PSD-95 binding dynamically regulates NLGN1 trafficking and function

Abstract

PSD-95 is a scaffolding protein that regulates the synaptic localization of many receptors, channels, and signaling proteins. The NLGN gene family encodes single-pass transmembrane postsynaptic cell adhesion molecules that are important for synapse assembly and function. At excitatory synapses, NLGN1 mediates transsynaptic binding with neurexin, a presynaptic cell adhesion molecule, and also binds to PSD-95, although the relevance of the PSD-95 interaction is not clear. We now show that disruption of the NLGN1 and PSD-95 interaction decreases surface expression of NLGN1 in cultured neurons. Furthermore, PKA phosphorylates NLGN1 on S839, near the PDZ ligand, and dynamically regulates PSD-95 binding. A phosphomimetic mutation of NLGN1 S839 significantly reduced PSD-95 binding. Impaired NLGN1/PSD-95 binding diminished synaptic NLGN1 expression and NLGN1-mediated synaptic enhancement. Our results establish a phosphorylation-dependent molecular mechanism that regulates NLGN1 and PSD-95 binding and provides insights into excitatory synaptic development and function.

Keywords: NLGN1; PKA; PSD-95; phosphorylation.

Fig. 1.

NLGN1 and PSD-95 binding regulates NLGN1 surface levels. (A) Cultured cortical neurons were transduced with shPSD-95 lentivirus at DIV 12 to 15. The surface biotinylation assays were performed 7 d after viral transduction. Surface proteins were analyzed by immunoblotting. Graph indicates mean ± SEM (n = 3). ***P = 0.0002 using one-way ANOVA Bonferroni’s multiple comparison test. n.s., not significant. (B) PSD-95 knockdown plasmid was cotransfected with NLGN miRs and HA-NLGN1 WT in cultured hippocampal neurons (n = 14 for CTL and n = 17 for PSD-95 knockdown). (Scale bar, 5 µm.) (C) HA-NLGN1 or HA-NLGN1 ∆PDZ were cotransfected with NLGN miRs in cultured hippocampal neurons (n = 13 for WT and n = 15 for ∆PDZ). (Scale bar, 25 µm.) (B and C) Surface labeling assays were performed as described in Materials and Methods. Regions from three dendrites per each neuron were collected for analysis. Graph indicates mean ± SEM **P = 0.0015 and ****P = 0.000032 using unpaired t test. (D and E) Cultured cortical neurons were transduced with shPSD-95 lentivirus or treated with 100 μM 2-BP for 20 h. The Triton-soluble extrasynaptic region and Triton-insoluble synaptic region were fractionated from the neurons. The fractions were analyzed by immunoblotting. The NLGN1/tubulin ratio was analyzed. The graph indicates mean ± SEM (n = 3). *P = 0.028 and ***P = 0.0004 using unpaired t test.

Fig. 2.

PKA phosphorylates NLGN1 on S839. (A) Sequence alignment for the C-tails of the NLGN isoforms. The pS839 antibody epitope is underlined in red. Schematic figures denote motifs within HA-NLGN1 and GST-NLGN1. (B) MS/MS spectrum of the phosphorylated NLGN1 peptide found in GST-NLGN1 fusion proteins incubated with ATP and purified PKA. Samples were digested with trypsin and analyzed using the LC/MS/MS method. (C) Autoradiography analysis of GST fusion proteins that were incubated with purified PKA and [γ-³²P] ATP. Quantitative graph represents mean ± SEM (n = 3). **P = 0.0016 using an unpaired t test. (D) Immunoblot analysis of GST-NLGN1 WT and S839A fusion proteins that were phosphorylated in vitro with purified PKA and probed with pS839 antibody. Arrows in C and D denote GST-NLGN1 position in blots. (E) Endogenous phospho-S839 NLGN1 was enriched from WT mouse brain P2 lysates by immunoprecipitation using pS839 antibody with NLGN1 KO mouse brain as control. The immunoprecipitates were analyzed by immunoblotting; β-tubulin was used as a protein loading control for the input. (F) (Left) Immunoblot analysis of HA-NLGN1 WT and S839A transfected or cotransfected with constitutively active PKA in HEK293 cells. Arrows in E and F denote the NLGN1-specific band. (G) (Top) Quantitative graph represents mean ± SEM (n = 3). **P = 0.0017 using an unpaired t test. (F) (Right) PP assays were performed on the HA antibody immunoprecipitates from HEK293 cells expressing HA-NLGN1 WT and constitutively active PKA. The phosphatase assay samples were analyzed by immunoblotting. (G) (Bottom) Quantitative graph represents mean ± SEM (n = 3). ***P = 0.0004 using unpaired t test. (H) The primary visual cortex was macrodissected from a mouse dark-rearing paradigm as described in Materials and Methods. Endogenous phospho-S839 NLGN1 was enriched from P2 lysates by immunoprecipitation using pS839 antibody. Graph indicates mean ± SEM (n = 6). The statistical significance between the mean of LR and the mean of each condition was calculated using one-way ANOVA with Tukey’s multiple comparison test. **P = 0.0098 (LR vs. DR+LE). n.s., not significant.

Fig. 3.

NLGN1 S839 phosphorylation attenuates PSD-95 binding. (A and B) GST-NLGN1 fusion proteins were incubated with adult rat brain P2 fraction lysates for GST pulldown experiments. PSD-95/GST-NLGN1 fusion proteins were analyzed. Graph indicates mean ± SEM (n = 3 to 4). ***P = 0.0002 and ****P = 0.0001. (C and D) HA-NLGN1 (WT, S839A, S839E, or ∆PDZ) and PSD-95-myc were transfected in HEK293 cells as indicated in the figures. PSD-95-myc in the immunoprecipitates with HA antibody were analyzed by immunoblotting. The ratio of coimmunoprecipitated PSD-95-myc/immunoprecipitated NLGN1 was analyzed. Graph indicates mean ± SEM (n = 3 to 5). **P = 0.001824 for C. **P = 0.003735 and ****P = 0.0001 for D. (E) HA-NLGN1 (WT, S839A, or S839E) and PSD-95-myc were transfected in cultured cortical neurons. Graph indicates mean ± SEM (n = 3). **P = 0.005729. The statistical significance between the mean of WT and the mean of each condition was calculated using one-way ANOVA with Dunnett’s multiple comparison test.

Fig. 4.

NLGN1 S839 phosphorylation state regulates NLGN1 surface levels. (A) Cultured cortical neurons were treated with forskolin and surface biotinylation assays were performed as described in Materials and Methods. Proteins were resolved by SDS/PAGE and analyzed by immunoblotting. Surface NLGN1/total NLGN1 was analyzed. Graph indicates mean ± SEM (n = 4). **P = 0.0031 using unpaired t test. (B−D) NLGN miRs (green) and HA-NLGN1 (WT or S839E) were coexpressed in cultured hippocampal neurons. Enlarged images of the boxed regions are shown below each panel. Regions from three dendrites per each neuron were collected for analysis. HA-NLGN1 S839E was normalized to HANLGN1 WT. (Scale bar, 25 μm.) Graph indicates mean ± SEM ****P < 0.0001 using unpaired t test. (B) Surface HA-NLGN1 was labeled with anti-HA and Alexa 555-conjugated secondary antibody (red). After fixation and permeabilization, intracellular HA-NLGN1 was stained with anti-HA and Alexa 647-conjugated secondary antibody (white, n = 16 for WT and n = 14 for S839E). (C) For antibody-feeding assays, the neurons were incubated with HA antibody in the media at 37 °C for 30 min to feed antibody. Surface HA-NLGN1 (red) and endocytosed HA-NLGN1 (white) were visualized as above (n = 23 for WT and S839E). (D) For recycling assays, the transfected neurons were incubated with anti-HA antibody at RT and allowed internalization at 37 °C for 30 min. Excess amounts of anti-IgG were added to remove surface bound anti-HA antibody. The neurons were transferred back to 37 °C for 60 min for recycling. Recycled HA-NLGN1 (red) and internal HA-NLGN1 (white) were visualized as above (n = 18 for WT and S839E).

Fig. 5.

NLGN1 S839 phosphorylation reduces synaptic enhancement. (A) Cultured hippocampal neurons were transfected with NLGN miRs and HA-NLGN1 (WT or S839E) at DIV 5. After fixation and permeabilization, HA-NLGN1 was stained with anti-HA and Alexa 555-conjugated secondary antibody (red). Regions from secondary dendrites per each neuron were collected for spine number counting. Arrow heads indicate spines. (Scale bar, 5 µm.) Graph indicates mean ± SEM (n = 18). The statistical significance between every condition was calculated using one-way ANOVA with Tukey’s multiple comparison test. *P = 0.024866, and ****P < 0.000001. (B) Regions from secondary dendrites for each neuron were collected to categorize spine morphology using Neurolucida software. Thin (red), mushroom (blue), and stubby (white) spines are indicated. Graph indicates mean ± SEM (n = 6). The statistical significance between every condition was calculated using two-way ANOVA with Tukey’s multiple comparison test. **P = 0.0032 and ***P = 0.0010 for thin spines. *P = 0.0439 and ***P = 0.0006 for mushroom spines. n.s., not significant. (C) NLGN miRs and HA-NLGN1 (WT or S839E) were coexpressed in cultured hippocampal neurons at DIV 12. Endogenous PSD-95 were labeled with anti−PSD-95 and Alexa 555-conjugated secondary antibody (red), and endogenous VGLUT1 were labeled with anti-VGLUT1 and Alexa 647-conjugated secondary antibody (green). (Scale bar, 5 µm.) The selected regions in the merged image were enlarged. (Scale bar, 2.5 µm.) Regions from three dendrites per each neuron were analyzed for Pearson’s coefficient. Graph indicates mean ± SEM (n = 23). ***P = 0.0007 using unpaired t test.

Fig. 6.

NLGN1 S839 phosphorylation reduces synaptic transmission. (A) Representative AMPAR mEPSC traces recorded in cultured hippocampal neurons expressing either NLGN miRs or NLGN miRs and HA-NLGN1 (WT or S839E). (B) Spontaneous AMPAR mEPSC mean frequency and cumulative probability. **P = 0.001525, ***P = 0.000235, and ****P < 0.000001. (C) Spontaneous AMPAR mEPSC mean amplitude and cumulative probability. *P = 0.024275. (B and C) Bar graph indicates mean ± SEM (n = 14 to 15). The statistical significance between every condition was calculated using one-way ANOVA with Tukey’s multiple comparison test. n.s., not significant.

Fig. 7.

Model of PKA regulation of NLGN1. (A) NLGN1 and PSD-95 associate at synaptic regions and form a stable complex. (B) PKA phosphorylates S839 in the cytoplasmic region of NLGN1. (C) S839 phosphorylation disrupts the binding between NLGN1 and PSD-95, (D) which results in reduced surface levels of NLGN1. (E) Consequently, spine number and excitatory synaptic transmission are impaired upon destabilized NLGN1 surface expression.