Netrin-1 Is an Important Mediator in Microglia Migration

Abstract

Microglia migrate to the cerebral cortex during early embryonic stages. However, the precise mechanisms underlying microglia migration remain incompletely understood. As an extracellular matrix protein, Netrin-1 is involved in modulating the motility of diverse cells. In this paper, we found that Netrin-1 promoted microglial BV2 cell migration in vitro. Mechanism studies indicated that the activation of GSK3β activity contributed to Netrin-1-mediated microglia migration. Furthermore, Integrin α6/β1 might be the relevant receptor. Single-cell data analysis revealed the higher expression of Integrin α6 subunit and β1 subunit in microglia in comparison with classical receptors, including Dcc, Neo1, Unc5a, Unc5b, Unc5c, Unc5d, and Dscam. Microscale thermophoresis (MST) measurement confirmed the high binding affinity between Integrin α6/β1 and Netrin-1. Importantly, activation of Integrin α6/β1 with IKVAV peptides mirrored the microglia migration and GSK3 activation induced by Netrin-1. Finally, conditional knockout (CKO) of Netrin-1 in radial glial cells and their progeny led to a reduction in microglia population in the cerebral cortex at early developmental stages. Together, our findings highlight the role of Netrin-1 in microglia migration and underscore its therapeutic potential in microglia-related brain diseases.

Keywords: GSK3β; Integrin α6β1; Netrin-1; cerebral cortex; microglia migration.

Figure 1

Promotional effect of Netrin-1 on BV2 cell migration. (A) Model of Netrin-1 conditioned medium preparation. (B) Representative images showing BV2 cell morphology in response to Netrin-1 stimulation (6 h). (C) Quantification of the percentage of elongated BV2 cells. Student’s t test, p = 0.0001. (D) Trans-well assay showing the migration of BV2 cells in application of Netrin-1 (18 h). (E) Quantification of the number of migrated BV2 cells. Student’s t test, p < 0.0001. The data are from at least three independent experiments. *** p < 0.001.

Figure 2

The increase of GSK3β activity by Netrin-1. (A) Western blotting showing the activity of kinases as indicated after Netrin-1 stimulation. (B) Quantification of the phosphorylated levels of indicated kinases. Student’s t test, p < 0.01 for pGSK3β. Data are presented as the mean ± SEM. The data are from at least three independent experiments. ns, no significant difference; ** p < 0.01.

Figure 3

Suppression of Netrin-1 mediated BV2 migration by GSK3β inhibitor. (A) Representative images showing BV2 cell morphology after Netrin-1 stimulation with or without LiCl (6 h). (B) Quantification of the percentage of elongated BV2 cells. One-way ANOVA, for Control and Netrin-1 groups, p < 0.0001; for Control and Netrin-1 + LiCl groups, p = 0.3314; for Netrin-1 and Netrin-1 + LiCl groups, p < 0.0001. (C) Trans-well assay showing the migration of BV2 cells after Netrin-1 stimulation with or without LiCl (18 h). (D) Quantification of the number of migrated BV2 cells. One-way ANOVA, for Control and Netrin-1 groups, p < 0.0001; for Control and Netrin-1 + LiCl groups, p = 0.0102; for Netrin-1 and Netrin-1 + LiCl groups, p < 0.0001. The data are from at least three independent experiments. ns, no significant difference; * p < 0.05; *** p < 0.001.

Figure 4

The high expression of Integrin α6β1 in microglia as well as the interaction between Integrin α6β1 and Netrin-1. (A) Single-cell data from Tabula Muris showing the expression level of Netrin-1 traditional receptors, involving Dcc, Neo1, Unc5a, Unc5b, Unc5c, Unc5d, and Dscam in mouse microglia. (B) Single-cell data from Tabula Muris showing the expression level of Integrin subunits associated with Netrin-1. (C) Multiple MST traces for different mixture ratios of Integrin α6β1 and Netrin-1. (D) Dose–response analysis of the MST traces for determination of the steady-state affinity of the Integrin α6β1–Netrin-1 interaction.

Figure 5

IKVAV mimicking the effects of Netrin-1 on BV2 migration and GSK3β activation. (A) Representative images showing BV2 cell morphology in response to IKVAV stimulation (6 h). (B) Quantification of the percentage of elongated BV2 cells. Student’s t test, p < 0.0001. (C) Trans-well assay showing the migration of BV2 cells in application of IKVAV (18 h). (D) Quantification of the number of migrated BV2 cells. Student’s t test, p < 0.0001. (E) Western blotting showing the activity of AKT and GSK3 after IKVAV stimulation. (F) Quantification of the phosphorylated levels of AKT. Student’s t test, p = 0.0052. (G) Quantification of the phosphorylated levels of GSK3β. Student’s t test, p = 0.0059. Data are presented as the mean ± SEM. The data are from at least three independent experiments. ** p < 0.01; *** p < 0.001.

Figure 6

The expression and distribution of Netrin-1 in developing cerebral cortex. (A) Western blotting showing the expression level of Netrin-1 at indicated developmental stages. (B) The distribution of extracellular Netrin-1 in E15.5 brain sections.

Figure 7

The decrease of Iba1⁺ microglia in the developing cerebral cortex of Ntn1 cKO mice. (A) Immunostaining showing the number of Iba1⁺ microglia in the cortical brain at indicated developmental stages. (B) Quantification of the microglia numbers. For each group, 10 sections from 3 mice were used for statistical analysis. Student’s t test, for E18.5, p < 0.0001; for P5, p < 0.0001; for P14, p = 0.1159; for P30, p = 0.2692; for P30, p = 0.3057. Data are presented as the mean ± SEM. The data are from at least three independent experiments. ns, no significant difference; *** p < 0.001.