Disruption of AMPA receptor GluR2 clusters following long-term depression induction in cerebellar Purkinje neurons

Abstract

Cerebellar long-term depression (LTD) is thought to play an important role in certain types of motor learning. However, the molecular mechanisms underlying this event have not been clarified. Here, using cultured Purkinje cells, we show that stimulations inducing cerebellar LTD cause phosphorylation of Ser880 in the intracellular C-terminal domain of the AMPA receptor subunit GluR2. This phosphorylation is accompanied by both a reduction in the affinity of GluR2 to glutamate receptor interacting protein (GRIP), a molecule known to be critical for AMPA receptor clustering, and a significant disruption of postsynaptic GluR2 clusters. Moreover, GluR2 protein released from GRIP is shown to be internalized. These results suggest that the dissociation of postsynaptic GluR2 clusters and subsequent internalization of the receptor protein, initiated by the phosphorylation of Ser880, are the mechanisms underlying the induction of cerebellar LTD.

Fig. 1. Induction of cerebellar LTD by PKC activation. Whole-cell voltage-clamp recording was performed from a Purkinje cell at 25 days in vitro. EPSC was evoked by extracellular stimulation of a single granule cell. (A) Amplitudes of the evoked EPSC, plotted as a function of time relative to the beginning of TPA application (200 nM TPA at 0 min). Each data point represents the amplitude of one evoked EPSC, as a percentage of the control amplitude. The average of the EPSC amplitude before TPA application was set at 100%. Insets show the representative current traces taken at times (a), (b) and (c), respectively. (B) Each point represents the normalized EPSC amplitude from five Purkinje cells (recording condition as above). The EPSC amplitudes were averaged over a 2 min time window. Bars indicate SD.

Fig. 2. Effect of PKC activation on the co-localization of GluR2 and GRIP in transfected HEK 293 cells. (A and B) HEK cells expressing either GluR2 (FITC labeled) (A) or GRIP_419–673 (rhodamine labeled) (B). (C and D) HEK cells simultaneously expressing GluR2 and the GRIP fragment prior to (C) and after (D) TPA stimulation (200 nM, 20 min). (E) A cell expressing GluR2 and the GRIP_419–673 20 min after treatment with TPA and GF109203X (200 nM), a selective PKC inhibitor. (F) A cell expressing mutant GluR2 (S880D) and GRIP_419–673.

Fig. 3. Disruption of GluR2 clusters in response to PKC activation. A cerebellar neuronal culture at 14 DIV was used for the following analysis. (A) Co-localization of GluR2 and a presynaptic marker synaptophysin. The cerebellar neuronal culture was double immunostained with anti-GluR2 N-terminal (left) and anti-synaptophysin (right) antibodies. Arrows indicate the spots representing immunoreactivity for both antibodies. Arrowheads indicate the GluR2- but not synaptophysin-immunoreactive spots. (B and C) Disruption of the postsynaptic GluR2 clusters following TPA application. Purkinje cells were treated with 200 nM TPA for 20 min, followed by staining with anti-GluR2 N-terminal antibodies (B) or rhodamine–phalloidin (C). The GluR2 and F-actin clusters were defined after filtering the images, followed by quantification of the maximum intensity of each cluster (see Materials and methods). Images were obtained from the dendritic regions of control (left) and TPA-treated (right) cells (scale bars 5 µm). Insets in (Ba) show fluorescence images for synaptophysin. Graphs (Bb) are cumulative plots of the maximum intensities for GluR2 (B) and F-actin (C) clusters. Solid and dashed lines represent the results from control and TPA-treated cells, respectively. The Kolmogorov–Smirnov test revealed a significant decrease in the intensity of GluR2 clusters following TPA stimulation (p <0.0001). Bar graphs in the inset show the mean of maximum cluster intensities obtained from five different cells. The intensity of the control culture was taken as 100%. Asterisks indicate that the mean intensity of the GluR2 clusters in TPA-treated cells was significantly lower than that in control cells (**p <0.01). (D) Phosphorylation of Ser880 in GluR2 following application of TPA. The membrane fraction of cultured Purkinje cells treated or not treated with TPA (200 nM, 20 min) was subjected to SDS–PAGE and immunoblotting with anti-GluR2 N-terminal antibodies (a) or antibodies specific for the phosphoSer880 residue in GluR2 (anti-P-Ser880 antibodies) (b). (E) Parallel decrease in the affinity of GluR2 for GRIP. Immunoprecipitation was performed using polyclonal anti-GRIP antibodies. Immunoprecipitated GRIP (a) and co-immunoprecipitated GluR2 (b) were detected by immunoblotting with anti-GRIP and anti-GluR2 N-terminal antibodies, respectively. Bar graphs in (D) and (E) show the mean of the band intensities (n = 3). Error bars indicate SD.

Fig. 4. Long-lasting disruption of GluR2 clusters in association with persistent phosphorylation of the Ser880 residue in GluR2 following conjunctive stimulation with TPA and AMPA. (A) Time course of changes in the density of GluR2 clusters. Cerebellar neuronal cultures were stimulated with TPA (200 nM, 20 min), AMPA (100 µM, 1 min) or TPA+AMPA, and incubated for 0, 30 and 120 min after the removal of the drugs. Then, the cells were fixed and immunostained for GluR2. Upper panels indicate the disruption of GluR2 immunoreactivity on Purkinje cell dendrites at 30 min after removal of the drugs (scale bar 5 µm). For quantitative analysis of the effect of TPA on GluR2 cluster density, the mean of the maximum cluster intensities was calculated from five different cells. The results are plotted on the graph, with the mean intensity in the control culture being 100%. (B) Time course of changes in Ser880 phosphorylation levels following various stimulations. Cerebellar neuronal cultures were treated as described above. After a set period of incubation, the cells were solubilized, and the membrane fractions were subjected to SDS–PAGE, followed by immunoblotting with anti-P-Ser880 antibodies (insets). The means of the band intensities from three independent experiments are plotted on the graph. The band intensities obtained from TPA-treated cultures without further incubation were defined as 100%. Error bars indicate SD. (C) No significant effect of VGCC blockers on TPA+AMPA induced disruption of the GluR2 clusters and phosphorylation of Ser880 in GluR2. TPA+AMPA stimulation was applied in the presence or absence of VGCC blockers, verapamil (10 µM) and ω-agatoxin TK (50 nM), and incubation was carried out for 30 min after the removal of drugs. Then, mean cluster intensity (a) and the phosphorylation level (b) of GluR2 were quantified. Asterisks indicate the significant difference as compared with control samples (*p <0.05, **p <0.01).

Fig. 5. Disruption of GluR2 clusters concurrently with phosphorylation of the Ser880 residue in GluR2 following KCl + glutamate stimulation. A cerebellar neuronal culture was stimulated with 50 mM KCl together with 10 µM glutamate for 4 min. Then, the drugs were removed and used for subsequent analysis immediately or after 2 h of incubation. (A) Quantitative analysis of GluR2 cluster intensities. The graph shows the means of the maximal cluster intensities obtained from five different cells fixed immediately or 2 h after KCl + glutamate stimulation (white columns). Black columns show the results from control samples. The mean cluster intensities obtained from control cultures were defined as 100%. Asterisks indicate that the mean intensity of the GluR2 clusters in the stimulated cells was statistically significantly weaker than that in control cells (*p <0.05). (B) Phosphorylation of the Ser880 residue in GluR2 following KCl + glutamate stimulation. Cerebellar neuronal cultures treated (white columns) or not treated (black columns) with KCl + glutamate, and incubated for 0 or 120 min after removal of the drugs. The membrane fractions were subjected to SDS–PAGE and subjected to immunoblotting with anti-P-Ser880 antibodies (insets). The lower graph shows the mean of the band intensities after normalization with loaded GluR2 protein (n = 3). Similar experiments with TPA stimulation (200 nM, 20 min, no further incubation) were performed simultaneously and the band intensities were taken as 100%. Error bars indicate SD.

Fig. 6. Internalization of GluR2 protein following PKC activation. (A) Increase in intensity of immunoreactive intradendritic GluR2. Upper panels show respective confocal optical sections of the dendritic regions in control and TPA-treated Purkinje cells immunostained after permeabilization treatment with anti-GluR2 N-terminal antibodies (scale bar 5 µm). White bars indicate the region whose immunoreactive intensities were quantified. The graph indicates results of the quantitative analysis of intracellular immunoreactive intensities (five cells, total ∼120 µm dendritic length). The mean intracellular intensities obtained from control cultures were defined as 100%. (B) Decrease in the amount of extracellularly expressed GluR2 following TPA stimulation. Cell surface proteins were biotinylated followed by immunoprecipitation for GluR2. Then, the biotinylated GluR2 protein was detected by immunoblotting with HRP-conjugated streptavidin. Inset graph indicates the total amount of immunoprecipitated GluR2 protein. (C) Increase in the amount of GluR2 protein contained in the 150 000 g fraction following TPA stimulation. A 150 000 g fraction containing clathrin-coated vesicles was obtained as described in Materials and methods. The amount of GluR2 in this fraction was quantified by immunoblot analysis using anti-GluR2 N-terminal antibodies. The inset graph indicates the results of the quantitative analysis of total GluR2 protein contained in respective cultures. The band intensities obtained from control (B) or TPA-treated (C) cultures were taken as 100%. Asterisks in (A)–(C) indicate that the difference in GluR2 immunoreaction between control and TPA-treated samples was statistically significant (*p <0.05, **p <0.01).

Fig. 7. A model depicting LTD of AMPA receptor response at parallel fiber–Purkinje cell synapses. The left part of the figure shows the basal state of the synapse. The intracellular C-terminal tail of GluR2 is bound to GRIP, and thus immobilized at the postsynaptic membrane. The right part of the figure shows the synapse after induction of LTD. Ser880 in the C-terminal domain of GluR2 is phosphorylated, which causes dissociation of GluR2 from GRIP, resulting in a decrease in the density of functional AMPA receptors in the postsynaptic membrane. PSD, postsynaptic density; GRIP, glutamate receptor interacting protein.