PSD-95 Binding Dynamically Regulates NLGN1 Trafficking and Function

PSD-95 binding dynamically regulates NLGN1 trafficking and function

Jaehoon Jeonga, Saurabh Pandeya, Yan Lia, John D. Badger IIa, Wei Lua, and Katherine W. Rochea,1

aNational Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892

Edited by Solomon H. Snyder, Johns Hopkins University School of Medicine, Baltimore, MD, and approved May 3, 2019 (received for review December 20, 2018) PSD-95 is a scaffolding protein that regulates the synaptic localization necessary for maintaining presynaptic release probability (15). of many receptors, channels, and signaling proteins. The NLGN gene Meanwhile, synaptic targeting of NLGN1 is known to be in- family encodes single-pass transmembrane postsynaptic cell adhesion dependent of PSD-95, as PSD-95 is recruited to the plasma molecules that are important for synapse assembly and function. At membrane after NLGN1 (13, 16, 17). In addition, non-PDZ excitatory synapses, NLGN1 mediates transsynaptic binding with ligand-dependent NLGN1 and NLGN3 functions have been in- neurexin, a presynaptic cell adhesion molecule, and also binds to vestigated (18), suggesting a complicated physiological interplay PSD-95, although the relevance of the PSD-95 interaction is not clear. between NLGNs and PSD-95. We now show that disruption of the NLGN1 and PSD-95 interaction Posttranslational modifications have been reported for several decreases surface expression of NLGN1 in cultured neurons. Further- NLGN isoforms and are postulated to confer distinct properties (11, more, PKA phosphorylates NLGN1 on S839, near the PDZ ligand, and 12). For example, phosphorylation of the cytoplasmic region of dynamically regulates PSD-95 binding. A phosphomimetic mutation NLGN1 has recently emerged as a mechanism for isoform-specific of NLGN1 S839 significantly reduced PSD-95 binding. Impaired function (19, 20). Here we identified a phosphorylation site on NLGN1/PSD-95 binding diminished synaptic NLGN1 expression and NLGN1 near the PDZ ligand. We show that serine 839 (S839) of NLGN1-mediated synaptic enhancement. Our results establish a NLGN1 is phosphorylated by cAMP-dependent protein kinase phosphorylation-dependent molecular mechanism that regulates (PKA) and observed that phosphorylation reduces NLGN1 and NLGN1 and PSD-95 binding and provides insights into excitatory syn- PSD-95 binding. Additionally, a phosphomimetic NLGN1 mutant aptic development and function. shows reduced surface expression and impaired trafficking. To- NEUROSCIENCE gether, these results demonstrate an NLGN1 regulatory mechanism PKA | NLGN1 | phosphorylation | PSD-95 consisting of PKA phosphorylation, which modulates NLGN1 binding to PSD-95, consequently affecting NLGN1 localization and SD-95, a member of membrane-associated guanylate kinases function at excitatory synapses. P(MAGUK) protein family, is a major constituent of glutamatergic excitatory synapses and is specifically enriched at the Results postsynaptic density (PSD) (1). A large number of channels, PSD-95 Regulates NLGN1 Surface Expression. Does PSD-95 regulate receptors, and adhesion molecules bind to the PSD-95/Discs NLGN1 surface expression? To answer this question, we exam- large/ZO-1 (PDZ), Src-homology-3, and guanylate kinase (GK) do- ined NLGN1 surface levels in cultured neurons under PSD-95 mains of PSD-95 (1, 2). As a scaffolding protein at excitatory syn- knockdown conditions. We transduced shPSD-95 lentivirus in apses, PSD-95 has been intensively studied vis a vis the organization cultured cortical neurons and performed surface biotinylation as- of glutamatergic postsynaptic signaling. In fact, synaptic expression says as described previously. In neurons with PSD-95 knockdown, and transmission of N-methyl-D-aspartate receptors (NMDARs) the surface NLGN1 level was significantly decreased (Fig. 1A), and α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptors (AMPARs) are regulated by PSD-95 via direct physical in- Significance teraction or coordination with auxiliary proteins (3–6). Numerous studies have demonstrated critical roles of PSD-95 in the forma- PSD-95 is a key scaffolding protein that is localized to the tion and maintenance of dendritic spines, and recent reports have postsynaptic density of excitatory synapses where it regulates focused on PSD-95 and the molecular mechanisms underlying a wide variety of receptors, channels, and signaling molecules. synapse maturation and plasticity. However, the role of PSD-95 binding to the cell adhesion Neuroligins (NLGNs) are type I membrane proteins and post- molecules, neuroligins, has been less clear. We now report that synaptic cell adhesion molecules. NLGNs were initially reported as PSD-95 specifically regulates NLGN1 among the various neu- endogenous neurexin (NRXN) ligands (7, 8). NLGNs and NRXNs roligin isoforms. In addition, we characterize a dynamic in- form a transsynaptic interaction, which is important for spinogenesis teraction between NLGN1 and PSD-95 that is regulated by the and functional synapse formation (9, 10). Five NLGN isoforms phosphorylation of NLGN1 by PKA. Phosphorylation on S839 of (NLGN1, NLGN2, NLGN3, NLGN4X, and NLGN4Y) are iden- NLGN1 blocks PSD-95 binding, reduces surface and synaptic tified in humans (7, 11). In fact, all identified NLGN isoforms show expression of NLGN1, and inhibits NLGN1-mediated enhance- high similarity in amino acid sequence. In addition, the defined ment of excitatory synaptic transmission. protein binding domains in the cytoplasmic region, including the PDZ ligand, are well conserved in all NLGN isoforms (11, 12). Author contributions: J.J., S.P., W.L., and K.W.R. designed research; J.J., S.P., Y.L., and However, isoform-specific mechanisms regulating synaptic locali- J.D.B. performed research; W.L. contributed new reagents/analytic tools; J.J., S.P., Y.L., zation are not fully understood. and K.W.R. analyzed data; and J.J. and K.W.R. wrote the paper. Because NLGN1 and PSD-95 are both localized to excitatory The authors declare no conflict of interest. synapses, and their direct interaction via third PDZ domain of This article is a PNAS Direct Submission. PSD-95 was identified in an early study (13), PSD-95 and Published under the PNAS license. NLGN1 have been investigated together in many studies for 1To whom correspondence should be addressed. Email: [email protected]. their physiological roles in the glutamatergic signaling pathway. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. For example, PSD-95 can restrict NLGN1 localization to excit- 1073/pnas.1821775116/-/DCSupplemental. atory synapses (14), and PSD-95 and NLGN1 are known to be

www.pnas.org/cgi/doi/10.1073/pnas.1821775116 PNAS Latest Articles | 1of10 Downloaded by guest on October 4, 2021 1% Total lysate Surface A B Surface Intracellular NLGNmiRs 150 PSD-95 KD -+ -+ 100

150 CTL ****

(% CTL) 50

NLGN1 NLGN1 Surface/Intracellular 100 0

PSD-95 KD PSD-95 CTL PSD-95 KD NLGN2 NLGNmiRs + NLGN1 100

NLGN3 C Surface Intracellular NLGNmiRs 150 100 Fig. 1. NLGN1 and PSD-95 binding regulates 100 NLGN1 surface levels. (A) Cultured cortical neurons PSD-95 ** 50 were transduced with shPSD-95 lentivirus at DIV

(% of WT) 50 (kDa) NLGN1 12 to 15. The surface biotinylation assays were per-

actin Surface/Intracellular formed 7 d after viral transduction. Surface proteins (WB) 0 WT PDZ were analyzed by immunoblotting. Graph indicates ± = = CTL NLGNmiRs + NLGN1 mean SEM (n 3). ***P 0.0002 using one-way 150 PSD-95 KD 150 ANOVA Bonferroni’s multiple comparison test. n.s., not significant. (B) PSD-95 knockdown plasmid was 100 n.s. n.s. PDZ 100 cotransfected with NLGN miRs and HA-NLGN1 WT in *** cultured hippocampal neurons (n = 14 for CTL and (% of CTL) 50 (% of WT) 50 Total NLGN1 Total NLGN1 n = 17 for PSD-95 knockdown). (Scale bar, 5 μm.) (C) 0 Δ Surface NLGN/Total NLGN Surface NLGN/Total HA-NLGN1 or HA-NLGN1 PDZ were cotransfected NLGN1 NLGN2 NLGN3 0 WT PDZ with NLGN miRs in cultured hippocampal neurons NLGNmiRs + NLGN1 (n = 13 for WT and n = 15 for ΔPDZ). (Scale bar, 25 μm.) (B and C) Surface labeling assays were per- 150 150 SPM SPM formed as described in Materials and Methods. Re- D E gions from three dendrites per each neuron were Triton sol Triton insol Triton sol Triton insol 100 100 *** collected for analysis. Graph indicates mean ± SEM * PSD-95 KD -+ -+ 2-BP -+ -+ **P = 0.0015 and ****P = 0.000032 using unpaired (% of CTL) (% of CTL) 50 50 150 150 Synaptic NLGN1 Synaptic NLGN1 t test. (D and E) Cultured cortical neurons were NLGN1 0 NLGN1 0 transduced with shPSD-95 lentivirus or treated with CTL 2-BP CTL PSD-95 KD 100 μM 2-BP for 20 h. The Triton-soluble extra- 150 100 150 100 synaptic region and Triton-insoluble synaptic region PSD-95 100 PSD-95 100 were fractionated from the neurons. The fractions

50 50 were analyzed by immunoblotting. The NLGN1/tu- (% of CTL) (kDa) (% of CTL) 50 (kDa) 50 bulin ratio was analyzed. The graph indicates mean ± tubulin tubulin Extrasynaptic NLGN1 (WB) (WB) Extra synaptic NLGN1 SEM (n = 3). *P = 0.028 and ***P = 0.0004 using 0 0 CTL PSD-95 KD CTL 2-BP unpaired t test.

whereas other NLGN isoforms were not significantly affected. the extrasynaptic fraction were comparable. These results indicate These data indicate PSD-95 is required for efficient NLGN1 synaptic NLGN1 is particularly sensitive to PSD-95 modulation. surface expression and show specificity for NLGN1 among the NLGN isoforms. PKA Phosphorylates NLGN1 on S839. NLGNs are regulated by various We also used immunofluorescence confocal microscopy to evaluate intracellular signaling molecules on their cytoplasmic tails. In NLGN1 surface expression upon PSD-95 knockdown. We cotrans- particular, PKAs have been shown to target various NLGNs. PKA fected the PSD-95 knockdown plasmid along with HA-NLGN1 WT is known to directly phosphorylate many synaptic proteins, in- – and NLGN microRNAs (miRs) in cultured hippocampal neurons. As cluding receptors, channels, and scaffolding proteins (21 24). To with our previous experiments (19), we performed these analyses on a determine whether PKA is able to phosphorylate NLGNs, we incubated GST-NLGN C-tail fusion proteins with purified PKA and triple NLGN knockdown condition (NLGNmiRs) to prevent di- ′ merization of our HA-NLGN1 with endogenous WT NLGNs (18, adenosine 5 -triphosphate (ATP) in vitro. We analyzed the samples using liquid chromatography coupled to tandem mass spec- 19). Consistently, PSD-95 knockdown also reduced surface HA- trometry (LC/MS/MS) and found NLGN1 was phosphorylated by NLGN1 WT levels (Fig. 1B). These immunocytochemistry data in- PKA on S839 (Fig. 2B), which is located adjacent to the PDZ li- dicate PSD-95 affects HA-NLGN1 WT surface expression. We next gand (Fig. 2A). When S839 is mutated to alanine, a phosphodeficient examined the effects of deleting the PDZ ligand. We transfected mutation, PKA phosphorylation assessed by incorporation of radio- cultured hippocampal neurons with NLGN miRs and HA-NLGN1 Δ Δ active ATP was markedly reduced in an in vitro kinase assay (Fig. WT or HA-NLGN1 PDZ. We observed that HA-NLGN1 PDZ is 2C), confirming that this residue is a PKA phosphorylation site. GST- able to go to the surface, but to a significantly lesser extent than HA- GluA1 was used as a positive control for PKA phosphorylation. NLGN1 WT (Fig. 1C). To characterize the phosphorylation of NLGN1 S839 further, To better understand how PSD-95 regulates NLGN1, we we generated a phosphorylation state-specific antibody, pS839 transduced cultured cortical neurons with shPSD-95 lentivirus to antibody, targeted against the NLGN1 C-terminal residues 833 knockdown PSD-95 or treated with 2-BP (2-bromopalmitate; pal- to 843 (Fig. 2A). We verified the specificity of pS839 antibody by mitoylation inhibitor) to inhibit palmitoylation, which is necessary incubating GST-NLGN1 and GST-NLGN1 S839A with PKA for synaptic localization of PSD-95. As NLGN1 mostly exerts its enzyme and ATP in vitro. Samples were resolved by sodium dodecyl function at postsynaptic sites, the extrasynaptic regions (Triton X- sulfate polyacrylamide gel electrophoresis (SDS/PAGE) and ana- 100−soluble) and synaptic regions (Triton X-100−insoluble) were lyzed by immunoblotting with pS839 antibody. We observed sig- fractionated from the neurons. We observed reduced NLGN1 levels nificantly diminished immunoreactivity for GST-NLGN1 S839A in the synaptic fraction with PSD-95 knockdown or 2-BP treatment (Fig. 2D). To verify the specificity of pS839 antibody, crude syn- compared with control (Fig. 1 D and E), whereas NLGN1 levels in aptosomal fractions (P2 pellets) were prepared from WT and

2of10 | www.pnas.org/cgi/doi/10.1073/pnas.1821775116 Jeong et al. Downloaded by guest on October 4, 2021 Fig. 2. PKA phosphorylates NLGN1 on S839. (A)Se- A PDZ ligand B NLGN1 TL PHPHPHPHSHSTTRV 843 100 quence alignment for the C-tails of the NLGN isoforms. 821TGGQNNTLPHPHPHPHSHPSTTRV843 NLGN2 A TSHNNTL P H PHSTTRV 835 The pS839 antibody epitope is underlined in red. Sche- NLGN3 TG-- - - - LP HSHS TTRV 828 80 matic figures denote motifs within HA-NLGN1 and GST- NLGN4X TN-- - - - LPHGHSTTRV 816 60 NLGN1. (B) MS/MS spectrum of the phosphorylated NLGN4Y TN-- - - - LPHGHSTTRV 816 NLGN1 peptide found in GST-NLGN1 fusion proteins 40 PDZ ligand incubated with ATP and purified PKA. Samples were HA

Relative Abundance 20 HA-NLGN1 TM S-TTRV 120 kDa digested with trypsin and analyzed using the LC/MS/MS method. (C) Autoradiography analysis of GST fusion 0 GST-NLGN1 GST S-TTRV 42 kDa 400 800 1200 1600 2000 proteins that were incubated with purified PKA and m/z [γ-32P] ATP. Quantitative graph represents mean ± SEM Extracellular Intracellular (n = 3). **P = 0.0016 using an unpaired t test. (D)Im- 1.5% IP: Input pS839 munoblot analysis of GST-NLGN1 WT and S839A fusion GST- GST-NLGN1 C D GST- NLGN1 E proteins that were phosphorylated in vitro with purified NLGN1 S839A T T O W KO W K PKA and probed with pS839 antibody. Arrows in C and PKA kinase GluA1 1 1 - PKA kinase N N reaction 839A G GN1 GST WT S 150 reaction L L D denote GST-NLGN1 position in blots. (E) Endogenous GST - + - + NLGN1N NLG N 50 50 phospho-S839 NLGN1 was enriched from WT mouse 150 37 100 brain P2 lysates by immunoprecipitation using pS839 37 25 100 antibody with NLGN1 KO mouse brain as control. The

(% of WT) 50 ** pS839 Autoradiograph P] Incorporation NLGN1 immunoprecipitates were analyzed by immunoblotting; 32 [ 50 50 β-tubulin was used as a protein loading control for the 0 50 37 WT S839A 37 (kDa) input. (F)(Left) Immunoblot analysis of HA-NLGN1 WT 25 (kDa) (kDa) GST tubulin and S839A transfected or cotransfected with constitu- CBB (WB) (WB) tively active PKA in HEK293 cells. Arrows in E and F

N1 DR DR+LE denote the NLGN1-specific band. (G)(Top) Quantitative LG myc NLGN1 -N - F -NLGN1 - A G H graph represents mean ± SEM (n = 3). **P = 0.0017 us- H HA HA S839A 150 + PKA CTL CTL ing an unpaired t test. (F)(Right) PP assays were per- PKA-myc Phosphatase 200 - - + - + - - + ** IP: pS839 formed on the HA antibody immunoprecipitates from reaction NLGN1 150 150 150 HEK293 cells expressing HA-NLGN1 WT and consti- 150 tutively active PKA. The phosphatase assay samples pS839 pS839 100 were analyzed by immunoblotting. (G)(Bottom) NEUROSCIENCE (% of WT) IP: HA 150 IP: HA 150 50 NLGN1 Quantitative graph represents mean ± SEM (n = 3).

1.5% Input 50 pNLGN1/Total NLGN1 pNLGN1/Total = HA HA 0 (kDa) ***P 0.0004 using unpaired t test. (H) The primary CTL PKA 150 150 visual cortex was macrodissected from a mouse dark- actin (WB) rearing paradigm as described in Materials and 150 150 HA HA Methods. Endogenous phospho-S839 NLGN1 was 37 37 n.s. 100 100 LR enriched from P2 lysates by immunoprecipitation us- ** ± = 1.5% Input PKA 1.5% Input PKA ing pS839 antibody. Graph indicates mean SEM (n (% of LR) 50 50 (% of CTL) 50 *** 50 6). The statistical significance between the mean of LR (kDa) (kDa) and the mean of each condition was calculated using actin NLGN1 pNLGN1/Total 0 0 actin NLGN1 pS839 NLGN1/Total ’ (WB) (WB) CTL PP DR DR+LE one-way ANOVA with Tukey s multiple comparison test. **P = 0.0098 (LR vs. DR+LE). n.s., not significant.

NLGN1 KO mouse brains, and phosphorylated NLGN1 was To investigate the dynamic regulation of NLGN1 S839 phos- immunoprecipitated using the pS839 antibody. The immunopre- phorylation in various conditions, we assessed NLGN1 S839 cipitates were analyzed by immunoblotting with NLGN1 antibody, phosphorylation upon physiological stimulation and in different and a band corresponding to the predicted molecular weight of developmental stages. We used a dark rearing paradigm to ac- endogenous NLGN1 was enriched only in the immunoprecipitates tivate neurons in vivo as described in Materials and Methods. The from WT mouse brain lysates, whereas a nonspecific uncharac- primary visual cortex was macrodissected and the P2 pellet terized band was observed for NLGN1 KO mouse brain lysates (crude synaptosomal fraction) was prepared for analysis as de- (Fig. 2E). All these data show the specificity of the pS839 antibody scribed previously. Intriguingly, neuronal activation in vivo re- for NLGN1 S839 phosphorylation. duced S839 phosphorylation in vivo (Fig. 2H). Furthermore, we also To determine whether full-length NLGN1 can be phosphory- observed a decrease in NLGN1 S839 phosphorylation upon KCl or lated by PKA, we cotransfected HA-NLGN1 WT or HA-NLGN1 glutamate treatment on cultured cortical neurons (SI Appendix,Fig. ’ S839A and the constitutively active PKA catalytic subunit (PKA S3 A and B), although we didn t observe robust changes of the T198E) in HEK293 cells. HA-NLGN1 WT and HA-NLGN1 phosphorylation in the mouse brain tissues from the different de- S839A were immunoprecipitated, and the immunoprecipitates velopmental stages (SI Appendix,Fig.S3C). All these results indicate were analyzed by immunoblotting with pS839 antibody (Fig. 2 F, that NLGN1 S839 phosphorylation can be dynamically regulated by various forms of neuronal activity. Left and Fig. 2 G, Top). The immunoblots showed increased immunoreactivity for NLGN1 WT upon PKA cotransfection, which NLGN1 S839 Phosphorylation Attenuates PSD-95 Binding. Multiple was not the case for the NLGN1 S839A mutant. Additionally, we papers have shown that PDZ interactions are regulated by phos- cotransfected HA-NLGN1 WT and the constitutively active PKA phorylation near the PDZ ligand in several proteins such as star- catalytic subunit in HEK293 cells. HA-NLGN1 were immuno- gazin, β1-adrenergic receptors, Kir5.1 channels, and the NR2 precipitated, and the immunoprecipitates were incubated with or subunit of NMDARs (24–27). Because S839 is close to the NLGN1 without lambda protein phosphatase (PP). The immunoprecipi- PDZ ligand, we tested whether S839 phosphorylation affects PSD- tates were analyzed by immunoblotting with pS839 antibody, and 95 binding with NLGN1. the immunoblots showed eliminated reactivity of pS839 antibody GST-NLGN1 WT, S839A, S839E, and ΔPDZ C-terminal fu- upon the phosphatase treatment (Fig. 2 F, Right and Fig. 2 G, sion proteins were incubated with rat brain P2 fraction lysates. The Bottom). These results indicate that PKA phosphorylates NLGN1 isolated beads were resolved by SDS/PAGE and analyzed by im- S839 in situ. munoblotting. The amount of GST fusion proteins was visualized

Jeong et al. PNAS Latest Articles | 3of10 Downloaded by guest on October 4, 2021 A GST-NLGN1 B GST-NLGN1

PDZ 0.1% InputBeadsGST WT S839AS839E 0.1% InputBeads GST WT S839E 150 150 100 100 100 100 (WB) PSD-95 (WB) PSD-95 50 50 (% of WT) (% of WT) 50 50 *** **** 37 37 **** PSD-95/GST-NLGN1 PSD-95/GST-NLGN1 25 0 25 0 WT S839A S839E WT S839E PDZ (kDa) (kDa) CBB CBB

HEK 293 cells HEK 293 cells cultured cortical neurons C D E Fig. 3. NLGN1 S839 phosphorylation attenuates 2.5% input IP: HA 2.5% input IP: HA 2.5% input IP: HA PSD-95 binding. (A and B) GST-NLGN1 fusion proteins HA-NLGN1 HA-NLGN1 HA-NLGN1 HA-NLGN1 HA-NLGN1 HA-NLGN1 were incubated with adult rat brain P2 fraction

A lysates for GST pulldown experiments. PSD-95/GST- L DZ L TL 839E T PDZ P T 839A839E CTL WT S839AS839E C WT S839AS CTL W S839E CT WT S839E CTL WT S839S839EC WT S S NLGN1 fusion proteins were analyzed. Graph indi- PSD-95-myc -++++-++++ PSD-95-myc -+++-+++ PSD-95-myc -+++-+++ cates mean ± SEM (n = 3 to 4). ***P = 0.0002 and = 100 100 100 ****P 0.0001. (C and D) HA-NLGN1 (WT, S839A, S839E, or ΔPDZ) and PSD-95-myc were transfected in myc myc PSD-95 150 150 HEK293 cells as indicated in the figures. PSD-95-myc in 150 the immunoprecipitates with HA antibody were analyzed HA HA HA by immunoblotting. The ratio of coimmunoprecipitated 50 50 50 PSD-95-myc/immunoprecipitated NLGN1 was ana- (kDa) (kDa) (kDa) ± = = actin actin actin lyzed. Graph indicates mean SEM (n 3to5).**P (WB) (WB) (WB) 0.001824 for C.**P = 0.003735 and ****P = 0.0001 200 150 150 for D.(E) HA-NLGN1 (WT, S839A, or S839E) and PSD- 95-myc were transfected in cultured cortical neurons. 150 100 100 ± = = ** Graph indicates mean SEM (n 3). **P 0.005729. 100 ** ** The statistical significance between the mean of WT (% of WT) (% of WT) (% of WT) 50 50 50 and the mean of each condition was calculated using **** one-way ANOVA with Dunnett’s multiple comparison PSD-95-myc/HA-NLGN1 PSD-95-myc/HA-NLGN1 PSD-95-myc/HA-NLGN1 0 0 0 WT S839A S839E WT S839E PDZ WT S839A S839E test.

by Coomassie Brilliant Blue staining in the same gel. As expected, NLGN1 S839 Phosphorylation and PSD-95 Binding Regulate NLGN1 Surface GST-NLGN1 WT showed strong binding with PSD-95 (Fig. 3A), Levels. We showed that PSD-95 knockdown affected NLGN1 surface whereas GST-NLGN1 ΔPDZ did not bind to PSD-95 (Fig. 3B). expression (Fig. 1 A, B,andD). PSD-95 plays important roles in Interestingly, the GST-NLGN1 S839A mutant showed compara- organizing and stabilizing postsynaptic channels, receptors, and ble PSD-95 binding to WT. Meanwhile, GST-NLGN1 S839E, a membrane proteins at synapses, and, in many cases, phosphorylation phosphomimetic mutant, showed significantly reduced binding is a dynamic regulator of PSD-95 effects (1). Our data showing the with PSD-95, comparable to that of GST-NLGN1 ΔPDZ (Fig. 3 A regulation of NLGN1 and PSD-95 binding by S839 phosphorylation led us to investigate the role of S839 phosphorylation in NLGN1 and B). These results indicate S839 is a critical regulatory site for trafficking and synaptic colocalization. NLGN1 and PSD-95 binding. To examine the effect of PKA activity on NLGN1 surface levels, To reexamine the pull-down results using coimmunoprecipi- we treated cultured cortical neurons with forskolin to activate tation in intact cells, we cotransfected full-length HA-NLGN1 Δ PKA and performed surface biotinylation assays. Interestingly, we WT, S839A, S839E, or PDZ and PSD-95-myc in HEK293 cells. observed a decrease in the surface expression of NLGN1 in The cells were lysed, full-length NLGNs were immunoprecipi- neurons treated with forskolin (Fig. 4A), indicating NLGN1 sur- tated and resolved by SDS/PAGE, and coimmunoprecipitated face expression is specifically susceptible to PKA activity. PSD-95-myc was analyzed by immunoblotting. As with the pull- We next examined the surface levels of HA-NLGN1 expressed down results, HA-NLGN1 S839E showed significantly reduced in cultured rat hippocampal neurons with immunofluorescence binding to PSD-95-myc compared with NLGN1 WT and the confocal microscopy. We transfected cultured hippocampal S839A mutant (Fig. 3C), whereas HA-NLGN1 ΔPDZ showed neurons with HA-NLGN1 (WT or S839E) and NLGN miRs for impaired binding with PSD-95-myc (Fig. 3D). We also confirmed NLGNs knockdown at day in vitro (DIV) 17. Surface and in- the specific regulation of S839 phosphorylation on NLGN1 and tracellular HA-NLGN1 were labeled at DIV 22, and the dendritic PSD-95 binding. We cotransfected full-length HA-NLGN1 WT, regions were imaged for analysis. Notably, the phosphomimetic S839A, or S839E and gephyrin-myc in HEK293 cells. The cells NLGN1 S839E mutation greatly reduced surface expression of were lysed, full-length NLGNs were immunoprecipitated and NLGN1 (Fig. 4B), indicating that S839 phosphorylation is impor- resolved by SDS/PAGE, and coimmunoprecipitated gephyrin- tant for the regulation of NLGN1 surface levels. All these data myc was analyzed by immunoblotting. The result showed that support an important role for PKA phosphorylation of NLGN1 S839 in NLGN1 surface expression. NLGN1 S839 phosphorylation does not affect NLGN1 and gephyrin binding (SI Appendix, Fig. S1). NLGN1 S839 Phosphorylation Affects NLGN1 Trafficking. Reduced We also cotransfected full-length HA-NLGN1 WT, S839A, or NLGN1 surface levels could be a consequence of decreased NLGN1 S839E and PSD-95-myc in cultured cortical neurons. Crude mem- protein levels or impaired NLGN1 trafficking. We didn’t observe any branes were prepared and lysed for coimmunoprecipitation. As with evidence of protein degradation of NLGN1 upon PSD-95 modulation the results in HEK293 cells, HA-NLGN1 S839E displayed reduced or PKA activation in the biochemical and immunocytochemistry re- binding to PSD-95-myc in cultured neurons (Fig. 3E). Together, all sults. Therefore, to examine the effect of S839 phosphorylation on of these data show that NLGN1 S839 phosphorylation regulates the NLGN1 trafficking, we performed an antibody-feeding and recycling NLGN1 and PSD-95 interaction. assay.

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A FSK -+ -+ B Surface Intracellular NLGNmiRs 150 150 100

NLGN1 **** 50 (% of WT) 50 NLGN1 Surface/Intracellular pCREB 0 WT S839E 37 (kDa) NLGNmiRs + NLGN1 GAPDH 150 (WB) 150 Fig. 4. NLGN1 S839 phosphorylation state regulates 100 NLGN1 surface levels. (A) Cultured cortical neurons 100 ** were treated with forskolin and surface biotinylation (% of WT) 50 assays were performed as described in Materials and NLGN1 S839E (% of CTL) 50 Total NLGN1 intensity Total Methods. Proteins were resolved by SDS/PAGE and 0 WT S839E analyzed by immunoblotting. Surface NLGN1/total 0

Surface NLGN1/Total NLGN1 Surface NLGN1/Total CTL FSK ± NLGNmiRs + NLGN1 NLGN1 was analyzed. Graph indicates mean SEM (n = 4). **P = 0.0031 using unpaired t test. (B−D) C D NLGN miRs (green) and HA-NLGN1 (WT or S839E) Surface Endocytosed NLGNmiRs Recycled Intracellular NLGNmiRs 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 NLGN1 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 = NEUROSCIENCE 16 for WT and n = 14 for S839E). (C) For antibody- NLGN1 S839E NLGN1 S839E 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 300 150 150 150 **** WT and S839E). (D) For recycling assays, the transfected neurons were incubated with anti-HA antibody at RT 200 100 100 100 **** and allowed internalization at 37 °C for 30 min. Excess amounts of anti-IgG were added to remove surface (% of WT) (% of WT) (% of WT) (% of WT) 100 50 50 50 Total NLGN1 Total Total NLGN1 Total

Recycled NLGN1 bound anti-HA antibody. The neurons were trans- Endocytosed NLGN1 0 0 0 0 ferred back to 37 °C for 60 min for recycling. Recycled WT S839E WT S839E WT S839E WT S839E HA-NLGN1 (red) and internal HA-NLGN1 (white) were NLGNmiRs + NLGN1 NLGNmiRs + NLGN1 NLGNmiRs + NLGN1 NLGNmiRs + NLGN1 visualized as above (n = 18 for WT and S839E).

Cultured hippocampal neurons were transfected with NLGN whether NLGN1-mediated synaptogenesis is regulated by S839 miRs and HA-NLGN1 (WT or S839E), and incubated with anti-HA phosphorylation. antibody in the media at 37 °C for 30 min to allow antibody binding Enhanced spine formation has been shown in young cultured and HA-NLGN1 endocytosis. Surface HA-NLGN1 and endocytosed neurons (DIV 8 to 12) upon NLGN1 overexpression (30, 31). HA-NLGN1 were labeled as described in Materials and Methods.For The cultured hippocampal neurons were transfected with HA- analysis, dendrites of transfected neurons were imaged and analyzed. NLGN1 (WT or S839E) and NLGN miRs at DIV 5 and pre- Although total HA-NLGN1 fluorescence intensities were comparable, pared at DIV 12 to count spine numbers. We observed HA- we observed that fluorescence intensity of endocytosed S839E mutant NLGN1 WT increases spine numbers, whereas HA-NLGN1 was significantly increased compared with WT (Fig. 4C). This result S839E has diminished increases in spine numbers compared suggests S839-phosphorylated NLGN1 undergoes robust endocytosis. with WT (Fig. 5A). Together with the previous biochemical re- We also examined whether S839 phosphorylation affects the sults, these data support the importance of the NLGN1 and NLGN1 recycling process. Briefly, neurons were prepared as PSD-95 interaction for NLGN1 synaptic localization and spino- above and incubated with anti-HA antibody at room temperature genesis during neuronal development. (RT) and allowed HA-NLGN1 internalization at 37 °C. An excess We further categorized spines based on morphology into thin, amount of anti-IgG was added later at RT to block any anti-HA stubby, and mushroom subtypes (Fig. 5B). Both HA-NLGN1 antibody bound to surface HA-NLGN1. The neurons were then WT and HA-NLGN1 S839E restored mushroom-type spines transferred to 37 °C for 60 min to allow HA-NLGN1 recycling. on NLGNmiRs conditions, although the HA-NLGN1 S839E Interestingly, we found that the HA-NLGN1 S839E mutant is rescue was to a lesser extent. Intriguingly, thin spines were fur- deficient in recycling to the surface compared with WT (Fig. 4D). ther recovered by HA-NLGN1 WT. Thin spines are immature This result indicates S839 phosphorylation also diminishes and have structural flexibility for AMPAR insertion and PSD- NLGN1 recycling to the surface. Together, these data demon- 95 recruitment, which gives a greater potential for synaptic strate that S839 affects both NLGN1 endocytosis and recycling. strengthening and plasticity (32, 33). These results indicate that HA-NLGN1 expression induces greater synaptic activity. NLGN1 S839 Phosphorylation Reduces NLGN1-Dependent Synaptic Next, we examined whether mutation of S839 would affect Enhancement. Synaptogenic properties of NLGN1 have been well functional synapse formation, in addition to the spine formation. characterized in cell culture systems (28, 29). We next examined Hippocampal cultured neurons were transfected with NLGN1

Jeong et al. PNAS Latest Articles | 5of10 Downloaded by guest on October 4, 2021 Fig. 5. NLGN1 S839 phosphorylation reduces synaptic NLGN1 * ABCTL NLGN1 S839E enhancement. (A) Cultured hippocampal neurons were NLGNmiRs **** **** transfected with NLGN miRs and HA-NLGN1 (WT or 6 S839E) at DIV 5. After fixation and permeabilization,

HA-NLGN1 was stained with anti-HA and Alexa 555- 4 conjugated secondary antibody (red). Regions from secondary dendrites per each neuron were collected for 2 spine number counting. Arrow heads indicate spines. NLGNmiRs + HA-NLGN1

(Scale bar, 5 μm.) Graph indicates mean ± SEM (n = 18). Spine number / 20µm 0 The statistical significance between every condition was ’ WT calculated using one-way ANOVA with Tukey s multiple S839E NLGNmiRs comparison test. *P = 0.024866, and ****P < 0.000001. NLGNmiRs NLGNmiRs + NLGN1 (B) Regions from secondary dendrites for each neuron were collected to categorize spine morphology using NLGNmiRs + HA-NLGN1 S839E Neurolucida software. Thin (red), mushroom (blue), and

stubby (white) spines are indicated. Graph indicates C -95 mean ± SEM (n = 6). The statistical significance between PSD VGLUT1 Merge NLGN1 NLGN1 S839E every condition was calculated using two-way ANOVA

with Tukey’s multiple comparison test. **P = 0.0032 and VGLUT1 * = = = 0.5 ***P 0.0010 for thin spines. *P 0.0439 and ***P n.s.

m *** ** *** NLGN1 0.0006 for mushroom spines. n.s., not significant. (C) PSD-95 0.4 NLGN miRs and HA-NLGN1 (WT or S839E) were coex- 0.8 0.3 pressed in cultured hippocampal neurons at DIV 12. *** Endogenous PSD-95 were labeled with anti−PSD-95 and 0.6 0.2 Alexa 555-conjugated secondary antibody (red), and 0.4 0.1

endogenous VGLUT1 were labeled with anti-VGLUT1 r=0.632 r=0.574 Spine numbers / and Alexa 647-conjugated secondary antibody (green). 0.2 0.0

NLGN1 S839E Thin Stubby Mushroom

μ (PSD-95 and VGLUT1) (Scale bar, 5 m.) The selected regions in the merged 0.0 image were enlarged. (Scale bar, 2.5 μm.) Regions from WT S839E NLGNmiRs three dendrites per each neuron were analyzed for NLGNmiRs + NLGN1 NLGNmiRs + NLGN1 NLGNmiRs + NLGN1 S839E Pearson’s coefficient. Graph indicates mean ± SEM (n = 23). ***P = 0.0007 using unpaired t test.

(WTorS839E)asdescribedpreviously and stained for endogenous 95 regulation of NLGN1. For example, NLGN1 and PSD-95 can PSD-95 and VGLUT1 to mark postsynaptic and presynaptic sites, mutually enhance the synaptic localization of each other (14, 30, respectively. The dendritic regions of transfected neurons were 34), which indicates a supportive role of PSD-95 in NLGN1 imaged and analyzed for colocalization of PSD-95 and VGLUT1. trafficking and stabilization at synapses. Nevertheless, it has also Interestingly, hippocampal cultured neurons expressing NLGN1 been suggested that the association of NLGN1 and PSD-95 with S839E displayed diminished colocalization of PSD-95 and VGLUT1, the dynein motor complex is involved in the retrograde transport indicating reduced functional synapse number (Fig. 5C). and deprivation of surface NLGN1 (35). Other studies have To investigate further any effect of S839 phosphorylation on reported that the membrane targeting of NLGN1 is independent synaptic transmission, we measured AMPAR miniature excit- of PSD-95 binding altogether (16, 36), and a dendritic sorting atory postsynaptic currents (mEPSC) in hippocampal cultured motif in the cytoplasmic region of NLGN1 may be sufficient to neurons expressing either NLGN miRs alone or NLGN miRs and target NLGN1 to dendrites, but not to synapses (37). These HA-NLGN1 (WT or S839E). Knockdown of endogenous NLGNs previous studies reveal a controversial role for PSD-95 in its induced a reduction in both mEPSC frequency and amplitude ability to regulate NLGN1 trafficking, which may vary according (Fig. 6). HA-NLGN1 fully rescued the reduced mEPSC frequency, to different experimental conditions. In particular, most studies but HA-NLGN1 S839E was not able to rescue the mEPSC fre- have used ectopic protein expression, with fewer studies precisely quency as efficiently as HA-NLGN1 WT (Fig. 6B). Meanwhile, investigating endogenous NLGN trafficking. To directly assess both HA-NLGN1 WT and S839E rescued the reduced mEPSC the effect of PSD-95 on endogenous NLGN trafficking, we amplitude (Fig. 6C). Thus, these findings suggest that NLGN1 evaluated endogenous NLGN1 upon knockdown of PSD-95 in S839 phosphorylation decreases excitatory synaptic transmission. cultured neurons and found a decrease in surface and synaptic All these results indicate S839 phosphorylation has an important NLGN1 (Fig. 1 A and D). Indeed, our results show that PSD- role in the synaptic enhancement by NLGN1. S839 phosphorylation 95 is specifically required for stabilizing synaptic NLGN1 ex- diminishes spine numbers (Fig. 5 A and B) and functional synapse pression, emphasizing a critical role for PSD-95 in the trafficking of number (Fig. 5C), consequently reducing excitatory synaptic trans- NLGN1. Furthermore, we show that NLGN1 and PSD-95 binding mission (Fig. 6). is under the control of PKA-regulated S839 phosphorylation, af- Overall, our findings support a model in which PKA phos- fecting NLGN1 trafficking (Fig. 4). phorylates S839 in the C-tail of NLGN1 and the phosphorylation NLGN1 is a key component of the postsynaptic complex of attenuates PSD-95 binding to NLGN1 (Fig. 7 A–C). As illus- proteins that regulate the assembly of excitatory synapses and trated in the model, S839 phosphorylation further regulates mediate excitatory synaptic transmission. NLGN1 is known to NLGN1 surface expression and trafficking (Fig. 7D). Ultimately, control synaptic incorporation and retention of NMDARs by S839 phosphorylation affects NLGN1-mediated spinogenesis direct binding via its extracellular domain, which is important for and excitatory synaptic transmission (Fig. 7E). NMDAR-mediated transmission in the CA1 region of hippo- campus (38). Impairment of long-term potential (LTP) in hip- Discussion pocampus and amygdala following NLGN1 manipulation has PDZ-mediated interactions are fundamental to the organization been reported (39–41), indicating the requirement of NLGN1 and signaling of the PSD. NLGN1 contains a conserved PDZ for excitatory synaptic plasticity. It also has been shown that ligand; however, its function and regulation have remained NLGN1 and PSD-95 coordinate AMPAR clusters and AMPAR- largely elusive. Many studies have investigated aspects of PSD- mediated mEPSCs for synapse maturation (42). A recent study

6of10 | www.pnas.org/cgi/doi/10.1073/pnas.1821775116 Jeong et al. Downloaded by guest on October 4, 2021 ** ABC*** n.s. n.s. 15 Cultured hippocampal neurons **** **** *** mEPSC 3.0 * Transfection Recording 10

2.0 DIV0 DIV5 DIV13-15 5 Amplitude (pA) 1.0 Fig. 6. NLGN1 S839 phosphorylation reduces syn- Frequency (Hz) CTL aptic transmission. (A) Representative AMPAR mEPSC 0.0 0 traces recorded in cultured hippocampal neurons CTL WT CTL WT NLGNmiRs S839E S839E expressing either NLGN miRs or NLGN miRs and HA- NLGNmiRs NLGNmiRs NLGNmiRs + NLGN1 NLGNmiRs + NLGN1 NLGN1 (WT or S839E). (B) Spontaneous AMPAR

1.0 1.0 mEPSC mean frequency and cumulative probability. NLGNmiRs + NLGN1 **P = 0.001525, ***P = 0.000235, and ****P < 0.000001. (C) Spontaneous AMPAR mEPSC mean = CTL CTL amplitude and cumulative probability. *P NLGNmiRs + NLGN1 S839E 0.5 0.5 NLGNmiRs NLGNmiRs 0.024275. (B and C) Bar graph indicates mean ± SEM NLGNmiRs + NLGN1 WT NLGNmiRs + NLGN1 WT (n = 14 to 15). The statistical significance between Cumulative probability Cumulative probability NLGNmiRs + NLGN1 S839E NLGNmiRs + NLGN1 S839E every condition was calculated using one-way 20 pA 0.0 0.0 ’ 1 s 010203040 0204060 ANOVA with Tukey s multiple comparison test. n.s., Interevent interval (s) Amplitude (pA) not significant.

reported that the NLGN1 and PSD-95 interaction might also be sion. For example, Shipman et al. (18) showed that a single residue important in aligning presynaptic release sites and AMPAR nano- in the critical region of the C-tail of NLGNs is required for the domains required for AMPAR-mediated synaptic transmission (43). enhancement of AMPAR or NMDAR currents and spine density The authors observed that both NLGN1-ΔCter [lacking the last by NLGNs, whereas surface expression of NLGN3 was not affected. 72 amino acids, but harboring the critical residue identified by Our previous work showed that phosphorylation on NLGN4X Shipman et al. (18)] or biomimetic ligands of 15 C-terminal amino T707 affects excitatory synaptic currents and spine number, but

acids of NLGN1 impair synaptic currents. Similarly, Letellier et al. doesn’t affect surface expression of NLGN4X (47). These studies NEUROSCIENCE (44) also showed that NLGN1-Δ5 (lacking the last five amino indicate that NLGN1 intrinsic properties for spinogenesis and acids) displays impaired AMPAR-dependent synaptic transmission NLGN1 surface expression can be regulated independently. More- and reduced spine density. In our current study, we observed a over, Kwon et al. (48) showed that spine density and NMDAR- defect in AMPAR-mediated mEPSC frequency by the NLGN1 mediated currents in a single cell are affected by the difference in S839E mutant compared with WT (Fig. 6 B and C). NLGN1 S839E NLGN1 expression levels relative to neighboring cells, which reveals does colocalize with PSD-95 (Fig.5C)andcanbindtoPSD-95(Fig. the complications in deciphering the functional correlation between 3 B and D), although this is significantly reduced compared with NLGN surface levels and spinogenesis in various NLGNs expression WT NLGN1. All of these studies indicate that NLGN1 expression backgrounds. In this study, we showed that a phosphomimetic mutant and its coordination with PSD-95 at synapses are important for of the PKA site, NLGN1 S839E, displays reduced surface expression, glutamate receptor signaling and synaptic plasticity. reduced spine number, and impaired mEPSC currents (Figs. 4–6). We previously showed that CaMKII-mediated phosphoryla- Our results support the model whereby PKA phosphorylation de- tion on NLGN1 T739 enhances NLGN1 surface expression (19). creases NLGN1 surface expression and thereby impairs spinogenesis In the current study, we reveal another precise molecular mechanism and excitatory synaptic transmission. that regulates NLGN1 surface expression, in this case, via PKA In addition to PSD-95, other trafficking machinery has been phosphorylation. Synaptic PKA activity has been reported to be implicated in the regulation of NLGN1 trafficking. Other members regulated by LTP, long-term depression, and homeostatic scaling (45, of the MAGUK family, such as SAP97, SAP102, and PSD-93, and 46). Since we show that NLGN1 S839 phosphorylation is dynamically the MAGUK with inverted orientation (MAGI) family, such as S- regulated upon neuronal activation in vivo and in cultured neurons SCAM, Magi1, and Magi3, are also known to interact with NLGN1 (Fig. 2H and SI Appendix,Fig.S3), it is possible that NLGN1 traf- via the PDZ ligand, although their functional relevance has not ficking can be dynamically regulated by NLGN1 S839 phosphoryla- been elucidated (36, 49, 50). It is likely that NLGN1 dynamically tion in response to a variety of neuronal stimuli. Meanwhile, Schapitz switches its interactors during its intracellular trafficking itinerary et al. (35) showed that chemical LTP using forskolin increases surface and following endocytosis. The promiscuous interaction of these NLGN1 in acute hippocampal slices and cultured hippocampal PDZ domain proteins to NLGN1 in different assays has made it neurons. This discrepancy could be the result of the different ex- difficult to definitively identify the precise functional significance of perimental conditions including different neuronal types, stage of these interactions. It is conceivable that phosphorylation of NLGN1 neuronal maturation, or specific preparation. It is possible that the at S839 not only breaks the interaction with PSD-95 but may fa- NLGN1 S839 phosphorylation mainly regulates NLGN1 surface cilitate a subsequent interaction with other PDZ domain proteins. levels during synaptic recycling, whereas the NLGN1 and PSD- Further intensive molecular studies will be required to identify the 95 interaction is required for retrieving NLGN1 by the dynein- physiological relevance of NLGN1 S839 phosphorylation for addi- mediated dendritic retrograde transport. Although both studies tional PDZ protein interactions. emphasize that the NLGN1 C-tail binding to PSD-95 is involved in Phosphorylation of PDZ ligands is a common conserved mecha- the regulation of NLGN1 surface expression, further molecular nism for regulating synaptic protein interactions with MAGUKs. For study to dissect the balance between the synaptic recycling and example, CK2 phosphorylation of GluN2B regulates NMDAR dendritic retrograde transport to modulate surface NLGN1 in re- binding to PSD-95 and stabilization at synapses (27). Interestingly, sponse to various physiological conditions will be required. this is exquisitely subunit-specific, and GluN2A is not phosphorylated NLGN-mediated spinogenesis and synapse formation have been by CK2 even though the PDZ ligand is conserved in GluN2A and widely studied in vivo or in cell lines by expressing WT or mutant GluN2B. In addition, K channels are phosphorylated by PKA in the exogenous NLGNs. However, it is not always clear whether reduced PDZligandtodisruptbindingtoPSD-95 (25). In these other cases of spine density and impaired glutamate currents following manipu- dynamic regulation of PSD-95 binding by receptor/channel phos- lations of NLGNs are the result of altered NLGN surface expres- phorylation, the phosphorylated residue is within the PDZ ligand at

Jeong et al. PNAS Latest Articles | 7of10 Downloaded by guest on October 4, 2021 NLGN1 NLGN1

PKA P P R C PSD-95 R C A B C NLGN1 and PSD-95 PKA phosphorylates S839 phosphorylation associate at the postsynaptic NLGN1 S839 attenuates NLGN1 binding membrane with PSD-95 E Spine number and synaptic transmission are reduced upon destabilized surface NLGN1 expression D P

Surface NLGN1 P

stabilization and NLGN1 N synaptogenic properties LGN1 are impaired upon S839 phosphorylation

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.

the −2 position. Our current findings support a similar role for PKA This study identifies a direct regulation of the PDZ ligand in regulation, but at a residue slightly upstream, for regulating NLGN1 NLGN1 by phosphorylation. Our present results provide a way interaction with PSD-95. of dynamically regulating NLGN1 at synapses, and pave the way Although the C-tails of all NLGNs show high similarity in for future research in the NLGN field. The specificity of NLGN amino acid sequence, isoform-specific phosphorylation has isoforms and compensation by other MAGUK family proteins emerged as a key regulator of isoform specificity. For example, have not been fully dissected. Understanding the dynamic reg- our laboratory revealed CaMKII-mediated phosphorylation on ulation of NLGNs and synaptic potentiation will allow a new NLGN1 T739 and its importance for NLGN1 surface expression avenue to be explored for treating psychiatric dysfunction. and glutamate currents, and this CaMKII phosphorylation event is not conserved in other isoforms (19). Moreover, phosphory- Materials and Methods lation on NLGN1 Y782, a site conserved in the gephyrin-binding In Vitro Kinase Assay and GST Pulldown. GST fusion proteins were phos- μ domain of all NLGNs, inhibits binding with gephyrin, thus phorylated in 10 mM Hepes, (pH 7.0), 20 mM MgCl2,50 M ATP, and 1 pmol γ 32 −1 inhibiting NLGN1 localization at inhibitory synapses (20). of [ - P] ATP (3,000 Ci mmol ) with 50 ng of purified PKA catalytic subunit (Promega). In vitro kinase assays were performed at 30 °C for 30 min. Pro- Phosphorylation on NLGN2 S714, which is also conserved in all teins were eluted from the glutathione-Sepharose resin and resolved by SDS/ NLGNs, negatively regulates binding with gephyrin by recruiting PAGE and analyzed by immunoblotting. Pin1 (51). Although some of these residues are conserved in the For GST pulldown, P2 pellet (crude synaptosomal fraction) was prepared other NLGN isoforms, these studies suggest the possibility that from adult rat brain as described previously and lysed in hypoosmotic buffer phosphorylation of distinct NLGNs can exert NLGN isoform- [20 mM Tris·HCl, pH 8.8, 5 mM ethylenediaminetetraacetic acid (EDTA), 1% specific regulation. In the same way, although the NLGN1 sodium deoxycholate (DOC)]. GST fusion proteins were incubated with the S839 residue is conserved in all NLGN isoforms and the analo- P2 lysate at 4 °C overnight. The samples were resolved by SDS/PAGE and an- gous S839 site in GST-NLGN3 can be phosphorylated by PKA in alyzed by immunoblotting vitro (SI Appendix, Fig. S2), S839 phosphorylation has a pro- found effect on the PSD-95 and NLGN1 interaction. Further- Lentiviral Production. HEK293 cells were transfected with the lentiviral vector FHUGW (FUGW variant) containing the short hairpin RNA sequence against more, knockdown of endogenous PSD-95 has a much more rat PSD-95 or NLGN miRs or HA-NLGN1 (WT or S839A or S839E) and the robust effect on NLGN1 expression compared with NLGN2 or packaging vector dsPAX2, and the vesicular stomatitis virus G envelope gly- NLGN3, revealing a high level of specificity. coprotein vector by using FUGENE HD (Promega, E2311) in UltraCULTURE In this study, we demonstrate that PSD-95 regulates NLGN1 medium (Lonza, 12-725F) containing 2 mM L-glutamine, 1 mM sodium pyru- trafficking and synaptogenic properties. We identified a PKA vate, and 0.075% sodium bicarbonate. Culture media containing lentiviral phosphorylation site, S839, adjacent to the PDZ ligand of particles were harvested and centrifuged at 100,000 × g for 2 h at 4 °C. The NLGN1, which we show controls the interaction with PSD-95. lentiviral particles were resuspended in phosphate-buffered saline (PBS), ali- − Phosphorylation of this key residue attenuates NLGN1 binding quoted, and kept at 80 °C. with PSD-95, thereby reducing its surface expression and Subcellular Fractionation of Brain Tissue and Cultured Neurons. Biochemical diminishing NLGN1-dependent enhancement of excitatory syn- fractionation was carried out as described in our previous studies (4, 19, 27). apses. Our findings confirm an indispensable role of PSD-95 in Briefly, mouse or rat brain tissue or cultured neurons were homogenized in the NLGN1 physiology and provide a molecular mechanism that ice-cold Tris/EDTA/vanadate/phosphatase (TEVP) buffer [320 mM sucrose, can dynamically regulate the NLGN1 and PSD-95 interaction. 10 mM Tris·HCl (pH 7.5), 5 mM EDTA, 1× protease inhibitor mixture (Roche,

8of10 | www.pnas.org/cgi/doi/10.1073/pnas.1821775116 Jeong et al. Downloaded by guest on October 4, 2021 11697498001), and phosphatase inhibitor mixture II (Sigma, P5726) and III Neurons were washed with PBS and fixed with 4% paraformaldehyde and (Sigma, P0044)]. Homogenates were centrifuged at 1,000 × g for 10 min at 4% sucrose in PBS for 10 min. After surface labeling of HA-NLGN1, the 4 °C. The supernatant was centrifuged at 10,000 × g for 20 min at 4 °C to get neurons were permeabilized with 0.25% Triton X-100 in PBS and blocked P2 pellet (crude synaptosomal fraction). The P2 pellet was lysed in an ap- with 10% goat serum. Intracellular HA-NLGN1 was labeled as above. propriate buffer for analysis or resuspended with ice-cold TEVP buffer Recycling assays were performed as described in our previous work (53). [35.6 mM sucrose, 10 mM Tris·HCl (pH 7.5), 5 mM EDTA, 1× protease in- Briefly, neurons were incubated with anti-HA antibody at RT for 10 min, and hibitor mixture, and phosphatase inhibitor mixture II and III] and centrifuged surface proteins were allowed to internalize at 37 °C for 30 min. Excess at 25,000 × g for 20 min to get synaptic plasma membrane (SPM). The SPM amount of anti-rabbit IgG (1 μg per 100 μL) was added later at RT for 20 min pellet was lysed in ice-cold TEVP buffer [1% Triton X-100, 10 mM Tris·HCl (pH and washed out with Neurobasal Media. The neurons were transferred back 7.5), 5 mM EDTA, 1× protease inhibitor mixture, and phosphatase inhibitor to 37 °C for 60 min for recycling. Recycled HA-NLGN1 and intracellular HA- mixture II and III]. The lysates were centrifuged at 33,000 × g for 30 min at 4 °C NLGN1 were labeled with appropriate antibodies as above. to obtain the synaptic regions (Triton X-100−insoluble) and extrasynaptic re- Neurons were cotransfected with NLGN miRs and HA-NLGN1 at DIV 5 to 6 gions (Triton X-100−soluble). and prepared at DIV 12 to count spine number. Total HA-NLGN1 was labeled with rat anti-HA antibody and Alexa 555-conjugated anti-rat secondary Surface Biotinylation. Cultured cortical neurons at DIV 17 to 20 were washed antibody.

three times with cold PBS (including 2 mM CaCl2 and 1 mM MgCl2). Neurons For endogenous PSD-95 and VGLUT1 staining, neurons were prepared as were incubated with 1 mg/mL biotin (EZLink Sulfo-NHS-LC-Biotin, Thermo above, then labeled with anti−PSD-95 (NeuroMab, clone K28/43) and anti- Fisher Scientific, #21335) in PBS (including 2 mM CaCl2 and 1 mM MgCl2)for VGLUT1 (Millipore, AB5905). 30 min at 4 °C and subsequently quenched free biotin by incubating neurons For analysis, regions from three dendrites per each neuron were collected with PBS containing 100 mM glycine for 20 min. After washing, neurons and quantified by the fluorescence intensity of target proteins. All images were lysed with RIPA buffer (150 mM NaCl, 50 mM Tris·HCl, pH 7.8, 1 mM were captured with a 63× objective on a Zeiss LSM 510 confocal microscope EDTA, 1% Triton X-100, 0.5% deoxycholic acid, 0.5% SDS). The lysates were and analyzed with the Image J and MetaMorph Version 7. vigorously sonicated, and supernatants were incubated with NeutrAvidin agarose resin (Thermo Fisher Scientific, #29202) for 1 h to 2 h. Proteins were Spine Morphology Analysis. Neurons were cotransfected with NLGN miRs and eluted from the agarose resin and resolved by SDS/PAGE, and analyzed HA-NLGN1 at DIV 5–6 and prepared at DIV 12 as described previously. For by immunoblotting. spine analysis, regions from three dendrites per each neuron were collected, and spine morphology was analyzed with the Neurolucida 360 software Dark-Rearing Experiments. Male and female C57BL/6J littermates were (MBF Bioscience). maintained as a previously published protocol (19, 52). Mice were housed in a normal light and dark cycle (12 h light and 12 h dark) from P0 to P26 (light- Electrophysiological Recording. AMPAR mEPSCs recordings were performed in reared, LR), were relocated to complete darkness from P21 to P26 (dark- dissociated rat hippocampal primary cultures (DIV 13 to 15). Recordings were NEUROSCIENCE reared, DR), or were kept in complete darkness from P21 to P26 but shif- done in artificial cerebrospinal fluid (ACSF) containing 119 mM NaCl, 2.5 mM ted back to light for 2 h before killing (DR plus light-exposed, DR+LE). The KCl, 26 mM NaHCO3, 1 mM Na2PO4, 11 mM glucose, 2.5 mM CaCl2,and primary visual cortex was macrodissected in the dark for the DR condition or 1.3 mM MgCl2; 0.1 mM Picrotoxin 0.1 and 0.5 μM TTX were added to the + in the light for the LR and DR LR conditions. P2 pellet (crude synaptosomal ACSF before recording. The intracellular solution for mEPSC recording con- fraction) was prepared for analysis as described previously. The National tained 135 mM CsMeSO4, 8 mM NaCl, 10 mM Hepes, 0.3 mM Na3GTP, 4 mM Institute of Neurological Disorders and Stroke (NINDS) Animal Care and MgATP, 0.3 mM EGTA, 5 mM QX-314, and 0.1 mM spermine. Osmolality of the Use Committee approved our use of experimental animals (protocol solutions were adjusted to 285 mOsm to 290 mOsm, and pH was buffered #1171). All animals were handled and the experiments were performed at 7.25 to 7.35. AMPAR mEPSCs were recorded at −70 mV, and the analysis according to these guidelines. of the mEPSCs was done semiautomatically, using in-house software Igor Pro (Wavemetrics) developed in Roger Nicoll’s laboratory at University of California, Immunocytochemistry. Cultured hippocampal neurons were grown on glass San Francisco (UCSF). All events were visually inspected to ensure they were coverslips precoated with poly-D-lysine (Sigma). Neurons were cotransfected mEPSCs during analysis, and those nonmEPSC traces were discarded. Series with pCAG-NLGN miRs-GFP (18) and pCAG-HA-NLGN1 (WT, S839A, or S839E) resistance was monitored and not compensated, and cells in which series re- plasmids with Lipofectamine2000 at DIV 12 to 15 and prepared for analysis at sistance varied by 25% during a recording session were discarded. Synaptic DIV 19 to 21, if not described otherwise. responses were collected with a Multiclamp 700B amplifier (Axon Instruments), To label surface HA-NLGN1, transfected neurons were labeled with rat filtered at 2 kHz, and digitized at 10 kHz. All pharmacological reagents were anti-HA antibody for 15 min at RT. Neurons were washed with PBS and fixed purchased from Abcam, and other chemicals were purchased from Sigma. with 4% paraformaldehyde and 4% sucrose in PBS for 10 min. The cells were incubated with Alexa 555-conjugated anti-rat secondary antibody (Molecular Statistical Analysis. Data analysis was carried out in Image Lab Software (Bio- Probes). After surface labeling, the neurons were permeabilized with 0.25% Rad) or GraphPad Prism (GraphPad Software). Triton X-100 in PBS and blocked with 10% goat serum. Intracellular HA- NLGN1 was labeled with rabbit anti-HA antibody and Alexa 647-conjugated ACKNOWLEDGMENTS. We thank Michael A. Bemben (UCSF) and all other anti-rabbit secondary antibody. lab colleagues for discussion and assistance. We thank the NINDS light im- For the antibody-feeding assay, the neurons were incubated with rat anti- aging facility for technical assistance. This research was supported by the HA antibody in the media at 37 °C for 30 min to allow antibody uptake. NINDS Intramural Research Program (J.J., S.P., W.L., and K.W.R.).

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