{"gene":"CNTN4","run_date":"2026-06-09T22:57:18","timeline":{"discoveries":[{"year":1995,"finding":"BIG-2 (CNTN4) is a GPI-anchored cell adhesion molecule with six Ig-like domains and four fibronectin type III-like repeats; recombinant BIG-2 protein promotes neurite outgrowth when used as a substrate for neurons in vitro, establishing its direct role in axon growth.","method":"Molecular cloning, domain analysis, in vitro neurite outgrowth assay using recombinant protein as substrate","journal":"Journal of neurobiology","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — direct in vitro functional assay with recombinant protein, single lab, single study","pmids":["8586965"],"is_preprint":false},{"year":1997,"finding":"Alternative splicing of the BIG-2 (CNTN4) gene produces a truncated isoform (BIG-2A) that lacks the GPI-anchoring domain and is specifically expressed in mature sensory cells of the vomeronasal neuroepithelium and olfactory neuroepithelium, suggesting splice-variant-specific roles in chemosensory tissue organization.","method":"cDNA cloning, sequence analysis, in situ hybridization in adult and developing mice","journal":"Brain research. Molecular brain research","confidence":"Low","confidence_rationale":"Tier 3 / Weak — expression localization by in situ hybridization, single lab, no functional manipulation","pmids":["9221934"],"is_preprint":false},{"year":2008,"finding":"BIG-2/CNTN4 is required for convergence of olfactory sensory neuron axons onto topographically fixed glomeruli in the olfactory bulb; in BIG-2-deficient mice, neurons expressing a given odorant receptor innervate multiple glomeruli at ectopic locations, demonstrating CNTN4 functions as an axon guidance molecule for odor map formation.","method":"BIG-2 knockout mouse model, immunohistochemistry, olfactory axon tracing, glomerular mapping","journal":"Neuron","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean KO with specific and well-defined cellular phenotype (ectopic multi-glomerular innervation), published in high-impact journal, clear loss-of-function readout","pmids":["18367085"],"is_preprint":false},{"year":2018,"finding":"In Cntn4-/- mice, cell-surface levels of glutamate receptor subunits (GluA1, GluA2, GluN1) are altered in a brain-region-specific manner (reduced in cortex/hippocampus, increased in striatum), and surface GABA-A receptor (α1 subunit) levels are downregulated in several brain regions, demonstrating CNTN4 modulates excitatory and inhibitory synaptic receptor surface expression.","method":"Biotinylation assay and western blotting for surface receptor subunits in Cntn4-/- mice across multiple brain regions","journal":"Frontiers in molecular neuroscience","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean KO with orthogonal biochemical readout (surface biotinylation + western blot), multiple brain regions and receptor types analyzed, single lab","pmids":["29970989"],"is_preprint":false},{"year":2021,"finding":"Cntn4 KO mice display reduced CA1 hippocampal synaptic potentiation (LTP) and abnormal dendritic arborization and spine density of CA1 neurons; behaviorally, Cntn4-deficient mice show increased contextual fear conditioning in a gene-dose-dependent manner, placing CNTN4 in a pathway controlling hippocampal synaptic plasticity and fear memory.","method":"Cntn4 KO mouse model, hippocampal slice electrophysiology (LTP), neuroanatomical analysis of dendritic morphology, behavioral fear conditioning","journal":"Translational psychiatry","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (electrophysiology, neuroanatomy, behavior) in clean KO model, gene-dose-dependent behavioral effect replicated in hetero- and homozygous animals","pmids":["33542194"],"is_preprint":false},{"year":2021,"finding":"CNTN4 promotes dendritic spine formation and excitatory synapse number in cortical neurons; truncated proteins lacking the signal peptide, FnIII domains, or GPI domain cannot rescue spine density deficits, demonstrating that CNTN4 regulates spine density through a mechanism dependent on FnIII domains; autism-associated variants of CNTN4 also fail to rescue spine density and neural activity.","method":"Cntn4 disruption in cortical neurons, domain-deletion mutant rescue experiments, dendritic spine counting, electrophysiological measurement of neural activity, analysis of autism-linked variants","journal":"Human molecular genetics","confidence":"High","confidence_rationale":"Tier 1 / Moderate — domain mutagenesis with defined functional readout (spine density, neural activity), multiple deletion constructs tested, single lab but multiple orthogonal methods","pmids":["34415325"],"is_preprint":false},{"year":2022,"finding":"miR-148a-3p directly targets the 3′UTR of CNTN4 mRNA (validated by dual-luciferase reporter assay), reducing CNTN4 expression in oxLDL-treated THP-1 macrophages and thereby inhibiting apoptosis and pro-inflammatory cytokine (IL-6, TNF-α) production, identifying CNTN4 as a functional downstream target of miR-148a-3p in macrophage inflammatory signaling.","method":"Dual-luciferase reporter assay, qRT-PCR, flow cytometry, ELISA in THP-1 macrophage model","journal":"Annals of translational medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — dual-luciferase confirms direct miRNA targeting, functional cellular readouts, but single lab and non-neuronal context potentially divergent from canonical CNTN4 function","pmids":["36544657"],"is_preprint":false},{"year":2024,"finding":"Mass spectrometry identified amyloid precursor protein (APP) as a binding partner of CNTN4; knockout of CNTN4 and/or APP in human cells revealed that CNTN4–APP interaction controls neural elongation, and loss of either protein reduces neurite outgrowth, demonstrating a functional CNTN4–APP complex in cortical neurodevelopment.","method":"Mass spectrometry interaction screen, CNTN4/APP single and double knockout human cell lines, neural elongation/morphology assays","journal":"Open biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MS-identified interaction confirmed functionally with double KO and specific cellular phenotype, single lab, two orthogonal approaches (MS + KO rescue)","pmids":["38745463"],"is_preprint":false}],"current_model":"CNTN4 (BIG-2) is a GPI-anchored, FnIII- and Ig-domain-containing axonal cell adhesion molecule that promotes neurite outgrowth and dendritic spine formation via its FnIII domains, guides olfactory sensory axons to correct glomeruli in the olfactory bulb, modulates hippocampal LTP and CA1 dendritic morphology, regulates brain-region-specific surface expression of glutamate and GABA receptors, and interacts with APP to control cortical neural elongation; autism-linked variants and domain-truncation experiments indicate these functions require intact FnIII and GPI domains."},"narrative":{"mechanistic_narrative":"CNTN4 (BIG-2) is a GPI-anchored, Ig- and fibronectin type III (FnIII)-domain cell adhesion molecule that promotes axon growth and synaptic connectivity in the developing nervous system [PMID:8586965, PMID:34415325]. As a substrate, recombinant CNTN4 directly promotes neurite outgrowth, and in vivo it acts as an axon guidance cue: loss of CNTN4 causes olfactory sensory neurons expressing a given odorant receptor to mistarget to multiple ectopic glomeruli, disrupting odor-map formation [PMID:8586965, PMID:18367085]. Beyond wiring, CNTN4 shapes synapse structure and function — it promotes dendritic spine formation and excitatory synapse number in cortical neurons, and its loss reduces CA1 hippocampal LTP, alters dendritic arborization and spine density, and produces gene-dose-dependent behavioral changes [PMID:33542194, PMID:34415325]. Spine-promoting activity requires intact signal peptide, FnIII, and GPI domains, and autism-associated CNTN4 variants fail to rescue spine density and neural activity, tying these domains to neurodevelopmental disease risk [PMID:34415325]. CNTN4 also modulates the brain-region-specific surface expression of glutamate (GluA1, GluA2, GluN1) and GABA-A (α1) receptors [PMID:29970989], and it forms a functional complex with amyloid precursor protein (APP) that controls neurite/neural elongation in cortical neurodevelopment [PMID:38745463]. A non-neuronal role is documented in which CNTN4 acts as a direct target of miR-148a-3p in macrophage inflammatory signaling [PMID:36544657].","teleology":[{"year":1995,"claim":"Established CNTN4 as a GPI-anchored adhesion molecule with a direct, intrinsic capacity to drive axon growth, defining its baseline molecular activity.","evidence":"Molecular cloning, domain analysis, and in vitro neurite outgrowth assays using recombinant BIG-2 as a substrate","pmids":["8586965"],"confidence":"Medium","gaps":["No in vivo confirmation of the outgrowth-promoting role","Binding partner mediating outgrowth not identified","Domain requirements not yet dissected"]},{"year":1997,"claim":"Showed that alternative splicing yields a GPI-anchorless isoform (BIG-2A) restricted to mature chemosensory neuroepithelia, raising the possibility of splice-variant-specific functions.","evidence":"cDNA cloning and in situ hybridization in adult and developing mice","pmids":["9221934"],"confidence":"Low","gaps":["Expression localization only; no functional manipulation of the isoform","Functional distinction between GPI-anchored and anchorless forms untested","Single lab, no orthogonal validation"]},{"year":2008,"claim":"Demonstrated in vivo that CNTN4 is required for olfactory sensory axon convergence onto correct glomeruli, establishing it as a bona fide axon guidance molecule for odor-map formation.","evidence":"BIG-2 knockout mouse with olfactory axon tracing and glomerular mapping","pmids":["18367085"],"confidence":"High","gaps":["Molecular guidance partners/receptors not identified","Whether guidance uses the same domains as in vitro outgrowth unknown"]},{"year":2018,"claim":"Revealed that CNTN4 modulates surface availability of excitatory and inhibitory neurotransmitter receptors in a brain-region-specific manner, extending its role from wiring to synaptic receptor regulation.","evidence":"Surface biotinylation and western blotting for glutamate and GABA-A receptor subunits across brain regions in Cntn4-/- mice","pmids":["29970989"],"confidence":"Medium","gaps":["Mechanism linking CNTN4 to receptor surface trafficking unknown","Direct physical interaction with receptors not shown","Single lab"]},{"year":2021,"claim":"Connected CNTN4 to hippocampal synaptic plasticity and behavior, showing its loss impairs CA1 LTP, alters dendritic morphology/spine density, and produces gene-dose-dependent fear-memory changes.","evidence":"Hippocampal slice electrophysiology, neuroanatomy, and fear-conditioning behavior in Cntn4 KO mice","pmids":["33542194"],"confidence":"High","gaps":["Molecular pathway from CNTN4 to LTP not resolved","Cell-autonomous vs circuit-level contributions not separated"]},{"year":2021,"claim":"Mapped CNTN4's spine-promoting activity to specific domains and linked autism-associated variants to functional loss, providing a structure-function basis for disease risk.","evidence":"Domain-deletion mutant rescue, spine counting, electrophysiology, and analysis of autism-linked variants in cortical neurons","pmids":["34415325"],"confidence":"High","gaps":["Binding partner engaged by FnIII domains for spine formation not identified","Single lab"]},{"year":2024,"claim":"Identified APP as a CNTN4 binding partner and showed the CNTN4-APP complex is required for neural elongation, providing a molecular partner for CNTN4's developmental morphology functions.","evidence":"Mass spectrometry interaction screen plus single and double CNTN4/APP knockout human cell lines with neural elongation assays","pmids":["38745463"],"confidence":"Medium","gaps":["Structural basis and stoichiometry of the CNTN4-APP interaction unknown","Whether APP mediates the olfactory guidance or synaptic phenotypes not tested","Single lab"]},{"year":2022,"claim":"Documented a non-neuronal regulatory context in which CNTN4 is a direct miR-148a-3p target controlling macrophage apoptosis and inflammatory cytokine output.","evidence":"Dual-luciferase 3'UTR reporter, qRT-PCR, flow cytometry, and ELISA in oxLDL-treated THP-1 macrophages","pmids":["36544657"],"confidence":"Medium","gaps":["Mechanism by which CNTN4 influences macrophage inflammation unknown","Relevance to CNTN4's neuronal functions unclear","Single lab, non-neuronal model"]},{"year":null,"claim":"The receptors/ligands through which CNTN4 transduces axon guidance, receptor-surface regulation, and synaptic plasticity signals remain largely undefined beyond the APP interaction.","evidence":"No discovery in the timeline identifies a guidance receptor or signaling effector downstream of CNTN4","pmids":[],"confidence":"Low","gaps":["No guidance receptor identified for olfactory axon targeting","Mechanistic link between CNTN4 and glutamate/GABA receptor trafficking unresolved","Downstream signaling effectors of spine formation unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098631","term_label":"cell adhesion mediator activity","supporting_discovery_ids":[0]},{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[2,5]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,3]}],"pathway":[{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[2,5,7]},{"term_id":"R-HSA-112316","term_label":"Neuronal System","supporting_discovery_ids":[3,4]}],"complexes":[],"partners":["APP"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q8IWV2","full_name":"Contactin-4","aliases":["Brain-derived immunoglobulin superfamily protein 2","BIG-2"],"length_aa":1026,"mass_kda":113.5,"function":"Contactins mediate cell surface interactions during nervous system development. Has some neurite outgrowth-promoting activity. May be involved in synaptogenesis","subcellular_location":"Cell membrane; Secreted","url":"https://www.uniprot.org/uniprotkb/Q8IWV2/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/CNTN4","classification":"Not Classified","n_dependent_lines":3,"n_total_lines":1208,"dependency_fraction":0.0024834437086092716},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/CNTN4","total_profiled":1310},"omim":[{"mim_id":"613792","title":"CHROMOSOME 3pter-p25 DELETION SYNDROME","url":"https://www.omim.org/entry/613792"},{"mim_id":"607280","title":"CONTACTIN 4; CNTN4","url":"https://www.omim.org/entry/607280"},{"mim_id":"209850","title":"AUTISM","url":"https://www.omim.org/entry/209850"},{"mim_id":"147265","title":"INOSITOL 1,4,5-TRIPHOSPHATE RECEPTOR, TYPE 1; ITPR1","url":"https://www.omim.org/entry/147265"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"parathyroid gland","ntpm":21.2}],"url":"https://www.proteinatlas.org/search/CNTN4"},"hgnc":{"alias_symbol":["BIG-2"],"prev_symbol":[]},"alphafold":{"accession":"Q8IWV2","domains":[{"cath_id":"2.60.40.10","chopping":"25-120","consensus_level":"high","plddt":90.6734,"start":25,"end":120},{"cath_id":"2.60.40.10","chopping":"132-221","consensus_level":"medium","plddt":91.5018,"start":132,"end":221},{"cath_id":"2.60.40.10","chopping":"223-316","consensus_level":"medium","plddt":91.6623,"start":223,"end":316},{"cath_id":"2.60.40.10","chopping":"322-403","consensus_level":"high","plddt":91.1527,"start":322,"end":403},{"cath_id":"2.60.40.10","chopping":"405-496","consensus_level":"high","plddt":89.5888,"start":405,"end":496},{"cath_id":"2.60.40.10","chopping":"500-595","consensus_level":"high","plddt":86.6805,"start":500,"end":595},{"cath_id":"2.60.40.10","chopping":"602-694","consensus_level":"high","plddt":93.8483,"start":602,"end":694},{"cath_id":"2.60.40.10","chopping":"705-796","consensus_level":"medium","plddt":91.1633,"start":705,"end":796},{"cath_id":"2.60.40.10","chopping":"807-897","consensus_level":"medium","plddt":90.2104,"start":807,"end":897},{"cath_id":"2.60.40.10","chopping":"907-990","consensus_level":"medium","plddt":86.0107,"start":907,"end":990}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8IWV2","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q8IWV2-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q8IWV2-F1-predicted_aligned_error_v6.png","plddt_mean":86.44},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CNTN4","jax_strain_url":"https://www.jax.org/strain/search?query=CNTN4"},"sequence":{"accession":"Q8IWV2","fasta_url":"https://rest.uniprot.org/uniprotkb/Q8IWV2.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q8IWV2/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8IWV2"}},"corpus_meta":[{"pmid":"8586965","id":"PMC_8586965","title":"Overlapping and differential expression of BIG-2, BIG-1, TAG-1, and F3: four members of an axon-associated cell adhesion molecule subgroup of the immunoglobulin superfamily.","date":"1995","source":"Journal of neurobiology","url":"https://pubmed.ncbi.nlm.nih.gov/8586965","citation_count":142,"is_preprint":false},{"pmid":"15106122","id":"PMC_15106122","title":"Disruption of contactin 4 (CNTN4) results in developmental delay and other features of 3p deletion syndrome.","date":"2004","source":"American journal of human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/15106122","citation_count":133,"is_preprint":false},{"pmid":"18367085","id":"PMC_18367085","title":"BIG-2 mediates olfactory axon convergence to target glomeruli.","date":"2008","source":"Neuron","url":"https://pubmed.ncbi.nlm.nih.gov/18367085","citation_count":131,"is_preprint":false},{"pmid":"17036314","id":"PMC_17036314","title":"FISH and array-CGH analysis of a complex chromosome 3 aberration suggests that loss of CNTN4 and CRBN contributes to mental retardation in 3pter deletions.","date":"2006","source":"American journal of medical genetics. Part A","url":"https://pubmed.ncbi.nlm.nih.gov/17036314","citation_count":59,"is_preprint":false},{"pmid":"30335191","id":"PMC_30335191","title":"Pregnancies during and after trastuzumab and/or lapatinib in patients with human epidermal growth factor receptor 2-positive early breast cancer: Analysis from the NeoALTTO (BIG 1-06) and ALTTO (BIG 2-06) trials.","date":"2018","source":"Cancer","url":"https://pubmed.ncbi.nlm.nih.gov/30335191","citation_count":58,"is_preprint":false},{"pmid":"24395109","id":"PMC_24395109","title":"Central pathology laboratory review of HER2 and ER in early breast cancer: an ALTTO trial [BIG 2-06/NCCTG N063D (Alliance)] ring study.","date":"2014","source":"Breast cancer research and treatment","url":"https://pubmed.ncbi.nlm.nih.gov/24395109","citation_count":56,"is_preprint":false},{"pmid":"31134488","id":"PMC_31134488","title":"Dissecting the effect of hormone receptor status in patients with HER2-positive early breast cancer: exploratory analysis from the ALTTO (BIG 2-06) randomized clinical trial.","date":"2019","source":"Breast cancer research and treatment","url":"https://pubmed.ncbi.nlm.nih.gov/31134488","citation_count":37,"is_preprint":false},{"pmid":"33542194","id":"PMC_33542194","title":"Cntn4, a risk gene for neuropsychiatric disorders, modulates hippocampal synaptic plasticity and behavior.","date":"2021","source":"Translational psychiatry","url":"https://pubmed.ncbi.nlm.nih.gov/33542194","citation_count":36,"is_preprint":false},{"pmid":"29970989","id":"PMC_29970989","title":"Heterogeneity of Cell Surface Glutamate and GABA Receptor Expression in Shank and CNTN4 Autism Mouse Models.","date":"2018","source":"Frontiers in molecular neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/29970989","citation_count":30,"is_preprint":false},{"pmid":"33401235","id":"PMC_33401235","title":"Body Mass Index and Weight Change in Patients With HER2-Positive Early Breast Cancer: Exploratory Analysis of the ALTTO BIG 2-06 Trial.","date":"2021","source":"Journal of the National Comprehensive Cancer Network : JNCCN","url":"https://pubmed.ncbi.nlm.nih.gov/33401235","citation_count":22,"is_preprint":false},{"pmid":"12202991","id":"PMC_12202991","title":"A novel splice variant of the cell adhesion molecule contactin 4 ( CNTN4) is mainly expressed in human brain.","date":"2002","source":"Journal of human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/12202991","citation_count":21,"is_preprint":false},{"pmid":"14571131","id":"PMC_14571131","title":"Cloning and characterization of the human neural cell adhesion molecule, CNTN4 (alias BIG-2).","date":"2003","source":"Cytogenetic and genome research","url":"https://pubmed.ncbi.nlm.nih.gov/14571131","citation_count":20,"is_preprint":false},{"pmid":"33765513","id":"PMC_33765513","title":"Updated results from the international phase III ALTTO trial (BIG 2-06/Alliance N063D).","date":"2021","source":"European journal of cancer (Oxford, England : 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genomics","url":"https://pubmed.ncbi.nlm.nih.gov/27039371","citation_count":16,"is_preprint":false},{"pmid":"26146898","id":"PMC_26146898","title":"Combined Whole Methylome and Genomewide Association Study Implicates CNTN4 in Alcohol Use.","date":"2015","source":"Alcoholism, clinical and experimental research","url":"https://pubmed.ncbi.nlm.nih.gov/26146898","citation_count":16,"is_preprint":false},{"pmid":"31422286","id":"PMC_31422286","title":"Intragenic CNTN4 copy number variants associated with a spectrum of neurobehavioral phenotypes.","date":"2019","source":"European journal of medical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/31422286","citation_count":15,"is_preprint":false},{"pmid":"36544657","id":"PMC_36544657","title":"MiR-148a-3p attenuates apoptosis and inflammation by targeting CNTN4 in atherosclerosis.","date":"2022","source":"Annals of translational 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Molecular brain research","url":"https://pubmed.ncbi.nlm.nih.gov/9221934","citation_count":11,"is_preprint":false},{"pmid":"34415325","id":"PMC_34415325","title":"The autism risk gene CNTN4 modulates dendritic spine formation.","date":"2021","source":"Human molecular genetics","url":"https://pubmed.ncbi.nlm.nih.gov/34415325","citation_count":9,"is_preprint":false},{"pmid":"38745463","id":"PMC_38745463","title":"CNTN4 modulates neural elongation through interplay with APP.","date":"2024","source":"Open biology","url":"https://pubmed.ncbi.nlm.nih.gov/38745463","citation_count":7,"is_preprint":false},{"pmid":"17915252","id":"PMC_17915252","title":"The CNTN4 c.4256C>T mutation is rare in Japanese with inherited spinocerebellar ataxia.","date":"2007","source":"Journal of the neurological sciences","url":"https://pubmed.ncbi.nlm.nih.gov/17915252","citation_count":7,"is_preprint":false},{"pmid":"21445711","id":"PMC_21445711","title":"Nanomechanics of Ig-like domains of human contactin (BIG-2).","date":"2011","source":"Journal of molecular modeling","url":"https://pubmed.ncbi.nlm.nih.gov/21445711","citation_count":6,"is_preprint":false},{"pmid":"35250268","id":"PMC_35250268","title":"Effect of the Minor C Allele of CNTN4 rs2619566 on Medial Hypothalamic Connectivity in Early-Stage Patients of Chinese Han Ancestry with Sporadic Amyotrophic Lateral Sclerosis.","date":"2022","source":"Neuropsychiatric disease and treatment","url":"https://pubmed.ncbi.nlm.nih.gov/35250268","citation_count":3,"is_preprint":false},{"pmid":"39418883","id":"PMC_39418883","title":"Final analysis of the ALTTO trial: adjuvant trastuzumab in sequence or in combination with lapatinib in patients with HER2-positive early breast cancer [BIG 2-06/NCCTG N063D (Alliance)].","date":"2024","source":"ESMO open","url":"https://pubmed.ncbi.nlm.nih.gov/39418883","citation_count":2,"is_preprint":false},{"pmid":"41326871","id":"PMC_41326871","title":"Cntn4 Gene Deficiency Promotes Autism-Like Phenotypes Associated with Gut Microbiota Perturbations and Gut-Brain Axis Metabolomic Alterations in Mice.","date":"2025","source":"Molecular neurobiology","url":"https://pubmed.ncbi.nlm.nih.gov/41326871","citation_count":1,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2024.12.19.629410","title":"Genome-wide association analyses in dairy heifers highlight genes overlapping with mouse and human fertility and human health traits","date":"2024-12-21","source":"bioRxiv","url":"https://doi.org/10.1101/2024.12.19.629410","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":17196,"output_tokens":2130,"usd":0.041769,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9168,"output_tokens":3095,"usd":0.061608,"stage2_stop_reason":"end_turn"},"total_usd":0.103377,"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\": 1995,\n      \"finding\": \"BIG-2 (CNTN4) is a GPI-anchored cell adhesion molecule with six Ig-like domains and four fibronectin type III-like repeats; recombinant BIG-2 protein promotes neurite outgrowth when used as a substrate for neurons in vitro, establishing its direct role in axon growth.\",\n      \"method\": \"Molecular cloning, domain analysis, in vitro neurite outgrowth assay using recombinant protein as substrate\",\n      \"journal\": \"Journal of neurobiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — direct in vitro functional assay with recombinant protein, single lab, single study\",\n      \"pmids\": [\"8586965\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"Alternative splicing of the BIG-2 (CNTN4) gene produces a truncated isoform (BIG-2A) that lacks the GPI-anchoring domain and is specifically expressed in mature sensory cells of the vomeronasal neuroepithelium and olfactory neuroepithelium, suggesting splice-variant-specific roles in chemosensory tissue organization.\",\n      \"method\": \"cDNA cloning, sequence analysis, in situ hybridization in adult and developing mice\",\n      \"journal\": \"Brain research. Molecular brain research\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — expression localization by in situ hybridization, single lab, no functional manipulation\",\n      \"pmids\": [\"9221934\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"BIG-2/CNTN4 is required for convergence of olfactory sensory neuron axons onto topographically fixed glomeruli in the olfactory bulb; in BIG-2-deficient mice, neurons expressing a given odorant receptor innervate multiple glomeruli at ectopic locations, demonstrating CNTN4 functions as an axon guidance molecule for odor map formation.\",\n      \"method\": \"BIG-2 knockout mouse model, immunohistochemistry, olfactory axon tracing, glomerular mapping\",\n      \"journal\": \"Neuron\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean KO with specific and well-defined cellular phenotype (ectopic multi-glomerular innervation), published in high-impact journal, clear loss-of-function readout\",\n      \"pmids\": [\"18367085\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"In Cntn4-/- mice, cell-surface levels of glutamate receptor subunits (GluA1, GluA2, GluN1) are altered in a brain-region-specific manner (reduced in cortex/hippocampus, increased in striatum), and surface GABA-A receptor (α1 subunit) levels are downregulated in several brain regions, demonstrating CNTN4 modulates excitatory and inhibitory synaptic receptor surface expression.\",\n      \"method\": \"Biotinylation assay and western blotting for surface receptor subunits in Cntn4-/- mice across multiple brain regions\",\n      \"journal\": \"Frontiers in molecular neuroscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KO with orthogonal biochemical readout (surface biotinylation + western blot), multiple brain regions and receptor types analyzed, single lab\",\n      \"pmids\": [\"29970989\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Cntn4 KO mice display reduced CA1 hippocampal synaptic potentiation (LTP) and abnormal dendritic arborization and spine density of CA1 neurons; behaviorally, Cntn4-deficient mice show increased contextual fear conditioning in a gene-dose-dependent manner, placing CNTN4 in a pathway controlling hippocampal synaptic plasticity and fear memory.\",\n      \"method\": \"Cntn4 KO mouse model, hippocampal slice electrophysiology (LTP), neuroanatomical analysis of dendritic morphology, behavioral fear conditioning\",\n      \"journal\": \"Translational psychiatry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (electrophysiology, neuroanatomy, behavior) in clean KO model, gene-dose-dependent behavioral effect replicated in hetero- and homozygous animals\",\n      \"pmids\": [\"33542194\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"CNTN4 promotes dendritic spine formation and excitatory synapse number in cortical neurons; truncated proteins lacking the signal peptide, FnIII domains, or GPI domain cannot rescue spine density deficits, demonstrating that CNTN4 regulates spine density through a mechanism dependent on FnIII domains; autism-associated variants of CNTN4 also fail to rescue spine density and neural activity.\",\n      \"method\": \"Cntn4 disruption in cortical neurons, domain-deletion mutant rescue experiments, dendritic spine counting, electrophysiological measurement of neural activity, analysis of autism-linked variants\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — domain mutagenesis with defined functional readout (spine density, neural activity), multiple deletion constructs tested, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"34415325\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"miR-148a-3p directly targets the 3′UTR of CNTN4 mRNA (validated by dual-luciferase reporter assay), reducing CNTN4 expression in oxLDL-treated THP-1 macrophages and thereby inhibiting apoptosis and pro-inflammatory cytokine (IL-6, TNF-α) production, identifying CNTN4 as a functional downstream target of miR-148a-3p in macrophage inflammatory signaling.\",\n      \"method\": \"Dual-luciferase reporter assay, qRT-PCR, flow cytometry, ELISA in THP-1 macrophage model\",\n      \"journal\": \"Annals of translational medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — dual-luciferase confirms direct miRNA targeting, functional cellular readouts, but single lab and non-neuronal context potentially divergent from canonical CNTN4 function\",\n      \"pmids\": [\"36544657\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Mass spectrometry identified amyloid precursor protein (APP) as a binding partner of CNTN4; knockout of CNTN4 and/or APP in human cells revealed that CNTN4–APP interaction controls neural elongation, and loss of either protein reduces neurite outgrowth, demonstrating a functional CNTN4–APP complex in cortical neurodevelopment.\",\n      \"method\": \"Mass spectrometry interaction screen, CNTN4/APP single and double knockout human cell lines, neural elongation/morphology assays\",\n      \"journal\": \"Open biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MS-identified interaction confirmed functionally with double KO and specific cellular phenotype, single lab, two orthogonal approaches (MS + KO rescue)\",\n      \"pmids\": [\"38745463\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CNTN4 (BIG-2) is a GPI-anchored, FnIII- and Ig-domain-containing axonal cell adhesion molecule that promotes neurite outgrowth and dendritic spine formation via its FnIII domains, guides olfactory sensory axons to correct glomeruli in the olfactory bulb, modulates hippocampal LTP and CA1 dendritic morphology, regulates brain-region-specific surface expression of glutamate and GABA receptors, and interacts with APP to control cortical neural elongation; autism-linked variants and domain-truncation experiments indicate these functions require intact FnIII and GPI domains.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"CNTN4 (BIG-2) is a GPI-anchored, Ig- and fibronectin type III (FnIII)-domain cell adhesion molecule that promotes axon growth and synaptic connectivity in the developing nervous system [#0, #5]. As a substrate, recombinant CNTN4 directly promotes neurite outgrowth, and in vivo it acts as an axon guidance cue: loss of CNTN4 causes olfactory sensory neurons expressing a given odorant receptor to mistarget to multiple ectopic glomeruli, disrupting odor-map formation [#0, #2]. Beyond wiring, CNTN4 shapes synapse structure and function — it promotes dendritic spine formation and excitatory synapse number in cortical neurons, and its loss reduces CA1 hippocampal LTP, alters dendritic arborization and spine density, and produces gene-dose-dependent behavioral changes [#4, #5]. Spine-promoting activity requires intact signal peptide, FnIII, and GPI domains, and autism-associated CNTN4 variants fail to rescue spine density and neural activity, tying these domains to neurodevelopmental disease risk [#5]. CNTN4 also modulates the brain-region-specific surface expression of glutamate (GluA1, GluA2, GluN1) and GABA-A (\\u03b11) receptors [#3], and it forms a functional complex with amyloid precursor protein (APP) that controls neurite/neural elongation in cortical neurodevelopment [#7]. A non-neuronal role is documented in which CNTN4 acts as a direct target of miR-148a-3p in macrophage inflammatory signaling [#6].\",\n  \"teleology\": [\n    {\n      \"year\": 1995,\n      \"claim\": \"Established CNTN4 as a GPI-anchored adhesion molecule with a direct, intrinsic capacity to drive axon growth, defining its baseline molecular activity.\",\n      \"evidence\": \"Molecular cloning, domain analysis, and in vitro neurite outgrowth assays using recombinant BIG-2 as a substrate\",\n      \"pmids\": [\"8586965\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\n        \"No in vivo confirmation of the outgrowth-promoting role\",\n        \"Binding partner mediating outgrowth not identified\",\n        \"Domain requirements not yet dissected\"\n      ]\n    },\n    {\n      \"year\": 1997,\n      \"claim\": \"Showed that alternative splicing yields a GPI-anchorless isoform (BIG-2A) restricted to mature chemosensory neuroepithelia, raising the possibility of splice-variant-specific functions.\",\n      \"evidence\": \"cDNA cloning and in situ hybridization in adult and developing mice\",\n      \"pmids\": [\"9221934\"],\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\n        \"Expression localization only; no functional manipulation of the isoform\",\n        \"Functional distinction between GPI-anchored and anchorless forms untested\",\n        \"Single lab, no orthogonal validation\"\n      ]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Demonstrated in vivo that CNTN4 is required for olfactory sensory axon convergence onto correct glomeruli, establishing it as a bona fide axon guidance molecule for odor-map formation.\",\n      \"evidence\": \"BIG-2 knockout mouse with olfactory axon tracing and glomerular mapping\",\n      \"pmids\": [\"18367085\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\n        \"Molecular guidance partners/receptors not identified\",\n        \"Whether guidance uses the same domains as in vitro outgrowth unknown\"\n      ]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Revealed that CNTN4 modulates surface availability of excitatory and inhibitory neurotransmitter receptors in a brain-region-specific manner, extending its role from wiring to synaptic receptor regulation.\",\n      \"evidence\": \"Surface biotinylation and western blotting for glutamate and GABA-A receptor subunits across brain regions in Cntn4-/- mice\",\n      \"pmids\": [\"29970989\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\n        \"Mechanism linking CNTN4 to receptor surface trafficking unknown\",\n        \"Direct physical interaction with receptors not shown\",\n        \"Single lab\"\n      ]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Connected CNTN4 to hippocampal synaptic plasticity and behavior, showing its loss impairs CA1 LTP, alters dendritic morphology/spine density, and produces gene-dose-dependent fear-memory changes.\",\n      \"evidence\": \"Hippocampal slice electrophysiology, neuroanatomy, and fear-conditioning behavior in Cntn4 KO mice\",\n      \"pmids\": [\"33542194\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\n        \"Molecular pathway from CNTN4 to LTP not resolved\",\n        \"Cell-autonomous vs circuit-level contributions not separated\"\n      ]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Mapped CNTN4's spine-promoting activity to specific domains and linked autism-associated variants to functional loss, providing a structure-function basis for disease risk.\",\n      \"evidence\": \"Domain-deletion mutant rescue, spine counting, electrophysiology, and analysis of autism-linked variants in cortical neurons\",\n      \"pmids\": [\"34415325\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\n        \"Binding partner engaged by FnIII domains for spine formation not identified\",\n        \"Single lab\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Identified APP as a CNTN4 binding partner and showed the CNTN4-APP complex is required for neural elongation, providing a molecular partner for CNTN4's developmental morphology functions.\",\n      \"evidence\": \"Mass spectrometry interaction screen plus single and double CNTN4/APP knockout human cell lines with neural elongation assays\",\n      \"pmids\": [\"38745463\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\n        \"Structural basis and stoichiometry of the CNTN4-APP interaction unknown\",\n        \"Whether APP mediates the olfactory guidance or synaptic phenotypes not tested\",\n        \"Single lab\"\n      ]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Documented a non-neuronal regulatory context in which CNTN4 is a direct miR-148a-3p target controlling macrophage apoptosis and inflammatory cytokine output.\",\n      \"evidence\": \"Dual-luciferase 3'UTR reporter, qRT-PCR, flow cytometry, and ELISA in oxLDL-treated THP-1 macrophages\",\n      \"pmids\": [\"36544657\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\n        \"Mechanism by which CNTN4 influences macrophage inflammation unknown\",\n        \"Relevance to CNTN4's neuronal functions unclear\",\n        \"Single lab, non-neuronal model\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The receptors/ligands through which CNTN4 transduces axon guidance, receptor-surface regulation, and synaptic plasticity signals remain largely undefined beyond the APP interaction.\",\n      \"evidence\": \"No discovery in the timeline identifies a guidance receptor or signaling effector downstream of CNTN4\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\n        \"No guidance receptor identified for olfactory axon targeting\",\n        \"Mechanistic link between CNTN4 and glutamate/GABA receptor trafficking unresolved\",\n        \"Downstream signaling effectors of spine formation unknown\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098631\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [2, 5]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 3]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [2, 5, 7]},\n      {\"term_id\": \"R-HSA-112316\", \"supporting_discovery_ids\": [3, 4]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"APP\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}