Figure 7.
Inhibition of Cdk5 in neurons decreases the binding of NR2B with the AP-2 complex and the endocytosis of NMDARs. A, The level of binding of NR2B with the AP-2 complex was monitored in neuronal cortical cultures treated with roscovitine (5 μm) or DMSO (as control) for 45 min and with roscovitine (or DMSO) and 100 μm glycine for 5 min. NR2B immunoprecipitated from lysates of cultures was analyzed for the amount of β2-adaptin coimmunoprecipitated with NR2B. In neuronal cortical cultures there is a significant decrease in the binding of NR2B with β2-adaptin in cultures treated with roscovitine (42.76 ± 13.81% of control; p = 0.01; n = 5). B, The levels of surface expression of NR2B were evaluated in neuronal cortical cultures treated with roscovitine (5 μm) or DMSO (as control) for 45 min, stimulated by 5 min in 100 μm glycine (with roscovitine or DMSO) followed by additional 5 min in 100 μm glycine and 50 μm NMDA (with roscovitine or DMSO). Live neuronal cultures were then treated with 0.05 mg/ml chymotrypsin for 10 min at 37°C to cleave the extracellular domain of the NMDARs on the membrane surface (D+, R+) or left untreated (D−, R−). The lysates derived from these neuronal cultures were analyzed for the expression of uncleaved NR2B and actin (as loading control). Chymo, Chymotrypsin. Quantification indicates that, under control conditions, after stimulation with glycine/NMDA (D+) 89.19 ± 5.14% of NR2B immunoreactivity is maintained (compared with D−), whereas in cultures treated with roscovitine (R) and stimulated with glycine/NMDA (R+) 47.05 ± 6.96% of NR2B immunoreactivity is present (p = 0.002; n = 3). C, The levels of surface expression of NR2B were evaluated in neuronal cortical cultures treated with control medium (DMSO) (D, a–c) or with control medium and glycine/NMDA (D+, d–f), or 5 μm roscovitine (R, g–i), or 5 μm roscovitine and glycine/NMDA (R+, j–l). Neurons were immunostained for NR1 to visualize surface NMDARs before being permeabilized (a, d, g, j) and immunostained for NR2B after permeabilization to visualize all NR2B-containing NMDARs (b, e, h, k). Overlays of NR1 and NR2B immunostaining (c, f, i, l). D, Quantification of staining conducted on coded material using MetaMorph software. In naive control cultures (D, Ca–Cc), 81.50 ± 7.30% of NR2B-containg NMDARs are present at the cell surface, whereas after treatment with glycine/NMDA, 46.85 ± 4.61% of NR2B puncta were also positive for NR1 (D+, Cd–Cf). In roscovitine-treated cultures (R, Cg–Ci) 73.79 ± 0.51% of NR2B-containg NMDARs are present at the cell surface. After glycine/NMDA stimulation of these cultures (R+, Cj–Cl), 67.14 ± 5.17% of NR2B-containg NMDARs are present at the cell surface. A two-way ANOVA was performed to compare the average number of puncta in the four experimental conditions. The overall effect was statistically significant (F(3,21) = 23.13; p < 0.0001), as also were the overall effect of roscovitine versus DMSO (86.65 ± 1.85% and 75.32 ± 1.92%, respectively; F(2,12) = 12.3; p < 0.001) and the main effect of glycine-NMDA stimulation (with, 72.32 ± 1.84% vs without, 87.65 ± 1.91%; F(2,12) = 33.1; p < 0.0001) and the interaction (roscovitine by glycine-NMDA stimulation, F = 26.16, p < 0.0001). Post hoc pairwise comparisons were evaluated using the Tukey's HSD test. Statistically significant (p < 0.05) differences were found between the average number of puncta of the DMSO-glycine/NMDA (D+) and the DMSO nonstimulated (D) groups and between the average number of puncta of the DMSO-glycine/NMDA (D+) and the roscovitine-glycine/NMDA (R+) groups (D). Total puncta: D, 391; D+, 500; R, 350; R+, 366. Error bars indicate SEM.