{"gene":"NEO1","run_date":"2026-06-10T05:19:52","timeline":{"discoveries":[{"year":1997,"finding":"Human neogenin (NEO1) protein consists of four immunoglobulin-like domains followed by six fibronectin type III domains, a transmembrane domain, and an intracellular domain, and is expressed in at least two isoforms derived from alternative splicing in the intracellular domain.","method":"cDNA cloning, sequencing, Northern blot analysis","journal":"Genomics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct molecular characterization by sequencing and Northern blot, single lab, multiple orthogonal methods","pmids":["9169140"],"is_preprint":false},{"year":2015,"finding":"A missense variant (p.Arg1130Cys) at the nuclear localization signal (NLS) domain of neogenin leads to defective nuclear translocation of the neogenin protein, as shown by in silico and functional analyses.","method":"Functional analysis of NLS mutant, in silico prediction, nuclear translocation assay","journal":"Behavioural brain research","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, functional assay described but limited methodological detail in abstract","pmids":["26518331"],"is_preprint":false},{"year":2019,"finding":"Rgma promotes NEO1 glycosylation and intramembrane proteolysis in zebrafish, resulting in production of a transient nuclear intracellular fragment (NeoICD) that is required for neuroepithelial cell elongation and neural tube morphogenesis; partial rescue by overexpressing neoICD supports proteolytic cleavage as the key signaling event.","method":"Zebrafish loss-of-function (morpholino knockdown), cell transplantation, overexpression rescue, reporter assays","journal":"The Journal of neuroscience","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — cell transplantation establishes cell autonomy, rescue experiments support mechanism, single lab with multiple orthogonal approaches","pmids":["31399534"],"is_preprint":false},{"year":2020,"finding":"Netrin-1 (NET1) signals through NEO1 and co-receptor UNC5B to co-regulate Wnt and MAPK pathways in mouse embryonic stem cells: NEO1 binding induces FAK kinase to inactivate GSK3α/β and stabilize β-catenin, while UNC5B signals through a PPP2R2C-containing PP2A complex to reduce ERK1/2 activity, promoting naive pluripotency.","method":"mESC culture with 2i/LIF, receptor knockdown/overexpression, kinase activity assays, co-immunoprecipitation, phospho-Western blot","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (Co-IP, kinase assays, phosphatase activity, genetic rescue), replicated in both mouse and human ESCs","pmids":["32231305"],"is_preprint":false},{"year":2021,"finding":"NEO1 simultaneously binds both NET1 and RGM ligands to form a ternary NEO1-NET1-RGM complex that assembles into a 'trimer-of-trimers' super-assembly in the cell membrane; this super-complex inhibits both RGMA-NEO1-mediated growth cone collapse and RGMA- or NET1-NEO1-mediated neuron migration by preventing formation of signaling-compatible RGM-NEO1 complexes and NET1-induced NEO1 ectodomain clustering.","method":"Crystal structure determination (X-ray crystallography), cryo-EM, growth cone collapse assay, neuron migration assay, cell membrane complex analysis","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure plus cryo-EM plus functional validation (growth cone collapse, migration assays) with mechanistic mutagenesis support","pmids":["33740419"],"is_preprint":false},{"year":2024,"finding":"Astrocytic NEO1 is required for blood-brain barrier integrity after subarachnoid hemorrhage; conditional knockout of NEO1 in astrocytes (NEO1-GFAP-Cre mice) leads to increased endothelial cell proliferation, altered BBB structure, and increased BBB permeability, which can be reversed by hepcidin treatment.","method":"Astrocyte-specific conditional knockout (GFAP-Cre), Evans Blue/dextran leakage assay, transmission electron microscopy, immunostaining, SAH mouse model","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — conditional KO with multiple readouts (TEM, BBB permeability, cell density), single lab","pmids":["39107268"],"is_preprint":false},{"year":2025,"finding":"NEO1 in astrocytes mediates A1 astrocyte polarization after subarachnoid hemorrhage through the cPLA2-MAVS-NF-κB signaling pathway; NEO1 knockout reduces cPLA2 and MAVS mRNA, inhibits A1 polarization and inflammatory factor release, and cPLA2 overexpression reverses these effects.","method":"Astrocyte-specific conditional knockout (GFAP-Cre), transcriptome sequencing, lentiviral cPLA2 overexpression, in vitro astrocyte assays, SAH mouse model","journal":"Journal of neuroinflammation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — conditional KO plus transcriptomics plus epistatic rescue with cPLA2 overexpression, single lab","pmids":["41345945"],"is_preprint":false},{"year":2026,"finding":"Netrin-1 signals through its receptor NEO1 in pancreatic epithelial cells to activate focal adhesion kinase (FAK), upregulate ZEB1 and SOX9, and promote epithelial-mesenchymal transition and cancer stemness; NEO1 also mediates netrin-1-promoted axonogenesis of sympathetic neurons in ex vivo celiac ganglia culture.","method":"Pancreatic organoid culture, KrasG12D mouse model, NEO1 knockout/knockdown, recombinant NTN1 treatment, netrin-1 neutralizing antibody, FAK phosphorylation assay, ex vivo celiac ganglia culture","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple in vitro and in vivo models, genetic KO plus antibody neutralization, single lab with several orthogonal approaches","pmids":["41474982"],"is_preprint":false},{"year":2025,"finding":"Cryo-EM reveals that phosphatidylinositol-4-phosphate (PI4P) binds NEO1 (yeast Neo1) within the substrate translocation pathway; Neo1 flips PI4P from the lumenal leaflet to the cytosolic leaflet in the Golgi, and loss of Neo1 activity leads to secretion of extracellular PI4P that serves as a neomycin receptor facilitating its endocytic uptake.","method":"Cryo-EM structure determination, yeast Neo1 mutant analysis, neomycin sensitivity assays, PI4P localization assays, Sac1 phosphatase epistasis","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — cryo-EM structure with substrate bound, supported by genetic epistasis and functional assays, single lab, preprint","pmids":[],"is_preprint":true}],"current_model":"NEO1 (neogenin) is a multifunctional transmembrane receptor that integrates axon guidance cues (Netrin-1 and RGM ligands) through formation of ternary and higher-order super-complexes at the cell surface, triggering downstream FAK-GSK3/β-catenin and MAPK/ERK signaling; ligand-induced intramembrane proteolysis generates a nuclear ICD fragment required for neuroepithelial morphogenesis; in astrocytes NEO1 regulates inflammatory polarization via a cPLA2-MAVS-NF-κB pathway and BBB integrity; its yeast ortholog Neo1 functions as a P4-ATPase phospholipid flippase that translocates PS, PE, and PI4P from the exofacial to cytosolic leaflet of Golgi/endosomal membranes to maintain membrane asymmetry essential for vesicle trafficking and viability."},"narrative":{"mechanistic_narrative":"NEO1 (neogenin) is a multidomain transmembrane receptor that transduces axon guidance and morphogenetic cues to control neural development, epithelial fate, and tissue barrier integrity [PMID:33740419, PMID:32231305]. Its ectodomain comprises four immunoglobulin-like domains and six fibronectin type III repeats followed by a transmembrane and intracellular segment, with isoform diversity generated by alternative splicing of the intracellular domain [PMID:9169140]. NEO1 simultaneously engages Netrin-1 and RGM ligands, assembling a ternary NEO1-NET1-RGM complex that oligomerizes into a 'trimer-of-trimers' super-assembly; this higher-order arrangement is inhibitory, blocking RGMA-NEO1 growth cone collapse and NET1- or RGMA-driven neuron migration by preventing formation of signaling-competent receptor clusters [PMID:33740419]. Productive Netrin-1-NEO1 signaling activates focal adhesion kinase (FAK): in embryonic stem cells, with co-receptor UNC5B, this inactivates GSK3α/β to stabilize β-catenin and dampens ERK1/2 via a PPP2R2C-PP2A complex to promote naive pluripotency [PMID:32231305], while in pancreatic epithelium FAK activation upregulates ZEB1 and SOX9 to drive epithelial-mesenchymal transition and cancer stemness [PMID:41474982]. RGM-induced glycosylation and intramembrane proteolysis of NEO1 release a nuclear intracellular fragment (NeoICD) required for neuroepithelial cell elongation and neural tube morphogenesis [PMID:31399534]. In astrocytes, NEO1 maintains blood-brain barrier integrity and governs inflammatory A1 polarization after subarachnoid hemorrhage through a cPLA2-MAVS-NF-κB pathway [PMID:39107268, PMID:41345945].","teleology":[{"year":1997,"claim":"Established the domain architecture of human neogenin, defining it as an Ig/FnIII-containing transmembrane receptor with splice variation in its intracellular domain.","evidence":"cDNA cloning, sequencing, and Northern blot of human NEO1","pmids":["9169140"],"confidence":"Medium","gaps":["No ligand or signaling partner identified at this stage","Functional role of intracellular splice isoforms not resolved"]},{"year":2015,"claim":"Implicated nuclear translocation of neogenin as functionally important by showing an NLS missense variant abolishes nuclear import.","evidence":"Functional NLS-mutant nuclear translocation assay with in silico prediction","pmids":["26518331"],"confidence":"Low","gaps":["Single-variant functional assay with limited methodological detail","Downstream consequences of failed nuclear import not characterized","Link to a physiological phenotype not established"]},{"year":2019,"claim":"Connected RGM ligand to regulated intramembrane proteolysis, showing the released NeoICD fragment is the morphogenetic effector for neural tube development.","evidence":"Zebrafish morpholino knockdown, cell transplantation, and NeoICD overexpression rescue","pmids":["31399534"],"confidence":"Medium","gaps":["Protease responsible for cleavage not identified","Nuclear targets of NeoICD unknown","Only partial rescue achieved by NeoICD"]},{"year":2020,"claim":"Defined a productive Netrin-1-NEO1 signaling output, coupling NEO1 to FAK-GSK3-β-catenin and, via UNC5B, to PP2A-ERK modulation to control stem cell pluripotency.","evidence":"mESC 2i/LIF culture, receptor knockdown/overexpression, kinase and phosphatase assays, Co-IP, replicated in human ESCs","pmids":["32231305"],"confidence":"High","gaps":["Stoichiometry of NEO1/UNC5B receptor partitioning not resolved","Direct FAK-NEO1 contact not structurally defined"]},{"year":2021,"claim":"Provided the structural basis for ligand integration, showing NEO1 binds NET1 and RGM simultaneously into an inhibitory super-complex that suppresses guidance signaling.","evidence":"X-ray crystallography, cryo-EM, growth cone collapse and neuron migration assays with mutagenesis","pmids":["33740419"],"confidence":"High","gaps":["In vivo prevalence of the super-complex versus signaling-competent complexes unquantified","How clustering state is set in physiological membranes unknown"]},{"year":2024,"claim":"Extended NEO1 function beyond neurons, showing astrocytic NEO1 is required for blood-brain barrier integrity after hemorrhage.","evidence":"Astrocyte GFAP-Cre conditional knockout, BBB leakage assays, TEM, SAH mouse model","pmids":["39107268"],"confidence":"Medium","gaps":["Molecular link between NEO1 and endothelial proliferation not defined","Mechanism of hepcidin rescue unresolved"]},{"year":2025,"claim":"Identified the inflammatory pathway downstream of astrocytic NEO1, placing it upstream of cPLA2-MAVS-NF-κB to drive A1 astrocyte polarization.","evidence":"Astrocyte conditional knockout, transcriptomics, lentiviral cPLA2 rescue, SAH model","pmids":["41345945"],"confidence":"Medium","gaps":["Direct biochemical link between NEO1 receptor and cPLA2 induction not shown","Ligand triggering this astrocytic pathway unidentified"]},{"year":2026,"claim":"Showed NEO1 mediates Netrin-1-driven epithelial-mesenchymal transition and cancer stemness in pancreatic tissue via FAK-ZEB1/SOX9, and supports sympathetic axonogenesis.","evidence":"Pancreatic organoids, KrasG12D mice, NEO1 KO/knockdown, NTN1 treatment and neutralizing antibody, ex vivo ganglia culture","pmids":["41474982"],"confidence":"Medium","gaps":["Whether super-complex formation modulates this oncogenic signaling untested","Contribution of UNC5B co-receptor in this context unknown"]},{"year":2025,"claim":"Defined a phospholipid flippase activity for the yeast ortholog Neo1, capturing PI4P in the substrate translocation pathway by cryo-EM and linking flippase loss to extracellular PI4P-mediated drug uptake.","evidence":"Cryo-EM with bound PI4P, yeast mutant and neomycin sensitivity assays, Sac1 epistasis (preprint)","pmids":[],"confidence":"Medium","gaps":["Activity demonstrated for yeast Neo1, not human NEO1","Preprint, not peer-reviewed","Relationship between flippase function and the receptor signaling roles of vertebrate NEO1 unestablished"]},{"year":null,"claim":"How the structurally defined inhibitory super-complex is dynamically converted to productive FAK-coupled signaling in vivo, and whether vertebrate NEO1 retains any lipid-handling activity, remain open.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structure of the active signaling-competent NEO1 complex with FAK","Protease and nuclear NeoICD targets unidentified","Flippase activity not demonstrated for human NEO1"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[3,4,7]},{"term_id":"GO:0048018","term_label":"receptor ligand activity","supporting_discovery_ids":[4]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,4]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[1,2]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[3,4,7]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[2,4]}],"complexes":["NEO1-NET1-RGM ternary super-complex"],"partners":["NTN1","RGMA","UNC5B","FAK"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q92859","full_name":"Neogenin","aliases":["Immunoglobulin superfamily DCC subclass member 2"],"length_aa":1461,"mass_kda":160.0,"function":"Multi-functional cell surface receptor regulating cell adhesion in many diverse developmental processes, including neural tube and mammary gland formation, myogenesis and angiogenesis. Receptor for members of the BMP, netrin, and repulsive guidance molecule (RGM) families. Netrin-Neogenin interactions result in a chemoattractive axon guidance response and cell-cell adhesion, the interaction between NEO1/Neogenin and RGMa and RGMb induces a chemorepulsive response","subcellular_location":"Cell membrane","url":"https://www.uniprot.org/uniprotkb/Q92859/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/NEO1","classification":"Not Classified","n_dependent_lines":11,"n_total_lines":1208,"dependency_fraction":0.009105960264900662},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/NEO1","total_profiled":1310},"omim":[{"mim_id":"618859","title":"NEURODEVELOPMENTAL DISORDER WITH OR WITHOUT AUTISTIC FEATURES AND/OR STRUCTURAL BRAIN ABNORMALITIES; NEDASB","url":"https://www.omim.org/entry/618859"},{"mim_id":"616810","title":"IMMUNOGLOBULIN SUPERFAMILY, DCC SUBCLASS, MEMBER 4; IGDCC4","url":"https://www.omim.org/entry/616810"},{"mim_id":"612687","title":"RGM DOMAIN FAMILY, MEMBER B; RGMB","url":"https://www.omim.org/entry/612687"},{"mim_id":"607362","title":"RGM DOMAIN FAMILY, MEMBER A; RGMA","url":"https://www.omim.org/entry/607362"},{"mim_id":"604184","title":"PUTATIVE NEURONAL CELL ADHESION MOLECULE; PUNC","url":"https://www.omim.org/entry/604184"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"},{"location":"Plasma membrane","reliability":"Approved"},{"location":"Golgi apparatus","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/NEO1"},"hgnc":{"alias_symbol":["NGN","HsT17534","IGDCC2","NTN1R2"],"prev_symbol":[]},"alphafold":{"accession":"Q92859","domains":[{"cath_id":"2.60.40.10","chopping":"50-150","consensus_level":"medium","plddt":79.8403,"start":50,"end":150},{"cath_id":"2.60.40.10","chopping":"154-340","consensus_level":"medium","plddt":86.5989,"start":154,"end":340},{"cath_id":"2.60.40.10","chopping":"347-430","consensus_level":"medium","plddt":88.9244,"start":347,"end":430},{"cath_id":"2.60.40.10","chopping":"435-532","consensus_level":"high","plddt":84.4141,"start":435,"end":532},{"cath_id":"2.60.40.10","chopping":"547-628","consensus_level":"high","plddt":86.0804,"start":547,"end":628},{"cath_id":"2.60.40.10","chopping":"642-728","consensus_level":"high","plddt":88.8611,"start":642,"end":728},{"cath_id":"2.60.40.10","chopping":"745-831_855-949","consensus_level":"medium","plddt":80.7232,"start":745,"end":949},{"cath_id":"2.60.40.10","chopping":"959-1052","consensus_level":"medium","plddt":81.5316,"start":959,"end":1052}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q92859","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q92859-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q92859-F1-predicted_aligned_error_v6.png","plddt_mean":68.0},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=NEO1","jax_strain_url":"https://www.jax.org/strain/search?query=NEO1"},"sequence":{"accession":"Q92859","fasta_url":"https://rest.uniprot.org/uniprotkb/Q92859.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q92859/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q92859"}},"corpus_meta":[{"pmid":"15576411","id":"PMC_15576411","title":"The SWI/SNF chromatin remodeling protein Brg1 is required for vertebrate neurogenesis and mediates transactivation of Ngn and NeuroD.","date":"2004","source":"Development (Cambridge, England)","url":"https://pubmed.ncbi.nlm.nih.gov/15576411","citation_count":164,"is_preprint":false},{"pmid":"17611227","id":"PMC_17611227","title":"Helt determines GABAergic over glutamatergic neuronal fate by repressing Ngn genes in the developing mesencephalon.","date":"2007","source":"Development (Cambridge, England)","url":"https://pubmed.ncbi.nlm.nih.gov/17611227","citation_count":87,"is_preprint":false},{"pmid":"27235400","id":"PMC_27235400","title":"The Essential Neo1 Protein from Budding Yeast Plays a Role in Establishing Aminophospholipid Asymmetry of the Plasma Membrane.","date":"2016","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/27235400","citation_count":49,"is_preprint":false},{"pmid":"32231305","id":"PMC_32231305","title":"Netrin-1 promotes naive 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Neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/31399534","citation_count":14,"is_preprint":false},{"pmid":"26518331","id":"PMC_26518331","title":"Unmasking a novel disease gene NEO1 associated with autism spectrum disorders by a hemizygous deletion on chromosome 15 and a functional polymorphism.","date":"2015","source":"Behavioural brain research","url":"https://pubmed.ncbi.nlm.nih.gov/26518331","citation_count":14,"is_preprint":false},{"pmid":"32273286","id":"PMC_32273286","title":"ngn-1/neurogenin Activates Transcription of Multiple Terminal Selector Transcription Factors in the Caenorhabditis elegans Nervous System.","date":"2020","source":"G3 (Bethesda, Md.)","url":"https://pubmed.ncbi.nlm.nih.gov/32273286","citation_count":12,"is_preprint":false},{"pmid":"30824614","id":"PMC_30824614","title":"The PQ-loop protein Any1 segregates Drs2 and Neo1 functions required for viability and plasma membrane phospholipid asymmetry.","date":"2019","source":"Journal of lipid 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2007)","url":"https://pubmed.ncbi.nlm.nih.gov/28921917","citation_count":5,"is_preprint":false},{"pmid":"38133504","id":"PMC_38133504","title":"Retinal Ganglion Cell Fate Induction by Ngn-Family Transcription Factors.","date":"2023","source":"Investigative ophthalmology & visual science","url":"https://pubmed.ncbi.nlm.nih.gov/38133504","citation_count":4,"is_preprint":false},{"pmid":"41474982","id":"PMC_41474982","title":"Netrin-1 Promotes Pancreatic Tumorigenesis and Innervation through NEO1.","date":"2026","source":"Cancer research","url":"https://pubmed.ncbi.nlm.nih.gov/41474982","citation_count":3,"is_preprint":false},{"pmid":"41345945","id":"PMC_41345945","title":"NEO1 modulates the A1 astrocyte polarization in subarachnoid hemorrhage through the cPLA2-MAVS signaling pathway.","date":"2025","source":"Journal of neuroinflammation","url":"https://pubmed.ncbi.nlm.nih.gov/41345945","citation_count":1,"is_preprint":false},{"pmid":"38898351","id":"PMC_38898351","title":"Predictive 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Northern blot analysis\",\n      \"journal\": \"Genomics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct molecular characterization by sequencing and Northern blot, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"9169140\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"A missense variant (p.Arg1130Cys) at the nuclear localization signal (NLS) domain of neogenin leads to defective nuclear translocation of the neogenin protein, as shown by in silico and functional analyses.\",\n      \"method\": \"Functional analysis of NLS mutant, in silico prediction, nuclear translocation assay\",\n      \"journal\": \"Behavioural brain research\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, functional assay described but limited methodological detail in abstract\",\n      \"pmids\": [\"26518331\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Rgma promotes NEO1 glycosylation and intramembrane proteolysis in zebrafish, resulting in production of a transient nuclear intracellular fragment (NeoICD) that is required for neuroepithelial cell elongation and neural tube morphogenesis; partial rescue by overexpressing neoICD supports proteolytic cleavage as the key signaling event.\",\n      \"method\": \"Zebrafish loss-of-function (morpholino knockdown), cell transplantation, overexpression rescue, reporter assays\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — cell transplantation establishes cell autonomy, rescue experiments support mechanism, single lab with multiple orthogonal approaches\",\n      \"pmids\": [\"31399534\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Netrin-1 (NET1) signals through NEO1 and co-receptor UNC5B to co-regulate Wnt and MAPK pathways in mouse embryonic stem cells: NEO1 binding induces FAK kinase to inactivate GSK3α/β and stabilize β-catenin, while UNC5B signals through a PPP2R2C-containing PP2A complex to reduce ERK1/2 activity, promoting naive pluripotency.\",\n      \"method\": \"mESC culture with 2i/LIF, receptor knockdown/overexpression, kinase activity assays, co-immunoprecipitation, phospho-Western blot\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (Co-IP, kinase assays, phosphatase activity, genetic rescue), replicated in both mouse and human ESCs\",\n      \"pmids\": [\"32231305\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"NEO1 simultaneously binds both NET1 and RGM ligands to form a ternary NEO1-NET1-RGM complex that assembles into a 'trimer-of-trimers' super-assembly in the cell membrane; this super-complex inhibits both RGMA-NEO1-mediated growth cone collapse and RGMA- or NET1-NEO1-mediated neuron migration by preventing formation of signaling-compatible RGM-NEO1 complexes and NET1-induced NEO1 ectodomain clustering.\",\n      \"method\": \"Crystal structure determination (X-ray crystallography), cryo-EM, growth cone collapse assay, neuron migration assay, cell membrane complex analysis\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure plus cryo-EM plus functional validation (growth cone collapse, migration assays) with mechanistic mutagenesis support\",\n      \"pmids\": [\"33740419\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Astrocytic NEO1 is required for blood-brain barrier integrity after subarachnoid hemorrhage; conditional knockout of NEO1 in astrocytes (NEO1-GFAP-Cre mice) leads to increased endothelial cell proliferation, altered BBB structure, and increased BBB permeability, which can be reversed by hepcidin treatment.\",\n      \"method\": \"Astrocyte-specific conditional knockout (GFAP-Cre), Evans Blue/dextran leakage assay, transmission electron microscopy, immunostaining, SAH mouse model\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional KO with multiple readouts (TEM, BBB permeability, cell density), single lab\",\n      \"pmids\": [\"39107268\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"NEO1 in astrocytes mediates A1 astrocyte polarization after subarachnoid hemorrhage through the cPLA2-MAVS-NF-κB signaling pathway; NEO1 knockout reduces cPLA2 and MAVS mRNA, inhibits A1 polarization and inflammatory factor release, and cPLA2 overexpression reverses these effects.\",\n      \"method\": \"Astrocyte-specific conditional knockout (GFAP-Cre), transcriptome sequencing, lentiviral cPLA2 overexpression, in vitro astrocyte assays, SAH mouse model\",\n      \"journal\": \"Journal of neuroinflammation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional KO plus transcriptomics plus epistatic rescue with cPLA2 overexpression, single lab\",\n      \"pmids\": [\"41345945\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"Netrin-1 signals through its receptor NEO1 in pancreatic epithelial cells to activate focal adhesion kinase (FAK), upregulate ZEB1 and SOX9, and promote epithelial-mesenchymal transition and cancer stemness; NEO1 also mediates netrin-1-promoted axonogenesis of sympathetic neurons in ex vivo celiac ganglia culture.\",\n      \"method\": \"Pancreatic organoid culture, KrasG12D mouse model, NEO1 knockout/knockdown, recombinant NTN1 treatment, netrin-1 neutralizing antibody, FAK phosphorylation assay, ex vivo celiac ganglia culture\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple in vitro and in vivo models, genetic KO plus antibody neutralization, single lab with several orthogonal approaches\",\n      \"pmids\": [\"41474982\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Cryo-EM reveals that phosphatidylinositol-4-phosphate (PI4P) binds NEO1 (yeast Neo1) within the substrate translocation pathway; Neo1 flips PI4P from the lumenal leaflet to the cytosolic leaflet in the Golgi, and loss of Neo1 activity leads to secretion of extracellular PI4P that serves as a neomycin receptor facilitating its endocytic uptake.\",\n      \"method\": \"Cryo-EM structure determination, yeast Neo1 mutant analysis, neomycin sensitivity assays, PI4P localization assays, Sac1 phosphatase epistasis\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — cryo-EM structure with substrate bound, supported by genetic epistasis and functional assays, single lab, preprint\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"NEO1 (neogenin) is a multifunctional transmembrane receptor that integrates axon guidance cues (Netrin-1 and RGM ligands) through formation of ternary and higher-order super-complexes at the cell surface, triggering downstream FAK-GSK3/β-catenin and MAPK/ERK signaling; ligand-induced intramembrane proteolysis generates a nuclear ICD fragment required for neuroepithelial morphogenesis; in astrocytes NEO1 regulates inflammatory polarization via a cPLA2-MAVS-NF-κB pathway and BBB integrity; its yeast ortholog Neo1 functions as a P4-ATPase phospholipid flippase that translocates PS, PE, and PI4P from the exofacial to cytosolic leaflet of Golgi/endosomal membranes to maintain membrane asymmetry essential for vesicle trafficking and viability.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"NEO1 (neogenin) is a multidomain transmembrane receptor that transduces axon guidance and morphogenetic cues to control neural development, epithelial fate, and tissue barrier integrity [#4, #3]. Its ectodomain comprises four immunoglobulin-like domains and six fibronectin type III repeats followed by a transmembrane and intracellular segment, with isoform diversity generated by alternative splicing of the intracellular domain [#0]. NEO1 simultaneously engages Netrin-1 and RGM ligands, assembling a ternary NEO1-NET1-RGM complex that oligomerizes into a 'trimer-of-trimers' super-assembly; this higher-order arrangement is inhibitory, blocking RGMA-NEO1 growth cone collapse and NET1- or RGMA-driven neuron migration by preventing formation of signaling-competent receptor clusters [#4]. Productive Netrin-1-NEO1 signaling activates focal adhesion kinase (FAK): in embryonic stem cells, with co-receptor UNC5B, this inactivates GSK3\\u03b1/\\u03b2 to stabilize \\u03b2-catenin and dampens ERK1/2 via a PPP2R2C-PP2A complex to promote naive pluripotency [#3], while in pancreatic epithelium FAK activation upregulates ZEB1 and SOX9 to drive epithelial-mesenchymal transition and cancer stemness [#7]. RGM-induced glycosylation and intramembrane proteolysis of NEO1 release a nuclear intracellular fragment (NeoICD) required for neuroepithelial cell elongation and neural tube morphogenesis [#2]. In astrocytes, NEO1 maintains blood-brain barrier integrity and governs inflammatory A1 polarization after subarachnoid hemorrhage through a cPLA2-MAVS-NF-\\u03baB pathway [#5, #6].\",\n  \"teleology\": [\n    {\n      \"year\": 1997,\n      \"claim\": \"Established the domain architecture of human neogenin, defining it as an Ig/FnIII-containing transmembrane receptor with splice variation in its intracellular domain.\",\n      \"evidence\": \"cDNA cloning, sequencing, and Northern blot of human NEO1\",\n      \"pmids\": [\"9169140\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No ligand or signaling partner identified at this stage\", \"Functional role of intracellular splice isoforms not resolved\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Implicated nuclear translocation of neogenin as functionally important by showing an NLS missense variant abolishes nuclear import.\",\n      \"evidence\": \"Functional NLS-mutant nuclear translocation assay with in silico prediction\",\n      \"pmids\": [\"26518331\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single-variant functional assay with limited methodological detail\", \"Downstream consequences of failed nuclear import not characterized\", \"Link to a physiological phenotype not established\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Connected RGM ligand to regulated intramembrane proteolysis, showing the released NeoICD fragment is the morphogenetic effector for neural tube development.\",\n      \"evidence\": \"Zebrafish morpholino knockdown, cell transplantation, and NeoICD overexpression rescue\",\n      \"pmids\": [\"31399534\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Protease responsible for cleavage not identified\", \"Nuclear targets of NeoICD unknown\", \"Only partial rescue achieved by NeoICD\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Defined a productive Netrin-1-NEO1 signaling output, coupling NEO1 to FAK-GSK3-\\u03b2-catenin and, via UNC5B, to PP2A-ERK modulation to control stem cell pluripotency.\",\n      \"evidence\": \"mESC 2i/LIF culture, receptor knockdown/overexpression, kinase and phosphatase assays, Co-IP, replicated in human ESCs\",\n      \"pmids\": [\"32231305\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry of NEO1/UNC5B receptor partitioning not resolved\", \"Direct FAK-NEO1 contact not structurally defined\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Provided the structural basis for ligand integration, showing NEO1 binds NET1 and RGM simultaneously into an inhibitory super-complex that suppresses guidance signaling.\",\n      \"evidence\": \"X-ray crystallography, cryo-EM, growth cone collapse and neuron migration assays with mutagenesis\",\n      \"pmids\": [\"33740419\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo prevalence of the super-complex versus signaling-competent complexes unquantified\", \"How clustering state is set in physiological membranes unknown\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Extended NEO1 function beyond neurons, showing astrocytic NEO1 is required for blood-brain barrier integrity after hemorrhage.\",\n      \"evidence\": \"Astrocyte GFAP-Cre conditional knockout, BBB leakage assays, TEM, SAH mouse model\",\n      \"pmids\": [\"39107268\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular link between NEO1 and endothelial proliferation not defined\", \"Mechanism of hepcidin rescue unresolved\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Identified the inflammatory pathway downstream of astrocytic NEO1, placing it upstream of cPLA2-MAVS-NF-\\u03baB to drive A1 astrocyte polarization.\",\n      \"evidence\": \"Astrocyte conditional knockout, transcriptomics, lentiviral cPLA2 rescue, SAH model\",\n      \"pmids\": [\"41345945\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct biochemical link between NEO1 receptor and cPLA2 induction not shown\", \"Ligand triggering this astrocytic pathway unidentified\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Showed NEO1 mediates Netrin-1-driven epithelial-mesenchymal transition and cancer stemness in pancreatic tissue via FAK-ZEB1/SOX9, and supports sympathetic axonogenesis.\",\n      \"evidence\": \"Pancreatic organoids, KrasG12D mice, NEO1 KO/knockdown, NTN1 treatment and neutralizing antibody, ex vivo ganglia culture\",\n      \"pmids\": [\"41474982\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether super-complex formation modulates this oncogenic signaling untested\", \"Contribution of UNC5B co-receptor in this context unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Defined a phospholipid flippase activity for the yeast ortholog Neo1, capturing PI4P in the substrate translocation pathway by cryo-EM and linking flippase loss to extracellular PI4P-mediated drug uptake.\",\n      \"evidence\": \"Cryo-EM with bound PI4P, yeast mutant and neomycin sensitivity assays, Sac1 epistasis (preprint)\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Activity demonstrated for yeast Neo1, not human NEO1\", \"Preprint, not peer-reviewed\", \"Relationship between flippase function and the receptor signaling roles of vertebrate NEO1 unestablished\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the structurally defined inhibitory super-complex is dynamically converted to productive FAK-coupled signaling in vivo, and whether vertebrate NEO1 retains any lipid-handling activity, remain open.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structure of the active signaling-competent NEO1 complex with FAK\", \"Protease and nuclear NeoICD targets unidentified\", \"Flippase activity not demonstrated for human NEO1\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [3, 4, 7]},\n      {\"term_id\": \"GO:0048018\", \"supporting_discovery_ids\": [4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 4]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [1, 2]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [3, 4, 7]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [2, 4]}\n    ],\n    \"complexes\": [\"NEO1-NET1-RGM ternary super-complex\"],\n    \"partners\": [\"NTN1\", \"RGMA\", \"UNC5B\", \"FAK\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}