LGI1-ADAM22-MAGUK configures transsynaptic nanoalignment for synaptic transmission and epilepsy prevention

Abstract

Physiological functioning and homeostasis of the brain rely on finely tuned synaptic transmission, which involves nanoscale alignment between presynaptic neurotransmitter-release machinery and postsynaptic receptors. However, the molecular identity and physiological significance of transsynaptic nanoalignment remain incompletely understood. Here, we report that epilepsy gene products, a secreted protein LGI1 and its receptor ADAM22, govern transsynaptic nanoalignment to prevent epilepsy. We found that LGI1-ADAM22 instructs PSD-95 family membrane-associated guanylate kinases (MAGUKs) to organize transsynaptic protein networks, including NMDA/AMPA receptors, Kv₁ channels, and LRRTM4-Neurexin adhesion molecules. Adam22^ΔC5/ΔC5 knock-in mice devoid of the ADAM22-MAGUK interaction display lethal epilepsy of hippocampal origin, representing the mouse model for ADAM22-related epileptic encephalopathy. This model shows less-condensed PSD-95 nanodomains, disordered transsynaptic nanoalignment, and decreased excitatory synaptic transmission in the hippocampus. Strikingly, without ADAM22 binding, PSD-95 cannot potentiate AMPA receptor-mediated synaptic transmission. Furthermore, forced coexpression of ADAM22 and PSD-95 reconstitutes nano-condensates in nonneuronal cells. Collectively, this study reveals LGI1-ADAM22-MAGUK as an essential component of transsynaptic nanoarchitecture for precise synaptic transmission and epilepsy prevention.

Keywords: AMPA receptor; LGI1–ADAM22; MAGUK; epilepsy; transsynaptic nanocolumn.

Fig. 1.

ADAM22 participates in transsynaptic protein networks involving MAGUKs. (A) In vivo ADAM22-associated protein complex purified from Adam22^FAH/FAH knock-in mouse brain. Protein names identified by in-gel digestion-based mass spectrometry are shown. Asterisks indicate immunoglobulin heavy chain. FAH, a tandem-tag composed of FLAG, AU1, and HA epitope tags; IP, immunoprecipitation. (B) Synaptic ADAM22-associated protein network under the high stringency condition. Volcano plot of protein enrichments between Adam22^FAH/FAH knock-in and wild-type mice (negative control). Two hundred thirty-four proteins were significantly enriched in ADAM22-FAH samples (n = 4 replicates). Thresholds for enrichment are shown by gray dashed lines. Color codes for classification are indicated (and apply to A–D). Smaller light gray dots, proteins with subthreshold values; gray dots above thresholds, proteins in other categories. All protein data (348 proteins) for the plot are shown in Dataset S2. (C and D) The identified specific proteins under the high stringency condition are lined up based on the protein functions and Mascot scores. Error bars show ± SEM (n = 4). (E) ADAM22-FAH and LGI1 colocalize with postsynaptic PSD-95 and presynaptic CASK in the hippocampus of an Adam22^FAH/FAH mouse. Arrowheads indicate synaptic colocalization of ADAM22-FAH (labeled by HA antibody), PSD-95 or CASK, and LGI1 in the stratum lucidum (SL) in the CA3 region. (Scale bars: 20 μm [magnified, 5 μm].) SR, stratum radiatum; Py, stratum pyramidale; SO, stratum oriens. (F) Two-color (2C) STED imaging reveals coclusters of ADAM22-FAH (labeled by HA antibody) and LGI1 that are positioned at the synaptic cleft in the CA1 region. Bassoon (Bsn) and PSD-95 represent the presynaptic active zone and PSD, respectively. Asterisks indicate postsynaptic nanodomains of PSD-95. The graph shows the distances between cluster peaks of two tested molecules (red and green). P values were determined by Kruskal–Wallis with post hoc Scheffé test. *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant. n = 60 pairs (HA–LGI1); 107 (HA–PSD-95); 78 (HA–Bsn); 67 (Bsn–PSD-95). (Scale bar: 200 nm.)

Fig. 2.

Loss of the ADAM22 PDZ ligand causes lethal epileptic seizures in mice. (A) Schematic presentation of ADAM22ΔC5, lacking a C-terminal PDZ-binding motif (–WETSI). Pro, prodomain; MP, inactive metalloprotease domain; DI, disintegrin domain; CR, cysteine-rich domain; EGF, EGF-like domain; TM, transmembrane domain. (B) Kaplan–Meier survival curve of Adam22^ΔC5/ΔC5 mice (n = 32), showing completely penetrant lethality around postnatal 2 to 8 mo. (C) A typical example of an epidural EEG recording during a spontaneous seizure of an Adam22^ΔC5/ΔC5 mouse (P178). A triangle, the onset timing of seizure activity; lines with numbers, recording at a faster sweep speed. At the beginning, repetitive large sharp spike waves were observed (line with “1”). Later, spike wave amplitude decreased (line with “2”), pauses appeared (line with “3”), and finally the seizure activity stopped. This mouse died on P182 (mouse #2 in D). (D) Frequency of epileptic discharges in the epidural EEGs (mice #1 and 2) and hippocampal (HP) LFPs (mice #3 to 5) of Adam22^ΔC5/ΔC5 mice. The arrows indicate dates of death. (E) Typical examples of LFPs recorded simultaneously from the hippocampus (Upper) and sensorimotor cortex (Lower) at the early (P141, Left) and late (P147, Right) stages of the one and the same Adam22^ΔC5/ΔC5 mouse (#3 in D). This mouse died on P151. Raw LFPs, time-frequency maps, and power spectral densities are shown. Hz, hertz. (F) c-Fos expression was robustly up-regulated in the dentate granule cells and hilar interneurons of the hippocampus of an Adam22^ΔC5/ΔC5 mouse (P131) at 30 min after the first generalized seizure event. (Scale bars: [Upper] 1 mm; [Lower] 0.5 mm.)

Fig. 3.

Transsynaptic nanoalignment is disordered in the Adam22^ΔC5/ΔC5 hippocampus. (A) Representative 2C-STED images of LGI1 and PSD-95 in the CA1 region of wild-type (^+/+) or Adam22^ΔC5/ΔC5 mice. Asterisks denote two or three adjoining nanodomains. (Scale bar: 500 nm.) (B–D) Quantification of the fluorescence mean intensity (B), size (C), and number (D) of PSD-95 nanodomains. (C and D) Nanodomains, when not completely separated, were quantified en bloc. P values were determined by a paired t test; n = 5 experiments (878 and 759 nanodomains were analyzed) (B) and by Mann–Whitney U tests; n = 5 experiments (C and D). (E) 2C-STED imaging unveils the disordered alignment between RIM2 (green) and PSD-95 (red) in the CA1 region of Adam22^ΔC5/ΔC5 mice. (Right) Intensity profiles of the nearest neighbor pair of RIM2 and PSD-95 were generated (e.g., along white dashed lines in the top images), and individual peak distances on the obtained intensity profiles were measured. (Scale bars: 500 nm [200 nm, magnified].) (F and G) Histograms, cumulative distributions (F, CA1 region), and dot plots (G, CA1 and DG) of the nearest neighbor distances between RIM2 and PSD-95, showing significantly larger distances between pre- and postsynaptic nanodomains in Adam22^ΔC5/ΔC5 (gray) than in wild-type mice (^+/+, black). ***P < 0.001 (Kolmogorov–Smirov test) (F) and Mann–Whitney U test (G; n = 4 experiments for RIM2–PSD-95; n = 3 experiments for RIM2–Homer1). Median distances (nm) are shown (G, red lines). Data with a <50-nm distance (gray area in G) were excluded.

Fig. 4.

Excitatory synaptic transmission decreases in Adam22^ΔC5/ΔC5 mice. (A and B) Simultaneous dual recording of AMPAR-mediated synaptic transmission in CA1 of ADAM22^ΔC5/ΔC5 mice (A). When ADAM22 was overexpressed (green) in ADAM22^ΔC5/ΔC5 neurons, AMPAR EPSC size greatly increased, as compared to the neighbor, nontransfected control ADAM22^ΔC5/ΔC5 neurons (black). The same setup was used to examine NMDAR-mediated synaptic transmission, and similar results were obtained (B). Scatterplots show individual dual recordings (open circles) and mean ± SEM (filled circle). (Insets) Representative EPSC traces. (Scale bars, 50 ms and 25 pA.) Bar graphs showing the normalized mean EPSC ± SEM. ***P = 0.0006, n = 10 pairs (A); ***P = 0.0007, n = 11 pairs (B). (C) Overexpression of ADAM22 does not increase AMPAR EPSCs in CA1 neurons of wild-type mice. n.s., not significant; n = 12 pairs.

Fig. 5.

ADAM22-PDZ binding is required for PSD-95 potentiation of excitatory synapses. (A) Overexpression of PSD-95 greatly increases AMPAR-mediated EPSCs in wild-type neurons. Scatterplot of AMPAR EPSC amplitudes recorded from wild-type control and PSD-95–overexpressing neurons. (B) Overexpression of PSD-95 does not increase AMPAR-mediated EPSCs in Adam22^ΔC5/ΔC5 neurons. Scatterplot of AMPAR EPSC amplitudes recorded from Adam22^ΔC5/ΔC5 control and Adam22^ΔC5/ΔC5 neurons overexpressing PSD-95. Representative traces of dual recordings are shown as Insets: bars, 50 ms and 20 pA (A and B). Bar graphs showing the normalized mean AMPAR EPSC ± SEM (A and B, Right). ***P = 0.00009, n = 8 (A); P = 0.59, n = 11 (B). (C) No difference in hippocampal LTP is observed between wild-type (black) and Adam22^ΔC5/ΔC5 (green) slices. P = 0.051. n.s., not significant.

Fig. 6.

Roles of ADAM22–PSD-95 binding in PSD-95 nano-condensation and human epilepsy. (A) Structure of the complex of ADAM22 PDZ ligand and PSD-95 PDZ3. The C-terminal PDZ ligand of ADAM22 (red) binds to the groove of PSD-95 PDZ3 (blue). (B and C) Close-up view of the interface between the ADAM22 C-terminal tail and PSD-95 PDZ3. The yellow represents choline, binding to the side chain of W902 of ADAM22. Hydrogen bonds are shown as dotted lines. Electron density map of the interface (2F_o-F_c map contoured at 1.0 σ level) is shown (C). (D and E) ADAM22 and PSD-95 interdependently make cell-surface clusters (D). (Scale bars: 20 μm [2 μm, magnified].) The coclustering activity of ADAM22 selectively requires the PDZ3-SH3-GK domain of PSD-95, whereas SynGAP1 acts on either the PDZ1-2 or PDZ3-SH3-GK domain (E). (F) 2C-STED imaging of extracellularly labeled ADAM22 (red) and PSD-95-GFP (green) in COS7 cells. (Inset in the Left) The image of ADAM22ΔC5-transfected cell at the same magnification as for the wild-type ADAM22-transfected cell. Dashed square area in the Left is magnified (Right). (Scale bars: 2 μm [200 nm, Right].) (G) Human epilepsy mutation of ADAM22 p.R896* causes the C-terminal 11-amino-acid deletion (ADAM22ΔC11). (H and I) ADAM22ΔC11 as well as ADAM22ΔC5 bind to LGI1 on the cell surface (H), but neither interacted with PSD-95 (I) in COS-7 cells when LGI1-FLAG or PSD-95-FLAG cotransfected with ADAM22 variants. Cell-surface bound LGI1-FLAG (red) via ADAM22 (green) was live-labeled by anti-FLAG antibody (H). Blue, nuclei labeled by Hoechst 33342. (Scale bar: 20 μm.) Arrow and arrowhead indicate the positions of immature and mature forms of ADAM22, respectively (I, Upper). The data shown are representative of two independent experiments.