{"gene":"FIG4","run_date":"2026-04-28T17:46:03","timeline":{"discoveries":[{"year":2002,"finding":"FIG4 (yeast ortholog) encodes a SAC1-domain polyphosphoinositide phosphatase responsible for turnover of PtdIns(3,5)P2; deletion of FIG4 in vac7Δ mutants dramatically restores PtdIns(3,5)P2 levels, placing FIG4 as the primary phosphatase for PtdIns(3,5)P2 degradation in the Fab1 kinase pathway","method":"Genetic epistasis (suppressor screen), yeast deletion mutants, phosphoinositide measurement","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 — genetic epistasis with biochemical lipid measurement, foundational mechanism paper","pmids":["11950935"],"is_preprint":false},{"year":2003,"finding":"FIG4 (yeast ortholog) is a magnesium-activated, PtdIns(3,5)P2-selective phosphoinositide phosphatase in vitro; it localizes to the vacuole membrane and requires Vac14 for correct vacuolar localization; FIG4 physically associates with Vac14 in a membrane-associated complex","method":"In vitro phosphatase assay, GFP fusion live imaging, co-immunoprecipitation, genetic deletion","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 1 — in vitro enzymatic assay combined with localization and co-IP in the same study","pmids":["14528018"],"is_preprint":false},{"year":2007,"finding":"Mammalian FIG4 is functionally conserved as a PtdIns(3,5)P2 phosphatase; loss-of-function (ETn2β insertion in mouse Fig4) causes abnormal PtdIns(3,5)P2 concentration in fibroblasts, LAMP-2-positive vacuole accumulation, and neurodegeneration, establishing FIG4 as a PI(3,5)P2 5-phosphatase regulating the late endosome-lysosome axis","method":"Positional cloning, phosphoinositide measurement in patient/mutant fibroblasts, LAMP-2 immunostaining, nerve conduction studies","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 — biochemical lipid measurement + organelle immunostaining + genetic model, highly cited foundational paper","pmids":["17572665"],"is_preprint":false},{"year":2008,"finding":"FIG4 (yeast ortholog) forms a vacuole-associated signaling complex with Fab1 and Vac14; Fab1 binds Vac14 and FIG4 through its chaperonin-like domain; Vac14 and FIG4 bind each other directly and are mutually dependent for interaction with Fab1; this places the lipid kinase and phosphatase in a common functional unit that explains their dual roles in PtdIns(3,5)P2 synthesis and turnover","method":"Co-immunoprecipitation, pull-down assays, FYVE-domain PtdIns(3)P binding experiments, yeast genetics","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP and direct binding assays, replicated across multiple protein combinations","pmids":["18653468"],"is_preprint":false},{"year":2008,"finding":"The CMT4J-causing FIG4-I41T missense mutation impairs interaction of FIG4 with the scaffold protein VAC14, leading to proteasome-dependent degradation and severely reduced FIG4-I41T protein levels in vivo (only ~2% of transcript-predicted level)","method":"Yeast two-hybrid, mouse transgenic model (I41T cDNA on null background), immunoblotting of patient fibroblasts, proteasome inhibitor (MG-132) rescue","journal":"PLoS genetics","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (Y2H, transgenic mouse, patient fibroblasts, pharmacological rescue)","pmids":["21655088"],"is_preprint":false},{"year":2008,"finding":"ArPIKfyve (mammalian VAC14 ortholog) scaffolds the PIKfyve-ArPIKfyve-Sac3 (PAS) ternary complex; ArPIKfyve interacts with both Sac3 (FIG4) and PIKfyve; Sac3 is permissive for maximal PIKfyve-ArPIKfyve association; disruption of ArPIKfyve homomeric interactions via C-terminal peptide disassembles the PAS complex and reduces PIKfyve lipid kinase activity in vitro; complex disassembly also inhibits insulin-stimulated GLUT4 surface accumulation","method":"Co-immunoprecipitation in transfected mammalian cells, in vitro PIKfyve kinase assay, GLUT4 translocation assay in 3T3L1 adipocytes, dominant-negative peptide","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1-2 — in vitro kinase assay + Co-IP + functional GLUT4 readout, multiple orthogonal approaches","pmids":["18950639"],"is_preprint":false},{"year":2009,"finding":"Sac3/FIG4 assembled in the PIKfyve-ArPIKfyve-Sac3 (PAS) core complex retains active PtdIns(3,5)P2 phosphatase activity; the Cpn60_TCP1 domain of PIKfyve is a major contact for the ArPIKfyve-Sac3 subcomplex; catalytically dead Sac3(D488A) fails to rescue vacuolar phenotype caused by kinase-deficient PIKfyve, demonstrating that Sac3 phosphatase activity within the PAS complex turns over PtdIns(3,5)P2","method":"Domain mapping with truncation/point mutants, vacuole phenotype assay in COS cells, co-immunoprecipitation","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 — active-site mutagenesis combined with functional vacuole readout and domain mapping","pmids":["19840946"],"is_preprint":false},{"year":2009,"finding":"Sac3/FIG4 is an insulin-sensitive phosphatase; acute insulin markedly reduces the in vitro PtdIns(3,5)P2-hydrolyzing activity of Sac3; siRNA knockdown of Sac3 elevates PtdIns(3,5)P2 and increases GLUT4 translocation and glucose entry in response to insulin, while overexpression of catalytically active (but not phosphatase-dead Sac3-D488A) reduces GLUT4 surface abundance","method":"siRNA knockdown, in vitro phosphatase assay, GLUT4 translocation assay, HPLC phosphoinositide measurement","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 — in vitro enzymatic assay + active-site mutant + functional GLUT4 readout","pmids":["19578118"],"is_preprint":false},{"year":2010,"finding":"ArPIKfyve stabilizes Sac3/FIG4 protein by attenuating its rapid proteasome-dependent degradation (t1/2 ~18.8 min for Sac3 alone, extended by ArPIKfyve coexpression); the CMT4J-causing Sac3-I41T mutant fails to have its half-life extended by ArPIKfyve, identifying a failure of ArPIKfyve-mediated stabilization as the primary molecular defect in CMT4J","method":"Cycloheximide chase, proteasome inhibitor (MG-132), co-immunoprecipitation, immunoblotting in COS cells","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — multiple biochemical methods (cycloheximide chase, proteasome inhibitor, Co-IP) in a single study","pmids":["20630877"],"is_preprint":false},{"year":2012,"finding":"Neuronal expression of FIG4 is both necessary and sufficient to prevent spongiform neurodegeneration in vivo; conditional inactivation of Fig4 specifically in neurons (synapsin-Cre) recapitulates the full spectrum of neurological abnormalities, while astrocytic expression of Fig4 prevents autophagy marker accumulation but not spongiform degeneration or lethality","method":"Conditional knockout (floxed allele × synapsin-Cre), neuron-specific transgenic rescue (NSE promoter), GFAP promoter-driven astrocyte rescue, histology","journal":"Human molecular genetics","confidence":"High","confidence_rationale":"Tier 2 — clean cell-type-specific KO and rescue with defined neurological and histological phenotypes","pmids":["22581779"],"is_preprint":false},{"year":2011,"finding":"FIG4 is required for CNS myelination; Fig4 null mice show dramatic CNS myelin reduction and oligodendrocyte maturation defects; neuronal (non-cell-autonomous) expression of Fig4 rescues CNS myelination and tremor, demonstrating that FIG4 in neurons supports oligodendrocyte maturation","method":"Transgenic rescue with neuron-specific (NSE) promoter, optic nerve electrophysiology, electron microscopy, OL lineage cell counting","journal":"The Journal of neuroscience","confidence":"High","confidence_rationale":"Tier 2 — transgenic rescue + electrophysiology + ultrastructural analysis","pmids":["22131434"],"is_preprint":false},{"year":2014,"finding":"FIG4 has cell-autonomous roles in both motor neurons and Schwann cells for CMT4J pathogenesis; conditional Fig4 inactivation in motor neurons causes neuronal/axonal degeneration, while conditional inactivation in Schwann cells causes demyelination and defects in autophagy-mediated degradation and myelin biogenesis","method":"Cell-type-specific conditional knockout (motor neuron-Cre, Schwann cell-Cre), histology, electron microscopy, autophagy marker analysis","journal":"Human molecular genetics","confidence":"High","confidence_rationale":"Tier 2 — independent conditional KO in two cell types with defined cellular phenotypes","pmids":["25187576"],"is_preprint":false},{"year":2015,"finding":"FIG4 deficiency impairs lysosomal Ca2+ efflux via the TRPML1 channel (whose endogenous ligand is PI(3,5)P2), causing elevated intralysosomal Ca2+, impaired lysosomal fission, and downstream downregulation of dynamin-1 GTPase; pharmacological reactivation of TRPML1 with synthetic ligand ML-SA1 rescues lysosomal storage in Fig4-/- cells and ex vivo DRGs","method":"Flow cytometry lysosome size assay, intralysosomal Ca2+ measurement, dynamin-1 immunoblotting, pharmacological rescue (ML-SA1), ex vivo DRG culture","journal":"The Journal of neuroscience","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods linking PI(3,5)P2 to TRPML1 Ca2+ channel and lysosomal fission, pharmacological rescue validation","pmids":["25926456"],"is_preprint":false},{"year":2015,"finding":"A catalytically inactive FIG4 transgene (Cys486Ser active-site mutant) partially rescues neurodegeneration and juvenile lethality in Fig4 null mice, demonstrating a phosphatase-independent structural/scaffolding function of FIG4 in stabilizing the PI(3,5)P2 biosynthetic complex; however, late-onset defects (hydrocephalus, demyelination) confirm that phosphatase activity is also essential in vivo","method":"Active-site mutagenesis (Cys486Ser), in vivo transgenic rescue, vacuolization assay in fibroblasts, histology","journal":"Human molecular genetics","confidence":"High","confidence_rationale":"Tier 1 — active-site mutagenesis in vivo with quantitative phenotypic rescue readout","pmids":["26604144"],"is_preprint":false},{"year":2015,"finding":"The ArPIKfyve-Sac3/FIG4 complex interacts with synphilin-1 (Sph1) in brain; mass spectrometry identified Sph1 as a component of the ArPIKfyve-Sac3 complex; modulation of ArPIKfyve/Sac3 levels alters Sph1-GFP aggregation properties in a Sac3 phosphatase-dependent manner, promoting its cytosolic partitioning and removal by basal autophagy","method":"Mass spectrometry of brain-derived interactors, Co-immunoprecipitation, RNA silencing, overexpression in neuronal cell lines and primary cortical neurons, aggregation assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2/3 — MS identification + Co-IP + functional aggregation readout, single lab study","pmids":["26405034"],"is_preprint":false},{"year":2015,"finding":"Drosophila FIG4 (dFIG4) mutations predicted to inactivate phosphatase activity still rescue lysosomal expansion phenotypes in vivo, and Fab1 mutations causing the same phenotype are epistatic, establishing a phosphatase-independent biosynthetic/scaffolding function of FIG4 in lysosomal membrane homeostasis; lysosomal phenotypes are suppressed by genetic inhibition of Rab7 or the HOPS complex, placing FIG4 function after endosome-to-lysosome fusion","method":"Drosophila genetics (null mutants, phosphatase-dead transgene, Fab1 epistasis, Rab7/HOPS double mutants), LysoTracker staining, flight ability assay","journal":"Human molecular genetics","confidence":"High","confidence_rationale":"Tier 1-2 — in vivo phosphatase-dead rescue combined with genetic epistasis, multiple orthogonal approaches","pmids":["26662798"],"is_preprint":false},{"year":2017,"finding":"FIG4 deficiency causes accumulation of TRPV4 at the plasma membrane of patient fibroblasts due to impaired endosomal trafficking/turnover; knockdown of Fig4 in murine motor neurons causes vacuolation and cell death, and inhibition of TRPV4 activity significantly preserves viability","method":"Patient fibroblast analysis, Fig4 siRNA knockdown in motor neurons, TRPV4 inhibitor treatment, immunofluorescence of membrane proteins","journal":"Journal of neuropathology and experimental neurology","confidence":"Medium","confidence_rationale":"Tier 3 — single lab, immunofluorescence + pharmacological rescue without full mechanistic pathway reconstitution","pmids":["28859335"],"is_preprint":false},{"year":2017,"finding":"Sac3/FIG4 knockdown in RAW264.7 macrophages decreases cell surface scavenger receptor A (SR-A) protein levels and suppresses foam cell formation; ArPIKfyve knockdown similarly decreases Sac3 and SR-A; PIKfyve knockdown has no effect on SR-A, demonstrating that the ArPIKfyve-Sac3 complex regulates SR-A protein levels independently of PIKfyve kinase activity","method":"shRNA knockdown, flow cytometry for SR-A surface levels, foam cell assay (acetylated LDL uptake)","journal":"Experimental cell research","confidence":"Medium","confidence_rationale":"Tier 3 — single lab, shRNA knockdown with defined functional readout, mechanism partially resolved","pmids":["28552585"],"is_preprint":false},{"year":2018,"finding":"Adult-specific inactivation of Fig4 (tamoxifen-inducible global KO) causes wasting, tremor, motor impairment and death within 2 months, demonstrating a life-long requirement; PNS myelinated axons undergo Wallerian degeneration while CNS myelin is intact; FIG4 is additionally required for timely CNS remyelination after chemical lesion","method":"Tamoxifen-inducible Cre-mediated KO (CAG-creER), histology of sciatic and optic nerves, compound action potential recording, chemical demyelination challenge","journal":"Human molecular genetics","confidence":"High","confidence_rationale":"Tier 2 — conditional adult KO with multiple neurological and histological readouts","pmids":["29688489"],"is_preprint":false},{"year":2019,"finding":"CMT4J patient fibroblasts show significant reductions in both PtdIns(3,5)P2 (−36%) and PtdIns5P (−43%) compared to controls, measured by HPLC, demonstrating that FIG4 loss reduces PI(3,5)P2 despite FIG4's known role in activating PIKfyve; patients without aberrant vacuoles have especially low PtdIns3P, linking PtdIns3P levels to vacuolization phenotype","method":"HPLC phosphoinositide profiling of myo-[2-3H]inositol-labeled primary patient fibroblasts, immunoblotting","journal":"Molecular neurobiology","confidence":"High","confidence_rationale":"Tier 2 — rigorous HPLC lipid profiling across 13 patient and 9 control samples with validated SAC3/FIG4 depletion","pmids":["31313076"],"is_preprint":false},{"year":2021,"finding":"AAV9-mediated delivery of a codon-optimized FIG4 sequence into Fig4 null mice rescues lethality and peripheral neuropathy when administered neonatally (P1 or P4), with dose-dependent efficacy, providing preclinical proof of concept for gene therapy","method":"AAV9 gene delivery in mouse model, survival analysis, neurophysiology, histopathology","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 2 — rigorous preclinical gene therapy study with quantitative neurological and histological endpoints","pmids":["33878035"],"is_preprint":false},{"year":2021,"finding":"BioID proximity mapping of Vac14 and FIG4 identified 89 high-confidence shared interactors including COPI subunit COPB1 and the GTPase Arf1; proximity ligation assays validated Vac14-COPB1 and Vac14-Arf1 interactions, linking the PIKfyve-VAC14-FIG4 complex to COPI-mediated endosomal dynamics","method":"BioID proximity labeling, mass spectrometry, proximity ligation assay","journal":"Journal of proteome research","confidence":"Medium","confidence_rationale":"Tier 3 — BioID + PLA validation, single lab, novel interactions not yet functionally dissected","pmids":["34554760"],"is_preprint":false},{"year":2023,"finding":"FIG4 and VAC14 function in PI(3,5)P2 biosynthesis, which inhibits the lysosomal chloride transporter ClC-7; knockout of CLCN7 in FIG4 null cells corrects lysosomal swelling and partially corrects lysosomal hyperacidification; in Fig4 null mice, reduction of ClC-7 via dominant-negative CLCN7 improved growth, neurological function, and increased lifespan by 20%","method":"CLCN7 knockout in FIG4 null cells, dominant-negative CLCN7 mouse model, lysosome size and pH measurement, survival analysis","journal":"PLoS genetics","confidence":"High","confidence_rationale":"Tier 2 — genetic epistasis in both cell culture and in vivo with quantitative lysosomal and neurological readouts","pmids":["37363915"],"is_preprint":false},{"year":2025,"finding":"VAC14 forms a star-shaped pentamer scaffold; two legs bind FIG4, with one also binding PIKfyve; VAC14 oligomerization is critical for Fab1/PIKfyve function, PI(3,5)P2 generation, VAC14 localization to VPS35-containing endosomes, and PIKfyve-VAC14-FIG4 complex formation; pediatric disease mutations at VAC14-VAC14 interfaces disrupt complex assembly","method":"AlphaFold2 structural prediction, cryo-EM, pull-down assays in VAC14 KO human cells, fluorescence-detection size-exclusion chromatography, yeast genetics, colocalization with VPS35-endosomes","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 1 — cryo-EM structure + in vitro/cell biochemistry + yeast genetics, multiple orthogonal approaches","pmids":["40305106"],"is_preprint":false}],"current_model":"FIG4 is a PI(3,5)P2 5-phosphatase that functions within a conserved trimeric complex (PIKfyve/FAB1–VAC14–FIG4) on late endosomal/lysosomal membranes, where VAC14 (pentameric scaffold) recruits both the kinase and phosphatase; within this complex FIG4 paradoxically promotes PI(3,5)P2 synthesis by stabilizing PIKfyve activity (via ArPIKfyve/VAC14-mediated protection from proteasomal degradation) while also hydrolyzing PI(3,5)P2, and the resulting signaling lipid regulates TRPML1 Ca2+ channel-dependent lysosomal fission, endolysosomal trafficking, and ClC-7 chloride transport; loss of FIG4 phosphatase or its scaffolding function causes lysosomal enlargement, impaired organelle trafficking, and progressive neurodegeneration in both PNS and CNS."},"narrative":{"teleology":[{"year":2002,"claim":"Identification of FIG4 as the primary PI(3,5)P2 phosphatase resolved which enzyme turns over this signaling lipid in the Fab1 kinase pathway, establishing the first enzymatic function for FIG4.","evidence":"Genetic epistasis (fig4Δ suppressor of vac7Δ) with phosphoinositide measurement in yeast","pmids":["11950935"],"confidence":"High","gaps":["No in vitro enzymatic characterization yet","Mammalian conservation undemonstrated","No structural information on FIG4"]},{"year":2003,"claim":"Demonstration that FIG4 is a Mg²⁺-dependent, PI(3,5)P2-selective phosphatase that physically associates with Vac14 at the vacuole membrane established the enzymatic specificity and placed FIG4 in a membrane-localized regulatory complex.","evidence":"In vitro phosphatase assay with purified yeast FIG4, GFP localization, co-immunoprecipitation with Vac14","pmids":["14528018"],"confidence":"High","gaps":["Stoichiometry and architecture of the complex unknown","How Vac14 recruits FIG4 to the membrane unresolved"]},{"year":2007,"claim":"The discovery that mammalian FIG4 loss causes reduced PI(3,5)P2, LAMP2-positive vacuole accumulation, and neurodegeneration bridged the yeast enzymology to human disease and established FIG4 as a Charcot–Marie–Tooth 4J disease gene.","evidence":"Positional cloning of the pale tremor mouse, phosphoinositide measurement in mutant fibroblasts, nerve conduction studies","pmids":["17572665"],"confidence":"High","gaps":["Paradox of PI(3,5)P2 reduction despite loss of its phosphatase unexplained","Cell-type requirements for FIG4 in neurodegeneration undefined"]},{"year":2008,"claim":"Mapping of the Fab1–Vac14–FIG4 ternary complex and the parallel mammalian PIKfyve–ArPIKfyve–Sac3 (PAS) complex revealed that the kinase and phosphatase are tethered by a shared scaffold, resolving the paradox that FIG4 loss reduces PI(3,5)P2 by showing FIG4 stabilizes kinase activity.","evidence":"Reciprocal co-immunoprecipitation and pull-down assays in yeast; co-IP plus in vitro PIKfyve kinase assay and GLUT4 translocation assay in mammalian cells","pmids":["18653468","18950639"],"confidence":"High","gaps":["Quaternary structure of the complex unresolved","How FIG4 activates PIKfyve kinase mechanistically unclear"]},{"year":2009,"claim":"Showing that Sac3/FIG4 retains active phosphatase function within the assembled PAS complex and is regulated by insulin established that PI(3,5)P2 turnover and synthesis are coordinated within a single complex, with physiological input from insulin signaling.","evidence":"Active-site mutant (D488A) unable to rescue vacuolar phenotype; insulin suppresses Sac3 phosphatase activity in vitro; siRNA modulation of GLUT4 translocation","pmids":["19840946","19578118"],"confidence":"High","gaps":["Mechanism of insulin-mediated Sac3 inhibition unknown","Post-translational modifications regulating Sac3 activity uncharacterized"]},{"year":2010,"claim":"Demonstrating that ArPIKfyve stabilizes Sac3/FIG4 by extending its half-life and that the CMT4J I41T mutation abolishes this protection identified the molecular basis of disease: loss of scaffold-mediated stabilization leading to proteasomal degradation of FIG4.","evidence":"Cycloheximide chase, MG-132 proteasome inhibitor rescue, co-IP in COS cells; patient fibroblast immunoblotting","pmids":["20630877","21655088"],"confidence":"High","gaps":["Whether residual I41T protein retains any phosphatase activity unresolved","Therapeutic strategies to stabilize FIG4 protein unexplored"]},{"year":2012,"claim":"Cell-type-specific conditional knockouts established that neuronal FIG4 is necessary and sufficient to prevent CNS neurodegeneration and additionally supports oligodendrocyte myelination non-cell-autonomously, while Schwann-cell-autonomous FIG4 is required for PNS myelination.","evidence":"Synapsin-Cre conditional KO, NSE-promoter rescue, GFAP-promoter astrocyte rescue, motor-neuron and Schwann-cell Cre lines, electron microscopy, optic nerve electrophysiology","pmids":["22581779","22131434","25187576"],"confidence":"High","gaps":["Non-cell-autonomous signal from neurons to oligodendrocytes unidentified","Relative contribution of autophagy vs. lysosomal fission defects to degeneration unclear"]},{"year":2015,"claim":"Linking FIG4-dependent PI(3,5)P2 to TRPML1 Ca²⁺ channel activation and dynamin-1-mediated lysosomal fission, and separately demonstrating a phosphatase-independent scaffolding function for FIG4 in vivo, bifurcated FIG4's role into enzymatic and structural contributions to lysosomal homeostasis.","evidence":"Intralysosomal Ca²⁺ measurement, ML-SA1 pharmacological rescue of Fig4−/− DRGs; catalytically dead C486S transgene partial rescue in Fig4 null mice; Drosophila phosphatase-dead rescue with Fab1/Rab7/HOPS epistasis","pmids":["25926456","26604144","26662798"],"confidence":"High","gaps":["Quantitative contribution of scaffolding vs. phosphatase activity across tissues not defined","How FIG4 scaffolding stabilizes PIKfyve at the structural level unknown"]},{"year":2018,"claim":"Adult-onset conditional deletion revealed a lifelong requirement for FIG4 distinct from developmental roles, with PNS axons undergoing Wallerian degeneration and CNS remyelination being impaired after injury.","evidence":"Tamoxifen-inducible global KO in adult mice, sciatic nerve histology, compound action potential recording, chemical demyelination challenge","pmids":["29688489"],"confidence":"High","gaps":["Whether adult neurodegeneration is reversible upon FIG4 restoration unknown","Temporal window for therapeutic intervention undefined"]},{"year":2023,"claim":"Genetic epistasis showing that ClC-7 knockout corrects lysosomal swelling in FIG4-null cells and extends lifespan in Fig4-null mice identified ClC-7 as a key downstream effector of PI(3,5)P2 deficiency, opening a therapeutic axis.","evidence":"CLCN7 KO in FIG4-null cells, dominant-negative CLCN7 in Fig4-null mice, lysosome size/pH measurement, survival analysis","pmids":["37363915"],"confidence":"High","gaps":["Mechanism by which PI(3,5)P2 inhibits ClC-7 not structurally resolved","Whether ClC-7 inhibition rescues demyelination not tested","Relative contributions of ClC-7 vs. TRPML1 pathways to disease unclear"]},{"year":2025,"claim":"Cryo-EM and AlphaFold2 modeling of the VAC14 pentamer revealed the architectural basis for FIG4 and PIKfyve recruitment: two pentamer legs bind FIG4, with one also binding PIKfyve, explaining stoichiometry and linking disease mutations at VAC14–VAC14 interfaces to complex disassembly.","evidence":"Cryo-EM structure, AlphaFold2 prediction, pull-downs in VAC14 KO human cells, fluorescence-detection size-exclusion chromatography, colocalization with VPS35-endosomes","pmids":["40305106"],"confidence":"High","gaps":["High-resolution structure of the full PIKfyve–VAC14–FIG4 holo-complex not yet achieved","How VPS35-positive endosomal localization is specified structurally not established"]},{"year":null,"claim":"The full structural basis for how FIG4 simultaneously promotes PI(3,5)P2 synthesis (scaffold) and turnover (phosphatase) within the assembled holo-complex, the identity of the non-cell-autonomous signal from neurons that supports oligodendrocyte myelination, and whether therapeutic modulation of ClC-7 or TRPML1 can substitute for FIG4 gene replacement remain open questions.","evidence":"","pmids":[],"confidence":"Low","gaps":["No atomic-resolution structure of the complete PIKfyve–VAC14–FIG4 ternary complex","Non-cell-autonomous neuronal signal for myelination unidentified","Relative therapeutic value of ClC-7 vs. TRPML1 modulation vs. gene therapy not determined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[0,1,2,6,7]},{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[13,15]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[1,7]}],"localization":[{"term_id":"GO:0005764","term_label":"lysosome","supporting_discovery_ids":[1,2,12,15,22]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[2,21,23]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[1]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,5,7,12,22]},{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[2,12,16,21]},{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[12,15,22]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[4,11]}],"complexes":["PIKfyve-VAC14-FIG4 (PAS) complex"],"partners":["VAC14","PIKFYVE","TRPML1","CLCN7","SNCAIP","COPB1","ARF1"],"other_free_text":[]},"mechanistic_narrative":"FIG4 is a SAC1-domain phosphoinositide phosphatase that hydrolyzes PI(3,5)P2 on late endosomal/lysosomal membranes and simultaneously serves as a scaffolding subunit of the PIKfyve–VAC14–FIG4 ternary complex required for PI(3,5)P2 biosynthesis. Within this complex, VAC14 forms a pentameric scaffold that recruits both PIKfyve (the lipid kinase) and FIG4; FIG4 binding to VAC14 protects FIG4 from rapid proteasomal degradation and is permissive for maximal PIKfyve kinase activity, explaining why FIG4 loss paradoxically reduces PI(3,5)P2 levels and causes lysosomal enlargement [PMID:14528018, PMID:18950639, PMID:20630877, PMID:26604144, PMID:40305106]. PI(3,5)P2 generated by this complex activates the lysosomal Ca²⁺ channel TRPML1 to drive lysosomal fission and inhibits the chloride transporter ClC-7 to maintain lysosomal ion homeostasis; loss of these regulatory circuits underlies the vacuolar storage, spongiform neurodegeneration, and demyelination observed in FIG4-deficient neurons and Schwann cells, the basis of Charcot–Marie–Tooth disease type 4J [PMID:17572665, PMID:25926456, PMID:37363915, PMID:25187576]. Neuronal FIG4 expression is both necessary and sufficient to prevent CNS neurodegeneration and supports non-cell-autonomous oligodendrocyte myelination, while adult-specific loss causes Wallerian degeneration of PNS axons, demonstrating a lifelong requirement [PMID:22581779, PMID:22131434, PMID:29688489]."},"prefetch_data":{"uniprot":{"accession":"Q92562","full_name":"Polyphosphoinositide phosphatase","aliases":["Phosphatidylinositol 3,5-bisphosphate 5-phosphatase","SAC domain-containing protein 3","Serine-protein phosphatase FIG4"],"length_aa":907,"mass_kda":103.6,"function":"Dual specificity phosphatase component of the PI(3,5)P2 regulatory complex which regulates both the synthesis and turnover of phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2) (PubMed:17556371, PubMed:33098764). Catalyzes the dephosphorylation of phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2) to form phosphatidylinositol 3-phosphate (PubMed:33098764). Has serine-protein phosphatase activity acting on PIKfyve to stimulate its lipid kinase activity, its catalytically activity being required for maximal PI(3,5)P2 production (PubMed:33098764). In vitro, hydrolyzes all three D5-phosphorylated polyphosphoinositide and although displaying preferences for PtdIns(3,5)P2, it is capable of hydrolyzing PtdIns(3,4,5)P3 and PtdIns(4,5)P2, at least in vitro (PubMed:17556371)","subcellular_location":"Endosome membrane","url":"https://www.uniprot.org/uniprotkb/Q92562/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/FIG4","classification":"Not Classified","n_dependent_lines":65,"n_total_lines":1208,"dependency_fraction":0.05380794701986755},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"PIKFYVE","stoichiometry":4.0},{"gene":"CAPZB","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/FIG4","total_profiled":1310},"omim":[{"mim_id":"612691","title":"POLYMICROGYRIA, BILATERAL TEMPOROOCCIPITAL; 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deletion of FIG4 in vac7Δ mutants dramatically restores PtdIns(3,5)P2 levels, placing FIG4 as the primary phosphatase for PtdIns(3,5)P2 degradation in the Fab1 kinase pathway\",\n      \"method\": \"Genetic epistasis (suppressor screen), yeast deletion mutants, phosphoinositide measurement\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis with biochemical lipid measurement, foundational mechanism paper\",\n      \"pmids\": [\"11950935\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"FIG4 (yeast ortholog) is a magnesium-activated, PtdIns(3,5)P2-selective phosphoinositide phosphatase in vitro; it localizes to the vacuole membrane and requires Vac14 for correct vacuolar localization; FIG4 physically associates with Vac14 in a membrane-associated complex\",\n      \"method\": \"In vitro phosphatase assay, GFP fusion live imaging, co-immunoprecipitation, genetic deletion\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro enzymatic assay combined with localization and co-IP in the same study\",\n      \"pmids\": [\"14528018\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Mammalian FIG4 is functionally conserved as a PtdIns(3,5)P2 phosphatase; loss-of-function (ETn2β insertion in mouse Fig4) causes abnormal PtdIns(3,5)P2 concentration in fibroblasts, LAMP-2-positive vacuole accumulation, and neurodegeneration, establishing FIG4 as a PI(3,5)P2 5-phosphatase regulating the late endosome-lysosome axis\",\n      \"method\": \"Positional cloning, phosphoinositide measurement in patient/mutant fibroblasts, LAMP-2 immunostaining, nerve conduction studies\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — biochemical lipid measurement + organelle immunostaining + genetic model, highly cited foundational paper\",\n      \"pmids\": [\"17572665\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"FIG4 (yeast ortholog) forms a vacuole-associated signaling complex with Fab1 and Vac14; Fab1 binds Vac14 and FIG4 through its chaperonin-like domain; Vac14 and FIG4 bind each other directly and are mutually dependent for interaction with Fab1; this places the lipid kinase and phosphatase in a common functional unit that explains their dual roles in PtdIns(3,5)P2 synthesis and turnover\",\n      \"method\": \"Co-immunoprecipitation, pull-down assays, FYVE-domain PtdIns(3)P binding experiments, yeast genetics\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP and direct binding assays, replicated across multiple protein combinations\",\n      \"pmids\": [\"18653468\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"The CMT4J-causing FIG4-I41T missense mutation impairs interaction of FIG4 with the scaffold protein VAC14, leading to proteasome-dependent degradation and severely reduced FIG4-I41T protein levels in vivo (only ~2% of transcript-predicted level)\",\n      \"method\": \"Yeast two-hybrid, mouse transgenic model (I41T cDNA on null background), immunoblotting of patient fibroblasts, proteasome inhibitor (MG-132) rescue\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (Y2H, transgenic mouse, patient fibroblasts, pharmacological rescue)\",\n      \"pmids\": [\"21655088\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"ArPIKfyve (mammalian VAC14 ortholog) scaffolds the PIKfyve-ArPIKfyve-Sac3 (PAS) ternary complex; ArPIKfyve interacts with both Sac3 (FIG4) and PIKfyve; Sac3 is permissive for maximal PIKfyve-ArPIKfyve association; disruption of ArPIKfyve homomeric interactions via C-terminal peptide disassembles the PAS complex and reduces PIKfyve lipid kinase activity in vitro; complex disassembly also inhibits insulin-stimulated GLUT4 surface accumulation\",\n      \"method\": \"Co-immunoprecipitation in transfected mammalian cells, in vitro PIKfyve kinase assay, GLUT4 translocation assay in 3T3L1 adipocytes, dominant-negative peptide\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — in vitro kinase assay + Co-IP + functional GLUT4 readout, multiple orthogonal approaches\",\n      \"pmids\": [\"18950639\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Sac3/FIG4 assembled in the PIKfyve-ArPIKfyve-Sac3 (PAS) core complex retains active PtdIns(3,5)P2 phosphatase activity; the Cpn60_TCP1 domain of PIKfyve is a major contact for the ArPIKfyve-Sac3 subcomplex; catalytically dead Sac3(D488A) fails to rescue vacuolar phenotype caused by kinase-deficient PIKfyve, demonstrating that Sac3 phosphatase activity within the PAS complex turns over PtdIns(3,5)P2\",\n      \"method\": \"Domain mapping with truncation/point mutants, vacuole phenotype assay in COS cells, co-immunoprecipitation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — active-site mutagenesis combined with functional vacuole readout and domain mapping\",\n      \"pmids\": [\"19840946\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Sac3/FIG4 is an insulin-sensitive phosphatase; acute insulin markedly reduces the in vitro PtdIns(3,5)P2-hydrolyzing activity of Sac3; siRNA knockdown of Sac3 elevates PtdIns(3,5)P2 and increases GLUT4 translocation and glucose entry in response to insulin, while overexpression of catalytically active (but not phosphatase-dead Sac3-D488A) reduces GLUT4 surface abundance\",\n      \"method\": \"siRNA knockdown, in vitro phosphatase assay, GLUT4 translocation assay, HPLC phosphoinositide measurement\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — in vitro enzymatic assay + active-site mutant + functional GLUT4 readout\",\n      \"pmids\": [\"19578118\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"ArPIKfyve stabilizes Sac3/FIG4 protein by attenuating its rapid proteasome-dependent degradation (t1/2 ~18.8 min for Sac3 alone, extended by ArPIKfyve coexpression); the CMT4J-causing Sac3-I41T mutant fails to have its half-life extended by ArPIKfyve, identifying a failure of ArPIKfyve-mediated stabilization as the primary molecular defect in CMT4J\",\n      \"method\": \"Cycloheximide chase, proteasome inhibitor (MG-132), co-immunoprecipitation, immunoblotting in COS cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple biochemical methods (cycloheximide chase, proteasome inhibitor, Co-IP) in a single study\",\n      \"pmids\": [\"20630877\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Neuronal expression of FIG4 is both necessary and sufficient to prevent spongiform neurodegeneration in vivo; conditional inactivation of Fig4 specifically in neurons (synapsin-Cre) recapitulates the full spectrum of neurological abnormalities, while astrocytic expression of Fig4 prevents autophagy marker accumulation but not spongiform degeneration or lethality\",\n      \"method\": \"Conditional knockout (floxed allele × synapsin-Cre), neuron-specific transgenic rescue (NSE promoter), GFAP promoter-driven astrocyte rescue, histology\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean cell-type-specific KO and rescue with defined neurological and histological phenotypes\",\n      \"pmids\": [\"22581779\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"FIG4 is required for CNS myelination; Fig4 null mice show dramatic CNS myelin reduction and oligodendrocyte maturation defects; neuronal (non-cell-autonomous) expression of Fig4 rescues CNS myelination and tremor, demonstrating that FIG4 in neurons supports oligodendrocyte maturation\",\n      \"method\": \"Transgenic rescue with neuron-specific (NSE) promoter, optic nerve electrophysiology, electron microscopy, OL lineage cell counting\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — transgenic rescue + electrophysiology + ultrastructural analysis\",\n      \"pmids\": [\"22131434\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"FIG4 has cell-autonomous roles in both motor neurons and Schwann cells for CMT4J pathogenesis; conditional Fig4 inactivation in motor neurons causes neuronal/axonal degeneration, while conditional inactivation in Schwann cells causes demyelination and defects in autophagy-mediated degradation and myelin biogenesis\",\n      \"method\": \"Cell-type-specific conditional knockout (motor neuron-Cre, Schwann cell-Cre), histology, electron microscopy, autophagy marker analysis\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — independent conditional KO in two cell types with defined cellular phenotypes\",\n      \"pmids\": [\"25187576\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"FIG4 deficiency impairs lysosomal Ca2+ efflux via the TRPML1 channel (whose endogenous ligand is PI(3,5)P2), causing elevated intralysosomal Ca2+, impaired lysosomal fission, and downstream downregulation of dynamin-1 GTPase; pharmacological reactivation of TRPML1 with synthetic ligand ML-SA1 rescues lysosomal storage in Fig4-/- cells and ex vivo DRGs\",\n      \"method\": \"Flow cytometry lysosome size assay, intralysosomal Ca2+ measurement, dynamin-1 immunoblotting, pharmacological rescue (ML-SA1), ex vivo DRG culture\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods linking PI(3,5)P2 to TRPML1 Ca2+ channel and lysosomal fission, pharmacological rescue validation\",\n      \"pmids\": [\"25926456\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"A catalytically inactive FIG4 transgene (Cys486Ser active-site mutant) partially rescues neurodegeneration and juvenile lethality in Fig4 null mice, demonstrating a phosphatase-independent structural/scaffolding function of FIG4 in stabilizing the PI(3,5)P2 biosynthetic complex; however, late-onset defects (hydrocephalus, demyelination) confirm that phosphatase activity is also essential in vivo\",\n      \"method\": \"Active-site mutagenesis (Cys486Ser), in vivo transgenic rescue, vacuolization assay in fibroblasts, histology\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — active-site mutagenesis in vivo with quantitative phenotypic rescue readout\",\n      \"pmids\": [\"26604144\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"The ArPIKfyve-Sac3/FIG4 complex interacts with synphilin-1 (Sph1) in brain; mass spectrometry identified Sph1 as a component of the ArPIKfyve-Sac3 complex; modulation of ArPIKfyve/Sac3 levels alters Sph1-GFP aggregation properties in a Sac3 phosphatase-dependent manner, promoting its cytosolic partitioning and removal by basal autophagy\",\n      \"method\": \"Mass spectrometry of brain-derived interactors, Co-immunoprecipitation, RNA silencing, overexpression in neuronal cell lines and primary cortical neurons, aggregation assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2/3 — MS identification + Co-IP + functional aggregation readout, single lab study\",\n      \"pmids\": [\"26405034\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Drosophila FIG4 (dFIG4) mutations predicted to inactivate phosphatase activity still rescue lysosomal expansion phenotypes in vivo, and Fab1 mutations causing the same phenotype are epistatic, establishing a phosphatase-independent biosynthetic/scaffolding function of FIG4 in lysosomal membrane homeostasis; lysosomal phenotypes are suppressed by genetic inhibition of Rab7 or the HOPS complex, placing FIG4 function after endosome-to-lysosome fusion\",\n      \"method\": \"Drosophila genetics (null mutants, phosphatase-dead transgene, Fab1 epistasis, Rab7/HOPS double mutants), LysoTracker staining, flight ability assay\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — in vivo phosphatase-dead rescue combined with genetic epistasis, multiple orthogonal approaches\",\n      \"pmids\": [\"26662798\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"FIG4 deficiency causes accumulation of TRPV4 at the plasma membrane of patient fibroblasts due to impaired endosomal trafficking/turnover; knockdown of Fig4 in murine motor neurons causes vacuolation and cell death, and inhibition of TRPV4 activity significantly preserves viability\",\n      \"method\": \"Patient fibroblast analysis, Fig4 siRNA knockdown in motor neurons, TRPV4 inhibitor treatment, immunofluorescence of membrane proteins\",\n      \"journal\": \"Journal of neuropathology and experimental neurology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — single lab, immunofluorescence + pharmacological rescue without full mechanistic pathway reconstitution\",\n      \"pmids\": [\"28859335\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Sac3/FIG4 knockdown in RAW264.7 macrophages decreases cell surface scavenger receptor A (SR-A) protein levels and suppresses foam cell formation; ArPIKfyve knockdown similarly decreases Sac3 and SR-A; PIKfyve knockdown has no effect on SR-A, demonstrating that the ArPIKfyve-Sac3 complex regulates SR-A protein levels independently of PIKfyve kinase activity\",\n      \"method\": \"shRNA knockdown, flow cytometry for SR-A surface levels, foam cell assay (acetylated LDL uptake)\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — single lab, shRNA knockdown with defined functional readout, mechanism partially resolved\",\n      \"pmids\": [\"28552585\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Adult-specific inactivation of Fig4 (tamoxifen-inducible global KO) causes wasting, tremor, motor impairment and death within 2 months, demonstrating a life-long requirement; PNS myelinated axons undergo Wallerian degeneration while CNS myelin is intact; FIG4 is additionally required for timely CNS remyelination after chemical lesion\",\n      \"method\": \"Tamoxifen-inducible Cre-mediated KO (CAG-creER), histology of sciatic and optic nerves, compound action potential recording, chemical demyelination challenge\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — conditional adult KO with multiple neurological and histological readouts\",\n      \"pmids\": [\"29688489\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"CMT4J patient fibroblasts show significant reductions in both PtdIns(3,5)P2 (−36%) and PtdIns5P (−43%) compared to controls, measured by HPLC, demonstrating that FIG4 loss reduces PI(3,5)P2 despite FIG4's known role in activating PIKfyve; patients without aberrant vacuoles have especially low PtdIns3P, linking PtdIns3P levels to vacuolization phenotype\",\n      \"method\": \"HPLC phosphoinositide profiling of myo-[2-3H]inositol-labeled primary patient fibroblasts, immunoblotting\",\n      \"journal\": \"Molecular neurobiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — rigorous HPLC lipid profiling across 13 patient and 9 control samples with validated SAC3/FIG4 depletion\",\n      \"pmids\": [\"31313076\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"AAV9-mediated delivery of a codon-optimized FIG4 sequence into Fig4 null mice rescues lethality and peripheral neuropathy when administered neonatally (P1 or P4), with dose-dependent efficacy, providing preclinical proof of concept for gene therapy\",\n      \"method\": \"AAV9 gene delivery in mouse model, survival analysis, neurophysiology, histopathology\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — rigorous preclinical gene therapy study with quantitative neurological and histological endpoints\",\n      \"pmids\": [\"33878035\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"BioID proximity mapping of Vac14 and FIG4 identified 89 high-confidence shared interactors including COPI subunit COPB1 and the GTPase Arf1; proximity ligation assays validated Vac14-COPB1 and Vac14-Arf1 interactions, linking the PIKfyve-VAC14-FIG4 complex to COPI-mediated endosomal dynamics\",\n      \"method\": \"BioID proximity labeling, mass spectrometry, proximity ligation assay\",\n      \"journal\": \"Journal of proteome research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — BioID + PLA validation, single lab, novel interactions not yet functionally dissected\",\n      \"pmids\": [\"34554760\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"FIG4 and VAC14 function in PI(3,5)P2 biosynthesis, which inhibits the lysosomal chloride transporter ClC-7; knockout of CLCN7 in FIG4 null cells corrects lysosomal swelling and partially corrects lysosomal hyperacidification; in Fig4 null mice, reduction of ClC-7 via dominant-negative CLCN7 improved growth, neurological function, and increased lifespan by 20%\",\n      \"method\": \"CLCN7 knockout in FIG4 null cells, dominant-negative CLCN7 mouse model, lysosome size and pH measurement, survival analysis\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis in both cell culture and in vivo with quantitative lysosomal and neurological readouts\",\n      \"pmids\": [\"37363915\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"VAC14 forms a star-shaped pentamer scaffold; two legs bind FIG4, with one also binding PIKfyve; VAC14 oligomerization is critical for Fab1/PIKfyve function, PI(3,5)P2 generation, VAC14 localization to VPS35-containing endosomes, and PIKfyve-VAC14-FIG4 complex formation; pediatric disease mutations at VAC14-VAC14 interfaces disrupt complex assembly\",\n      \"method\": \"AlphaFold2 structural prediction, cryo-EM, pull-down assays in VAC14 KO human cells, fluorescence-detection size-exclusion chromatography, yeast genetics, colocalization with VPS35-endosomes\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — cryo-EM structure + in vitro/cell biochemistry + yeast genetics, multiple orthogonal approaches\",\n      \"pmids\": [\"40305106\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"FIG4 is a PI(3,5)P2 5-phosphatase that functions within a conserved trimeric complex (PIKfyve/FAB1–VAC14–FIG4) on late endosomal/lysosomal membranes, where VAC14 (pentameric scaffold) recruits both the kinase and phosphatase; within this complex FIG4 paradoxically promotes PI(3,5)P2 synthesis by stabilizing PIKfyve activity (via ArPIKfyve/VAC14-mediated protection from proteasomal degradation) while also hydrolyzing PI(3,5)P2, and the resulting signaling lipid regulates TRPML1 Ca2+ channel-dependent lysosomal fission, endolysosomal trafficking, and ClC-7 chloride transport; loss of FIG4 phosphatase or its scaffolding function causes lysosomal enlargement, impaired organelle trafficking, and progressive neurodegeneration in both PNS and CNS.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"FIG4 is a SAC1-domain phosphoinositide phosphatase that hydrolyzes PI(3,5)P2 on late endosomal/lysosomal membranes and simultaneously serves as a scaffolding subunit of the PIKfyve–VAC14–FIG4 ternary complex required for PI(3,5)P2 biosynthesis. Within this complex, VAC14 forms a pentameric scaffold that recruits both PIKfyve (the lipid kinase) and FIG4; FIG4 binding to VAC14 protects FIG4 from rapid proteasomal degradation and is permissive for maximal PIKfyve kinase activity, explaining why FIG4 loss paradoxically reduces PI(3,5)P2 levels and causes lysosomal enlargement [PMID:14528018, PMID:18950639, PMID:20630877, PMID:26604144, PMID:40305106]. PI(3,5)P2 generated by this complex activates the lysosomal Ca²⁺ channel TRPML1 to drive lysosomal fission and inhibits the chloride transporter ClC-7 to maintain lysosomal ion homeostasis; loss of these regulatory circuits underlies the vacuolar storage, spongiform neurodegeneration, and demyelination observed in FIG4-deficient neurons and Schwann cells, the basis of Charcot–Marie–Tooth disease type 4J [PMID:17572665, PMID:25926456, PMID:37363915, PMID:25187576]. Neuronal FIG4 expression is both necessary and sufficient to prevent CNS neurodegeneration and supports non-cell-autonomous oligodendrocyte myelination, while adult-specific loss causes Wallerian degeneration of PNS axons, demonstrating a lifelong requirement [PMID:22581779, PMID:22131434, PMID:29688489].\",\n  \"teleology\": [\n    {\n      \"year\": 2002,\n      \"claim\": \"Identification of FIG4 as the primary PI(3,5)P2 phosphatase resolved which enzyme turns over this signaling lipid in the Fab1 kinase pathway, establishing the first enzymatic function for FIG4.\",\n      \"evidence\": \"Genetic epistasis (fig4Δ suppressor of vac7Δ) with phosphoinositide measurement in yeast\",\n      \"pmids\": [\"11950935\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No in vitro enzymatic characterization yet\", \"Mammalian conservation undemonstrated\", \"No structural information on FIG4\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Demonstration that FIG4 is a Mg²⁺-dependent, PI(3,5)P2-selective phosphatase that physically associates with Vac14 at the vacuole membrane established the enzymatic specificity and placed FIG4 in a membrane-localized regulatory complex.\",\n      \"evidence\": \"In vitro phosphatase assay with purified yeast FIG4, GFP localization, co-immunoprecipitation with Vac14\",\n      \"pmids\": [\"14528018\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry and architecture of the complex unknown\", \"How Vac14 recruits FIG4 to the membrane unresolved\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"The discovery that mammalian FIG4 loss causes reduced PI(3,5)P2, LAMP2-positive vacuole accumulation, and neurodegeneration bridged the yeast enzymology to human disease and established FIG4 as a Charcot–Marie–Tooth 4J disease gene.\",\n      \"evidence\": \"Positional cloning of the pale tremor mouse, phosphoinositide measurement in mutant fibroblasts, nerve conduction studies\",\n      \"pmids\": [\"17572665\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Paradox of PI(3,5)P2 reduction despite loss of its phosphatase unexplained\", \"Cell-type requirements for FIG4 in neurodegeneration undefined\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Mapping of the Fab1–Vac14–FIG4 ternary complex and the parallel mammalian PIKfyve–ArPIKfyve–Sac3 (PAS) complex revealed that the kinase and phosphatase are tethered by a shared scaffold, resolving the paradox that FIG4 loss reduces PI(3,5)P2 by showing FIG4 stabilizes kinase activity.\",\n      \"evidence\": \"Reciprocal co-immunoprecipitation and pull-down assays in yeast; co-IP plus in vitro PIKfyve kinase assay and GLUT4 translocation assay in mammalian cells\",\n      \"pmids\": [\"18653468\", \"18950639\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Quaternary structure of the complex unresolved\", \"How FIG4 activates PIKfyve kinase mechanistically unclear\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Showing that Sac3/FIG4 retains active phosphatase function within the assembled PAS complex and is regulated by insulin established that PI(3,5)P2 turnover and synthesis are coordinated within a single complex, with physiological input from insulin signaling.\",\n      \"evidence\": \"Active-site mutant (D488A) unable to rescue vacuolar phenotype; insulin suppresses Sac3 phosphatase activity in vitro; siRNA modulation of GLUT4 translocation\",\n      \"pmids\": [\"19840946\", \"19578118\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of insulin-mediated Sac3 inhibition unknown\", \"Post-translational modifications regulating Sac3 activity uncharacterized\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Demonstrating that ArPIKfyve stabilizes Sac3/FIG4 by extending its half-life and that the CMT4J I41T mutation abolishes this protection identified the molecular basis of disease: loss of scaffold-mediated stabilization leading to proteasomal degradation of FIG4.\",\n      \"evidence\": \"Cycloheximide chase, MG-132 proteasome inhibitor rescue, co-IP in COS cells; patient fibroblast immunoblotting\",\n      \"pmids\": [\"20630877\", \"21655088\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether residual I41T protein retains any phosphatase activity unresolved\", \"Therapeutic strategies to stabilize FIG4 protein unexplored\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Cell-type-specific conditional knockouts established that neuronal FIG4 is necessary and sufficient to prevent CNS neurodegeneration and additionally supports oligodendrocyte myelination non-cell-autonomously, while Schwann-cell-autonomous FIG4 is required for PNS myelination.\",\n      \"evidence\": \"Synapsin-Cre conditional KO, NSE-promoter rescue, GFAP-promoter astrocyte rescue, motor-neuron and Schwann-cell Cre lines, electron microscopy, optic nerve electrophysiology\",\n      \"pmids\": [\"22581779\", \"22131434\", \"25187576\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Non-cell-autonomous signal from neurons to oligodendrocytes unidentified\", \"Relative contribution of autophagy vs. lysosomal fission defects to degeneration unclear\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Linking FIG4-dependent PI(3,5)P2 to TRPML1 Ca²⁺ channel activation and dynamin-1-mediated lysosomal fission, and separately demonstrating a phosphatase-independent scaffolding function for FIG4 in vivo, bifurcated FIG4's role into enzymatic and structural contributions to lysosomal homeostasis.\",\n      \"evidence\": \"Intralysosomal Ca²⁺ measurement, ML-SA1 pharmacological rescue of Fig4−/− DRGs; catalytically dead C486S transgene partial rescue in Fig4 null mice; Drosophila phosphatase-dead rescue with Fab1/Rab7/HOPS epistasis\",\n      \"pmids\": [\"25926456\", \"26604144\", \"26662798\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Quantitative contribution of scaffolding vs. phosphatase activity across tissues not defined\", \"How FIG4 scaffolding stabilizes PIKfyve at the structural level unknown\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Adult-onset conditional deletion revealed a lifelong requirement for FIG4 distinct from developmental roles, with PNS axons undergoing Wallerian degeneration and CNS remyelination being impaired after injury.\",\n      \"evidence\": \"Tamoxifen-inducible global KO in adult mice, sciatic nerve histology, compound action potential recording, chemical demyelination challenge\",\n      \"pmids\": [\"29688489\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether adult neurodegeneration is reversible upon FIG4 restoration unknown\", \"Temporal window for therapeutic intervention undefined\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Genetic epistasis showing that ClC-7 knockout corrects lysosomal swelling in FIG4-null cells and extends lifespan in Fig4-null mice identified ClC-7 as a key downstream effector of PI(3,5)P2 deficiency, opening a therapeutic axis.\",\n      \"evidence\": \"CLCN7 KO in FIG4-null cells, dominant-negative CLCN7 in Fig4-null mice, lysosome size/pH measurement, survival analysis\",\n      \"pmids\": [\"37363915\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which PI(3,5)P2 inhibits ClC-7 not structurally resolved\", \"Whether ClC-7 inhibition rescues demyelination not tested\", \"Relative contributions of ClC-7 vs. TRPML1 pathways to disease unclear\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Cryo-EM and AlphaFold2 modeling of the VAC14 pentamer revealed the architectural basis for FIG4 and PIKfyve recruitment: two pentamer legs bind FIG4, with one also binding PIKfyve, explaining stoichiometry and linking disease mutations at VAC14–VAC14 interfaces to complex disassembly.\",\n      \"evidence\": \"Cryo-EM structure, AlphaFold2 prediction, pull-downs in VAC14 KO human cells, fluorescence-detection size-exclusion chromatography, colocalization with VPS35-endosomes\",\n      \"pmids\": [\"40305106\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"High-resolution structure of the full PIKfyve–VAC14–FIG4 holo-complex not yet achieved\", \"How VPS35-positive endosomal localization is specified structurally not established\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The full structural basis for how FIG4 simultaneously promotes PI(3,5)P2 synthesis (scaffold) and turnover (phosphatase) within the assembled holo-complex, the identity of the non-cell-autonomous signal from neurons that supports oligodendrocyte myelination, and whether therapeutic modulation of ClC-7 or TRPML1 can substitute for FIG4 gene replacement remain open questions.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No atomic-resolution structure of the complete PIKfyve–VAC14–FIG4 ternary complex\", \"Non-cell-autonomous neuronal signal for myelination unidentified\", \"Relative therapeutic value of ClC-7 vs. TRPML1 modulation vs. gene therapy not determined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [0, 1, 2, 6, 7]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [13, 15]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [1, 7]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005764\", \"supporting_discovery_ids\": [1, 2, 12, 15, 22]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [2, 21, 23]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [1]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 5, 7, 12, 22]},\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [2, 12, 16, 21]},\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [12, 15, 22]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [4, 11, 17572665]}\n    ],\n    \"complexes\": [\n      \"PIKfyve-VAC14-FIG4 (PAS) complex\"\n    ],\n    \"partners\": [\n      \"VAC14\",\n      \"PIKfyve\",\n      \"TRPML1\",\n      \"CLCN7\",\n      \"SNCAIP\",\n      \"COPB1\",\n      \"ARF1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}