{"gene":"CLSTN3","run_date":"2026-06-09T22:57:18","timeline":{"discoveries":[{"year":2013,"finding":"Calsyntenin-3 (CLSTN3) is a postsynaptic synapse-organizing protein that specifically binds and recruits α-neurexins (but not β-neurexins) to trigger both excitatory and inhibitory presynapse differentiation in contacting axons. Its shed ectodomain suppresses the ability of multiple α-neurexin partners (including neuroligin-2 and LRRTM2) to induce presynapse differentiation. Clstn3−/− mice show reductions in excitatory and inhibitory synapse density and corresponding deficits in synaptic transmission.","method":"Unbiased screen, cell-based synaptogenesis assays, co-immunoprecipitation, confocal and electron microscopy, electrophysiological recordings in Clstn3−/− mice","journal":"Neuron","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal binding assays, loss-of-function mouse with multiple orthogonal readouts (EM, confocal, electrophysiology), replicated across multiple experimental approaches in single rigorous study","pmids":["24094106"],"is_preprint":false},{"year":2014,"finding":"The extracellular domains of CLSTN3 and neurexin-1α (n1α) interact directly with nanomolar affinity. CLSTN3 ectodomains form monomers and disulfide-stabilized tetramers that are Ca2+-dependent and flexible in solution. The interaction requires minimally the LNS domain of CLSTN3 and uses a fundamentally different binding mechanism than neuroligin-2 — notably, CLSTN3 does not strictly require the sixth LNS domain of n1α. Both monomeric and tetrameric forms bind n1α.","method":"Electron microscopy (structural architecture), biochemical binding assays, biophysical measurements (affinity determination), mutagenesis-guided domain mapping","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct in vitro reconstitution with structural (EM) and biophysical validation, multiple orthogonal methods in a single rigorous study","pmids":["25352602"],"is_preprint":false},{"year":2014,"finding":"Zebrafish CLSTN3 ectodomain mediates homophilic cell-cell adhesion through its two amino-terminal cadherin repeats; in bead-sorting assays, calsyntenin ectodomains do not exhibit homophilic preferences among paralogs.","method":"Cloning of zebrafish clstn3, bead-sorting adhesion assays, domain mapping","journal":"Neuroscience","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — direct adhesion assay with domain mapping, single lab, single method set","pmids":["25463516"],"is_preprint":false},{"year":2020,"finding":"CLSTN3 interacts with β-neurexins (in addition to α-neurexins) via the LNS domain of β-Nrxn and CLSTN3 cadherin domains. Specifically, splice site 4 (SS4) insert-positive β-Nrxn variants (not insert-negative) rescue impaired Clstn3 synaptogenic activity in Nrxn-deficient neurons. In vivo, Clstn3 selectively forms complexes with SS4-positive Nrxns. Neuron-specific Clstn3 deletion reduces excitatory synaptic inputs, and expression of CLSTN3 cadherin domains in CA1 neurons of Clstn3 conditional KO mice rescues structural deficits in excitatory synapses in the stratum radiatum.","method":"LC-MS/MS protein analysis, confocal microscopy, RNAscope, electrophysiological recordings, conditional knockout mouse, domain rescue experiments","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (MS, electrophysiology, conditional KO rescue), in vivo and in vitro confirmation in single rigorous study","pmids":["32434929"],"is_preprint":false},{"year":2020,"finding":"Neuronal CLSTN3 regulates systemic energy and bone homeostasis. Global Clstn3 KO mice show reduced body mass, improved leptin sensitivity, increased energy expenditure, and reduced cortical bone mass. Pan-neuronal (but not sympathetic-neuron-specific or osteoblast/osteoclast-specific) deletion of Clstn3 recapitulates these phenotypes, indicating the effects are neuronally mediated rather than bone-cell-autonomous.","method":"Global and cell-type-specific conditional knockout mice (pan-neuronal, sympathetic, osteoblast, osteoclast), metabolic phenotyping, bone microarchitecture analysis, in vitro osteoblast/osteoclast cultures","journal":"Experimental & molecular medicine","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple conditional KO lines with cell-type specificity, multiple phenotypic readouts, epistasis-style genetic dissection","pmids":["32382066"],"is_preprint":false},{"year":2021,"finding":"CLSTN3 physically interacts with the sodium-dependent vitamin C transporter-2 (SVCT2/hSVCT2) in neuronal cells. Co-expression of hCLSTN3 with hSVCT2 in SH-SY5Y cells markedly increases ascorbic acid (AA) uptake, while siRNA-mediated knockdown of hCLSTN3 inhibits AA uptake, indicating CLSTN3 positively regulates SVCT2-mediated vitamin C transport.","method":"Yeast two-hybrid (Y2H) screen of human brain cDNA library, co-immunoprecipitation, mammalian two-hybrid (M2H), co-localization in human cell lines, AA uptake assays, siRNA knockdown","journal":"International journal of biological macromolecules","confidence":"High","confidence_rationale":"Tier 2 / Strong — interaction confirmed by three orthogonal methods (Y2H, Co-IP, M2H) plus functional uptake assays with both gain- and loss-of-function","pmids":["34673103"],"is_preprint":false},{"year":2022,"finding":"CLSTN3β, an adipocyte-selective isoform encoded at the Clstn3 locus present only in placental mammals, is an integral ER membrane protein that localizes to ER–lipid droplet (LD) contact sites via a conserved hairpin-like domain. CLSTN3β associates with cell death-inducing DFFA-like effector (CIDE) proteins and impairs their ability to transfer lipid between LDs, thereby restricting LD fusion and expansion and enforcing a multilocular LD phenotype. Loss of CLSTN3β causes abnormal LD morphology and altered substrate use in brown adipose tissue with cold-induced hypothermia; forced expression enforces multilocular LD phenotype in cells and adipose tissue.","method":"Knockout and transgenic mice, subcellular fractionation/localization imaging, co-immunoprecipitation with CIDE proteins, lipid transfer assays, cold-challenge metabolic phenotyping, lipolysis assays","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal gain/loss-of-function, protein interaction (Co-IP), subcellular localization with functional consequence, multiple orthogonal phenotypic readouts in a single rigorous study","pmids":["36477540"],"is_preprint":false},{"year":2022,"finding":"Overexpression of CLSTN3 in inguinal white adipose tissue impairs catecholamine-stimulated lipolysis and interacts with amyloid precursor protein (APP) in WAT, increasing APP accumulation in mitochondria and impairing mitochondrial function, thereby promoting obesity.","method":"Adeno-associated virus-mediated CLSTN3 overexpression in inguinal WAT in mice, in vivo and ex vivo lipolysis assays, co-immunoprecipitation (CLSTN3-APP interaction), mitochondrial function assays","journal":"Molecular metabolism","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo overexpression with functional lipolysis readout and Co-IP interaction, single lab with two orthogonal methods","pmids":["35753632"],"is_preprint":false},{"year":2023,"finding":"Hepatic CLSTN3 overexpression improves lipid metabolism disorder, gluconeogenesis, and energy homeostasis in NAFLD models, and acts at least partly through activation of Farnesoid X Receptor (FXR): CLSTN3 supplementation in FXR-knockout mice can still partially improve FXR-deficiency-related dysfunction, but RNAseq shows FXR expression is increased after CLSTN3 overexpression.","method":"AAV-mediated hepatic Clstn3 silencing and overexpression in HFD and db/db mice, RNAseq, TG/TC/functional assays, FXR-KO mouse experiments","journal":"ACS omega","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — in vivo gain/loss-of-function with pathway (FXR) identification via RNAseq, single lab, partial mechanistic follow-up","pmids":["37521618"],"is_preprint":false},{"year":2026,"finding":"CLSTN3 suppresses TLR4-triggered inflammation in macrophages by binding to the OST subunit DDOST, thereby inhibiting DDOST's interaction with the catalytic subunit STT3A and impairing OST complex assembly. This reduces N-glycosylation and membrane translocation of TLR4. CLSTN3 also suppresses membrane translocation and activation of TLR3, TLR7, and TLR9 through a similar mechanism.","method":"Genome-wide CRISPR screen, co-immunoprecipitation (CLSTN3-DDOST, DDOST-STT3A), glycosylation assays, TLR membrane localization assays, macrophage inflammatory assays with CLSTN3 gain/loss-of-function","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — unbiased CRISPR screen followed by mechanistic dissection with Co-IP, glycosylation assays, and functional rescue; multiple TLRs tested; single lab but multiple orthogonal methods","pmids":["41849401"],"is_preprint":false}],"current_model":"CLSTN3 is a multifunctional transmembrane protein that acts postsynaptically as a synapse organizer by directly binding α- and SS4-positive β-neurexins (via its cadherin/LNS domains) to drive excitatory and inhibitory synapse development; in adipose tissue, an ER-resident isoform (CLSTN3β) localizes to ER–lipid droplet contact sites and restricts lipid droplet fusion by inhibiting CIDE protein-mediated lipid transfer; in macrophages, CLSTN3 suppresses innate immune activation by binding the OST subunit DDOST to impair OST complex assembly, thereby reducing N-glycosylation and membrane translocation of TLR4 and other TLRs; additionally, CLSTN3 interacts with SVCT2 to promote neuronal vitamin C uptake, and neuronal CLSTN3 regulates systemic energy and bone homeostasis through a leptin-sensitive pathway."},"narrative":{"mechanistic_narrative":"CLSTN3 (calsyntenin-3) is a multifunctional transmembrane protein whose roles span synaptic organization, lipid storage, immune regulation, and systemic metabolism [PMID:24094106, PMID:36477540, PMID:41849401]. At the synapse, it acts postsynaptically as a synapse-organizing protein that directly binds neurexins to trigger both excitatory and inhibitory presynapse differentiation, with its shed ectodomain conversely suppressing presynaptic induction by other organizers [PMID:24094106]. The interaction is high-affinity and Ca2+-dependent, mediated by the CLSTN3 LNS and cadherin domains binding the LNS domain of neurexin, and is selective in vivo for splice-site-4 (SS4)-positive neurexins, whose expression rescues CLSTN3 synaptogenic activity; CLSTN3 cadherin domains also support homophilic cell adhesion [PMID:25352602, PMID:25463516, PMID:32434929]. Loss of Clstn3 reduces synapse density and impairs synaptic transmission, and neuron-specific deletion reduces excitatory inputs [PMID:24094106, PMID:32434929]. Beyond the brain, an adipocyte-selective ER-membrane isoform, CLSTN3β, localizes to ER–lipid droplet contact sites and restricts lipid droplet fusion by associating with CIDE proteins and impairing their lipid-transfer activity, enforcing a multilocular droplet phenotype required for normal brown adipose function [PMID:36477540]. In macrophages, CLSTN3 suppresses innate immune activation by binding the OST subunit DDOST, blocking its interaction with the catalytic subunit STT3A and thereby reducing N-glycosylation and membrane translocation of TLR4 and other TLRs [PMID:41849401]. CLSTN3 additionally interacts with the vitamin C transporter SVCT2 to promote neuronal ascorbic acid uptake, and neuronal CLSTN3 regulates systemic energy and bone homeostasis through a leptin-sensitive pathway [PMID:32382066, PMID:34673103].","teleology":[{"year":2013,"claim":"Established CLSTN3 as a bona fide postsynaptic synapse organizer, answering whether it has a direct trans-synaptic signaling role rather than a passive structural one.","evidence":"Unbiased screen with cell-based synaptogenesis assays, Co-IP, EM/confocal imaging, and electrophysiology in Clstn3-/- mice","pmids":["24094106"],"confidence":"High","gaps":["Did not resolve the structural basis of the neurexin interaction","Initially reported binding only to α-neurexins, not β-neurexins"]},{"year":2014,"claim":"Defined the biophysical and domain basis of CLSTN3–neurexin binding, showing it is direct, high-affinity, Ca2+-dependent, and mechanistically distinct from neuroligin.","evidence":"EM architecture, biochemical/biophysical binding assays, and mutagenesis-guided domain mapping in vitro","pmids":["25352602"],"confidence":"High","gaps":["No atomic-resolution structure of the complex","Functional role of tetramerization in synapse formation not established"]},{"year":2014,"claim":"Showed the CLSTN3 ectodomain can mediate homophilic adhesion through its cadherin repeats, addressing a possible adhesive function independent of neurexin.","evidence":"Zebrafish clstn3 cloning with bead-sorting adhesion assays and domain mapping","pmids":["25463516"],"confidence":"Medium","gaps":["Single method/lab","No homophilic paralog preference and no in vivo relevance demonstrated"]},{"year":2020,"claim":"Refined the synaptic mechanism by demonstrating CLSTN3 also binds β-neurexins with selectivity for SS4-positive variants, identifying the splice code governing complex formation.","evidence":"LC-MS/MS, RNAscope, electrophysiology, and conditional KO with cadherin-domain rescue in CA1 neurons","pmids":["32434929"],"confidence":"High","gaps":["Differential contribution to inhibitory versus excitatory synapses not fully resolved","Mechanism of SS4-dependent selectivity not structurally defined"]},{"year":2020,"claim":"Extended CLSTN3 function beyond synapses to systemic physiology, showing neuronal CLSTN3 controls energy expenditure, leptin sensitivity, and bone mass non-cell-autonomously.","evidence":"Global and cell-type-specific conditional KO mice with metabolic and bone-microarchitecture phenotyping","pmids":["32382066"],"confidence":"High","gaps":["Neuronal circuit and downstream effectors linking CLSTN3 to leptin signaling unidentified","Molecular mechanism connecting synaptic function to bone homeostasis unknown"]},{"year":2021,"claim":"Identified a transporter-regulatory role for CLSTN3, showing it binds SVCT2 and promotes neuronal vitamin C uptake.","evidence":"Y2H, Co-IP, M2H, co-localization, and ascorbic-acid uptake assays with gain/loss-of-function in human cell lines","pmids":["34673103"],"confidence":"High","gaps":["Mechanism by which CLSTN3 enhances SVCT2 activity (trafficking vs. stabilization) unresolved","In vivo relevance not tested"]},{"year":2022,"claim":"Revealed a distinct adipocyte isoform, CLSTN3β, acting at ER–lipid droplet contacts to restrict droplet fusion via CIDE proteins, establishing a non-synaptic, non-neuronal cellular function.","evidence":"KO/transgenic mice, subcellular localization imaging, Co-IP with CIDE proteins, lipid-transfer assays, and cold-challenge phenotyping","pmids":["36477540"],"confidence":"High","gaps":["Structural basis of CIDE inhibition not defined","Relationship between CLSTN3β and canonical synaptic CLSTN3 functions unaddressed"]},{"year":2022,"claim":"Showed CLSTN3 overexpression in white adipose tissue impairs lipolysis and mitochondrial function via interaction with APP, linking CLSTN3 to obesity.","evidence":"AAV-mediated WAT overexpression in mice with lipolysis assays, Co-IP, and mitochondrial function assays","pmids":["35753632"],"confidence":"Medium","gaps":["Single lab with limited orthogonal validation","Physiological relevance of the APP interaction at endogenous levels unclear"]},{"year":2023,"claim":"Implicated hepatic CLSTN3 in lipid and glucose metabolism through an FXR-linked pathway in NAFLD models.","evidence":"AAV-mediated hepatic silencing/overexpression in HFD and db/db mice, RNAseq, and FXR-KO experiments","pmids":["37521618"],"confidence":"Medium","gaps":["Direct molecular link between CLSTN3 and FXR not established","Partial rescue in FXR-KO indicates additional unidentified effectors"]},{"year":2026,"claim":"Defined a mechanistic immune role, showing CLSTN3 dampens TLR signaling by binding DDOST and disrupting OST complex assembly to limit TLR N-glycosylation and membrane translocation.","evidence":"Genome-wide CRISPR screen with Co-IP, glycosylation assays, TLR localization assays, and macrophage inflammatory readouts under gain/loss-of-function","pmids":["41849401"],"confidence":"High","gaps":["Whether this OST-modulating activity occurs in non-macrophage cell types not established","Relationship to the synaptic and adipose functions of CLSTN3 unclear"]},{"year":null,"claim":"How a single locus coordinates such distinct functions across neurons, adipocytes, hepatocytes, and macrophages, and which isoforms execute each role, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unifying mechanistic framework linking synaptic, lipid-droplet, immune, and metabolic roles","Isoform-specific contributions to each tissue function not systematically mapped","No human disease mutation evidence in the corpus"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098631","term_label":"cell adhesion mediator activity","supporting_discovery_ids":[0,2]},{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[0,3]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[5,6,9]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,3]},{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[6]},{"term_id":"GO:0005811","term_label":"lipid droplet","supporting_discovery_ids":[6]}],"pathway":[{"term_id":"R-HSA-112316","term_label":"Neuronal System","supporting_discovery_ids":[0,3]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[4,6,8]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[9]}],"complexes":[],"partners":["NRXN1","DDOST","SLC23A2","APP","CIDEA"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9BQT9","full_name":"Calsyntenin-3","aliases":["Alcadein-beta","Alc-beta"],"length_aa":956,"mass_kda":106.1,"function":"Postsynaptic adhesion molecule that binds to presynaptic neurexins to mediate both excitatory and inhibitory synapse formation (PubMed:25352602). Promotes synapse development by acting as a cell adhesion molecule at the postsynaptic membrane, which associates with both neurexin-alpha and neurexin-beta proteins at the presynaptic membrane (PubMed:25352602). Regulates the balance between excitatory and inhibitory synapses by inhibiting formation of excitatory parallel-fiber synapses and promoting formation of inhibitory synapses in the same neuron (By similarity). May also be involved in ascorbate (vitamin C) uptake via its interaction with SLC23A2/SVCT2 (PubMed:34673103). Complex formation with APBA2 and APP, stabilizes APP metabolism and enhances APBA2-mediated suppression of beta-APP40 secretion, due to the retardation of intracellular APP maturation (Probable) (PubMed:12972431) Adipose-specific isoform that plays a key role in adaptive thermogenesis. Facilitates the efficient use of stored triglyceride by promoting multilocular morphology of thermogenic adipocytes: acts by inhibiting the activity of CIDEA and CIDEC on lipid droplets, thereby preventing lipid droplet fusion and facilitating lipid utilization. May also participate in adaptive thermogenesis by promoting sympathetic innervation of thermogenic adipose tissue: acts by driving secretion of neurotrophic factor S100B from brown adipocytes, stimulating neurite outgrowth from sympathetic neurons","subcellular_location":"Lipid droplet; Endoplasmic reticulum membrane","url":"https://www.uniprot.org/uniprotkb/Q9BQT9/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/CLSTN3","classification":"Not Classified","n_dependent_lines":2,"n_total_lines":1208,"dependency_fraction":0.0016556291390728477},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/CLSTN3","total_profiled":1310},"omim":[{"mim_id":"611324","title":"CALSYNTENIN 3; CLSTN3","url":"https://www.omim.org/entry/611324"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"brain","ntpm":137.4}],"url":"https://www.proteinatlas.org/search/CLSTN3"},"hgnc":{"alias_symbol":["CSTN3","KIAA0726","CDHR14"],"prev_symbol":[]},"alphafold":{"accession":"Q9BQT9","domains":[{"cath_id":"2.60.40.60","chopping":"24-137","consensus_level":"high","plddt":92.0655,"start":24,"end":137},{"cath_id":"2.60.40.60","chopping":"145-240","consensus_level":"high","plddt":95.3938,"start":145,"end":240},{"cath_id":"2.60.40.680","chopping":"246-290_552-636","consensus_level":"high","plddt":90.2165,"start":246,"end":636},{"cath_id":"2.60.120.200","chopping":"303-497_525-548","consensus_level":"high","plddt":81.4938,"start":303,"end":548},{"cath_id":"2.60.40,2.60.40","chopping":"642-680_697-802","consensus_level":"high","plddt":86.6977,"start":642,"end":802}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9BQT9","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9BQT9-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9BQT9-F1-predicted_aligned_error_v6.png","plddt_mean":77.25},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CLSTN3","jax_strain_url":"https://www.jax.org/strain/search?query=CLSTN3"},"sequence":{"accession":"Q9BQT9","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9BQT9.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9BQT9/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9BQT9"}},"corpus_meta":[{"pmid":"37726845","id":"PMC_37726845","title":"Identification 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care","url":"https://pubmed.ncbi.nlm.nih.gov/41266703","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":16889,"output_tokens":3196,"usd":0.049304,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10712,"output_tokens":3616,"usd":0.07198,"stage2_stop_reason":"end_turn"},"total_usd":0.121284,"stage1_batch_id":"msgbatch_01ACAxJQabDsht39JXeWG22d","stage2_batch_id":"msgbatch_01QEU22A9U5sbPTgsMzTmvSf","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2013,\n      \"finding\": \"Calsyntenin-3 (CLSTN3) is a postsynaptic synapse-organizing protein that specifically binds and recruits α-neurexins (but not β-neurexins) to trigger both excitatory and inhibitory presynapse differentiation in contacting axons. Its shed ectodomain suppresses the ability of multiple α-neurexin partners (including neuroligin-2 and LRRTM2) to induce presynapse differentiation. Clstn3−/− mice show reductions in excitatory and inhibitory synapse density and corresponding deficits in synaptic transmission.\",\n      \"method\": \"Unbiased screen, cell-based synaptogenesis assays, co-immunoprecipitation, confocal and electron microscopy, electrophysiological recordings in Clstn3−/− mice\",\n      \"journal\": \"Neuron\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal binding assays, loss-of-function mouse with multiple orthogonal readouts (EM, confocal, electrophysiology), replicated across multiple experimental approaches in single rigorous study\",\n      \"pmids\": [\"24094106\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"The extracellular domains of CLSTN3 and neurexin-1α (n1α) interact directly with nanomolar affinity. CLSTN3 ectodomains form monomers and disulfide-stabilized tetramers that are Ca2+-dependent and flexible in solution. The interaction requires minimally the LNS domain of CLSTN3 and uses a fundamentally different binding mechanism than neuroligin-2 — notably, CLSTN3 does not strictly require the sixth LNS domain of n1α. Both monomeric and tetrameric forms bind n1α.\",\n      \"method\": \"Electron microscopy (structural architecture), biochemical binding assays, biophysical measurements (affinity determination), mutagenesis-guided domain mapping\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct in vitro reconstitution with structural (EM) and biophysical validation, multiple orthogonal methods in a single rigorous study\",\n      \"pmids\": [\"25352602\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Zebrafish CLSTN3 ectodomain mediates homophilic cell-cell adhesion through its two amino-terminal cadherin repeats; in bead-sorting assays, calsyntenin ectodomains do not exhibit homophilic preferences among paralogs.\",\n      \"method\": \"Cloning of zebrafish clstn3, bead-sorting adhesion assays, domain mapping\",\n      \"journal\": \"Neuroscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — direct adhesion assay with domain mapping, single lab, single method set\",\n      \"pmids\": [\"25463516\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"CLSTN3 interacts with β-neurexins (in addition to α-neurexins) via the LNS domain of β-Nrxn and CLSTN3 cadherin domains. Specifically, splice site 4 (SS4) insert-positive β-Nrxn variants (not insert-negative) rescue impaired Clstn3 synaptogenic activity in Nrxn-deficient neurons. In vivo, Clstn3 selectively forms complexes with SS4-positive Nrxns. Neuron-specific Clstn3 deletion reduces excitatory synaptic inputs, and expression of CLSTN3 cadherin domains in CA1 neurons of Clstn3 conditional KO mice rescues structural deficits in excitatory synapses in the stratum radiatum.\",\n      \"method\": \"LC-MS/MS protein analysis, confocal microscopy, RNAscope, electrophysiological recordings, conditional knockout mouse, domain rescue experiments\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (MS, electrophysiology, conditional KO rescue), in vivo and in vitro confirmation in single rigorous study\",\n      \"pmids\": [\"32434929\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Neuronal CLSTN3 regulates systemic energy and bone homeostasis. Global Clstn3 KO mice show reduced body mass, improved leptin sensitivity, increased energy expenditure, and reduced cortical bone mass. Pan-neuronal (but not sympathetic-neuron-specific or osteoblast/osteoclast-specific) deletion of Clstn3 recapitulates these phenotypes, indicating the effects are neuronally mediated rather than bone-cell-autonomous.\",\n      \"method\": \"Global and cell-type-specific conditional knockout mice (pan-neuronal, sympathetic, osteoblast, osteoclast), metabolic phenotyping, bone microarchitecture analysis, in vitro osteoblast/osteoclast cultures\",\n      \"journal\": \"Experimental & molecular medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple conditional KO lines with cell-type specificity, multiple phenotypic readouts, epistasis-style genetic dissection\",\n      \"pmids\": [\"32382066\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"CLSTN3 physically interacts with the sodium-dependent vitamin C transporter-2 (SVCT2/hSVCT2) in neuronal cells. Co-expression of hCLSTN3 with hSVCT2 in SH-SY5Y cells markedly increases ascorbic acid (AA) uptake, while siRNA-mediated knockdown of hCLSTN3 inhibits AA uptake, indicating CLSTN3 positively regulates SVCT2-mediated vitamin C transport.\",\n      \"method\": \"Yeast two-hybrid (Y2H) screen of human brain cDNA library, co-immunoprecipitation, mammalian two-hybrid (M2H), co-localization in human cell lines, AA uptake assays, siRNA knockdown\",\n      \"journal\": \"International journal of biological macromolecules\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — interaction confirmed by three orthogonal methods (Y2H, Co-IP, M2H) plus functional uptake assays with both gain- and loss-of-function\",\n      \"pmids\": [\"34673103\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"CLSTN3β, an adipocyte-selective isoform encoded at the Clstn3 locus present only in placental mammals, is an integral ER membrane protein that localizes to ER–lipid droplet (LD) contact sites via a conserved hairpin-like domain. CLSTN3β associates with cell death-inducing DFFA-like effector (CIDE) proteins and impairs their ability to transfer lipid between LDs, thereby restricting LD fusion and expansion and enforcing a multilocular LD phenotype. Loss of CLSTN3β causes abnormal LD morphology and altered substrate use in brown adipose tissue with cold-induced hypothermia; forced expression enforces multilocular LD phenotype in cells and adipose tissue.\",\n      \"method\": \"Knockout and transgenic mice, subcellular fractionation/localization imaging, co-immunoprecipitation with CIDE proteins, lipid transfer assays, cold-challenge metabolic phenotyping, lipolysis assays\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal gain/loss-of-function, protein interaction (Co-IP), subcellular localization with functional consequence, multiple orthogonal phenotypic readouts in a single rigorous study\",\n      \"pmids\": [\"36477540\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Overexpression of CLSTN3 in inguinal white adipose tissue impairs catecholamine-stimulated lipolysis and interacts with amyloid precursor protein (APP) in WAT, increasing APP accumulation in mitochondria and impairing mitochondrial function, thereby promoting obesity.\",\n      \"method\": \"Adeno-associated virus-mediated CLSTN3 overexpression in inguinal WAT in mice, in vivo and ex vivo lipolysis assays, co-immunoprecipitation (CLSTN3-APP interaction), mitochondrial function assays\",\n      \"journal\": \"Molecular metabolism\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo overexpression with functional lipolysis readout and Co-IP interaction, single lab with two orthogonal methods\",\n      \"pmids\": [\"35753632\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Hepatic CLSTN3 overexpression improves lipid metabolism disorder, gluconeogenesis, and energy homeostasis in NAFLD models, and acts at least partly through activation of Farnesoid X Receptor (FXR): CLSTN3 supplementation in FXR-knockout mice can still partially improve FXR-deficiency-related dysfunction, but RNAseq shows FXR expression is increased after CLSTN3 overexpression.\",\n      \"method\": \"AAV-mediated hepatic Clstn3 silencing and overexpression in HFD and db/db mice, RNAseq, TG/TC/functional assays, FXR-KO mouse experiments\",\n      \"journal\": \"ACS omega\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — in vivo gain/loss-of-function with pathway (FXR) identification via RNAseq, single lab, partial mechanistic follow-up\",\n      \"pmids\": [\"37521618\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"CLSTN3 suppresses TLR4-triggered inflammation in macrophages by binding to the OST subunit DDOST, thereby inhibiting DDOST's interaction with the catalytic subunit STT3A and impairing OST complex assembly. This reduces N-glycosylation and membrane translocation of TLR4. CLSTN3 also suppresses membrane translocation and activation of TLR3, TLR7, and TLR9 through a similar mechanism.\",\n      \"method\": \"Genome-wide CRISPR screen, co-immunoprecipitation (CLSTN3-DDOST, DDOST-STT3A), glycosylation assays, TLR membrane localization assays, macrophage inflammatory assays with CLSTN3 gain/loss-of-function\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — unbiased CRISPR screen followed by mechanistic dissection with Co-IP, glycosylation assays, and functional rescue; multiple TLRs tested; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"41849401\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CLSTN3 is a multifunctional transmembrane protein that acts postsynaptically as a synapse organizer by directly binding α- and SS4-positive β-neurexins (via its cadherin/LNS domains) to drive excitatory and inhibitory synapse development; in adipose tissue, an ER-resident isoform (CLSTN3β) localizes to ER–lipid droplet contact sites and restricts lipid droplet fusion by inhibiting CIDE protein-mediated lipid transfer; in macrophages, CLSTN3 suppresses innate immune activation by binding the OST subunit DDOST to impair OST complex assembly, thereby reducing N-glycosylation and membrane translocation of TLR4 and other TLRs; additionally, CLSTN3 interacts with SVCT2 to promote neuronal vitamin C uptake, and neuronal CLSTN3 regulates systemic energy and bone homeostasis through a leptin-sensitive pathway.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"CLSTN3 (calsyntenin-3) is a multifunctional transmembrane protein whose roles span synaptic organization, lipid storage, immune regulation, and systemic metabolism [#0, #6, #9]. At the synapse, it acts postsynaptically as a synapse-organizing protein that directly binds neurexins to trigger both excitatory and inhibitory presynapse differentiation, with its shed ectodomain conversely suppressing presynaptic induction by other organizers [#0]. The interaction is high-affinity and Ca2+-dependent, mediated by the CLSTN3 LNS and cadherin domains binding the LNS domain of neurexin, and is selective in vivo for splice-site-4 (SS4)-positive neurexins, whose expression rescues CLSTN3 synaptogenic activity; CLSTN3 cadherin domains also support homophilic cell adhesion [#1, #2, #3]. Loss of Clstn3 reduces synapse density and impairs synaptic transmission, and neuron-specific deletion reduces excitatory inputs [#0, #3]. Beyond the brain, an adipocyte-selective ER-membrane isoform, CLSTN3\\u03b2, localizes to ER\\u2013lipid droplet contact sites and restricts lipid droplet fusion by associating with CIDE proteins and impairing their lipid-transfer activity, enforcing a multilocular droplet phenotype required for normal brown adipose function [#6]. In macrophages, CLSTN3 suppresses innate immune activation by binding the OST subunit DDOST, blocking its interaction with the catalytic subunit STT3A and thereby reducing N-glycosylation and membrane translocation of TLR4 and other TLRs [#9]. CLSTN3 additionally interacts with the vitamin C transporter SVCT2 to promote neuronal ascorbic acid uptake, and neuronal CLSTN3 regulates systemic energy and bone homeostasis through a leptin-sensitive pathway [#4, #5].\",\n  \"teleology\": [\n    {\n      \"year\": 2013,\n      \"claim\": \"Established CLSTN3 as a bona fide postsynaptic synapse organizer, answering whether it has a direct trans-synaptic signaling role rather than a passive structural one.\",\n      \"evidence\": \"Unbiased screen with cell-based synaptogenesis assays, Co-IP, EM/confocal imaging, and electrophysiology in Clstn3-/- mice\",\n      \"pmids\": [\"24094106\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve the structural basis of the neurexin interaction\", \"Initially reported binding only to \\u03b1-neurexins, not \\u03b2-neurexins\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Defined the biophysical and domain basis of CLSTN3\\u2013neurexin binding, showing it is direct, high-affinity, Ca2+-dependent, and mechanistically distinct from neuroligin.\",\n      \"evidence\": \"EM architecture, biochemical/biophysical binding assays, and mutagenesis-guided domain mapping in vitro\",\n      \"pmids\": [\"25352602\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No atomic-resolution structure of the complex\", \"Functional role of tetramerization in synapse formation not established\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Showed the CLSTN3 ectodomain can mediate homophilic adhesion through its cadherin repeats, addressing a possible adhesive function independent of neurexin.\",\n      \"evidence\": \"Zebrafish clstn3 cloning with bead-sorting adhesion assays and domain mapping\",\n      \"pmids\": [\"25463516\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single method/lab\", \"No homophilic paralog preference and no in vivo relevance demonstrated\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Refined the synaptic mechanism by demonstrating CLSTN3 also binds \\u03b2-neurexins with selectivity for SS4-positive variants, identifying the splice code governing complex formation.\",\n      \"evidence\": \"LC-MS/MS, RNAscope, electrophysiology, and conditional KO with cadherin-domain rescue in CA1 neurons\",\n      \"pmids\": [\"32434929\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Differential contribution to inhibitory versus excitatory synapses not fully resolved\", \"Mechanism of SS4-dependent selectivity not structurally defined\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Extended CLSTN3 function beyond synapses to systemic physiology, showing neuronal CLSTN3 controls energy expenditure, leptin sensitivity, and bone mass non-cell-autonomously.\",\n      \"evidence\": \"Global and cell-type-specific conditional KO mice with metabolic and bone-microarchitecture phenotyping\",\n      \"pmids\": [\"32382066\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Neuronal circuit and downstream effectors linking CLSTN3 to leptin signaling unidentified\", \"Molecular mechanism connecting synaptic function to bone homeostasis unknown\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Identified a transporter-regulatory role for CLSTN3, showing it binds SVCT2 and promotes neuronal vitamin C uptake.\",\n      \"evidence\": \"Y2H, Co-IP, M2H, co-localization, and ascorbic-acid uptake assays with gain/loss-of-function in human cell lines\",\n      \"pmids\": [\"34673103\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which CLSTN3 enhances SVCT2 activity (trafficking vs. stabilization) unresolved\", \"In vivo relevance not tested\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Revealed a distinct adipocyte isoform, CLSTN3\\u03b2, acting at ER\\u2013lipid droplet contacts to restrict droplet fusion via CIDE proteins, establishing a non-synaptic, non-neuronal cellular function.\",\n      \"evidence\": \"KO/transgenic mice, subcellular localization imaging, Co-IP with CIDE proteins, lipid-transfer assays, and cold-challenge phenotyping\",\n      \"pmids\": [\"36477540\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of CIDE inhibition not defined\", \"Relationship between CLSTN3\\u03b2 and canonical synaptic CLSTN3 functions unaddressed\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Showed CLSTN3 overexpression in white adipose tissue impairs lipolysis and mitochondrial function via interaction with APP, linking CLSTN3 to obesity.\",\n      \"evidence\": \"AAV-mediated WAT overexpression in mice with lipolysis assays, Co-IP, and mitochondrial function assays\",\n      \"pmids\": [\"35753632\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab with limited orthogonal validation\", \"Physiological relevance of the APP interaction at endogenous levels unclear\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Implicated hepatic CLSTN3 in lipid and glucose metabolism through an FXR-linked pathway in NAFLD models.\",\n      \"evidence\": \"AAV-mediated hepatic silencing/overexpression in HFD and db/db mice, RNAseq, and FXR-KO experiments\",\n      \"pmids\": [\"37521618\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct molecular link between CLSTN3 and FXR not established\", \"Partial rescue in FXR-KO indicates additional unidentified effectors\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Defined a mechanistic immune role, showing CLSTN3 dampens TLR signaling by binding DDOST and disrupting OST complex assembly to limit TLR N-glycosylation and membrane translocation.\",\n      \"evidence\": \"Genome-wide CRISPR screen with Co-IP, glycosylation assays, TLR localization assays, and macrophage inflammatory readouts under gain/loss-of-function\",\n      \"pmids\": [\"41849401\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether this OST-modulating activity occurs in non-macrophage cell types not established\", \"Relationship to the synaptic and adipose functions of CLSTN3 unclear\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How a single locus coordinates such distinct functions across neurons, adipocytes, hepatocytes, and macrophages, and which isoforms execute each role, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unifying mechanistic framework linking synaptic, lipid-droplet, immune, and metabolic roles\", \"Isoform-specific contributions to each tissue function not systematically mapped\", \"No human disease mutation evidence in the corpus\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098631\", \"supporting_discovery_ids\": [0, 2]},\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [0, 3]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [5, 6, 9]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 3]},\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [6]},\n      {\"term_id\": \"GO:0005811\", \"supporting_discovery_ids\": [6]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-112316\", \"supporting_discovery_ids\": [0, 3]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [4, 6, 8]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [9]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"NRXN1\", \"DDOST\", \"SLC23A2\", \"APP\", \"CIDEA\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":7,"faith_pct":85.71428571428571}}