Canonical versus non-canonical transsynaptic signaling of neuroligin 3 tunes development of sociality in mice

ABSTRACT Neuroligin 3 (NLGN3) and neurexins (NRXNs) constitute a canonical transsynaptic cell-adhesion pair, which has been implicated in autism. In autism spectrum disorder (ASD)

development of sociality can be impaired. However, the molecular mechanism underlying NLGN3-mediated social development is unclear. Here, we identify non-canonical interactions between NLGN3

and protein tyrosine phosphatase δ (PTPδ) splice variants, competing with NRXN binding. NLGN3-PTPδ complex structure revealed a splicing-dependent interaction mode and competition mechanism

between PTPδ and NRXNs. Mice carrying a NLGN3 mutation that selectively impairs NLGN3-NRXN interaction show increased sociability, whereas mice where the NLGN3-PTPδ interaction is impaired

exhibit impaired social behavior and enhanced motor learning, with imbalance in excitatory/inhibitory synaptic protein expressions, as reported in the _Nlgn3_ R451C autism model. At neuronal

level, the autism-related _Nlgn3_ R451C mutation causes selective impairment in the non-canonical pathway. Our findings suggest that canonical and non-canonical NLGN3 pathways compete and

regulate the development of sociality. SIMILAR CONTENT BEING VIEWED BY OTHERS A COMBINATORIAL CODE OF NEUREXIN-3 ALTERNATIVE SPLICING CONTROLS INHIBITORY SYNAPSES VIA A TRANS-SYNAPTIC

DYSTROGLYCAN SIGNALING LOOP Article Open access 30 March 2023 ANALYSES OF THE AUTISM-ASSOCIATED NEUROLIGIN-3 R451C MUTATION IN HUMAN NEURONS REVEAL A GAIN-OF-FUNCTION SYNAPTIC MECHANISM

Article 24 October 2022 IMBALANCED POST- AND EXTRASYNAPTIC SHANK2A FUNCTIONS DURING DEVELOPMENT AFFECT SOCIAL BEHAVIOR IN SHANK2-MEDIATED NEUROPSYCHIATRIC DISORDERS Article Open access 21

May 2021 INTRODUCTION Social behaviors for properly communicating with others are mediated by various cortical and subcortical neural circuits, including the medial prefrontal cortex (mPFC),

amygdala, anterior insula, anterior cingulate cortex, inferior frontal gyrus, and superior temporal sulcus. Developmental dysregulation of these social brain circuits is associated with the

pathophysiology of autism spectrum disorders (ASDs), a group of neurodevelopmental disorders with core symptoms of impaired social interaction and communication accompanied by restricted

interests and repetitive behaviors1,2. Genetic and genomic studies have revealed that distortions of transsynaptic signaling mediated by synaptic cell adhesion proteins are closely

associated with the pathogenesis of ASDs3,4,5,6. The X-linked _neuroligin 3_ (_NLGN3_) gene encodes the postsynaptic adhesion molecule NLGN3, which interacts with presynaptic neurexins

(NRXNs) to organize synaptogenesis. _NLGN3_ is one of the best-characterized sociality-related genes, with various mutations including deletions and an arginine-to-cysteine missense (R451C)

mutation linked to ASDs7,8,9,10. _Nlgn3_ knock-in mice harboring the autism-related R451C mutation (_Nlgn3__R/C_) and _Nlgn3_ knockout (KO) mice display enhanced ability to remain on the

accelerating rotarod associated with the repetitive behaviors of ASD11. In contrast, decreased social interaction accompanied by imbalances in the excitatory and inhibitory synaptic protein

expression and transmissions in the cerebral cortex and hippocampus is observed exclusively in the _Nlgn3__R/C_ mice, and not in the KO mice, even though the R451C mutation seems to be a

loss-of-function mutation that causes reduced affinity to the canonical binding partners, NRXNs, and intracellular retention of the mutant protein in the endoplasmic reticulum12,13,14,15.

Therefore, the molecular mechanism underlying the NLGN3-mediated social development, as well as the pathogenic mechanism of ASD-linked NLGN3 mutations, remains a mystery. In fact,

intriguingly, NLGN3 KO and the R451C mutation differentially impact synaptic transmission in a circuit-, neuronal cell-, or synapse-type-specific manner, and the R451C mutation

differentially affects two different types of input onto the same postsynaptic cell11,16,17,18, implying that NLGN3 may utilize different types of transsynaptic signaling. Mutations in

_NRXN1_, one of the three members of the _NRXN_ gene family, have also been detected in ASD-affected individuals, although they appear to predispose to a wider range of neurodevelopmental

and psychiatric disorders, probably because NRXNs function as a presynaptic hub to interact with multifarious postsynaptic partners6. The canonical postsynaptic partners of NRXNs include

NLGNs, leucine-rich repeat transmembrane neuronal proteins, and cerebellin precursor proteins-GluDs (ref. 6). Type IIA receptor protein tyrosine phosphatases (RPTPs; PTPδ, PTPσ, and

leukocyte common antigen-related (LAR) in mammals) are another group of presynaptic hub proteins5. Type IIA RPTPs exist in multiple isoforms generated by alternative splicing of microexons

at the sites corresponding to a loop within the extracellular second immunoglobulin (Ig)-like domain (mini-exon A; meA) and the junction between the second and third Ig-like domains

(mini-exon B; meB), with the greatest variation having been observed in PTPδ (Fig. 1a) (refs. 19,20). The postsynaptic ligands for PTPδ include interleukin-1 receptor accessory protein

(IL-1RAcP), IL-1RAcP-like-1 (IL1RAPL1), Slit- and Trk-like proteins (Slitrks), and synaptic adhesion-like molecules (SALMs), most of which prefer PTPδ splice variants containing the meB

peptide21,22,23,24,25. Here, we identified a non-canonical synapse-organizing interaction between NLGN3 and PTPδ splice variants lacking the meB peptide, which competed with NRXNs by steric

hindrance. The crystal structure of the NLGN3–PTPδ complex revealed the splicing-dependent interaction mechanism for synapse organization. Based on the atomic-level comparison of NLGN3–PTPδ

and the reported NLGN1/4–NRXN1 interfaces, we designed _Nlgn3_ knock-in mutations in mice to dissect the physiological roles of the canonical and non-canonical pathways. Our results suggest

that a balance between the canonical and non-canonical NLGN3 pathways may contribute to the development of sociality, with the autism-related R451C mutation disrupting this balance to

diminish the non-canonical PTPδ–NLGN3 pathway. RESULTS PTPΔ SPLICE VARIANTS HAVE DISTINCT SYNAPTOGENIC PROPERTIES The developing mouse brain expresses at least eight Ig domain-splice

variants of PTPδ (Fig. 1a)20. We examined the postsynapse-inducing activity of these PTPδ splice variants using an artificial synaptogenic assay. Cortical neurons were incubated with

magnetic beads coated with equal amounts of the respective extracellular domains (ECDs) of the PTPδ splice variants, followed by immunostaining for the excitatory and inhibitory postsynaptic

scaffold proteins Shank2 and gephyrin, respectively (Fig. 1b). Selective accumulation of Shank2, but not of gephyrin, was detected on the cortical neurons in contact with the beads coated

with meB-peptide-containing PTPδ variants (PTPδ-meB(+)s; PTPδA9B+, PTPδA6B+, PTPδA3B+, and PTPδA–B+). In contrast, accumulation of both Shank2 and gephyrin was detected on the beads coated

with ECDs of PTPδ variants lacking the meB peptide (PTPδ-meB(–)s; PTPδA9B–, PTPδA6B–, PTPδA3B–, and PTPδA–B–). Moreover, the signal intensities for Shank2 and gephyrin were dependent on the

size of the meA peptides, i.e., the longer the meA peptides, the greater the accumulation of the postsynaptic proteins on the beads. These results suggest that PTPδ-meB(+)s selectively

induce the excitatory postsynaptic differentiation, whereas PTPδ-meB(–)s are involved in both excitatory and inhibitory postsynaptic differentiation. IDENTIFICATION OF NLGN3 AS A SELECTIVE

LIGAND FOR PTPΔ-MEB(–)S The differential postsynapse-inducing properties of PTPδ splice variants imply the existence of diverse postsynaptic ligands selective for each PTPδ splice variant.

The previously characterized PTPδ ligands IL1RAPL1, IL-1RAcP, Slitrks, and SALMs, preferentially interact with PTPδ-meB(+)s20,21,22,23,24,25. In fact, the excitatory postsynaptic

differentiation induced by PTPδA9B+, a PTPδ-meB(+) isoform abundantly expressed in the developing mouse brain20, was reduced by ~70% in cultured cortical neurons from IL1RAPL1/IL-1RAcP

double-knockout (DKO) mice (Supplementary Fig. 1a, b), suggesting IL1RAPL1 and IL-1RAcP are major postsynaptic ligands for PTPδA9B+ to induce excitatory synapse formation in the cortical

neurons. In contrast, IL1RAPL1/IL-1RAcP DKO had little effect on the postsynaptic differentiation induced by PTPδA3B–, the most abundant PTPδ-meB(–) isoform (Supplementary Fig. 1c, d),

suggesting that PTPδ-meB(–)s utilize other ligand(s) for postsynaptic differentiation. To identify the ligands that mediate PTPδ-meB(–)-induced postsynaptic differentiation, we employed in

situ proteomic screening (Fig. 2a)26. After postsynaptic differentiation of cultured cortical neurons was induced on beads conjugated with the ECD of PTPδA3B–, postsynaptic proteins that

extracellularly interacted with PTPδA3B– were chemically cross-linked and recovered. Liquid chromatography-tandem mass spectrometry of the PTPδA3B– complex identified four transmembrane

proteins, including NLGN3 (Supplementary Table 1). We confirmed the direct interaction between PTPδ splice variants and NLGN3 using a cell surface binding (CSB) assay and a cell aggregation

assay. Among the mouse NLGN3 splice variants containing (NLGN3A) or lacking (NLGN3(–)) the splice segment (SS) A in the ECD, the NLGN3A isoform was used in this study, and “NLGN3” indicates

this isoform, henceforth. HEK293T cells expressing NLGN3 were incubated with the soluble ECDs of PTPδ splice variants fused to Fc and then stained with an anti-Fc antibody, to quantify the

cell surface-bound Fc fusion proteins (Fig. 2b, c). We detected staining signals on cells incubated with the ECDs of PTPδ-meB(–)s, but not those of PTPδ-meB(+)s. Correspondingly, HEK293T

cells expressing NLGN3 selectively aggregated with those expressing PTPδ-meB(–)s (Fig. 2d). NLGN3 also interacted with LAR and PTPσ splice variants lacking the meB peptide, although the

interactions were much weaker than the interaction with PTPδ A3B– (Supplementary Fig. 2a). In contrast, among the four murine NLGN family members, NLGN3 splice variants alone showed

significant binding to PTPδA3B– (Supplementary Fig. 2b), suggesting that PTPδ-meB(–)s interact exclusively with NLGN3. We further confirmed the NLGN3-PTPδ interaction in vivo by

coimmunoprecipitation assay using anti-NLGN3 antibody in the synaptosomal fraction of striatums where highest _PtprdA3B__–_ expression was observed (Fig. 2e and Supplementary Fig. 2c). Next,

we examined the involvement of NLGN3 in PTPδ-meB(–)-induced postsynaptic differentiation by coculture assays using cortical neurons from neuron-specific NLGN3 KO mice that lacked the exon 7

by Cre recombinase under the control of the nestin promoter (Supplementary Fig. 3a–c). Exon 7 encodes a half of the ECD, and skipping of this exon is predicted to lead to a frame shift and

a premature stop codon. In NLGN3 KO neurons, no accumulation of Shank2 or gephyrin was detected on the beads coated with PTPδ-meB(–)s, indicating that PTPδ-meB(–)s require NLGN3 to induce

postsynaptic differentiation (Fig. 2f, g, and Supplementary Fig. 3d–f). In contrast, NLGN3 KO had a negligible effect on the synaptogenic activities of PTPδ-meB(+)s. These results suggest

that the selective interaction between NLGN3 and PTPδ-meB(–)s organizes both excitatory and inhibitory synaptogenesis. PTPΔ AND NRXN1 COMPETE FOR BINDING TO NLGN3 The identification of the

non-canonical NLGN3–PTPδ-meB(–) interaction prompted us to examine its physiological and pathological roles in brain development for sociality by designing and analyzing NLGN3 mutants that

dissect the canonical NRXN- and non-canonical PTPδ-meB(–)-mediated synapse-organizing pathways. Therefore, we examined the relationship between PTPδ-meB(–)s and NRXN1β for binding to NLGN3.

An NRXN1β isoform lacking SS4 showing the strongest NLGN3 binding among the NRXN family members was used in this study27. In a CSB assay using soluble PTPδA3B–-Fc and NLGN3-expressing

HEK293T cells, cell surface-bound PTPδA3B–-Fc signals were reduced by the addition of similar concentrations of hexahistidine-tagged PTPδA3B–-ECD or NRXN1β-ECD but not of alkaline

phosphatase (Fig. 2h). Furthermore, preformed NLGN3-NRXN1β complex on the cell surface was replaced by the NLGN3-PTPδA3B– complex by adding PTPδA3B–-Fc (Supplementary Fig. 2d). Consistently,

isothermal titration calorimetric (ITC) analyses showed the binding affinity between NLGN3 and PTPδA3B– (_K_D = 4.4 µM) is comparable to that between NLGN3 and NRXN1β (_K_D = 1.8 µM in the

presence of 1 mM Ca2+) (Fig. 2i). Furthermore, the NLGN3 mutant with three alanine substitution mutations at Leu374, Asn375, and Asp377 in the putative interface with NRXN1β (NLGN3 LND)28

failed to interact with PTPδA3B– (Supplementary Fig. 2e). These results suggest that PTPδA3B– and NRXN1β compete for binding to NLGN3 by sharing interfaces. This is further supported by the

fact that, MDGA1, known as an NLGN3 interactor that inhibits NRXN interaction29, also interfered with the NLGN3-PTPδ interaction in competitive CSB assay (Supplementary Fig. 2f). Therefore,

atomic-level structural information at their interfaces is required to design genetic tools to separately dissect the canonical and non-canonical transsynaptic signaling pathways of NLGN3.

STRUCTURAL BASIS OF THE NLGN3–PTPΔA3B– INTERACTION To elucidate the structural mechanism of the NLGN3–PTPδ-meB(–) interaction, we determined the crystal structures of mouse apo-NLGN3 ECD

(residues 35–684) at 2.76 Å resolution (Supplementary Table 2 and Supplementary Fig. 4a) and NLGN3 ECD in complex with PTPδA3B– Ig1–Fn1 at 3.85 Å resolution (Supplementary Table 2 and Fig. 

3). The NLGN3 structure adopts an α/β-hydrolase fold, which is topologically identical to the reported structures of NLGN1, 2, and 428,30,31,32. The electron densities corresponding to

residues 148–173 and 555–567 were invisible, likely owing to the structural disorder. The first disordered region contains a 19-residue insertion at SS-A. The asymmetric unit of the

apo-NLGN3 crystal contains two NLGN3 molecules, which form a dimer (Supplementary Fig. 4a). The molar mass of the recombinant mouse NLGN3 ECD determined by size-exclusion chromatography

coupled with multiangle light scattering (SEC-MALS) analysis (Supplementary Fig. 4b) is 160 kDa, which corresponds to the mouse NLGN3 ECD dimer. All reported NLGN dimer structures, including

our NLGN3 structure, exhibit a similar dimerization interface, where two helices from each protomer form a four-helix bundle28,30,31,32. In the crystal of the NLGN3–PTPδA3B– complex, two

adjacent NLGN3 molecules that are related by twofold crystallographic symmetry form a dimer in a manner similar to apo-NLGN3 ECD. Consequently, PTPδA3B– and NLGN3 form a W-shaped complex

with a 2:2 stoichiometry (Fig. 3a and Supplementary Fig. 5a). The Ig1–Ig3 domains of PTPδ interact extensively with the α/β-hydrolase-fold core of NLGN3 (Fig. 3b, c). The Ig2 domain of PTPδ

also contacts the C-terminal tail of NLGN3 ECD (Fig. 3b). The NLGN3–PTPδ interface buries a surface of 1540 Å2 in total. Phe170 and the aliphatic portions of Arg75, Glu77, Arg90, and Gln92

in PTPδ Ig1 form a hydrophobic pocket to accommodate the side chain of Leu320 in NLGN3 (Fig. 4a and Supplementary Fig. 5b). Leu141, Pro221, and Tyr225 of PTPδ Ig2 form a hydrophobic

interface with Tyr302, Val305, and Ile355 in the α/β-hydrolase-fold core of NLGN3 (Fig. 4b and Supplementary Fig. 5c). In addition, the C-terminal tail of NLGN3 ECD lies on the meA3

insertion in PTPδ Ig2 (Fig. 4c and Supplementary Fig. 5d). The C-terminal tail of NLGN3 ECD is disordered in the apo-NLGN3 ECD structure, suggesting that it undergoes a disorder-to-order

transition upon binding to PTPδ. Met614 and Phe615 of NLGN3 appear likely to form hydrophobic interactions with Leu153, Leu185, Ile190, and the aliphatic portions of Thr151 and Ser187 in

PTPδ Ig2. Ile190 is included in the meA3 insertion. The contribution of the meA3 insertion to the binding is consistent with the results of our CSB and cell aggregation assays (Fig. 2b–d),

which showed lower binding of PTPδA–B– to NLGN3 compared with PTPδA3B–. The relative orientation of Ig3 to Ig2 in the NLGN3-bound PTPδA3B– is totally different from that in the apo-PTPδA3B–

(ref. 24): the Ig3 domain of PTPδ rotates by ~160 degrees upon binding to NLGN3 (Fig. 3c)33. Val257, Met261, and Ile281 of PTPδ Ig3 form a hydrophobic patch to interact with Gly371, Leu374,

and Tyr474 of NLGN3 (Fig. 4d). Ser236 and Arg283 of PTPδ may form hydrogen bonds with Asn375 and the main chain of Gly475 in NLGN3, respectively. In addition, PTPδ Arg234 is positioned close

enough to form a hydrogen bond with Glu372 of NLGN3. The domain rearrangement of PTPδ Ig3 allows both PTPδ Ig2 and Ig3 to simultaneously interact with NLGN3. The relative spacing between

the Ig2 and Ig3 domains appears to be adjusted by the length of the meB-lacking linker connecting these two domains; the meB-containing linker is likely to be more flexible with an extended

spacing (9 Å longer than the meB-lacking linker), as deduced from the comparison with the IL1RAPL1- and IL-1RAcP-bound PTPδA9B+ (Fig. 3c)24. DISSECTION OF THE NLGN3–PTPΔ AND NLGN3–NRXN

PATHWAYS The amino-acid sequences of the NRXN-binding site are highly conserved among all NLGNs including NLGN3 (Supplementary Fig. 4c). The (potential) NRXN-binding site of NLGN3 comprises

His279–Glu281, Asp362, and Gln370–Asp377 (Fig. 4d), which correspond to Tyr295–Glu297, Asp387, and Gln395–Asp402 of NLGN1 (Fig. 4e) or Tyr268–Glu270, Asp351, and Gln359–Asp366 of NLGN4 (Fig.

 4f), respectively. The binding site of NLGN3 for PTPδ Ig3 overlaps with the (potential) NRXN-binding site. The bound PTPδ Ig3 covers Gln370–Asp377 (Fig. 4d). Specifically, Gly371, Glu372,

Leu374, and Asn375 of NLGN3 interact directly with PTPδ Ig3. Moreover, Asp362 of NLGN3, which is equivalent to Asp387 of NLGN1, contacts Val143 of PTPδ in close proximity to the PTPδ

Ig2–NLGN3 interface (Fig. 4b). These structural features are consistent with the results of the abovementioned cell surface-binding assay of NLGN3 LND (Supplementary Fig. 2e) and the ITC

experiments mentioned below. Based on the crystal structures of the NLGN–NRXN and NLGN3–PTPδ complexes, we designed three distinct types of NLGN3 mutants to dissect the NRXN- and

PTPδ-meB(–)-mediated pathways. The first type is the H279A S280A E281A triple mutation (hereafter referred to as HSE/AAA). His279, Ser380, and Glu281, which comprise a loop in the proximity

of the common interaction site for PTPδ and NRXNs, appear to contribute to binding to NRXNs (Fig. 4d) but not to PTPδ. NLGN1 Glu297 and NLGN4 Glu270, which are equivalent to NLGN3 Glu281,

form a hydrogen bond with NRXN1β Arg109 (Fig. 4e, f)34,35, which is conserved in NRXN2 and NRXN3. In fact, the NLGN3 HSE/AAA mutation abolished the interaction with all NRXNs without

affecting the affinity to PTPδA3B– (_K_D = 1.4 µM) (Figs. 2i, 4g, and Supplementary Fig. 6c–e). The second group of mutants contains the M614I F615S or M614A F615A double mutation (hereafter

referred to as MF/IS or MF/AA, respectively), which disturbs the interface between PTPδ Ig2 and the C-terminal tail of NLGN3 ECD without any effect on the interaction with NRXNs. In fact,

PTPδA3B– failed to pull down the MF/IS and MF/AA mutants of NLGN3 (Fig. 4g). The _K_D of the NLGN3 MF/IS mutant for PTPδA3B– was 27 µM, which is 6 times lower than that of wild-type NLGN3,

whereas that for NRXN1β was 2.3 µM, which is comparable to the affinity of wild-type NLGN3 (Fig. 2i and Supplementary Fig. 6a). The MF/AA mutation more severely blocked the PTPδA3B–

interaction than the MF/IS in the CSB assay (Supplementary Fig. 6e). The third is the D362A E372A N375A triple mutation (hereafter referred to as DEN/AAA), which disturbs a common

interaction site for PTPδ and NRXNs. No binding of the NLGN3 DEN/AAA mutant to NRXN1β or PTPδA3B– was detected in the pulldown and ITC experiments (Fig. 4g and Supplementary Fig. 6b, e).

Altogether, we successfully obtained NLGN3 mutants with different binding selectivity for PTPδA3B– and NRXN1β as potential molecular tools for dissecting the canonical and non-canonical

transsynaptic signaling of NLGN3. DIFFERENTIAL EFFECTS ON SOCIALITY BY BLOCKADE OF THE CANONICAL AND NON-CANONICAL NLGN3 SIGNALING To address the physiological roles of the canonical

NLGN3–NRXN and non-canonical NLGN3–PTPδ pathways in social development, we generated knock-in mutant mice on a pure C57BL/6 genetic background with the HSE/AAA mutation (_Nlgn3__hse_ line)

and MF/AA mutation (_Nlgn3__mf_ line), respectively, using CRISPR/Cas9 genome editing (Supplementary Fig. 7). The HSE/AAA and MF/AA mutations did not alter the stability of the NLGN3 protein

itself in the mouse brain (Supplementary Fig. 7d, g). We confirmed the impairment of the canonical and non-canonical synaptogenic pathways in these mutant mice by coculture assays using

NRXN1β and PTPδA3B– beads, respectively. These mutations did not seem to change background staining signals for Shank2 and gephyrin in cultured neurons (Supplementary Fig. 7h, i). In

cortical neurons from _Nlgn3__hse_ male mice, NRXN1β-beads failed to induce accumulation of NLGN3 while Shank2 and gephyrin signals on the NRXN1β-beads were attenuated but not completely

abolished (Fig. 5a, c). In contrast, the PTPδA3B–-mediated accumulation of NLGN3 and gephyrin, but not Shank2, was rather upregulated (Fig. 5a, c). The residual postsynapse-inducing activity

of NRXN1β beads in the _Nlgn3__hse_ cortical neurons was probably mediated by other NRXN1β ligands such as NLGN1, NLGN2, NLGN4 and LRRTMs. In cortical neurons from _Nlgn3__mf_ male mice,

PTPδA3B– beads failed to induce NLGN3, Shank2, and gephyrin accumulation, while NRXN1β-induced NLGN3 and gephyrin accumulation was markedly enhanced (Fig. 5b, d). Consistently, anti-NLGN3

antibody failed to coimmunoprecipitate PTPδ in the _Nlgn3__mf_ mice brain, whereas it coimmunoprecipitated more PTPδ in the brains of the _Nlgn3__hse_ mutants than in those of wild-type

littermates (Fig. 5e). These results suggest that the canonical NLGN3–NRXN and non-canonical NLGN3–PTPδ transsynaptic pathways are actually impaired in the _Nlgn3__hse_ and _Nlgn3__mf_

lines, respectively. Furthermore, the canonical and non-canonical pathways seem to counterbalance each other for inhibitory synapse formation. Next, the development of social behaviors in

these mutant mice was assessed using the three-chamber social approach test36. In this test, a wire cage with a stranger mouse was placed in one side chamber and an empty cage was placed in

the other side chamber. Both _Nlgn3__hse_ mutants and their wild-type littermates spent more time in the chamber with the stranger mouse than in the chamber with the non-social empty cage

(Fig. 5f). However, unexpectedly, _Nlgn3__hse_ mutants spent significantly more time in the chamber with the stranger (Fig. 5f) and more preferentially stayed around the stranger cage than

the littermate controls (Fig. 5g and Supplementary Fig. 8a–c, g), suggesting that disruption of the canonical NLGN3–NRXN interaction enhances sociability development. In contrast, in

_Nlgn3__mf_ mutant mice impairing the non-canonical PTPδ-pathway, the time spent in the chamber with the stranger mouse was not significantly longer than that with non-social empty cage,

whereas their wild-type littermates spent more time in the chamber with the stranger (Fig. 5h), suggesting no obvious enhancement of social preference in the _Nlgn3__mf_ mutants. Although

the social-behavioral phenotype of _Nlgn3__mf_ mice was not robust, _Nlgn3__mf_ mice showed significantly shorter duration in each stranger-cage-sniffing behavior than their wild-type

littermates (Fig. 5h,i and Supplementary Fig. 8d–f, h). To further characterize the social behavior of the mutant mice, the reciprocal social interaction was recorded and analyzed using a

3D-video-based markerless motion capture system (Fig. 5j–n)37. In the test, both _Nlgn3__hse_ and _Nlgn3__mf_ mutant mice spent more time in proximity with the other mice than their

wild-type littermates (Fig. 5j, l and Supplementary Fig. 8i, j). We then examined a ‘defensive upright posture’38 by measuring elevated head-head contacts during the test. Both _Nlgn3__hse_

and their littermate wild-type mice showed comparable numbers of the elevated head-head contacts throughout the test period while _Nlgn3__mf_ mice exhibited the contacts more frequently than

their wild-type littermates in the middle of the test session (Fig. 5m, n). These results suggest both _Nlgn3__hse_ and _Nlgn3__mf_ mutants showed increased social interaction but their

qualities seem different. Regarding ASD-related behavioral phenotypes other than social abnormalities, enhanced motor routine learning is observed in _Nlgn3_ KO and _Nlgn3__R/C_ mice11. We

therefore subjected our mutant mice to the accelerating rotarod test, in which the rotation speed increased up to 80 rpm over 300 s (Fig. 5o, p). _Nlgn3__mf_ mice showed a significantly

higher performance than wild-type littermates in this accelerating condition, while _Nlgn3__hse_ mice exhibited a comparable performance to that of their wild-type littermates, suggesting

that non-canonical NLGN3-PTPδ signaling is responsible for the enhanced motor learning. THE AUTISM-RELATED _NLGN3_ R/C MUTATION DISRUPTS THE NON-CANONICAL NLGN3–PTPΔ PATHWAY The molecular

pathogenic mechanism of the ASD-related R451C mutation remains unclear. Therefore, we examined the effect of the R451C mutation on the canonical and non-canonical synaptogenic pathways of

NLGN3. Arg448 in mouse NLGN3, equivalent to Arg451 in human NLGN3, is located in the helix next to the dimer interface (Fig. 6a and Supplementary Fig. 4a). The side chain of Arg448 faces the

protein core and forms a stacking interaction with Trp495. The R/C mutation seems to affect this local structure in close proximity to the dimer interface. In SEC purification, the R/C

mutant of mouse NLGN3 was eluted as two peaks: the first, broad peak corresponds to higher-molecular-weight species larger than the wild-type dimer, whereas the second, sharp peak

corresponds to the monomer (100 kDa; determined by SEC-MALS; Supplementary Fig. 4b). Structurally, the R/C mutation may affect the stability and dimeric assembly of NLGN3. Functionally, the

R/C mutation reduced the pulldown efficiency by NRXN1β and PTPδA3B– to 40% and 20% of wild-type, respectively (Fig. 6b). In the beads-neuron coculture assay, the staining signals of both

Shank2 and gephyrin on the PTPδA3B– beads were abolished in cortical neurons from _Nlgn3__R/C_ mutant mice (Fig. 6c). In contrast, the NRXN1β-induced accumulation of Shank2 and gephyrin was

unaltered and significantly increased in _Nlgn3__R/C_ cortical neurons, respectively. These results suggest that the _Nlgn3_ R/C mutation impairs the non-canonical pathway. The resemblance

of the synaptogenic properties of _Nlgn3__R/C_ and _Nlgn3__mf_ cortical neurons led us to further explore whether _Nlgn3__hse_ and _Nlgn3__mf_ mutant mice exhibit the ASD-related

endophenotypes observed in the _Nlgn3__R/C_ mice, such as selective increase in the expression of inhibitory synaptic proteins in the forebrain and GABAA receptor-mediated inhibitory

synaptic transmission in the cerebral cortical layer 2/3 neurons15. In _Nlgn3__hse_ mutant mice, no obvious changes in the expression levels of synaptic proteins in the forebrain and

synaptic transmissions in the cerebral cortical layer 2/3 neurons were observed (Fig. 6d, e). In contrast, in the forebrain of _Nlgn3__mf_ mutant mice, the expression of inhibitory synaptic

proteins vesicular GABA transporter (VGAT) and gephyrin, was selectively upregulated, while those of the excitatory synaptic proteins vesicular glutamate transporter 1 (VGluT1) and PSD95

remained unaltered (Fig. 6d). Consistently, immunostaining signal ratios for VGAT compared to VGluT1 in the cerebral cortex were higher in the _Nlgn3__mf_ mutants than littermate controls

(Supplementary Fig. 7j, k). On the contrary, the frequencies of both miniature excitatory and inhibitory postsynaptic currents (mEPSC and mIPSC) were increased in _Nlgn3__mf_ mutants (Fig. 

6f). The biochemical and histochemical phenotypes of _Nlgn3__mf_ mice, but not of _Nlgn3__hse_ mutants, seem similar to those of the _Nlgn3__R/C_ ASD model mice, though electrophysiological

ones are likely different. DISCUSSION NRXNs and type IIA RPTPs are the two major presynaptic hub molecules for organizing and specifying synapses through interactions with their cognate

postsynaptic ligands5,6. In this study, we identified an interaction between PTPδ and NLGN3, one of the best-characterized postsynaptic binding partners of NRXNs39,40. This non-canonical

interaction between NLGN3 and PTPδ is exclusively dependent on the deletion of the meB peptide and prefers meA peptide insertions in the Ig-like domains of PTPδ (Fig. 2). Our structural

analyses suggest that the deletion of the meB peptide confines the spacing between Ig2 and Ig3 of PTPδ for simultaneous interaction with NLGN3, whereas the meA3 peptide in PTPδ Ig2

contributes to direct hydrophobic interaction with the C-terminal tail of the NLGN3 ECD. In fact, among the PTPδ-meB(–)s, the meA3-containing one showed the strongest NLGN3-binding activity

in the CSB assay (Fig. 2c). Conversely, the synaptogenic activity of the PTPδ-meB(–)s became stronger as the length of the meA peptide increased (Fig. 1b), despite the observation that all

the PTPδ-meB(–)s required NLGN3 for their synaptogenic activities (Supplementary Fig. 3). Therefore, additional factors may exist that reconcile this discrepancy between NLGN3-binding

activity and the synaptogenic activity of PTPδ-meB(–)s. The NRXN-binding interface of NLGN3 overlapped with the PTPδ-binding interface and the shared site was crucial for binding to both

proteins (Fig. 4d–f). This steric hindrance prevents NLGN3 from simultaneous interaction with NRXNs and PTPδ-meB(–)s, which may provide a molecular basis for the mutual counterbalancing of

the canonical and non-canonical NLGN3 transsynaptic signaling for the developmental regulation of sociality, as discussed below. Structural comparison between the NLGN3–PTPδA3B– and

NLGN1–MDGA1 complexes show an overlap of their binding interfaces (Supplementary Fig. 2g), and MDGA1 can weakly interfere with the binding between NLGN3 and PTPδA3B–, as suggested by the

competitive cell surface-binding assay mentioned above (Supplementary Fig. 2f). MDGA1 might function as a negative regulator for the PTPδ-meB(–)s–NLGN3 pathway as well as for the NRXN–NLGN

pathway. The _Nlgn3__hse_ mutants impairing the canonical NLGN3-NRXN signaling showed increased social preference, whereas impairment of non-canonical signaling in the _Nlgn3__mf_ mutants

caused a decrease in social preference and rather negative social response as defensive upright postures (Fig. 5f–n and Supplementary Fig. 8), suggesting the canonical and non-canonical

NLGN3 signaling pathways contribute to social development in a different direction. Given that PTPδ-meB(–)s and NRXNs exhibited comparable _K_D values and sterically competed with each other

for binding to NLGN3, PTPδ-meB(–)-mediated synaptogenesis is expected to be rather enhanced without the NLGN3–NRXN interaction in _Nlgn3__hse_ mutant mice. In fact, more NLGN3-PTPδ complex

was formed in the synaptosomal fraction of the _Nlgn3__hse_ mouse brain and PTPδA3B–-mediated inhibitory synaptogenesis was increased in cultured _Nlgn3__hse_ neurons (Fig. 5a–e).

Conversely, NRXN1-mediated inhibitory synaptogenesis was increased in the _Nlgn__mf_ neurons, which lacked PTPδA3B–-mediated synaptogenesis. These observations support the idea that the

canonical and non-canonical synaptogenic pathways of NLGN3 actually compete and counterbalance with each other at synapse-level in the developing brain. Therefore, it may be reasonable to

assume that the competition between the canonical and non-canonical NLGN3 synaptogenic signaling shapes local circuits within some brain regions, which, in turn, contributes to bidirectional

regulation of social development. Cortical neurons from the humanized _Nlgn3__R/C_ ASD model mice showed similar synaptogenic properties to those from _Nlgn3__mf_ mice, i.e., impaired

PTPδ-meB(–)-mediated synaptogenesis and enhanced NRXN1-mediated inhibitory synaptogenesis (Fig. 6c). Furthermore, _Nlgn3__mf_ mice partly recapitulated the biochemical and histochemical

features observed in the _Nlgn3__R/C_ ASD model, such as increased expression of inhibitory synaptic proteins in the forebrain; however, the synaptic phenotype in the cerebral cortices

seemed different between the _Nlgn3__R/C_ mice and _Nlgn3__mf_ mice, in which the enhancement of synaptic transmission was not restricted to inhibitory synapses (Fig. 6d, f). Therefore, the

selective disruption of the non-canonical NLGN3 pathway and the concomitant upregulation of the canonical pathway may cause the ASD-related endophenotypes of _Nlgn3__R/C_ mice. The R/C

mutation causes ~90% decrease of the amount of NLGN3 protein in the mouse brain, probably due to disruption of proper dimeric conformation (Supplementary Fig. 4b) and missorting and

degradation of the mutated proteins12,15. The upregulation of NRXN1-mediated inhibitory synaptogenesis in _Nlgn3__R/C_ neurons, despite of the decreased surface expression and NRXN1 binding

affinity of the NLGN3 R451C protein, may suggest some ligand-level compensation mechanism involving other inhibitory postsynaptic NRXN1 ligands such as NLGN2 and Cblns. In contrast to the

redundant interactions of NRXNs to all the NLGN family members, PTPδ-meB(–)s selectively interact with NLGN3 among NLGN family members and the synaptogenic activity of PTPδ-meB(–)s depends

thoroughly on NLGN3 (Supplementary Fig. 3). Therefore, the substantial reduction of NLGN3 protein by the R/C mutation may selectively impair the non-canonical NLGN3 pathway with no

functional compensation by other NLGNs. The robust increases of inhibitory synaptic proteins in the forebrain of _Nlgn3__R/C_ or _Nlgn3__mf_ mutants, despite of proportionally very low

contribution of the non-canonical synaptogenic pathway, imply some circuit or system-level mechanisms to compensate for the lack of the non-canonical NLGN3 pathway in these mice. The

non-canonical versus canonical NLGN3 signaling model for social development could account for the phenotypic differences between _Nlgn3__R/C_ and _Nlgn3_ KO mice15, in which both NLGN3

signaling pathways are expected to be abolished. The social phenotype of the _Nlgn3__R/C_ model seems to depend largely on the genetic background, as social deficits are observed on a mixed

129Sv/C57BL/6 background, but not on a pure C57BL/6 background15,41,42,43, which may relate to the non-robust social defect in _Nlgn3__mf_ mutants on a C57BL/6 background in this study. In

contrast, enhanced learning of motor routines, another aspect of the ASD-related behavioral phenotype observed in the _Nlgn3_ KO and _Nlgn3__R/C_ mice, likely regardless of genetic

background, was selectively recapitulated by disruption of the non-canonical pathway in _Nlgn3__mf_ mice (Fig. 5o,p). Therefore, the balancing mechanism of the canonical and non-canonical

NLGN3 signaling associated with social development and the non-canonical pathway-selective regulatory mechanism for repetitive behaviors would be potential therapeutic targets for ASD.

Alternative splicing of ultra-short exons, called microexons, is most abundantly regulated in neuronal cells and is thought to modify the function of interaction domains of proteins in a

switch-like manner for neuronal differentiation and function44. Misregulation of neural-specific alternative splicing of microexons is observed in individuals with ASD45. In line with this,

we found that the meB and meA peptides of PTPδ regulated excitatory/inhibitory (E/I) synaptic target selection and the extent of synaptogenic activities, respectively. Dysregulation of meA/B

splicing would therefore result in the disturbance of neuronal wiring including an imbalance of E/I synaptogenesis and a change in the non-canonical NLGN3-mediated synapse organization for

ASD pathogenesis. Since the amount of the _Ptprd_ meB(–) mRNAs in the developing mouse brain is <5% of total _Ptprd_ mRNA20, only a small number of neurons may express _Ptprd_ meB(–)

variants. The identification and characterization of the subset of synapses expressing PTPδ-meB(–)s in certain brain regions of wild-type and our mutant mice will help to understand the

roles of the canonical and non-canonical NLGN3 signaling in each synapse as well as circuit basis of social development and repetitive behaviors linked to ASD pathogenesis. _Nlgn3_ KO and

_Nlgn3__R/C_ genetic mutations in mice have been shown to differently affect synaptic transmission at the calyx of Held synapse46 and to exert input-selective effects in the hippocampal CA1

pyramidal neurons17, implying a multiple transsynaptic ligand system for NLGN3 to regulate synaptic transmission and specificity in these synapses. Therefore, the competition mechanism

between NRXNs and PTPδ may underlie the NLGN3-mediated developmental and functional regulation of these synapses. The mPFC and nucleus accumbens (NAc) are reported to be implicated in social

behavioral changes in the _Nlgn3__R/C_ and _Nlgn3_ KO models18,47 and _Nlgn3__mf_ mutants exhibited a change of VGAT and VGluT1 expression ratio in the mPFC (Supplementary Fig. 7k).

Furthermore, impaired synaptic inhibition onto D1-dopamine receptor-expressing neurons in NAc is known to be responsible for the enhanced motor learning in both _Nlgn3__R/C_ and _Nlgn3_ KO

models11. Synaptic functional analyses of _Nlgn3__mf_ mice in these brain regions and neurons would help to clarify the causal linkage between the lack of the non-canonical pathway and

behavioral changes in sociability and motor learning. Recent structural studies have revealed that pre- and postsynaptic organizers form complexes in various combinations in a mutually

exclusive manner by steric clashes22,23,24,25,48. Therefore, multiple synapse organizer pathways interact with each other, providing a molecular basis for the resilient and plastic

development of neural networks. METHODS ANIMAL EXPERIMENTS Animal care and experimental protocols were approved by the Animal Experiment Committee of the University of Toyama (Authorization

No. A2016MED-58 and A2016OPR-3) and conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals of the University of Toyama. Mice were housed in a room with a 12-h

light/dark cycle (lights on at 7:00 a.m.) at 23 ± 3 °C and 30–60% humidity with access to food and water ad libitum. Wild-type and mutant mice were generated by crossing wild-type male mice

and heterozygous female mice. All biochemical, immunohistochemical, electrophysiological, and behavioral analyses were carried out with male mice. For behavioral testing, the male offspring

of mating pairs were weaned around one month, genotyped, and housed 4 (two pairs of wild-type and mutant mice) per cage. CONSTRUCTION OF EXPRESSION VECTORS The coding sequences of mouse

NLGN2, NLGN3, and NLGN4 were amplified by RT-PCR using total RNA prepared from the forebrains of mice at postnatal day (P) 11 with primer sets, Nlgn2-Cla-S/ Nlgn2-Sal-A,

Nlgn3-ERI-S2/Nlgn3-Sal-A, and Nlgn4-ERI-S/Nlgn4-RV-A, respectively, and cloned into pFLAG-CMV-1 (Sigma) to yield N-terminally FLAG-tagged expression vectors. The FLAG-NLGN1 expression vector

was previously described20. The coding sequences of ECDs of mouse PTPδ and NLGN splice variants were cloned into pEB6-Igκ-Fc49 to yield expression vectors for Fc fusion proteins. Expression

vectors for the mutated forms of NLGN3 were generated by PCR-based mutagenesis using the FLAG-, Fc- or hexahistidine (His6)-tagged NLGN3A (see “Crystallization”) as templates. The coding

sequences of ECD of mouse MDGA1, myc epitope, and His6 were cloned into pEBMulti-Neo (Wako chemicals) to yield expression vector for myc-His-tagged MDGA1-ECD. Other expression vectors were

previously reported20,21,50. Primer sequences used in this study are presented in Supplementary Table 3. CELL CULTURES AND COCULTURE ASSAY Primary cortical cultures were prepared from mice

at embryonic day (E) 18 or postnatal day (P) 0 essentially as described previously20. Cortical cells were placed on coverslips coated with 30 μg/mL poly-L-lysine and 10 μg/mL mouse laminin

at a density of 5 × 105 cells per well in a 24-well-dish for coculture assay. The cells were cultured in Neurobasal-A supplemented with 2% B-27 supplement (Invitrogen), 5% fetal calf serum

(FCS), 100 U/mL penicillin, 100 μg/mL streptomycin and 0.5 mM L-glutamine for 24 h, and then cultured in the same medium without FCS. Cultures of HEK293T cells were maintained as previously

described51. Expression vectors for Fc fusion proteins were transfected into HEK293T cells using NanoJuice transfection reagent (Novagen). After 5 days of culture, the culture media

containing 1–30 μg/mL Fc fusion proteins were filtrated and incubated with Protein A-conjugated magnetic particles (smooth surface, 4.0–4.5 μm diameter; Spherotech). Beads coupled with Fc or

Fc fusion proteins were added to cortical neurons at days in vitro (DIV) 13 to induce postsynaptic differentiation. After 24 h, cocultures were fixed and immunostained with rabbit

anti-Shank2 (Frontier Institute, Shank2-Rb-Af750, 1:200), mouse anti-gephyrin (Synaptic Systems, 47011, 1:1000) or mouse anti-NLGN3 (Biolegend, MMS-5166-100, 1:200) antibodies. At least two

independent neuronal cultures for each experimental condition were used for coculture experiments. Quantification of fluorescent signals of cocultures from _Nlgn3_ mutant mice was conducted

in a blind manner with respect to genotype. SCREENING OF PROTEINS INTERACTING WITH PTPΔA3B– Primary cortical cultures were prepared from ICR mice at P0 as previously described20 and were

placed on a cell culture dish coated with poly-L-lysine and laminin at a density of 1.5 × 107 cells per 100-mm dish. Magnetic beads coated with Fc or the ECD of PTPδA3B– fused to Fc were

added to cultured cortical neurons at DIV13 at a density of 5 × 107 beads per 100-mm dish. After 24 h, cultures were cross-linked with DTSSP (Pierce) and lysed according to previous

protocols26. Bead-bound proteins were washed and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The gel lanes were excised into two pieces, one including

the bait proteins and the other, reduced, alkylated, and digested with trypsin. The resulting peptides were analyzed by LC-MS/MS using an ESI ion trap mass spectrometer (LCQ Fleet, Thermo

Fisher Scientific). CSB ASSAY Expression vectors for FLAG-tagged NLGNs, PTPδA3B– and NRXN1β were transfected into HEK293T cells. Transfected cells were incubated with Fc fusion recombinant

proteins or control Fc (0.1 μM) in DMEM containing 10% FCS, 2 mM CaCl2, and 1 mM MgCl2 for 30 min at room temperature. After washing, cells were fixed with 4% PFA, immunostained with mouse

anti-FLAG (Sigma) and rabbit anti-human IgG (Rockland) antibodies, and then visualized with Alexa Fluor 488-conjugated donkey anti-mouse IgG and Alexa Fluor 555-conjugated donkey anti-rabbit

IgG antibodies (Invitrogen). For the competition assay, alkaline phosphatase tagged with Myc and His6 epitopes (AP-MH), ECDs of MDGA1, NRXN1β and PTPδA3B– tagged with Myc and His6 epitopes

(MDGA1-MH, NRXN1β-MH and PTPδA3B–-MH, respectively), and PTPδA3B–-Fc were added at concentrations of 0.01, 0.03, 0.1, 0.3, 1.0, and 3.0 μM. At least two independent experiments were

performed for each experimental condition. PULLDOWN ASSAY Soluble recombinant proteins were mixed at a concentration of 0.03 μM each in Hank’s balanced salt solution (HBSS) containing 2 mM

CaCl2 and 1 mM MgCl2 and incubated for 3 h followed by incubation with Protein A-Sepharose Fast Flow (GE Healthcare) for 1 h at room temperature. Subsequently, the sepharose suspensions were

washed five times with HBSS containing 2 mM CaCl2 and 1 mM MgCl2. Bound proteins were eluted by boiling in SDS sample buffer, separated by a 5–20% gradient SDS-PAGE gel, and analyzed by

Western blotting with horseradish peroxidase-conjugated mouse anti-His (MBL, M089-3, 1:1000). CELL AGGREGATION ASSAY HEK293T cells transfected with expression vectors for EGFP and PTPδ

splice variants and those with expression vectors for RFP and NLGN3 were mixed at a density of 4.0 × 106 cells/mL and incubated at room temperature with rotation. After 15 min, fluorescence

images were taken using a fluorescence stereomicroscope (M165 FC, Leica Microsystems). At least two independent experiments were conducted. CRYSTALLIZATION The gene encoding mouse NLGN3 ECD

(residues 1–684) with the C-terminal His6 tag was amplified by PCR from cDNA (accession No. NM_172932.4) with primers, Xho-Nlgn3-S and Not-Nlgn3-2052 (Supplementary Table 3), and cloned into

the pEBMulti-Neo vector. The genes encoding the various lengths of mouse PTPδA3B– ECD were cloned into the pEBMulti-Neo vector as described previously24. All proteins were transiently

expressed using FreeStyle 293-F cells (Invitrogen). The proteins were purified from culture media by Ni-NTA (Qiagen) metal affinity resin following the standard protocol, and dialyzed

against a buffer containing 20 mM Tris-HCl buffer (pH 8.0) containing 150 mM NaCl. The purified proteins were concentrated to ~100 µM, flash frozen in liquid N2, and stored at –80 ˚C until

use. For crystallization of apo-NLGN3 ECD, the protein solution was mixed at a 1:1 ratio with reservoir solution containing 15% PEG3350, 0.2 M ammonium chloride, and 0.1 M MES-Na (pH 6.0).

The crystals of apo-NLGN3 ECD were grown at 20 °C by the sitting-drop vapor diffusion method. The crystals were cryoprotected by transferring crystals into a reservoir solution supplemented

with 30% ethylene glycol. For crystallization of the NLGN3 ECD–PTPδ Ig1–Fn1 complex, each protein was mixed at an equimolar ratio. This mixture was further mixed at a 1:1 ratio with a

reservoir solution containing 10% PEG3350, 0.1 M ammonium iodide, and 0.1 M MES-Na (pH 6.0). The crystals of the NLGN3–PTPδ Ig1–Fn1 complex were grown at 20  °C by the sitting-drop vapor

diffusion method. The crystals were cryoprotected by transferring crystals into reservoir solution supplemented with 25% ethylene glycol and flash frozen in liquid N2. CRYSTALLOGRAPHY All

diffraction data were collected at 100 K at BL41XU in SPring-8 and processed with the HKL200052 and CCP4 program suite53 (Supplementary Table 2). The apo-NLGN3 ECD structure was determined

by the molecular replacement method using the NLGN1 ECD (PDB 3BIX) as the search model with the program Molrep54. The NLGN3 ECD–PTPδ Ig1–Fn1 complex structure was determined by the molecular

replacement method using the apo-NLGN3 ECD, PTPδ Ig1–Ig2 (PDB 2YD6)55, PTPδ Ig3 (PDB 4YFD)24 and PTPδ Fn1 (PDB 4YFE)24 as the search models. Model building and refinement were carried out

using the programs Coot56 and Phenix57, respectively. The final model of apo-NLGN3 ECD was refined at 2.76 Å to _R_work and _R_free of 0.218 and 0.251, respectively. The final model of the

NLGN3 ECD–PTPδ Ig1–Fn1 complex was refined at 3.85 Å to _R_work and _R_free of 0.253 and 0.289, respectively. Even in this moderate resolution, positional refinement could be applied without

secondary structure restraint or reference structure restraint for the final model. During the initial iterative cycles of model improvement and refinement, reference structure restraint

was used to improve the main chain geometry. Buried surface areas were calculated by the program PISA58. The stereochemistry of the final models was assessed by the program MolProbity59. No

residues are in the disallowed region for both structures. Structural figures were prepared with the program PyMol (Schrödinger, LLC). ITC ANALYSIS The NLGN3 ECD (wild-type or mutant) and

PTPδ Ig1–Fn1 (A3B– or A9B+) were dialyzed against 20 mM Tris-HCl buffer (pH 8.0) containing 150 mM NaCl. This dialysis process was repeated at least three times. For the measurement of the

interaction between NLGN3 and NRXN1β, both proteins were dialyzed against the same buffer in the presence of 1 mM CaCl2. The ITC measurements were performed at 25 °C with MicroCal Auto-iTC

(GE Healthcare). Either wild-type or mutant NLGN3 (20 µM) was placed in the cell. PTPδ or NRXN1β solution (200 µM each) was added to the cell in a series of injections. Binding constants

were calculated by fitting the data with a one-site binding model using the software Origin (MicroCal). Titration was performed twice with similar kinetics for wild-type proteins but once

for NLGN3 mutant proteins due to technical difficulty related to protein expression, purification, and concentration. SEC-MALS Purified NLGN3 ECD (wild-type or R/C mutant) was concentrated

to 1.5 or 1.1 g/L, respectively, and applied onto an ENrich SEC 650 (10 × 300 mm) column (Bio-Rad) pre-equilibrated with 20 mM Tris-HCl buffer (pH 7.5) containing 150 mM NaCl. The MALS data

were collected on a DAWN HELEOS 8+ detector (Wyatt Technology) with an RF-20A UV detector (Shimadzu) and analyzed by the program ASTRA (Wyatt Technology). GENERATION OF NEURON-SPECIFIC NLGN3

KO MICE Neuron-specific _Nlgn3_ exon 7-deleted mice were obtained by crossing heterozygous female XX_Nlgn3_fR/C mice15 with nestin-Cre transgenic male mice (+/_nestin-cre_) (Jackson

Laboratories). The autism-related _Nlgn3__R/C_ mice were generated by the same crossing. Immediately after genotyping by PCR using tail-tip DNA with primers for _Nlgn3_ (Nlgn3-GT-A,

Nlgn3-GT-B, and Nlgn3-GT-C, Supplementary Table 3) and _Cre_ (Cre-S and Cre-A, Supplementary Table 3) at P0, the mice were sacrificed for cerebral cortical cultures. GENERATION OF _NLGN3_

_MF_ AND _NLGN3_ _HSE_ KNOCK-IN MICE The injection of guide RNAs, Cas9 RNA, and ssODNs into C57BL/6N mouse zygotes was performed as previously described60. The combinations of guide RNA and

ssODN were as follows: gRNA-E6T2 and E6-ssODN1, and gRNA-E8T2 and E8-ssODN for the HSE/AAA and MF/AA mutations, respectively (For nucleotide sequences, see Supplementary Table 3). The tail

or ear tip of the F0 mice were alkaline lysed and subjected to cleaved amplified polymorphic sequence analyses using the following primer sets and restriction enzymes:

Nlgn3-E6-U2/Nlgn3-E6-L2 and PvuII, and Nlgn3-E8-U2/Nlgn3-E8-L2 and BspHI, for the detection of the HSE/AAA and MF/AA mutations, respectively. The F0 mosaic mice were crossed with wild-type

C57BL/6N mice to generate F1 founder mice. Exon 6, exon 8, and intron 6 of the _Nlgn3_ gene of the F1 mice were sequenced to confirm the precise knock-in events. The F2 heterozygous female

mice were mated with wild-type C57BL/6N male mice to generate male mice for phenotypic analyses. WESTERN BLOTTING The S1 fractions were prepared from 6-week-old male mice as previously

described61, and protein concentrations were quantified using the BCA protein assay kit (PIERCE, Rockford, USA). Equal amounts of protein were separated by SDS-PAGE, transferred to PVDF

membranes, and probed with primary antibodies followed by horseradish-peroxidase-conjugated secondary antibodies. The blots were developed and imaged using a Luminescent Image Analyzer

LAS-4000 mini (Fujifilm, Tokyo, Japan). Blots were stripped and reprobed with antibodies against β-actin to normalize for differences in loading. The relative levels of each protein were

determined by densitometric analysis using serially diluted protein samples on the same blots. The primary antibodies used in this study were as follows: mouse anti-NLGN3 (BioLegend

(MMS-5166-100), 1:200 dilution), mouse anti-Gephyrin (Synaptic Systems (147111), 1:5000 dilution), mouse anti-VAMP2 (Synaptic Systems (104211), 1:2000 dilution), rabbit anti-actin (Santa

Cruz Biotechnology (sc-1616-R), 1:1000 dilution), rabbit anti-PSD95 (a gift from Dr. Watanabe, Hokkaido University, 1:600 dilution), goat anti-VGAT (a gift from Dr. Watanabe, Hokkaido

University, 1:600 dilution) and rabbit anti-VGluT1 (custom-made antibody for amino acid residues 542–560 of rat VGluT1 (GATHSTVQPPRPPPPVRDY), Eurofins Genomics, 1:1000 dilution). For both

_Nlgn3__mf_ and _Nlgn3__hse_, four independent experiments using two litters (eight litters in total) were performed and pooled for densitometric analysis. Densitometric analysis of western

blots was performed using Image J 1.46 software62 in a genotype-blind manner. Uncropped blots are presented in the Source Data file. REAL-TIME PCR Quantitative real-time PCR was performed

using cDNAs prepared from olfactory bulb, cerebral cortex, hippocampus, striatum, thalamus, medulla oblongata, and cerebellum of 2 month-old mice with primer sets, Ptprd-725-S/Ptprd-941-A

and Gapdh-S/Gapdh-A for total _Ptprd_ and _Gapdh_, respectively. For amplification of the _PtprdA3B__–_, the cDNAs were first subjected to 28 cycles of PCR with primers Ptprd-460-S and

Ptprd-1907-A, and treated with Alu I and Rsa I to digest meA6 and/or meB-including _Ptprd_ splice variants. Secondary PCR was performed using primers, PtprdA3-S and Ptprd-B-A. All the

quantitative real-time PCR reaction was carried out on MX3000P (Stratagene) with GeneAce SYBR qPCR kit (Nippongene) to quantify the relative expression levels using the comparative threshold

cycle (Ct) method. Ct values of total _Ptprd_ were normalized by those of _Gapdh_. The relative expression of _PtprdA3B__–_ in each brain region was quantified by comparing Ct values for

_PtprdA3B__–_ and total _Ptprd_. For primer sequences, see Supplementary Table 3. COIMMUNOPRECIPITATION The crude synaptosomal (S2′) fractions were prepared from striatums of 2–3 male mice

of 4-week old as previously described61 and treated with 1 mM DTSSP in a buffer containing 0.32 M sucrose, 4 mM HEPES (pH7.4) and 3 mM CaCl2 for 30 min followed by incubation with 50 mM

Tris-HCl (pH7.5) for 20 min. Synaptic proteins were solubilized with radioimmunoprecipitation (RIPA) buffer (50 mM Tris-HCl, pH8.0, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1%

Nonidet P-40, 3 mM CaCl2) containing protease inhibitors (Complete EDTA-free; Roche). Soluble fractions containing 0.8 mg protein were incubated with 3 μg rabbit anti-NLGN3 (Frontier

Institute, Nlgn3-Rb-Af1010) or control rabbit IgG (Rockland, 609-4103) for 8 h followed by 4-h incubation with ~50 μg protein-G sepharose. The sepharose was washed five times with RIPA

buffer. The bound proteins were boiled in SDS sample buffer containing 0.1 M DTT for 15 min to cleave DTSSP and to elute from the sepharose. The eluates were separated by SDS-PAGE, and

analyzed by Western blotting with mouse anti-NLGN3 (Biolegend, 1:400 dilution) and mouse anti-PTPRD (Abcam (ab233806), 1:500 dilution) antibodies. For each _Nlgn3__mf_ and _Nlgn3__hse_ line,

four independent experiments using one litter (4 litters in total) were performed and pooled for densitometric analysis. Uncropped blots are presented in the Source Data file.

IMMUNOHISTOCHEMISTRY Under deep isoflurane anesthesia, mice were perfused transcardially with 4% paraformaldehyde (PFA) in 0.1 M sodium phosphate buffer. After post-fixation with 4% PFA for

2 h, 50 μm sections were prepared with microslicer (VT1000S; Leica). Sections were incubated with rabbit anti-VGluT1 (a gift from Dr. Watanabe, Hokkaido University, 1:600 dilution) and goat

anti-VGAT (a gift from Dr. Watanabe, Hokkaido University, 1:600 dilution), followed by incubation with Alexa Fluor 555-conjugated and Alexa Fluor 647-conjugated secondary antibodies

(Invitrogen). For each _Nlgn3__mf_ and _Nlgn3__hse_ line, two independent experiments using one litter (2 liters in total) were performed and pooled for densitometric analysis. Images of

double immunofluorescence were taken with a confocal laser-scanning microscope (SP5II; Leica) in a genotype-blind manner. SLICE PREPARATIONS Mice (13-16-days old) were anesthetized with

sevoflurane anesthesia. After decapitation, their brains were removed from the skulls and rapidly submerged in an ice cold, oxygenated (95% O2–5% CO2 gas mixture) cutting solution containing

(in mM) 252 sucrose, 3 KCl, 1.24 NaH2PO4, 26 NaHCO3, 0.5 CaCl2, 6.3 MgSO4, 0.2 ascorbic acid, and 25 glucose. Coronal slices (300-µm thick) were cut on a vibrating blade microtome (PRO 7;

Dosaka EM, Kyoto, Japan) in ice-cold oxygenated cutting solution. The slices, containing layer 2/3 of the somatosensory cortex, were incubated in oxygenated standard artificial cerebrospinal

fluid (ACSF) containing (in mM) 126 NaCl, 3 KCl, 1.25 NaH2PO4, 26 NaHCO3, 2 CaCl2, 2 MgSO4, and 25 glucose at 34 °C for 30 min. Recordings were performed after following incubation at room

temperature for at least 30 min. ELECTROPHYSIOLOGICAL RECORDINGS The slices were transferred to a recording chamber with oxygenated ACSF continuously perfused at a flow rate of 1 ml/min at

room temperature. Whole-cell patch-clamp recording pipettes were pulled from 1.5-mm thin-wall glass capillaries on an electrode puller (PP-830; Narishige, Tokyo, Japan). The whole cell

recording pipettes contained (in mM) 145 KCl, 5 NaCl, 10 HEPES, 0.1 CaCl2, 10 EGTA, 4 Mg-ATP, and 0.3 Na-GTP, and 10 QX-314 with pH adjusted to 7.3 with KOH. The pipette resistance was 3–6 

MΩ. Neurons and recording pipettes were visualized using an infrared-differential interference contrast microscope (BX50WI; Olympus, Tokyo, Japan) with a ×40 water immersion objective and

charge-coupled device camera (C2741-79; Hamamatsu Photonics, Hamamatsu, Japan) and a real-time differential video microscopy processor (XL-20; Olympus). Pyramidal neurons in layer 2/3 of the

somatosensory cortex were identified by soma size and the single apical dendrite. Whole-cell voltage-clamp recording of the pyramidal neurons was performed with a holding potential of –70 

mV. Data were corrected and digitized with a sampling rate of 20 kHz using a patch-clamp amplifier and an A/D converter board (Axopatch 200B and Digidata 1200B; Axon Instruments, Union City,

CA). Signals were filtered at 1 kHz, and recorded on a hard disk using data acquisition and analysis software (pCLAMP 8; Axon Instruments). An Ag/AgCl reference electrode was placed near

the intermediate position between the inlet and outlet of the chamber. To isolate miniature synaptic events, tetrodotoxin (0.5 µM) was contained in the ACSF at all times. In addition, the

ACSF contained picrotoxin (50 µM) and 2-chloradenosine (2 µM) for measurements of excitatory postsynaptic currents (EPSCs), or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 20 µM) for

measurements of inhibitory postsynaptic currents (IPSCs). Miniature postsynaptic currents with an amplitude of >15 pA were detected on recording lasting 5 min or longer. Series resistance

compensation was performed as much as possible by the amplifier. Series resistance was measured by application of a voltage step pulse (15 ms duration) every 10 s. Recordings with a series

resistance of >20 MΩ or a leak current of >200 pA were discarded. All recordings and data analyses were performed in a genotype-blind manner. THREE-CHAMBER SOCIABILITY TEST The test

for sociability was conducted as previously described36. The apparatus comprised a rectangular, three-chambered box and a lid containing an infrared video camera (Ohara & Co.)63. Each

chamber was 20 × 40 × 22 cm and the dividing walls were made from clear Plexiglas, with small square openings (5 × 3 cm) allowing access into each chamber. One day before testing, the

subject mice were individually placed in the middle chamber and allowed to freely explore the entire apparatus for 10 min. In the sociability test, an unfamiliar C57BL/6J male (stranger)

that had no prior contact with the subject mouse was placed in one of the side chambers. The placement of the stranger in the left or right side chamber was systematically alternated between

trials. The stranger mouse was enclosed in a small, circular wire cage that allowed nose contact between the bars, but prevented fighting. The cage was 11 cm high, with a bottom diameter of

9 cm and bars spaced 0.5 cm apart. An identical empty wire cage was placed in the opposite chamber. A weighted plastic cup was placed on top of each cage to prevent the mouse from climbing

to the top. The subject mouse was first placed in the middle chamber and allowed to explore the three chambers for 10 min. The amount of time spent in each chamber and in each area within 5 

cm of the wire cage, the number of entries into each chamber and each area within 5 cm of the wire cage, total distance traveled, and average speed were recorded using Image J 1.47a software

(NIH). Cage sniffing behavior during each 10-min test period was manually scored from video images of 600 frames (1 s/frame) by an observer blind to genotype. Sniffing time was defined as

each frame in which a nose of a subject touched the wire cage. RECIPROCAL SOCIAL INTERACTION TEST AND 3D VIDEO-BASED ANALYSIS OF THE SOCIAL BEHAVIOR The 3D video of the behavior of pairs of

freely interacting mice was recorded and analyzed using the 3DTracker system37 (www.3dtracker.org). With the aid of the system, a 3D image of mice in a transparent acrylic recording chamber

(21  × 21 × 40 (H) cm), was reconstructed by integrating images captured by four depth cameras (Realsense R200, Intel Corp.) surrounding the chamber. Then, 3D positions of body parts (the

center of head, neck, trunk, and hip) of each mouse in each video frame were estimated by fitting skeleton models of the mouse to the 3D images semi-automatically. The 3D video-based

analysis enables quick and objective analysis of complex social behavior during the reciprocal social interaction. For the test, adult male mutant mice and their wild-type littermates were

used. The male mice were housed two to three animals per cage with their male littermates (wild-type and/or mutant mice) after weaning. A pair of mice for the test was randomly selected

under the following constraints: (1) the mice had the same genotype; (2) the mice had not been housed together; (3) the body weight difference between the mice was <10%. Prior to the test

day, the subject mice were individually placed in the recording chamber and allowed to freely explore for 20 min. In the test, a pair of mice were put in the recording chamber

simultaneously, and were allowed to freely interact for 30 min. Some of the mice were tested once again in a different day after changing the pair combination. In the data analysis, to check

the amount of the general social interaction, proximity events (distance of trunks of mice <7 cm) were counted. Although no obvious fighting was observed during the test, a proximity

event could occur with negative (aggressive) as well as positive (affiliative) interaction. To further analyze the quality of the interaction in the proximity, taking advantages of the 3D

video analysis, we also counted the defensive upright posture38, as measured by elevated head-head contacts, where both mice were standing (height of head – height of hip > 3 cm) and

facing in proximity (the distance of heads of mice <3 cm). The behavioral parameters were compared with two-way repeated measured ANOVA among six conditions: two groups (control and

mutant groups) × 3-time intervals (0–10, 10–20, 20–30 min after the onset of the test). Subsequent multiple post hoc comparisons were performed with simple main effect analyses. ROTAROD TEST

Rotarod test was conducted using an accelerating rotarod apparatus (UGO Basile, Comerio-Varese, Italy) according to a previous report11. Mice were placed on rotating drum and the rotating

speed (rpm) to fall was recorded with 5 min cutoff. Rotarod testing consisted of 12 trials with 3 trials per day over the course of 4 days. The speed of the drum accelerated from 4 to 40 rpm

over a 5 min period for the first 6 trials, then from 8 to 80 rpm over a 5 min period for the next 6 trials. IMAGE ACQUISITION, QUANTIFICATION, AND STATISTICS Image acquisition and

quantification were performed as described previously20,49. Statistical significance was evaluated by the Mann–Whitney _U_-test, Student’s _t_-test or one-way ANOVA followed by Tukey’s or

Dunnett’s post hoc test using JMP Pro software (version 15.0.0, SAS Institute Inc.). All the statistical tests used in this study were two-sided. Statistical significance was assumed when

_p_ < 0.05. The exact _p_ values for statistical tests used in this study are provided in Supplementary Table 4. REPORTING SUMMARY Further information on research design is available in

the Nature Research Reporting Summary linked to this article. DATA AVAILABILITY The coordinates and structure factors of apo-NLGN3 ECD and the NLGN3 ECD–PTPδ Ig1–Fn1 complex have been

deposited in the Protein Data Bank under the accession codes 7CEE and 7CEG, respectively. Other data, mouse strains, and custom-made rabbit anti-VGluT1 antibody generated in this study are

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repeatedly treated with propofol. _Transl. Regul. Sci._ 1, 46–57 (2019). Google Scholar  Download references ACKNOWLEDGEMENTS We thank Mses. Yumie Koshidaka, Emi Matsuura, Kayoko Awamori,

Shoko Kawakami, Mizuki Sendo, and Maina Demura for technical assistance, Prof. Masahiko Watanabe (Hokkaido University) for antibodies against PSD95, VGluT1, and VGAT, and Prof. Toshihide

Tabata (University of Toyama) for helpful suggestions. We also thank the beam-line staff at BL41XU of SPring-8 (Hyogo, Japan) for technical help during data collection and Enago

(www.enago.jp) for English language review. This work was supported in part by grants from JSPS/MEXT KAKENHI (JP25293057, JP19H04744, JP20H03350, JP20K21444 and JP16H06276 (AdAMS) to T.Y.,

JP16H04749 to A.Y., JP16H04676 to M.M., JP16H06534 to J.M., and JP24247014 to S.F.), JST PRESTO and the Takeda Science Foundation to T.Y. and JST CREST (JPMJCR12M5) to T.U. and S.F. AUTHOR

INFORMATION AUTHORS AND AFFILIATIONS * Department of Molecular Neuroscience, Faculty of Medicine, University of Toyama, Toyama, Japan Tomoyuki Yoshida, Ayako Imai, Hironori Izumi, Shiho

Iwasawa-Okamoto, Kenji Azechi & Hisashi Mori * Research Center for Idling Brain Science, University of Toyama, Toyama, Japan Tomoyuki Yoshida, Jumpei Matsumoto, Hisao Nishijo, Keizo

Takao & Hisashi Mori * JST PRESTO, Saitama, Japan Tomoyuki Yoshida & Katsuhiko Tabuchi * RIKEN Center for Biosystems Dynamics Research, Kanagawa, Japan Atsushi Yamagata * Division of

Bio-Information Engineering, Faculty of Engineering, University of Toyama, Toyama, Japan Juhyon Kim * Department of System Emotional Science, Faculty of Medicine, University of Toyama,

Toyama, Japan Shogo Nakashima, Jumpei Matsumoto & Hisao Nishijo * Department of Anatomy, Kitasato University School of Medicine, Kanagawa, Japan Tomoko Shiroshima * Research Institute

for Diseases of Old Age, Juntendo University Graduate School of Medicine, Tokyo, Japan Asami Maeda * Center for Research and Education on Drug Discovery, Faculty of Pharmaceutical Sciences,

Hokkaido University, Sapporo, Japan Fumina Osaka, Takashi Saitoh & Katsumi Maenaka * Laboratory of Biomolecular Science, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo,

Japan Katsumi Maenaka * SHIMADZU Bioscience Research Partnership, Innovation Center, Shimadzu Scientific Instruments, Bothell, WA, USA Takashi Shimada * Division of Membrane Physiology,

Department of Molecular and Cellular Physiology, National Institute for Physiological Sciences, National Institutes of Natural Sciences, Aichi, Japan Yuko Fukata & Masaki Fukata * Life

Science Research Center, University of Toyama, Toyama, Japan Keizo Takao * Department of Cellular Neurobiology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan Shinji

Tanaka & Shigeo Okabe * Department of Molecular and Cellular Physiology, Institute of Medicine, Academic Assembly, Shinshu University, Nagano, Japan Katsuhiko Tabuchi * Institute for

Biomedical Sciences, Interdisciplinary Cluster for Cutting Edge Research, Shinshu University, Nagano, Japan Katsuhiko Tabuchi & Takeshi Uemura * Division of Gene Research, Research

Center for Supports to Advanced Science, Shinshu University, Nagano, Japan Takeshi Uemura * Brain Science Laboratory, Research Organization of Science and Technology, Ritsumeikan University,

Shiga, Japan Masayoshi Mishina * Department of Chemistry, Graduate School of Science, Kyoto University, Kyoto, Japan Shuya Fukai Authors * Tomoyuki Yoshida View author publications You can

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can also search for this author inPubMed Google Scholar * Shuya Fukai View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS T.Y., T.S., A.I.,

S.I.-O. T.U., and K.A. performed biochemical, immunocytochemical, and cell biological analyses. T.Sh., Y.F., and M.F. performed mass spectrometry analysis. H.I., A.I., and T.Y. generated

gene-edited _Nlgn3__hse_ and _Nlgn3__mf_ mice. J.K. performed electrophysiological experiments and data processing. A.I., S.N., J.M., and K.Tak. performed behavioral experiments and data

analysis. A.Y., T.S., and A.M. performed sample preparation and crystallization. A.Y. and S.F. collected diffraction data, analyzed the collected data, and determined the structures. F.O.,

T.Sa., and K.M. performed ITC measurement. S.T., K.Tab. and S.O. helped to prepare and analyzing _Nlgn3__R/C_ and _Nlgn3_ KO mice. T.Y., A.Y., J.K., J.M., and S.F. wrote the manuscript with

editing by K.M., M.F., H.N., K.Tak, H.M., A.Y., M.M., S.F., and T.Y. T.Y. and S.F. designed and supervised the study. CORRESPONDING AUTHORS Correspondence to Tomoyuki Yoshida or Shuya Fukai.

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ARTICLE Yoshida, T., Yamagata, A., Imai, A. _et al._ Canonical versus non-canonical transsynaptic signaling of neuroligin 3 tunes development of sociality in mice. _Nat Commun_ 12, 1848

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