Research ArticleCELLULAR NEUROSCIENCE

New neurons use Slit-Robo signaling to migrate through the glial meshwork and approach a lesion for functional regeneration

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Science Advances  12 Dec 2018:
Vol. 4, no. 12, eaav0618
DOI: 10.1126/sciadv.aav0618
  • Fig. 1 Reactive astrocytes inhibit the ability of neuroblasts to approach the lesion in the poststroke brain.

    3D reconstruction of SBF-SEM images of a chain of neuroblasts (A) in the poststroke striatum and the same chain with a single (A′) or all (A″) of the surrounding astrocytes tightly enwrapping an adjacent blood vessel (BV). (B and C) Z-stacked confocal images of neuroblasts (Dcx, red), astrocytes (GFAP, green), and mature neurons (NeuN, blue) in an 18-day poststroke brain section. Panels in (C) are higher-magnification images of the neuroblast chain boxed in (B). The white broken line in (B) indicates the boundary of the infarct area. The pink broken line in (B) indicates the boundary of the reactive astrocyte–rich and –free areas. (D) Distribution of Dcx+ neuroblasts in the reactive astrocyte (RA)–free area and the reactive astrocyte–rich area at different distances from the reactive astrocyte–free and reactive astrocyte–rich border (<100, 100 to 200, 200 to 500, and >500 μm lateral to the boundary) in 12-day [wild-type (WT), n = 6 mice; knockout (KO), 11 mice], 18-day (WT, n = 14 mice; KO, n = 12 mice), and 35-day poststroke mice (WT, n = 17 mice; KO, n = 10 mice). Comparison among the time points (12, 18, and 35 days): one-way analysis of variance (ANOVA); comparison between WT and KO: two-tailed unpaired t test. (E and F) Localization of Slit1 and Robo2 in intact and 18-day poststroke striatum. Higher-magnification images (E′ and F′) show Dcx+ neuroblasts (red) and GFAP+ reactive astrocytes (blue), which expressed Slit1 and Robo2 (green), respectively. Box plots show the median (dot), upper and lower quartiles (box), maximal and minimal values excluding outliers (whiskers), and outliers (blue squares). *P < 0.05, **P < 0.01. Scale bars, 100 μm (B, E, and F) and 20 μm (C, E′, and F′).

  • Fig. 2 Slit-Robo signaling controls neuronal migration through stroke-activated astrocytes.

    (A and B) Distribution of Dcx+ neuroblasts in 18-day poststroke controls (Cnt) and Slit1-KO Gfap-EGFP mouse striatum. (B) shows the percentage of Dcx+ neuroblasts in areas at different distances from the V-SVZ (two-tailed Mann-Whitney U test; Cnt, n = 12 mice; KO, n = 15 mice). (C to E) Quantitative analyses of the time-lapse imaging (movie S3) of migrating neuroblasts in the Cnt and Slit1-KO poststroke striatum, which was enriched with reactive astrocytes. Graphs show the migration speed [C, Steel-Dwass test; reactive astrocyte (RA)–free area: Cnt, n = 77 cells; KO, n = 56 cells; reactive astrocyte–rich area: Cnt, n = 99 cells; KO, n = 97 cells] and the moving speed (D) and length (E) of the leading process (two-tailed one-way ANOVA and Bonferroni test; reactive astrocyte–free: Cnt, n = 14 cells; KO, n = 19 cells; reactive astrocyte–rich: Cnt, n = 24 cells; KO, n = 23 cells). (F and G) Electron microscopy of neuroblast chains (red) and their surrounding astrocytic processes (blue) in 14-day poststroke striatum (arrows indicate astrocytic processes inserted into the chain). (G) shows the percentage of the chain-forming neuronal membrane in contact with astrocytes (two-tailed Mann-Whitney U test; Cnt, n = 36 cells; KO, n = 73 cells). (H to K′) Distribution of Slit1-KO Dcx-EGFP neuroblasts (green) 5 days after transplantation into WT poststroke brain and their association with surrounding reactive astrocytes (red; two-tailed unpaired t test; Cnt, n = 8 mice; KO, n = 7 mice). (L to O′) Distribution of neuroblasts in the striatum with Robo2-knockdown astrocytes [area containing noninfected astrocytes (area 1) and areas enriched with shRNA-expressing astrocytes (area 2: <200 μm from area 1; area 3: ≥200 μm from area 1); two-tailed unpaired t test; Cnt, n = 7 mice; Robo2-knockdown (KD), n = 6 mice]. tGFP+, turbo-GFP–positive. Box plots show the median (dot), upper and lower quartiles (box), maximal and minimal values excluding outliers (whiskers), and outliers (blue squares). *P < 0.05, **P < 0.01, ***P < 0.001. Scale bars, 100 μm (A, J, K, L, N, N′, O, and O′), 20 μm (J′ and K′), and 2 μm (F). MCAO, middle cerebral artery occlusion.

  • Fig. 3 Neuroblast-induced morphological dynamics of reactive astrocytes involve the inhibition of Cdc42-regulated actin polymerization.

    (A) 3D reconstruction of SBF-SEM images of the surface of reactive astrocytes (blue) surrounding a chain of neuroblasts (transparent pink). (A′-1) and (A′-2) show higher-magnification images of the boxed area. (B to E) Time-lapse imaging of F-actin visualized with Lifeact (green) in hypoxia-treated striatal astrocytes embedded in collagen gel with Dcx-DsRed neuroblasts (red) (C). (D) shows the extension of processes [arrowheads in (B)] for 18 min before (pre) and during/after neuroblast contact (two-tailed paired t test; Cnt, n = 9 areas; KO, n = 10 areas). (E) shows the frequency of F-actin–rich protrusion [arrowheads in (B)] formation at the surface of astrocytes before and during neuroblast contact (two-tailed paired t test; Cnt, n = 9 areas; KO, n = 10 areas). (F) Quantification procedure in (H) to (L). (G) Migration speed of neuroblasts in contact with hypoxia-treated astrocytes (two-tailed Mann-Whitney U test; WT neuroblasts, n = 35 cells; KO neuroblasts, n = 32 cells; WT-Cdc42, n = 50 cells; CA-Cdc42, n = 51 cells). (H and I) Time-lapse imaging of F-actin (Lifeact) in striatal astrocytes with migrating neuroblasts (asterisks) on a culture dish (H) and relative F-actin level in the astrocytes before, during, and after neuroblast contact (I) (WT, n = 42 areas; KO, n = 45 areas; within groups, Friedman test followed by pairwise comparison of Scheffé; WT versus KO, Mann-Whitney U test). (J) Relative F-actin level in astrocytes transfected with WT-Cdc42 or CA-Cdc42 during neuroblast contact (two-tailed unpaired t test; WT-Cdc42, n = 37 areas; CA-Cdc42, n = 39 areas). (K and L) Time-lapse imaging of Cdc42 biosensor-transfected astrocytes (rectangles, moving processes) with neuroblasts (asterisks) (K) and the relative Cdc42 activity in these astrocytes before, during, and after neuroblast contact (L) (WT, n = 74 areas; KO, n = 73 areas; within groups, Friedman test followed by pairwise comparison of Scheffé; WT versus KO, Mann-Whitney U test). Box plots show the median (dot), upper and lower quartiles (box), maximal and minimal values excluding outliers (whiskers), and outliers (blue squares). Dot plots show each value (small dots), and line graph values represent the mean ± standard error of the mean. *P < 0.05, **P < 0.01, ***P < 0.001, #P < 0.05, ##P < 0.01, ###P < 0.001. Scale bars, 20 μm (H and K) and 5 μm (A to C). Asterisk indicates a significant difference between Pre and each of the other time points in the same cell group; number sign indicates a significant difference between groups at one time point.

  • Fig. 4 Enhanced migration promotes neuronal regeneration around the lesion.

    (A) Experimental procedure. (B to D) Transplant-derived Slit1-OE cells expressing DARPP32, parvalbumin, and calretinin [arrows in (B) to (D)]. (E to H) Electrophysiological properties of transplant-derived neurons. In response to depolarizing current injection, transplant-derived cells showed early outward rectification and slowly developing ramp depolarization (E, bottom) (pulse amplitudes, 100, 200, and 300 pA) and a long latency to spike discharge (E, top) (pulse amplitude, 350 pA). (F) shows the change in voltage from the beginning (measured at 80 ms from the onset) to the end of a current injection just below spike threshold [ΔV in (E)] in transplant (TP)–derived and host neurons (two-tailed unpaired t test; transplanted, n = 4 cells; host, n = 5 cells). sEPSCs were observed in the transplant-derived cells (G) (the averaged sEPSC is presented in the box). (H) shows the comparison of the quantitative data between the transplant-derived and host striatal projection neurons (mean ± standard error of the mean, two-tailed unpaired t test). (I to K) Morphology of a transplant-derived striatal projection neuron intracellularly labeled with biocytin. Higher-magnification images of spiny dendrites (rectangles) and the lentivirus-labeled soma (arrow) are shown in (J), (J′), and (K), respectively. (L and M) Cholera toxin subunit B (CTB)–mediated labeling of Slit1-OE transplant-derived neurons projecting into the globus pallidus [arrows in (M)]. (N and O) Immunoelectron microscopy of synaptic structures formed by the transplant-derived cells. A postsynaptic area in the striatum (N) and a presynaptic area in the globus pallidus (O) labeled with gold particles (arrows) are shown. (N′) and (O′) are higher-magnification images of the boxed areas in (N) and (O), respectively. (P and Q) Percentage of NeuN colabeled transplanted cells distributed at various distances from the transplant site in the host striatum (Q) [Cnt (DsRed+), n = 20 mice; Slit1-OE (Venus+), n = 21 mice]. (R and S) Axonal projection of Cnt and Slit1-OE striatal new neurons into the globus pallidus, a major target area of the striatal projection neurons. Slit1-OE axons were more widely distributed throughout the globus pallidus compared with the Cnt axons (R). The percentage of projections into the lateral part of the globus pallidus was significantly higher in the Slit1-OE group (S) (two-tailed unpaired t test; Cnt, n = 6 mice; Slit1-OE, n = 6 mice). Box plots show the median (dot), upper and lower quartiles (box), maximal and minimal values excluding outliers (whiskers), and outliers (blue squares). **P < 0.01, ***P < 0.001. Scale bars, 100 μm (P and R), 10 μm (B to D, I, K, and M), 2 μm (J), 0.2 μm (N and O), and 0.1 μm (N′ and O′).

  • Fig. 5 Enhanced neuroblast migration promotes functional recovery.

    (A to C) Neurological assessments performed before (7 days after stroke) and 1 to 4 weeks after V-SVZ cell transplantation. The percentage of right swings in the elevated body swing test (EBST) (A) (Friedman test followed by pairwise comparison of Scheffé; transplant (TP) Cnt, n = 20 mice; TP Slit1-OE, n = 19 mice; no TP, n = 16 mice; TP Slit1–OE and DTA, n = 15 mice), the percentage of all forepaw slips that were left forepaw slips (B), and the percentage of all paw slips that were left forepaw and hindpaw slips (C) in the foot-fault test (FFT) (TP Cnt, n = 20 mice; TP Slit1-OE, n = 19 mice; no TP, n = 16 mice; TP Slit1–OE and DTA, n = 15 mice) are presented. Comparison between 7 and 35 days in each group: two-tailed Wilcoxon’s signed-rank test, *P < 0.05, **P < 0.01; comparison between the no-TP group and every other group at 35 days: Steel test, #P < 0.05, ##P < 0.01. (D to G) Scatterplots and regression lines showing the correlation of behavioral improvement in the elevated body swing test and foot-fault test with the total number of transplant-derived NeuN+ neurons (D and E) or the percentage of transplant-derived NeuN+ cells that moved more than 100 μm toward the injured area from the transplanted site (F and G) in each mouse (n = 34). (H to J) Neurological assessments performed before and 4 and 10 weeks after V-SVZ cell transplantation (7, 35, and 77 days after stroke, respectively). The percentage of right swings in the elevated body swing test (H) (Friedman test followed by pairwise comparison of Scheffé), the percentage of all forepaw slips that were left forepaw slips (I), and the percentage of all paw slips that were left forepaw and hindpaw slips (J) in the foot-fault test are presented (TP Cnt, n = 19 mice; TP Slit1-OE, n = 20 mice; TP Slit1–OE and DTA, n = 14 mice). Comparison between day points in each group: Friedman test followed by pairwise comparison of Scheffé, *P < 0.05, **P < 0.01; comparison between TP Cnt and every other group at 77 days: Steel test, #P < 0.05, ##P < 0.01. (K) Schematic summary of the findings. St, striatum; GP, globus pallidus; OB, olfactory bulb. Dot plots represent each value (small dot) with lines representing mean ± standard error of the mean.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/12/eaav0618/DC1

    Supplementary Materials and Methods

    Fig. S1. Neuroblast migration in the 18-day poststroke brain.

    Fig. S2. Impaired neuroblast migration by Slit-Robo signaling inhibition.

    Fig. S3. Neuroblast-induced cytoskeletal modification in reactive astrocytes.

    Fig. S4. Effects of Slit1 overexpression on the migration, proliferation, and apoptosis of neuroblasts.

    Movie S1. SBF-SEM and 3D-reconstructed images of migrating neuroblasts and surrounding astrocytes in the poststroke brain.

    Movie S2. Time-lapse imaging of migrating neuroblasts and reactive astrocytes in a poststroke brain slice.

    Movie S3. Time-lapse imaging of neuroblasts migrating in a reactive astrocyte–enriched area in poststroke brain slices.

    Movie S4. Altered actin polymerization in astrocytes making contact with migrating neuroblasts.

    Movie S5. Alteration in Cdc42 activity in astrocytes induced by neuroblast contact.

    Movie S6. Dynamic alteration of Cdc42 activity and protrusion formation at the surface of astrocytes.

    Movie S7. Time-lapse imaging of Cnt and Slit1-OE neuroblasts in the poststroke brain.

    References (59, 60)

  • Supplementary Materials

    The PDF file includes:

    • Supplementary Materials and Methods
    • Fig. S1. Neuroblast migration in the 18-day poststroke brain.
    • Fig. S2. Impaired neuroblast migration by Slit-Robo signaling inhibition.
    • Fig. S3. Neuroblast-induced cytoskeletal modification in reactive astrocytes.
    • Fig. S4. Effects of Slit1 overexpression on the migration, proliferation, and apoptosis of neuroblasts.
    • Legends for movies S1 to S7
    • References (59, 60)

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mov format). SBF-SEM and 3D-reconstructed images of migrating neuroblasts and surrounding astrocytes in the poststroke brain.
    • Movie S2 (.mov format). Time-lapse imaging of migrating neuroblasts and reactive astrocytes in a poststroke brain slice.
    • Movie S3 (.mov format). Time-lapse imaging of neuroblasts migrating in a reactive astrocyte–enriched area in poststroke brain slices.
    • Movie S4 (.mov format). Altered actin polymerization in astrocytes making contact with migrating neuroblasts.
    • Movie S5 (.mp4 format). Alteration in Cdc42 activity in astrocytes induced by neuroblast contact.
    • Movie S6 (.mov format). Dynamic alteration of Cdc42 activity and protrusion formation at the surface of astrocytes.
    • Movie S7 (.mov format). Time-lapse imaging of Cnt and Slit1-OE neuroblasts in the poststroke brain.

    Files in this Data Supplement:

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