Neuroligin-1 controls synaptic abundance of NMDA-type glutamate receptors through extracellular coupling

Abstract

Despite the pivotal functions of the NMDA receptor (NMDAR) for neural circuit development and synaptic plasticity, the molecular mechanisms underlying the dynamics of NMDAR trafficking are poorly understood. The cell adhesion molecule neuroligin-1 (NL1) modifies NMDAR-dependent synaptic transmission and synaptic plasticity, but it is unclear whether NL1 controls synaptic accumulation or function of the receptors. Here, we provide evidence that NL1 regulates the abundance of NMDARs at postsynaptic sites. This function relies on extracellular, NL1 isoform-specific sequences that facilitate biochemical interactions between NL1 and the NMDAR GluN1 subunit. Our work uncovers NL1 isoform-specific cis-interactions with ionotropic glutamate receptors as a key mechanism for controlling synaptic properties.

Fig. 1.

NL1-specific recruitment of NMDARs. (A) Hippocampal neurons transfected with eGFP or NL expression vectors encoding HA-tagged NL1, NL2, or NL3. Dendritic segments of cells triple immunostained with anti-eGFP or anti-HA, anti-GluN1, and anti-vGluT1 antibodies. (Scale bar: 5 μm.) (B) Quantification of cells expressing eGFP, NL1, NL2, or NL3 (n ≥ 10 for each group, P values for comparisons to eGFP controls). (C) EPSCs were elicited at WT hippocampal neurons nontransfected (−) or transfected with NL1. Sample traces of EPSCs evoked by local extracellular stimulation, with the measurement of NMDAR (Upper, at +40 mV, small red circles) and AMPAR (Lower, at −70 mV, large red circles) EPSCs. A summary histogram for mean NMDAR/AMPAR ratios (n ≥ 18 cells for each group, *P < 0.05). (D) Hippocampal neurons were cotransfected with an expression construct for eGFP, HA-tagged NL1 (HA NL1), or a C-terminal deletion mutant of NL1 (HA NL1∆C) and negative control (shRNA DsRed) or shRNA to PSD95 (shRNA PSD95). (Scale bar: 5 μm.) (E) Quantification in neurons cotransfected with eGFP, NL1, or NL1∆C and either shRNA DsRed or shRNA PSD95 (n ≥ 10 for each group, P values for comparisons to eGFP controls, **P < 0.01, ***P < 0.001).

Fig. 2.

NL1 cholinesterase domains are determinants of selectivity for NMDARs. (A) Dendritic segments of hippocampal neurons transfected with eGFP or HA-tagged NL1, NL2, NL1-2, or NL2-1, triple immunostained with anti-eGFP or anti-HA, anti-GluN1, and anti-vGluT1 antibodies. (Scale bar: 5 µm.) (B) Schematic depiction of NL1-NL2 chimeric proteins containing NL1 (black) and NL2 (gray) sequences and quantification of GluN1 puncta density (n ≥ 10 for each group, P values for comparisons to eGFP controls). (C) NL1, NL2, or NL3 was immunoprecipitated from total brain lysates probed with GluN1 or GluA2/3 antibodies. Preimmune serum (Pre) and protein A beads without antibody (Bds) were used as negative controls. The panels shown for input (10%) and immunoprecipitate are derived from the same blotting membrane and identical exposure times. (D) HEK293 cells were cotransfected with HA-tagged NLs (NL1 or NL2) and NMDAR (GluN1/GluN2A/GluN2B) or AMPAR (GluA1/GluA2) expression vectors. Protein complexes immunoprecipitated (IP) with anti-HA antibodies probed with anti-GluN1 or anti-GluA2 antibodies. Comparable expression of transfected proteins confirmed by analysis of the cell lysates. (E) HEK293 cells cotransfected with HA-tagged NL variants and NMDAR (GluN1/GluN2A/GluN2B) expression vectors. Protein complexes immunoprecipitated (IP) with anti-HA antibodies probed in Western blotting with anti-GluN1 or anti-HA antibodies. (F) COS7 cells cotransfected with cDNAs encoding GluN subunits together with NL1. Images show PLA signal from nonpermeabilized cells and subsequent immunofluorescence detection of the individual proteins performed after completion of the PLA reaction and cell permeabilization. (Scale bar: 50 μm.) (G) Quantification of average intensity of PLA signals (mean and SEM, n > 40 cells per condition per experiment, ***P < 0.001).

Fig. 3.

Synaptic recruitment of endogenous NMDARs by NL1. (A) Hippocampal slices were perfused in 0.25 mM Mg²⁺ artificial cerebrospinal fluid (ACSF) containing 5 µM NBQX, and the perfusion rate was increased to 5 mL/min. Shaded bar indicates the period of MK-801 inclusion. (Insets) Representative traces of NMDAR EPSCs from CA1 pyramidal neurons of WT mice (−), noninfected (−), and infected neurons of NL1 KO mice. The numbers of traces denote the time points when individual recordings were made. (B) MK-801 wash-out experiment after viral delivery of chimeric NLs into hippocampi of NL1 KO mice. Shaded bar indicates the period of MK-801 inclusion. (C) Summary histogram of % EPSC recovery for each group (n ≥ 8 for each group, see SI Materials and Methods for calculation of % EPSC recovery, **P < 0.01).

Fig. 4.

Localization of NLs to the neurotransmitter receptor–containing PSD. (A–E) Synaptic localization of NLs detected with a panNL antibody that recognizes all NL isoforms (5-nm gold particles, one example in A marked with a gray arrowhead) colabeled with anti-PSD95 antibody (10-nm gold particles, one example in A marked with a black arrowhead; A–C) or GluN1 antibody (5-nm gold particles, examples marked with black arrowheads, D and E) in Stratum radiatum of mouse hippocampal CA1 area. Synaptic transmembrane protein complexes are visible as intramembrane particles (IMPs). P-face of a spine synapse with cross-fractured presynaptic terminal (A), a perforated glutamatergic synapse (B), and a PSD95-negative presumptive GABAergic synapse (C). D and E show E- and P-faces of single synapses, labeled with anti-GluN1 and anti-panNL antibodies, respectively (in both cases, the antigen is detected with 5-nm gold particles). The proximity of synaptic territory occupied by GluN1 and NL1 is illustrated by overlaying the position of gold particles with a delineation of a postsynaptic area (solid line) based on an IMP cluster on the E-face (Right). [Scale bars: 200 (A–C) and 100 nm (D and E).] (F) Quantitative assessment of anti-GluN1 labeling in CA1 reveals a significant reduction in GluN1 density at postsynaptic sites. In the same preparation, no significant change in the density of IMPs was detected (n = 3 pairs of heterozygous control and NL1 KO animals, total of 100 synapses analyzed, mean ± SEM, *P < 0.05).

Fig. 5.

ChE domain–dependent NL1-NMDAR coupling for normal synaptic properties. (A–C) Scatter plots show individual amplitudes of AMPAR (holding at −70 mV, Left) and NMDAR-EPSCs (holding at +40 mV, Center) for each recording pair and filled symbols indicate the means. Insets show representative traces of EPSCs from noninfected (black lines) or infected (green lines) NL1 KO neurons. NMDAR/AMPAR ratios are presented for noninfected (−) or NL1 KO neurons infected with lentiviruses encoding NL1, NL1-2, or NL2-1, respectively (Right). n.s., nonsignificant; ***P < 0.001. (D–F) Mean EPSC amplitudes before and after a pairing protocol (arrows denote the time point for the pairing) are shown after the normalization to prepairing levels for noninfected or NL1 KO neurons expressing NL1, NL1-2, or NL2-1 (n ≥ 8 pairs for each group).