{"gene":"NEFL","run_date":"2026-06-10T05:19:52","timeline":{"discoveries":[{"year":1989,"finding":"NF-L and NF-M polypeptides expressed in non-neuronal fibroblasts (L cells) were efficiently assembled into intermediate filament arrays, demonstrating competence of both subunits to assemble in vivo without neuron-specific factors. NF-L-containing filaments appeared to be copolymers with vimentin, as evidenced by altered solubility of endogenous vimentin filaments.","method":"Stable DNA transfection into L cells; immunofluorescence localization; vimentin solubility co-fractionation","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct reconstitution in non-neuronal cells with two orthogonal readouts (IF and biochemical fractionation), single lab but rigorous controls","pmids":["2493000"],"is_preprint":false},{"year":1991,"finding":"Immunofluorescence of rat dorsal root ganglia identified three neuronal subpopulations defined by NF-L and peripherin expression: large NF-L-positive/peripherin-negative neurons and small peripherin-positive/NF-L-negative neurons, with a minor dual-positive population. NF-L-positive large neurons rarely contained neuropeptides, whereas peripherin-positive small neurons predominantly expressed substance P and CGRP.","method":"Double immunofluorescence with antibodies against NF-L and peripherin on rat DRG sections; neuropeptide co-localization","journal":"Journal of neuroscience research","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct localization experiment with functional context (cell-type classification), replicated across developmental ages and in culture","pmids":["1795410"],"is_preprint":false},{"year":1995,"finding":"Characterization of the rat NF-L gene promoter identified basic promoter activity between -407 and +75 bp, an NGF-responsive element between -38 and +75 bp, and a functional cAMP-responsive element between -97 and -38 bp. NGF treatment of PC12 cells stimulated NF-L promoter-driven CAT expression up to 12-fold, while EGF had no effect.","method":"Reporter gene (CAT) transfection assays with serial promoter deletion constructs in PC12 and non-neural cell lines; DNase I hypersensitivity mapping; transcription factor binding assays","journal":"Journal of neuroscience research","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro promoter dissection with deletion constructs and functional readout, multiple cell lines tested","pmids":["7745611"],"is_preprint":false},{"year":2003,"finding":"Mutations in functionally defined domains of the NEFL protein (six missense mutations and one 3-bp in-frame deletion) cause autosomal dominant Charcot-Marie-Tooth neuropathy (CMT2E/CMT1F), with nerve biopsy showing axonal pathology including axonal regeneration clusters and onion bulb formations.","method":"Mutation screening of 323 CMT patients by direct sequencing; sural nerve biopsy with histopathology","journal":"Brain : a journal of neurology","confidence":"High","confidence_rationale":"Tier 2 / Strong — large patient cohort, direct sequencing, pathological confirmation by nerve biopsy, multiple independent mutations identified","pmids":["12566280"],"is_preprint":false},{"year":2006,"finding":"14-3-3 proteins (beta, zeta, tau, gamma, and eta isoforms) bind directly to the NFL mRNA 3' UTR via two hexanucleotide motifs. Mutation of one or both motifs decreased 14-3-3 interaction, altered predicted mRNA structure, and changed mRNA stability, establishing 14-3-3 as a novel regulator of NFL mRNA stability.","method":"LC/MS/MS of NFL 3'UTR-interacting proteins from human spinal cord; in vitro expression of isoforms; gel shift assay; 3'UTR motif mutagenesis","journal":"Molecular and cellular neurosciences","confidence":"High","confidence_rationale":"Tier 1 / Moderate — biochemical reconstitution (gel shift), mass spectrometry identification, and mutagenesis of binding motifs in one study","pmids":["17098443"],"is_preprint":false},{"year":2007,"finding":"CMT2E-linked NFL mutations and CMT2F-linked HSPB1 (S135F) mutations share a common pathogenic pathway: disruption of the neurofilament network with aggregation of NFL protein leads to progressive motor neuron degeneration. Wild-type HSPB1 physically associates with NFL, can reverse CMT mutant NFL aggregates, and reduces CMT mutant NFL-induced motor neuron death. Deletion of NFL markedly reduces degeneration caused by S135F HSPB1.","method":"Motor neuron culture; co-expression of mutant and wild-type proteins; co-immunoprecipitation (HSPB1–NFL association); loss-of-function (NFL deletion); cell viability assays","journal":"Human molecular genetics","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal genetic and biochemical approach (Co-IP, loss-of-function rescue, gain-of-function), multiple orthogonal methods in single study","pmids":["17881652"],"is_preprint":false},{"year":2007,"finding":"CMT-linked mutations in MTMR2 (G103E, R283W) cause NFL aggregation in SW13vim(-) cells lacking endogenous intermediate filaments, and mutations in MTM1 also induce NFL abnormalities in these cells. MTMR2 mutants G103E and R283W are unable to form dimers and undergo phosphorylation in vivo, implicating impaired myotubularin complex formation in NFL misassembly.","method":"Transfection of SW13vim(-) cells; immunofluorescence for NFL aggregation; in vivo phosphorylation and dimerization assays for MTMR2 mutants","journal":"Journal of neurochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — cell-based assembly assay with defined cell line plus biochemical characterization, single lab","pmids":["17973976"],"is_preprint":false},{"year":2009,"finding":"TDP-43 binds NFL mRNA 3'UTR via ribonucleotide (UG) motifs on stem loops, using its RRM1 and RRM2 motifs. Ex vivo, TDP-43, 14-3-3, and SOD1 interact to modulate NFL mRNA stability. In ALS motor neurons, NFL mRNA is preferentially sequestered to stress granules (TIA-1-positive) and processing bodies (P-bodies; XRN-1-positive) that co-localize with TDP-43, whereas these compartments are more prevalent in ALS than in control neurons.","method":"Ex vivo mRNA stability assays; RNA-immunoprecipitation-PCR; immunofluorescence co-localization; TDP-43 domain mutagenesis (RRM1/RRM2)","journal":"Brain research","confidence":"High","confidence_rationale":"Tier 1 / Moderate — mutagenesis of TDP-43 RRM domains, RNA-IP-PCR, and immunofluorescence co-localization, multiple orthogonal methods in one study","pmids":["19815002"],"is_preprint":false},{"year":2009,"finding":"Phosphorylation of NFL head domain regulates neurofilament axonal transport. Mutation of three or four head domain phosphorylation sites to phosphomimetic residues inhibited axonal transport and disrupted neurofilament assembly, while precluding phosphorylation at those sites had no effect on transport.","method":"Site-directed mutagenesis of four phosphorylation sites in NFL head domain; monitoring of axonal transport of phosphorylation mutants in neurons","journal":"European journal of cell biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — mutagenesis (phosphoablation and phosphomimetic) with direct functional readout (axonal transport), systematic comparison across four sites","pmids":["19147253"],"is_preprint":false},{"year":2009,"finding":"A homozygous nonsense mutation (E210X) in NEFL causes autosomal recessive, severe early-onset axonal neuropathy. The E210X mutant protein fails to form an intermediate filament network in SW13vim(-) cells but does not interfere with filament formation by wild-type NEFL. Sural nerve biopsy from an affected patient confirmed absence of intermediate filaments in remaining myelinated axons and smaller axonal caliber, consistent with neurofilaments being the main determinant of axonal caliber.","method":"SW13vim(-) cell transfection assay for filament-forming ability; sural nerve biopsy with electron microscopy and immunohistochemistry; genetic analysis (homozygosity mapping)","journal":"Annals of neurology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro filament assembly assay plus patient nerve biopsy ultrastructural confirmation, loss-of-function mechanism established","pmids":["20039262"],"is_preprint":false},{"year":2010,"finding":"Human RGNEF (a homologue of mouse p190RhoGEF) binds NFL mRNA in vitro via gel shift assay, and this interaction is detectable in ALS tissue lysates but not in control lysates, suggesting that the RGNEF–NFL mRNA interaction is disease-state dependent.","method":"Gel shift assays (in vitro and in tissue lysates); IP-RT-PCR; RT-PCR expression analysis in ALS and control spinal cord","journal":"Amyotrophic lateral sclerosis","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — reciprocal gel shift and IP-RT-PCR, single lab, interaction in ALS lysates only","pmids":["19488899"],"is_preprint":false},{"year":2013,"finding":"NEFL mutations in different functional domains of the protein have heterogeneous effects on neurofilament assembly and chaperone susceptibility: Q333P causes reversible misfolding and forms coiled-coil dimers in vitro; it can be refolded by chaotropic agent and its aggregation in motor neurons prevented by HSPA1 induction by celastrol. P8R is sensitive to HSPB1 but not HSPA1 chaperoning, and celastrol is protective against P8R toxicity only in large sensory neurons, not motor neurons.","method":"SW13vim(-) cell transfection assay; in vitro refolding with chaotropic agents; celastrol treatment of cultured motor and sensory neurons; chaperone induction assays","journal":"The international journal of biochemistry & cell biology","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro reconstitution and mutagenesis with cell culture validation, single lab","pmids":["23618875"],"is_preprint":false},{"year":2014,"finding":"Two novel miRNAs (miR-b1336 and miR-b2403) are downregulated in ALS spinal cord and stabilize NEFL mRNA, as demonstrated by reporter gene assay and RT-PCR; inhibition with anti-miRs further confirmed their role in NEFL mRNA stability.","method":"Small RNA library sequencing from ALS and control spinal cords; real-time PCR; reporter gene (luciferase) assay; anti-miR functional assays","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — reporter assay plus anti-miR functional validation, single lab, novel miRNAs not previously characterized","pmids":["24454911"],"is_preprint":false},{"year":2015,"finding":"miR-381 directly targets NEFL (identified by 2-D DIGE, MALDI-TOF/TOF-MS/MS, and functional validation); suppressing miR-381 or enforcing NEFL expression sensitizes glioblastoma cells to temozolomide by inhibiting multidrug resistance factors (ABCG2, ABCC3, ABCC5) and stemness factors (ALDH1, CD44, SOX2, etc.). NEFL-siRNA reverses the TMZ sensitization caused by LNA-anti-miR-381.","method":"2-D DIGE and mass spectrometry protein identification; miR-381 overexpression/inhibition; NEFL overexpression and siRNA knockdown; cell viability assays; Western blot","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — proteomic identification plus functional rescue/knockdown experiments, two orthogonal manipulations (miR inhibition and NEFL siRNA)","pmids":["25605243"],"is_preprint":false},{"year":2015,"finding":"miR-25 directly targets NEFL in glioblastoma cells; miR-25 overexpression promotes and its antisense oligos inhibit cell proliferation and invasion. NEFL-siRNA attenuates the inhibitory effects of miR-25 knockdown on proliferation and invasion via the mTOR signaling pathway.","method":"miR-25 overexpression/knockdown in U251 cells; NEFL siRNA; proliferation and invasion assays; mTOR pathway analysis","journal":"Molecular and cellular biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — direct target validation with functional rescue, single lab","pmids":["26209061"],"is_preprint":false},{"year":2015,"finding":"In CMT2E patients carrying NEFL Glu396Lys mutation, skin biopsy showed markedly reduced or absent neurofilament (NF) proteins in axons by immunofluorescence and Western blot, with normal α-tubulin expression. Electron microscopy confirmed absence of NF aggregates in dermal axons and smaller axonal caliber, demonstrating that decreased NF abundance (not aggregation) correlates with reduced axonal caliber in CMT2E.","method":"Skin biopsy; indirect immunofluorescence; Western blot; electron microscopy; morphometric analysis of axonal caliber","journal":"Neurology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (IF, WB, EM) on patient tissue, direct functional correlation with axon caliber","pmids":["26109717"],"is_preprint":false},{"year":2016,"finding":"Human small heat shock protein HspB1 physically associates with NFL and modulates NFL assembly: wild-type HspB1 decreased the quantity of NFL in pellets and increased NFL in supernatant. HspB1 decreased the rate of NFL polymerization from tetramers to filaments. At saturation, ~1 mol HspB1 monomer bound per 2 mol NFL. HspB5 (αB-crystallin) also decreased NFL polymerization rate, while HspB6 and HspB8 were less effective.","method":"Differential centrifugation; analytical ultracentrifugation; fluorescent spectroscopy of NFL polymerization; binding stoichiometry determination","journal":"Cell stress & chaperones","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with multiple biochemical methods (centrifugation, AUC, fluorescence), quantitative binding stoichiometry established","pmids":["28000086"],"is_preprint":false},{"year":2018,"finding":"Patient-specific iPSC-derived motor neurons carrying a homozygous nonsense NEFL mutation showed nearly total loss of NEFL mRNA by qPCR, leading to complete absence of NEFL protein by immunocytochemistry and Western blot. Nonsense-mediated decay (NMD) caused the mRNA loss, as NMD inhibition partially rescued NEFL mRNA. Neurons still formed neurofilaments and neuronal networks in the absence of NEFL, indicating the recessive mechanism is loss-of-function.","method":"iPSC differentiation to motor neurons; qPCR; Western blot; immunocytochemistry; electron microscopy; single-cell transcriptomics; NMD inhibitor treatment","journal":"Neurology. Genetics","confidence":"High","confidence_rationale":"Tier 1 / Moderate — patient-derived neurons, multiple orthogonal methods (qPCR, WB, ICC, EM, scRNA-seq), NMD inhibitor rescue experiment","pmids":["29888333"],"is_preprint":false},{"year":2016,"finding":"miR-183 directly targets NEFL in glioblastoma cells; miR-183 overexpression promotes and its antisense oligos inhibit cell proliferation and invasion in U251 cells. NEFL-siRNA attenuates the inhibitory effects of miR-183 knockdown via the mTOR signaling pathway.","method":"miR-183 overexpression/knockdown in glioblastoma cells; NEFL siRNA; proliferation and invasion assays; mTOR pathway analysis","journal":"Cellular and molecular neurobiology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — functional target validation with rescue experiment, single lab, mTOR pathway placement by siRNA","pmids":["26879754"],"is_preprint":false},{"year":2005,"finding":"NFL-deficient mice (NFL-/-) develop transient NF aggregates in motor neurons, reactive microgliosis, progressive motor neuron loss, significant astrogliosis, and HSP-70 upregulation in motor neurons and astrocytes. In contrast, hNFL(+/+) overexpression mice showed NF aggregates and microgliosis but no significant astrogliosis. This indicates that the etiology of NF aggregate formation determines the glial response pattern.","method":"Longitudinal histological analysis of transgenic mice (1–18 months); immunohistochemistry for microglia, astrocytes, and HSP-70; motor neuron counting","journal":"Glia","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo loss-of-function (NFL-/-) and gain-of-function (hNFL+/+) mouse models with longitudinal histology, two orthogonal genetic models","pmids":["15920739"],"is_preprint":false},{"year":2022,"finding":"MiR-30b-5p directly targets NEFL mRNA (confirmed by dual-luciferase reporter gene assay), negatively regulating its mRNA and protein levels. NEFL activates mTOR signaling. In SCI cell and rat models, miR-30b-5p inactivates mTOR signaling by negatively regulating NEFL, thereby attenuating the inflammatory response and facilitating functional recovery.","method":"Dual-luciferase reporter assay; RT-qPCR and Western blot; NEFL silencing in LPS-stimulated PC12 cells; SCI rat model; mTOR pathway analysis","journal":"Brain and behavior","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — luciferase reporter validates direct targeting, in vivo rat model with mTOR pathway placement, single lab","pmids":["36282532"],"is_preprint":false},{"year":1987,"finding":"The human neurofilament light chain gene (NEFL) was localized to chromosome band 8p21 by Southern blotting of hybrid cell panels and in situ hybridization to metaphase chromosomes.","method":"Southern blotting of somatic cell hybrid panels; in situ hybridization to metaphase chromosomes","journal":"Cytogenetics and cell genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — two orthogonal methods (Southern blot and in situ hybridization), replicated by a second study (PMID 3145240)","pmids":["3036423","3145240"],"is_preprint":false},{"year":2021,"finding":"SNAIL regulates NEFL expression in gastric cancer: microarray analysis after SNAIL knockdown in HGC27 cells identified NEFL as one of the most downregulated genes. Upregulation of NEFL or downregulation of CCN3 impaired SNAIL-dependent EMT activity (tumorigenicity, chemoresistance), placing NEFL in the SNAIL/CCN3/NEFL axis governing gastric cancer stemness.","method":"Microarray analysis; SNAIL knockdown; NEFL modulation; tumorigenicity and chemoresistance assays in gastric cancer cell lines and gastric oncospheres","journal":"Carcinogenesis","confidence":"Low","confidence_rationale":"Tier 3 / Weak — microarray-based identification with functional assays, pathway placement inferred rather than mechanistically dissected, single lab","pmids":["33313663"],"is_preprint":false}],"current_model":"NEFL encodes the light subunit of neuronal neurofilaments, an obligate component of the hetero-polymeric intermediate filament network in axons: it co-assembles with NF-M and NF-H (and can copolymerize with vimentin in non-neuronal cells) to form filaments whose abundance determines axonal caliber; head-domain phosphorylation of NFL inhibits both neurofilament assembly and axonal transport; NFL mRNA stability is post-transcriptionally regulated by a complex of RNA-binding proteins including TDP-43 (via UG-motif stem loops and its RRM domains), 14-3-3 isoforms, SOD1, and RGNEF, with pathological redistribution of NFL mRNA into stress granules and P-bodies occurring in ALS; small heat shock proteins HspB1 and HspB5 bind NFL and retard its polymerization from tetramers to filaments; dominant missense mutations in NEFL cause CMT neuropathy by disrupting neurofilament assembly and inducing NFL aggregation (a shared mechanism with HSPB1 mutations), while homozygous nonsense mutations cause a recessive loss-of-function neuropathy through nonsense-mediated mRNA decay and absence of NEFL protein; and in cancer contexts NEFL acts as a tumor suppressor whose expression is silenced by oncomiRs (miR-381, miR-25, miR-183) that place it upstream of mTOR signaling."},"narrative":{"mechanistic_narrative":"NEFL encodes the light subunit of neuronal neurofilaments, an intermediate filament protein that assembles into the axonal cytoskeleton and is the principal determinant of axonal caliber [PMID:2493000, PMID:20039262]. The NF-L subunit is intrinsically competent to polymerize into intermediate filament arrays, copolymerizing with vimentin in non-neuronal cells and forming filaments without requiring neuron-specific factors [PMID:2493000]; it marks a discrete population of large-caliber neurons distinct from peripherin-expressing small neurons [PMID:1795410]. Assembly and axonal transport of neurofilaments are negatively regulated by phosphorylation of the NF-L head domain, where phosphomimetic substitutions inhibit both transport and filament formation [PMID:19147253], and by small heat-shock proteins HspB1 and HspB5, which bind NF-L and retard its polymerization from tetramers to filaments [PMID:28000086]. NEFL mRNA abundance is governed post-transcriptionally by a 3'UTR-binding regulatory network: 14-3-3 isoforms bind hexanucleotide motifs, while TDP-43 engages UG-motif stem loops through its RRM1/RRM2 domains and acts together with 14-3-3 and SOD1 to modulate transcript stability; in ALS motor neurons this mRNA is pathologically sequestered into TDP-43-positive stress granules and P-bodies [PMID:17098443, PMID:19815002]. Dominant missense mutations in NEFL cause Charcot-Marie-Tooth neuropathy (CMT2E/CMT1F) by disrupting the neurofilament network and inducing NF-L aggregation, a mechanism shared with HSPB1 mutations and reversible by chaperone activity [PMID:12566280, PMID:17881652], whereas homozygous nonsense mutations cause a recessive loss-of-function neuropathy via nonsense-mediated decay and complete absence of NF-L protein [PMID:20039262, PMID:29888333]. In cancer, NEFL behaves as a tumor suppressor silenced by oncogenic miRNAs (miR-381, miR-25, miR-183) and positioned upstream of mTOR signaling [PMID:25605243, PMID:26209061, PMID:26879754].","teleology":[{"year":1989,"claim":"Established that NF-L is an autonomous intermediate filament-forming subunit, answering whether neuron-specific machinery is required for neurofilament assembly.","evidence":"Stable transfection of NF-L into non-neuronal L cells with immunofluorescence and vimentin co-fractionation","pmids":["2493000"],"confidence":"High","gaps":["Does not address obligate heteropolymer requirements with NF-M/NF-H in neurons","Vimentin copolymerization is a non-neuronal context"]},{"year":1991,"claim":"Defined NF-L as a marker distinguishing large-caliber neurons from peripherin-positive small neurons, linking subunit expression to neuronal cell identity.","evidence":"Double immunofluorescence on rat DRG with neuropeptide co-localization","pmids":["1795410"],"confidence":"High","gaps":["Correlative classification, not mechanistic","Does not explain caliber control"]},{"year":1995,"claim":"Mapped transcriptional control elements of the NF-L gene, showing NGF- and cAMP-responsive regulation of its promoter.","evidence":"Serial promoter deletion CAT reporter assays in PC12 cells; DNase I hypersensitivity mapping","pmids":["7745611"],"confidence":"High","gaps":["Identifies responsive regions but not all bound transcription factors","Rat promoter; human conservation not addressed"]},{"year":2003,"claim":"Identified NEFL as a CMT disease gene, establishing that dominant mutations cause axonal neuropathy.","evidence":"Mutation screening of 323 CMT patients by direct sequencing with sural nerve biopsy","pmids":["12566280"],"confidence":"High","gaps":["Genotype identified but molecular consequences of each mutation not dissected"]},{"year":2006,"claim":"Revealed post-transcriptional control of NFL mRNA stability by 14-3-3 proteins binding the 3'UTR, opening a regulatory layer beyond transcription.","evidence":"LC/MS/MS of 3'UTR-interacting proteins, gel shift, and motif mutagenesis from human spinal cord","pmids":["17098443"],"confidence":"High","gaps":["In vitro binding; physiological stability changes not quantified in neurons","Functional consequence of each isoform unresolved"]},{"year":2007,"claim":"Demonstrated a shared pathogenic pathway between NFL and HSPB1 CMT mutations centered on NFL aggregation, and a chaperone role for HspB1.","evidence":"Motor neuron culture, co-expression, Co-IP, NFL deletion rescue, and viability assays","pmids":["17881652"],"confidence":"High","gaps":["Mechanism of HspB1-mediated disaggregation not resolved at structural level"]},{"year":2007,"claim":"Extended mutational converging on NFL misassembly to MTMR2/MTM1, linking myotubularin dimerization defects to neurofilament abnormalities.","evidence":"SW13vim(-) transfection assembly assay with MTMR2 dimerization and phosphorylation analysis","pmids":["17973976"],"confidence":"Medium","gaps":["Cell-line based, single lab","Direct biochemical link between myotubularin and NFL not shown"]},{"year":2009,"claim":"Showed head-domain phosphorylation governs neurofilament assembly and axonal transport, defining a post-translational switch.","evidence":"Phosphoablation and phosphomimetic mutagenesis of four head-domain sites with axonal transport readout","pmids":["19147253"],"confidence":"High","gaps":["Responsible kinases not identified in this corpus","In vivo physiological relevance not established"]},{"year":2009,"claim":"Defined the TDP-43/14-3-3/SOD1 mRNA-stability network and its pathological redistribution into stress granules and P-bodies in ALS.","evidence":"Ex vivo stability assays, RNA-IP-PCR, TDP-43 RRM mutagenesis, and immunofluorescence co-localization","pmids":["19815002"],"confidence":"High","gaps":["Causal direction between mRNA sequestration and neurodegeneration unresolved"]},{"year":2009,"claim":"Established a recessive loss-of-function mechanism via a homozygous nonsense mutation, confirming neurofilaments as the main determinant of axonal caliber.","evidence":"SW13vim(-) filament assays, sural nerve biopsy EM, and homozygosity mapping","pmids":["20039262"],"confidence":"High","gaps":["mRNA fate of the nonsense allele not yet defined at this stage"]},{"year":2010,"claim":"Added RGNEF as a disease-state-dependent NFL mRNA-binding protein detectable in ALS but not control tissue.","evidence":"Gel shift assays in vitro and in tissue lysates with IP-RT-PCR","pmids":["19488899"],"confidence":"Medium","gaps":["Functional effect on mRNA stability not measured","Single lab"]},{"year":2013,"claim":"Showed CMT NEFL mutations differ in misfolding behavior and chaperone susceptibility, refining therapeutic chaperone strategies.","evidence":"SW13vim(-) assays, in vitro refolding, and celastrol/chaperone induction in motor and sensory neurons","pmids":["23618875"],"confidence":"Medium","gaps":["Single lab","Cell-type-specific chaperone responses mechanistically unexplained"]},{"year":2014,"claim":"Identified ALS-downregulated miRNAs that stabilize NEFL mRNA, expanding the mRNA-stability regulatory layer.","evidence":"Small RNA sequencing, luciferase reporter, and anti-miR functional assays","pmids":["24454911"],"confidence":"Medium","gaps":["Novel miRNAs uncharacterized beyond this study","Single lab"]},{"year":2015,"claim":"Defined a tumor-suppressor role for NEFL silenced by miR-381, linking its loss to chemoresistance and stemness in glioblastoma.","evidence":"2-D DIGE/MS identification with miR-381 and NEFL gain/loss-of-function and TMZ viability assays","pmids":["25605243"],"confidence":"Medium","gaps":["Mechanism by which NEFL suppresses resistance factors not resolved"]},{"year":2015,"claim":"Placed NEFL upstream of mTOR signaling via miR-25, connecting its loss to glioblastoma proliferation and invasion.","evidence":"miR-25 gain/loss and NEFL siRNA rescue with mTOR pathway analysis in U251 cells","pmids":["26209061"],"confidence":"Medium","gaps":["Molecular link between NEFL and mTOR not biochemically defined","Single lab"]},{"year":2015,"claim":"Demonstrated in CMT2E patient tissue that reduced neurofilament abundance, not aggregation, correlates with reduced axonal caliber.","evidence":"Skin biopsy IF, Western blot, EM, and axonal caliber morphometry","pmids":["26109717"],"confidence":"High","gaps":["Single mutation (E396K); generalizability across CMT alleles not shown"]},{"year":2016,"claim":"Quantified the biochemical chaperone interaction, showing HspB1 and HspB5 bind NFL and slow tetramer-to-filament polymerization.","evidence":"Differential centrifugation, analytical ultracentrifugation, and fluorescent polymerization assays with stoichiometry","pmids":["28000086"],"confidence":"High","gaps":["In vitro reconstitution; in-cell relevance of stoichiometry not addressed"]},{"year":2016,"claim":"Added miR-183 to the oncomiR set repressing NEFL upstream of mTOR in glioblastoma.","evidence":"miR-183 gain/loss and NEFL siRNA rescue with mTOR analysis in U251 cells","pmids":["26879754"],"confidence":"Medium","gaps":["Direct NEFL-mTOR molecular link unresolved","Single lab"]},{"year":2018,"claim":"Established that the recessive nonsense allele triggers nonsense-mediated decay, proving a true loss-of-function mechanism in patient-derived neurons.","evidence":"iPSC-derived motor neurons, qPCR, Western blot, ICC, EM, scRNA-seq, and NMD inhibitor rescue","pmids":["29888333"],"confidence":"High","gaps":["Why neurons still form networks without NF-L not mechanistically explained"]},{"year":2022,"claim":"Extended the NEFL-mTOR axis to spinal cord injury, with miR-30b-5p repression of NEFL modulating inflammation and recovery.","evidence":"Dual-luciferase reporter, NEFL silencing in PC12 cells, and SCI rat model with mTOR analysis","pmids":["36282532"],"confidence":"Medium","gaps":["Single lab","NEFL-mTOR coupling mechanism still undefined"]},{"year":null,"claim":"The molecular mechanism by which cytoplasmic NEFL activates mTOR signaling and acts as a tumor suppressor remains undefined.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No biochemical link between NF-L and mTOR pathway components identified","Kinases controlling head-domain phosphorylation not identified in this corpus","Structural basis of chaperone-mediated assembly control unresolved"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[0,9,15]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[0]}],"localization":[{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[0,9]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[16]}],"pathway":[{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[3,5,9,17]},{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[4,7]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[14,18,20]}],"complexes":["neurofilament"],"partners":["NEFM","VIM","HSPB1","HSPB5","TARDBP","SOD1","YWHAZ","RGNEF"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P07196","full_name":"Neurofilament light polypeptide","aliases":["68 kDa neurofilament protein","Neurofilament triplet L protein"],"length_aa":543,"mass_kda":61.5,"function":"Neurofilaments usually contain three intermediate filament proteins: NEFL, NEFM, and NEFH which are involved in the maintenance of neuronal caliber. 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neurochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/17973976","citation_count":19,"is_preprint":false},{"pmid":"40551285","id":"PMC_40551285","title":"Diagnostic performance of plasma Aβ42/40 ratio, p-tau181, GFAP, and NfL along the continuum of Alzheimer's disease and non-AD dementias: An international multi-center study.","date":"2025","source":"Alzheimer's & dementia : the journal of the Alzheimer's Association","url":"https://pubmed.ncbi.nlm.nih.gov/40551285","citation_count":17,"is_preprint":false},{"pmid":"35273370","id":"PMC_35273370","title":"Combined GFAP, NFL, Tau, and UCH-L1 panel increases prediction of outcomes in neonatal encephalopathy.","date":"2022","source":"Pediatric research","url":"https://pubmed.ncbi.nlm.nih.gov/35273370","citation_count":17,"is_preprint":false},{"pmid":"21168446","id":"PMC_21168446","title":"Sensorimotor and cognitive function of a NEFL(P22S) mutant model of Charcot-Marie-Tooth disease type 2E.","date":"2010","source":"Behavioural brain 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psychiatry","url":"https://pubmed.ncbi.nlm.nih.gov/33373836","citation_count":16,"is_preprint":false},{"pmid":"33313663","id":"PMC_33313663","title":"SNAIL regulates gastric carcinogenesis through CCN3 and NEFL.","date":"2021","source":"Carcinogenesis","url":"https://pubmed.ncbi.nlm.nih.gov/33313663","citation_count":15,"is_preprint":false},{"pmid":"35041115","id":"PMC_35041115","title":"Serum NfL associated with anti-NMDA receptor encephalitis.","date":"2022","source":"Neurological sciences : official journal of the Italian Neurological Society and of the Italian Society of Clinical Neurophysiology","url":"https://pubmed.ncbi.nlm.nih.gov/35041115","citation_count":15,"is_preprint":false},{"pmid":"33851572","id":"PMC_33851572","title":"Elevated sTREM2 and NFL levels in patients with sepsis associated encephalopathy.","date":"2021","source":"The International journal of neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/33851572","citation_count":15,"is_preprint":false},{"pmid":"40009787","id":"PMC_40009787","title":"Comparative Performances of 4 Serum NfL Assays, pTau181, and GFAP in Patients With Amyotrophic Lateral Sclerosis.","date":"2025","source":"Neurology","url":"https://pubmed.ncbi.nlm.nih.gov/40009787","citation_count":15,"is_preprint":false},{"pmid":"35236877","id":"PMC_35236877","title":"Salivary S100 calcium-binding protein beta (S100B) and neurofilament light (NfL) after acute exposure to repeated head impacts in collegiate water polo players.","date":"2022","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/35236877","citation_count":15,"is_preprint":false},{"pmid":"27177578","id":"PMC_27177578","title":"The Neurofilament-Derived Peptide NFL-TBS.40-63 Targets Neural Stem Cells and Affects Their Properties.","date":"2016","source":"Stem cells translational medicine","url":"https://pubmed.ncbi.nlm.nih.gov/27177578","citation_count":15,"is_preprint":false},{"pmid":"39866832","id":"PMC_39866832","title":"The role of Neurofilament light (NfL) and glial fibrillary acidic protein (GFAP) in MS and AQP4-NMOSD: Advancing clinical applications.","date":"2025","source":"eNeurologicalSci","url":"https://pubmed.ncbi.nlm.nih.gov/39866832","citation_count":14,"is_preprint":false},{"pmid":"40566826","id":"PMC_40566826","title":"Plasma NfL and GFAP for predicting VCI and related brain changes in community and clinical cohorts.","date":"2025","source":"Alzheimer's & dementia : the journal of the Alzheimer's Association","url":"https://pubmed.ncbi.nlm.nih.gov/40566826","citation_count":13,"is_preprint":false},{"pmid":"36301211","id":"PMC_36301211","title":"Plasma tau, NfL, GFAP and UCHL1 as candidate biomarkers of alcohol withdrawal-associated brain damage: A pilot study.","date":"2022","source":"Addiction biology","url":"https://pubmed.ncbi.nlm.nih.gov/36301211","citation_count":13,"is_preprint":false},{"pmid":"19286384","id":"PMC_19286384","title":"The neurofilament light chain gene (NEFL) mutation Pro22Ser can be associated with mixed axonal and demyelinating neuropathy.","date":"2009","source":"Journal of clinical neuroscience : official journal of the Neurosurgical Society of Australasia","url":"https://pubmed.ncbi.nlm.nih.gov/19286384","citation_count":13,"is_preprint":false},{"pmid":"32477099","id":"PMC_32477099","title":"Increased CSF NFL in Non-demented Parkinson's Disease Subjects Reflects Early White Matter Damage.","date":"2020","source":"Frontiers in aging neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/32477099","citation_count":13,"is_preprint":false},{"pmid":"35044100","id":"PMC_35044100","title":"Phenotypic heterogeneity in patients with NEFL-related Charcot-Marie-Tooth disease.","date":"2022","source":"Molecular genetics & genomic 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Effects on regional brain metabolism.","date":"2005","source":"Journal of neuroscience research","url":"https://pubmed.ncbi.nlm.nih.gov/15742362","citation_count":12,"is_preprint":false},{"pmid":"37418931","id":"PMC_37418931","title":"NfL and GFAP in serum are associated with microstructural brain damage in progressive multiple sclerosis.","date":"2023","source":"Multiple sclerosis and related disorders","url":"https://pubmed.ncbi.nlm.nih.gov/37418931","citation_count":11,"is_preprint":false},{"pmid":"36282532","id":"PMC_36282532","title":"MiR-30b-5p attenuates the inflammatory response and facilitates the functional recovery of spinal cord injury by targeting the NEFL/mTOR pathway.","date":"2022","source":"Brain and behavior","url":"https://pubmed.ncbi.nlm.nih.gov/36282532","citation_count":11,"is_preprint":false},{"pmid":"38292036","id":"PMC_38292036","title":"Serum GFAP, NfL, and tau concentrations are associated with worse neurobehavioral functioning following mild, moderate, and severe TBI: a cross-sectional multiple-cohort study.","date":"2024","source":"Frontiers in neurology","url":"https://pubmed.ncbi.nlm.nih.gov/38292036","citation_count":11,"is_preprint":false},{"pmid":"39048548","id":"PMC_39048548","title":"An exploratory study of the damage markers NfL, GFAP, and t-Tau, in cerebrospinal fluid and other findings from a patient cohort enriched for suspected autoimmune psychiatric disease.","date":"2024","source":"Translational psychiatry","url":"https://pubmed.ncbi.nlm.nih.gov/39048548","citation_count":11,"is_preprint":false},{"pmid":"35287127","id":"PMC_35287127","title":"Plasma Cystatin C Correlates with Plasma NfL Levels and Predicts Disease Progression in Parkinson's Disease.","date":"2022","source":"Neuro-degenerative diseases","url":"https://pubmed.ncbi.nlm.nih.gov/35287127","citation_count":11,"is_preprint":false},{"pmid":"38578900","id":"PMC_38578900","title":"Novel Genetic and Biochemical Insights into the Spectrum of NEFL-Associated Phenotypes.","date":"2024","source":"Journal of neuromuscular diseases","url":"https://pubmed.ncbi.nlm.nih.gov/38578900","citation_count":10,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":58432,"output_tokens":6184,"usd":0.134028,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":15021,"output_tokens":4990,"usd":0.099928,"stage2_stop_reason":"end_turn"},"total_usd":0.233956,"stage1_batch_id":"msgbatch_013a8chtv3AWyTqv4z4sEF4C","stage2_batch_id":"msgbatch_01AHsuW63vinsvuTgzmQ3n62","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1989,\n      \"finding\": \"NF-L and NF-M polypeptides expressed in non-neuronal fibroblasts (L cells) were efficiently assembled into intermediate filament arrays, demonstrating competence of both subunits to assemble in vivo without neuron-specific factors. NF-L-containing filaments appeared to be copolymers with vimentin, as evidenced by altered solubility of endogenous vimentin filaments.\",\n      \"method\": \"Stable DNA transfection into L cells; immunofluorescence localization; vimentin solubility co-fractionation\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct reconstitution in non-neuronal cells with two orthogonal readouts (IF and biochemical fractionation), single lab but rigorous controls\",\n      \"pmids\": [\"2493000\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1991,\n      \"finding\": \"Immunofluorescence of rat dorsal root ganglia identified three neuronal subpopulations defined by NF-L and peripherin expression: large NF-L-positive/peripherin-negative neurons and small peripherin-positive/NF-L-negative neurons, with a minor dual-positive population. NF-L-positive large neurons rarely contained neuropeptides, whereas peripherin-positive small neurons predominantly expressed substance P and CGRP.\",\n      \"method\": \"Double immunofluorescence with antibodies against NF-L and peripherin on rat DRG sections; neuropeptide co-localization\",\n      \"journal\": \"Journal of neuroscience research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct localization experiment with functional context (cell-type classification), replicated across developmental ages and in culture\",\n      \"pmids\": [\"1795410\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"Characterization of the rat NF-L gene promoter identified basic promoter activity between -407 and +75 bp, an NGF-responsive element between -38 and +75 bp, and a functional cAMP-responsive element between -97 and -38 bp. NGF treatment of PC12 cells stimulated NF-L promoter-driven CAT expression up to 12-fold, while EGF had no effect.\",\n      \"method\": \"Reporter gene (CAT) transfection assays with serial promoter deletion constructs in PC12 and non-neural cell lines; DNase I hypersensitivity mapping; transcription factor binding assays\",\n      \"journal\": \"Journal of neuroscience research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro promoter dissection with deletion constructs and functional readout, multiple cell lines tested\",\n      \"pmids\": [\"7745611\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Mutations in functionally defined domains of the NEFL protein (six missense mutations and one 3-bp in-frame deletion) cause autosomal dominant Charcot-Marie-Tooth neuropathy (CMT2E/CMT1F), with nerve biopsy showing axonal pathology including axonal regeneration clusters and onion bulb formations.\",\n      \"method\": \"Mutation screening of 323 CMT patients by direct sequencing; sural nerve biopsy with histopathology\",\n      \"journal\": \"Brain : a journal of neurology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — large patient cohort, direct sequencing, pathological confirmation by nerve biopsy, multiple independent mutations identified\",\n      \"pmids\": [\"12566280\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"14-3-3 proteins (beta, zeta, tau, gamma, and eta isoforms) bind directly to the NFL mRNA 3' UTR via two hexanucleotide motifs. Mutation of one or both motifs decreased 14-3-3 interaction, altered predicted mRNA structure, and changed mRNA stability, establishing 14-3-3 as a novel regulator of NFL mRNA stability.\",\n      \"method\": \"LC/MS/MS of NFL 3'UTR-interacting proteins from human spinal cord; in vitro expression of isoforms; gel shift assay; 3'UTR motif mutagenesis\",\n      \"journal\": \"Molecular and cellular neurosciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — biochemical reconstitution (gel shift), mass spectrometry identification, and mutagenesis of binding motifs in one study\",\n      \"pmids\": [\"17098443\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"CMT2E-linked NFL mutations and CMT2F-linked HSPB1 (S135F) mutations share a common pathogenic pathway: disruption of the neurofilament network with aggregation of NFL protein leads to progressive motor neuron degeneration. Wild-type HSPB1 physically associates with NFL, can reverse CMT mutant NFL aggregates, and reduces CMT mutant NFL-induced motor neuron death. Deletion of NFL markedly reduces degeneration caused by S135F HSPB1.\",\n      \"method\": \"Motor neuron culture; co-expression of mutant and wild-type proteins; co-immunoprecipitation (HSPB1–NFL association); loss-of-function (NFL deletion); cell viability assays\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal genetic and biochemical approach (Co-IP, loss-of-function rescue, gain-of-function), multiple orthogonal methods in single study\",\n      \"pmids\": [\"17881652\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"CMT-linked mutations in MTMR2 (G103E, R283W) cause NFL aggregation in SW13vim(-) cells lacking endogenous intermediate filaments, and mutations in MTM1 also induce NFL abnormalities in these cells. MTMR2 mutants G103E and R283W are unable to form dimers and undergo phosphorylation in vivo, implicating impaired myotubularin complex formation in NFL misassembly.\",\n      \"method\": \"Transfection of SW13vim(-) cells; immunofluorescence for NFL aggregation; in vivo phosphorylation and dimerization assays for MTMR2 mutants\",\n      \"journal\": \"Journal of neurochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — cell-based assembly assay with defined cell line plus biochemical characterization, single lab\",\n      \"pmids\": [\"17973976\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"TDP-43 binds NFL mRNA 3'UTR via ribonucleotide (UG) motifs on stem loops, using its RRM1 and RRM2 motifs. Ex vivo, TDP-43, 14-3-3, and SOD1 interact to modulate NFL mRNA stability. In ALS motor neurons, NFL mRNA is preferentially sequestered to stress granules (TIA-1-positive) and processing bodies (P-bodies; XRN-1-positive) that co-localize with TDP-43, whereas these compartments are more prevalent in ALS than in control neurons.\",\n      \"method\": \"Ex vivo mRNA stability assays; RNA-immunoprecipitation-PCR; immunofluorescence co-localization; TDP-43 domain mutagenesis (RRM1/RRM2)\",\n      \"journal\": \"Brain research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — mutagenesis of TDP-43 RRM domains, RNA-IP-PCR, and immunofluorescence co-localization, multiple orthogonal methods in one study\",\n      \"pmids\": [\"19815002\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Phosphorylation of NFL head domain regulates neurofilament axonal transport. Mutation of three or four head domain phosphorylation sites to phosphomimetic residues inhibited axonal transport and disrupted neurofilament assembly, while precluding phosphorylation at those sites had no effect on transport.\",\n      \"method\": \"Site-directed mutagenesis of four phosphorylation sites in NFL head domain; monitoring of axonal transport of phosphorylation mutants in neurons\",\n      \"journal\": \"European journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — mutagenesis (phosphoablation and phosphomimetic) with direct functional readout (axonal transport), systematic comparison across four sites\",\n      \"pmids\": [\"19147253\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"A homozygous nonsense mutation (E210X) in NEFL causes autosomal recessive, severe early-onset axonal neuropathy. The E210X mutant protein fails to form an intermediate filament network in SW13vim(-) cells but does not interfere with filament formation by wild-type NEFL. Sural nerve biopsy from an affected patient confirmed absence of intermediate filaments in remaining myelinated axons and smaller axonal caliber, consistent with neurofilaments being the main determinant of axonal caliber.\",\n      \"method\": \"SW13vim(-) cell transfection assay for filament-forming ability; sural nerve biopsy with electron microscopy and immunohistochemistry; genetic analysis (homozygosity mapping)\",\n      \"journal\": \"Annals of neurology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro filament assembly assay plus patient nerve biopsy ultrastructural confirmation, loss-of-function mechanism established\",\n      \"pmids\": [\"20039262\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Human RGNEF (a homologue of mouse p190RhoGEF) binds NFL mRNA in vitro via gel shift assay, and this interaction is detectable in ALS tissue lysates but not in control lysates, suggesting that the RGNEF–NFL mRNA interaction is disease-state dependent.\",\n      \"method\": \"Gel shift assays (in vitro and in tissue lysates); IP-RT-PCR; RT-PCR expression analysis in ALS and control spinal cord\",\n      \"journal\": \"Amyotrophic lateral sclerosis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — reciprocal gel shift and IP-RT-PCR, single lab, interaction in ALS lysates only\",\n      \"pmids\": [\"19488899\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"NEFL mutations in different functional domains of the protein have heterogeneous effects on neurofilament assembly and chaperone susceptibility: Q333P causes reversible misfolding and forms coiled-coil dimers in vitro; it can be refolded by chaotropic agent and its aggregation in motor neurons prevented by HSPA1 induction by celastrol. P8R is sensitive to HSPB1 but not HSPA1 chaperoning, and celastrol is protective against P8R toxicity only in large sensory neurons, not motor neurons.\",\n      \"method\": \"SW13vim(-) cell transfection assay; in vitro refolding with chaotropic agents; celastrol treatment of cultured motor and sensory neurons; chaperone induction assays\",\n      \"journal\": \"The international journal of biochemistry & cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro reconstitution and mutagenesis with cell culture validation, single lab\",\n      \"pmids\": [\"23618875\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Two novel miRNAs (miR-b1336 and miR-b2403) are downregulated in ALS spinal cord and stabilize NEFL mRNA, as demonstrated by reporter gene assay and RT-PCR; inhibition with anti-miRs further confirmed their role in NEFL mRNA stability.\",\n      \"method\": \"Small RNA library sequencing from ALS and control spinal cords; real-time PCR; reporter gene (luciferase) assay; anti-miR functional assays\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — reporter assay plus anti-miR functional validation, single lab, novel miRNAs not previously characterized\",\n      \"pmids\": [\"24454911\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"miR-381 directly targets NEFL (identified by 2-D DIGE, MALDI-TOF/TOF-MS/MS, and functional validation); suppressing miR-381 or enforcing NEFL expression sensitizes glioblastoma cells to temozolomide by inhibiting multidrug resistance factors (ABCG2, ABCC3, ABCC5) and stemness factors (ALDH1, CD44, SOX2, etc.). NEFL-siRNA reverses the TMZ sensitization caused by LNA-anti-miR-381.\",\n      \"method\": \"2-D DIGE and mass spectrometry protein identification; miR-381 overexpression/inhibition; NEFL overexpression and siRNA knockdown; cell viability assays; Western blot\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — proteomic identification plus functional rescue/knockdown experiments, two orthogonal manipulations (miR inhibition and NEFL siRNA)\",\n      \"pmids\": [\"25605243\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"miR-25 directly targets NEFL in glioblastoma cells; miR-25 overexpression promotes and its antisense oligos inhibit cell proliferation and invasion. NEFL-siRNA attenuates the inhibitory effects of miR-25 knockdown on proliferation and invasion via the mTOR signaling pathway.\",\n      \"method\": \"miR-25 overexpression/knockdown in U251 cells; NEFL siRNA; proliferation and invasion assays; mTOR pathway analysis\",\n      \"journal\": \"Molecular and cellular biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — direct target validation with functional rescue, single lab\",\n      \"pmids\": [\"26209061\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"In CMT2E patients carrying NEFL Glu396Lys mutation, skin biopsy showed markedly reduced or absent neurofilament (NF) proteins in axons by immunofluorescence and Western blot, with normal α-tubulin expression. Electron microscopy confirmed absence of NF aggregates in dermal axons and smaller axonal caliber, demonstrating that decreased NF abundance (not aggregation) correlates with reduced axonal caliber in CMT2E.\",\n      \"method\": \"Skin biopsy; indirect immunofluorescence; Western blot; electron microscopy; morphometric analysis of axonal caliber\",\n      \"journal\": \"Neurology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (IF, WB, EM) on patient tissue, direct functional correlation with axon caliber\",\n      \"pmids\": [\"26109717\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Human small heat shock protein HspB1 physically associates with NFL and modulates NFL assembly: wild-type HspB1 decreased the quantity of NFL in pellets and increased NFL in supernatant. HspB1 decreased the rate of NFL polymerization from tetramers to filaments. At saturation, ~1 mol HspB1 monomer bound per 2 mol NFL. HspB5 (αB-crystallin) also decreased NFL polymerization rate, while HspB6 and HspB8 were less effective.\",\n      \"method\": \"Differential centrifugation; analytical ultracentrifugation; fluorescent spectroscopy of NFL polymerization; binding stoichiometry determination\",\n      \"journal\": \"Cell stress & chaperones\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with multiple biochemical methods (centrifugation, AUC, fluorescence), quantitative binding stoichiometry established\",\n      \"pmids\": [\"28000086\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Patient-specific iPSC-derived motor neurons carrying a homozygous nonsense NEFL mutation showed nearly total loss of NEFL mRNA by qPCR, leading to complete absence of NEFL protein by immunocytochemistry and Western blot. Nonsense-mediated decay (NMD) caused the mRNA loss, as NMD inhibition partially rescued NEFL mRNA. Neurons still formed neurofilaments and neuronal networks in the absence of NEFL, indicating the recessive mechanism is loss-of-function.\",\n      \"method\": \"iPSC differentiation to motor neurons; qPCR; Western blot; immunocytochemistry; electron microscopy; single-cell transcriptomics; NMD inhibitor treatment\",\n      \"journal\": \"Neurology. Genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — patient-derived neurons, multiple orthogonal methods (qPCR, WB, ICC, EM, scRNA-seq), NMD inhibitor rescue experiment\",\n      \"pmids\": [\"29888333\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"miR-183 directly targets NEFL in glioblastoma cells; miR-183 overexpression promotes and its antisense oligos inhibit cell proliferation and invasion in U251 cells. NEFL-siRNA attenuates the inhibitory effects of miR-183 knockdown via the mTOR signaling pathway.\",\n      \"method\": \"miR-183 overexpression/knockdown in glioblastoma cells; NEFL siRNA; proliferation and invasion assays; mTOR pathway analysis\",\n      \"journal\": \"Cellular and molecular neurobiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — functional target validation with rescue experiment, single lab, mTOR pathway placement by siRNA\",\n      \"pmids\": [\"26879754\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"NFL-deficient mice (NFL-/-) develop transient NF aggregates in motor neurons, reactive microgliosis, progressive motor neuron loss, significant astrogliosis, and HSP-70 upregulation in motor neurons and astrocytes. In contrast, hNFL(+/+) overexpression mice showed NF aggregates and microgliosis but no significant astrogliosis. This indicates that the etiology of NF aggregate formation determines the glial response pattern.\",\n      \"method\": \"Longitudinal histological analysis of transgenic mice (1–18 months); immunohistochemistry for microglia, astrocytes, and HSP-70; motor neuron counting\",\n      \"journal\": \"Glia\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo loss-of-function (NFL-/-) and gain-of-function (hNFL+/+) mouse models with longitudinal histology, two orthogonal genetic models\",\n      \"pmids\": [\"15920739\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"MiR-30b-5p directly targets NEFL mRNA (confirmed by dual-luciferase reporter gene assay), negatively regulating its mRNA and protein levels. NEFL activates mTOR signaling. In SCI cell and rat models, miR-30b-5p inactivates mTOR signaling by negatively regulating NEFL, thereby attenuating the inflammatory response and facilitating functional recovery.\",\n      \"method\": \"Dual-luciferase reporter assay; RT-qPCR and Western blot; NEFL silencing in LPS-stimulated PC12 cells; SCI rat model; mTOR pathway analysis\",\n      \"journal\": \"Brain and behavior\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — luciferase reporter validates direct targeting, in vivo rat model with mTOR pathway placement, single lab\",\n      \"pmids\": [\"36282532\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1987,\n      \"finding\": \"The human neurofilament light chain gene (NEFL) was localized to chromosome band 8p21 by Southern blotting of hybrid cell panels and in situ hybridization to metaphase chromosomes.\",\n      \"method\": \"Southern blotting of somatic cell hybrid panels; in situ hybridization to metaphase chromosomes\",\n      \"journal\": \"Cytogenetics and cell genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — two orthogonal methods (Southern blot and in situ hybridization), replicated by a second study (PMID 3145240)\",\n      \"pmids\": [\"3036423\", \"3145240\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"SNAIL regulates NEFL expression in gastric cancer: microarray analysis after SNAIL knockdown in HGC27 cells identified NEFL as one of the most downregulated genes. Upregulation of NEFL or downregulation of CCN3 impaired SNAIL-dependent EMT activity (tumorigenicity, chemoresistance), placing NEFL in the SNAIL/CCN3/NEFL axis governing gastric cancer stemness.\",\n      \"method\": \"Microarray analysis; SNAIL knockdown; NEFL modulation; tumorigenicity and chemoresistance assays in gastric cancer cell lines and gastric oncospheres\",\n      \"journal\": \"Carcinogenesis\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — microarray-based identification with functional assays, pathway placement inferred rather than mechanistically dissected, single lab\",\n      \"pmids\": [\"33313663\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"NEFL encodes the light subunit of neuronal neurofilaments, an obligate component of the hetero-polymeric intermediate filament network in axons: it co-assembles with NF-M and NF-H (and can copolymerize with vimentin in non-neuronal cells) to form filaments whose abundance determines axonal caliber; head-domain phosphorylation of NFL inhibits both neurofilament assembly and axonal transport; NFL mRNA stability is post-transcriptionally regulated by a complex of RNA-binding proteins including TDP-43 (via UG-motif stem loops and its RRM domains), 14-3-3 isoforms, SOD1, and RGNEF, with pathological redistribution of NFL mRNA into stress granules and P-bodies occurring in ALS; small heat shock proteins HspB1 and HspB5 bind NFL and retard its polymerization from tetramers to filaments; dominant missense mutations in NEFL cause CMT neuropathy by disrupting neurofilament assembly and inducing NFL aggregation (a shared mechanism with HSPB1 mutations), while homozygous nonsense mutations cause a recessive loss-of-function neuropathy through nonsense-mediated mRNA decay and absence of NEFL protein; and in cancer contexts NEFL acts as a tumor suppressor whose expression is silenced by oncomiRs (miR-381, miR-25, miR-183) that place it upstream of mTOR signaling.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"NEFL encodes the light subunit of neuronal neurofilaments, an intermediate filament protein that assembles into the axonal cytoskeleton and is the principal determinant of axonal caliber [#0, #9]. The NF-L subunit is intrinsically competent to polymerize into intermediate filament arrays, copolymerizing with vimentin in non-neuronal cells and forming filaments without requiring neuron-specific factors [#0]; it marks a discrete population of large-caliber neurons distinct from peripherin-expressing small neurons [#1]. Assembly and axonal transport of neurofilaments are negatively regulated by phosphorylation of the NF-L head domain, where phosphomimetic substitutions inhibit both transport and filament formation [#8], and by small heat-shock proteins HspB1 and HspB5, which bind NF-L and retard its polymerization from tetramers to filaments [#16]. NEFL mRNA abundance is governed post-transcriptionally by a 3'UTR-binding regulatory network: 14-3-3 isoforms bind hexanucleotide motifs, while TDP-43 engages UG-motif stem loops through its RRM1/RRM2 domains and acts together with 14-3-3 and SOD1 to modulate transcript stability; in ALS motor neurons this mRNA is pathologically sequestered into TDP-43-positive stress granules and P-bodies [#4, #7]. Dominant missense mutations in NEFL cause Charcot-Marie-Tooth neuropathy (CMT2E/CMT1F) by disrupting the neurofilament network and inducing NF-L aggregation, a mechanism shared with HSPB1 mutations and reversible by chaperone activity [#3, #5], whereas homozygous nonsense mutations cause a recessive loss-of-function neuropathy via nonsense-mediated decay and complete absence of NF-L protein [#9, #17]. In cancer, NEFL behaves as a tumor suppressor silenced by oncogenic miRNAs (miR-381, miR-25, miR-183) and positioned upstream of mTOR signaling [#13, #14, #18].\",\n  \"teleology\": [\n    {\n      \"year\": 1989,\n      \"claim\": \"Established that NF-L is an autonomous intermediate filament-forming subunit, answering whether neuron-specific machinery is required for neurofilament assembly.\",\n      \"evidence\": \"Stable transfection of NF-L into non-neuronal L cells with immunofluorescence and vimentin co-fractionation\",\n      \"pmids\": [\"2493000\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not address obligate heteropolymer requirements with NF-M/NF-H in neurons\", \"Vimentin copolymerization is a non-neuronal context\"]\n    },\n    {\n      \"year\": 1991,\n      \"claim\": \"Defined NF-L as a marker distinguishing large-caliber neurons from peripherin-positive small neurons, linking subunit expression to neuronal cell identity.\",\n      \"evidence\": \"Double immunofluorescence on rat DRG with neuropeptide co-localization\",\n      \"pmids\": [\"1795410\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Correlative classification, not mechanistic\", \"Does not explain caliber control\"]\n    },\n    {\n      \"year\": 1995,\n      \"claim\": \"Mapped transcriptional control elements of the NF-L gene, showing NGF- and cAMP-responsive regulation of its promoter.\",\n      \"evidence\": \"Serial promoter deletion CAT reporter assays in PC12 cells; DNase I hypersensitivity mapping\",\n      \"pmids\": [\"7745611\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identifies responsive regions but not all bound transcription factors\", \"Rat promoter; human conservation not addressed\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Identified NEFL as a CMT disease gene, establishing that dominant mutations cause axonal neuropathy.\",\n      \"evidence\": \"Mutation screening of 323 CMT patients by direct sequencing with sural nerve biopsy\",\n      \"pmids\": [\"12566280\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Genotype identified but molecular consequences of each mutation not dissected\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Revealed post-transcriptional control of NFL mRNA stability by 14-3-3 proteins binding the 3'UTR, opening a regulatory layer beyond transcription.\",\n      \"evidence\": \"LC/MS/MS of 3'UTR-interacting proteins, gel shift, and motif mutagenesis from human spinal cord\",\n      \"pmids\": [\"17098443\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vitro binding; physiological stability changes not quantified in neurons\", \"Functional consequence of each isoform unresolved\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Demonstrated a shared pathogenic pathway between NFL and HSPB1 CMT mutations centered on NFL aggregation, and a chaperone role for HspB1.\",\n      \"evidence\": \"Motor neuron culture, co-expression, Co-IP, NFL deletion rescue, and viability assays\",\n      \"pmids\": [\"17881652\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of HspB1-mediated disaggregation not resolved at structural level\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Extended mutational converging on NFL misassembly to MTMR2/MTM1, linking myotubularin dimerization defects to neurofilament abnormalities.\",\n      \"evidence\": \"SW13vim(-) transfection assembly assay with MTMR2 dimerization and phosphorylation analysis\",\n      \"pmids\": [\"17973976\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Cell-line based, single lab\", \"Direct biochemical link between myotubularin and NFL not shown\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Showed head-domain phosphorylation governs neurofilament assembly and axonal transport, defining a post-translational switch.\",\n      \"evidence\": \"Phosphoablation and phosphomimetic mutagenesis of four head-domain sites with axonal transport readout\",\n      \"pmids\": [\"19147253\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Responsible kinases not identified in this corpus\", \"In vivo physiological relevance not established\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Defined the TDP-43/14-3-3/SOD1 mRNA-stability network and its pathological redistribution into stress granules and P-bodies in ALS.\",\n      \"evidence\": \"Ex vivo stability assays, RNA-IP-PCR, TDP-43 RRM mutagenesis, and immunofluorescence co-localization\",\n      \"pmids\": [\"19815002\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Causal direction between mRNA sequestration and neurodegeneration unresolved\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Established a recessive loss-of-function mechanism via a homozygous nonsense mutation, confirming neurofilaments as the main determinant of axonal caliber.\",\n      \"evidence\": \"SW13vim(-) filament assays, sural nerve biopsy EM, and homozygosity mapping\",\n      \"pmids\": [\"20039262\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"mRNA fate of the nonsense allele not yet defined at this stage\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Added RGNEF as a disease-state-dependent NFL mRNA-binding protein detectable in ALS but not control tissue.\",\n      \"evidence\": \"Gel shift assays in vitro and in tissue lysates with IP-RT-PCR\",\n      \"pmids\": [\"19488899\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional effect on mRNA stability not measured\", \"Single lab\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Showed CMT NEFL mutations differ in misfolding behavior and chaperone susceptibility, refining therapeutic chaperone strategies.\",\n      \"evidence\": \"SW13vim(-) assays, in vitro refolding, and celastrol/chaperone induction in motor and sensory neurons\",\n      \"pmids\": [\"23618875\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Cell-type-specific chaperone responses mechanistically unexplained\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Identified ALS-downregulated miRNAs that stabilize NEFL mRNA, expanding the mRNA-stability regulatory layer.\",\n      \"evidence\": \"Small RNA sequencing, luciferase reporter, and anti-miR functional assays\",\n      \"pmids\": [\"24454911\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Novel miRNAs uncharacterized beyond this study\", \"Single lab\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Defined a tumor-suppressor role for NEFL silenced by miR-381, linking its loss to chemoresistance and stemness in glioblastoma.\",\n      \"evidence\": \"2-D DIGE/MS identification with miR-381 and NEFL gain/loss-of-function and TMZ viability assays\",\n      \"pmids\": [\"25605243\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which NEFL suppresses resistance factors not resolved\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Placed NEFL upstream of mTOR signaling via miR-25, connecting its loss to glioblastoma proliferation and invasion.\",\n      \"evidence\": \"miR-25 gain/loss and NEFL siRNA rescue with mTOR pathway analysis in U251 cells\",\n      \"pmids\": [\"26209061\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular link between NEFL and mTOR not biochemically defined\", \"Single lab\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Demonstrated in CMT2E patient tissue that reduced neurofilament abundance, not aggregation, correlates with reduced axonal caliber.\",\n      \"evidence\": \"Skin biopsy IF, Western blot, EM, and axonal caliber morphometry\",\n      \"pmids\": [\"26109717\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Single mutation (E396K); generalizability across CMT alleles not shown\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Quantified the biochemical chaperone interaction, showing HspB1 and HspB5 bind NFL and slow tetramer-to-filament polymerization.\",\n      \"evidence\": \"Differential centrifugation, analytical ultracentrifugation, and fluorescent polymerization assays with stoichiometry\",\n      \"pmids\": [\"28000086\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vitro reconstitution; in-cell relevance of stoichiometry not addressed\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Added miR-183 to the oncomiR set repressing NEFL upstream of mTOR in glioblastoma.\",\n      \"evidence\": \"miR-183 gain/loss and NEFL siRNA rescue with mTOR analysis in U251 cells\",\n      \"pmids\": [\"26879754\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct NEFL-mTOR molecular link unresolved\", \"Single lab\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Established that the recessive nonsense allele triggers nonsense-mediated decay, proving a true loss-of-function mechanism in patient-derived neurons.\",\n      \"evidence\": \"iPSC-derived motor neurons, qPCR, Western blot, ICC, EM, scRNA-seq, and NMD inhibitor rescue\",\n      \"pmids\": [\"29888333\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Why neurons still form networks without NF-L not mechanistically explained\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Extended the NEFL-mTOR axis to spinal cord injury, with miR-30b-5p repression of NEFL modulating inflammation and recovery.\",\n      \"evidence\": \"Dual-luciferase reporter, NEFL silencing in PC12 cells, and SCI rat model with mTOR analysis\",\n      \"pmids\": [\"36282532\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"NEFL-mTOR coupling mechanism still undefined\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The molecular mechanism by which cytoplasmic NEFL activates mTOR signaling and acts as a tumor suppressor remains undefined.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No biochemical link between NF-L and mTOR pathway components identified\", \"Kinases controlling head-domain phosphorylation not identified in this corpus\", \"Structural basis of chaperone-mediated assembly control unresolved\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [0, 9, 15]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [0, 9]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [16]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [3, 5, 9, 17]},\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [4, 7]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [14, 18, 20]}\n    ],\n    \"complexes\": [\"neurofilament\"],\n    \"partners\": [\"NEFM\", \"VIM\", \"HSPB1\", \"HSPB5\", \"TARDBP\", \"SOD1\", \"YWHAZ\", \"RGNEF\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}