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. 2017 Oct 10;114(41):E8760-E8769.
doi: 10.1073/pnas.1620153114. Epub 2017 Sep 25.

Proteasome-independent Polyubiquitin Linkage Regulates Synapse Scaffolding, Efficacy, and Plasticity

Affiliations
Free PMC article

Proteasome-independent Polyubiquitin Linkage Regulates Synapse Scaffolding, Efficacy, and Plasticity

Qi Ma et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Ubiquitination-directed proteasomal degradation of synaptic proteins, presumably mediated by lysine 48 (K48) of ubiquitin, is a key mechanism in synapse and neural circuit remodeling. However, more than half of polyubiquitin (polyUb) species in the mammalian brain are estimated to be non-K48; among them, the most abundant is Lys 63 (K63)-linked polyUb chains that do not tag substrates for degradation but rather modify their properties and activity. Virtually nothing is known about the role of these nonproteolytic polyUb chains at the synapse. Here we report that K63-polyUb chains play a significant role in postsynaptic protein scaffolding and synaptic strength and plasticity. We found that the postsynaptic scaffold PSD-95 (postsynaptic density protein 95) undergoes K63 polyubiquitination, which markedly modifies PSD-95's scaffolding potentials, enables its synaptic targeting, and promotes synapse maturation and efficacy. TNF receptor-associated factor 6 (TRAF6) is identified as a direct E3 ligase for PSD-95, which, together with the E2 complex Ubc13/Uev1a, assembles K63-chains on PSD-95. In contrast, CYLD (cylindromatosis tumor-suppressor protein), a K63-specific deubiquitinase enriched in postsynaptic densities, cleaves K63-chains from PSD-95. We found that neuronal activity exerts potent control of global and synaptic K63-polyUb levels and, through NMDA receptors, drives rapid, CYLD-mediated PSD-95 deubiquitination, mobilizing and depleting PSD-95 from synapses. Silencing CYLD in hippocampal neurons abolishes NMDA-induced chemical long-term depression. Our results unveil a previously unsuspected role for nonproteolytic polyUb chains in the synapse and illustrate a mechanism by which a PSD-associated K63-linkage-specific ubiquitin machinery acts on a major postsynaptic scaffold to regulate synapse organization, function, and plasticity.

Keywords: CYLD; PSD-95; TRAF6; long-term depression; lysine 63-linked ubiquitination.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
K63-polyUb is present at excitatory synapses and targets PSD-95 as a substrate. (A) Immunoblot (IB) analysis of K63-polyUb species in forebrain lysates prepared from mice at different postnatal stages. (B) Total ubiquitin and K63-polyUb (after stripping and reprobing) species in mouse forebrains (P30) and cultured rat hippocampal neurons at 21 d in vitro (DIV21) after immunoprecipitation (IP) with an anti-ubiquitin antibody. (C) Immunostaining of K63-polyUb and PSD-95 in cultured rat hippocampal neurons. [Scale bars, 20 μm (Upper) and 5 μm (Lower).] Arrowheads denote colocalized PSD-95 and K63-polyUb clusters, and arrows indicate K63-polyUb clusters lacking PSD-95 costaining. (D) Correlation plot of PSD-95 and K63-polyUb cluster intensities in C. Pearson’s r is shown. (E) K63-polyubiquitination of endogenous PSD-95 in mouse brain. WCL, whole-cell lysate. (F) K63-polyubiquitination of PSD-95–Flag in transfected HEK293FT cells. (G) Schematic showing ubiquitin lysine mutants used in this study. (H) Effects of HA-ubiquitin and mutants in promoting K63 ubiquitination of PSD-95–Flag in HEK293FT cells. Experiments were performed in the absence of proteasome inhibitors.
Fig. S1.
Fig. S1.
Additional characterizations of K63-polyUb presence in neurons and synapses. (A) Immunostaining of K63-polyUb and PSD-95 on mouse hippocampal sections. Hoechst 3342 was used to label neuronal nucleus. (Scale bar, 10 μm.) (B) Spine localization for HA-UbK63, but significantly less for HA-UbR63, in cotransfected cultured rat hippocampal neurons. (Scale bars, 10 μm.) (C) Quantification of HA fluorescence pixel intensities for dendritic spines or protrusions and shafts in B. n = 16 shafts and 534 spines for HA-UbK63; n = 11 shafts and 285 spines for HA-UbR63. ***P < 0.001; unpaired t tests.
Fig. S2.
Fig. S2.
Additional characterizations of PSD-95 polyubiquitination. (A and B) Simultaneous detection of both nonubiquitinated (∼95 kDa band) and ubiquitinated PSD-95 (higher molecular weight smears) in transfected HEK293FT cell (A) and mouse brain (B) lysates with anti-Flag (A) or anti–PSD-95 (B) following immunoprecipitation with anti-Flag (A) or anti–PSD-95 (B). The percentage of ubiquitinated PSD-95 compared with the total PSD-95 in the immunoprecipitate, defined as [high molecular weight smear intensity/(high molecular weight smear intensity + 95 kDa band intensity)] × 100, is shown at the bottom. Experiments were performed on denatured lysates in the absence of proteasome inhibitors. (C) K63-linked polyubiquitination of endogenous PSD-95 in Neuro2a cells, cultured rat hippocampal neurons, and cultured WT or PSD-95–KO mouse neurons. (D) K63 polyubiquitination of PSD-95 and mutants, analyzed by immunoprecipitation and immunoblotting using lysed HEK293FT cells transfected with the indicated plasmids. (E and F) Conservation of identified lysine sites in PSD-95 SH3 and GK domains across species (E) and in the MAGUK family (F). K491, K544, K558, and K672 (red) represent the lysine residue locations in rat PSD-95. (G) Quantification of K48 polyubiquitination in HEK293FT cells cotransfected with Flag-tagged WT PSD-95 or mutants and HA-UbK48. Cell lysates were immunoprecipitated with anti-Flag and immunoblotted with anti-HA for K48-linked polyubiquitination. Data are based on gel density analysis of high molecular weight ubiquitin smears for each K-to-R mutant and normalized to WT PSD-95 (+UbK48). Substantially impaired mutants are shown in red. n = 3–11 experiments. *P < 0.05; unpaired t tests vs. WT(+UbK48) control. (H) Severely impaired K63 polyubiquitination of K558R in rat hippocampal neurons. Rat hippocampal cultures were infected at DIV4 with lentiviruses expressing Myc-GFP, Myc–PSD-95, or Myc-K558R and were analyzed by immunoprecipitation with anti–PSD-95 and immunoblotting with anti-UbK63 antibody to asses K63 polyubiquitination. (I) The K558R mutation does not affect K48-linked polyubiquitination in neurons. The experiment was similar to that in H except that immunoblotting was done with anti-UbK48 antibody to assess K48 polyubiquitination. MG132 (10 μM) was used in this experiment. (J and K) Myc-544R, Myc-558R, and Myc-672R all nearly abolished K63 polyubiquitination of PSD-95, whereas only Myc-544R and Myc-672R severely impaired K48 polyubiquitination of PSD-95 in cultured rat hippocampal neurons infected with the indicated lentiviruses. Immunoprecipitation was done with Myc-conjugated agarose beads.
Fig. 2.
Fig. 2.
Mapping ubiquitination sites on PSD-95. (A) Modular structure of PSD-95. GK, guanylate kinase-like; PDZ, PSD-95, Dlg, ZO-1; SH3, Src-homology 3. Within the NT, cysteine palmitoylation residues (C3, C5), the PEST motif, and the Src-binding region are shown. Within GK, four key lysine residues identified and used in this study are indicated. (BD) K63- or K48-linked ubiquitination of PSD-95–Flag and mutants in HEK293FT cells. WCL, whole-cell lysate. (E) Quantification of K63-polyUb levels normalized to WT PSD-95 (+UbK63). n = 3–13 experiments. ***P < 0.001, **P < 0.01, *P < 0.05; unpaired t tests vs. WT(+UbK63). (F) Mutagenesis summary; “+” in dark blue or red indicates an essential contribution; “+” in light blue indicates a modest contribution; “−” indicates little or no contribution.
Fig. S3.
Fig. S3.
Additional experiments characterizing the E2/E3/DUB complex for K63 polyubiquitination of PSD-95. (A) Dendritic distributions of Ubc13, Uev1A, TRAF6, and CYLD in cultured hippocampal neurons and their localizations relative to PSD-95. (Scale bars, 5 μm.) (B) TRAF6 promotes K63-linked polyubiquitination of WT PSD-95 but not K558R. Lysates from HEK293FT cells transfected with the indicated plasmids were analyzed by immunoprecipitation and immunoblotting. (C) Effects of TRAF6 and C70A overexpression on PSD-95 K63 ubiquitination in cultured rat hippocampal neurons. Neurons were infected with lentiviruses expressing the indicated proteins, followed by immunoprecipitation and immunoblotting assays. Quantification is shown to the right. n = 3 independent experiments. *P < 0.05, **P < 0.01; unpaired t tests. (D) Interaction between TRAF6 and PSD-95 in HEK293FT cells. Cells were transfected with plasmids encoding PSD-95–Flag or Myc-TRAF6 or both, followed by immunoprecipitation and immunoblotting assays. (E) Lack of interaction between TRAF6 and PSD-95 mutants PSD-95ΔNT and C3,C5S in HEK293FT cells. Cells transfected with the indicated plasmids were analyzed by immunoprecipitation and immunoblotting. (FI) Association of TRAF6 (F), Ubc13 (G), Uev1A (H), and CYLD (I) with PSD-95 in the mouse brain, analyzed by coimmunoprecipitation. Forebrain tissues were homogenated and used in immunoprecipitation and immunoblotting assays. IgG was used as a control in immunoprecipitation experiments.
Fig. 3.
Fig. 3.
Identification of an E2/E3/DUB complex for PSD-95. (A) Immunoblots showing the presence of indicated proteins in subcellular fractions of the rat forebrain. H, homogenates; LP1, synaptosome fraction; P2, crude synaptosomal membranes; PSD (I), PSD fraction after one Triton X-100 extraction; S3, cytosol; SPM, synaptic plasma membrane (P3); SVE, synaptic vesicle enriched (S4). (B) Effects of TRAFs in promoting K63 ubiquitination of PSD-95 in HEK293FT cells. (C) TRAF6 promotes K63 but not K48 ubiquitination of PSD-95. (D) Impaired K63 ubiquitination of PSD-95ΔNT. (E) In vitro ubiquitination assay. (F) Immunoblots showing inhibition of TRAF6-promoted PSD-95 K63 ubiquitination by CYLD in HEK293FT cells. (G) In vitro deubiquitination assay. (H) Efficiency of TRAF6 and CYLD knockdown in cultured hippocampal neurons. (IL) Effects of TRAF6 and CYLD overexpression (I and K) or knockdown (J and L) on PSD-95 K63 ubiquitination in neurons. (M and N) Quantification of IL. n = 3 or 4 experiments; ***P < 0.001, **P < 0.01, *P < 0.05; unpaired t tests vs. controls.
Fig. 4.
Fig. 4.
K63-polyUb regulates GK-target interactions. (A) Immunoblots showing lack of interaction between K558R and SPAR in HEK293FT cells. (B) Severely impaired interaction between K558R and SPAR-C in yeasts. (C) Quantification of B. n = 3 experiments; **P < 0.01; one-way ANOVA with post hoc Tukey’s test. (D, Upper) Immunoblots confirming expression of purified GST, GST–PSD-95, GST-K558R, and His–SPAR-C from E. coli. (Lower) GST pulldown showing that both purified GST–PSD-95 and GST-K558R do not interact with His–SPAR-C. (E) In vitro reconstitution of PSD-95–SPAR interaction. (F) Immunoblots showing markedly weakened interaction between transfected K558R-Flag and endogenous GKAP in HeLa cells. (G) Immunoblots showing unaltered K558–SAP102 interaction in cotransfected HEK293FT cells. (H) A ribbon diagram showing the structure of PSD-95 GK in complex with a phosphorylated target peptide derived from Lgl2. Lys558 (shown in the spherical model) is located in the α1/β2 loop opposite the GK target-binding groove.
Fig. 5.
Fig. 5.
K63-polyUb regulates PSD-95 targeting to and clustering at dendritic spines. (A) PSD-95–KO mouse neurons transfected with Myc–PSD-95 or Myc-K558R and immunostained with anti-Myc. Arrowheads indicate PSD-95 and K558R clusters along dendrites. [Scale bars, 20 μm (Left) and 5 μm (Right).] (B) Spines at higher resolution. (Scale bars, 1 μm.) (C, Left) Quantification of spine/shaft Myc fluorescence ratio. n = 32–41 cells; ***P < 0.001, *P < 0.05; unpaired t tests. (Right) Cumulative probability of spine/shaft fluorescence ratios for all spines from each group. (D) Effects of TRAF6 or CYLD knockdown on PSD-95 clustering. (E) Quantification of PSD-95 clustering intensity and spine/shaft ratio. n = 22–34 cells. **P < 0.01, ***P < 0.001, unpaired t tests.
Fig. S4.
Fig. S4.
Additional experiments using expanded N-terminal and GK-domain mutants to show that K63 ubiquitination is important for PSD-95–SPAR interaction and is independent of PSD-95 palmitoylation. (A) Impaired K63 polyubiquitination of C3,5S-Flag, C5S-Flag, and V7S-Flag mutants, analyzed by immunoprecipitation and immunoblotting using lysed HEK293FT cells transfected with the indicated plasmids. (B) Severely impaired interactions between C3,5S, C5S, or V7S and SPAR-C in the yeast two-hybrid system. Yeast strain MaV203 was cotransformed with plasmids encoding DB–PSD-95, DB-C3,5S, DB-C5S, or DB-V7S and AD–SPAR-C. Protein–protein interaction was assayed by growing the yeast on selective medium lacking leucine and tryptophan. DB-p53 and AD-T were cotransformed as a positive control. DB and AD–SPAR-C, DB–PSD-95 and AD, DB-C3,5S, DB-C5S, or DB-V7S and AD were cotransformed as negative controls. Unlabeled yeast colonies at the lower left corner were unrelated experiments. (C) Severely impaired interactions between C3,5S-Flag, C5S-Flag, or V7S-Flag and Myc-SPAR in HEK293FT cells. Cells were transfected with indicated plasmids and analyzed by immunoprecipitation and immunoblotting. (D) Severely impaired interactions between K544R-Flag, K558R-Flag, or K672R-Flag and Myc-SPAR in HEK293FT cells. Cells were transfected with the indicated plasmids and analyzed by immunoprecipitation and immunoblotting. (E) Effects of several palmitoylation-regulating mutants on K63 polyubiquitination of PSD-95 analyzed by immunoprecipitation and immunoblotting using lysed HEK293FT cells transfected with the indicated plasmids.
Fig. S5.
Fig. S5.
Additional data showing that K63-polyUb regulates PSD-95–target interactions in a GK domain-preferred, target-dependent manner, using several K63-polyUb-deficient mutants. (A) GST pulldown analysis of 14 known PSD-95–binding proteins on mouse brain homogenates using purified GST, GST–PSD-95, and GST-K558R. Western blot analysis of each protein in total lysates is shown at the bottom. (B) Quantification of GST pulldown assays. Gel densitometric analysis was performed for individual proteins, and protein–protein interaction strength was presented as normalized levels. For all proteins except SPAR, GKAP, and AKAP, data were normalized to respective GST–PSD-95 controls. Protein bands for SPAR, GKAP, and AKAP were undetectable and not quantifiable and thus were assigned the value zero and included in the graph to indicate the lack of interaction between them and PSD-95 or K558R. The absence of these bands even for the WT full-length PSD-95 protein is consistent with our hypothesis that K63-linked ubiquitination of PSD-95, which does not occur for bacteria-expressed GST fusion proteins, is essential for PSD-95 to interact with SPAR, GKAP, and AKAP. n = 3 experiments; ***P < 0.001 vs. GST, ###P < 0.001 vs. GST–PSD-95; one-way ANOVA with Tukey’s post hoc tests. (C) Schematic summary of PSD-95–interacting proteins examined in A and B. The module(s) on PSD-95 to which each protein binds are depicted. The relative importance of K63-linked polyubiquitination on each interaction is color-coded: red indicates essential importance, light red indicates nonessential but facilitating, light blue indicates slightly inhibitory, and white indicates no effect. (D, Upper) GST pulldown analysis of three GK-binding and five non–GK-binding proteins on mouse brain homogenates, using purified GST, GST–PSD-95, GST-K544R, or GST-K672R. Western blot analysis of each protein in total lysates is shown at the bottom. (Lower) Pulldown analysis of interactions between K544R or K672R and three GK-binding and five non–GK-binding proteins on mouse brain homogenates using purified K544R-Flag or K672R-Flag from denatured HEK293FT cell lysates. Western blot analysis of each protein in total lysates is shown at the bottom. (E) Computational modeling shows that the K–R substitution at K558, K544, or K672 is not expected to significantly impact the GK structure. The orange model displays the X-ray crystallography model (Protein Data Bank ID code: 1KJW). The cyan model displays the mutant model as predicted by Phyre. Labels indicate positions of α-helices and β-sheets within each domain. Red/blue residues indicate lysine/arginine, respectively. The rmsd scores are under the X-ray model resolution of 1.8 Å, indicating highly similar structures (66). (F) Summary of PSD-95–target interaction literature details. IF, immunofluorescence; IH, immunohistochemistry; N.A., not available; PD, GST pulldown assay; Y2H, yeast two-hybrid assay.
Fig. S6.
Fig. S6.
Additional experiments showing that PSD-95 K63 ubiquitination is required for dendritic spine targeting of PSD-95 and SPAR. (A) A mouse hippocampal neuron transfected with Myc–PSD-95 (red) illustrating the selection strategy of proximal, medial, and distal dendritic segments. (Scale bar, 40 μm.) Proximal, medial, and distal dendrites were selected based on their distance from the soma or branching points. Specifically, proximal segments were within 100 μm of soma on the primary dendrites; medial segments were within 100 μm of the first branching points on the secondary dendrites; distal segments were on a tertiary or higher-order dendrite. (B) Impaired spine targeting of K558R in cultured PSD-95–KO hippocampal mouse neurons transfected with Myc–PSD-95 or Myc-K558R. (Scale bar, 5 μm.) (C) Quantification of the spine/shaft fluorescence ratio in B. n = 6–23 cells; **P < 0.01, *P < 0.05; unpaired t tests vs. respective controls. (D) Impaired spine targeting of K558R in cultured rat hippocampal neurons transfected with Myc–PSD-95 or Myc-K558R. (Scale bar, 5 μm.) (E) Quantification of the spine/shaft fluorescence ratio in D. n = 3–8 cells; **P < 0.01, *P < 0.05; unpaired t tests vs. respective controls. (F) Impaired spine targeting of K544R and K672R but not K703R in cultured rat hippocampal neurons transfected with Myc–PSD-95, Myc-K544R, Myc-K672R, or Myc-K703R. (Scale bar, 5 μm.) (G) Quantification of the spine/shaft fluorescence ratio in F. n = 10–16 cells; **P < 0.01; post hoc Dunnett’s tests following one-way ANOVA. (H) SPAR clustering is markedly diminished in cultured PSD-95–KO hippocampal mouse neurons, revealed by SPAR immunofluorescence. (Scale bar, 5 μm.) (I) PSD-95–Flag, but not K558R-Flag, significantly restored clustering of HA-SPAR in cotransfected cultured PSD-95–KO hippocampal mouse neurons. (Scale bar, 5 μm.) (J) Quantification of SPAR clustering intensity in I. n = 12–14 cells; **P < 0.01; post hoc Dunnett’s tests following one-way ANOVA.
Fig. 6.
Fig. 6.
K63-polyUb facilitates synapse maturation and strengthening. (A) sGluA1 clusters (red) from untransfected neurons or neurons transfected with PSD-95–Flag or K558R-Flag (green). (Scale bar, 5 μm.) (B) Quantification of sGluA1 cluster intensity, normalized to untransfected neurons. n = 28–29 cells; ***P < 0.001. (C) Synapsin I clusters (green) from untransfected and PSD-95–Flag or K558R-Flag transfected (red) neurons. (Scale bar, 5 μm.) (D) Quantification of synapsin I cluster intensity, normalized to untransfected neurons. n = 14–26 cells; ***P < 0.001, *P < 0.05. (E) GFP images of dendrites and spines from neurons (co)transfected with the indicated plasmids. (Scale bar, 5 µm.) (F) Quantification of spine density (Upper) and size (Lower) in E. n = 7–12 cells. **P < 0.01, *P < 0.05. (G) Representative mEPSCs from neurons transfected with indicated plasmids. (H) Mean mEPSC frequency (Left) and amplitude (Right). n = 27–38 cells. **P < 0.01. (I) Lentivirus expression of Myc–PSD-95 or Myc-K558R in the hippocampus in vivo. Viruses expressing Myc–PSD-95, Myc-K558R, or Myc-GFP and GFP within the same dual promoter vector (Fig. S7C) were injected into P20 PSD-95–KO brains. Nuclei are stained with Hoechst (blue). (Scale bar, 500 µm.) (J) Examples of apical dendrites (GFP) of infected pyramidal neurons. (Scale bar, 5 µm.) (K) Quantification of spine density in J. n = 28–31 cells. **P < 0.01. (L) Example PSD-95 and synapsin I puncta in infected striatum radiatum. (Scale bar, 5 μm.) (M) Quantification of synapsin I cluster intensity in L. n = 16–18 cells. ***P < 0.001 vs. Myc-GFP; ###P < 0.001 vs. Myc–PSD-95; unpaired t tests in B and D, one-way ANOVA with Dunnett’s’ test vs. GFP in F, H, and K, and one-way ANOVA with Tukey’s test in M.
Fig. S7.
Fig. S7.
Additional data on K544R and K672R supporting the notion that K63 ubiquitination of PSD-95 promotes dendritic spine formation and maturation in cultured rat hippocampal neurons. (A) sGluA1 clusters (red) from untransfected neurons or from neurons transfected with PSD-95–Flag, K544R-Flag, or K672R-Flag (green). (Scale bar, 5 μm.) (B) Quantification of sGluA1 cluster intensity, normalized to untransfected neurons. n = 9–29 cells; ***P < 0.001, **P < 0.01. PSD-95–Flag transfected and untransfected data are replotted from Fig. 6B for direct comparisons. (C) Plasmids containing dual-promoter expression cassettes. Syn, synapsin I promoter. (D) Representative GFP images of dendrites and spines from DIV21 neurons transfected with the indicated plasmids. (Scale bar, 5 µm.) (E) Quantification of spine density in D. n = 15–25 cells; **P < 0.01, *P < 0.05; one-way ANOVA followed by Dunnett’s test vs. GFP.
Fig. 7.
Fig. 7.
Activity-dependent regulation of K63-polyUb conjugation in cultured rat hippocampal neurons. (A) Rapid and global loss of K63-polyUb signals following NMDA application. Neurons were treated with NMDA (30 μM), NMDA + AP5 (50 μM), or control solution for 3 or 9 min before immunostaining for K63-polyUb. (Scale bar, 40 μm.) (B) Quantification of A. n = 19–36 cells. ***P < 0.001, *P < 0.05; unpaired t tests. (C) Immunoblots showing levels of K63 polyubiquitinated PSD-95 at various time points after treatment with NMDA (30 μM), NMDA+AP5 (50 μM), NMDA in Ca2+-free artificial cerebrospinal (ACSF) fluid, AP5, TTX (2 μM), or bicuculline (Bic; 40 μM). (D) Quantification of C and G. K63-ubiquitinated and total PSD-95 in immunoprecipitation eluates following treatments were normalized to respective untreated controls. n = 3 or 4 experiments; ***P < 0.001, **P < 0.01, *P < 0.05; unpaired t tests vs. controls. (E and F) Immunoblots (E) and summary (F) showing prolonged loss of K63-polyUb from PSD-95 induced by NMDA. n = 3; **P < 0.01, one-way ANOVA with Tukey’s post hoc tests. (G) Immunoblots showing effects of AP5 on PSD-95 K63 ubiquitination in neurons infected with sh-control or sh-TRAF6 lentiviruses. (H) Immunoblots showing CYLD knockdown abolished NMDA-induced PSD-95 deubiquitination. (I) Summary of H. n = 6–8 experiments; **P < 0.01; paired t tests vs. controls.
Fig. S8.
Fig. S8.
Activity-dependent regulation of K63 ubiquitination of PSD-95 in mouse brain slices. (AF) Immunoblots of K63 polyubiquitination of PSD-95 in mouse prefrontal cortical slices following various stimulations. Acutely cut slices (containing the anterior cingulated cortex and the prelimbic cortex) were treated with the specified agonists or antagonists for the time indicated, lysed, and analyzed by immunoprecipitation and immunoblotting assays. In CE, a positive control (NMDA treatment for 3 min) was also included. Drugs were included in ACSF at the following concentrations: NMDA (30 μM), AP5 (50 μM), TTX (2 μM), and bicuculline (40 μM). Each experiment was performed using pooled slices from an independent PSD-95 WT mouse. (G) Quantification of AF. Ubiquitinated and total PSD-95 in immunoprecipitation eluates following treatments were normalized to respective untreated controls. n = 3–5 experiments; *P < 0.05; unpaired t tests vs. controls.
Fig. 8.
Fig. 8.
CYLD mediates NMDA-induced rapid PSD-95 declustering from spines. (A and B) NMDA-induced, proteasome-independent PSD-95 declustering on dendrites. (A) Neurons were treated with NMDA (30 μM), NMDA + MG132 (20 μM), or control solution for 3 or 9 min before immunostaining for PSD-95 or K63-polyUb. (B) Neurons were incubated with MG132 for 4 h before 9-min NMDA stimulation. (Scale bars, 5 μm.) (C) Quantification of dendritic PSD-95 and K63-polyUb fluorescence intensities in A. n = 22–24 cells; ***P < 0.001; unpaired t tests vs. no-treatment control. (D) Quantification of B. n = 20 cells; ***P < 0.001, unpaired t tests. (E) Effects of CYLD knockdown on NMDA-induced endogenous PSD-95 declustering. Neurons infected with the indicated vectors were treated with NMDA or control solution for 9 min before immunostaining for PSD-95. (Scale bar, 5 μm.) (F) Quantifications of E. n = 10–34 cells; ***P < 0.001, **P < 0.01, *P < 0.05; unpaired t tests.
Fig. S9.
Fig. S9.
Additional data on NMDA-triggered PSD-95 declustering and sGluA1 internalization in cultured rat (AC) and mouse (DG) hippocampal neurons. (A) Declustering of K703R-Flag, but not K558R-Flag, following NMDA stimulation. Neurons transfected with the indicated vectors were treated with NMDA or control solution for 9 min before immunostaining for Flag. Cotransfected GFP was used to identify positive cells. (Scale bar, 5 μm.) (B) Quantification of dendritic PSD-95 (Flag) fluorescence intensities in A. n = 13–22 cells; ***P < 0.001, *P < 0.05; unpaired t tests vs. respective no-treatment controls. (C) Representative images showing the coinfected CYLD-601A mutant failed to rescue the sh-CYLD abolishment of NMDA-triggered PSD-95 declustering. Neurons infected with sh-CYLD and C601A-GFP were treated with NMDA or control solution for 9 min before immunostaining for PSD-95. This experiment was run as a part of the experiments shown in Fig. 8 E and F. (Scale bar, 5 μm.) (D) NMDA-triggered AMPAR internalization assay in PSD-95 WT neurons. Representative images show surface and internalized GluA1 (iGluA1) receptors following NMDA (50 μM, 9 min) or control treatment. (E) Quantified sGluA1 internalization ratio in D. n = 20–22 cells; ***P < 0.001; unpaired t test. (F) NMDA-triggered sGluA1 internalization in PSD-95–KO neurons infected with Myc-GFP, Myc–PSD-95 (and GFP), or Myc-K558 (and GFP) (expressed with dual-Syn promoter vectors). (G) Quantification of NMDA-triggered sGluA1 internalization in F normalized to respective no-NMDA controls. n = 2646 cells; ***P < 0.001; unpaired t tests.
Fig. 9.
Fig. 9.
CYLD is required for cLTD. (A) NMDA (50 μM, 9 min)-triggered sGluA1 internalization in cultured rat hippocampal neurons infected with indicated plasmids. (Scale bar, 30 μm.) (B) Quantification of A, normalized to respective no-NMDA controls. n = 13–19 cells; ***P < 0.001; unpaired t tests. (C) NMDA-triggered depression of mEPSCs in cultured rat hippocampal neurons transfected with indicated plasmids. Intracellular BAPTA: 15 mM. (D) Quantification of mEPSC frequency and amplitude. n = 6–8 cells; ***P < 0.001, **P < 0.01, *P < 0.05; one-way ANOVA with post hoc Tukey’s test. (E) Model for roles of nonproteolytic K63 linkage in the synapse using PSD-95 as a proof of principle. (Upper) An E2/E3/DUB complex controls the balance of K63 ubiquitination/deubiquitination of PSD-95. (Lower) K63-polyUb plays an essential role in synaptic maintenance and orderly organization of PSD-95 and LTD. PSD-95 is constitutively conjugated by K63-polyUb chains (by Ubc13/Uev1A–TRAF6), which maintains and compartmentalizes the protein (perhaps combined with other posttranslational modifications) to specific subdomains within the PSD. Synaptic activity opens NMDARs, allowing Ca2+ influx, and recruits/activates CYLD (dashed arrow), which subsequently removes K63-polyUb from PSD-95. Deubiquitinated PSD-95 translocates away from PSD, destabilizing the PSD and weakening the synapse. Palmitoylated PSD-95 is targeted to the synapse membrane but may not be incorporated in a particular scaffolding/signaling complex that depends on PSD-95 K63 ubiquitination.

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