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PSD-95 Binding Dynamically Regulates NLGN1 Trafficking and Function

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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 bind- ing 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 gluta- matergic 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 recep- tors (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
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