Research Article

Identification of an elaborate complex mediating postsynaptic inhibition

See allHide authors and affiliations

Science  09 Sep 2016:
Vol. 353, Issue 6304, pp. 1123-1129
DOI: 10.1126/science.aag0821
  • Fig. 1 Development of iBioID for synaptic proteomics.

    (A) Schematic of the iBioID approach for synapses. Vglut, vesicular glutamate transporter; Vgat, vesicular GABA transporter. (B) Validation of BirA constructs in hippocampal slice using markers labeled in (A): (i) BirA-gephyrin and (ii) PSD-95–BirA (white arrows point to colocalized puncta); and (iii) BirA alone nonspecifically labels proteins. Scale bar, 2 μm. (C) Outline of iBioID method in mice. (D) Successful biotinylation of proteins in vivo following intraperitoneal biotin administration. Scale bar, 0.5 mm. (E) Biotinylation is specific to regions expressing BirA-gephyrin (coronal section). (Insets) From thalamus (e1) and hippocampus (e2) and insets of e2 are shown. Scale bars: top right, 0.5 mm; e1 and e2, 20 μm. Electron micrographs verify enrichment of biotinylation at (F and f′) inhibitory or (G and g′) excitatory PSD substructures. Large gold beads, streptavidin labeling; small gold beads, immunolabel for GABA. Scale bar, 250 nm. (H) Specific purification of known PSD proteins for each BirA fusion protein.

  • Fig. 2 Scale-free graph of the iPSD proteome.

    (A) InSyn1 (blue), gephyrin (green), and arhgef9 (yellow) BirA-dependent iBioID identify a rich network of known and previously unknown proteins enriched at the iPSD. Node titles correspond to gene name; size represents fold-enrichment over negative control. Edges are shaded according to the types of interactions (gray, iBioID; black, protein-protein interactions previously reported). (B) Clustergram topology of iPSD proteins (red) in selected functional categories.

  • Fig. 3 Validation of selected iPSD proteins.

    (A) Colocalization of iPSD proteins (column 1), some Flag-tagged, some hemagglutinin tagged, and Myc–PX-RICS (Myc epitope–tagged Phox domain–containing isoform of RhoGAP involved in β-catenin–N-cadherin and N-methyl-d-aspartate receptor signaling) with endogenous gephyrin (column 2) in hippocampal neurons. EBFP, enhanced blue fluorescent protein. Scale bar, 10 μm. (B) Each iPSD protein significantly colocalizes with gephyrin in dendrites compared with PSD-95 (n > 9 dendritic regions of interest). (C to F) iPSD proteins coimmunoprecipitate with gephyrin when coexpressed in human embryonic kidney 293 (HEK293) cells. ***P < 0.001 one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparisons test (B). Error bars ±SEM.

  • Fig. 4 Abnormal synaptic inhibition follows loss of the iPSD protein InSyn1.

    (A) Experimental schematic for (C) to (F). DIV, days in vitro. (B) Validation of InSyn1 gRNAs (#12 and #17) and rescue constructs by cotransfection of 293T cells and immunoblotting. (C and D) GABAA-dependent miniature inhibitory postsynaptic currents (GABAA mIPSCs) recorded from CA1 pyramidal cells. (Inset) GABAA mIPSC waveform averages. InSyn1 depletion did not alter mIPSC kinetics (rise: controls, 3.6 ± 0.6 ms; InSyn1, 3.4 ± 0.6 ms; P = 0.51; decay: controls, 9.7 ± 2.1 ms; InSyn1, 10.3 ± 1.8 ms; P = 0.42). IEIs of InSyn1-depleted GABAA mIPSCs (red) are specifically increased compared with control (black) and rescue (blue) neurons. (E and F) AMPAR-dependent mEPSCs are not altered in InSyn1-depleted neurons. (G) Time-line schematic for local field potential (LFP) recordings in acute slices from Cas9 knock-in (KI) mice infected with AAV:Cre/Insyn1 gRNA. Representative extent of AAV infection in hippocampus. (H) Representative LFP activities recorded in hippocampal area CA3 in the presence of 10 µM carbachol to model “awake state” gamma rhythm. Top trace, Pure 30 to 40 Hz gamma oscillation; middle trace, 3 to 5 Hz spike-wave discharges; bottom trace, ictal-like burst—the latter two indicative of hyperexcitable or epileptiform activity. (I) InSyn1 gRNA–expressing slices exhibit increased–epileptiform activity. (J) Averaged power spectra showing signal energy in the InSyn1 gRNA -expressing slices is increased in the 0 to 15 Hz frequency band and decreased in the 20 to 50 Hz frequency bands. *P < 0.05, **P < 0.01; ***P < 0.001; n.s., not statistically significant. Error bars ± SEM.

  • Fig. 5 InSyn1 functionally associates with the dystrophin complex at the iPSD.

    (A) Network analysis of affinity-purified InSyn1-GFP proteome from mouse brain. Affinity purification–mass spectrometry (AP/MS) interaction. (B and C) Clustergram topologies of InSyn1-associated proteins in selected functional categories. (D) Colocalization of InSyn1 with α-dystroglycan (α-DG) is diminished after depleting InSyn1 with Cas9 conditional knock-in mouse hippocampal neurons infected with AAV:Cre/Insyn1 gRNA. EGFP, enhanced GFP. Scale bar, 10 µm. (E) InSyn1 is essential for GABAAR and α-DG cluster density. Hippocampal neurons from Cas9 KI mice were stained for GABAA subunit Rγ2 and α-DG following infection with control AAV:Cre/(-)gRNA (top panel, control); AAV:Cre/Insyn1 gRNA (middle panel, knockdown), or AAV:Cre/Insyn1 gRNA, and transfected with InSyn1 gRNA–resistant plasmid (bottom panels, Rescue). (Insets) Higher magnification regions with open (colocalized puncta) and closed (noncolocalized puncta) arrows. (F to H) Quantification of α-DG and GABAARγ2 puncta colocalization or density (n = 16 to 18). *P < 0.05, **P < 0.01, ***P < 0.001 one-way ANOVA followed by Tukey's multiple comparisons test (F) to (H). Error bars ± SEM. Scale bar, 10 µm. (I) Loss of InSyn1 does not alter PSD-95 puncta density. Cas9 knock-in mouse hippocampal neurons infected with AAV:Cre/Insyn1 gRNA were stained with PSD-95 and quantified (n = 18 to 20 neurons); n.s., not significant, by two-tailed t test (I).

Supplementary Materials

  • Identification of an elaborate complex mediating postsynaptic inhibition

    Akiyoshi Uezu, Daniel J. Kanak, Tyler W. A. Bradshaw, Erik J. Soderblom, Christina M. Catavero, Alain C. Burette, Richard J. Weinberg, Scott H. Soderling

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

    Download Supplement
    • Materials and Methods
    • Supplementary Text
    • Figs. S1 to S5
    • Table legends S1 to S6
    • References
    Table S1
    Filtered proteins based on EM staining
    Table S2
    Enriched biotinylated proteins from iBioID pilot
    Table S3
    Enriched biotinylated proteins from PSD-95â€"BirA probe utilizing quantitative mass spectrometry analysis
    Table S4
    Enriched biotinylated proteins from three independent iPSD targeted BirA probes
    Table S5
    Proteins common to ePSD and iPSD datasets
    Table S6
    InSyn1 GFP-trap data

Navigate This Article