and maintained for 14 days, and purified OPCs were added to the DRG explant culture at DIV 0. Co-cultures were treated with MTAs (2 nM) at DIV 12 and fixed at DIV 14. Myelination proceeds through three stages, contacting, extending, and wrapping. (B) Representative images of co-cultures stained with anti-neurofilament (NF) and anti-MBP antibodies. Scale bar, 25 µm. (C) Quantification of myelination. Myelinating oligodendrocytes were categorized as depicted in A. Graph shows mean ± SEM. n = 4. * p < 0.05, ** p < 0.01, and *** p < 0.001, Mann–Whitney test.
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