An Autism-Associated Mutation Impairs Neuroligin-4 Glycosylation and Enhances Excitatory Synaptic Transmission in Human Neurons

Autistic individual carries a missense mutation in NLGN4

The patient we describe in this study is a male child who began to manifest signs of pervasive developmental abnormality at ∼1 year of age and was diagnosed with autism when he was 2 years old. He was first seen in a neurology clinic for the evaluation of recurring unresponsive staring spells. At 8 years of age, EEG recordings identified abnormal activity with independent epileptiform discharges in the cerebral cortex. He was since treated with Trileptal and Lamictal, and subsequently showed subjectively enhanced social response including elevated nonverbal interactions and spontaneous greetings. He is partially capable of communicating with others using rapid prompting method aids and often demonstrates emotional intelligence and the ability to comprehend social cues by responding accordingly. However, despite these improvements, the subject continues to lack verbal expression, and struggles with repetitive behavior and social detachment.

At the age of 4 years, his social responsiveness was evaluated using SRS scales, and his overall test score met the rating criteria for severe autism (Fig. 1Ai). By 14 years of age, he showed moderate improvement in social awareness and cognition but remained equally impaired for social communication, placing him into the moderate autism category (Fig. 1Ai). The patient was also assessed for his aberrant behavior in residential-, educational-, and community-based facilities. ABC-2 test subdomains ranked him as severely autistic, as he continued to struggle with hyperactivity, irritability, and repetitive interest or stereotyped behavior (Fig. 1Aii). The Vineland-3 test was performed to evaluate his adaptive behavior skills. The composite score suggested that his communication, socialization, and daily living skills range well below the expected age classification (Fig. 1Aiii). In addition, when assessed by the CARS-2 test, he scored beyond the cutoff value to be characterized as severely autistic (Fig. 1Aiv).

To determine whether any underlying genetic mutations could help explain the clinical symptoms of this child, a high-throughput WES test was conducted privately in a clinic. This genomic analysis followed by bidirectional Sanger sequencing identified the presence of two mutations in the patient exome; a maternally inherited missense variant 302G>A creating a hemizygous R101Q substitution in NLGN4 CDS, and a paternally inherited frameshift variant causing heterozygous deletion in GLI2 (Fig. 1B). Both parents are heterozygous for their corresponding mutations and are asymptomatic, suggesting compensatory effects from their second alleles. Although the male patient is also heterozygous for his autosomal GLI2 deletion, he lacks a second allele to compensate for the X-linked NLGN4 mutation. Therefore, we hypothesized that the NLGN4X missense mutation could manifest pathogenic potential. Moreover, several mutations in NLGN4X gene have been previously reported in autistic probands, making the R101Q substitution a likely candidate leading to ASD diagnosis in this individual.

The R101 residue is located within the esterase homology domain of NLGN4, adjacent to a consensus N-linked glycosylation sequon (N102), and proximal to a downstream cysteine-loop (Fig. 1C). Interestingly, the R101Q mutation is also situated next to a region with multiple previously identified ASD mutations (e.g., G84R, R87W, and G99S), some with mostly unknown mechanistic consequences (Fig. 1C; Fabrichny et al., 2007; Zhang et al., 2009; Xu et al., 2017; Schepici et al., 2019; Nguyen et al., 2020). A structural model of the extracellular domain of NLGN4 predicted that the R101Q mutation is spatially oriented opposite to its dimerization domain and away from its NRXN contact site (Fig. 1D; Fabrichny et al., 2007; Leone et al., 2010).

R101Q mutation impairs NLGN4 maturation and its trafficking to cell surface

We first inquired whether R101Q substitution has any impact on the maturation of NLGN4 protein. We transfected HEK293 cells with constructs expressing HA-tagged NLGN4 WT versus R101Q variant followed by either an IRES-EGFP or IRES-mOrange reporter (Fig. 2A). We extracted total cell lysate proteins after 48 h and performed quantitative immunoblotting with HA antibody. The molecular weight (MW) of WT NLGN4 is ∼92 kDa, but it generated a predominantly high MW product (>100 kDa) which likely reflected a maturely glycosylated version, as described for other NLGNs (Fig. 2B; also see Boucard et al., 2005; Budreck and Scheiffele, 2007; Zhang et al., 2009; Poulopoulos et al., 2012). The WT NLGN4 also generated a minor, low MW (<100 kDa) version, which indicated an immaturely glycosylated product that was possibly undergoing post-translational modifications (PTMs) in intracellular organelles (Fig. 2B; also see Zhang et al., 2009; Poulopoulos et al., 2012). We noticed that both products were similarly present in R101Q condition, however, their relative abundance was significantly altered (Fig. 2B). R101Q substitution reduced the total NLGN4 protein content, considerably decreased its mature version, and increased the immature form, when normalized to coexpressed EGFP levels (Fig. 2C).

Because the R101Q mutation partially inhibited NLGN4 maturation, we next asked whether it influenced the surface trafficking of NLGN4, an important feature of all synaptic CAMs. We immunostained transfected HEK293 cells after 48 h with HA antibody under nonpermeabilized conditions and normalized the signal to a coexpressed intracellular mOrange intensity. We noticed that R101Q substitution considerably diminished the localization of NLGN4 at the cell surface, suggestive of a major trafficking defect (Fig. 2D).

We next asked whether R101Q mutation alters other critical properties of NLGN4, e.g., dimerization or NRXN-binding. We first confirmed the specificity of HA antibody as it failed to probe any similar MW product in cell lysates from untransfected control; moreover, both NLGN4 WT and R101Q variants did not exhibit any nonspecific binding when immunoprecipitated with the HA antibody versus a control IgG (Fig. 2Ei). To monitor NLGN4 dimerization, we next coexpressed FLAG-tagged and HA-tagged NLGN4 in HEK293 cells, lysed them after 48 h, immunoprecipitated NLGN4 (WT vs R101Q mutant) using HA antibody, and immunolabeled the products with HA and FLAG antibodies of two different species (Fig. 2Eii). We observed that both mature and immature versions of NLGN4 WT and R101Q mutant were able to form homodimers in similar proportions, as evidenced by HA and FLAG colabeling (Fig. 2Eii,F).

To examine whether R101Q substitution affects the ability of NLGN4 to bind NRXN, we transfected HEK293 cells with a NRXN1β^-SS4 construct followed by IRES-mOrange reporter, and EGFP-only control, NLGN4 WT, or R101Q mutant constructs followed by IRES-EGFP reporter (Fig. 2G). At 48 h post-transfection, we dissociated the NRXN-expressing and NLGN4-expressing cells, mixed them together, and evaluated their capacity to form aggregates via molecular interaction. We found that, with respect to control condition, expression of both NLGN4 WT and R101Q mutant substantially increased the formation of multicellular clusters, as identified by colocalization of mOrange and EGFP-positive cells (Fig. 2H,I). This result suggests that the NLGN4 R101Q variant can interact with NRXN. However, when compared with WT, the R101Q condition showed a small but significant reduction in cell aggregation, likely because of the lower surface export of mutant protein (Fig. 2H,I).

In summary, our data revealed that the R101Q mutation is pathogenic, inhibits maturation of NLGN4 protein and its transport to the cell surface, but does not severely alter its ability to dimerize or its affinity for NRXN.

R101Q mutation causes glycosylation defects in NLGN4 and increases its retention at endoplasmic reticulum and Golgi

The vicinity of the R101 residue of NLGN4 to a potential N-glycosylation sequon (N102; Fig. 1C,D) and the appearance of a lower MW product in R101Q conditions (Fig. 2B) made us hypothesize that the R101Q mutation could indirectly impair glycosylation at the adjacent N102 residue. To probe this, we first substituted the N102 residue for an alanine (N102A) to directly eliminate its glycosylation (Fig. 3A). We expressed this HA-tagged mutant construct in HEK293 cells for 48 h and performed Western blot with HA antibody under a denaturing condition. We found that the N102A mutation similarly generated a prominent immature product, and phenocopied the R101Q variant by substantially decreasing mature NLGN4 level (Fig. 3B).

To further characterize the glycosylation profile of mature versus immature NLGN4 products, we next treated the WT and R101Q variant with glycosidases: Endo H (which cleaves only the ER-associated, immature, high-mannose oligosaccharides from N-linked glycoproteins) or PNGase F (which removes all N-linked oligosaccharides from glycoproteins, including both complex and hybrid forms as well as immature high-mannose oligosaccharides). We noticed that Endo H treatment specifically downshifted the immature NLGN4 bands of lower MW without affecting the position of mature NLGN4 bands of higher MW; whereas PNGase F treatment displaced both high- and low-MW NLGN4 products for both the WT and R101Q conditions (Fig. 3C,D). Therefore, the low- and high-MW bands, respectively, correspond with immaturely and maturely glycosylated versions of NLGN4, and the ratio between these two was significantly altered by R101Q substitution.

Because R101Q mutation impaired the maturation of NLGN4 and prevented its transport to the cell surface (Fig. 2), we postulated that the mutant protein might accumulate in intracellular organelles (e.g., ER and Golgi, which play major roles in PTM pathways including glycosylation). We transfected HEK293 cells with HA-tagged NLGN4 WT versus R101Q followed by an IRES-mOrange reporter, and coimmunostained them for ER-specific marker calnexin and HA antibody under permeabilized conditions. We noticed that the R101Q variant exhibited higher localization at ER, when compared with WT NLGN4 (Fig. 3E). We also observed a similar increase in colocalization between the NLGN4 R101Q variant and the Golgi-specific marker GM130 (Fig. 3F). These data indicate that the R101Q substitution inhibits the glycosylation of NLGN4 and increases its retention at intracellular compartments.

R101Q-equivalent mutations trigger trafficking defects in all NLGNs with conserved glycosylation sites

The R101 residue and its adjacent glycosylation site are highly conserved among all NLGN genes from different species, except mouse NLGN4 (also referred to as NLGN4-like), which contains an aspartic acid residue (D85) instead of asparagine in the equivalent location, eliminating the consensus sequence for N-linked glycosylation (Fig. 4A). Therefore, we next introduced R101Q-equivalent mutations in HA-tagged versions of rat NLGN1, rat NLGN2, and mouse NLGN3, and asked whether that can similarly affect their molecular properties (Fig. 4B). Our immunoblotting experiments from HEK293 cells transfected with NLGN1 R108Q mutant indicated a definitive increase in immature product and a decrease in mature product (Fig. 4C). This effect was similarly reproduced by R97Q mutation in NLGN2 and R94Q mutation in NLGN3 (Fig. 4D,E). Of note, R101Q-equivalent mutations also decreased the total protein content of NLGN3 (similar to NLGN4; Fig. 2B), but not that of NLGN1 or NLGN2 (Fig. 4C–E), which contained a higher number of N-glycosylation sites (Fig. 4A). Thus, R101Q-equivalent mutations similarly affected the maturation of all NLGNs and also impaired the stability of different NLGNs depending on their potential glycosylation profiles.

To further assess whether these mutation-induced deficiencies in NLGN1/2/3 maturation can also affect their ability to localize at the cell surface, we conducted immunostaining of NLGN-expressing HEK293 cells under nonpermeabilized conditions. We found that NLGN1 R108Q, NLGN2 R97Q, and NLGN3 R94Q substitutions displayed significant reduction in surface intensity, with respect to their corresponding WT versions (Fig. 4F–H). These results demonstrate that the N102-equivalent glycosylation sites play a critical role in the maturation and surface trafficking of all NLGNs, and that the R101-equivalent upstream residues can strongly influence this process.

R101Q mutation blocks the ability of NLGN4 to induce excitatory synaptogenesis

After we used HEK293 cells as a reduced system to establish that R101Q substitution is not benign and directly impairs the molecular properties of NLGN4, we next inquired whether this pathogenic mutation affects the morphologic and/or functional properties of human neurons. To this end, we first performed dual-SMAD inhibition in human ES cells to generate NSCs, then differentiated the NSCs into neurons, and subsequently cocultured them with mouse primary astrocytes, as described previously (Fig. 5A; Chambers et al., 2009; Chanda et al., 2019). During this period, the cells gradually changed their identity in terms of relevant marker expression, from Oct3/4-positive ES cells to Nestin-positive NSCs to Dcx-positive neurons (Fig. 5C). We infected the neurons with lentivirus expressing either a control EGFP or NLGN4 WT versus R101Q mutant constructs under hSyn1 promoter and followed by IRES-driven EGFP reporter (Fig. 5B). After an additional 30 d, i.e. at 60 d postinfection, we observed robust EGFP fluorescence in these neurons, confirming successful transgene expression (Fig. 5C).

To monitor synapse formation in NSC-derived neurons, we immunostained them for cytoskeletal marker Tuj1 (class III β-tubulin) and synaptic marker Synapsin. We found that the NLGN4 R101Q mutation did not affect the cell number or total neurite volume, but substantially diminished Synapsin puncta intensity (Fig. 5D). Further analysis confirmed that, compared with WT NLGN4, the R101Q substitution considerably reduced both the density and size of synapses formed along Map2-positive dendrites (Fig. 5E).

To gain insight into the type of synapses affected by NLGN4 R101Q mutation, we immunolabeled the neurons with GABAergic or glutamatergic synapse markers (i.e., vGAT or VGluT, respectively). Compared to neurons expressing a control vector, the NLGN4 WT and R101Q conditions displayed a minor increase in GABAergic synapse density without significantly affecting synapse size (Fig. 5F). However, for GABAergic synapses, there was no apparent change in either synaptic parameters between the WT and R101Q conditions (Fig. 5F). For glutamatergic synapses, the expression of WT NLGN4 substantially elevated both the number and size of synaptic puncta, presumably by recruiting excitatory presynaptic terminals via the NRXN interaction, whereas R101Q mutant failed to demonstrate any sizable impact compared with the control condition (Fig. 5G). These data indicate that R101Q substitution substantially impairs the ability of human NLGN4 to induce excitatory synapses when overexpressed, again suggesting a loss-of-function phenotype.

R101Q mutation inhibits NLGN4-mediated suppression of excitatory synaptic transmission

To investigate the effects of NLGN4 R101Q mutation on the functional properties of human neurons, we made electrophysiological recordings from NSC-derived neurons expressing either a control vector or NLGN4 WT versus R101Q constructs (Fig. 5B). Current-clamp recordings detected no noticeable differences among the control versus NLGN4 WT versus R101Q conditions in terms of their ability to fire repetitive APs (Fig. 6A). Furthermore, the membrane capacitance (C_m), membrane resistance (R_m), and resting membrane potential (V_m) did not significantly differ across conditions (Fig. 6B). Voltage-clamp recordings revealed no effect on the amplitude or frequency of GABAergic mIPSCs by either NLGN4 WT or R101Q mutant (Fig. 6C,D). However, the expression of NLGN4 WT but not the R101Q mutant attenuated the frequency of AMPAR-mediated mEPSCs and augmented their interevent interval without affecting mEPSC amplitude (Fig. 6E,F).

The kinetic parameters of mEPSC events were not considerably altered by NLGN4 WT/R101Q expression, suggesting no apparent impact on AMPAR composition (Fig. 6G). Finally, the overexpression of NLGN4 WT but not the R101Q mutant also suppressed the amplitude of AMPAR-evoked EPSCs without affecting presynaptic release probability [as estimated by paired-pulse ratios (PPRs)], when measured under similar recording conditions [as assessed by series resistance (R_s) parameters; Fig. 6H]. These results suggest that NLGN4 overexpression can negatively modulate the strength of excitatory synapses in human neurons by a potentially postsynaptic mechanism, and the R101Q substitution causes a major loss of this NLGN4-induced synaptic phenotype.

To test whether our conclusions apply to neurons generated by a different protocol, we transdifferentiated human ES cells into cortical glutamatergic neurons by ectopic expression of a single TF, Ngn2 (Fig. 7A; Zhang et al., 2013). We coinfected the neurons with lower-titer lentivirus encoding either a control vector or HA-tagged NLGN4 WT versus R101Q mutant, and detected no noticeable difference in their cell viability (Fig. 7A,B). We next identified infected neurons by coexpressed fluorescent reporters and made patch-clamp recordings to evaluate their functional properties (Fig. 7A,C). Similar to NSC-derived neurons, the Ngn2-induced human neurons expressing WT NLGN4 showed a significantly lower amplitude of AMPAR-mediated evoked EPSCs; whereas the R101Q variant largely reversed this phenotype (Fig. 7D). Furthermore, R101Q substitution in NLGN4 also elevated the AMPAR mEPSC frequency compared with WT version, with a relatively minor effect on mEPSC amplitude (Fig. 7E,F). Thus, both NLGN4 WT and R101Q variant had highly consistent and reproducible effects on the excitatory synaptic transmission in human neurons irrespective of their differentiation or reprogramming methods.

R101Q mutation prevents NLGN4-mediated silencing of excitatory postsynapses

We aimed to understand how loss of NLGN4 function by R101Q mutation can contribute to aberrant synaptic transmission. We first used IRES-driven EGFP fluorescence to visualize the morphology of Ngn2-induced neurons from control, WT, and R101Q conditions, but failed to observe any difference in their soma size, primary dendrite number, or total neurite length (Fig. 8A). To determine NLGN4 levels in these neurons, we performed qPCR analysis, and found that cells overexpressing WT versus R101Q versions displayed an equivalent increase in mRNA levels relative to control condition (Fig. 8B). We next tracked the distribution of NLGN4 protein along dendrites by immunostaining the cells with HA antibody under nonpermeabilized conditions, and then permeabilized them to counterstain for Synapsin. We noticed that R101Q mutation caused a substantial reduction in the density of NLGN4 clusters along the surface of dendritic branches (Fig. 8C) and thus decreased the colocalization of NLGN4 with the Synapsin signal (Fig. 8D). Therefore, R101Q substitution inhibited the surface trafficking of NLGN4 in human neurons, even to a greater extent than our observations in HEK293 cells (compare with Fig. 2D) and simultaneously diminished its targeting at synapses.

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Figure 8.

R101Q mutation impairs NLGN4 synaptic localization and inhibits NLGN4-mediated reduction in synaptic AMPAR clusters. A, Representative images (left) of Ngn2-induced human neurons expressing either a control (Ctrl) vector or NLGN4 WT versus R101Q variant, as their morphologic parameters were assessed using an EGFP reporter. Summary graphs (right) of soma size, primary dendrite number, and average neurite length per cell. B, NLGN4 mRNA levels were estimated by quantitative real-time PCR and normalized to those of GAPDH (internal control) for Ngn2-induced neurons expressing either a control vector, or NLGN4 WT versus R101Q mutant constructs. The average bar graphs combine values from two independent primer sets (set 1: circles; n = 6 Ctrl, 6 WT, and 5 for R101Q; and set 2: squares; n = 6 Ctrl, 6 WT, and 5 for R101Q). C, Sample images (left) of EGFP-expressing dendritic segments (pseudocolored for better visibility) from NLGN4 WT (top) versus R101Q (bottom) conditions, when immunostained with anti-HA antibody (arrowheads) under nonpermeabilized state. Summary graphs (right) of surface HA-NLGN4 puncta density and size. D, Representative images (left) of EGFP-labeled dendritic segments from WT (top) versus R101Q (bottom) conditions, when incubated with anti-HA antibody under nonpermeabilized state, then permeabilized and coimmunostained with Synapsin antibody. The channel colors were artificially reassigned to provide better visibility for synaptic signals; arrowheads point at synaptic puncta. Summary graphs (right) indicate synaptic distribution of NLGN4, characterized using normalized Mander's colocalization coefficients between HA-NLGN4 and Synapsin signals. E, F, Dendritic sections of mOrange-expressing human neurons from control, NLGN4 WT, and NLGN4 R101Q conditions immunostained with either Synapsin (E, left) or VGluT (F, left) antibody, and integrated intensity of Synapsin (E, right) or VGluT (F, right) puncta normalized with respect to the dendrite length. G, Neurons expressed either a control vector or NLGN4 WT versus R101Q mutant followed by mOrange reporter. Sample images (left; pseudocolor) of dendritic branches immunolabeled for surface AMPARs (SEP-GluA1) using anti-GFP antibody under nonpermeabilized condition, subsequently permeabilized and stained with anti-Synapsin antibody. Arrowheads indicate synaptic clusters; note that many lack copresence of SEP-GluA1 in WT condition. Summary graphs (right) of normalized Mander's coefficient indicating a Synapsin (Syn) puncta fraction containing SEP-GluA1 signal and the intensity of SEP-GluA1 puncta colocalized with Synapsin. Average bar graphs represent the mean ± SEM, with the number of frames analyzed/number of experiments, or only the number of independent batches (for B). Statistical significance was assessed by two-tailed, unpaired (one-tailed and paired for B), Student's t test. ***p < 0.005; *p < 0.05; ns, not significant (p > 0.05).

To further evaluate the consequences of reduced NLGN4 content at excitatory synapses, we immunostained Ngn2-induced neurons with Synapsin and VGluT antibodies. We found that, compared with control condition, the overexpression of WT NLGN4 but not the R101Q version caused a considerable increase in both Synapsin and VGluT intensities at dendritic branches (Fig. 8E,F), mimicking our observations in NSC-derived neurons (Fig. 5G). Although these results suggest that overexpression of NLGN4 WT but not the R101Q mutant can induce the formation of glutamatergic synapses, their function depends on the availability of postsynaptic AMPARs. To directly assess that, we infected Ngn2-induced neurons with lentiviruses expressing SEP-GluA1, and either control or NLGN4 WT versus R101Q constructs followed by an IRES-mOrange reporter. We noticed that, compared with control condition, WT NLGN4 significantly reduced the localization and signal intensity of SEP-GluA1 at Synapsin-positive synaptic clusters, whereas the R101Q variant alleviated these phenotypes (Fig. 8G). Together, these results indicate that R101Q substitution increases excitatory synaptic transmission (Fig. 7C–F) by preventing NLGN4-induced loss of postsynaptic AMPARs.