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Synaptic adhesion molecule IgSF11 regulates synaptic transmission and plasticity

Nature Neuroscience volume 19, pages 84–93 (2016)Cite this article

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Abstract

Synaptic adhesion molecules regulate synapse development and plasticity through mechanisms that include trans-synaptic adhesion and recruitment of diverse synaptic proteins. We found that the immunoglobulin superfamily member 11 (IgSF11), a homophilic adhesion molecule that preferentially expressed in the brain, is a dual-binding partner of the postsynaptic scaffolding protein PSD-95 and AMPA glutamate receptors (AMPARs). IgSF11 required PSD-95 binding for its excitatory synaptic localization. In addition, IgSF11 stabilized synaptic AMPARs, as determined by IgSF11 knockdown–induced suppression of AMPAR-mediated synaptic transmission and increased surface mobility of AMPARs, measured by high-throughput, single-molecule tracking. IgSF11 deletion in mice led to the suppression of AMPAR-mediated synaptic transmission in the dentate gyrus and long-term potentiation in the CA1 region of the hippocampus. IgSF11 did not regulate the functional characteristics of AMPARs, including desensitization, deactivation or recovery. These results suggest that IgSF11 regulates excitatory synaptic transmission and plasticity through its tripartite interactions with PSD-95 and AMPARs.

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Figure 1: IgSF11 interacts with PSD-95 and is targeted to excitatory synapses in a PDZ interaction–dependent manner.
Figure 2: IgSF11 interacts with AMPARs and recruits both PSD-95 and AMPARs to sites of homophilic IgSF11 adhesion.
Figure 3: IgSF11 knockdown reduces surface clustering of AMPARs without affecting PSD-95 clustering.
Figure 4: IgSF11 knockdown suppresses mEPSCs and evoked EPSCs.
Figure 5: IgSF11 knockdown increases surface mobility and endocytosis of AMPARs.
Figure 6: Dominant-negative inhibition of IgSF11 suppresses mEPSC amplitude in DG granule cells.
Figure 7: IgSF11 has no effect on the functional properties of AMPARs.
Figure 8: Reduced mEPSC amplitude in DG cells and suppressed LTP at SC-CA1 synapses in the Igsf11−/− hippocampus.

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Acknowledgements

We would like to thank R. Neve for the kind gift of the HSV system, J.-R. Lee and K. Han for the PSD fraction sample, J.W. Um and J. Ko for the subcellular fraction sample, M.-H. Kim for the mini analysis program, and Mihyeon Kim for helping with IgSF11 antibody generation. This work was supported by the Institute for Basic Science (to E.K.), the Brain Research Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Science, ICT & Future Planning (2013-056732 to H.K.), National Honor Scientist Program of the NRF (to B.K.K.), and Wellcome Trust and UK MRC Neurodegenerative Disease Initiative Programme (to K.C.).

Author information

Author notes

  1. Seil Jang, Daeyoung Oh, Yeunkum Lee and Eric Hosy: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Korea

    Seil Jang, Seung Min Um, Seok-kyu Kwon, Jooyeon Woo & Eunjoon Kim

  2. Department of Biomedical Sciences, KAIST, Daejeon, Korea

    Daeyoung Oh & Hyewon Shin

  3. Department of Psychiatry, CHA Bundang Medical Center, CHA University, Seoul, Korea

    Daeyoung Oh

  4. Center for Synaptic Brain Dysfunctions, Institute for Basic Science (IBS), Daejeon, Korea

    Yeunkum Lee, Doyoun Kim, Sun Gyun Kim, Junyeop Daniel Roh & Eunjoon Kim

  5. University of Bordeaux, Interdisciplinary Institute for Neuroscience, Bordeaux, France

    Eric Hosy & Daniel Choquet

  6. CNRS UMR 5297, Bordeaux, France

    Eric Hosy & Daniel Choquet

  7. Department of Molecular Neurobiology, Max Planck Institute of Experimental Medicine, Göttingen, Germany

    Christoph van Riesen & Jeong-Seop Rhee

  8. School of Clinical Sciences, Faculty of Medicine and Dentistry, University of Bristol, Bristol, UK

    Daniel Whitcomb, Julia M Warburton, Jihoon Jo & Kwangwook Cho

  9. Centre for Synaptic Plasticity, University of Bristol, Bristol, UK

    Daniel Whitcomb & Kwangwook Cho

  10. Department of Biomedical Sciences, Chonnam National University Medical School, Gwangju, South Korea

    Jihoon Jo

  11. Department of Physiology, Seoul National University College of Medicine, Seoul, Republic of Korea

    Myoung-Hwan Kim

  12. Seoul National University Bundang Hospital, Seongnam, Gyeonggi, Republic of Korea

    Myoung-Hwan Kim

  13. Biological Sciences, College of Natural Sciences, Seoul National University, Seoul, Korea

    Heejung Jun & Bong-Kiun Kaang

  14. Department of Anatomy and Division of Brain Korea 21 Biomedical Science, College of Medicine, Korea University, Seoul, Korea

    Dongmin Lee & Hyun Kim

  15. Department of Anatomy and Neurobiology, School of Dentistry, Kyungpook National University, Daegu, Korea

    Won Mah

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  1. Seil Jang
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Contributions

D.L. and H.K. performed the in situ hybridization analysis. S.J., D.O., Y.L., H.S., D.K. and S.G.K. performed immunoblot and co-immunoprecipitation experiments. D.O., S.K.K., J.W. and H.S. performed neuron culture, co-culture, co-clustering experiments, homophilic binding assay and antibody feeding experiments. D.O., Y.L. and H.S. performed neuronal shRNA knockdown experiments. S.J., C.V.R., D.W., J.M.W., J.J., S.G.K., S.M.U., S.K.K., M.H.K., J.D.R., H.J., W.M. and K.C. performed electrophysiological experiments and analyzed the data. E.H. performed uPAINT experiments. Y.L. and J.D.R. carried out HSV experiments. S.J. generated and characterized Igsf11–/– (LacZ) and Igsf11–/– mice. D.K. modeled IgSF11 structures. H.K., B.K.K., K.C., J.S.R., D.C. and E.K. supervised the project and wrote the manuscript.

Corresponding authors

Correspondence to Jeong-Seop Rhee, Daniel Choquet or Eunjoon Kim.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Amino acid sequence alignment of IgSF11 proteins from various vertebrate species.

Amino acids in black and gray backgrounds indicate fully and partially conserved residues. The following amino acid sequences were used for comparison; human (NP_001015887.1), chimpanzee (XP_001161500.1), macaque monkey (NP_001244835.1), mouse (NP_733548.2), rat (NP_001013138.1), chicken (XP_416568.3), finch (ENSTGUP00000013797), lizard (ENSACAP00000002845), Xenopus (ENSXETP00000049789), and zebrafish (XP_001343768.4).

Supplementary Figure 2 Amino acid sequence alignment of CAR subgroup members and their in situ hybridization patterns in the brain.

(a) Amino acids in black and gray backgrounds indicate fully and partially conserved residues. The following mouse amino acid sequences were used for comparison; IgSF11 (NP_733548.2), CAR (NP_001020363.1), ESAM (NP_081378.1), and CLMP (NP_598494.2). (b) In situ mRNA distribution patterns of IgSF11, CAR, ESAM, and CLMP in adult (8 weeks) male mouse brains obtained from the Allen Brain Atlas (IgSF11, http://mouse.brain-map.org/experiment/show?id=68861816, CAR, http://mouse.brain-map.org/experiment/show?id=77454980, ESAM, http://mouse.brain-map.org/experiment/show?id=69262331, and CLMP, http://mouse.brain-map.org/experiment/show?id=70445538). Scale bar, 1 mm.

Supplementary Figure 3 IgSF11 interacts with PSD-95 in yeast two-hybrid assays.

The C-terminal PDZ-binding motif of IgSF11 interacts with PDZ domains of PSD-95 family proteins (PSD-95, PSD-93/chapsyn-110 and SAP97) in yeast two-hybrid assays. pBHA, bait vector; pGAD10, prey vector; WT, wild type (last seven residues); VA, a mutant IgSF11 in which the last residue (valine) was changed to alanine. PDZ domains from GRIP2, Shank1 and NHERF were used as controls. β-Galactosidase (β-gal) activity: +++, < 45 min; ++, 45–90 min; +, 90–240 min; –, no significant β-gal activity. Three independent experiments were performed.

Supplementary Figure 4 IgSF11 does not induce clustering of pre- or postsynaptic proteins in contacting axons and dendrites, and does not interact with other synaptic adhesion molecules.

(a-c) Cultured rat hippocampal neurons were cocultured with HEK293T cells expressing rat IgSF11 (untagged), or EGFP (control; DIV 10–13), followed by staining for synapsin I (a presynaptic protein), PSD-95 (an excitatory postsynaptic protein), and gephyrin (an inhibitory postsynaptic protein). Scale bar, 30 μm. (d) IgSF11 does not interact with other synaptic adhesion molecules, as shown by the lack of binding of the ectodomain of IgSF11 fused to human Fc (IgSF11-ecto-Fc) to known synaptic adhesion molecules expressed in HEK293T cells. IgSF11-expressing HEK293T cells were used as a control. Scale bar, 10 μm. Three independent experiments were performed.

Supplementary Figure 5 The transmembrane domain of IgSF11 is important for GluA1 binding.

(a and b) HEK293T cells were cotransfected with GluA1 and the indicated deletion variants of HA-IgSF11, followed by immunoprecipitation with HA antibodies and immunoblotting with HA and GluA1 antibodies. Three independent experiments were performed. Full-length blots are presented in Supplementary Figure 17c.

Supplementary Figure 6 Characterization of IgSF11-knockdown and IgSF11 rescue constructs.

(a) Acute knockdown of IgSF11 expression in heterologous cells. HEK293T cells were doubly transfected with rIgSF11 (rat IgSF11) and sh-vec (empty vector), sh-IgSF11#1, or sh-IgSF11#1* (a mutant incapable of knocking down IgSF11) for two days, and the cell lysates were characterized by immunoblotting for IgSF11 and α-tubulin (loading control). n = 3, * p < 0.05, ** p < 0.01, ns, not significant, one-way ANOVA. Full-length blots are presented in Supplementary Figure 17d.

(b) Acute knockdown of IgSF11 expression by an independent IgSF11 knockdown construct (sh-IgSF11#2). HEK293T cells were transfected with doubly transfected with IgSF11 and sh-vec (empty vector) or sh-IgSF11#2 for two days, and the cell lysates were characterized by immunoblotting for IgSF11 and α-tubulin (loading control). n = 3, *** p < 0.001, Student’s t-test. Full-length blots are presented in Supplementary Figure 17e.

(c and d) Characterization of an sh-IgSF11#1-resistant IgSF11 expression construct. HEK293T cells were doubly transfected with IgSF11 (rat) + sh-vec, IgSF11 + sh-IgSF11#1, or sh-IgSF11#1-resistant IgSF11 (WT or ΔC3) + sh-IgSF11#1, followed by immunoblotting with IgSF11 antibodies. Three independent experiments were performed. Full-length blots are presented in Supplementary Figure 17f,g.

Supplementary Figure 7 IgSF11 knockdown in cultured neurons suppresses surface clustering of GluA1 and GluA2 but has no effect on excitatory synapses.

(a-c) Acute knockdown of IgSF11 decreases surface clustering of GluA1 in cultured hippocampal neurons. Cultured neurons were transfected with sh-vec (empty vector), sh-IgSF11#1, or sh-IgSF11#1* (DIV 14–17), and stained for surface GluA1. au, arbitrary unit; n = 18 for sh-vec, 17 for sh-IgSF11#1, and 17 for sh-IgSF11#1*, *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA. Scale bar, 10 μm.

(d-g) IgSF11 knockdown has no effect on excitatory synapses, defined by synapsin I-positive PSD-95 clusters. Cultured neurons were transfected with sh-vec, sh-IgSF11#1, or sh-IgSF11#1* (DIV14–17), and immunostained for PSD-95 and synapsin I. n = 22 for sh-vec, 23 for sh-IgSF11#1, and 21 for sh-IgSF11#1*, ns, not significant, one-way ANOVA. Scale bar, 10 μm.

(h-k) Independent IgSF11 knockdown suppresses surface GluA2 clustering but has no effect on PSD-95 clusters. Cultured hippocampal neurons were transfected with sh-vec (empty vector) or sh-IgSF11#2 (DIV 14–17), and stained for surface GluA2 or PSD-95. au, arbitrary unit; n = 18 for sh-vec, 17 for sh-IgSF11#2, ***p < 0.001, ns, not significant, Student’s t-test. Scale bar, 10 μm.

Supplementary Figure 8 IgSF11 knockdown reduces steady-state levels of surface GluA2.

Cultured hippocampal neurons doubly transfected with N-terminally HA-tagged GluA2 (HA-GluA2) and sh-vec, sh-IgSF11#1, or IgSF11#1* (DIV 14–17) were measured of surface and internal levels of GluA2. n = 19 for sh-vec, 19 for sh-IgSF11#1, and 18 for sh-IgSF11#1*, *p < 0.05, **p < 0.01, one-way ANOVA. Scale bar, 30 μm.

Supplementary Figure 9 Generation and characterization of Igsf11−/− (LacZ) and Igsf11−/− mice.

(a) Schematic diagram showing the strategy for the generation of the gene-trapped Igsf11−/− (LacZ) mice and exon 3-deleted Igsf11−/− mice. Note that exon 2 is captured by the inserted cassette to yield a truncated protein fused to β-geo (β-galactosidase + neomycin), and that crossing of Igsf11 floxed mice with protamine-Cre mice leads to the deletion of exon 3 in the IgSF11 gene. Primer locations for genotyping are indicated. (b) Genotyping of wild-type (Igsf11+/+; WT), Igsf11+/− (LacZ), and Igsf11−/− (LacZ) mice by PCR. Full-length gels are presented in Supplementary Figure 17h. (c) Igsf11−/− (LacZ) mice show no detectable levels of IgSF11 proteins in the brain. α-Tubulin blot was used as a control. Full-length blots are presented in Supplementary Figure 17i.

Supplementary Figure 10 Spatiotemporal IgSF11 expression revealed by X-Gal staining.

(a and b) The spatial expression pattern of IgSF11 was characterized by X‑Gal staining of sagittal brain slices from Igsf11−/− (LacZ) mice at P5 and postnatal weeks 2, 3, and 12. Scale bars, 1 mm for whole brain, 200 μm for hippocampus. Three independent experiments were performed.(c) No differences are observed in left and right Igsf11−/− (LacZ) hippocampal regions at 8 weeks. (d) IgSF11 protein levels in mouse brains are rapidly increased during the first and second weeks and are maintained at later stages. Three independent experiments were performed. Full-length blots are presented in Supplementary Figure 17j.

Supplementary Figure 11 Normal expression levels of synaptic proteins in the Igsf11−/− brain.

(a and b) Hippocampal samples from WT and Igsf11−/− (LacZ) brains (3 weeks) were immunoblotted for the synaptic proteins indicated, followed by quantification. Protein amounts in Igsf11−/− brains normalized to α-tubulin were normalized to those of WT brains. n = 3 mice for WT and KO, ns, not significant, Student’s t-test. Full-length blots are presented in Supplementary Figure 17k.

Supplementary Figure 12 Igsf11−/− hippocampal CA1 pyramidal neurons display unaltered levels extrasynaptic AMPAR currents.

(a and b) Igsf11−/− (LacZ) DG granule cells in acute hippocampal slices (3 weeks) were treated with AMPA (1 μM), a condition known to induce extrasynaptic AMPAR-mediated currents, and measured of evoked EPSCs. n = 8 slices (2 mice) for WT and KO, ns, not significant, Student’s t-test.

Supplementary Figure 13 Normal levels of basal transmission and paired pulse ratio at Igsf11−/− MPP-DG and SC-CA1 synapses.

(a and b) Basal transmission (input-out relationship) and paired pulse ratio at WT and Igsf11−/− (LacZ) MPP-DG synapses (3–6 months). n = 12 slices (6 mice) for WT, and 10, 6 for KO. (c and d) Basal transmission and paired pulse ratio at WT and Igsf11−/− (LacZ) SC-CA1 synapses (3–6 months). n = 10, 6 for WT and KO.

Supplementary Figure 14 The Igsf11−/− hippocampus shows normal levels of immature GluA2.

Hippocampal lysates from WT and Igsf11−/− (LacZ) mice were digested with endoglycosidase H (Endo H) or peptide N-glycosidase F (PNGase F), and measured of the ratio of Endo H-sensitive GluA2 over total (Endo H-resistant + Endo H-sensitive) GluA2. PNGase F, which completely removes all types of N-linked oligosaccharides on GluA2, was used as a control. n = 3 mice for WT and KO (3 weeks), ns, not significant, Student’s t-test. Full-length blots are presented in Supplementary Figure 17l.

Supplementary Figure 15 Molecular modeling of IgSF11-dependent homophilic adhesion.

Three possible molecular models of homophilic and trans-cellular adhesion mediated by the two Ig domains of IgSF11. These models were based on the X-ray crystal structure of CAR (a relative of IgSF11) 49.

Supplementary Figure 16 Full-length images of immunoblots and gels.

(a) Full-length immunoblots for Fig. 1d. (b) Fig. 1e. (c) Fig. 1f. (d) Fig. 1g. (e) Fig. 1h. (f) Fig. 1i. (g) Fig. 2a. (h) Fig. 2b.

Supplementary Figure 17 Full-length images of immunoblots and gels.

(a) A full-length gel for Fig. 8a. (b) Full-length immunoblots for Fig. 8b. (c) Supplementary Fig. 5b. (d) Supplementary Fig. 6a. (e) Supplementary Fig. 6b. (f) Supplementary Fig. 6c. (g) Supplementary Fig. 6d. (h) A full-length gel for Supplementary Fig. 9b. (i) Full-length immunoblots for Supplementary Fig. 9c. (j) Supplementary Fig. 10d. (k) Supplementary Fig. 11a. The blackened immunoblot panels indicate overexposed films, which could be properly exposed in other immunoblot experiments. (l) A full-length immunoblot for Supplementary Fig. 14a.

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Jang, S., Oh, D., Lee, Y. et al. Synaptic adhesion molecule IgSF11 regulates synaptic transmission and plasticity. Nat Neurosci 19, 84–93 (2016). https://doi.org/10.1038/nn.4176

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