Calsyntenin-3 interacts with both a - and b -neurexins in the regulation of excitatory synaptic innervation in specific Schaffer collateral pathways

Calsyntenin-3 (Clstn3) is a postsynaptic adhesion molecule that induces presynaptic differentiation via presynaptic neurexins (Nrxns), but whether Nrxns directly bind to Clstn3 has been a matter of debate. Here, using LC – MS/MS – based protein analysis, confocal microscopy, RNAscope assays, and electrophysiological recordings, we show that b -Nrxns directly interact via their LNS domain with Clstn3 and Clstn3 cadherin

Calsyntenin-3 (Clstn3) is a postsynaptic adhesion molecule that induces presynaptic differentiation via presynaptic neurexins (Nrxns), but whether Nrxns directly bind to Clstn3 has been a matter of debate. Here, using LC-MS/MS-based protein analysis, confocal microscopy, RNAscope assays, and electrophysiological recordings, we show that b-Nrxns directly interact via their LNS domain with Clstn3 and Clstn3 cadherin domains. Expression of splice site 4 (SS4) insert-positive b-Nrxn variants, but not insert-negative variants, reversed the impaired Clstn3 synaptogenic activity observed in Nrxn-deficient neurons. Consistently, Clstn3 selectively formed complexes with SS4-positive Nrxns in vivo. Neuron-specific Clstn3 deletion caused significant reductions in number of excitatory synaptic inputs. Moreover, expression of Clstn3 cadherin domains in CA1 neurons of Clstn3 conditional knockout mice rescued structural deficits in excitatory synapses, especially within the stratum radiatum layer. Collectively, our results suggest that Clstn3 links to SS4-positive Nrxns to induce presynaptic differentiation and orchestrate excitatory synapse development in specific hippocampal neural circuits, including Schaffer collateral afferents.
Synaptogenic adhesion molecules, a class of synaptic transmembrane proteins that induce synaptic differentiation in vitro (1)(2)(3), are central to various aspects of synapse development, but their precise roles in synapse assembly, validation, and/or plasticity in vivo are only beginning to be revealed (1,4,5). Presynaptic neurexins (Nrxns) and leukocyte common antigenrelated receptor protein-tyrosine phosphatases (LAR-RPTPs) are among the synaptogenic adhesion molecules that have emerged as key platforms that facilitate convergence of diverse signals from multifarious postsynaptogenic ligands at mammalian synapses (5,6). Of particular note is the fact that, although Nrxns and LAR-RPTPs are evolutionarily conserved, only a subset of their ligands in mammals has homologs in invertebrate species that also play crucial roles in various aspects of central nervous system development, hinting at the possibility that Nrxns and LAR-RPTPs serve fundamental functions through these selective adhesion pathways.
Calsyntenins (Clstns) are evolutionarily conserved synaptogenic adhesion proteins of the cadherin superfamily that are expressed most highly in the brain (7). Synaptic functions of the three vertebrate Clstn family members have recently been reported. For example, juvenile Clstn1-deficient mice exhibit compromised excitatory synaptic transmission, possibly because of disrupted targeting of N-methyl-D-aspartate (NMDA) receptor subunits (8). They also show increased synaptic levels of GluN2B subunit-containing NMDA receptors, enhanced long-term potentiation (LTP), and greater filopodia-like dendritic protrusions in the hippocampus, but decreased dendritic arborization, suggesting that Clstn1 mediates dendritic transport of NMDA receptor subunits and regulates spine maturation during early development (8). In addition, Clstn1 regulates guidance receptor trafficking by shuttling Rab11-positive vesicles, leading to switching of commissural axon responsiveness (9). Clstn1 also contributes to peripheral sensory axon arborization, branching, endosomal dynamics, and microtubule polarity in zebrafish (10,11). Clstn2, on the other hand, plays a nonredundant role in inhibitory synapse development and influences a subset of cognitive abilities (12,13). Clstn3 was identified as a postsynaptogenic adhesion molecule that acts through presynaptic Nrxns (14,15). However, in contrast to the report of Pettem et al. (14), we did not detect direct interactions between Clstn3 and a-Nrxns.
In the present study, we revisited these issues. Strikingly, utilizing newly engineered Nrxn1 expression vectors to increase Nrxn1b expression levels, we found that recombinant Clstn3 bound both Nrxn1b and Nrxn1a, with a slight preference for splice site 4 (SS4) insert-positive variants, requiring an Nrxn splice variant containing an insert at SS4 as a functional receptor for its presynaptic differentiation-inducing activity. Conditional This article contains supporting information. * For correspondence: Jaewon Ko, jaewonko@dgist.ac.kr; Ji Won Um, jiwonum@dgist.ac.kr.
deletion of Clstn3 in neurons led to drastic reductions in excitatory synapse structures. Finally, adeno-associated virus-mediated expression of Nrxn-binding CST-3 cadherin domains was sufficient to rescue decreased excitatory synapse puncta density in CA1 stratum radiatum layers of Clstn3-deficient mice. Viewed together, our results revise our previous molecular model, showing that Clstn3 directly interacts with SS4-positive Nrxn splice variants to induce presynaptic differentiation, and suggesting synapse-organizing functions of Clstn3 that may control specific synaptic inputs from Schaffer-collateral afferents in the hippocampus.

Clstn3 directly binds b-Nrxns
Our previous cell surface-binding assays used a variety of Nrxn1a deletion variants derived from the bovine Nrxn1a gene or Nrxn1b variants derived from the rat Nrxn1b gene (15). These vectors have long been used to characterize Nrxn interactions with neuroligins (NLs) and other ligands, including leucine-rich repeat transmembrane neuronal proteins (LRRTMs), neurexophilins, and latrophilin-1 (16)(17)(18). However, using Nrxn vectors constructed from mouse Nrxn genes, Craig and colleagues (14) reported that Nrxn1a, but not Nrxn1b, binds to Clstn3. To resolve this discrepancy, we performed affinity chromatography of solubilized mouse synaptosomes using immobilized recombinant IgClstn3 followed by MS. Intriguingly, among the captured proteins was a tryptic peptide unique to Nrxn1b (in addition to Nrxn1a peptides) (Fig. 1, A-D; Table S1). To confirm binding between Clstn3 and Nrxn1b, we engineered mouse Nrxn1b expression constructs and performed cell surface-binding assays. We found robust binding of IgClstn3 to HEK293T cells expressing C terminally FLAG-tagged mNrxn1b lacking (mNrxn1b -SS4 -FLAG) or containing (mNrxn1b 1SS4 -FLAG) the SS4 insert ( Fig. 1, E and F). In assays measuring dimeric ligand binding to mNrxn-expressing cell surfaces, IgClstn3 interacted with both mNrxn1b -SS4 and mNrxn1b 1SS4 with nanomolar affinity (Fig. 1, G (Fig. 1, G and H). These findings indicate that Clstn3 binds Nrxn1b with high affinity and exhibits a slight preference for SS4 insert-positive splice variants. To investigate differences between bovine and rat Nrxn1 plasmids (bNrxn1a and rNrxn1b) used in our previous studies and the mouse Nrxn1 plasmids (mNrxn1a and mNrxn1b) used in the current study, we compared total protein expression levels produced by the different vectors by immunoblot analysis using serially diluted lysates from HEK293T cells transfected with expression plasmids for mNrxn and bNrxn1a or rNrxn1b (Fig. S1). Surprisingly, expression levels of mNrxn vectors were ;100-fold higher than those of bNrxn and rNrxn vectors (Fig. S1A). This difference in expression was not likely attributable to differences in the position of the FLAG epitope between rNrxn1b (N terminus) and our original mNrxn1b construct, as evidenced by the comparably robust binding of a newly generated N terminally FLAG-tagged mNrxn1b 1SS4 (FLAG-mNrxn1b 1SS4 ) and our original C terminally FLAG-tagged mNrxn1b 1SS4 (mNrxn1b 1SS4 -FLAG) to IgClstn3 in cell surface-binding assays (Fig. S1, B and C).

Cadherin domains of Clstn3 mediate direct binding to b-Nrxns
In addition to cell surface-binding assays, in vitro pulldown assays clearly showed binding of Clstn3 to IgmNrxn1b 1SS4 and IgmNrxn1a 1SS4 . To identify a minimal Clstn3 domain involved in Nrxn binding, we used three different FLAG-tagged Clstn3 constructs: Full (full-length Clstn3), Cad (containing tandem cadherin domains, the transmembrane segment and intracellular residues of Clstn3), and DCad (full-length Clstn3 lacking the tandem cadherin domains) (see Fig. 2A for schematic diagram of Clstn3 constructs). IgmNrxn1a 1SS4 and IgmNrxn1b 1SS4 both pulled down Clstn3 Cad, but not Clstn3 DCad (Fig. 2, B and C), suggesting that cadherin domains of Clstn3 mediate binding to Nrxns. The results of these and aforementioned cell-based surface-binding assays do not exclude the possibility that intermediate(s) expressed in HEK293T cells may bridge indirect associations of Nrxns with Clstn3. However, binding assays performed using purified recombinant IgNrxn1 (IgNrxn1b -SS4 , IgNrxn1b 1SS4 , IgNrxn1a -SS4 , or IgNrxn1a 1SS4 ) and recombinant His-HA-Clstn3 (Clstn3 Ecto, Clstn3 Cad, or Clstn3 DCad) (Fig. 2D) showed that His-HA-Clstn3 Ecto directly bound to recombinant aand b-Nrxns (Fig. 2, E and F). Moreover, Clstn3 Cad, but not Clstn3 DCad, directly bound to recombinant b-Nrxns (Fig.  2F). These data suggest that cadherin domains of Clstn3 mediate direct interactions with Nrxns, consistent with our previous observation that the cadherin domains of Clstn3 are necessary and sufficient to induce presynaptic differentiation (15).

Clstn3 binds to an LNS domain of mNrxn1b in a Ca 21 -dependent manner
We next investigated which Nrxn sequences mediate Clstn3 binding. To this end, we generated the following FLAG-tagged mNrxn1b-deletion constructs: mNrxn1b DHRD, which lacks a b-Nrxn-unique histidine-rich sequence; mNrxn1b DLNS, which lacks a b-Nrxn LNS domain; and DStalk1, which lacks the entire stalk region (Fig. 3A). We found that IgClstn3 bound comparably to HEK293T cells expressing mNrxn1b DHRD or mNrxn1b WT, but did not bind HEK293T cells expressing mNrxn1b DLNS (Fig. 3, B and C), indicating that the Nrxn1b LNS domain is necessary for Clstn3 binding. Interestingly, deletion of the entire stalk region (mNrxn1b DStalk1) diminished Clstn3 binding as well as NL-2 binding (Fig. 3, B and C). The stalk region contains ;40 residues with several putative Olinked glycosylated sites and a short cysteine-loop sequence composed of two conserved cysteines flanking an 8-residue acidic sequence (19). We thus hypothesized that O-linked glycosylation of mNrxnb regulates Clstn3 binding and that mNrxn1b DStalk1 displayed weak Clstn3 binding because it lacks O-linked glycosylation. To test this, we generated mNrxn1b constructs containing point mutations or deletions in the conserved stalk region. Clstn3 binding was retained in mNrxn1b constructs in which putative O-glycosylated threonines or serines were replaced with glycines (mNrxn1b DCHO), the cysteine-loop sequence was deleted (mNrxn1b DCysL), the stalk region was partially deleted (mNrxn1b DStalk2), or the conserved serine residue for attaching heparan sulfate chains was replaced with alanine (mNrxn1b DHS) (Fig. 4, A-D and F). However, Clstn3 did not bind Nrxn1g, a newly identified Nrxn1 isoform (19), or other heparan sulfate proteogylcans, such as glypicans or syndecans (Figs. 3, B and C, and 4, E and F). Control experiments showed that surface expression levels of individual mNrxn1b deletion constructs in HEK293T cells were comparable with those of WT protein (Fig. S2). These findings suggest that the Nrxn1b LNS domain is a Clstn3-binding site. We further found that treatment with the Ca 21 chelator EGTA prevented these interactions (Fig. S3).

Clstn3 requires specific Nrxn splice variants for presynaptic differentiation
To delineate the molecular mechanisms that link Nrxns to Clstn3 and support its synaptogenic activity, we performed heterologous synapse-formation assays in cultured hippocampal neu-rons in which all three Nrxns were down-regulated using small hairpin RNAs (15,20). We found that Nrxns triple-knockdown (TKD) significantly reduced the synaptogenic activity of Clstn3 (Fig. 5, A and B) (15). Synaptogenic activity was completely restored by coexpression of Nrxn1a 1SS4 or Nrxn1b 1SS4 , but not Nrxn1a -SS4 or Nrxn1b -SS4 , suggesting that SS4-positive Nrxns act as functional receptors for Clstn3 (Fig. 5, A and 5B, Fig. S4, A and B). Moreover, other SS4-positive b-Nrxns (Nrxn2b 1SS4 and Nrxn3b 1SS4 ) also rescued the lost synaptogenic activity in Nrxn-TKD neurons (Fig. S4, C and D). Similarly, re-expression of the respective Nrx splice variants rescued deficits in the synaptogenic activities of NL-1 (neuroligin-1) and LRRTM2 (leucine-rich repeat transmembrane neuronal 2) (Fig. 5, A and B). Subsequent pulldown assays showed that IgmNrxn1a 1SS4 and IgmNrxn1b 1SS4 , but not IgmNrxn1a -SS4 or IgmNrxn1b -SS4 , were capable of capturing Clstn3 from mouse synaptosomal membrane fractions (Fig. 5, C and D). Moreover, re-expression of Nrxn1b 1SS4 in Nrxn-TKD neurons triggered clustering of both the inhibitory presynaptic marker GAD67 (glutamic acid decarboxylase 67) and the excitatory presynaptic marker VGLUT1 (vesicular glutamate transporter  Cad, but not FLAG-Clstn3 DCad. Input, 5%. C, IgNrxn1a 1SS4 , IgNrxn1b 1SS4 , or IgC used for pulldown assays were analyzed by direct comparison of bands revealed by parallel Ponceau S staining. D, diagrams of His-HAtagged extracellular Clstn3 variants used in direct-binding assays. E and F, purified His-tagged Clstn3 proteins were incubated with purified IgNrxn proteins, as indicated. Precipitates obtained using Talon resin were analyzed by immunoblotting with human IgG or HA antibodies. Input, 10%. Interaction of Clstn3 with a/b-Nrxns 1) in contacting axons of co-cultured neurons (Fig. S5), suggesting that Clstn3 exerts its synaptogenic activities through Nrxn SS4positive splice variants at both excitatory and inhibitory synapses.

Generation and characterization of Clstn3 conditional knockout (cKO) mice
To elucidate the physiological significance of Clstn3-Nrxn interactions in vivo, we used Clstn3 tm1a(EUCOMM)Hmgu mice in which a targeting cassette harboring FRT, lacZ, and loxP sites was inserted between exon 7 and exon 8, resulting in a "knockout-first" lacZ-reporter-tagged Clstn3 tm1a insertion allele with conditional potential (21). We then crossed Clstn3 tm1a/tm1a mice with an FLPe knock-in strain to remove contaminating transgenes and the neomycin resistance cassette to generate Clstn3 tm1c(EUCOMM)Hmgu mice. To decide which Cre driver lines to cross, for the current study, we first analyzed Clstn3 expres-sion patterns in the mouse brain. For this, we performed RNAscope-based in situ hybridization analysis using a Clstn3-specific probe. This analysis showed that Clstn3 mRNA was strongly expressed in most pyramidal cell layers and interneurons in the mouse hippocampus (Fig. 6A), with particularly prominent expression observed in pyramidal cell layers in the CA3 region (Fig. 6, A and C). We further found that Clstn3 mRNA was also expressed in parvalbumin (PV)-and somatostatin (SST)-positive GABAergic interneurons in the hippocampus, as previously reported (14) (Fig. 6B). Moreover, immunofluorescence analyses using a Clstn3-specific antibody (JK091) showed that the expression pattern of the Clstn3 protein was similar to that of Clstn3 mRNA in the hippocampus (Fig. 6D). Based on these results, the Nestin-Cre (Nestin-Clstn3) driver line was chosen to further cross with Clstn3 tm1c(EUCOMM)Hmgu mice to generate Clstn3-cKO mice (Fig. 7, A and B). Loss of Clstn3 protein in Nestin-Clstn3 mice was verified by the absence of detectable Clstn3 immunoreactivity to Clstn3-specific antibodies (JK091; Fig. 7, G and H, and Fig. S6). These mice were largely indistinguishable from control littermates (Clstn3 f/f ; Ctrl) in terms of birth rate, although Nestin-Clstn3 mice weighed marginally (but significantly) less at both postnatal day 30 (P30) and P54, which is a generalized metabolic phenotype of the Nestin-Cre driver line (Fig. 7C, Table S2). In addition, gross morphology (as assessed by Nissl staining) and neuron numbers (as assessed by NeuN staining) in Nestin-Clstn3 mice were comparable with those of control littermates (Fig. 7, D-F). Semi-quantitative immunoblot analyses showed that the relative expression levels of various synaptic proteins in the Interaction of Clstn3 with a/b-Nrxns hippocampus (Fig. 7G) and cortex (Fig. 7H) of Nestin-Clstn3 mice were unchanged compared with littermate controls (Fig. 7, G-I). Collectively, these data suggest that Clstn3 is not essential for mouse survival or breeding, and does not affect the expression levels of other synaptic proteins.
Cadherin domains of Clstn3 are required for restoration of the impaired excitatory innervations observed in Clstn3-cKO hippocampal neurons We next analyzed the effect of Clstn3 deletion on the intensity of excitatory and inhibitory synaptic puncta in various layers of the hippocampal CA1 region using Nestin-Clstn3 (Figs. 8 and 9). We found a significant decrease in the intensity of VGLUT1 and PSD-95 (excitatory postsynaptic marker) puncta in most layers of hippocampal CA1 regions (stratum oriens (SO) and stratum radiatum (SR)), but not in the stratum lacunosum moleculare (SLM) layer, in Clstn3-cKO (Fig. 8, B-E). In contrast, the intensity of GAD67, GAD65, and GABA A Rg2 puncta was unchanged in all examined layers of hippocampal CA1 regions (SO, SR, stratum pyramidale (SP), and SLM) ( Fig. 9, A-D). To investigate the physiological significance of Clstn3-Nrxn interactions in vivo, we constructed AAVs encoding full-length Clstn3 (Full), cadherin domains plus intracellular regions (Cad), or a cadherin domain-deleted Clstn3 fragment (DCad) (Fig. 8A, Fig. S7). We then stereotactically injected AAVs expressing the indicated Clstn3 protein into the CA1 hippocampus of ;9-week-old Nestin-Clstn3 mice and performed quantitative immunofluorescence analyses after 2 weeks using antibodies to VGLUT1 or PSD-95 ( Fig.  8, A-E). Expression of Clstn3 Full restored the decreased VGLUT1 and PSD-95 puncta density to WT mouse levels in SO and SR layers of Nestin-Clstn3 mice (Fig. 8, B-E). Strikingly, expression of Clstn3 Cad, but not Clstn3 DCad, also restored the decreased VGLUT1 puncta density in the SR layer, but neither Clstn3 Cad nor Clstn3 DCad expression exerted a rescue effect in the SO layer of Nestin-Clstn3 mice (Fig. 8, B-E). These results suggest that Clstn3 organizes the development of hippocampal CA1 excitatory synapses, and likely acts through cadherin domains-mediated interactions with presynaptic Nrxns to control the specific excitatory synaptic projections involving the SR layer.
To complement these anatomical analyses, we performed whole cell electrophysiological recordings of miniature excitatory and inhibitory postsynaptic currents (mEPSCs and mIPSCs) in brain slices from Nestin-Clstn3 and littermate WT mice (Fig. S8). Surprisingly, no differences in the amplitude or frequency of mEPSCs and mIPSCs were detected between Nestin-Clstn3 and Ctrl mice (Fig. S8). These results suggest that Clstn3 is not required for basal excitatory synaptic transmission in CA1 hippocampal pyramidal neurons.

Discussion
The present study was initiated with the goal of reconciling discrepancies surrounding molecular mechanisms of Clstn3mediated synapse development. For this, we revisited the most critical (and controversial) issues using newly engineered Nrxns expression vectors and recombinant proteins, and a newly developed Clstn3-cKO mice. Taken together with our previous findings, the three principal observations of the current study provide plausible explanations for discrepancies surrounding the molecular mechanisms of Clstn3-mediated synapse development.

Interaction of Clstn3 with a/b-Nrxns
Second, re-expressing select Nrxn1 variants in Nrxn-deficient neurons rescued impaired Clstn3 synaptogenic activity (Fig. 5). Previously, we reported that presynaptic Nrxns serve as functional receptors for postsynaptic Clstn3, but do not directly interact with them (15). Our new data indicate that our previous model should incorporate direct binding of b-Nrxns to Clstn3 (Figs. 1-5). In support of this interpretation, BAM-2, a Nrxn-related Caenorhabditis elegans homolog, was recently reported to bind CASY-1 and mediate neural circuit wiring of male-specific hook-sensory HOA neurons in C. elegans (24). Intriguingly, CASY-1 does not directly interact with NRX-1, a canonical C. elegans Nrxn ortholog (24). Although BAM-2 exhibits considerable sequence homology/similarity with a-Nrxns (25), whether these vertebrate and worm genes are functionally homologous remains to be determined. Nrxns interact with various postsynaptogenic proteins, including NLs, LRRTMs, Cblns, and latrophilins (3). These interactions are dynamically modulated by the alternative splicing status of Interaction of Clstn3 with a/b-Nrxns Nrxns, mainly at the canonical SS4 splice site. Strikingly, the synapse-promoting activity of Clstn3 required Nrxn splice variants containing the SS4 insert (Fig. 5), as has been shown for other Nrxn ligands (i.e. Cblns) (3). These observations are consistent with slightly stronger binding of Nrxn1b 1SS4 to Clstn3, which contrasts with the exclusive binding of Nrxn1b 1SS4 to Cblns (Fig. 1, G and H). Pulldown experiments in mouse brains also showed that greater enrichment of Clstn3 using IgNrxn1a 1SS4 than was obtained using IgNrxn1a -SS4 (Fig. 5, C and D) (15). Additionally, the shed ectodomain of Clstn3 suppresses the synaptogenic activity of NL-2 and LRRTM2, suggesting that Clstn3 competes with these Nrxn ligands (14). However, LRRTM2 interacts only with Nrxn-SS4-negative splice variants (16), whereas Clstn3 activity requires Nrxn-SS4-positive splice variants; moreover, a b-Nrxn LNS domain (identical to the sixth LNS domain-sixth LNS domain in a-Nrxns) is sufficient for Clstn3 binding. Although we still failed to detect clear interactions of a-Nrxns with Clstn3 in cell surface-binding assays, we observed robust Nrxn1a-Clstn3 interactions in brain pulldown and direct binding assays (Figs. 2E and 5D). It is possible that the surface-binding assays employed here were too stringent to observe this interaction, or the cell surface presentation of a-Nrxns was not optimal for binding. Thus, we conclude that Clstn3 binds to both aand b-Nrxns through a common LNS domain, but a subset of Clstn3 synaptic functions might require other unidentified ligands in vivo. Moreover, given the activitydependent regulation of Nrxn alternative splicing at the SS4 site (26), future studies should investigate whether Clstn3-Nrxn interactions could be finetuned depending on distinct activity patterns in vivo. Furthermore, given a recent report that different SS4-positive splice variants of two different Nrxns (Nrxn1 versus Nrxn3) differentially control different postsynaptic responses (27), it is possible that Clstn3 preferentially partners with a specific Nrxn in vivo.
Third, re-expressing Clstn3 cadherin domains was sufficient to completely rescue the impaired excitatory synapse structures in hippocampal CA1 neurons from Nestin-Clstn3 mice (Fig. 8), but only in the SR layer. In contrast, the structural deficits in other hippocampal CA1 layers were rescued by expression of full-length Clstn3 protein, but not by expression of partial Clstn3 proteins (Fig. 8). Although it is possible that other unidentified proteins could also bind to Clstn3 cadherin domains, it is likely that presynaptic neurexins and postsynaptic Clstn3 control the properties of excitatory synapse development in specific Schaffer-collateral projections within the SR layer. A recent paper highlighted the fact that the distinctive features of two different Schaffer-collateral projections differentially regulate mushroom spine density and high-magnitude LTP in the SO layer, organized by heterophilic type II cadherins (28). Moreover, Nrxn genes show differential, but overlapping, isoform-and region-dependent expression in different classes of neurons, and undergo highly distinctive, cell type-specific alternative splicing (29,30). One plausible scenario would be that CA3 neurons projecting to CA1 neurons in the SR layer, but not the SO layer, express higher levels of SS4-positive Nrxns at their nerve terminals. A recent extensive chromogenic and fluorescent in situ hybridization analysis showed that the levels of Nrxn mRNAs in hippocampal subfields are significantly lower in GABAergic neurons than excitatory neurons (31), which might account for preferential deficits on excitatory synapses in CA1 hippocampal regions of Clstn3 KO mice. Another possible scenario is that major glutamatergic axon fibers of Schaffer-collateral (SC) pathways are targeted to CA1 pyramidal neurons in the SR layer, whereas a subpopulation of axon fibers of SC pathways innervates both bistratified GABAergic interneurons in the SO layer and CA1 pyramidal neurons. Additional studies will also be required to determine the identity of neurons in vivo that are responsible for Nrxn-Clstn3 interactions in other neural circuits inside and outside of the hippocampus. Notably, in stark contrast to the previous report that decreased inhibitory synapse structure and transmission in Clstn3-KO mice (14), we found that inhibitory synapses in CA1 hippocampal layers and the cortex were morphologically normal in our Clstn3-KO mice (Fig. 9). The reason for this discrepancy is currently unclear, but it is likely that Clstn family proteins are functionally redundant in the maintenance of basal synaptic transmission; alternatively, Clstn3 may be specifically required for certain forms of synaptic plasticity. A notable difference between the study of Pettem et al. (14) and the current study is that the former targeted exons 2 and 3 of the mouse Clstn3 gene, whereas our study targeted exon 8 (Fig. 7). In addition, Pettem et al. (14) employed an EIIa-Cre driver line that permits germ line deletion in both excitatory and inhibitory neurons by driving expression of Cre recombinase in the early mouse embryo, whereas we used a pan-neuronal Nestin-Cre driver line was that presumed to be similar to EIIa-Cre. It is also possible that specifically deleting Clstn3 in GABAergic inhibitory neurons may produce marked deficits in GABAergic synapse development, as previously documented (14), but this did not clearly manifest in our Clstn3-cKO mice. A more system-atic, follow-up investigation to probe cell type-specific contributions of Clstn3 to distinct anatomical and electrophysiological phenotypes should address these important, but puzzling, observations. Future studies should also probe how Nrxn-Clstn3 complexes mediate synaptic specificity involving the SR layer of the hippocampal CA1 region. A complete understanding of how Clstn3 functions will also require investigation of the potential contributions of other Clstn3 domains (e.g. LNS domain, intracellular region) to the functions of Clstn3, as was similarly shown in C. elegans (24,32,33). In summary, the present study unambiguously establishes that Clstn3 plays a role in specifying the proper-ties of a specific hippocampal CA1 neural circuit, in part by regulating excitatory synapse development through formation of physical complexes with specific Nrxn splice variants.

Animals
Clstn3 tm1a(EUCOMM)Hmgu mice, in which a targeting cassette harboring FRT, lacZ, and loxP sites is inserted between exon 7 Interaction of Clstn3 with a/b-Nrxns and exon 8, resulting in a "knockout-first" lacZ reporter-tagged Clstn3 tm1a insertion allele with conditional potential, were obtained from the European Mouse Mutagenesis Consortium (EUCOMM). To generate conditional Clstn3 KO mice (Clstn3 flox/flox ), we first bred Clstn3 tm1a/tm1a mice in a C57BL/6J background with FLPO recombinase driver mice (C57BL/ 6N-Tg(CAG-Flpo)1Afst/Mmucd, MMRRC) to generate Clstn3 tmc1c/tm1c mice. Male homozygous Clstn3 tmc1c/tm1c mice in a C57BL/6J genetic background were crossed with female heterozygous Clstn3 tm1c/1 mice (for Nestin conditional) carrying the Cre transgene. A Nestin-Cre (003771) line in a C57BL/ 6N genetic background, generated by crossing the original Credriver lines purchased from the Jackson Laboratory with C57BL/6J for more than five generations, was obtained from Dr. Albert Chen (DUKE-NUS, Singapore). Cre-negative Clstn3 flox/flox littermates were used as controls in all experiments. Mice were housed and bred at the animal facility of Daegu Gyeongbuk Institute of Science and Technology (DGIST), and experimental use was approved by the Institutional Animal Care and Use Committee of DGIST (DGIST-IACUC-19052110-00). Mice were given ad libitum access to food and water, and were maintained in a controlled environment with a 12-h light/dark cycle. Mice were weaned at the age of P24-27, and mixed-genotype littermate mice were grouphoused (4-5 mice per cage) until experiments. Genotyping was performed on genomic DNA extracted from tail biopsies using the 23 Taq

Production of recombinant lentiviruses and adeno-associated viruses (rAAVs)
Lentivirus production-Recombinant lentiviruses were produced as previously described (34). In brief, HEK293T cells were transfected with three plasmids: lentivirus vectors, psPAX2, and pMD2G, at a 2:2:1 ratio using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer's protocol. After 72 h, lentiviruses were harvested by collecting the medium from transfected HEK293T cells and briefly centrifuging at 1,000 3 g to remove cellular debris. Filtered media containing 5% sucrose were centrifuged at 117,969 3 g for 2 h; supernatants were then removed and the virus pellet was washed with ice-cold PBS and resuspended in 80 ml of PBS.
AAV production-HEK293T cells were co-transfected with the indicated AAV vectors and pHelper and pRC1-DJ vectors. Seventy-two h later, transfected HEK293T cells were collected, lysed, and mixed with 40% PEG and 2.5 M NaCl, and centrifuged at 2,000 3 g for 30 min. The cell pellets were resuspended in HEPES buffer (20 mM HEPES; 115 mM NaCl, 1.2 mM CaCl 2 , 1.2 mM MgCl 2 , 2.4 mM KH 2 PO 4 ) and an equal volume of chloroform was added. The mixture was centrifuged at 400 3 g for 5 min, and concentrated three times with a Centriprep centrifugal filter (Millipore) at 1,220 3 g for 5 min each and with an Amicon Ultra centrifugal filter (Millipore) at 16,000 3 g for 10 min. Before titering AAVs, contaminating plasmid DNA was eliminated by treating 1 ml of concentrated, sterile-filtered AAVs with 1 ml of DNase I (Sigma-Aldrich) for 30 min at 37°C. After treatment with 1 ml of stop solution (50 mM EDTA) for 10 min at 65°C, 10 mg of protease K (Sigma-Aldrich) was added and AAVs were incubated for 1 h at 50°C. Reactions were inactivated by incubating samples for 20 min at 95°C. The final virus titer was quantified by quantitative RT-PCR detection of EGFP sequences and subsequent reference to a standard curve generated using the pAAV-U6-EGFP plasmid. All plasmids were purified using a Plasmid Maxi Kit (Qiagen GmbH).

In-gel digest
Gel bands were excised, after which proteins in bands were reduced by treating with DTT, alkylated by treating with indole-3 acetic acid, and then digested by treating with trypsin. Specifically, bands were washed with 10 mM ammonium bicarbonate and 50% acetonitrile, swollen in digestion buffer containing 50 mM ammonium bicarbonate, 5 mM CaCl 2 , and 1 mg of trypsin, and then incubated at 37°C for 16 h. Peptides were recovered by two cycles of extraction with 50 mM ammonium bicarbonate and 100% acetonitrile. The resulting peptide extracts for each band were lyophilized and stored at 220°C until mass spectrometric analysis.

LC-MS/MS protein analysis
Peptides were analyzed using a nanoflow LC-MS/MS system consisting of an Easy nLC 1000 system (Thermo Fisher) and an LTQ Orbitrap Elite mass spectrometer (Thermo Fisher) equipped with a nano-electrospray source. Peptide solutions (5-ml aliquots) were loaded onto a C 18 trap column (20 3 75 mM, 3 mm particle size; Thermo Fisher) using an autosampler. Peptides were desalted and concentrated on the column at a flow rate of 5 ml/min. Trapped peptides were then separated on a 150-mm custom-built microcapillary column consisting of C 18 (particle size, 3 mM; Aqua Science, Yokohama, Japan) packed in 100-mm silica tubing with a 6-mm inner diameter orifice. The mobile phases A and B were composed of 0 and 100% acetonitrile, respectively, and each contained 0.1% formic acid. The LC gradient began with 5% B for 5 min and was increased to 15% B over 5 min, 50% B over 55 min, and 95% B over 5 min, then remained at 95% B over 5 min, followed by 5% B for an additional 5 min. The column was re-equilibrated with 5% B for 15 min between runs. A voltage of 2.2 kV was applied to produce the electrospray. In each mass analysis duty cycle, one high-mass resolution (60,000) MS spectrum was acquired using the Orbitrap analyzer, followed by 10 data-dependent MS/MS scans using the linear ion trap analyzer. For MS/MS analysis, a normalized collision energy (35%) was used throughout the collision-induced dissociation phase. All MS and MS/MS spectra were acquired using the following parameters: no sheath and auxiliary gas flow; ion-transfer tube temperature, 200°C; activation Q, 0.25; and activation time, 20 ms. Dynamic exclusion was employed with a repeat count of 1, a repeat duration of 30 s, an exclusion list size of 500, an exclusion duration of 60 s, and an exclusion mass width of 61.5 m/z.

MS data analysis
MS/MS spectra were analyzed using the following analysis protocol, referencing the UniProt mouse database (04-08-2020 release; 17,333 entries). Briefly, each protein's reversed sequence was appended onto the database to calculate the false discovery rate. Peptides were identified using ProLuCID (version 1.3.5.1) (40) in Integrated Proteomics Pipeline software, IP2, with a precursor mass error of 25 ppm and a fragment ion mass error of 600 ppm. Trypsin was used as the protease, and two potential missed cleavages were allowed. Carbamidomethylation at cysteine was chosen as a static modification, and methionine oxidation was chosen as a variable modification. Protein lists consisting of two or more peptide assignments for protein identification (false-positive rate ,0.01) were prepared by filtering and sorting output data files.
Primary neuron culture, immunocytochemistry, image acquisition, and quantitative analysis The indicated analyses were performed using cultured, E18derived, rat hippocampal neurons and confocal microscopy, as previously described (15,41,42). Rat hippocampal neurons were prepared from E18 rat brains and cultured on coverslips coated with poly-D-lysine in Neurobasal media supplemented with B-27 (Thermo Fisher), 0.5% fetal bovine serum, 0.5 mM GlutaMax (Thermo Fisher), and sodium pyruvate (Thermo Fisher). For immunocytochemistry, cultured neurons were fixed with 4% formaldehyde, 4% sucrose, permeabilized with 0.2% Triton X-100 in PBS, and immunostained with primary antibodies and Cy3-or FITC-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA). Images were acquired using a confocal microscope (LSM700; Carl Zeiss) equipped with a 633 objective lens. All image settings were kept constant. Z-stack images were converted to maximal projection. All images were quantitatively analyzed in a blinded manner using MetaMorph software (Molecular Devices).

Cell surface-binding assays
Ig-fusion proteins of Clstn3, IgNrxn1b splice variants, and IgC alone (Control) were produced in HEK293T cells. Soluble Ig-fused proteins were purified using protein A-Sepharose beads (GE Healthcare). Bound proteins were eluted with 0.1 M glycine (pH 2.5) and immediately neutralized with 1 M Tris-HCl (pH 8.0). Transfected HEK293T cells expressing the indicated plasmids were incubated with 10 mg/ml of Ig-fused proteins for 2 h at 37°C. Images were acquired using a confocal microscope (LSM700; Carl Zeiss).

Direct protein-interaction assays
For direct interaction assays, 10 mg of IgC (control), IgNrxn1a -SS4 , IgNrxn1a 1SS4 , IgNrxn1b -SS4 , or IgNrxn1b 1SS4 was incubated with 4 mg of purified His-HA-Clstn3 Full, His-HA-Clstn3 Cad, or His-HA-Clstn3 DCad for 2 h at 4°C in binding buffer (25 mM Tris, pH 7.5, 30 mM MgCl 2 , 40 mM NaCl, and 0.5% Triton X-100). Talon metal affinity resins (Clontech) beads were then added to purified protein mixtures as indicated, and incubated for 2 h at 4°C. Beads were washed three times with binding buffer, solubilized in SDS sample buffer, and loaded onto SDS-PAGE gels for immunoblot analyses. Anti-human IgG was used for immunoblotting.

Heterologous synapse-formation assays
Heterologous synapse-formation assays were performed using recombinant Clstn3 fusion proteins as previously described (43). Briefly, HEK293T cells were transfected with EGFP (negative control) or the indicated Clstn3 constructs using Lipofectamine 2000 (Thermo Fisher). After 48 h, the transfected HEK293T cells were trypsinized, seeded onto in vitro day 10 (DIV10) hippocampal neurons, co-cultured for an additional 72 h, and double-immunostained on DIV13 with antibodies against EGFP, HA, and the indicated synaptic markers (synapsin, VGLUT1, or GAD67). All images were acquired using a confocal microscope (LSM700; Zeiss). For quantification, the contours of transfected HEK293T cells were chosen as the region of interest. Fluorescence intensities of synaptic markers in each region of interest were quantified for both red and green channels using MetaMorph software (Molecular Devices). Normalized synapse density on transfected HEK293T cells was expressed as the ratio of red to green fluorescence.

Semiquantitative immunoblot analysis
For semiquantitative immunoblot analysis, brains from P42 WT, Clstn3 fl/fl , or Clstn3 fl/fl ::Nestin-Cre (Nestin-Clstn3) mice were homogenized in 0.32 M sucrose, 1 mM MgCl 2 containing a protease inhibitor mixture (Thermo Fisher Scientific) using a Precellys Evolution tissue homogenizer (Bertin Co.). After centrifuging homogenates at 1,000 3 g for 10 min, the supernatant was transferred to a fresh microcentrifuge tube and centrifuged at 15,000 3 g for 30 min. The resulting synaptosome-enriched pellet (P2) was resuspended in lysis buffer and centrifuged at 20,800 3 g, after which the supernatant was analyzed by Western blotting. Quantitation was performed using ImageJ software (National Institutes of Health).

Nissl staining
Mice were perfused first with PBS and then with 4% paraformaldehyde by cardiac injection. Fixed brain tissue was isolated, post-fixed for 12 h at 4°C, and dehydrated in 30% sucrose for 1-2 days. Thereafter, brain tissue was embedded in OCT (optimal cutting temperature) compound and stored at 280°C. The frozen tissue was mounted and cut at a thickness of 20 mm using a cryostat (Leica CM5120). Slices were mounted on glass slides, washed three times with PBS (15 min each), and permeabilized with 0.1% Triton X-100 in PBS for 10 min. Permeabilized slices were washed twice with PBS (5 min each), then incubated for 20 min in 200 ml of NeuroTrace 500/525 Green Fluorescent Nissl Stain (Molecular Probes), diluted 1:100 in PBS before use. Thereafter slices were washed in PBS containing 0.1% Triton X-100 for 10 min, then washed twice in PBS (5 min each) and incubated in PBS for 2 h. Slides were dried and mounted using Vectashield mounting medium containing 4',6diamidino-2-phenylindole (DAPI; Vector Laboratories). Green fluorescence was imaged by slide scanner microscopy (AxioScan.Z1; Zeiss).

Fluorescent in situ hybridization (RNAscope assay)
Frozen sections (14 mm thick) were cut coronally through the hippocampal formation and thaw-mounted onto Superfrost Plus microscope slides (Advanced Cell Diagnostics). Sections were fixed in 4% formaldehyde for 10 min, dehydrated in increasing concentrations of ethanol for 5 min, air dried, and then pretreated with protease for 10 min at room temperature. For RNA detection, sections were incubated in different amplifier solutions in a HybEZ hybridization oven (Advanced Cell Diagnostics) at 40°C. Three synthetic oligonucleotides complementary to the sequence corresponding to nucleotide residues 302-1210 of Mm-Clstn3-tv1, 896-1986 of Mm-CaMKIIa-C2, 18-407 of Mm-SST-C3, and 2-885 of Mm-Pvalb-C3 (Advanced Cell Diagnostics) were used as probes. The labeled probes were conjugated to Alexa Fluor 488, Altto 550, or Altto 647, after which labeled probe mixtures were hybridized by incubating with slide-mounted sections for 2 h at 40°C. Nonspecifically hybridized probes were removed by washing the sections three times for 2 min each with 13 wash buffer at room temperature, followed by incubation with Amplifier 1-FL for 30 min, Amplifier 2-FL for 15 min, Amplifier 3-FL for 30 min, and Amplifier 4 Alt B-FL for 15 min at 40°C. Each amplifier was removed by washing with 13 wash buffer for 2 min at room temperature. Slides were imaged with an LSM700 microscope (Zeiss) and analyzed using MetaMorph software (Molecular Devices).

Stereotaxic injection of rAAVs
Adult (;9-week-old) male mice were anesthetized with Avertin (400 mg/kg body weight) by intraperitoneal injection. rAAV solutions (titers 1 3 10 11 viral genomes/ml) were injected with a NanoFil syringe (World Precision Instruments) at a flow rate of 0.1 ml/min. The coordinates used for the CA1 region of the dorsal hippocampus were AP 22.5 mm, ML 61.5 mm, DV 11.5 mm (from the dura). The site at DV 11.5 mm received a 1-ml injection. Injected mice were allowed to recover for at least 14 days following surgery prior to use in experiments.

Statistical analysis
All data are expressed as mean 6 S.E., and significance is indicated with asterisk (compared with a value from control group) or hashtag (compared with a value from experimental group). N values are denoted in the individual figure legends. For cell surface-binding and heterologous synapse-formation assays, cells used for analysis were obtained from 5 to 10 images from at least three independent experiments. The normality of data distributions was evaluated using the Shapiro-Wilk test, followed by nonparametric Kruskal-Wallis test with Dunn's multiple comparison test for post hoc group comparisons or ANOVA with Tukey's test, using cell numbers or the number of experiments as the basis for n.

Data availability
All data except MS data are contained within this article and the supporting information. All raw MS and supporting data files from the current study have been deposited to the repository of MassIVE with identifier PXD018433.