Autism-associated mutation inhibits protein kinase C-mediated neuroligin-4X enhancement of excitatory synapses

Abstract

Autism spectrum disorders (ASDs) comprise a highly heritable, multifarious group of neurodevelopmental disorders, which are characterized by repetitive behaviors and impairments in social interactions. Point mutations have been identified in X-linked Neuroligin (NLGN) 3 and 4X genes in patients with ASDs and all of these reside in their extracellular domains except for a single point mutation in the cytoplasmic domain of NLGN4X in which an arginine is mutated to a cysteine (R704C). Here we show that endogenous NLGN4X is robustly phosphorylated by protein kinase C (PKC) at T707, and R704C completely eliminates T707 phosphorylation. Endogenous NLGN4X is intensely phosphorylated on T707 upon PKC stimulation in human neurons. Furthermore, a phospho-mimetic mutation at T707 has a profound effect on NLGN4X-mediated excitatory potentiation. Our results now establish an important interplay between a genetic mutation, a key posttranslational modification, and robust synaptic changes, which can provide insights into the synaptic dysfunction of ASDs.

Keywords: autism; development; neuroligin; synaptic adhesion molecule; synaptogenesis.

Fig. 1.

Autism-associated mutation eliminates PKC phosphorylation of NLGN4X. (A) Alignment of the transmembrane domains and partial c-tails of human NLGNs 1, 2, 3, 4X, and 4Y. The PKC phosphorylation site, T707, is boxed in orange; autism mutation, R704, boxed in blue; and pT707-Ab epitope boxed in yellow. The CaMKII phosphorylation site on NLGN1 is boxed in gray. (B and D) Purified PKC and [γ-P-32]ATP were incubated with GST-fusion proteins and analyzed by autoradiography. CBB protein staining was used to visualize total protein, and GST (negative) and GST-GluA1 c-tail (positive) functioned as phosphorylation controls. (C) ETD MS/MS spectrum of chymotrypsin digested phosphorylated NLGN4X peptide 704-RHETHRRPSPQ-714 found in GST-NLGN4X fusion proteins that were incubated with ATP and purified PKC. Samples were analyzed using the LC/MS/MS method. (E) Means ± SEM of phosphorylated NLGN4X by PKC normalized to WT (n = 4) for NLGN4X R704C (P = 0.0013, n = 4) and NLGN4X T707A (P = 0.0030, n = 4). (F) GST, GST-NLGNs 1, 2, 3, and 4X (WT, R704C, and T707A) were incubated with PKC, and phosphorylation was evaluated by immunoblotting with pT707-Ab. Total protein was evaluated with a GST-Ab. (G) GFP, FLAG-GluA1, or NLGN4X (WT, R704C, T707A, or T707D) were transfected in COS cells and treated with DMSO or PMA, a PKC activator. GluA1 pS831 served as control for PKC activation and the arrow denotes the NLGN4X specific band. (H) Means ± SEM of phosphorylated NLGN4X pT707 achieved by PMA activation normalized to WT (n = 3) and NLGN4X R704C (P = 0.0310, n = 3). (I) Means ± SEM of phosphorylated GluA1 S831 achieved by PMA activation (P = 0.0187, n = 3) normalized to no treatment (n = 3). Immunoblots (WB) were probed with indicated antibodies in F and G. *P < 0.05 **P < 0.01.

Fig. 2.

NLGN4X phosphorylation at T707 induces synaptogenesis. (A) Coexpression of NLmiRs and NLGN4X (WT, R704C, T707A, or T707D) in rat hippocampal neurons. Surface and intracellular receptors were labeled with an anti-hemagglutinin (HA) antibody, which recognized a tag inserted downstream of the signal peptide. (Scale bar, 20 μm.) (B) Means ± SEM normalized to NLGN4X (n = 27), NLGN4X R704C (P > 0.05, n = 29), NLGN4X T707A (P > 0.05, n = 30), and NLGN4X T707D (P > 0.05, n = 29). (C) Means of spine number ± SEM normalized to NLGN4X (n = 30), NLGN4X R704C (P > 0.05, n = 30), NLGN4X T707A (P > 0.05, n = 30), and NLGN4X T707D (P = 0.001, n = 30). (D) Same transfections as in A except NLGN4X constructs did not contain an HA tag. Endogenous VGLUT1 and PSD-95 were stained and visualized as described in Experimental Procedures. (E) Means ± SEM of VGLUT1 normalized to NLGN4X (n = 30) for NLGN4X R704C (P > 0.05, n = 30), NLGN4X T707A (P > 0.05, n = 30), and NLGN4X T707D (P = 0.0466, n = 30). (F) Means ± SEM of PSD-95 normalized to NLGN4X (n = 30) for NLGN4X R704C (P > 0.05, n = 27), NLGN4X T707A (P > 0.05, n = 28), and NLGN4X T707D (P = 0.0049, n = 30). *P < 0.05 **P < 0.01, ***P < 0.001.

Fig. 3.

NLGN4X T707D dramatically enhances excitatory postsynaptic currents. (A) AMPAR-mediated EPSC scatter plots. Expression of either NLGN4X or NLGN4X T707D results in a potentiation of AMPAR-mediated currents compared with control, untransfected cells (P = 0.0023, n = 18; P = 0.0010, n = 11). The enhancement was absent in NLGN4X R704C (P > 0.05, n = 13) and NLGN4X T707A (P > 0.05, n = 11) expressing cells. Experiments were performed in rat organotypic slice cultures on a reduced NLGN background (NLmiRs). Open circles are individual pairs; filled (in red) are mean ± SEM. Black sample traces are control; green are transfected. (Scale bars, 15 pA and 10 ms.) (B) Summary graph of data in A. Expression of NLGN4X T707D resulted in a greater enhancement of AMPAR-mediated currents compared with NLGN4X (P = 0.0143), NLGN4X R704C (P = 0.0025), or NLGN4X T707A (P = 0.0066). (C) NMDAR-mediated EPSC scatter plots. Expression of NLGN4X (n = 12) or phosphodeficient mutants, NLGN4X R704C (n = 11), or NLGN4X T707A (n = 13), did not enhance NMDAR-mediated currents compared with control untransfected cells (P > 0.05), whereas expression of NLGN4X T707D significantly potentiated NMDA-mediated currents (P = 0.0117, n = 9). Open circles are individual pairs, filled (in red) are mean ± SEM. Black sample traces are control; green are transfected. (Scale bars, 30 pA and 20 ms.) (D) Summary graph of data in C. Expression of NLGN4X T707D resulted in an enhancement of NMDAR-mediated currents compared with NLGN4X (P = 0.0409), NLGN4X R704C (P = 0.0250), and NLGN4X T707A (P = 0.0138). *P < 0.05 **P < 0.01, ***P < 0.001.

Fig. 4.

PKC phosphorylates endogenous NLGN4X in human neurons. (A) Immunofluorescence image of human embryonic neurons stained with the neuronal marker MAP2. (B) Regulation of NLGN4X phosphorylation at T707 in human embryonic neurons ± PMA treatment. Arrow denotes the NLGN4X pT707-specific band. Immunoblots (WB) were probed with indicated antibodies. (C) Means ± SEM of phosphorylated NLGN4X T707 achieved by PMA activation normalized to no treatment (n = 4) and + PMA treatment (P = 0.0148, n = 4).