Cdk5-dependent Mst3 phosphorylation and activity regulate neuronal migration through RhoA inhibition

Abstract

The radial migration of newborn neurons is critical for the lamination of the cerebral cortex. Proper neuronal migration requires precise and rapid reorganization of the actin and microtubule cytoskeleton. However, the underlying signaling mechanisms controlling cytoskeletal reorganization are not well understood. Here, we show that Mst3, a serine/threonine kinase highly expressed in the developing mouse brain, is essential for radial neuronal migration and final neuronal positioning in the developing mouse neocortex. Mst3 silencing by in utero electroporation perturbed the multipolar-to-bipolar transition of migrating neurons and significantly retards radial migration. Although the kinase activity of Mst3 is essential for its functions in neuronal morphogenesis and migration, it is regulated via its phosphorylation at Ser79 by a serine/threonine kinase, cyclin-dependent kinase 5 (Cdk5). Our results show that Mst3 regulates neuronal migration through modulating the activity of RhoA, a Rho-GTPase critical for actin cytoskeletal reorganization. Mst3 phosphorylates RhoA at Ser26, thereby negatively regulating the GTPase activity of RhoA. Importantly, RhoA knockdown successfully rescues neuronal migration defect in Mst3-knockdown cortices. Our findings collectively suggest that Cdk5-Mst3 signaling regulates neuronal migration via RhoA-dependent actin dynamics.

Keywords: Rho-GTPase; actin; cyclin-dependent kinase; neuronal migration.

Figure 1.

Mst3 is important for radial migration in the mouse cerebral cortex. A, Developmental expression of Mst3 protein in the mouse brain. Whole-brain lysates at various developmental stages (E12 to adult) were subjected to Western blotting for Mst3 with GAPDH as a loading control. B, Developmental regulation of Mst3 activity in the mouse brain. Whole-brain lysates at different developmental stages were subjected to a kinase activity assay using histone H1 as the substrate. C, Knockdown of endogenous Mst3 expression in neurons by pSUPER-Mst3 shRNA constructs. Cultured cortical neurons at 0 DIV were transfected with one of three different pSUPER-Mst3 shRNAs or scrambled shRNA. Protein lysates were collected at 4 DIV and subjected to Western blotting for Mst3 and GAPDH as a control. D–I, Mst3 knockdown arrested migrating cortical neurons in the intermediate zone (IZ) in the early stage. E14 mouse embryos were electroporated with GFP expression construct and pSUPER, Mst3 shRNA, or Mst3 scramble shRNA (Mst3 Scr) by in utero electroporation. Mouse brain sections were collected at E17 (D–G) and E18 (H, I). D, Representative images stained for GFP (green) and CS-56 (subplate marker, red), and counterstained with TO-PRO-3 (nuclear marker, blue). SVZ, Subventricular zone; VZ, ventricular zone; CP, cortical plate. Scale bar, 100 μm. E, Quantification of the distribution of GFP⁺ neurons in D. **p < 0.01 versus pSUPER, n = 3 experiments, Student's t test, mean ± SEM. More than 500 GFP⁺ neurons from six brains were analyzed in each group. F, Mst3 knockdown resulted in impaired morphology of migrating neurons in the IZ. Asterisks indicate representative neurons with unipolar/bipolar morphology in the pSUPER, Mst3 Scr, and Mst3-WT Res groups in contrast to no process morphology in Mst3 shRNA group. Scale bar, 25 μm. G, Quantification of the percentages of neurons with unipolar/bipolar, no, and multiple processes. **p < 0.01 versus pSUPER, n = 3, Student's t test, mean ± SEM. More than 200 GFP⁺ neurons from six brains were analyzed in each group. H, Representative images of E18 cortical sections stained with GFP antibody (green) and TO-PRO-3 (blue). I, Quantification of the distribution of GFP⁺ neurons in H. *p < 0.05, n = 3, Student's t test, mean ± SEM. More than 300 GFP⁺ neurons from four brains were analyzed in each group.

Figure 2.

Mst3 knockdown perturbs neuronal positioning and dendritogenesis. A, B, Mst3 knockdown perturbed the final positions of layer II–IV neurons. E14 mouse embryos were electroporated with GFP expression construct and pSUPER or Mst3 shRNA by in utero electroporation. Mouse brains were collected at P2 (A) or P5 (B) and stained for GFP (green), Cutl1 (layer II–IV marker, red), and TO-PRO-3 (nuclear marker, blue) (A) or GFP alone (B). Scale bar, 100 μm. C, Mst3 knockdown altered neuronal morphology and reduced apical dendrite length. E14 mouse embryos were electroporated with GFP expression construct and pSUPER or Mst3 shRNA by in utero electroporation. Brains were collected at P5 and stained for GFP alone. Asterisks indicate representative neurons. Scale bar, 50 μm. D, Quantification of the distance from GFP⁺ neuron cell bodies to the pial surface in A and B. ***p < 0.001, n = 3, Student's t test, mean ± SEM. More than 200 GFP⁺ neurons from three brains were analyzed in each group. E, Quantification of the length of apical dendrites of GFP⁺ neurons in C. ***p < 0.001, n = 3, Student's t test, mean ± SEM. More than 200 GFP⁺ neurons from three brains were analyzed in each group. F–H, Spontaneous glutamatergic synaptic activity [i.e., spontaneous EPSCs (sEPSCs)] was altered in Mst3-knockdown neurons. F, Representative traces of sEPSCs were recorded in layer II–III pyramidal neurons of pSUPER- and Mst3 shRNA-electroporated cortices from P15–P20. Mean frequencies (G) and mean amplitudes (H) of sEPSCs in layer II–III pyramidal neurons of pSUPER- and Mst3 shRNA-electroporated sections. *p < 0.05, n = 3, Student's t test, mean ± SEM. Six to eight neurons from three mice were analyzed in each group.

Figure 3.

Mst3 activity is required for proper radial migration. A, The KD mutant (Lys53 → Arg, K53R) of Mst3 lacked kinase activity. In vitro kinase assay using histone H1 as a substrate. B, Overexpression of RNAi-resistant Mst3 (WT Res or KD Res) restored Mst3 protein expression in HEK293T cells expressing Mst3 shRNA. C–F, Kinase activity of Mst3 is important for radial migration. C, Expression of Mst3 WT Res, but not the Mst3 KD Res, rescued the delayed migration of cortical neurons upon Mst3 knockdown. Scale bar, 100 μm. D, Images of neurons in the upper intermediate zone (IZ) stained with GFP antibody (green) and Mst3 antibody (red). Scale bar, 25 μm. E, Quantification of the distribution of GFP⁺ neurons in the brain sections electroporated with Mst3 shRNA together with Mst3 WT Res or KD Res. ***p < 0.001, n = 3, Student's t test, mean ± SEM. F, Quantification of the percentages of neurons with unipolar/bipolar, no, or multiple processes. ***p < 0.001, n = 3, Student's t test, mean ± SEM. More than 200 GFP⁺ neurons from six brains were analyzed in each group. G, Expression of Mst3 WT or Mst3 KD had no effect on neuronal migration. Scale bar, 100 μm. H, Quantification of the distribution of GFP⁺ neurons in the brain sections electroporated with pCAG, Mst3 WT, or Mst3 KD. n = 3, Student's t test, mean ± SEM.

Figure 4.

Cdk5-dependent phosphorylation of Mst3 at Ser79 is important for radial migration. A, Cdk5 inhibition abolishes Mst3 activation. Cultured cortical neurons were treated for 1 h with different specific kinase inhibitors: 200 nm BIO (GSK3), 10 μm Ros (Cdk5), 10 μm SP600125 (JNK), 100 nm UO126 (MEK), or 20 μm H89 (PKA). Mst3 kinase activity was examined by Western blot analysis for Mst3 autophosphorylation at Thr178 (p-Mst3). B, Mst3 kinase activity was reduced in Cdk5^−/− mouse brains. The brain lysates of E18 Cdk5^−/− mice were immunoprecipitated with anti-Mst3 antibody and subjected to a kinase assay (n = 3; p-H1: 0.53 ± 0.05-fold in Cdk5^−/− brains, mean ± SEM, p < 0.001, Student's t test) or Western blot analysis with p-Mst3 antibody (n = 4; p-Mst3: 0.49 ± 0.07-fold in Cdk5^−/− brains, mean ± SEM, p < 0.001, Student's t test). C, Direct phosphorylation of Mst3 by Cdk5 in an in vitro phosphorylation assay. D, Serine phosphorylation of Mst3 was dramatically reduced in Cdk5^−/− brains blotted with p-(Ser) CDK substrate antibody. E, Schematic diagram indicating the domains of Mst3 and mutation sites described in the text. F, Cdk5 phosphorylated Mst3 at Ser79. Western blot analysis was performed using anti-phospho-serine (p-Ser CDK substrate) antibody. G, Generation of a phosphospecific antibody against Mst3 at Ser79 (p-S79 Mst3). Mst3 protein was overexpressed in HEK293T cells, immunoprecipitated by anti-Mst3 antibody, and incubated with Cdk5/p35 complex for the in vitro phosphorylation assay. Phosphorylated Mst3 was detected by a phosphospecific antibody targeting the Ser79 site of Mst3. H, Calf intestinal phosphatase (CIP) treatment abolished Ser79 phosphorylation of Mst3. I, Mst3 phosphorylation at Ser79 was attenuated in Cdk5^−/− mouse brains. (n = 5; p-S79 Mst3: 0.58 ± 0.01-fold in Cdk5^−/− brains, mean ± SEM, p < 0.001; Student's t test). J–L, The Ser79 phosphodeficient mutant of Mst3 (S79A) failed to rescue the delayed migration of cortical neurons upon Mst3 knockdown. J, Representative images at E17 were stained with anti-GFP antibody (green) and TO-PRO-3 (blue). Scale bar, 100 μm. K, Quantification of the distribution of GFP⁺ neurons. ***p < 0.001 versus pCAG+Mst3 shRNA; n = 3, Student's t test, mean ± SEM. L, Quantification of the percentages of neurons with unipolar/bipolar, no, and multiple processes. More than 200 GFP⁺ neurons from five brains were analyzed in each group. ***p < 0.001, n = 3, Student's t test, mean ± SEM. M, The expression of Mst3 WT or Mst3 S79A had no effect on neuronal migration. Scale bar, 100 μm. N, Quantification of the distribution of GFP⁺ neurons in the brain sections electroporated with pCAG, Mst3 WT, or Mst3 S79A. n = 3, Student's t test, mean ± SEM.

Figure 5.

Mst3 regulates actin dynamics and RhoA activity. A, Mst3 was concentrated in the F-actin-rich region. Cultured cortical neurons at 3 DIV were stained with anti-Mst3 (green) and anti-Tau1 (axon marker, blue) antibodies and fluorescently labeled phalloidin (F-actin marker, red). B, C, The G/F-actin ratio was lower in Mst3-knockdown cortical neurons. Cortical neurons were transfected with pSUPER or Mst3 shRNA. The F-actin and G-actin fractions were separated by SDS-PAGE and actin was quantified by Western blotting using anti-actin antibody. The F-actin (F; pellet fraction) and G-actin (G; supernatant fraction) pools are shown. C, Quantification of G/F-actin ratio in the Mst3-knockdown cortical neurons in B. n = 4, G/F-actin ratio, ∼50% reduction, **p < 0.01; Student's t test, mean ± SEM. D, E, Mst3 knockdown reduced the number of intact growth cones. Cortical neurons, transfected with GFP (green) and pSUPER or Mst3 shRNA, were treated with RI (Y27632). The fixed neurons were stained with anti-GFP (green) and anti-Tuj1 (βIII-tubulin marker, blue) antibodies and phalloidin (F-actin marker, red). E, Quantification of the number of intact growth cones in D. ***p < 0.001 versus pSUPER in the control condition; #p < 0.05 versus pSUPER in the RI condition. n = 3, Student's t test, mean ± SEM. More than 300 GFP⁺ neurons from three different experiments were analyzed in each group. F, RhoA interacted with Mst3 in HEK293T cells. HEK293T cells expressing FLAG-Mst3 and RhoA were lysed and immunoprecipitated with anti-FLAG antibody. Mouse IgG was used as a negative control. G, RhoA activity was elevated in Mst3-knockdown cortical neurons. n = 3; GTP-RhoA: 2.02 ± 0.10-fold in Mst3 shRNA, mean ± SEM, p < 0.001, Student's t test. H, Ectopic Mst3 expression reduced RhoA activity in cortical neurons. n = 3, GTP-RhoA: 0.19 ± 0.09-fold in Mst3 overexpressing condition, mean ± SEM, p < 0.001, Student's t test.

Figure 6.

Mst3 regulates neuronal migration via the modulation of RhoA activity. A, Mst3 phosphorylated RhoA at serine residue(s). In vitro phosphorylation assay (top) and Western blotting for p-Ser antibody (middle), and total RhoA (bottom). B, Mst3 phosphorylated RhoA at Ser26. The Mst3 phosphorylation site on Ser26 site is shown. WT and two phosphodeficient mutants of RhoA at putative Mst3 phosphorylation sites (S26A and S160A) were subjected to an in vitro phosphorylation assay. C, Blockade of RhoA phosphorylation at Ser26 increased the GTPase activity of RhoA. HEK293T cells expressing WT, S26A, or S160A mutants of RhoA were pulled down by anti-FLAG antibody and subjected to GTPase activity assay. Fold change of GTP-RhoA (mean ± SEM, *p < 0.01, n = 3, Student's t test). D, Mst3-dependent RhoA phosphorylation is important for radial migration. E14 mouse brains were electroporated with pCAG vector, RhoA WT, or RhoA S26A. Representative mouse brain sections were stained with anti-GFP antibody (green) and TO-PRO-3 (blue). Scale bar, 100 μm. E, Quantification of the distribution of GFP⁺ neurons. ***p < 0.001, **p < 0.01 versus pCAG; ###p < 0.001 versus RhoA WT; n = 3, Student's t test, mean ± SEM. F, Quantification of the percentages of neurons with unipolar/bipolar, no, and multiple processes. ***p < 0.001 versus pCAG; #p < 0.05, ^##p < 0.01 versus RhoA WT, n = 3, Student's t test, mean ± SEM. More than 300 GFP⁺ neurons from five brains were analyzed in each group. G, RhoA knockdown rescued the Mst3-dependent delayed migration. E14 mouse brains were electroporated with GFP construct together with pSUPER, Mst3 shRNA, or Mst3 shRNA together with RhoA shRNA. Representative sections of E17 brains were stained with anti-GFP antibody (green) and TO-PRO-3 (blue). Scale bar, 100 μm. H, Quantification of the distribution of GFP⁺ neurons. ***p < 0.001 versus pSUPER; ###p < 0.001 versus Mst3 shRNA, n = 3, Student's t test, mean ± SEM. I, Quantification of the percentages of neurons with unipolar/bipolar, no, and multiple processes. ***p < 0.001 versus pSUPER; ##p < 0.01 versus Mst3 shRNA, n = 3, Student's t test, mean ± SEM. More than 300 GFP⁺ neurons from six brains were analyzed in each group.