Fig 1: SUSD4 binds NEDD4 ubiquitin ligases, known regulators of AMPA receptor turnover and degradation.(A) Mass spectrometry identification of SUSD4 interactors. Left: Affinity purification from cerebellar synaptosomes was performed using either GFP-SUSD4 as a bait or GFP as a control. Proteins were then resolved using SDS-PAGE followed by immunoblot for anti-GFP and coomassie staining of proteins. Right: Gene ontology (GO) enrichment analysis network (Molecular Function category) of the 28 candidate proteins (Cytoscape plugin ClueGO) identified in affinity purified samples (A) by liquid chromatography with tandem mass spectrometry (LC MS/MS). The ubiquitin ligase activity term is significantly enriched in particular due to the identification of several members of the NEDD4 family of HECT ubiquitin ligases. See also Table 1 (n = 3 independent experiments). (B) Immunoblot confirmation of SUSD4 interaction with NEDD4 ubiquitin ligases. Affinity purification from cerebellar synaptosomes was performed using full-length SUSD4 (HA-tagged, HA-SUSD4), a mutant lacking the C-terminal tail (HA-SUSD4?CT), or GFP as a bait. Proteins were then resolved using SDS-PAGE followed by immunoblot for NEDD4, ITCH, WWP1, or HA-SUSD4 (anti-HA). HA-SUSD4 interacts with all three members of the NEDD4 family. This interaction is lost when the C-terminal tail of SUSD4 is deleted or when GFP is used instead of SUSD4 as a control. (C) Schematic representation of HA-tagged SUSD4 and different mutant constructs: SUSD4?CT (lacking the cytoplasmic tail), SUSD4?NT (lacking the extracellular domain), SUSD4NT (lacking the transmembrane and intracellular domains), SUSD4?PY (point mutation of the PPxY site), SUSD4?LY (point mutation of the LPxY), and SUSD4?PY/LY (double mutant at both PPxY and LPxY). (D) SUSD4 interaction with GluA2 and NEDD4 was assessed by co-immunoprecipitation using HEK293 cells transfected with SEP-GluA2 together with PVRL3a as a control or one of the HA-SUSD4 constructs represented in (C). Affinity purification was performed with an anti-HA antibody and extracts were probed for co-immunoprecipitation of GluA2 (with an anti-GluA2 antibody) and of the HECT ubiquitin ligase NEDD4 (anti-NEDD4 antibody). Co-immunoprecipitated GluA2 levels are normalized to input GluA2 and then represented as relative to the immunoprecipitated levels for each SUSD4 construct. N = 3 independent experiments. (E) Potential interactors of SUSD4 control several parameters of AMPA receptor turnover. Three different pools of AMPA receptors are found in dendrites and spines: synaptic, extrasynaptic, and intracellular. AMPA receptors are synthetized and delivered close to the synaptic spine to reach the synaptic surface. At the surface, AMPA receptors can move laterally (lateral diffusion) or vertically by endocytosis and exocytosis. Endocytosis can be mediated by clathrin (CM-endocytosis) or be clathrin-independent (CI-endocytosis). CM-endocytosis is often related to activity-dependent processes. After endocytosis, AMPA receptors can choose between two different pathways from the early endosomes, one for recycling and the other for degradation. Potential molecular partners of SUSD4 identified by our proteomic analysis could regulate AMPA receptor turnover at several levels of this cycle (in red).Figure 5—source data 1.Numerical data to support graphs in Figure 5.
Fig 2: Western blot analysis of the GluA2 and GluA3 subunits surface densities in cortical terminals that were incubated with the anti-GluA2 and the anti-GluA3 antibodies. (A) Western blot analysis of the GluA2 (upper) and of the GluA3 (lower) immunoreactivity in the total synaptosomal lysate (L), the biotin-untreated synaptosomal lysate (B), the control biotin-treated synaptosomal lysate (C), the lysates from biotin-treated synaptosomes incubated with the anti-GluA2 and the anti-GluA3. The blot is representative of three different experiments runs on different days. Changes in GluA2 (white bar) and in GluA3 (grey bar) subunits surface density in anti-GluA2 (B) or anti-GluA3 (C) incubated synaptosomes. The results are expressed as percentage of the respective control and are reported as mean ± SEM. *p < 0.05 versus respective control; **p < 0.01 versus respective control.
Fig 3: Upregulation of NMDAR pathways underlying ASD, learning/memory and epilepsy. (A) Protein interaction network for ASD (ASD Network). ASD candidate genes were mapped onto the hippocampal interactome to extract a network of ASD candidate genes and their first co-expressed neighbors. Most of the ASD candidate genes (light pink) show no expression changes. Red node: upregulated; blue node: downregulated; light pink node: without expression change; the node with green border: co-expressed neighbor; gray line: protein-protein interaction (PPI); double lines: PPI and co-expression; node size: degree; nodes with degree ≥ 3 are labeled in the panel. (B) Protein interaction network for epilepsy (EP Network). EP candidate genes were mapped onto the hippocampal interactome to extract a network consisting of the EP candidate genes and their first co-expressed neighbors. Most of the EP candidate genes (yellow) show no expression changes. Red node: upregulated; blue node: downregulated; yellow node: without expression change; node with green border: co-expressed neighbor; gray line: protein-protein interaction (PPI); double lines: PPI and co-expression; node size: degree; nodes with degree ≥ 3 are labeled in the panel. (C) Protein interaction network for learning and memory (LM Network). LM-related genes were mapped onto the hippocampal interactome to extract a network including the LM-related genes and their first co-expressed neighbors. Most of the LM-related genes (cyan) show no expression changes. Red node: upregulated; blue node: downregulated; cyan node: without expression change; node with green border: co-expressed neighbor; gray line: protein-protein interaction (PPI); double lines: PPI and co-expression; node size: degree; nodes with degree ≥ 3 are labeled in the panel. (D) Overlaps between the networks and the converged network module. There are 21 genes overlapping in the ASD-, EP and LM Networks. Red nodes: up-regulated genes, gray nodes: not changed genes. (E) Co-immunoprecipitation of GluN1 with GluN2B, GluN2A and PSEN1. (F) Relative expression levels of NMDAR and AMPAR genes in the hippocampus of Htr3a-/- mice were compared to WT mice (Student's t test, p = 0.1056 for Gria1, p = 0.3168 for Gria2, p = 0.6752 for Gria3, p = 0.6313 for Gria4, p = 0.0377 for Grin1, p = 0.0299 for Grin2a, p = 0.0360 for Grin2b, p = 0.0375 for Grin2c, p = 0.1066 for Grin2d, p = 0.3296 for Grin3a, n = 8 for each group). (G) Expression levels of NMDAR subunit GluN1 and GluN2B were increased in the hippocampus of Htr3a-/- mice compared with WT mice (Student's t test, p = 0.7277 for AMPAR1, p = 0.6083 for AMPAR2, p = 0.0076 for GluN1, p = 0.9121 for GluN2A, p = 0.0162 for GluN2B, n = 5 for each group). Data are presented as boxplots (median and 5th-95th percentile whiskers), or mean ± SEM., * p < 0.05; ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Fig 4: Increased mEPSC amplitude and levels of excitatory postsynaptic receptors in model rats. (A) Diagram showing model generation and patch clamp/western blot analysis. (B) Representative mEPSC (left) and mIPSC (right) traces recorded in the ACC. (C) Statistical analysis indicating significantly increased amplitude but not frequency of mEPSCs in model rat ACC (frequency: Control, 1.92 ± 0.06 Hz, PP, 1.71 ± 0.124 Hz, p = 0.136; amplitude: Control, 12.26 ± 0.48 pA, PP, 14.22 ± 0.55 pA, p = 0.012; Control, n = 21 cells of five rats; PP, n = 18 cells of five rats). (D) Identical mIPSC frequency and amplitude in control and model ACC (frequency: Control, 2.00 ± 0.11 Hz, PP, 1.95 ± 0.11 Hz, p = 0.20; amplitude: Control,13.06 ± 0.59 pA, PP, 11.67 ± 0.55 pA, p = 0.09; Control, n = 22 cells of five rats; PP, n = 22 cells of five rats). (E) Example of immunoblots of ACC extracts probed with anti-GluR1, GluR2, GluR4, NR1, and NR2B antibodies and quantification of the immunoblots revealing significant increases in levels of GluR1 (p = 0.014) and NR1 (p = 0.024), but not GluR2, GluR4 and NR2B. (F) Representative mEPSC (left) and mIPSC (right) traces in the HIPP. (G) Statistical analysis showing significantly increased mEPSC amplitude, but not frequency, in model rats (frequency: Control, 1.36 ± 0.12 Hz, p = 0.23; PP, 1.31 ± 0.15 Hz, p = 0.23; amplitude: Control, 10.89 ± 0.78 pA, PP, 12.67 ± 0.84 pA, p = 0.02; Control, n = 21 cells of five rats; PP, n = 20 cells of five rats). (H) Identical mIPSC frequency and amplitude in HIPP of control and model rats (frequency: Control, 0.98 ± 0.14, PP, 0.76 ± 0.18, p = 0.43; amplitude: Control, 10.71 ± 0.53, PP, 9.82 ± 0.59, p = 0.36; Control, n = 22 cells of 5 rats; PP, n = 22 cells of five rats). (I) Example of immunoblots probed with anti-GluR1, GluR2, GluR4, NR1, and NR2B antibodies and quantification of the immunoblots revealing a significant increase in levels of NR1 (p = 0.029). Values represent mean ± SEM. One-way ANOVA was used for mEPSCs and mIPSCs, and two-sample t-test for western blotting. *p < 0.05, ***p < 0.001.
Fig 5: GluA receptor subunits colocalize with syntaxin 1A in mouse cortical synaptosomes. Confocal analysis of the GluA1 (red, B), GluA2 (red, E), GluA3 (red, H), and GluA4 (red, M) subunit immunoreactivities in syntaxin 1A-positive cortical nerve terminals (green, A, D, G, L, respectively) and their colocalization (merge, yellow, C, F, I, N, respectively). The figure shows representative images of three independent experiments.
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