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Two different approaches were used to study AMPA-receptor trafficking and to show the influence of ephrinB2 reverse signaling on this process.We used the biochemical technique of surface biotinylation to detect changes in the number of receptors present at the cell surface. A complementary immunofluorescence method we call “antibody feeding assay” was used to track receptor molecules moving in and out of the plasma membrane.

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5.2.1.1 EphrinB2 reverse signaling prevents endocytosis of GluR2 in 293 GluR2 cells

In our biochemical experiments, 293 HEK cells stably expressing the GluR2 subunit of the AMPA receptor were transfected with ephrinB2 YFP and AMPA-receptor trafficking under various stimulation conditions was analyzed using surface biotinylation (7 Material and Methods). Cells plated in 10 cm culture dishes were starved for 24 hours after transfection and treated with a thiol-cleavable amine-reactive biotin to mark all proteins currently at the cell surface. Afterwards, the biotin-labelled cells were stimulated with pre-clustered EphB4-Fc or Fc (control) for 30 minutes and/or with 100 µM AMPA for 5 minutes and their lysates analyzed for GluR2 internalization in Western blots (Figure 5-9).

Stimulation with AMPA resulted in a robust internalization of GluR2 indicated by a strong signal on the Western blot (Figure 5-9). In contrast, simultaneous activation of ephrinB2 by EphB4-Fc, the ephrinB2 specific receptor, completely inhibited AMPA-induced GluR2 internalization. EphB4-Fc stimulation alone slightly reduced the level of AMPA-receptor endocytosis compared to the control condition (Fc).

Figure 5-9: EphrinB2 activation inhibits AMPA-receptor internalization in 293 GluR2 cells. Surface biotinylation assay using 293 HEK GluR2 cells transfected with ephrinB2 YFP and stimulated as indicated. The cells were incubated with biotin to label cell surface proteins. After stimulation, the remaining surface biotin was removed and the internalized biotin-marked molecules precipitated from the cell lysates with straptavidin beads. AMPA-receptor internalization was analyzed by Western blot using anti-GluR2 antibodies. The strength of the signal correlates to the amount of internalized AMPA receptors.

70 5.2.1.2 EphrinB2 reverse signaling blocks AMPA-receptor endocytosis in

cultured hippocampal neurons

We next investigated the influence of ephrinB2 on AMPA receptors in a more physiological system using primary hippocampal neurons. First, in analogy to the 293 cell- experiment, we analyzed AMPA-receptor internalization as before by surface biotinylation (Figure 5-10, a). Hippocampal neurons isolated from E19 rats and cultivated for 14-18 DIV were stimulated with pre-clustered EphB4-Fc or Fc for 1 hour and 100 µM AMPA was added for the last 10 minutes. As expected, AMPA stimulation resulted in an increased level of AMPA-receptor internalization (GluR2) when compared to the control condition (Fc). Again, in a manner similar to that observed in 293 GluR2 cells, simultaneous activation of ephrinB2 reverse signaling resulted in a strong inhibition of the AMPA-induced AMPA-receptor endocytosis. Quantifications of four independent experiments revealed this inhibition to be highly significant.

The inhibitory effect of ephrinB2 was additionally analyzed using an ‘antibody feeding assay’ (Lin et al., 2000; Man et al., 2000). Here, a specific primary antibody (anti-GluR2 or GluR1) was applied to the cells to mark the surface pool of AMPA receptors. Thereafter, the cells were stimulated to allow receptor internalization followed by fixation with PFA. AMPA-receptors retained on the cell surface were labelled with a green-tagged secondary antibody. Following permeabilization, a red-tagged secondary antibody was used to mark receptors internalized during the experiment. Representative images of each condition are shown in Figure 5-10, b.

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Figure 5-10: EphrinB2 inhibits AMPA-receptor internalization in hippocampal neurons. (a) AMPA-receptor internalization (GluR2) in hippocampal neurons 21DIV under the indicated stimulation conditions analyzed using surface biotinylation assay (left panel) as in Figure 5-9. N-cadherin levels (N- Cad) were unaffected. Quantification of four independent experiments (right panel) indicated as fold increase compared to Fc (control) (SEM, ** P < 0.005). (b) AMPA-receptor internalization under various stimulation conditions (B4: pre-clustered EphB4-Fc; CNQX: specific AMPA-receptor antagonist) visualized using the ‘antibody feeding assay’. Neurons 15-21 DIV were labeled with primary antibodies (anti-GluR2), stimulated, fixed and incubated with a first secondary antibody (green), recognizing surface retained receptors. A second secondary antibody (red) was applied after permeabilization to mark internalized receptors. Scale bar, 5 µm. (c) Quantification of AMPA-receptor internalization based on fluorescence intensities, shown as the percentage of internalized GluR2 (red) versus total GluR2 (red + green). The conditions analyzed are those illustrated in b. (SEM, *** P < 0.0005). (d) AMPA-receptor internalization monitored as trafficking of GluR1 and visualized using the ‘antibody feeding assay’ as in b. Scale bar, 5 µm. (e) Quantification of GluR1 internalization as in c. Conditions analyzed are represented in d (SEM, *** P < 0.0001).

72 The internalization levels were quantified as percentage of internalized GluR2 versus total GluR2 (Figure 5-10, c). In control conditions, the basal level of AMPA-receptor internalization was 20.7 % ± 1.6 (green fluorescence signal Figure 5-10, b). Stimulation with 100 µM AMPA led to a strong AMPA-receptor internalization of 65.7 % ± 1.9 seen as an intense red signal (internalized GluR2) and a weak green signal (remnant surface receptors). Simultaneous ephrinB2 activation by pre-clustered EphB4-Fc significantly blocked AMPA-evoked internalization (36.8 % ± 3.3), to an extent comparable to that of a competitive antagonist of AMPA receptors (34.6 % ± 2.7), namely 6-cyano-7- nitoquinoxaline-2, 3-dione (CNQX).

AMPA receptors at active synapses are heteromers mainly composed of GluR1 and GluR2 subunits. We therefore confirmed our observations using antibodies against GluR1 (Figure 5-10, d+e). EphrinB2 activation resulted in a significant inhibition of AMPA- induced GluR1 endocytosis (40.0 % ± 2.2 compared to solely AMPA stimulation 55.1 % ± 2.4).

5.2.1.3 EphrinB2 inhibits also the AMPA-receptor endocytosis following NMDA stimulation

AMPA-receptor internalization is known to be induced in two different manners, first, by its direct activation through the specific agonist AMPA, and second indirectly via NMDA- receptor activation. The latter has been shown to be important for the expression of long- term depression (LTD) triggered by NMDA-receptor activation (Beattie et al., 2000). Therefore, we next tested whether ephrinB2 ligands would also inhibit AMPA-receptor endocytosis in neurons stimulated with NMDA. In these experiments, cultured hippocampal neurons were treated with pre-clustered EphB4-Fc or Fc for 1 hour while 50 µM NMDA was added shortly for 2 minutes (10 minutes before the reaction was stopped). AMPA receptor endocytosis was visualized by the antibody feeding assay method, as illustrated in Figure 5-11, a, and quantified as percentage of internalized GluR2 versus total GluR2 (Figure 5-11, b). The basal level of AMPA-receptor endocytosis of 29.8 % ± 2.2 was increased after NMDA application (51.8 % ± 1.6) and was efficiently blocked by ephrinB2 activation (33.1 % ± 1.8).

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Figure 5-11: EphrinB2 blocks NMDA-induced AMPA-receptor endocytosis. Hippocampal neurons (15-18DIV) stimulated as indicated and analyzed for AMPA-receptor (GluR2) internalization using the ‘antibody feeding assay’ as in Figure 5-9. (a) Neurons were incubated with Fc alone or with 50 µM NMDA for 2 minutes together with pre-clustered EphB4-Fc or Fc (control). Surface retained receptors appear in green, internalized in red puncta. Scale bar, 5 µm. (b) Quantification of AMPA- receptor internalization based on fluorescence intensities as in Figure 5-10 b. The conditions analyzed are those illustrated in b (SEM, *** P < 0.0001).

Taken together, these results suggest that ephrinB2 reverse signaling regulates AMPA- receptor trafficking by stabilizing the receptors at the cell membrane. Moreover, ephrinB2 reverse signaling was also seen to inhibit NMDA-induced AMPA-receptor internalization suggesting a broader role for ephrinBs in the regulation of glutamate-receptor trafficking.