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. 2012 Mar 16;287(12):9346-59.
doi: 10.1074/jbc.M111.302802. Epub 2012 Jan 26.

EphA signaling promotes actin-based dendritic spine remodeling through slingshot phosphatase

Lei Zhou  1 , Emma V Jones, Keith K Murai
Affiliations

EphA signaling promotes actin-based dendritic spine remodeling through slingshot phosphatase

Lei Zhou et al. J Biol Chem. .

Abstract

Actin cytoskeletal remodeling plays a critical role in transforming the morphology of subcellular structures across various cell types. In the brain, restructuring of dendritic spines through actin cytoskeleletal reorganization is implicated in the regulation of synaptic efficacy and the storage of information in neural circuits. However, the upstream pathways that provoke actin-based spine changes remain only partly understood. Here we show that EphA receptor signaling remodels spines by triggering a sequence of events involving actin filament rearrangement and synapse/spine reorganization. Rapid EphA signaling over minutes activates the actin filament depolymerizing/severing factor cofilin, alters F-actin distribution in spines, and causes transient spine elongation through the phosphatases slingshot 1 (SSH1) and calcineurin/protein phosphatase 2B (PP2B). This early phase of spine extension is followed by synaptic reorganization events that take place over minutes to hours and involve the relocation of pre/postsynaptic components and ultimately spine retraction. Thus, EphA receptors utilize discrete cellular and molecular pathways to promote actin-based structural plasticity of excitatory synapses.

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Figures

FIGURE 1.
FIGURE 1.
Time-dependent effects on spine morphology and synapses by EphA activation. A–L, hippocampal neurons cultured for 14 DIV and expressing EGFP-f were stimulated with Fc control or ephrin-A for 10 min (A, D, G, and J), 45 min (B, E, H, and K), and 4 h (C, F, I, and L) prior to fixation and staining for synapsin and PSD-95. Shown is EGFP-f to delineate the cell membrane and colabeled punctae of synapsin/PSD-95 to demarcate synapses. D–I, cumulative distribution plots and graphs displaying average values of various spine parameters for each stimulus condition and time period. D, *, p = 0.004, Mann Whitney U test. E, p = 0.456, two-tailed t test. F, *, p = 0.037, two-tailed t test. G, *, p = 0.003, two-tailed t test. H, *, p = 0.00015, two-tailed t test. I, *, p = 0.0005, Mann Whitney U test. J–L, the effect of ephrin stimulation on the distance of synapses to dendritic shafts following 10 min (J), 45 min (K), and 4 h (L) of EphA stimulation. J, p = 0.687 Mann Whitney U test. K, *, p = 0.002, Mann Whitney U test. L, p = 0.113, Mann Whitney U test. For the 10-min time point, n = 19 for Fc control and n = 23 for ephrin-A Fc; for the 45 time point, n = 18 for Fc control and n = 21 for ephrin-A Fc; and for the 4-h time point, n = 17 for Fc control and n = 21 for ephrin-A Fc. The data were collected from three independent experiments. M, schematic showing the time-dependent changes in spines and synapses following EphA activation. Scale bar, 5 μm. The error bars indicate S.E.
FIGURE 2.
FIGURE 2.
EphA stimulation leads to cofilin dephosphorylation and activation in HT22 cells and hippocampal neurons. A, stimulation of HT22 cells with ephrin-A for 45 min decreased the level of phosphorylated cofilin compared with cells treated with control Fc (*, p < 0.01, t test, n = 3). B, cofilin dephosphorylation was observed 2 and 5 min after initiating ephrin-A3-Fc stimulation of HT22 cells (*, p < 0.05, one-way ANOVA with Bonferroni test, n = 3). C, ephrin-A treatment of neurons (14 DIV) for 5 min caused a significant reduction in the level of phospho-cofilin as compared with neurons treated with control Fc (*, p < 0.05, unpaired t test, n = 5). Phospho-cofilin levels were corrected according to total cofilin levels in all graphs. The error bars indicate S.E. Cof, cofilin; P-Cof, phospho-cofilin.
FIGURE 3.
FIGURE 3.
SSH is required for EphA-mediated cofilin dephosphorylation and actin remodeling. A, expression of SSH(CS) blocked ephrin-A-induced cofilin dephosphorylation in HT22 cells and hippocampal neurons (14 DIV; 5 min stimulation, n = 3). B, knockdown of SSH1 expression with SSH1 siRNAs blocked the ability of ephrin-A to reduce phospho-cofilin levels in HT22 cells (*, p < 0.05, one-way ANOVA with Kruskal-Wallis test, n = 3). C, HT22 cells were treated with Fc or ephrin-A and cell morphology, and F-actin structures were visualized with Alexa 568-conjugated phalloidin. Ephrin-A treatment caused an increase in the number of shrunken cells when compared with Fc treatment (***, p < 0.001, t test). SSH1 siRNAs reduced the percentage of shrunk cells upon ephrin-A treatment (***, p < 0.001, t test). n = 3 independent experiments (10 images from randomly chosen areas for each condition for each experiment). Arrowheads indicate shrunken cells. D, expression of a siRNA-resistant form of SSH1 (human SSH1) was able to restore ephrin-A-induced cell rounding. **, p < 0.05, ANOVA with post hoc Holm-Sidak test, versus all other conditions. Scale bars, 10 μm. The error bars indicate S.E. Cof, cofilin; P-Cof, phospho-cofilin; n.s., not significant.
FIGURE 4.
FIGURE 4.
SSH1 is expressed in the developing and adult mouse hippocampus and is localized at dendritic spines. A, Western blot analysis showing that SSH1 protein levels peak during the first postnatal week and decline toward adulthood in mouse hippocampus. B, immunostaining of neurons (14 DIV) reveal SSH1 punctae (green) localized in neuronal processes, including dendrites and spine heads (arrows). Panels 1a–3c, SSH1 (green) showed partial colocalization with PSD-95 (red). C, expression of V5-tagged wild type SSH1 (SSHwt) in dissociated hippocampal neurons. SSH1 was concentrated in the head region of the dendritic spines, and immunostaining revealed colocalization with PSD-95 and F-actin (using phalloidin staining). Scale bars, 5 μm in B and C and 1 μm in panels 1a–3c. The error bars indicate S.E.
FIGURE 5.
FIGURE 5.
SSH1 is necessary for maintaining CA1 dendritic spine morphology in organotypic hippocampal slices. A–C, examples showing abnormal spine morphology after expression of SSH(CS) when compared with control and SSHwt-expressing CA1 cells. Membrane-targeted EGFP-f was expressed alone (control) or coexpressed with SSHwt or SSH(CS). D–G, quantification of spine parameters shows that spine properties were not affected with SSHwt expression. However, spine head length was increased, and spine head width was decreased after expression of SSH(CS). H, expression of SSH(CS) decreased the number of mushroom-shaped spines and increased the number of elongated spines (*, p < 0.05; **, p < 0.01; ANOVA with Student-Newman-Keul's test). n = 24 (428 spines total, control), n = 24 (419 spines total, SSHwt), and n = 24 (410 spines, SSH(CS)) from three independent experiments. Scale bars, 5 μm in A–C (1 μm in the insets). The error bars indicate S.E.
FIGURE 6.
FIGURE 6.
Calcineurin is required for EphA-mediated cofilin dephosphorylation and actin remodeling. A, HT22 cells or neurons were pretreated with the calcineurin inhibitor FK506 (10 mm) for 10 min and then treated with Fc or ephrin-A for 5 min. FK506 blocked the ability of ephrin-A to reduce cofilin phosphorylation (p > 0.05, t test, n = 3). B, FK506 significantly reduced the percentage of shrunk cells upon ephrin-A treatment (*, p < 0.05; ***, p < 0.01; one-way ANOVA with Bonferroni test). n = 3 independent experiments (10 images from randomly chosen areas for each condition for each experiment). Scale bars, 10 μm. The error bars indicate S.E. Cof, cofilin; P-Cof, phospho-cofilin; n.s., not significant.
FIGURE 7.
FIGURE 7.
EphA regulation of dendritic spine morphology requires SSH1 and calcineurin. A, examples showing spine morphology after 10 min Fc or ephrin-A treatment. Neurons were infected with SFV expressing EGFP-f to delineate spine morphology or EGFP-f and SSH(CS) to disrupt endogenous SSH1 function. B and C, ephrin-A treatment caused an increase in spine length and a reduction in spine head width (***, p < 0.001; **, p < 0.01; one-way ANOVA with Bonferroni test). The presence of FK506 or expression of SSH(CS) abolished ephrin-A-induced spine changes. D, spine density was not significantly affected in any group. n = 15 for Fc control, n = 15 for ephrin-A, n = 15 for Fc + FK506, n = 15 for ephrin-A + FK506, n = 15 for Fc + SSH(CS), and n = 15 for ephrin-A + SSH(CS). Scale bar, 5 μm. The error bars indicate S.E.
FIGURE 8.
FIGURE 8.
EphA-induced reorganization of postsynaptic F-actin requires calcineurin and SSH1. A, examples of dendrite segments from 14 DIV neurons treated with either Fc or ephrin-A in control conditions or in the presence of FK506 or following SSH(CS) expression. Neurons were stained with Alexa 568-conjugated phalloidin (red) and PSD-95 antibody (in blue) to visualize F-actin and postsynaptic structures in spines. All of the neurons were expressing EGFP-f to outline dendrite and spine morphology. B–G, F-actin distribution across the spine head, neck, and dendritic shaft region following Fc or ephrin-A treatment and with FK506 application or expression of SSH(CS). The plots shown to the right of each image show fluorescence intensity changes for F-actin, PSD-95, and EGFP-f along the magenta line laid over spines. Brackets labeled D indicate the dendritic shafts. H–J, quantitative analysis of F-actin cluster size, intensity, and circularity. F-actin cluster size and intensity were similar in all conditions (p > 0.05, Kruskal-Wallis). However, ephrin-A treatment decreased F-actin cluster circularity (*, p < 0.05, Kruskal-Wallis with Dunn's post test). Expression of SSH(CS) alone also significantly changed F-actin cluster circularity (*, p < 0.05, Kruskal-Wallis test with Dunn's post test). n = 6 (469 particles were analyzed from six dendritic segments across three independent experiments). Scale bars, 5 and 1 μm in insets and B–G, respectively. The error bars indicate S.E.
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