Regulation of spine development by semaphorin3A through cyclin-dependent kinase 5 phosphorylation of collapsin response mediator protein 1

Abstract

Collapsin response mediator protein 1 (CRMP1) is one of the CRMP family members that mediates signal transduction of axonal guidance and neuronal migration. We show here evidence that CRMP1 is involved in semaphorin3A (Sema3A)-induced spine development in the cerebral cortex. In the cultured cortical neurons from crmp1+/- mice, Sema3A increased the density of clusters of synapsin I and postsynaptic density-95, but this increase was markedly attenuated in crmp1-/- mice. This attenuation was also seen in cyclin-dependent kinase 5 (cdk5)-/- neurons. Furthermore, the introduction of wild-type CRMP1 but not CRMP1-T509A/S522A, (Thr 509 and Ser 522 were replaced by Ala), a mutant that cannot be phosphorylated by Cdk5, into crmp1-/- neurons rescued the defect in Sema3A responsiveness. The Golgi-impregnation method showed that the crmp1-/- layer V cortical neurons showed a lower density of synaptic bouton-like structures and that this phenotype had genetic interaction with sema3A. These findings suggest that Sema3A-induced spine development is regulated by phosphorylation of CRMP1 by Cdk5.

Figure 1.

Early and postnatal expression of crmp1, nrp1, and sema3A in the cortex. Mouse E16.5, P0, and P15 sagittal sections processed for in situ hybridization with probes of CRMP1, NRP1, and Sema3A. Cortical layers are shown on the left. Scale bars: 100 μm. MZ, Marginal zone; CP, cortical plate; IZ, intermediate zone; SVZ, subventricular zone; VZ, ventricular zone.

Figure 2.

Subcellular localization of CRMP1 in cultured cortical neurons. A, Immunocytochemistry with anti-CRMP1 (a) and anti-MAP2 (b) antibodies in cultured E16.5 mouse cortical neurons on 14 d in vitro (DIV). Merged image is shown in c. Scale bars: 10 μm. B–D, Cultured E16.5 mouse cortical neurons on 14 DIV doubled stained with CRMP1 antibody (B–D, a), and Alexa488-labeled phalloidin (B, b), anti-synapsin I (C, b), or PSD-95 (D,b) antibody. A merged image is shown in c, and magnified images of the boxed area in c are shown in d. Arrowheads shown in c represent typical merged clusters. Scale bars: 5 μm. E, Immunoblot analysis of CRMP1, synapsin I (Syn I), synaptophysin (Syn) and PSD-95 contents in homogenate, P2, non-PSD, synaptosomal and PSD fractions (PSD-IT, PSD-IIT, PSD-IT, S) prepared from the P15 mouse cortex. Molecular weight is shown on the right. In CRMP1 immunoblot, upper band (80 kDa) and lower band (62 kDa) were considered to be CRMP1A and CRMP1B, respectively (see Materials and Methods).

Figure 3.

The effect of Sema3A on the density and the area of synapsin I and PSD-95 clusters and on the morphology of spines in crmp1^+/− and crmp1^−/− cultured cortical neurons. A, The cultured crmp1^+/− (a, b, e, f) or crmp1^−/− (c, d, g, h) E18.5 cortical neurons at 11 d in vitro were applied with (b, d, f, h) or without (a, c, e, g) Sema3A (3 nm) for 24 h, fixed, and then double stained with Alexa594-labeled phalloidin (magenta) and anti-synapsin I (green; a–d) or anti-PSD-95 (green; e–h) antibodies. Arrowheads represent typical immunoreactive clusters at actin-rich protrusions. B, Quantitative analysis of the effects of Sema3A on the density of synapsin I (a) or PSD-95 (c) clusters, and the mean area of synapsin I (b) or PSD-95 (d) clusters in crmp1^+/− and crmp1^−/− cultured cortical neurons. C, Quantitative analysis of the effects of Sema3A on the density (a), the length (b), and the width (c) of phalloidin-positive spines in crmp1^+/− and crmp1^−/− cultured cortical neurons. The density was estimated as shown in Materials and Methods. Each value represents mean ± SEM from 60 neurons of three independent cultures. *p < 0.01 compared with vehicle control. Scale bars: 5 μm.

Figure 4.

Sema3A increases the double-positive clusters with anti-PSD-95 and synapsin I antibodies in crmp1^+/−, but not crmp1^−/− cultured cortical neurons. A, The cultured crmp1^+/− (a–d) or crmp1^−/− (e–h) E18.5 cortical neurons at 11 d in vitro were applied with (b, d, f, h) or without (a, c, e, g) Sema3A (3 nm) for 24 h, fixed, and then double stained with anti-PSD-95 (magenta) and anti-synapsin I (green) antibodies. Magnified images of boxed area in a, b, e, and f are shown in c, d, g, and h, respectively. Arrowheads represent typical double-positive clusters. Scale bars: a, b, e, f, 5 μm; c, d, g, h, 1 μm. B, Quantitative analysis of the effects of Sema3A on the ratio of the number of double-positive to synapsin I-positive clusters (see Materials and Methods). Each value represents mean ± SEM from 46 to 60 neurons of three independent cultures. *p < 0.01 compared with vehicle control.

Figure 5.

The effect of Sema3A on the density and the area of synapsin I and PSD-95 clusters in cdk5^+/− and cdk5^−/− cultured cortical neurons. A, The cultured cdk5^+/− (a, b, e, f) or cdk5^−/− (c, d, g, h) E18.5 cortical neurons at 11 d in vitro were applied with (b, d, f, h) or without (a, c, e, g) Sema3A (3 nm) for 24 h, fixed, and then double stained with Alexa594-labeled phalloidin (magenta) and anti-synapsin I (green; a–d) or anti-PSD-95 (green; e–h) antibodies. Arrowheads represent typical immunoreactive clusters at actin-rich protrusions. Scale bars: 5 μm. B, Quantitative analysis of the effects of Sema3A on the density of synapsin I (a) or PSD-95 (c) clusters, and the mean area of synapsin I (b) or PSD-95 (d) clusters in cdk5^+/− and cdk5^−/− cultured cortical neurons. Each value represents mean ± SEM from 54 to 60 neurons of three independent cultures. *p < 0.05; **p < 0.01 compared with vehicle control.

Figure 6.

Introduction of wild-type CRMP1 (CRMP1-WT), but not CRMP1-T509A/S522A, into crmp1^−/− neurons rescues the defect in Sema3A responsiveness. A, The cultured cortical neurons of crmp1^−/− mice at E18.5, transfected with CRMP1-WT (a, b, e, f) or CRMP1-T509A/S522A (c, d, g, h) at 11 d in vitro, were applied with (b, d, f, h) or without (a, c, e, g) Sema3A (3 nm) for 24 h, fixed, and then stained with anti-synapsin I (a–d) or anti-PSD-95 (e–h) antibodies. Arrowheads represent typical immunoreactive clusters at EGFP-positive protrusions. Scale bars: 5 μm. B, Quantitative analysis of the effects of Sema3A on the density of synapsin I (a) or PSD-95 (c) clusters, and the area of synapsin I (b) or PSD-95 (d) clusters in crmp1^−/− cultured cortical neurons transfected with CRMP1-WT or CRMP1-T509A/S522A. Each value represents mean ± SEM from 60 neurons of three independent cultures. *p < 0.01 compared with vehicle control.

Figure 7.

Aberrant neurite projection and dendritic spine morphology in the cortical neurons of crmp1^−/− mice. A, Photographs of Golgi impregnation of wild-type (wt) (a, d), crmp1^−/− (b, e), and sema3A^+/−;crmp1^+/− (c, f) mice. Coronal sections of the somatosensory cortex are presented. Magnified image of boxed area in a–c are shown in d–f, respectively. In crmp1^−/− cortex, some pyramidal neurons showed atypical morphology (arrow). Cortical layers are shown on the left. Scale bars: 100 μm. B, Drawings of Golgi impregnation of wt or crmp1^−/− mouse layer III and V pyramidal neurons observed with a ×20 objective. Dendrites that extended over 100 μm from the center of the cell body were defined as apical dendrites. Scale bars: 50 μm. C, Rate of neurons possessing the apical dendrites toward the pial surface. Layer III and V neurons from wt and crmp1^−/− mice were analyzed (n = 200 from two or three individual brains). *p < 0.05 compared with wt. D, Drawings of Golgi impregnation of wt, crmp1^−/−, and sema3A^+/−;crmp1^+/− mouse layer V dendrites observed with a ×100 objective. Unbranched dendritic protrusions with a head showing a bouton-like structure were defined as dendritic spines. Scale bars: 10 μm. E, Number of dendritic spines expressed on basal and apical branches of layer V pyramidal neurons of wt, sema3A^−/−, crmp1^−/−, sema3A^+/−, crmp1^+/−, and sema3A^+/−;crmp1^+/−. The number of spines per 100 μm of dendritic shaft was calculated (n = 20–24 from two or three individual brains). *p < 0.01 compared with wt.