The stress hormone corticosterone increases synaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors via serum- and glucocorticoid-inducible kinase (SGK) regulation of the GDI-Rab4 complex

Abstract

Corticosterone, the major stress hormone, plays an important role in regulating neuronal functions of the limbic system, although the cellular targets and molecular mechanisms of corticosteroid signaling are largely unknown. Here we show that a short treatment of corticosterone significantly increases alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-mediated synaptic transmission and AMPAR membrane trafficking in pyramidal neurons of prefrontal cortex, a key region involved in cognition and emotion. This enhancing effect of corticosterone is through a mechanism dependent on Rab4, the small GTPase-controlling receptor recycling between early endosome and plasma membrane. Guanosine nucleotide dissociation inhibitor (GDI), which regulates the cycle of Rab proteins between membrane and cytosol, forms an increased complex with Rab4 after corticosterone treatment. Corticosterone also triggers an increased GDI phosphorylation at Ser-213 by the serum- and glucocorticoid-inducible kinase (SGK). Moreover, AMPAR synaptic currents and surface expression and their regulation by corticosterone are altered by mutating Ser-213 on GDI. These results suggest that corticosterone, via SGK phosphorylation of GDI at Ser-213, increases the formation of GDI-Rab4 complex, facilitating the functional cycle of Rab4 and Rab4-mediated recycling of AMPARs to the synaptic membrane. It provides a potential mechanism underlying the role of corticosteroid stress hormone in up-regulating excitatory synaptic efficacy in cortical neurons.

FIGURE 1.

Rab4 is involved in corticosterone regulation of synaptic AMPAR currents. A, cumulative plots of the distribution of mEPSC amplitudes in untreated (control) or corticosterone (100 nm, 30 min)-treated cortical neurons transfected with GFP. B, cumulative plots of the distribution of mEPSC amplitudes in cultured neurons transfected with GFP alone, GFP plus dominant-negative Rab4 (DN-Rab4), or GFP plus constitutively active Rab4 (CA-Rab4). C and D, cumulative plots of the distribution of mEPSC amplitudes in untreated (control) or corticosterone-treated neurons transfected with DN-Rab4 (C) or CA-Rab4 (D). Insets (A, C, and D) show representative mEPSC traces. Scale bars, 30 pA, 1 s. E and F, bar graphs (mean ± S.E.) showing the mEPSC amplitude (mEPSC Amp) (E) and mEPSC frequency (mEPSC Freq) (F) in control versus corticosterone-treated neurons transfected with different constructs. *, p < 0.01, ANOVA.

FIGURE 2.

Rab4 is involved in corticosterone regulation of AMPAR surface expression. A–C, immunocytochemical images of surface GluR1 staining in untreated (control) or corticosterone (100 nm, 30 min)-treated cortical neurons cultures (DIV 20) transfected with GFP alone (A), GFP plus DN-Rab4 (B), or GFP plus CA-Rab4 (C). Enlarged versions of the boxed regions of dendrites are shown beneath each of the images. D–F, cumulative data (mean ± S.E.) showing the cluster density (D), cluster size (E), and fluorescence intensity (Fluo. intensity) (F) of surface GluR1 in control versus corticosterone-treated neurons transfected with different constructs. *, p < 0.01, ANOVA.

FIGURE 3.

Corticosterone treatment increases the formation of GDI-Rab4 complex, which is blocked by mutating Ser-213. A, co-immunoprecipitation (IP) blots showing the level of Rab4 that binds to GDI in HEK293 cells transfected with FLAG-tagged wild-type GDI or its three mutants, S45A, S121A, S213A. After transfection, cells were treated without or with corticosterone (100 nm) for 30 min. A control for the amount of FLAG-GDI effectively immunoprecipitated is also shown. WB, Western blot; HC, heavy chain. B, quantification showing the normalized level of GDI-bound Rab4 in control versus corticosterone-treated HEK293 cells transfected with different GDI constructs. *, p < 0.01, ANOVA. Data are mean ± S.E.

FIGURE 4.

Corticosterone treatment increases GDI phosphorylation by SGK1, which is blocked by mutating Ser-213. A, diagram of rat GDI-1 protein showing the three serine sites that face the outer surface and the substrate motif of SGK1. Only Ser-213 matches the motif. B, representative autoradiography of GDI in vivo phosphorylation assay in HEK293 cells transfected with HA-tagged wild-type GDI or its three mutants. After corticosterone treatment (100 nm, 30 min), [³²P]orthophosphate was incorporated into the cells. Cell lysates were immunoprecipitated (IP) with the HA antibody, and ³²P-labeled proteins were subjected to SDS-PAGE and visualized with autoradiography (upper panel). A loading control was shown in the HA blots (lower panel). WB, Western blot; HC, heavy chain. C, quantification showing the normalized radioactive intensity of ³²P-labeled GDI in control versus corticosterone-treated HEK293 cells transfected with different GDI constructs. *, p < 0.01, ANOVA. Data are mean ± S.E. D, representative autoradiography of GDI in vitro phosphorylation assay in HEK293 transfected without or with SGK1 small interfering RNA (siRNA). After corticosterone treatment (100 nm, 30 min), cell lysates were added to reaction tubes including 1 μCi of [γ-³²P]ATP and 1 μg of purified GST fusion protein of wild-type GDI or its three mutants. Phosphorylated GDI proteins were subjected to SDS-PAGE and visualized with autoradiography.

FIGURE 5.

Phosphorylation of GDI at Ser-213 is required for corticosterone regulation of synaptic AMPAR currents. A, cumulative plots of the distribution of mEPSC amplitudes in cultured cortical neurons transfected with GFP alone, GFP plus ^S213AGDI (non-phosphorylatable mutant), or GFP plus ^S213DGDI (phosphomimetic mutant). B and C, cumulative plots of the distribution of mEPSC amplitudes in untreated (control) or corticosterone (100 nm, 30 min)-treated neurons transfected with ^S213AGDI (B) or ^S213DGDI (C). Inset, representative mEPSC traces. Scale bars, 30 pA, 1 s. D and E, bar graphs (mean ± S.E.) showing the mEPSC amplitude (mEPSC Amp) (D) and mEPSC frequency (mEPSC Freq) (E) in control versus corticosterone-treated neurons transfected with different constructs. *, p < 0.01, ANOVA.

FIGURE 6.

Phosphorylation of GDI at Ser-213 is required for corticosterone regulation of AMPAR surface expression. A–C, immunocytochemical images of surface GluR1 staining in control or corticosterone (100 nm, 30 min)-treated neurons transfected with GFP alone (A), GFP plus ^S213AGDI (B), or GFP plus ^S213DGDI (C). D, cumulative data (mean ± S.E.) showing the surface GluR1 cluster density in control versus corticosterone-treated neurons transfected with different constructs. *, p < 0.01, ANOVA.