Stargazin regulates AMPA receptor trafficking through adaptor protein. complexes during long term depression

Transcription

1 Supplementary Information Stargazin regulates AMPA receptor trafficking through adaptor protein complexes during long term depression Shinji Matsuda, Wataru Kakegawa, Timotheus Budisantoso, Toshihiro Nomura, Kazuhisa Kohda, Michisuke Yuzaki, Contents: Supplementary Figures S1 S15 1

2 Supplementary Figure S1 TARPs bind to the subunits of AP-2 and AP-3A and recruit them to AMPA receptor complexes. (a) GST pull-down assay examining the interaction between stargazin (STG) and AP-2 ( 2) and AP-3A ( 3A). Lysates of HEK293 cells expressing Flag tagged 2 (upper panel) or Flag tagged 3A (lower panel) were applied to glutathione sepharose beads conjugated with no proteins (no GST), GST itself ( ), GST tagged wild-type STG (STG wt ), or GST tagged mutant STG in which all serine residues were replaced with aspartate (STG 9D ). The bound proteins were eluted and subjected immunoblot analyses using anti-flag antibody (IB). (b) GST pull-down assay examining the interaction between wild type or phosphor-mimetic 8 and AP-2 ( 2) and AP-3A ( 3A). (c) Coimmunoprecipitation assay indicates interaction between TARPs [ 2 (STG), 3, 4] and AP-2 ( 2; left panels) or AP-3A ( 3A; right panels). Lysates of HEK293 cells expressing the indicated HA tagged TARP and Flag tagged 2 or 3A were immunoprecipitated by an anti-ha antibody and analyzed using 2

3 an anti-flag antibody. (d) Coexpression of STG strongly enhances the coimmunoprecipitation of 2 and 3A with GluA1 and GluA2. Lysates of HEK293 cells expressing Flag tagged 2 or 3A with various combinations of GluA1, GluA2, and STG were immunoprecipitated using anti-glua1 antibody (left and middle panels) or anti-glua2 (right panel) antibody and immunoblotted with anti-flag antibody. Endogenous actin was not coimmunoprecipitated with GluA1 or GluA2 (lower two panels) in the absence or presence of STG. 3

4 Supplementary Figure S2 Endocytosis of AMPA receptors and LTD in neurons not overexpressing STG. (a) Immunocytochemical analysis of the NMDA induced removal of surface AMPA receptors in neurons not expressing any STG constructs. Cultured hippocampal neurons expressing N-terminal HA tagged GluA2 were treated with 50 M NMDA for 10 min, fixed, and stained for surface HA GluA2. After permeabilization of the plasma membranes with Triton X, the neurons were stained to detect the total HA GluA2 level. Scale bar, 20 m. (b) LTD induction in uninfected CA1 pyramidal neurons. Schaffer collateral evoked EPSC amplitudes at holding potential of 80 mv were normalized to the amplitudes immediately before low-frequency stimulation (LFS; 1 Hz, 300 stimuli at 40 mv) and averaged (n = 7). Error bars represent means + s.e.m. Inset, representative EPSCs recorded from uninfected neurons just before (solid lines) and 30 min after (grey lines) the application of LFS. 4

5 Supplementary Figure S3 Binding of STG to AP-2 and AP-3A is required for the NMDA induced removal of surface GluA1. (a d) Cultured hippocampal neurons expressing N-terminal HA tagged GluA1 and STG wt (a), STG 9D (b), STG AP2 (c), or STG AP3A (d) were treated with 50 M NMDA for 10 min, fixed, and stained for surface HA GluA1. After permeabilization of their plasma membranes with Triton X, 5

6 the neurons were stained to detect the total HA-GluA1 level. Representative images from two independent experiments are shown. Scale bar, 20 m. Graphs show the quantitative analysis of the NMDA induced changes in surface GluA1 immunoreactivity. The surface HA-GluA1 immunoreactivity was normalized to the total HA-GluA1 immunoreactivity, and its value in control neurons (i.e., not treated with NMDA) was arbitrarily defined as 100%. **, P < by Student s t-test; n = 7 9 cells for each STG construct from 3 independent experiments. Data represent means + s.e.m. 6

7 Supplementary Figure S4 STG s binding to AP-2 and AP-3A is not required for the AMPA induced removal of surface GluA2. (a c) Cultured hippocampal neurons expressing N-terminal HA tagged GluA2 and STG wt. (a), STG AP2 (b), or STG AP3A were treated with 100 M AMPA for 10 min, fixed, and stained for surface HA GluA2. 7

8 After permeabilization of the plasma membranes with Triton X, the neurons were stained to detect the total HA-GluA2 level. Representative images from two independent experiments are shown. Scale bars, 20 m. Graphs show the quantitative analysis of the AMPA induced changes in surface GluA2 immunoreactivity. The surface HA-GluA2 immunoreactivity was normalized to the total HA-GluA2 immunoreactivity, and its value in control neurons (i.e., no AMPA treatment) was arbitrarily defined as 100%. *, P < 0.05 by Student s t-test; n = 8 11 cells for each STG construct from 3 independent experiments. Error bars represent means + s.e.m. 8

9 Supplementary Figure S5 Effects of sirna on the expression of AP-2 and AP-3A proteins. (a) Immunoblot analyses of sirna against AP-2. HEK293 cells were transfected with sirna against AP-2 or control scrambled sirna together with a cdna encoding the HA tagged wild-type or sirna resistant AP-2. One day after the transfection, the amount of HA AP-2 was examined by immunoblot analysis using anti-ha antibody. (b) Immunoblot analyses of sirnas against AP-3. HEK293 cells were transfected with sirna against AP-3 or control scrambled sirna together with a cdna encoding the HA tagged wild-type or sirna resistant AP-3. One day after the transfection, the amount of AP-3 was assessed by immunoblot analysis using anti-ha antibody. (c f) Cultured hippocampal neurons were transfected with GFP and an sirna 9

10 against AP-2 (c, e) or AP3 (d, f) with or without a cdna encoding the sirna resistant mutant of AP-2 (e) or AP-3 (f) at 7 10 DIV. Seven days after the transfection, endogenous AP-2 or AP-3 and MAP2 were immunostained. The AP-2 immunoreactivity was significantly reduced in the GFP positive, MAP2 positive neurons (arrows), which were transfected with sirna (c); this effect was rescued by the coexpression of sirna resistant AP-2 (arrows, e). Similarly, the AP-3 immunoreactivity was significantly reduced by sirna (arrows, d), and this effect was rescued by the coexpression of sirna resistant AP-3 (arrows, f). Arrowheads indicate GFP negative neurons that show normal expression levels of AP-2 (c, e) or AP-3 (d, f). Scale bar, 20 m. 10

11 Supplementary Figure S6 Knockdown of AP-2 inhibits the NMDA induced removal of surface AMPA receptors. (a) Effect of sirna on the expression of AP-2 protein. Cultured hippocampal neurons were transfected with GFP and an sirna 11

12 against AP-2 at 7 10 DIV. Seven days after the transfection, the endogenous AP-2 was immunostained. The AP-2 immunoreactivity was significantly reduced in the GFP positive neurons (arrows), which were transfected with the sirna. Scale bar, 20 m. (b,c) Cultured hippocampal neurons at 7 10 DIV were transfected with N-terminal HA tagged GluA2 and sirna against Scale bars, 20 m. (b) or scrambled control sirna (c). At DIV, the neurons were treated with NMDA (50 M) for 10 min, and the surface and total HA-GluA2 were labeled before and after membrane permeabilization, respectively. The regions marked by white squares are shown at higher magnification in the lower panels. The NMDA induced reduction in surface GluA2 immunoreactivity was inhibited in neurons expressing the sirna against (b), but not in those expressing the scrambled control sirna (c). The graphs at right show the quantitative analysis of the NMDA induced changes in surface GluA2 immunoreactivity. **, P < 0.01 by Student s t-test; n = cells from 3 independent experiments. Error bars represent means + s.e.m. 12

13 Supplementary Figure S7 AP-1 interacts with STG but does not inhibit the NMDA induced removal of surface AMPA receptors. (a) Lysates of HEK293 cells expressing Flag tagged AP-1 ( 1) were pulled down by GST, the GST tagged C-terminus of 13

14 STG (GST STG wt ), or a GST STG mutant lacking the CT2 region (GST STG CT2 ), and immunoblotted (IB) with an anti-flag antibody. (b) Effects of sirna on the expression of AP-1 protein. Cultured hippocampal neurons were transfected with GFP and an sirna against AP-1 at 7 10 DIV. Seven days after the transfection, the endogenous AP-1 was immunostained. The AP-1 immunoreactivity was significantly reduced in the GFP positive neurons (arrows), which were transfected with the sirna. Scale bar, 20 m. (c,d) Cultured hippocampal neurons at 7 10 DIV were transfected with N-terminal HA tagged GluA2 and sirna against Scale bars, 20 m. (c) or scrambled control sirna (d). At DIV, the neurons were treated with NMDA (50 M) for 10 min, and the surface and total HA-GluA2 were labeled before and after membrane permeabilization, respectively. The NMDA induced reduction of surface GluA2 immunoreactivity was not inhibited in neurons expressing sirna against (c) or scrambled control sirna (d). The graphs at right show the quantitative analysis of the NMDA induced changes in surface GluA2 immunoreactivity. *, P < 0.05 by Student s t-test; n = 7 8 cells from 3 independent experiments. Error bars represent means + s.e.m. 14

15 Supplementary Figure S8 The NMDA induced removal of surface AMPA receptors is inhibited by the knock-down of AP-2 or AP-3A. Cultured hippocampal neurons at 7 10 DIV were cotransfected with N-terminal SEP tagged GluA2 and sirna against AP-2 (a; 2) or AP-3 (b; 3). Scale bars, 20 m. Scrambled sirna for each construct was used as a control. At 16 DIV, the SEP fluorescence in living neurons was examined before and 10 min. after NMDA treatment (50 M). NH 4 Cl was applied to visualize the SEP intensity in the acidic intracellular compartments. The bottom graphs show the quantitative analysis of the NMDA induced changes in the fluorescence intensity of SEP. *, P < 0.05 by Student s t-test; n = 4 cells. Error bars represent means + s.e.m. 15

16 Supplementary Figure S9 The binding of STG to AP-2, but not AP-3A, is required for the NMDA induced removal of surface AMPA receptors at 3 min after NMDA treatment. (a c) Immunocytochemical analysis of the effects of wild-type and mutant STG on the NMDA induced removal of surface AMPA receptors. Cultured hippocampal neurons coexpressing N-terminal HA tagged GluA2, GFP, and STG wt (a), 16

17 STG AP2 (b), or STG AP3A (c) were treated with 50 M NMDA for 3 min, fixed, and stained for surface HA-GluA2. The total HA-GluA2 was immunostained after permeabilization of the plasma membranes with Triton X. Scale bars, 20 m. Graphs at right show the quantitative analysis of the NMDA induced changes in surface GluA2 immunoreactivity. The surface HA-GluA2 immunoreactivity was normalized to the total HA-GluA2 immunoreactivity, and its value in control neurons (i.e., not NMDA treated) was arbitrarily defined as 100%. **, P < by Student s t-test; n = 8 10 cells for each STG construct from 3 independent experiments. Error bars represent means + s.e.m. 17

18 Supplementary Figure S10 Localization of STG and endosomal markers at basal states. Cultured hippocampal neurons at 7 10 DIV were transfected with HA tagged STG wt, STG AP3, or STG AP2 together with an early endosomal marker, GFP Rab4 Scale bar, 10 m. (a) or a late endosome/lysosome marker GFP Rab7 (b). At DIV, the neurons were fixed and immunostained for HA and GFP. No obvious colocalization of HA STG and GFP Rab4 (a) or GFP Rab7 (b) was observed. 18

19 Supplementary Figure S11 Localization of AMPA receptors compared with markers for early endosomes and late endosomes/lysosomes following NMDA induced AMPA receptor endocytosis. (a, b) Cultured hippocampal neurons at 7 10 DIV were transfected with HA tagged GluA2, an early endosome marker, GFP Rab4 (a), or a late endosome/lysosome marker, GFP Rab7 (b), together with untagged STG wt, STG AP3 or STG AP2 and E2-Crimson linked by self-cleaving 2A peptide. Scale bars, 10 m. At 17 DIV, the neurons were treated with NMDA (50 M) for 0 min, 3 min (a), 10 min and 20 min (b), fixed, and immunostained for HA and GFP. Arrows indicate the colocalization of HA GluA2 and GFP Rab4 (c) or HA STG and GFP Rab7 (d). At 17 DIV, the neurons were treated with NMDA (50 M) for 3 min (a), or 10 min (b) and immunostained for HA and GFP. Arrows indicate the colocalization of HA GluA2 and 19

20 GFP Rab. (c, d) Quantitative analysis of colocalization of GluA2 with Rab4 or Rab7 at various time points after NMDA application. At 3 min after NMDA treatment, HA GluA2 significantly colocalized with Rab4 in neurons coexpressing STG wt or STG AP3, but not in neurons coexpressing STG AP3. In contrast, at 10 and 20 min after NMDA treatment, HA GluA2 significantly colocalized with Rab7 in neurons coexpressing STG wt, but not in neurons coexpressing STG AP2 or STG AP3. *, P < 0.05 vs. time 0 by one-way ANOVA followed by Tukey test; n = 5 10 cells. Error bars represent means + s.e.m. 20

21 Supplementary Figure S12 AMPA receptors are degraded in lysosomes in an AP-3A-dependent manner. (a) Internalized AMPA receptors colocalize with the lysosome marker cathepsin. Cultured hippocampal neurons at 7 10 DIV were transfected with sirna against the subunit of AP-3 ( 3) or control scrambled sirna together with cdnas encoding HA tagged GluA2 and mcherry tagged cathepsin B. Neurons were incubated for 1 h with leupeptin (100 g/ml) to inhibit lysosomal degradation and an anti-ha antibody (1:100) to label the cell-surface GluA2. After washing out excess antibodies, the neurons were stimulated with 50 M NMDA for 20 min, fixed, and immunostained for HA. Arrows indicate cathepsin colocalized with internalized HA GluA2. Scale bar, 10 m. (b) Quantitative analysis of the 21

22 colocalization of internalized GluA2 with cathepsin. The proportion of HA GluA2 colocalized with mcherry cathepsin in untreated control neurons expressing scrambled sirna was arbitrarily defined as 100%. *, P < 0.05 by Student s t-test; n = 8 10 cells for each STG construct from 3 independent experiments. Error bars represent means + s.e.m. Significantly more internalized HA GluA2 was colocalized with mcherry cathepsin after NMDA application in neurons expressing scrambled RNA, but not in neurons expressing sirna against AP-3. (c) The AP-3-dependent lysosomal degradative pathway contributes to the total GluA2 protein level in neurons treated with NMDA. Cultured hippocampal neurons at 7 10 DIV were transfected with sirna against the subunit of AP-3 ( 3) or control scrambled sirna together with cdna encoding GFP. Neurons were treated with 50 M NMDA for 20 min, fixed, and immunostained for endogenous GluA2 and MAP2. The arrows indicate neurons expressing sirna (GFP). Among the neurons treated with NMDA, the GluA2 immunoreactivity was higher in the GFP-positive neurons expressing the sirna against AP-3 than in the GFP-negative neurons (yellow arrowheads). Scale bar, 20 m. (d) Quantitative analysis of the endogenous GluA2 immunoreactivity in neurons expressing sirna against 3 or scrambled RNA. *, P < 0.05 by Student s t-test; n = 6 cells from 2 independent experiments. Error bars represent means + s.e.m. 22

23 Supplementary Figure S13 Basic synaptic properties of infected and uninfected CA1 pyramidal neurons in wild-type mice. (a) No difference in input-output relationship of EPSC amplitude versus stimulus intensity at synapses between Shaffer collateral and CA1 pyramidal neurons that were either uninfected (WT: black, n = 5) or expressing STG wt (green; n = 6), STG ΔAP2 (blue, n = 7) or STG ΔAP3A (red, n = 7). Data are presented as mean ± SEM (One-way ANOVA followed by Bonferroni post-hoc analysis, P < 0.05). (b) No difference in miniature EPSC (mepsc) amplitude between the investigated groups in the presence of 0.5 µm TTX. Bar graph shows the population average of the mean amplitude (WT = 14.0 ± 0.4 pa (consistent with previous reports), n = 6; STG wt = 14.1 ± 0.9 pa, n = 5; STG ΔAP2 = 13.9 ± 2.1, n = 5; STG ΔAP3A = 13.6 ± 0.4, n = 6). Data are presented as mean ± SEM (one-way ANOVA followed by Bonferroni post-hoc analysis, P < 0.05). Below is shown the cumulative probability plot 23

24 of mepsc amplitude (Mann-Whitney test, P < 0.05). (c) No difference in AMPA/NMDA ratio estimated by dividing the EPSC peak amplitude at 90 mv by the amplitude at 200 ms from the onset at +30 mv(wt = 3.64 ± 0.56, n = 6; STG wt = 3.58 ± 0.40, n = 6; STG ΔAP2 = 3.86 ± 0.49, n = 11; STG ΔAP3A = 3.97 ± 0.33, n = 8). Data are presented as mean ± s.e.m. (one-way ANOVA followed by Bonferroni post-hoc analysis, P < 0.05). 24

25 Supplementary Figure S14 Effect of the R845A mutation of GluA2 on the interaction between STG and 2. (a) Coimmunoprecipitation assay to examine the interaction between wild-type or R845A mutant GluA2 and 2 in the presence and absence of STG. Lysates of HEK293 cells expressing wild-type (wt) or R845A mutant GluA2 and Flag-tagged 2 with or without STG were immunoprecipitated using the 25

26 anti GluA2 antibody and analyzed using the anti-flag antibody. *, P < 0.05 by Student's t-test (n = 5). Coexpression of STG strongly enhanced the coimmunoprecipitation of 2 with wild-type GluA2 but not with R845A mutant GluA2. Error bars represent as means + s.e.m. (b) Coimmunoprecipitation assay to examine the interaction between STG and wild-type or R845A mutant GluA2. Lysates of HEK293 cells coexpressing STG and wild-type (wt) or R845A mutant GluA2 were immunoprecipitated using the anti-glua2 antibody and analyzed using the anti-stg antibody. In the bar graph at bottom, the intensity of the band corresponding to STG that coimmunoprecipitated with GluA2 was normalized to its intensity in the input lysates. The intensity of STG that coimmunoprecipitated with wild-type GluA2 was arbitrarily established as 1.0. Error bars represent as means + s.e.m. (c) Schematic drawing of the STG GluA2 AP-2/AP-3 complex. The 2 or 3A subunit only weakly binds to GluA2 in a manner independent of the R845A mutation under our experimental conditions (upper panels). The 2 or 3A subunit is incorporated into the AMPA receptor complex by binding to STG (lower panels). The interaction between STG and 2/ 3A is likely to be inhibited allosterically by the GluA2 R845A mutation. 26

27 Supplementary Figure S15 Original scans of key western blots. 27