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. 2019 Dec 27;9(1):19882.
doi: 10.1038/s41598-019-56488-7.

Laminin α2, α4, and α5 Chains Positively Regulate Migration and Survival of Oligodendrocyte Precursor Cells

Nobuharu Suzuki  1   2 , Mai Hyodo  3 , Chikako Hayashi  4   3 , Yo Mabuchi  3   5 , Kaori Sekimoto  3 , Chinami Onchi  4 , Kiyotoshi Sekiguchi  6 , Chihiro Akazawa  7   8
Affiliations

Laminin α2, α4, and α5 Chains Positively Regulate Migration and Survival of Oligodendrocyte Precursor Cells

Nobuharu Suzuki et al. Sci Rep. .

Abstract

In the developing central nervous system (CNS), oligodendrocyte precursor cells (OPCs) migrate along blood vessels and are widely distributed in the CNS. Meanwhile, OPCs require survival factors from the extracellular microenvironment. In other tissues, laminins, heterotrimetric (αβγ) extracellular matrix proteins, promote cell migration and survival. However, the expression pattern and functions of laminins in OPC development remain poorly understood. In the present study, we first investigated the expression of laminin α chains, which bind to cell surface receptors such as integrins, in the postnatal murine brain. We found that laminin α1, α2, α4, and α5 chains were expressed around blood vessels and OPCs attached the laminin α chain-positive vessels. We then evaluated the effect of these laminins on OPCs activity using recombinant laminin E8s (LME8s) that are minimally active fragments of the laminin isoforms. OPCs attached on LM211E8, LM411E8, and LM511E8, containing laminin α2, α4, and α5 chains, respectively, through integrin β1. Further, these three LME8s promoted migration of OPCs, and OPC survival was prolonged on either LM411E8 or LM511E8 via the activation of focal adhesion kinase. Together, our findings suggest that laminins expressed surrounding blood vessels positively regulate migration and survival of OPCs through the integrin β1-FAK pathway.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Expression of laminin α chains in P2 Sox10-Venus and WT mouse brains. (a) Immunohistochemistry of laminin α chains (red) in the ventricular/subventricular zone of P2 Sox10-Venus mouse brains. (b) Higher magnification images of the boxes in (a). (c) Immunohistochemistry of laminin α chains (green) and Olig2 (red) in P2 WT mouse brains. (d) Immunohistochemistry of laminin α chains (green) and CD31 (red) in P2 WT mouse brains. (e) Immunohistochemistry of laminin α chains (red) in the pia of P2 Sox10-Venus mouse brains. DAPI staining (blue) was used to visualize nuclei in (a,b,d,e). Arrowheads: attachment of OPC to vessel. Scale bars: 20 μm (a); 10 μm (b–e). LM: laminin.
Figure 2
Figure 2
Cell attachment activity of OPCs on laminin E8 fragments. (a) Ratio of attached cell numbers on recombinant laminin E8 fragments (LM111E8, LM211E8, LM411E8, and LM511E8). The number of attached cells on non-coat as a control was set as 1.0. Error bars, s.e.m. (*p < 0.05; **p < 0.01, t test). (b–d) Ratio of attached cell numbers on LM211E8 (b), LM411E8 (c), and LM511 (d) with each inhibitory antibody. The number of attached cells with normal IgG as a control was set as 1.0. Error bars, s.e.m. (*p < 0.05; **p < 0.01, t test). At least triplicate experiments were independently performed. 111: LM111E8; 211: LM211E8; 411: LM411E8; 511: LM511E8; Itg: integrin.
Figure 3
Figure 3
The effect of laminin E8 fragments on OPC migration. Ratio of migrated OPC numbers on recombinant laminin E8 fragments (LM211E8, LM411E8, and LM511E8) and poly-D-lysine. The number of migrated cells on non-coat as a control was set as 1.0. Error bars, s.e.m. (*p < 0.05, t test). At least triplicate experiments were independently performed. PDL: Poly-D-lysine; 211: LM211E8; 411: LM411E8; 511: LM511E8.
Figure 4
Figure 4
The effect of laminin E8 fragments on OPC survival. (a) Ratio of total OPCs numbers on recombinant laminin E8 fragments (LM211E8, LM411E8, and LM511E8). The number of attached cells on poly-D-lysine as a control was set as 1.0. Error bars, s.e.m. (*p < 0.05, t test). (b) Ratio of cleaved caspase-3-positive OPC numbers on recombinant laminin E8 fragments (LM211E8, LM411E8, and LM511E8). The number of cleaved caspase-3-positive cells on poly-D-lysine as a control was set as 1.0. Error bars, s.e.m. (*p < 0.05, **p < 0.01, t test). (c) Ratio of Ki67-positive OPC numbers on recombinant laminin E8 fragments (LM211E8, LM411E8, and LM511E8). The number of Ki67-positive cells on poly-D-lysine as a control was set as 1.0. Error bars, s.e.m. (d) Western blotting of phosphorylated FAK (Tyr397) and total FAK in OPCs cultured on recombinant laminin E8 fragments (LM211E8, LM411E8, and LM511E8). These images were cropped from the results of the membrane blots as indicated in Fig. S2. (e) Quantification of phosphorylation levels of FAK. The intensity of Western blotting bands was measured and pFAK/FAK was calculated. The phosphorylation level in OPCs on poly-D-lysine as a control was set as 1.0. Error bars, s.e.m. (*p < 0.05, t test). (f,g) Ratio of cleaved caspase-3-positive OPC numbers on LM411E8 (f) and LM511E8 (g) in the presence of the FAK inhibitor. The number of cleaved caspase-3-positive cells with only dimethyl sulfoxide as a control was set as 1.0. Error bars, s.e.m. (*p < 0.05, t test). At least triplicate experiments were independently performed. PDL: poly-D-lysine; 211: LM211E8; 411: LM411E8; 511: LM511E8; DMSO: dimethyl sulfoxide.
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