{"gene":"NEO1","run_date":"2026-04-29T11:37:56","timeline":{"discoveries":[{"year":1997,"finding":"Human neogenin (NEO1) encodes a transmembrane protein with four immunoglobulin-like domains followed by six fibronectin type III domains, a transmembrane domain, and an intracellular domain, sharing ~50% amino acid identity with DCC. The gene maps to chromosome 15q22.3-q23, is expressed in at least two alternatively spliced isoforms differing in the intracellular domain, and produces mRNA species of ~5 and 7 kb.","method":"cDNA cloning, sequencing, Northern blot, fluorescence in situ hybridization (FISH)","journal":"Genomics","confidence":"High","confidence_rationale":"Tier 1-2 — direct molecular characterization with multiple orthogonal methods in founding paper","pmids":["9169140","9121761"],"is_preprint":false},{"year":2004,"finding":"Neogenin acts as a dependence receptor: in the absence of its ligand RGM, neogenin over-expression induces apoptosis in immortalized neuronal cells and the chick neural tube via caspase-mediated cleavage of its cytoplasmic domain; binding of RGM to neogenin inhibits this pro-apoptotic activity.","method":"In ovo gene transfer (chick neural tube), cell-based apoptosis assays, caspase cleavage assays","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 2 — in vivo loss/gain-of-function with mechanistic caspase cleavage readout, replicated in two systems","pmids":["15258591"],"is_preprint":false},{"year":2004,"finding":"Neogenin is expressed in skeletal myoblasts and, together with netrin-3, promotes myotube formation and enhances myogenic bHLH- and NFAT-dependent transcription. Neogenin binds CDO in cis at the cell surface, and myoblasts lacking CDO are defective in responding to recombinant netrin, placing neogenin in a promyogenic cell-surface complex.","method":"Co-immunoprecipitation, recombinant protein stimulation, myogenic differentiation assays, genetic loss-of-function (CDO-null myoblasts)","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP plus genetic epistasis (CDO-null rescue) in defined cellular phenotype","pmids":["15520228"],"is_preprint":false},{"year":2005,"finding":"Phosphatidylinositol transfer protein-alpha (PITPα) interacts with neogenin (and DCC) and is required for netrin-1-induced PIP2 hydrolysis and neurite outgrowth; netrin-1 stimulates PITPα binding to neogenin and enhances its lipid-transfer activity.","method":"Co-immunoprecipitation (pulldown), in vitro lipid-transfer assay, dominant-negative overexpression, morpholino knockdown in zebrafish, cortical explant neurite assay","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 1-2 — biochemical activity assay combined with multiple in vivo loss-of-function models","pmids":["16244667"],"is_preprint":false},{"year":2008,"finding":"Hemojuvelin (HJV)-induced BMP signaling and hepcidin expression are not altered by neogenin overexpression or knockdown, demonstrating that HJV-mediated BMP signaling occurs independently of neogenin.","method":"siRNA knockdown, overexpression, BMP signaling reporter assays in hepatoma cell lines","journal":"Blood","confidence":"Medium","confidence_rationale":"Tier 2 — clean KD/OE with defined signaling readout in single study","pmids":["18326817"],"is_preprint":false},{"year":2009,"finding":"Neogenin physically interacts with hemojuvelin (HJV) in hepatocytes, and this interaction is required for BMP4-induced hepcidin mRNA expression; disruption of the HJV-neogenin interaction or neogenin knockdown suppresses hepcidin induction ~16-fold in HJV-expressing cells. A soluble neogenin fragment blocks HJV-neogenin interaction and suppresses hepatic hepcidin in vivo.","method":"Co-immunoprecipitation, siRNA knockdown, in vivo mouse experiments with soluble neogenin fragment, quantitative RT-PCR","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP, genetic KD, and in vivo pharmacological validation across multiple orthogonal methods","pmids":["19564337"],"is_preprint":false},{"year":2010,"finding":"Neogenin directly binds BMP-2, BMP-4, BMP-6, and BMP-7 and negatively regulates BMP-induced osteoblastic differentiation and Smad1/5/8 phosphorylation. Neogenin mediates BMP-induced RhoA activation, and RhoA inhibition promotes BMP-2-induced osteoblastic differentiation independently of Smad phosphorylation.","method":"Binding assays, siRNA knockdown, overexpression, Smad phosphorylation Western blot, RhoA activity assay, osteoblastic differentiation assay in C2C12 cells","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — direct binding demonstrated with multiple BMP ligands, functional KD/OE with pathway dissection (Smad vs RhoA)","pmids":["21149453"],"is_preprint":false},{"year":2015,"finding":"Crystal structures of the N-terminal domains of all human RGM family members in complex with BMP2, and the ternary BMP2-RGM-NEO1 complex, were determined. RGM acts as a central structural bridge physically connecting NEO1 and BMP signaling pathways; BMP-induced clustering of the RGM-NEO1 complex was confirmed by solution scattering and live-cell super-resolution fluorescence microscopy.","method":"X-ray crystallography (ternary complex), small-angle X-ray scattering (SAXS), live-cell super-resolution fluorescence microscopy","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 — crystal structure of ternary complex plus orthogonal validation by SAXS and live-cell imaging","pmids":["25938661"],"is_preprint":false},{"year":2019,"finding":"Rgma promotes glycosylation and intramembrane proteolytic cleavage of Neo1 in zebrafish, generating a transient nuclear intracellular fragment (NeoICD). This proteolytic processing is essential for microtubule-mediated neuroepithelial cell elongation and neural tube morphogenesis, acting cell-autonomously and independently of establishing apical junctional complexes.","method":"Morpholino knockdown, cell transplantation (cell autonomy), overexpression of NeoICD rescue construct, immunostaining, zebrafish neurulation phenotype analysis","journal":"The Journal of neuroscience","confidence":"High","confidence_rationale":"Tier 2 — cell-autonomous rescue with defined proteolytic fragment, genetic KD + OE rescue with specific cellular phenotype readout","pmids":["31399534"],"is_preprint":false},{"year":2020,"finding":"Netrin-1 promotes naive pluripotency by co-regulating Wnt and MAPK pathways through a balance of its receptors NEO1 and Unc5B in mouse ESCs. Mechanistically, Netrin-1 induces FAK kinase to inactivate Gsk3α/β and stabilize β-catenin, while increasing Pp2a (Ppp2r2c-containing complex) phosphatase activity to reduce Erk1/2 activity; these opposing outputs depend on the relative receptor dosage of NEO1 vs Unc5B.","method":"Chemical inhibitor rescue, signaling pathway analysis (phospho-Western blot), receptor overexpression/knockdown, transcriptomic and epigenetic profiling of mESCs","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (biochemical, genetic, transcriptomic) establishing pathway mechanism in two ESC systems","pmids":["32231305"],"is_preprint":false},{"year":2021,"finding":"NEO1 simultaneously binds Netrin-1 (NET1) and repulsive guidance molecule (RGM) to form a ternary NEO1-NET1-RGM complex that assembles into a 'trimer-of-trimers' super-assembly in the cell membrane. Super-assembly formation results in reciprocal silencing: it inhibits RGMA-NEO1-mediated growth cone collapse and RGMA- or NET1-NEO1-mediated neuron migration by preventing formation of signaling-competent RGM-NEO1 complexes and blocking NET1-induced NEO1 ectodomain clustering.","method":"Crystal structure determination (ternary complex), cryo-electron microscopy, cell migration assays, growth cone collapse assay, super-resolution microscopy of membrane complexes","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1 — crystal structure + cryo-EM of super-assembly plus multiple functional assays establishing signaling outcome","pmids":["33740419"],"is_preprint":false},{"year":2015,"finding":"NEO1 gene variants (hemizygous deletion combined with intragenic missense p.Arg1130Cys in the nuclear localization signal domain) were identified in ASD patients; in silico and functional analyses showed that p.Arg1130Cys causes defective nuclear translocation of neogenin, implicating NEO1-mediated nuclear signaling in cortical interneuron development.","method":"Array CGH, Sanger sequencing, in silico domain analysis, functional nuclear translocation assay","journal":"Behavioural brain research","confidence":"Low","confidence_rationale":"Tier 3 — functional translocation assay in small patient cohort, single lab, limited mechanistic follow-up","pmids":["26518331"],"is_preprint":false},{"year":2024,"finding":"Astrocytic NEO1 is required for blood-brain barrier integrity after subarachnoid hemorrhage (SAH); conditional knockout of NEO1 in astrocytes (GFAP-Cre) increased endothelial cell proliferation and BBB permeability. Hepcidin administration reversed NEO1-cKO-induced endothelial dysfunction, linking astrocytic NEO1 to iron homeostasis and BBB maintenance.","method":"Conditional knockout mice (GFAP-Cre;NEO1fl/fl), Evans Blue and dextran permeability assays, transmission electron microscopy, immunostaining, CSF proteomics","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 — conditional KO with multiple in vivo permeability readouts, mechanistic rescue by hepcidin","pmids":["39107268"],"is_preprint":false},{"year":2025,"finding":"NEO1 in astrocytes mediates A1 astrocyte polarization after SAH through the cPLA2-MAVS-NF-κB signaling pathway; astrocyte-specific NEO1 knockout significantly reduced A1 polarization and inflammatory factor release, and this was reversed by lentiviral cPLA2 overexpression, placing NEO1 upstream of cPLA2 and MAVS in neuroinflammatory signaling.","method":"Astrocyte-specific conditional knockout (GFAP-Cre;NEO1fl/fl), single-cell RNA sequencing, transcriptome sequencing, lentiviral cPLA2 overexpression rescue, mouse SAH model","journal":"Journal of neuroinflammation","confidence":"Medium","confidence_rationale":"Tier 2 — conditional KO with transcriptomic pathway mapping and genetic rescue, single lab","pmids":["41345945"],"is_preprint":false},{"year":2026,"finding":"Netrin-1 promotes pancreatic tumor cell growth, EMT, and cancer stemness through NEO1-mediated activation of focal adhesion kinase (FAK), upregulating ZEB1 and SOX9; in vivo knockout of Neo1 in tumor cells reduced FAK phosphorylation, EMT markers, and liver metastasis progression. Netrin-1/NEO1 also promotes sympathetic axonogenesis of celiac ganglia neurons, indirectly supporting tumor growth.","method":"Pancreatic organoid culture, in vivo Pdx1-Cre;KrasG12D mouse model with Ntn1/Neo1 knockout, liver metastasis model with neutralizing antibody, ex vivo celiac ganglia axonogenesis assay, FAK phosphorylation Western blot","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal in vivo and ex vivo models with defined mechanistic readout (FAK phosphorylation, ZEB1/SOX9)","pmids":["41474982"],"is_preprint":false},{"year":2025,"finding":"G-quadruplex (G4) RNA structures in the 3'UTR of NEO1 mRNA regulate alternative polyadenylation site selection, affecting NEO1 transcript isoform length and protein synthesis; stabilization of G4 structures by RHPS4 modulates this selection.","method":"PolyAclick-seq, in vitro G4 assays, G4 mutagenesis constructs, RHPS4 ligand treatment","journal":"bioRxiv","confidence":"Low","confidence_rationale":"Tier 3 — preprint, single lab, post-transcriptional regulatory mechanism without functional protein-level validation","pmids":[],"is_preprint":true}],"current_model":"NEO1 (neogenin) is a transmembrane receptor with four Ig-like and six fibronectin type III domains that functions as a multifunctional signaling hub: it acts as a dependence receptor (inducing apoptosis via caspase cleavage of its cytoplasmic domain in the absence of ligand), binds Netrin-1, RGM family members, BMPs, and hemojuvelin, and integrates opposing guidance cues by forming a ternary NET1-NEO1-RGM super-assembly that silences both attractive and repulsive downstream signals; in different contexts NEO1 activates FAK/β-catenin/Wnt and RhoA pathways, undergoes Rgma-induced intramembrane proteolysis to generate a nuclear fragment required for neuroepithelial cell elongation, and in astrocytes regulates neuroinflammatory A1 polarization via a cPLA2-MAVS-NF-κB pathway and maintains blood-brain barrier integrity through iron homeostasis."},"narrative":{"teleology":[{"year":1997,"claim":"Cloning of human NEO1 established its domain architecture (4 Ig + 6 FNIII + TM + ICD) and ~50% identity to DCC, defining it as a distinct member of the DCC receptor family rather than a DCC orthologue.","evidence":"cDNA cloning, sequencing, Northern blot, and FISH mapping to 15q22.3-q23","pmids":["9169140","9121761"],"confidence":"High","gaps":["No ligand had been identified","Function of the intracellular domain was unknown"]},{"year":2004,"claim":"Demonstration that neogenin is a dependence receptor — inducing caspase-mediated apoptosis in the absence of its ligand RGM — revealed a survival-checkpoint function beyond guidance, while identification of neogenin–CDO complex formation in myoblasts showed it also operates outside the nervous system to promote netrin-dependent myogenesis.","evidence":"Chick neural tube electroporation and cell-based apoptosis/caspase cleavage assays (dependence receptor); co-immunoprecipitation and CDO-null myoblast differentiation assays (myogenesis)","pmids":["15258591","15520228"],"confidence":"High","gaps":["Downstream apoptotic cascade components beyond caspase cleavage were not identified","How CDO–neogenin cis-interaction modulates intracellular signaling remained unclear"]},{"year":2005,"claim":"Identification of PITPα as a netrin-1-recruited effector of neogenin/DCC linked receptor activation to phospholipid metabolism (PIP2 hydrolysis), providing the first intracellular signaling mechanism for netrin-1-induced neurite outgrowth.","evidence":"Co-immunoprecipitation, in vitro lipid-transfer assay, zebrafish morpholino knockdown, and cortical explant neurite assay","pmids":["16244667"],"confidence":"High","gaps":["Whether PITPα acts on neogenin and DCC independently or in a shared complex was unresolved","Downstream targets of PIP2 hydrolysis were not mapped"]},{"year":2009,"claim":"Resolution of the contested role of neogenin in iron homeostasis: neogenin physically interacts with hemojuvelin (HJV) and is required for BMP4-induced hepcidin expression in hepatocytes, placing it as a co-receptor in the HJV–BMP–hepcidin axis despite an earlier report of HJV-BMP signaling independence.","evidence":"Co-immunoprecipitation, siRNA knockdown, soluble neogenin fragment blocking in vivo, and qRT-PCR for hepcidin","pmids":["19564337","18326817"],"confidence":"High","gaps":["Whether neogenin contacts BMP ligands directly in this context was unknown","In vivo hepatic phenotype of neogenin loss was not characterized"]},{"year":2010,"claim":"Direct binding of neogenin to BMP-2, -4, -6, and -7 was demonstrated, and a signaling bifurcation was identified: neogenin suppresses Smad1/5/8-dependent osteoblastic differentiation while simultaneously activating RhoA, establishing it as a BMP co-receptor that redirects pathway output.","evidence":"Ligand binding assays, siRNA knockdown and overexpression, phospho-Smad and RhoA activity assays in C2C12 cells","pmids":["21149453"],"confidence":"High","gaps":["Structural basis of direct neogenin–BMP interaction was missing","How neogenin toggles between Smad suppression and RhoA activation was mechanistically unresolved"]},{"year":2015,"claim":"Crystal structures of RGM–BMP2 and the ternary BMP2–RGM–NEO1 complex revealed that RGM bridges NEO1 and BMP signaling as a central structural scaffold, and BMP-induced clustering of this complex was validated at the cell surface.","evidence":"X-ray crystallography, SAXS, and live-cell super-resolution fluorescence microscopy","pmids":["25938661"],"confidence":"High","gaps":["Signaling consequences of cluster formation were not directly measured","Stoichiometry and dynamics of full-length receptor assemblies in native membranes remained undefined"]},{"year":2019,"claim":"RGMa-induced intramembrane proteolysis of Neo1 generates a nuclear intracellular domain fragment (NeoICD) that is essential for microtubule-dependent neuroepithelial cell elongation during neural tube morphogenesis, revealing a regulated intramembrane proteolysis (RIP) signaling mode for neogenin.","evidence":"Zebrafish morpholino knockdown, cell transplantation for cell autonomy, NeoICD rescue construct overexpression","pmids":["31399534"],"confidence":"High","gaps":["Nuclear targets of NeoICD were not identified","The protease(s) mediating intramembrane cleavage were not characterized"]},{"year":2020,"claim":"Netrin-1–NEO1 signaling was placed at the center of naive pluripotency maintenance by showing that NEO1 activates FAK to inactivate GSK3 and stabilize β-catenin while modulating MAPK through PP2A, with the balance between NEO1 and UNC5B receptors determining pathway output.","evidence":"Chemical inhibitor rescue, phospho-Western blot signaling analysis, receptor OE/KD, transcriptomic profiling in mouse ESCs","pmids":["32231305"],"confidence":"High","gaps":["Direct physical interaction between NEO1 and PP2A was not demonstrated","Whether these pathways operate in human ESCs was untested"]},{"year":2021,"claim":"Structural determination of the Netrin-1–NEO1–RGM ternary complex and its higher-order 'trimer-of-trimers' super-assembly provided a molecular mechanism for reciprocal silencing: the super-assembly sequesters both attractive and repulsive signaling-competent receptor states, explaining how opposing guidance cues are integrated at the membrane.","evidence":"X-ray crystallography, cryo-electron microscopy, growth cone collapse and cell migration assays, super-resolution microscopy","pmids":["33740419"],"confidence":"High","gaps":["In vivo validation of the super-assembly in developing organisms was lacking","How cells dissolve the super-assembly to re-enable signaling was unknown"]},{"year":2024,"claim":"Astrocyte-specific NEO1 conditional knockout demonstrated that NEO1 maintains blood–brain barrier integrity after injury through iron–hepcidin homeostasis, and subsequent work showed NEO1 drives neuroinflammatory A1 astrocyte polarization via a cPLA2–MAVS–NF-κB pathway.","evidence":"GFAP-Cre;NEO1fl/fl conditional knockout mice, Evans Blue/dextran permeability assays, hepcidin rescue, scRNA-seq, lentiviral cPLA2 OE rescue in SAH model","pmids":["39107268","41345945"],"confidence":"Medium","gaps":["How NEO1 activates cPLA2 biochemically is undefined","Whether the iron-homeostasis and neuroinflammatory functions are mechanistically coupled is unknown","Findings are from a single laboratory"]},{"year":2025,"claim":"In pancreatic cancer, Netrin-1–NEO1 signaling through FAK was shown to drive EMT, cancer stemness (via ZEB1/SOX9), and liver metastasis, and to promote sympathetic axonogenesis that supports tumor growth — extending the FAK-dependent signaling axis to tumor biology.","evidence":"Pdx1-Cre;KrasG12D mouse model with Neo1 knockout, liver metastasis neutralizing antibody model, ex vivo celiac ganglia axonogenesis assay, FAK phosphorylation analysis","pmids":["41474982"],"confidence":"High","gaps":["Whether NEO1 is the sole Netrin-1 receptor mediating these tumor effects or acts redundantly with DCC/UNC5 is unclear","Therapeutic relevance of NEO1 inhibition versus FAK inhibition was not compared"]},{"year":null,"claim":"Key unresolved questions include the identity of nuclear targets of the NeoICD fragment, the protease(s) responsible for intramembrane cleavage, the structural basis for how the super-assembly is dynamically regulated in vivo, and whether the astrocytic iron-homeostasis and neuroinflammatory functions represent the same or distinct signaling branches.","evidence":"","pmids":[],"confidence":"High","gaps":["Nuclear targets of NeoICD unknown","Protease mediating RIP cleavage unidentified","Super-assembly dynamics in vivo uncharacterized","Relationship between iron-homeostasis and cPLA2-MAVS pathways in astrocytes unresolved"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[1,6,9,10,14]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[6,9]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[5,7]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,7,10]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[8]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[1,6,7,9,10,14]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[1]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[2,8]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[12,13]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[14]}],"complexes":["NEO1-RGM-BMP ternary complex","NET1-NEO1-RGM trimer-of-trimers super-assembly","NEO1-CDO promyogenic complex"],"partners":["RGMA","NTN1","HFE2","BMP2","BMP4","CDO","PITPNA","UNC5B"],"other_free_text":[]},"mechanistic_narrative":"Neogenin (NEO1) is a multifunctional transmembrane receptor that integrates guidance, morphogenetic, and survival signals by binding Netrin-1, RGM family members, and BMPs through its four immunoglobulin-like and six fibronectin type III extracellular domains [PMID:9169140, PMID:25938661]. In the absence of ligand, neogenin functions as a dependence receptor, triggering caspase-mediated apoptosis via cleavage of its intracellular domain, whereas RGM binding suppresses this pro-apoptotic output and instead drives intramembrane proteolysis to generate a nuclear fragment required for neuroepithelial cell elongation [PMID:15258591, PMID:31399534]. Formation of a ternary Netrin-1–NEO1–RGM super-assembly reciprocally silences both attractive and repulsive guidance signals, providing a structural mechanism for integrating opposing cues at the cell surface [PMID:33740419]. Beyond axon guidance, NEO1 activates FAK–β-catenin and RhoA pathways in contexts ranging from pluripotency maintenance in embryonic stem cells to promotion of epithelial-mesenchymal transition in pancreatic cancer, and in astrocytes it regulates blood–brain barrier integrity through iron-hepcidin homeostasis and controls neuroinflammatory A1 polarization via cPLA2–MAVS–NF-κB signaling [PMID:32231305, PMID:41474982, PMID:39107268, PMID:41345945]."},"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,"source_track":"pubmed_title"},{"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,"source_track":"pubmed_title"},{"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 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\"NEO1 acts as an attractive guidance receptor when Netrin-1 (NET1) binds and mediates repulsion via repulsive guidance molecule (RGM) ligands; simultaneous binding of NET1 and RGM forms a ternary NEO1-NET1-RGM 'trimer-of-trimers' super-assembly in the cell membrane that reciprocally silences downstream signaling, inhibiting RGMA-NEO1-mediated growth cone collapse and RGMA- or NET1-NEO1-mediated neuron migration.\",\n      \"method\": \"Structural determination (cryo-EM/X-ray crystallography), cell-based functional assays (growth cone collapse, neuron migration assays), biochemical complex formation\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — structural resolution of ternary complex plus multiple orthogonal functional assays\",\n      \"pmids\": [\"33740419\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Netrin-1 signals through NEO1 (and co-receptor Unc5B) to promote naive pluripotency in mESCs by inducing FAK kinase to inactivate Gsk3α/β and stabilize β-catenin, while increasing activity of a Ppp2r2c-containing PP2A complex to reduce Erk1/2 activity, thereby co-regulating Wnt and MAPK pathways.\",\n      \"method\": \"Genetic knockdown/knockout, pharmacological inhibitors, co-immunoprecipitation, phospho-western blotting, transcriptomic and epigenetic profiling\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal mechanistic methods in mouse and human ESCs, demonstrated receptor balance controls pathway output\",\n      \"pmids\": [\"32231305\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Rgma promotes NEO1 glycosylation and intramembrane proteolysis in zebrafish, generating a transient nuclear intracellular fragment (NeoICD); this proteolytic cleavage is essential for neuroepithelial cell microtubule-mediated elongation during neural tube morphogenesis, and NEO1 functions cell-autonomously in this process.\",\n      \"method\": \"Zebrafish loss-of-function (morpholino knockdown), cell transplantation, overexpression of NeoICD rescue, immunostaining for glycosylation and cleavage products\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — cell-autonomous function established by transplantation, rescue by NeoICD overexpression; single lab\",\n      \"pmids\": [\"31399534\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"The Arg1130Cys missense variant at the nuclear localization signal (NLS) domain of human Neogenin leads to defective nuclear translocation of the protein, as demonstrated by functional analysis of the variant.\",\n      \"method\": \"In silico analysis and functional nuclear translocation assay of NLS domain variant\",\n      \"journal\": \"Behavioural brain research\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — functional localization assay for single variant, single lab, limited methodological detail\",\n      \"pmids\": [\"26518331\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Astrocytic NEO1 is required for blood-brain barrier integrity after subarachnoid hemorrhage; astrocyte-specific NEO1 conditional knockout (GFAP-Cre) increases endothelial cell proliferation and BBB permeability, and hepcidin treatment reverses this EC dysfunction, implicating NEO1 in astrocyte-to-endothelium signaling that maintains BBB structure.\",\n      \"method\": \"Conditional knockout mice (NEO1-GFAP-Cre), Evans Blue/dextran leakage assays, transmission electron microscopy, immunostaining, pharmacological rescue with hepcidin\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — cell-type-specific KO with defined structural and functional BBB readouts; single lab\",\n      \"pmids\": [\"39107268\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"NEO1 in astrocytes regulates A1 astrocyte polarization after subarachnoid hemorrhage through the cPLA2-MAVS-NF-κB signaling pathway; NEO1 knockout reduces cPLA2 and MAVS mRNA levels and suppresses A1 polarization, while cPLA2 overexpression reverses NEO1-KO-mediated suppression of A1 polarization.\",\n      \"method\": \"Astrocyte-specific NEO1 cKO mice, transcriptome sequencing, lentiviral cPLA2 overexpression, in vitro primary astrocyte experiments\",\n      \"journal\": \"Journal of neuroinflammation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — epistasis established by rescue experiment; conditional KO with transcriptomic pathway identification; 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 and neurons to promote EMT, cancer stemness, and sympathetic innervation; NEO1-mediated activation of FAK upregulates ZEB1 and SOX9, and tumoral Neo1 knockout reduces FAK phosphorylation, EMT markers, and metastatic progression.\",\n      \"method\": \"NEO1 knockout in murine PDAC model, recombinant netrin-1 treatment of organoids, ex vivo celiac ganglia axonogenesis assay, FAK phosphorylation assays, liver metastasis model\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vivo KO with mechanistic FAK pathway readout, multiple model systems; single lab\",\n      \"pmids\": [\"41474982\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"ATP9A, the human ortholog of yeast Neo1, maintains phosphatidylinositol-4-phosphate (PI4P) asymmetry in the plasma membrane; knockdown of ATP9A in human cells exposes extracellular PI4P, linking the flippase activity of the Neo1/ATP9A subfamily to PI4P homeostasis and neomycin sensitivity.\",\n      \"method\": \"ATP9A knockdown in human cells, PI4P exposure assay, neomycin sensitivity assay; cryo-EM of yeast Neo1 with PI4P\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 — cryo-EM structure shows PI4P in translocation pathway; functional validation in human cells; preprint\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"G-quadruplex (G4) RNA structures in the 3'UTR of NEO1 mRNA regulate alternative polyadenylation site selection, influencing NEO1 isoform choice and protein synthesis.\",\n      \"method\": \"PolyAclick-seq, in vitro G4 assays, G4 mutagenesis constructs, RHPS4 ligand treatment\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — post-transcriptional regulatory mechanism demonstrated in vitro and in silico; preprint, single lab\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"NEO1 (Neogenin-1) is a transmembrane receptor with four Ig-like and six fibronectin type III extracellular domains that integrates opposing guidance cues: Netrin-1 binding promotes attraction and pluripotency signaling (via FAK-Gsk3/β-catenin and PP2A-ERK axes), while RGM binding mediates repulsion; simultaneous NET1 and RGM binding forms a 'trimer-of-trimers' super-assembly that silences both signals, and RGMA-induced intramembrane proteolysis of NEO1 generates a nuclear ICD fragment required for neuroepithelial cell elongation during neural tube morphogenesis; in astrocytes, NEO1 maintains BBB integrity and controls A1 polarization via a cPLA2-MAVS-NF-κB pathway.\"\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1997,\n      \"finding\": \"Human neogenin (NEO1) encodes a transmembrane protein with four immunoglobulin-like domains followed by six fibronectin type III domains, a transmembrane domain, and an intracellular domain, sharing ~50% amino acid identity with DCC. The gene maps to chromosome 15q22.3-q23, is expressed in at least two alternatively spliced isoforms differing in the intracellular domain, and produces mRNA species of ~5 and 7 kb.\",\n      \"method\": \"cDNA cloning, sequencing, Northern blot, fluorescence in situ hybridization (FISH)\",\n      \"journal\": \"Genomics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — direct molecular characterization with multiple orthogonal methods in founding paper\",\n      \"pmids\": [\"9169140\", \"9121761\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Neogenin acts as a dependence receptor: in the absence of its ligand RGM, neogenin over-expression induces apoptosis in immortalized neuronal cells and the chick neural tube via caspase-mediated cleavage of its cytoplasmic domain; binding of RGM to neogenin inhibits this pro-apoptotic activity.\",\n      \"method\": \"In ovo gene transfer (chick neural tube), cell-based apoptosis assays, caspase cleavage assays\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — in vivo loss/gain-of-function with mechanistic caspase cleavage readout, replicated in two systems\",\n      \"pmids\": [\"15258591\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Neogenin is expressed in skeletal myoblasts and, together with netrin-3, promotes myotube formation and enhances myogenic bHLH- and NFAT-dependent transcription. Neogenin binds CDO in cis at the cell surface, and myoblasts lacking CDO are defective in responding to recombinant netrin, placing neogenin in a promyogenic cell-surface complex.\",\n      \"method\": \"Co-immunoprecipitation, recombinant protein stimulation, myogenic differentiation assays, genetic loss-of-function (CDO-null myoblasts)\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP plus genetic epistasis (CDO-null rescue) in defined cellular phenotype\",\n      \"pmids\": [\"15520228\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Phosphatidylinositol transfer protein-alpha (PITPα) interacts with neogenin (and DCC) and is required for netrin-1-induced PIP2 hydrolysis and neurite outgrowth; netrin-1 stimulates PITPα binding to neogenin and enhances its lipid-transfer activity.\",\n      \"method\": \"Co-immunoprecipitation (pulldown), in vitro lipid-transfer assay, dominant-negative overexpression, morpholino knockdown in zebrafish, cortical explant neurite assay\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — biochemical activity assay combined with multiple in vivo loss-of-function models\",\n      \"pmids\": [\"16244667\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Hemojuvelin (HJV)-induced BMP signaling and hepcidin expression are not altered by neogenin overexpression or knockdown, demonstrating that HJV-mediated BMP signaling occurs independently of neogenin.\",\n      \"method\": \"siRNA knockdown, overexpression, BMP signaling reporter assays in hepatoma cell lines\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — clean KD/OE with defined signaling readout in single study\",\n      \"pmids\": [\"18326817\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Neogenin physically interacts with hemojuvelin (HJV) in hepatocytes, and this interaction is required for BMP4-induced hepcidin mRNA expression; disruption of the HJV-neogenin interaction or neogenin knockdown suppresses hepcidin induction ~16-fold in HJV-expressing cells. A soluble neogenin fragment blocks HJV-neogenin interaction and suppresses hepatic hepcidin in vivo.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, in vivo mouse experiments with soluble neogenin fragment, quantitative RT-PCR\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP, genetic KD, and in vivo pharmacological validation across multiple orthogonal methods\",\n      \"pmids\": [\"19564337\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Neogenin directly binds BMP-2, BMP-4, BMP-6, and BMP-7 and negatively regulates BMP-induced osteoblastic differentiation and Smad1/5/8 phosphorylation. Neogenin mediates BMP-induced RhoA activation, and RhoA inhibition promotes BMP-2-induced osteoblastic differentiation independently of Smad phosphorylation.\",\n      \"method\": \"Binding assays, siRNA knockdown, overexpression, Smad phosphorylation Western blot, RhoA activity assay, osteoblastic differentiation assay in C2C12 cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct binding demonstrated with multiple BMP ligands, functional KD/OE with pathway dissection (Smad vs RhoA)\",\n      \"pmids\": [\"21149453\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Crystal structures of the N-terminal domains of all human RGM family members in complex with BMP2, and the ternary BMP2-RGM-NEO1 complex, were determined. RGM acts as a central structural bridge physically connecting NEO1 and BMP signaling pathways; BMP-induced clustering of the RGM-NEO1 complex was confirmed by solution scattering and live-cell super-resolution fluorescence microscopy.\",\n      \"method\": \"X-ray crystallography (ternary complex), small-angle X-ray scattering (SAXS), live-cell super-resolution fluorescence microscopy\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure of ternary complex plus orthogonal validation by SAXS and live-cell imaging\",\n      \"pmids\": [\"25938661\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Rgma promotes glycosylation and intramembrane proteolytic cleavage of Neo1 in zebrafish, generating a transient nuclear intracellular fragment (NeoICD). This proteolytic processing is essential for microtubule-mediated neuroepithelial cell elongation and neural tube morphogenesis, acting cell-autonomously and independently of establishing apical junctional complexes.\",\n      \"method\": \"Morpholino knockdown, cell transplantation (cell autonomy), overexpression of NeoICD rescue construct, immunostaining, zebrafish neurulation phenotype analysis\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — cell-autonomous rescue with defined proteolytic fragment, genetic KD + OE rescue with specific cellular phenotype readout\",\n      \"pmids\": [\"31399534\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Netrin-1 promotes naive pluripotency by co-regulating Wnt and MAPK pathways through a balance of its receptors NEO1 and Unc5B in mouse ESCs. Mechanistically, Netrin-1 induces FAK kinase to inactivate Gsk3α/β and stabilize β-catenin, while increasing Pp2a (Ppp2r2c-containing complex) phosphatase activity to reduce Erk1/2 activity; these opposing outputs depend on the relative receptor dosage of NEO1 vs Unc5B.\",\n      \"method\": \"Chemical inhibitor rescue, signaling pathway analysis (phospho-Western blot), receptor overexpression/knockdown, transcriptomic and epigenetic profiling of mESCs\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (biochemical, genetic, transcriptomic) establishing pathway mechanism in two ESC systems\",\n      \"pmids\": [\"32231305\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"NEO1 simultaneously binds Netrin-1 (NET1) and repulsive guidance molecule (RGM) to form a ternary NEO1-NET1-RGM complex that assembles into a 'trimer-of-trimers' super-assembly in the cell membrane. Super-assembly formation results in reciprocal silencing: it inhibits RGMA-NEO1-mediated growth cone collapse and RGMA- or NET1-NEO1-mediated neuron migration by preventing formation of signaling-competent RGM-NEO1 complexes and blocking NET1-induced NEO1 ectodomain clustering.\",\n      \"method\": \"Crystal structure determination (ternary complex), cryo-electron microscopy, cell migration assays, growth cone collapse assay, super-resolution microscopy of membrane complexes\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure + cryo-EM of super-assembly plus multiple functional assays establishing signaling outcome\",\n      \"pmids\": [\"33740419\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"NEO1 gene variants (hemizygous deletion combined with intragenic missense p.Arg1130Cys in the nuclear localization signal domain) were identified in ASD patients; in silico and functional analyses showed that p.Arg1130Cys causes defective nuclear translocation of neogenin, implicating NEO1-mediated nuclear signaling in cortical interneuron development.\",\n      \"method\": \"Array CGH, Sanger sequencing, in silico domain analysis, functional nuclear translocation assay\",\n      \"journal\": \"Behavioural brain research\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — functional translocation assay in small patient cohort, single lab, limited mechanistic follow-up\",\n      \"pmids\": [\"26518331\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Astrocytic NEO1 is required for blood-brain barrier integrity after subarachnoid hemorrhage (SAH); conditional knockout of NEO1 in astrocytes (GFAP-Cre) increased endothelial cell proliferation and BBB permeability. Hepcidin administration reversed NEO1-cKO-induced endothelial dysfunction, linking astrocytic NEO1 to iron homeostasis and BBB maintenance.\",\n      \"method\": \"Conditional knockout mice (GFAP-Cre;NEO1fl/fl), Evans Blue and dextran permeability assays, transmission electron microscopy, immunostaining, CSF proteomics\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — conditional KO with multiple in vivo permeability readouts, mechanistic rescue by hepcidin\",\n      \"pmids\": [\"39107268\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"NEO1 in astrocytes mediates A1 astrocyte polarization after SAH through the cPLA2-MAVS-NF-κB signaling pathway; astrocyte-specific NEO1 knockout significantly reduced A1 polarization and inflammatory factor release, and this was reversed by lentiviral cPLA2 overexpression, placing NEO1 upstream of cPLA2 and MAVS in neuroinflammatory signaling.\",\n      \"method\": \"Astrocyte-specific conditional knockout (GFAP-Cre;NEO1fl/fl), single-cell RNA sequencing, transcriptome sequencing, lentiviral cPLA2 overexpression rescue, mouse SAH model\",\n      \"journal\": \"Journal of neuroinflammation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — conditional KO with transcriptomic pathway mapping and genetic rescue, single lab\",\n      \"pmids\": [\"41345945\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"Netrin-1 promotes pancreatic tumor cell growth, EMT, and cancer stemness through NEO1-mediated activation of focal adhesion kinase (FAK), upregulating ZEB1 and SOX9; in vivo knockout of Neo1 in tumor cells reduced FAK phosphorylation, EMT markers, and liver metastasis progression. Netrin-1/NEO1 also promotes sympathetic axonogenesis of celiac ganglia neurons, indirectly supporting tumor growth.\",\n      \"method\": \"Pancreatic organoid culture, in vivo Pdx1-Cre;KrasG12D mouse model with Ntn1/Neo1 knockout, liver metastasis model with neutralizing antibody, ex vivo celiac ganglia axonogenesis assay, FAK phosphorylation Western blot\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal in vivo and ex vivo models with defined mechanistic readout (FAK phosphorylation, ZEB1/SOX9)\",\n      \"pmids\": [\"41474982\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"G-quadruplex (G4) RNA structures in the 3'UTR of NEO1 mRNA regulate alternative polyadenylation site selection, affecting NEO1 transcript isoform length and protein synthesis; stabilization of G4 structures by RHPS4 modulates this selection.\",\n      \"method\": \"PolyAclick-seq, in vitro G4 assays, G4 mutagenesis constructs, RHPS4 ligand treatment\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — preprint, single lab, post-transcriptional regulatory mechanism without functional protein-level validation\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"NEO1 (neogenin) is a transmembrane receptor with four Ig-like and six fibronectin type III domains that functions as a multifunctional signaling hub: it acts as a dependence receptor (inducing apoptosis via caspase cleavage of its cytoplasmic domain in the absence of ligand), binds Netrin-1, RGM family members, BMPs, and hemojuvelin, and integrates opposing guidance cues by forming a ternary NET1-NEO1-RGM super-assembly that silences both attractive and repulsive downstream signals; in different contexts NEO1 activates FAK/β-catenin/Wnt and RhoA pathways, undergoes Rgma-induced intramembrane proteolysis to generate a nuclear fragment required for neuroepithelial cell elongation, and in astrocytes regulates neuroinflammatory A1 polarization via a cPLA2-MAVS-NF-κB pathway and maintains blood-brain barrier integrity through iron homeostasis.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"NEO1 (Neogenin-1) is a transmembrane guidance receptor that integrates opposing extracellular cues to control cell migration, morphogenesis, pluripotency, and tissue homeostasis. Netrin-1 binding to NEO1 activates FAK, which inactivates GSK3α/β to stabilize β-catenin and engages a PP2A complex to suppress ERK1/2, thereby co-regulating Wnt and MAPK pathways to promote naive pluripotency in embryonic stem cells and, in pancreatic epithelium, to drive EMT and cancer stemness via ZEB1/SOX9 upregulation [PMID:32231305, PMID:41474982]. Repulsive guidance molecule (RGM) binding triggers NEO1 intramembrane proteolysis, generating a nuclear intracellular domain fragment (NeoICD) required for microtubule-mediated neuroepithelial cell elongation during neural tube closure, while simultaneous NET1 and RGM binding assembles a trimer-of-trimers super-complex that reciprocally silences both attractive and repulsive outputs [PMID:31399534, PMID:33740419]. In astrocytes, NEO1 maintains blood–brain barrier integrity through hepcidin-sensitive astrocyte-to-endothelium signaling and controls neurotoxic A1 astrocyte polarization via the cPLA2–MAVS–NF-κB pathway [PMID:39107268, PMID:41345945].\",\n  \"teleology\": [\n    {\n      \"year\": 1997,\n      \"claim\": \"Molecular identity of NEO1 was established, revealing the Ig-FNIII-transmembrane architecture and existence of alternatively spliced intracellular domain isoforms, setting the stage for receptor-ligand studies.\",\n      \"evidence\": \"cDNA cloning and sequencing of human NEO1 with Northern blot isoform detection\",\n      \"pmids\": [\"9169140\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"No ligand identified at this stage\",\n        \"Functional significance of alternative splicing in the intracellular domain unknown\",\n        \"Single-lab characterization without independent replication\"\n      ]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"RGMa-induced intramembrane proteolysis of NEO1 was shown to generate a nuclear ICD fragment essential for neuroepithelial cell elongation, establishing that NEO1 functions as a regulated intramembrane proteolysis substrate with a cell-autonomous morphogenetic role during neural tube closure.\",\n      \"evidence\": \"Zebrafish morpholino knockdown, cell transplantation for cell-autonomy, NeoICD overexpression rescue\",\n      \"pmids\": [\"31399534\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Nuclear targets of NeoICD not identified\",\n        \"Protease(s) responsible for intramembrane cleavage not characterized\",\n        \"Single-lab study in zebrafish; mammalian validation lacking\"\n      ]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"NEO1 was identified as the Netrin-1 receptor through which FAK-mediated GSK3 inactivation and PP2A-mediated ERK suppression converge to maintain naive pluripotency, revealing how a guidance receptor co-regulates Wnt and MAPK signaling in stem cells.\",\n      \"evidence\": \"Genetic KO/knockdown, pharmacological inhibition, co-IP, phospho-western blots, and transcriptomic profiling in mouse and human ESCs\",\n      \"pmids\": [\"32231305\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Structural basis for NEO1–FAK interaction not resolved\",\n        \"Whether Unc5B co-receptor is required for all downstream branches unclear\",\n        \"Relevance to in vivo embryonic pluripotency maintenance not tested\"\n      ]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Structural resolution of the ternary NEO1–NET1–RGM complex as a trimer-of-trimers super-assembly explained how simultaneous ligand engagement reciprocally silences both attractive and repulsive signaling, providing a molecular switch model for guidance receptor integration.\",\n      \"evidence\": \"Cryo-EM/X-ray crystallography of the ternary complex, growth cone collapse assays, and neuron migration functional readouts\",\n      \"pmids\": [\"33740419\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Downstream signaling events silenced by the super-assembly not molecularly defined\",\n        \"Whether silencing complex forms at physiological ligand concentrations in vivo unclear\",\n        \"Dynamics of complex assembly and disassembly on the cell surface unknown\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Astrocytic NEO1 was shown to maintain blood–brain barrier integrity after injury, with conditional knockout increasing BBB permeability and endothelial proliferation — a phenotype rescued by hepcidin — revealing a non-neuronal, paracrine role for NEO1.\",\n      \"evidence\": \"Astrocyte-specific NEO1 cKO (GFAP-Cre), Evans Blue/dextran leakage, TEM, hepcidin pharmacological rescue in SAH model\",\n      \"pmids\": [\"39107268\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Molecular mechanism linking NEO1 to hepcidin regulation in astrocytes not defined\",\n        \"Whether NEO1 ligands (Netrin-1, RGMs) are involved in BBB maintenance not tested\",\n        \"Single-lab finding in one injury model\"\n      ]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"The downstream pathway by which astrocytic NEO1 controls neurotoxic A1 polarization was mapped to cPLA2–MAVS–NF-κB, establishing an inflammatory signaling axis dependent on NEO1 expression.\",\n      \"evidence\": \"Astrocyte-specific NEO1 cKO, transcriptome sequencing, lentiviral cPLA2 overexpression rescue in primary astrocytes\",\n      \"pmids\": [\"41345945\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"How NEO1 regulates cPLA2 transcription is unknown\",\n        \"Whether cPLA2–MAVS–NF-κB axis operates in non-injury contexts not tested\",\n        \"Single-lab study; independent validation needed\"\n      ]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"NEO1 was identified as the receptor through which Netrin-1 drives EMT, cancer stemness, and sympathetic innervation in pancreatic cancer via FAK–ZEB1/SOX9 signaling, extending the FAK-dependent mechanism from stem cells to tumor biology.\",\n      \"evidence\": \"NEO1 KO in murine PDAC model, recombinant Netrin-1 organoid treatment, ex vivo axonogenesis, liver metastasis model\",\n      \"pmids\": [\"41474982\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Whether Unc5 co-receptors modulate the tumoral NEO1 response not assessed\",\n        \"Patient-derived evidence for NEO1 as therapeutic target in PDAC lacking\",\n        \"Mechanism of NEO1-driven sympathetic innervation not fully dissected\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the identity of nuclear targets of NeoICD, the protease(s) executing NEO1 intramembrane proteolysis, whether the trimer-of-trimers silencing complex forms under physiological conditions in vivo, and how NEO1 transduces signals in astrocytes to regulate cPLA2 and hepcidin-dependent BBB maintenance.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Nuclear transcriptional targets of NeoICD remain unidentified\",\n        \"No structural or biochemical characterization of NEO1 intramembrane proteolysis machinery\",\n        \"In vivo evidence for ternary silencing complex formation under physiological conditions absent\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [1, 2, 7]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [2, 7]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 1, 3]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [3, 4]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [1, 2, 7]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [1, 3]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [6]}\n    ],\n    \"complexes\": [\n      \"NEO1-NET1-RGM trimer-of-trimers\"\n    ],\n    \"partners\": [\n      \"NTN1\",\n      \"RGMA\",\n      \"FAK\",\n      \"UNC5B\",\n      \"CTNNB1\",\n      \"MAVS\",\n      \"PLA2G4A\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"mechanistic_narrative\": \"Neogenin (NEO1) is a multifunctional transmembrane receptor that integrates guidance, morphogenetic, and survival signals by binding Netrin-1, RGM family members, and BMPs through its four immunoglobulin-like and six fibronectin type III extracellular domains [PMID:9169140, PMID:25938661]. In the absence of ligand, neogenin functions as a dependence receptor, triggering caspase-mediated apoptosis via cleavage of its intracellular domain, whereas RGM binding suppresses this pro-apoptotic output and instead drives intramembrane proteolysis to generate a nuclear fragment required for neuroepithelial cell elongation [PMID:15258591, PMID:31399534]. Formation of a ternary Netrin-1–NEO1–RGM super-assembly reciprocally silences both attractive and repulsive guidance signals, providing a structural mechanism for integrating opposing cues at the cell surface [PMID:33740419]. Beyond axon guidance, NEO1 activates FAK–β-catenin and RhoA pathways in contexts ranging from pluripotency maintenance in embryonic stem cells to promotion of epithelial-mesenchymal transition in pancreatic cancer, and in astrocytes it regulates blood–brain barrier integrity through iron-hepcidin homeostasis and controls neuroinflammatory A1 polarization via cPLA2–MAVS–NF-κB signaling [PMID:32231305, PMID:41474982, PMID:39107268, PMID:41345945].\",\n  \"teleology\": [\n    {\n      \"year\": 1997,\n      \"claim\": \"Cloning of human NEO1 established its domain architecture (4 Ig + 6 FNIII + TM + ICD) and ~50% identity to DCC, defining it as a distinct member of the DCC receptor family rather than a DCC orthologue.\",\n      \"evidence\": \"cDNA cloning, sequencing, Northern blot, and FISH mapping to 15q22.3-q23\",\n      \"pmids\": [\"9169140\", \"9121761\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No ligand had been identified\", \"Function of the intracellular domain was unknown\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Demonstration that neogenin is a dependence receptor — inducing caspase-mediated apoptosis in the absence of its ligand RGM — revealed a survival-checkpoint function beyond guidance, while identification of neogenin–CDO complex formation in myoblasts showed it also operates outside the nervous system to promote netrin-dependent myogenesis.\",\n      \"evidence\": \"Chick neural tube electroporation and cell-based apoptosis/caspase cleavage assays (dependence receptor); co-immunoprecipitation and CDO-null myoblast differentiation assays (myogenesis)\",\n      \"pmids\": [\"15258591\", \"15520228\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream apoptotic cascade components beyond caspase cleavage were not identified\", \"How CDO–neogenin cis-interaction modulates intracellular signaling remained unclear\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Identification of PITPα as a netrin-1-recruited effector of neogenin/DCC linked receptor activation to phospholipid metabolism (PIP2 hydrolysis), providing the first intracellular signaling mechanism for netrin-1-induced neurite outgrowth.\",\n      \"evidence\": \"Co-immunoprecipitation, in vitro lipid-transfer assay, zebrafish morpholino knockdown, and cortical explant neurite assay\",\n      \"pmids\": [\"16244667\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether PITPα acts on neogenin and DCC independently or in a shared complex was unresolved\", \"Downstream targets of PIP2 hydrolysis were not mapped\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Resolution of the contested role of neogenin in iron homeostasis: neogenin physically interacts with hemojuvelin (HJV) and is required for BMP4-induced hepcidin expression in hepatocytes, placing it as a co-receptor in the HJV–BMP–hepcidin axis despite an earlier report of HJV-BMP signaling independence.\",\n      \"evidence\": \"Co-immunoprecipitation, siRNA knockdown, soluble neogenin fragment blocking in vivo, and qRT-PCR for hepcidin\",\n      \"pmids\": [\"19564337\", \"18326817\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether neogenin contacts BMP ligands directly in this context was unknown\", \"In vivo hepatic phenotype of neogenin loss was not characterized\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Direct binding of neogenin to BMP-2, -4, -6, and -7 was demonstrated, and a signaling bifurcation was identified: neogenin suppresses Smad1/5/8-dependent osteoblastic differentiation while simultaneously activating RhoA, establishing it as a BMP co-receptor that redirects pathway output.\",\n      \"evidence\": \"Ligand binding assays, siRNA knockdown and overexpression, phospho-Smad and RhoA activity assays in C2C12 cells\",\n      \"pmids\": [\"21149453\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of direct neogenin–BMP interaction was missing\", \"How neogenin toggles between Smad suppression and RhoA activation was mechanistically unresolved\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Crystal structures of RGM–BMP2 and the ternary BMP2–RGM–NEO1 complex revealed that RGM bridges NEO1 and BMP signaling as a central structural scaffold, and BMP-induced clustering of this complex was validated at the cell surface.\",\n      \"evidence\": \"X-ray crystallography, SAXS, and live-cell super-resolution fluorescence microscopy\",\n      \"pmids\": [\"25938661\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Signaling consequences of cluster formation were not directly measured\", \"Stoichiometry and dynamics of full-length receptor assemblies in native membranes remained undefined\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"RGMa-induced intramembrane proteolysis of Neo1 generates a nuclear intracellular domain fragment (NeoICD) that is essential for microtubule-dependent neuroepithelial cell elongation during neural tube morphogenesis, revealing a regulated intramembrane proteolysis (RIP) signaling mode for neogenin.\",\n      \"evidence\": \"Zebrafish morpholino knockdown, cell transplantation for cell autonomy, NeoICD rescue construct overexpression\",\n      \"pmids\": [\"31399534\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Nuclear targets of NeoICD were not identified\", \"The protease(s) mediating intramembrane cleavage were not characterized\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Netrin-1–NEO1 signaling was placed at the center of naive pluripotency maintenance by showing that NEO1 activates FAK to inactivate GSK3 and stabilize β-catenin while modulating MAPK through PP2A, with the balance between NEO1 and UNC5B receptors determining pathway output.\",\n      \"evidence\": \"Chemical inhibitor rescue, phospho-Western blot signaling analysis, receptor OE/KD, transcriptomic profiling in mouse ESCs\",\n      \"pmids\": [\"32231305\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct physical interaction between NEO1 and PP2A was not demonstrated\", \"Whether these pathways operate in human ESCs was untested\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Structural determination of the Netrin-1–NEO1–RGM ternary complex and its higher-order 'trimer-of-trimers' super-assembly provided a molecular mechanism for reciprocal silencing: the super-assembly sequesters both attractive and repulsive signaling-competent receptor states, explaining how opposing guidance cues are integrated at the membrane.\",\n      \"evidence\": \"X-ray crystallography, cryo-electron microscopy, growth cone collapse and cell migration assays, super-resolution microscopy\",\n      \"pmids\": [\"33740419\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo validation of the super-assembly in developing organisms was lacking\", \"How cells dissolve the super-assembly to re-enable signaling was unknown\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Astrocyte-specific NEO1 conditional knockout demonstrated that NEO1 maintains blood–brain barrier integrity after injury through iron–hepcidin homeostasis, and subsequent work showed NEO1 drives neuroinflammatory A1 astrocyte polarization via a cPLA2–MAVS–NF-κB pathway.\",\n      \"evidence\": \"GFAP-Cre;NEO1fl/fl conditional knockout mice, Evans Blue/dextran permeability assays, hepcidin rescue, scRNA-seq, lentiviral cPLA2 OE rescue in SAH model\",\n      \"pmids\": [\"39107268\", \"41345945\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How NEO1 activates cPLA2 biochemically is undefined\", \"Whether the iron-homeostasis and neuroinflammatory functions are mechanistically coupled is unknown\", \"Findings are from a single laboratory\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"In pancreatic cancer, Netrin-1–NEO1 signaling through FAK was shown to drive EMT, cancer stemness (via ZEB1/SOX9), and liver metastasis, and to promote sympathetic axonogenesis that supports tumor growth — extending the FAK-dependent signaling axis to tumor biology.\",\n      \"evidence\": \"Pdx1-Cre;KrasG12D mouse model with Neo1 knockout, liver metastasis neutralizing antibody model, ex vivo celiac ganglia axonogenesis assay, FAK phosphorylation analysis\",\n      \"pmids\": [\"41474982\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether NEO1 is the sole Netrin-1 receptor mediating these tumor effects or acts redundantly with DCC/UNC5 is unclear\", \"Therapeutic relevance of NEO1 inhibition versus FAK inhibition was not compared\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the identity of nuclear targets of the NeoICD fragment, the protease(s) responsible for intramembrane cleavage, the structural basis for how the super-assembly is dynamically regulated in vivo, and whether the astrocytic iron-homeostasis and neuroinflammatory functions represent the same or distinct signaling branches.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Nuclear targets of NeoICD unknown\", \"Protease mediating RIP cleavage unidentified\", \"Super-assembly dynamics in vivo uncharacterized\", \"Relationship between iron-homeostasis and cPLA2-MAVS pathways in astrocytes unresolved\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [1, 6, 9, 10, 14]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [6, 9]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [5, 7]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 7, 10]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [8]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [1, 6, 7, 9, 10, 14]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [1]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [2, 8]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [12, 13]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [14]}\n    ],\n    \"complexes\": [\n      \"NEO1-RGM-BMP ternary complex\",\n      \"NET1-NEO1-RGM trimer-of-trimers super-assembly\",\n      \"NEO1-CDO promyogenic complex\"\n    ],\n    \"partners\": [\n      \"RGMA\",\n      \"NTN1\",\n      \"HFE2\",\n      \"BMP2\",\n      \"BMP4\",\n      \"CDO\",\n      \"PITPNA\",\n      \"UNC5B\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}