Liprin-α3 controls vesicle docking and exocytosis at the active zone of hippocampal synapses

Abstract

The presynaptic active zone provides sites for vesicle docking and release at central nervous synapses and is essential for speed and accuracy of synaptic transmission. Liprin-α binds to several active zone proteins, and loss-of-function studies in invertebrates established important roles for Liprin-α in neurodevelopment and active zone assembly. However, Liprin-α localization and functions in vertebrates have remained unclear. We used stimulated emission depletion superresolution microscopy to systematically determine the localization of Liprin-α2 and Liprin-α3, the two predominant Liprin-α proteins in the vertebrate brain, relative to other active-zone proteins. Both proteins were widely distributed in hippocampal nerve terminals, and Liprin-α3, but not Liprin-α2, had a prominent component that colocalized with the active-zone proteins Bassoon, RIM, Munc13, RIM-BP, and ELKS. To assess Liprin-α3 functions, we generated Liprin-α3-KO mice by using CRISPR/Cas9 gene editing. We found reduced synaptic vesicle tethering and docking in hippocampal neurons of Liprin-α3-KO mice, and synaptic vesicle exocytosis was impaired. Liprin-α3 KO also led to mild alterations in active zone structure, accompanied by translocation of Liprin-α2 to active zones. These findings establish important roles for Liprin-α3 in active-zone assembly and function, and suggest that interplay between various Liprin-α proteins controls their active-zone localization.

Keywords: Liprin-α; active zone; active zone assembly; synaptic vesicle exocytosis; vesicle docking.

Fig. 1.

STED microscopy establishes active-zone localization of Liprin-α3 at excitatory hippocampal synapses. (A) Schematic illustration of a synapse. (B) Example images of hippocampal excitatory synapses in side view in confocal and STED microscopy stained for Bassoon^C and PSD-95. The synaptic vesicle cluster (labeled for synapsin) was imaged by confocal microscopy in all experiments. The intensity profile normalized to peak fluorescence is shown for the profile scan (transparent white rectangle) perpendicular through the center of Bassoon. (C) Representative STED images of individual excitatory synapses in side view stained for test proteins, Bassoon, and the vesicle marker vGlut1 (for PSD-95, synapsin antibodies were used due to limitations in antibody compatibility). Fluorescence intensity profiles are shown below. Dashed line at zero indicates the peak localization of Bassoon^N. Overview images and confocal scans are provided in Fig. S1. (D) Axial positions of test proteins relative to Bassoon^N. Bassoon^N, n = 920 synapses/8 independent cultures; Bassoon^C, n = 231/3; RIM1, n = 130/3; ELKS2, n = 141/3; RIM-BP2, n = 84/3; Munc13-1, n = 92/3; Liprin-α2, n = 105/3; Liprin-α3, n = 234/3; PSD-95, n = 79/3. (E) Percentage of test protein clusters associated (yellow) or not associated (red) with the active zone. Bassoon^C, n = 985 synapses/5 images/3 independent cultures; RIM1, n = 795/5/3; ELKS2, n = 845/6/3; RIM-BP2, n = 1,298/6/3; Munc13-1, n = 1,935/6/3; Liprin-α2, n = 1,296/6/3; Liprin-α3, n = 1,425/6/3. Statistical significance determined by two-way ANOVA (localization significant at P ≤ 0.001; test protein not significant, interaction significant at P ≤ 0.001) followed by Holm–Šídák posttests (reported in figure) comparing each distribution vs. that of Bassoon^C. All data are means ± SEM (***P ≤ 0.001).

Fig. 2.

Generation of Liprin-α3–KO mice. (A) CRISPR/Cas9-mediated genome editing in single-cell zygotes. The sgRNA-targeting sequence is shown in bold, the protospacer-adjacent motif (PAM) is red, and the dashed line indicates the deletion. (B) Survival analysis of offspring of heterozygous matings. The dashed line represents a Mendelian distribution (n = 418 animals/54 litters). (C) Liprin-α3 expression in hippocampi analyzed by fluorescent Western blotting (quantification shown in Fig. S3 G and H). The Liprin-α3 antibody 3 cross-reacts with Liprin-α1–α4.

Fig. 3.

Synaptic vesicle exocytosis is impaired upon Liprin-α3 KO. (A and B) Example traces (A) and quantification (B) of mEPSCs. Liprin-α3^+/+, n = 47 cells/4 independent cultures; Liprin-α3^−/−, n = 48/4. (C) Pseudocolored images of peak fluorescence change in sypHy-expressing Liprin-α3^+/+ neurons, Liprin-α3^−/− neurons, and Liprin-α3^−/− neurons rescued with lentiviral expression of Liprin-α3 transduced at DIV 1. Neurons were stimulated with 40 or 200 action potentials and dequenched with NH₄Cl. (D) Quantification of the percentage of sypHy puncta responsive to 40 (Top) or 200 (Bottom) action potentials. (E–G) Quantification of sypHy mean fluorescence changes at active synapses stimulated with 40 action potentials expressed as percentage of the NH₄Cl-responsive vesicle pool (E), and the frequency distribution (F) and peak response (G) of the same data. Liprin-α3^+/+, n = 2,365 NH₄Cl-responsive synapses/1,338 active synapses/11 coverslips/6 independent cultures; Liprin-α3^−/− 2,371/1,093/11/6; Liprin-α3^−/− + Liprin-α3, n = 2,438/1,463/11/6. (H–J) Quantification as in E–G but for neurons stimulated with 200 action potentials. Liprin-α3^+/+, n = 2,353/1,485/11/6; Liprin-α3^−/−, n = 2,346/1,164/11/6; Liprin-α3^−/− + Liprin-α3, n = 2,427/1,598/11/6. All data are means ± SEM [*P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001, Student’s t tests (B; all P > 0.05) or one-way ANOVA (D, G, and J) followed by Holm–Šídák multiple comparisons test comparing each condition vs. Liprin-α3^−/−]. The number of coverslips was used as a basis for statistics in sypHy imaging.

Fig. 4.

Impaired synaptic vesicle docking and tethering after Liprin-α3 KO. (A) Representative EM images of synapses of cultured hippocampal neurons fixed with glutaraldehyde. (B–F) Quantification of vesicles per bouton (B), bouton size (C), PSD length (D), docked vesicles (E), and vesicles tethered within 100 nm (F). Liprin-α3^+/+, n = 150 synapses/3 independent cultures; Liprin-α3^−/−, n = 147/3. (G) Histogram of the vesicle distribution in the first 100 nm of the active zone in 10-nm bins expressed as a percentage of all synaptic vesicles. Numbers of synapses and cultures are as in B–F. (H–N) Same as A–G, but for neurons cryofixed by high-pressure freezing. Liprin-α3^+/+, n = 104/3; Liprin-α3^−/−, n = 121/3. Data are means ± SEM unless stated otherwise (*P ≤ 0.05 and ***P ≤ 0.001, unpaired Student’s t test).

Fig. 5.

STED analysis of Liprin-α3–KO synapses. (A) Representative STED images of side-view synapses and line profiles [as in Fig. 1 but showing arbitrary units (a.u.)]. (B) Axial positions of the test proteins relative to Bassoon: Bassoon^N (^+/+, n = 841/8; ^−/−, n = 817/8); Liprin-α3 (^+/+, n = 234/3); Liprin-α2 (^+/+, n = 105/3; ^−/−, n = 120/3); Bassoon^C (^+/+, n = 55/3; ^−/−, n = 28/3); Munc13-1 (^+/+, n = 92/3; ^−/−, n = 77/3); ELKS2 (^+/+, n = 141/3; ^−/−, n = 196/3); RIM1 (^+/+, n = 130/3; ^−/−, n = 114/3); RIM-BP2 (^+/+, n = 84/3; ^−/−, n = 107/3); and PSD-95 (^+/+, n = 79/3; ^−/−, n = 116/3). Statistical significance determined by two-way ANOVA (genotype significant at P ≤ 0.05; test protein significant at P ≤ 0.001; interaction significant at P ≤ 0.001) followed by Holm–Šídák multiple-comparisons posttests (reported in figure). (C) Peak intensity of line profiles normalized to the average of Liprin-α3^+/+ synapses: Liprin-α3 (^+/+, n = 234/3; ^−/−, n = 175/3); Bassoon^N (^+/+, n = 772/8; ^−/−, n = 805/8); Liprin-α2 (^+/+, n = 105/3; ^−/−, n = 120/3); Bassoon^C (^+/+, n = 231/3; ^−/−, n = 169/3); Munc13-1 (^+/+, n = 92/3; ^−/−, n = 77/3); ELKS2 (^+/+, n = 141/3; ^−/−, n = 175/3); RIM1 (^+/+, n = 130/3; ^−/−, n = 114/3); RIM-BP2 (^+/+, n = 84/3; ^−/−, n = 107/3); and PSD-95 (^+/+, n = 116/3; ^−/−, n = 158/3). Statistical significance determined by two-way ANOVA (genotype not significant; test protein significant at P ≤ 0.001; interaction significant at P ≤ 0.001) followed by Holm–Šídák multiple-comparisons posttests (reported in figure). All data are means ± SEM (*P ≤ 0.05 and ***P ≤ 0.001). The data were acquired simultaneously with identical settings in a blind experiment in Liprin-α3^+/+ control and Liprin-α3^−/− neurons, and the Liprin-α3^+/+ data are the same data that are shown in Fig. 1. A second, independent assessment of active-zone protein localization is shown in Fig. S2. Overview images and additional analyses are in Figs. S8 and S9.