β1 integrin signaling promotes neuronal migration along vascular scaffolds in the post-stroke brain

Abstract

Cerebral ischemic stroke is a main cause of chronic disability. However, there is currently no effective treatment to promote recovery from stroke-induced neurological symptoms. Recent studies suggest that after stroke, immature neurons, referred to as neuroblasts, generated in a neurogenic niche, the ventricular-subventricular zone, migrate toward the injured area, where they differentiate into mature neurons. Interventions that increase the number of neuroblasts distributed at and around the lesion facilitate neuronal repair in rodent models for ischemic stroke, suggesting that promoting neuroblast migration in the post-stroke brain could improve efficient neuronal regeneration. To move toward the lesion, neuroblasts form chain-like aggregates and migrate along blood vessels, which are thought to increase their migration efficiency. However, the molecular mechanisms regulating these migration processes are largely unknown. Here we studied the role of β1-class integrins, transmembrane receptors for extracellular matrix proteins, in these migrating neuroblasts. We found that the neuroblast chain formation and blood vessel-guided migration critically depend on β1 integrin signaling. β1 integrin facilitated the adhesion of neuroblasts to laminin and the efficient translocation of their soma during migration. Moreover, artificial laminin-containing scaffolds promoted neuroblast chain formation and migration toward the injured area. These data suggest that laminin signaling via β1 integrin supports vasculature-guided neuronal migration to efficiently supply neuroblasts to injured areas. This study also highlights the importance of vascular scaffolds for cell migration in development and regeneration.

Keywords: Blood vessel; Chain migration; Laminin; Stroke; Vasculature-guided migration; β1 integrin.

Graphical abstract

Fig. 1

Role of β1 integrin in neuroblast chain formation and efficient migration toward an injured site. a: Schema of an adult mouse sagittal brain section showing the migration route of neuroblasts (red arrows). V-SVZ: ventricular-subventricular zone, RMS: rostral migratory stream, OB: olfactory bulb. b: Percentage of DCX+ neuroblasts that were β1 integrin+ in each part of the migration route. EPL: external plexiform layer, GL: glomerular layer. c: Schema of a coronal section of post-stroke mouse brain showing the migration route of neuroblasts toward the injured area (red arrows). d–e: Section of 18 d-post-stroke brain immunostained for β1 integrin (green), DCX (red), and laminin (blue) (asterisks: a blood vessel). f–h: Percentage DCX+ cells that were β1 integrin+ in the striatum or V-SVZ (f), with or without blood-vessel contact (g), and migrating individually or in chains of various sizes (h). i: Experimental design. dps: days-post-stroke. j–k: Post-stroke brain sections stained for DCX (red), CD31 (green), and GFAP (blue). l–m: Mean migration distance of neuroblasts from the V-SVZ (l), aspect ratio of the neuronal chains (m), and percentage of neuroblasts integrated into small (2–5 cells), medium (6–9 cells), or large (> 10 cells) chains (n) in the post-stroke striatum. o–s: Electron microscopy. Chains of neuroblasts (red) in the post-stroke striatum in contact with astrocytes (blue) and blood vessels (green) (o–p’), and adherent-like junctions between neuroblasts (q–r’, arrowheads). Mean junction length in the Cnt and cKO groups is shown in (s). Data are the mean ± SEM; b, f–h: n = 4 mice, l–n: n = 6 mice (Cnt), n = 8 mice (cKO); *p < 0.05, **p < 0.01. Scale bars, 50 μm: j, 20 μm: j’, 10 μm: d, 5 μm: o, 0.5 μm: o’, q, 0.1 μm: q’.

Fig. 2

Effects of neuroblast-specific β1 integrin deletion on blood vessel-associated neuronal migration in cultured post-stroke brain slices. Time-lapse images of tdTomato-labeled (red) migrating neuroblasts (arrowheads) associated with blood vessels (green) in post-stroke control (Cnt) and Itgb1-cKO brain slices (a). Mean speed (b) and percent resting-phase duration (c) in the neuroblasts migrating along blood vessels. Data are the mean ± SEM; n = 15 cells from 11 mice (Cnt), n = 17 cells from 12 mice (cKO); *p < 0.05, **p < 0.01, Scale bar, 20 μm.

Fig. 3

Laminin-β1-integrin-mediated adhesion promotes neuroblast chain formation and migration. a: Phase-contrast images of control (Cnt) and Itgb1-cKO (cKO) neuroblasts migrating on a culture dish surface coated with or without laminin. See also Movie S3. b–c: Duration of adhesion with the dish surface (b) and speed (c) of migrating neurons. d–h: Saltatory movement of neuroblasts. Time-lapse imaging of Cnt and cKO neuroblasts migrating in contact with a laminin-coated dish surface (d, see Movie S4). Schema of saltatory neuronal migration (e), percentage of resting period (f), speed of the extending leading tip (g), and duration of swelling period (h). i–k: Migration of Cnt and cKO neuroblasts cultured on a monolayer of striatal astrocytes (i). Neuroblast migration speed (j) and efficiency (k). Data are the mean ± SEM; b–c: n = 18–20 cells, f–h: n = 7 cells, j–k: n = 11–20 cells, at least two independent experiments; *p < 0.05, **p < 0.01. Scale bars, 50 μm: a, 20 μm: d. Laminin-β1-integrin-mediated adhesion promotes neuroblast chain formation and migration. a: Phase-contrast images of control (Cnt) and Itgb1-cKO (cKO) neuroblasts migrating on a culture dish surface coated with or without laminin. See also Movie S3. b–c: Duration of adhesion with the dish surface (b) and speed (c) of migrating neurons. d–h: Saltatory movement of neuroblasts. Time-lapse imaging of Cnt and cKO neuroblasts migrating in contact with a laminin-coated dish surface (d, see Movie S4). Schema of saltatory neuronal migration (e), percentage of resting period (f), speed of the extending leading tip (g), and duration of swelling period (h). i–k: Migration of Cnt and cKO neuroblasts cultured on a monolayer of striatal astrocytes (i). Neuroblast migration speed (j) and efficiency (k). Data are the mean ± SEM; b–c: n = 18–20 cells, f–h: n = 7 cells, j–k: n = 11–20 cells, at least two independent experiments; *p < 0.05, **p < 0.01. Scale bars, 50 μm: a, 20 μm: d.

Fig. 4

Laminin scaffolds promote neuronal migration via β1 integrin. a–b: Migration of neuroblasts in contact with the sponge surface. DsRed-labeled control (Cnt) or Itgb1-cKO (cKO) neuroblasts embedded in collagen gels with fluorescein-labeled porous gelatin sponge containing (laminin-sp) or not containing (Cnt-sp) laminin. Both Cnt and cKO neuroblasts (red) contacted the laminin-sp. surface (green) (a). Migration speed of neuroblasts with or without sponge contact (b). c–f: Migration of neuroblasts along an artificial laminin scaffold toward an injured area. Experimental procedure (c). Number (d) and confocal images (e–f) of neuroblasts (red) migrating along the fluorescently labeled hydrogel (blue) without and with laminin in coronal sections of the post-stroke striatum. Insets show higher magnification images of the boxed area. Data are the mean ± SEM; b: n = 9–22 cells, 3 independent experiments, d: n = 7 mice; *p < 0.05, **p < 0.01. Scale bars, 20 μm: e, 10 μm: a.