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Semaphorin 3A Activates the Guanosine Triphosphatase Rab5 to Promote Growth Cone Collapse and Organize Callosal Axon Projections

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Semaphorin 3A Activates the Guanosine Triphosphatase Rab5 to Promote Growth Cone Collapse and Organize Callosal Axon Projections

Kong-Yan Wu et al. Sci Signal.

Abstract

Axon guidance (pathfinding) wires the brain during development and is regulated by various attractive and repulsive cues. Semaphorin 3A (Sema3A) is a repulsive cue, inducing the collapse of axon growth cones. In the mammalian forebrain, the corpus callosum is the major commissure that transmits information flow between the two hemispheres, and contralateral axons assemble into well-defined tracts. We found that the patterning of callosal axon projections in rodent layer II and III (L2/3) cortical neurons in response to Sema3A was mediated by the activation of Rab5, a small guanosine triphosphatase (GTPase) that mediates endocytosis, through the membrane fusion protein Rabaptin-5 and the Rab5 guanine nucleotide exchange factor (GEF) Rabex-5. Rabaptin-5 bound directly to Plexin-A1 in the Sema3A receptor complex [an obligate heterodimer formed by Plexin-A1 and neuropilin 1 (NP1)]; Sema3A enhanced this interaction in cultured neurons. Rabaptin-5 bridged the interaction between Rab5 and Plexin-A1. Sema3A stimulated endocytosis from the cell surface of callosal axon growth cones. In utero electroporation to reduce Rab5 or Rabaptin-5 impaired axon fasciculation or caused mistargeting of L2/3 callosal projections in rats. Overexpression of Rabaptin-5 or Rab5 rescued the defective callosal axon fasciculation or mistargeting of callosal axons caused by the loss of Sema3A-Plexin-A1 signaling in rats expressing dominant-negative Plexin-A1 or in NP1-deficient mice. Thus, our findings suggest that Rab5, its effector Rabaptin-5, and its regulator Rabex-5 mediate Sema3A-induced axon guidance during brain development.

Figures

Fig. 1
Fig. 1. Rabaptin-5 interacts with Plexin-A1
(A) Schematic illustration of mouse Plexin-A1 protein consisting of Sema, PSI (plexin-semaphorin-integrin), and IPT [immunoglobulin (Ig)–like, plexins, transcription factor] extracellular domains, and the transmembrane and intracellular domains. (B) Immunoblots for coimmunoprecipitation of Rabaptin-5 with Plexin-A1 in homogenate of postnatal day 5 (P5) rat upper cortical layers. Blots are representatives of three experiments. Ctrl, control. (C) Pull-down of Rabaptin-5 expressed in HEK293 cells by GST–cytoPlexin-A1 or GST beads. Blot is representative of three experiments. (D) Pull-down of purified His-tagged Rabaptin-5 by GST–cytoPlexin-A1 or GST beads. Blots are representatives of five experiments. (E and F) Immunoblots for cytoPlexin-A1 (by the HA tag), Rabaptin-5, and Rab5b before (Input) and after immunoprecipitation for Rabaptin-5 (E) or HA (F) in the lysates from HEK293 cells. Blots are representatives of five experiments. (G) Immunoblots for Rabaptin-5 and Plexin-A1 after immunoprecipitation for Rabaptin-5 in cultured cortical neurons treated with Sema3A (300 ng/ml) for indicated times. Blots are representatives of three experiments. (H and I) Immunostaining analysis for colocalization between endogenous Rab5 and Plexin-A1 in cultured L2/3 cortical neurons transfected with GFP, without or with treatment with Sema3A (300 ng/ml, 5 min). Data are means ± SEM from three experiments (29 neurons for control and 34 neurons for Sema3A). ***P < 0.001, Student’s t test. Scale bar, 5 μm.
Fig. 2
Fig. 2. Sema3A signaling activates Rab5
(A and B) Abundance of Rab5-GTP determined by pull-down using GST-R5BD in 3 DIV (days in vitro) L2/3 neurons treated with Sema3A (300 ng/ml) for the indicated time. Data are means ± SEM for the ratio of Rab5-GTP and total Rab5 from three experiments, normalized to control. *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA (analysis of variance) with Tukey HSD (honestly significant difference) post hoc test. a.u., arbitrary units. (C) Immunoblots for the efficiency of siRabaptin-5 in cultured cortical neurons with endogenous b-actin and vehicle-encoded GFP as loading controls. (D and E) Rab5-GTP marked by GST-R5BD in axonal growth cones of Satb2+ L2/3 neurons transfected with scrambled (control) or indicated siRNAs, treated with vehicle or Sema3A (300 ng/ml, 15 min). Vector-encoded GFP was used to mark growth cone morphology and volume. Data are means ± SEM from at least 23 neurons from three experiments. *P < 0.05, **P < 0.01, two-way ANOVA with post hoc comparisons using the Bonferroni correction. Scale bar, 10 μm. (F) Immunoblots for Rabaptin-5 and Rabex-5 after immunoprecipitation for Rabaptin-5 from homogenates of P5 rat upper-layer cortex. Blots are representatives of five experiments. (G and H) Immunoblots for the abundance of Rab5-GTP in the cortex or hippocampus from P30 mice with indicated genotypes. Data are means ± SEM of three experiments. ***P < 0.001, one-way ANOVA with Tukey HSD post hoc test.
Fig. 3
Fig. 3. Role of Rab5b in growth cone dynamics
(A and B) Immunoblots for the efficiencies of indicated siRNAs on the abundance of ectopic HA-Rab5b in the lysates of HEK293 cells (A) or endogenous Rab5b in cultured cortical neurons (B), with vectors encoding corresponding scrambled sequences as controls. Blots are representatives of three experiments. (C to E) Analysis for axonal growth cones of Satb2+ L2/3 neurons transfected with indicated vectors treated with or without Sema3A (300 ng/ml, 15 min). F-actin was marked by Alexa Fluor 555 phalloidin (C). Data are means ± SEM for the percentage of collapsed growth cones (D) or normalized growth cone area (E) from three experiments. **P < 0.01, ***P < 0.001, two-way ANOVA with Bonferroni correction post hoc test. Scale bar, 10 μm. (F to H) Analysis for axonal growth cones of Satb2+ L2/3 neurons transfected with indicated constructs. Data are means ± SEM for the percentage of axons without apparent growth cones (G) or normalized growth cone area (H) from three experiments. *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA with Tukey HSD post hoc test. Scale bar, 10 μm.
Fig. 4
Fig. 4. Silencing of Rab5 impedes Sema3A-induced membrane endocytosis
(A and C) Cultured L2/3 neurons transfected with indicated plasmids were incubated with Alexa-labeled dextran (A) or FM4-64 (C), in the presence or absence of Sema3A for 15 min. Vehicle plasmids (A) or pSUPER encoding scrambled siRNA (C) were set as control. Images show endocytosed dyes (red) after treatment with Sema3A. Scale bar, 10 μm. (B and D) Quantification for the uptake of dextran (B) or FM4-64 (D). The ratio between the intensity of dyes and GFP represents endocytosis activity. Data are means ± SEM of three experiments, normalized to control group. *P < 0.05, **P < 0.01, ***P < 0.001, two-way ANOVA with Bonferroni correction post hoc test.
Fig. 5
Fig. 5. Sema3A repels and inhibits axonal growth through Rab5
(A) E17.5 rat brain slices stained for Sema3A and Smi312, and with DAPI (4′,6-diamidino-2-phenylindole). NCx, neocortex; WM, white matter; VZ/SVZ, ventricular zone/subventricular zone; SCS, subcallosal sling; IGG, indusium griseum glia. Scale bar, 500 μm. (B and C) Axon patterns of DIV3 L2/3 neurons transfected with Rab5b siRNA (#503) or scrambled siRNA (control) on stripes of Sema3A or control proteins. Images are representatives of at least 127 neurons in each group (B). Data are means ± SEM of three experiments for the percentage of neurons with cell body and axon restricted in the same gap between two stripes. ***P < 0.001, two-way ANOVA with Bonferroni correction post hoc test. Scale bar, 60 μm. (D) Illustration of two-compartment microfluidic culture system, with magnified area showing microchannels between two compartments. (E to G) Axonal growth of L2/3 neurons transfected with Rab5b siRNA (#503) or scrambled siRNA into distal compartments without or with the presence of Sema3A (300 ng/ml) added at DIV4. Shown are representatives 48 hours after Sema3A application (E), percentage of axons that had crossed the channels (F), and average length of axons in distal compartment (G). Data are means ± SEM of three experiments. *P < 0.05, **P < 0.01, ***P < 0.001, two-way ANOVA with Bonferroni correction post hoc test. Scale bar, 100 μm.
Fig. 6
Fig. 6. Rab5 is essential for axon fasciculation in vivo
(A and B) Callosal axonal patterns of primary somatosensory cortex (S1) and secondary somatosensory cortex (S2) from P7 rats electroporated at E15.5 with indicated plasmids (dnRab5b or Rab5b, siRab5b#503 or siRab5b#503 plus siRab5a#2 and siRab5c#2; Fig. S7, A to C, shows specificity and efficiency of each) together with pCAG-IRES-EGFP, with DAPI indicating cortical layers and corpus callosum landmarks. Boxed areas show axonal tracts in the midline and contralateral side, 1400 μm away from the midline, with dashed white lines marking dorsal-ventral (DV) surfaces of the white matter and short white lines indicating borders of electroporated axonal bundles. Scale bar, 1000 μm. (C to F) Quantification of the ratio between width of electroporated axonal tracts and that of the entire white matter in the midline (C and E) or contralateral side (D and F) in each group, normalized with GFP intensity in electroporated S1 and S2 regions and that from control group set as 1. Data are means ± SEM from at least three rats in each group. *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA with Tukey HSD post hoc test. (G) Representative images for the effect of decreased Rab5b on callosal axons. Arrow indicates the invasion of axons into the ventral parenchyma underneath the corpus callosum. Scale bar, 1000 μm.
Fig. 7
Fig. 7. Role of Rabaptin-5 in axon fasciculation in vivo
(A and B) Callosal axonal patterns of S1 and S2 from P7 rats electroporated at E15.5 with indicated plasmids and pCAG-IRES-EGFP, with DAPI indicating cortical layers and corpus callosum landmarks. Borders of axonal tracts and the white matter are indicated by short and dashed white lines, respectively. Scale bar, 1000 μm. (C to F) Quantification for the ratio between the width of electroporated axonal bundles and that of the white matter in the midline (C and E) and contralateral side (D and F) of each group, normalized with GFP intensity in S1 and S2 regions. Data are means ± SEM from at least three rats in each group. *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA with Tukey HSD post hoc test (C and D) and two-way ANOVA with Bonferroni correction post hoc test (E and F).
Fig. 8
Fig. 8. Rab5 prevents axon defasciculation caused by NP1 knockout
(A) E15.5 NP1f/f mouse embryos were electroporated with vehicle vector, ubiqutin-Cre-2A-GFP, or ubiqutin-Cre-2A-GFP plus Rab5b, together with pCAG-IRES-EGFP. Callosal axons were analyzed at P7, with DAPI and Sema3A signals revealing the corpus callosum landmarks. Boxed areas show axonal tracts in the midline (a1, a3, a5) and contralateral side (a2, a4, a6), with short and dashed white lines indicating boundaries of electroporated axonal tracts and the white matter, respectively. Scale bar, 1000 μm. (B and C) Quantification for the ratio between the width of electroporated axonal tracts and that of the white matter in the corpus callosum midline (B) and contralateral side (C) in each group, normalized with GFP intensity in S1 and S2 regions. Data are means ± SEM of at least three mice in each group. ***P < 0.001, two-way ANOVA with Bonferroni correction post hoc test.
Fig. 9
Fig. 9. Inhibition of Rab5 causes mistargeting of contralateral callosal axon trajectories
(A to C) E15.5 mouse embryos were electroporated with vehicle, Rab5b, or dnRab5b plasmid, together with pCAG-IRES-EGFP. P14 mouse brains at the approximate level of bregma 1.58 mm were coronally sectioned and stained for Sema3A and DAPI to mark cortical layers and the corpus callosum. Boxed areas in (A) indicate contralateral S1 and S2 (B) and RSA and RSG (C) regions, respectively. RSA, retrosplenial agranular cortex; RSG, retrosplenial granular cortex. Scale bars, 1000 μm (A) and 500 μm (B and C). (D) Quantification for callosal axon projection index (API), calculated with the following formula: (GFP intensity at indicated region/GFP intensity of total axon bundles in contralateral hemisphere) × 100. Data are means ± SEM from at least four mice in each group. *P < 0.05, **P < 0.01, one-way ANOVA with Tukey HSD post hoc test.
Fig. 10
Fig. 10. Forced expression of Rab5 overcomes mistargeting of contralateral axon projections caused by NP1 knockout
(A to C) E15.5 NP1f/f mouse embryos were electroporated with vehicle vector, ubiqutin-Cre-2A-GFP, or ubiqutin-Cre-2A-GFP plus Rab5b, together with pCAG-IRES-EGFP. P14 mice brains were coronally sectioned and stained for Sema3A and DAPI to mark cortical layers and the corpus callosum. Boxed areas in (A) indicate contralateral S1 and S2 (B) and RSA and RSG (C) regions, respectively. Scale bars, 1000 μm (A) and 500 μm (B and C). (D) Quantification for API. Data are means ± SEM from at least three mice in each group. *P < 0.05, **P < 0.01, ***P < 0.001, two-way ANOVA with Bonferroni correction post hoc test.

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