Regulation of AMPA Receptor Trafficking and Function by Glycogen Synthase Kinase 3*

Accumulating evidence suggests that glycogen synthase kinase 3 (GSK-3) is a multifunctional kinase implicated in neuronal development, mood stabilization, and neurodegeneration. However, the synaptic actions of GSK-3 are largely unknown. In this study, we examined the impact of GSK-3 on AMPA receptor (AMPAR) channels, the major mediator of excitatory transmission, in cortical neurons. Application of GSK-3 inhibitors or knockdown of GSK-3 caused a significant reduction of the amplitude of miniature excitatory postsynaptic current (mEPSC), a readout of the unitary strength of synaptic AMPARs. Treatment with GSK-3 inhibitors also decreased surface and synaptic GluR1 clusters on dendrites and increased internalized GluR1 in cortical cultures. Rab5, the small GTPase controlling the transport from plasma membrane to early endosomes, was activated by GSK-3 inhibitors. Knockdown of Rab5 prevented GSK-3 inhibitors from regulating mEPSC amplitude. Guanyl nucleotide dissociation inhibitor (GDI), which regulates the cycle of Rab5 between membrane and cytosol, formed an increased complex with Rab5 after treatment with GSK-3 inhibitors. Blocking the function of GDI occluded the effect of GSK-3 inhibitors on mEPSC amplitude. In cells transfected with the non-phosphorylatable GDI mutant, GDI(S45A), GSK-3 inhibitors lost the capability to regulate GDI-Rab5 complex, mEPSC amplitude, and AMPAR surface expression. These results suggest that GSK-3, via altering the GDI-Rab5 complex, regulates Rab5-mediated endocytosis of AMPARs. It provides a potential mechanism underlying the role of GSK-3 in synaptic transmission and plasticity.


MATERIALS AND METHODS
Primary Neuronal Culture-Rat cortical cultures were prepared as described previously (19,20). In brief, frontal cortex was dissected from embryonic day 18 rat embryos, and cells were dissociated using trypsin and trituration through a Pasteur pipette. Neurons were plated on coverslips coated with poly-L-lysine in Dulbecco's modified Eagle's medium with 10% fetal calf serum at a density of 1 ϫ 10 5 cells/cm 2 . When neurons attached to the coverslips within 24 h, the medium was changed to Neurobasal medium with vitamin B 27 supplement (Invitrogen). Cytosine arabinoside (Arac, 5 M) was added at DIV 3 to stop glial proliferation. Culture neurons (DIV 14 -16) were transfected with various plasmids using Lipofectamine 2000 (Invitrogen) method.
Synaptic currents were analyzed with the Mini Analysis program (Synaptosoft, Leonia, NJ). The noise level is below 5 pA, and we usually used 10 pA as the threshold for mEPSC events. Two minutes of representative mEPSC recordings (300 -400 events) were used to generate the cumulative distribution plot. Statistical comparisons of synaptic currents were made using the Kolmogorov-Smirnov test. Summary data were presented as mean Ϯ S.E. ANOVA or Student's t tests were performed to compare groups subjected to different treatments.
The internalized AMPA receptors were detected as described previously (21). Briefly, surface GluR1 was labeled with a polyclonal anti-GluR1 antibody (1:100; Millipore, 07-660) in living cells for 20 min at 37°C in the culture medium. After washing, neurons were treated with SB216763 (10 M) or DMSO for 10 min at 37°C. Following the treatment, the antibody that binds to the remaining surface GluR1 was stripped off with an acid solution (0.5 M NaCl, 0.2 N acetic acid) at 4°C for 4 min. Cells were then washed, fixed, permeabilized, and incubated with a monoclonal anti-GluR1 antibody (1:200; Santa Cruz Biotechnology, sc-13152) for 2 h at room temperature. The internalized GluR1 (labeled with a polyclonal GluR1 antibody) was detected with the Alexa Fluor 594 (red)-conjugated anti-rabbit secondary antibody, whereas the total GluR1 (labeled with a monoclonal GluR1 antibody) was detected with the Alexa Fluor 488 (green)-conjugated anti-mouse secondary antibody.
Labeled cells were imaged using a 100ϫ objective with a cooled CCD camera mounted on a Nikon microscope. All specimens were imaged under identical conditions and analyzed using identical parameters. The surface GluR1 clusters and internalized GluR1 were measured using the ImageJ software according to our previously described procedures (19 -21). To define dendritic clusters, a single threshold was chosen manually so that clusters corresponded to puncta of at least 2-fold greater intensity than the diffuse fluorescence on the dendritic shaft. Three to four independent experiments for each of the treatments were performed. On each coverslip, the cluster density, size, and fluorescence intensity of 4 -6 neurons (2-3 dendritic segments of at least 50 m in length per neuron) were measured. Quantitative analyses were conducted blindly (without knowledge of experimental treatment).
DNA Constructs-Rat GDI-1 open reading frame was cloned from rat brain cDNA by PCR, and a FLAG tag was added in the N terminus of GDI in-frame. Generation of GDI mutants (WT, S45A, S121A, S213A) was carried out with the QuikChange site-directed mutagenesis kit (Stratagene). All constructs were verified by DNA sequencing. GDI constructs (WT, S45A) were co-transfected with enhanced GFP into cortical cultures (DIV 14 -16) using the Lipofectamine 2000 method. Two days after transfection, immunostaining or recordings were performed.
Given the effect of GSK-3 inhibitors on AMPARs, we would like to know the natural stimulus that could activate this pathway. Insulin has been found to induce GSK-3 inhibition via protein kinase B (PKB, also called AKT) signaling (22). Thus, we examined the effect of insulin on mEPSC. As shown in Fig. 1, F and G, application of insulin (0.5 M, 10 min) caused a significant reduction of mEPSC amplitude (control, 21.3 Ϯ 0.7 pA, n ϭ 23; insulin, 17.6 Ϯ 0.9 pA, n ϭ 12, p Ͻ 0.01, ANOVA) and occluded the effect of SB216763 (insulin ϩ SB216763, 16.6 Ϯ 0.5 pA, n ϭ 11). It suggests that  AUGUST  GSK-3 Regulates the Surface Expression and Internalization of AMPA Receptors-To test whether the GSK-3 regulation of mEPSC amplitude can be accounted for by the altered number of AMPA receptors on the cell membrane, we first performed surface biotinylation experiments to measure levels of surface GluR1. Surface proteins were labeled with sulfo-NHS-LC-biotin, and then biotinylated surface proteins were separated from non-labeled intracellular proteins by reaction with NeutrAvidin beads. Surface and total proteins were subjected to electrophoresis and probed with anti-GluR1 antibody. As shown in Fig. 2A, treatment of cortical neurons with SB216763 (10 M, 10 min) significantly decreased the level of surface GluR1 (54.9 Ϯ 2.3% of control, n ϭ 3; p Ͻ 0.01, t test) and surface GluR2 (45.3 Ϯ 10.0% of control, n ϭ 4; p Ͻ 0.01, t test), whereas the level of total GluR1 and GluR2 was unchanged. In contrast, surface GABA A receptors were significantly increased by SB216763 treatment (2.09-Ϯ 0.33fold of control, n ϭ 4; p Ͻ 0.05, t test), consistent with the recruitment of functional GABA A receptors to postsynaptic domains by insulin (23).

GSK-3 Regulation of AMPA Receptors
To further demonstrate the change in surface AMPARs by inhibiting GSK-3, we carried out a quantitative surface immunostaining assay in cortical cultures. The surface distribution of GluR1 was assessed by immunostaining with an antibody against the extracellular N-terminal domain of GluR1 in non-permeabilized conditions. As shown in Fig. 2B  test). These data suggest that inhibiting GSK-3 reduces AMPAR surface expression.
We also performed immunocytochemical experiments to detect AMPAR internalization in cultured cortical neurons. Surface AMPARs were first stained with an antibody to the extracellular region of GluR1 subunit, and then following the treatment with GSK-3 inhibitors, surface-bound antibodies were stripped away so that only internalized AMPARs were visualized. As shown in Fig. 2, F and G, SB216763 (10 M, 10 min) treatment caused a significant increase in the fluorescence intensity of internalized GluR1 on neuronal dendrites (control, 28.1 Ϯ 0.7, n ϭ 28; SB216763, 37.3 Ϯ 2.1, n ϭ 30; p Ͻ 0.01, t test). It suggests that inhibiting GSK-3 increases the internalization of AMPARs, which may result in the reduction of AMPARs at synaptic membrane.

GSK-3 Regulation of AMPARs Involves the Stimulation of the GDI-Rab5
Complex-To understand the potential mechanism underlying GSK-3 regulation of AMPAR internalization, we examined the role of Rab5, a key mediator of protein transport from plasma membrane to early endosomes during clathrindependent endocytosis (24). First, we examined whether GSK-3 could regulate the activity of this small GTPase. Because Rabaptin-5, a molecule identified as a Rab5-interacting protein, binds to only the GTP-bound, active form of Rab5 at its C terminus (25), we measured Rabaptin-5-bound Rab5 by co-immunoprecipitation experiments to indicate its activity level. As shown in Fig. 3A, SB216763 (10 M, 10 min) treatment of cortical slices induced a significant increase of Rab5 activity, as indicated by the elevated level of Rabaptin-5-bound Rab5 (2.02-Ϯ 0.19-fold of control, n ϭ 3, p Ͻ 0.01, t test). To test the specificity of Rab5 involvement, we also examined Rab4, which mediates receptor recycling between early endosomes and the plasma membrane. Rabaptin-5 binds to the active form of Rab4 at its N terminus (25). As shown in Fig. 3A, the Rabaptin-5-bound (active) Rab4 was not significantly changed by GSK-3 inhibition.
Next, we sought to determine the mechanism underlying GSK-3 regulation of Rab5-mediated AMPAR internalization. It is known that the recycling of Rab proteins between a membrane-bound and a cytosolic state is dependent on the GDP dissociation inhibitor (26). Previous studies have found that the formation of GDI-Rab complex can be altered by phosphorylation of GDI (27,28), leading to accelerated exocytosis or endocytosis. Thus, we tested the potential involvement of GDI in GSK-3 regulation of AMPARs.
First, we examined whether GSK-3 alters the formation of the GDI-Rab5 complex. As shown in the co-immunoprecipitation assay (Fig. 4A), SB216763 (10 M, 10 min) treatment significantly increased the amount of Rab5 that binds to GDI (1.63-Ϯ 0.13-fold of control, n ϭ 3, p Ͻ 0.01, t test). It suggests that inhibiting GSK-3 increases the formation of GDI-Rab5 complex, which may account for the increased endocytic trafficking of surface AMPA receptors.

GDI Phosphorylation at Ser-45 Is Required for GSK-3 Regulation of AMPAR Synaptic Activity and Surface Expression-GDI contains 26
Ser residues, and Ser-45, Ser-121, and Ser-213 have been predicted to face the outer surface of the molecule based on its three-dimensional structure (28,29). To identify the phosphorylation site that is critically involved in GSK-3 regulation of AMPARs, we transfected HEK293 cells with FLAG-tagged wild-type GDI or non-phosphorylatable GDI mutants, S45A, S121A, and S213A. After transfection, cells were treated with SB216763. Cell lysates were subjected to a co-immunoprecipitation assay to detect the GDI-Rab5 complex. As shown in Fig. 5A, SB216763 (10 M, 10 min) treatment significantly increased the amount of Rab5 that binds to WT-GDI, S121A-GDI, or S213A-GDI but not to S45A-GDI. It suggests that GSK-3 regulation of the GDI-Rab5 complex requires an intact Ser-45 phosphorylation site on GDI.
We further investigated whether GDI phosphorylation at Ser-45 could influence synaptic AMPAR activity and its regulation by GSK-3 in cortical cultures. As shown in Fig.  5B, when compared with neurons transfected with GFP alone, transfecting S45A GDI, but not WT GDI, caused a significant decrease of mEPSC amplitude. Furthermore,   Fig. 5E). These data suggest that GSK-3 regulation of synaptic AMPAR currents requires an intact Ser-45 phosphorylation site on GDI.
In summary, we have revealed a potential mechanism for GSK-3 regulation of AMPARs. Our results suggest that constitutively active endogenous GSK-3 plays an important role in maintaining AMPARs at the synaptic membrane. It is conceivable that dysregulation of glutamatergic transmission by impaired GSK-3 signaling may be a key pathophysiological mechanism for those mental illnesses involving GSK-3.