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Cited by 175Alzheimer's disease research and treatmentsAmyotrophic Lateral Sclerosis ResearchNeuroinflammation and Neurodegeneration Mechanisms

ELAVL4, splicing, and glutamatergic dysfunction precede neuron loss in MAPT mutation cerebral organoids

Open access
Bowles, Kathryn R., Silva, M. Catarina, Whitney, Kristen, Bertucci, Taylor, Berlind, Joshua E., Lai, Jesse D., Garza, Jacob C., Boles, Nathan C. +17 more·2021·Cell
DOI 10.1016/j.cell.2021.07.003PMC PMC8635409
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Abstract

Frontotemporal dementia (FTD) because of MAPT mutation causes pathological accumulation of tau and glutamatergic cortical neuronal death by unknown mechanisms. We used human induced pluripotent stem cell (iPSC)-derived cerebral organoids expressing tau-V337M and isogenic corrected controls to discover early alterations because of the mutation that precede neurodegeneration. At 2 months, mutant organoids show upregulated expression of MAPT, glutamatergic signaling pathways, and regulators, including the RNA-binding protein ELAVL4, and increased stress granules. Over the following 4 months, mutant organoids accumulate splicing changes, disruption of autophagy function, and build-up of tau and P-tau-S396. By 6 months, tau-V337M organoids show specific loss of glutamatergic neurons as seen in individuals with FTD. Mutant neurons are susceptible to glutamate toxicity, which can be rescued pharmacologically by the PIKFYVE kinase inhibitor apilimod. Our results demonstrate a sequence of events that precede neurodegeneration, revealing molecular pathways associated with glutamate signaling as potential targets for therapeutic intervention in FTD.

AI snapshot

Written from the paper’s full text.

TL;DR

Human iPSC-derived cerebral organoids carrying the tau-V337M MAPT mutation show early ELAVL4 upregulation, glutamatergic/synaptic pathway changes, stress granules, splicing dysregulation, and autophagy-lysosomal dysfunction before later tau/P-tau accumulation and selective glutamatergic neuron loss. Glutamate-induced death of mutant neurons is rescued by PIKFYVE inhibition with apilimod or PIKFYVE ASO.

Key findings

  • At 2 months, tau-V337M organoids show upregulated MAPT, glutamatergic signaling pathways, and ELAVL4, plus increased stress granules; by 6 months they show selective loss of excitatory neurons in deep-layer (ExDp2, p = 0.004), maturing (ExM, p = 0.003), and newborn (ExN, p = 0.0001) categories, with no effect on interneurons or astrocytes.
  • Splicing dysregulation increases from 190 differentially spliced junctions at 2 months to over 5,000 at 6 months; tau and P-tau S396/total tau ratio increase between 4 and 6 months, while LAMP1 and CTSD are increased at 2 months and p62 and LC3-II are reduced at 6 months.
  • ELAVL4 binds MAPT RNA and co-localizes with tau and stress granule markers TIA1/G3BP1; over 50% of differentially spliced genes at later time points overlap with neuronal ELAVL-bound RNA.
  • Repeated 5 mM glutamate triggers more rapid loss of tau-V337M neurons than isogenic controls (p < 0.0001); apilimod fully rescues this death (p < 0.0001), and PIKFYVE ASO improves survival in mutant (p < 0.0001) but not control neurons (p = 0.5369).

Methods

  • Human iPSC lines from three tau-V337M donors and isogenic CRISPR-corrected controls (seven lines total) were differentiated into cerebral organoids and assessed at 2, 4, and 6 months.
  • Bulk RNA-seq was performed on 239 organoids (V337M n = 99, V337V n = 140); cell-hashing scRNA-seq was performed on 339 organoids, yielding 379,064 cells (V337M n = 161,706, V337V n = 217,358).
  • Analyses included SingleR annotation, Seurat/SCTransform, UMAP, Monocle3 pseudotime, GSEA/IPA, LeafCutter splicing, nELAVL RIP-seq intersection, pseudobulk network analysis, IHC/immunoblot/EM, and longitudinal Synapsin1::GFP neuron survival with glutamate, apilimod, or PIKFYVE ASO.

Limitations

  • Stated: low 4R tau expression in the organoid model may miss effects of mutant 4R protein; generalizability to other MAPT mutations and sporadic tauopathies is unknown; microglia are not included; glutamatergic signaling findings require comprehensive electrophysiological validation; and the relative contribution of the identified changes remains to be determined.
  • Evident from the text: organoid models do not accurately reflect adult aging, and variability between lines and individual organoids increased over time, indicating donor and clonal effects on long-term maturation.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1016/j.cell.2021.07.003

Cites

112 of 112 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

49 of 61 cited sources checked · 21 supported · 1 flagged · accuracy not yet measured

  • Premisecitation

    iPSC-based models derived from individuals with a MAPT mutation indicate molecular mechanisms underlying tauopathies.

    “Despite this limitation, iPSC-based models derived from individuals with a MAPT mutation indicate molecular mechanisms underlying tauopathies (Gonzalez et al., 2018; lovino et al., 2015; Jiang et al., 2018; Nakamura et al., 2019; Silva et al., 2016, 2020; Wray, 2017).”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites Gonzalez et al., 2018 source

      The Results identify the patient-derived iPSC model as familial Alzheimer’s disease with a presenilin 1 mutation and Down syndrome, not a MAPT mutation. The Discussion explicitly says tau developed a pathological phenotype in these organoids “without any mutations in the tau gene.” No page number is available in our copy. (LLM, conf=0.88)

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured
    • cites lovino et al., 2015

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites Jiang et al., 2018 source
  • Findingcitation

    Organoid development followed the expected temporal changes.

    “Organoid development followed the expected temporal changes (Lancaster et al., 2013; Velasco et al., 2019; Watanabe et al., 2017; Yoon et al., 2019; Figures 1B–1D; Figure S1A).”
    Quote in the paperNot a substantial claim3 of 3 sources checked
    • cites Lancaster et al., 2013 source
    • cites Velasco et al., 2019 source
    • cites Watanabe et al., 2017 source
  • Premisecitation

    Little 4R tau is expressed in iPSC-derived models without a splice-site mutation.

    “This is important because little 4R tau is expressed in induced pluripotent stem cell (iPSC)-derived models without a splice-site mutation (lovino et al., 2015; Sato et al., 2018; Sposito et al., 2015; Verheyen et al., 2018).”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites lovino et al., 2015

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites Sato et al., 2018 source
    • cites Sposito et al., 2015 source

      The Results section reports no detectable 4R tau mRNA in control iPSC-derived cortical neurons through 100 days of differentiation, supporting the claim for those cultures. But the Results section on extended culture reports robust 4R expression in control neurons at 365 days, so the unqualified statement does not hold across all culture durations. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
  • Interpretationcitation

    The increased tau burden with age in tau-V337M organoids mirrors prior observations in 2D iPSC-derived neuronal models and in the human V337M brain.

    “The increased tau burden with age mirrors prior observations in 2D iPSC-derived neuronal models (Ehrlich et al., 2015; Fong et al., 2013; lovino et al., 2015; Silva et al., 2016, 2020; Wren et al., 2015) and in the human V337M brain (Spillantini et al., 1996; Spina et al., 2017).”
    Quote in the paperNot a substantial claim2 of 3 sources checked
    • cites Ehrlich et al., 2015 source

      The Results section, “Altered Expression of TAU in FTDP-17 iPSC-Derived Neurons” (page unavailable), reports increased tau fragments and increased AT8-positive phosphorylated tau in V337M non-dopaminergic iPSC-derived neurons compared with controls. It does not report that tau burden increased as the neurons aged. Ehrlich et al. is cited for the 2D neuronal-model comparison, not for the separate claim about the human V337M brain. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites Fong et al., 2013 source

      The Results section reports increased phosphorylated tau accumulation in 2D iPSC-derived neurons carrying MAPT A152T. It describes an increase with mutation gene dose, not with age, and does not study V337M. The human V337M brain observation is attributed to other citations in the citing sentence. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites lovino et al., 2015

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Premisecitation

    PIKFYVE inhibition improves proteostasis in neurons.

    “improves proteostasis in neurons (Shi et al., 2018; Staats et al., 2019).”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites Shi et al., 2018 source

      In “Small molecule and genetic regulators of endosomal trafficking rescue patient iMN survival” (page unavailable), the paper reports that PIKFYVE inhibition lowers glutamate-receptor levels and improves survival in C9ORF72 patient motor neurons. It proposes that increased autophagosome–lysosome fusion may help remove dipeptide repeat proteins, but does not establish a general improvement in neuronal proteostasis. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
    • cites Staats et al., 2019 source

      The Main text reports that PIKFYVE inhibition reduced dipeptide-repeat protein aggregates in hippocampal neurons of C9ORF72 mouse models and increased lysosome numbers in C9ORF72-deficient hippocampal neurons. These findings support improved handling of disease-associated proteins, but the paper does not establish a general improvement in neuronal proteostasis beyond those models. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
  • Classified as probably not a substantial claim. 35
  • May combine two claims. 23

  • Classified as probably not a substantial claim.

    Premisecitation

    In iPSC-derived induced neurons, tau-V337M disrupts neuronal activity regulation and excitability.

    “In iPSC-derived induced neurons, tau-V337M disrupts neuronal activity regulation and excitability (Sohn et al., 2019), spurring further studies in more complex models.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Sohn et al., 2019 source

  • Classified as probably not a substantial claim.

    Premisecitation

    FTD-tau is associated with glutamatergic neuronal loss in frontal and temporal cortices.

    “FTD-tau is associated with glutamatergic neuronal loss in frontal and temporal cortices (Benussi et al., 2019).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Benussi et al., 2019 source

      The Abstract reports depletion of glutamatergic pyramidal neurons in the frontal and temporal cortices of patients with FTD. It does not specify that this finding applies to FTD-tau; the Introduction identifies tau as one of several FTD pathologies. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim.

    Premisecitation

    Early alterations in brain development contribute to later manifestation of neurodegenerative diseases.

    “Increasing evidence showing that early alterations in brain development contribute to later manifestation of neurodegenerative diseases (Faravelli et al., 2020) further encourages use of iPSC-based organoid systems to study FTD-tau.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Faravelli et al., 2020 source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Pathologic studies reveal accumulation of filamentous, hyperphosphorylated tau protein in cerebral cortical and hippocampal neurons and glia, and tau redistribution from axons to the somatodendritic compartment.

    “Pathologic studies reveal accumulation of filamentous, hyperphosphorylated tau protein (P-tau) in cerebral cortical and hippocampal neurons and glia, and tau redistribution from axons to the somatodendritic compartment.”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites Bodea et al., 2016 source
    • cites Clare et al., 2010 source

      The 'Synaptic Proteins and AD' section mentions neurons containing neurofibrillary tangles, but does not identify their composition or describe tau pathology. Searches for hyperphosphorylated tau and its redistribution from axons to the somatodendritic compartment found no passage addressing the claim in our copy; page numbers were unavailable. (LLM, conf=0.72)

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    Brain organoids derived from iPSCs develop in culture along a trajectory mimicking key features of fetal brain development and can be patterned toward specific brain regions affected in tauopathy.

    “Brain organoids derived from iPSCs develop in culture along a trajectory mimicking key features of fetal brain development and can be patterned toward specific brain regions affected in tauopathy (Camp et al., 2015; Lancaster et al., 2017; Paşca et al., 2015; Velasco et al., 2019; Yoon et al., 2019).”
    Quote in the paperSubstantial claim1 of 3 sources checked
    • cites Camp et al., 2015 source
    • cites Lancaster et al., 2017 source

      The Online Methods (Preparation of EBs and enCORs) include an iPS cell line, and the main text reports that engineered organoids form forebrain tissue, including regions consistent with hippocampal identity. It also finds that 60-day organoids most closely match early-gestation human forebrain gene expression. The copy does not establish a developmental trajectory matching fetal brain development or connect the patterned regions to tauopathy. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites Paşca et al., 2015

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    These tau changes correlate with degeneration of synapses and neuronal loss in frontal and temporal cortices.

    “These changes correlate with degeneration of synapses and neuronal loss in frontal and temporal cortices (Bodea et al., 2016; Clare et al., 2010).”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites Bodea et al., 2016 source

      The section ‘Role of tau in dendritic spines and synaptic impairment’ links pathological tau to synaptic degeneration and reports neuronal loss in a tau mouse model. The paper also describes frontal and temporal cortical atrophy in FTLD, but it does not establish that the tau changes correlate with both synaptic degeneration and neuronal loss specifically in those cortices. The mouse-model comparison distinguishes tau changes associated with synaptic loss from those associated with neuronal loss. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
    • cites Clare et al., 2010 source

      The ‘Synaptic Proteins and AD’ section reports a correlation between synapse loss and neurons containing neurofibrillary tangles, but also notes that another synaptic marker showed no correlation with tangle density. The FTLD section reports synaptic reductions in frontal and temporal regions. Neither section establishes that tau changes correlate with neuronal loss in those cortices. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    The tau-V337M mutation is located in MAPT exon 12, expressed in iPSC-derived neurons, and associated with FTD with abundant neurofibrillary tangles in frontal and temporal cortices and the cingulate gyrus and atrophy throughout the hippocampus and amygdala.

    “The tau-V337M mutation selected for this study is located in MAPT exon 12, expressed in iPSC-derived neurons, and associated with FTD with abundant neurofibrillary tangles in frontal and temporal cortices and the cingulate gyrus and atrophy throughout the hippocampus and amygdala (Spillantini et al., 1996; Spina et al., 2017; Sumi et al., 1992).”
    Quote in the paperSubstantial claim1 of 3 sources checked
    • cites Spillantini et al., 1996 source
    • cites Spina et al., 2017 source

      Split into 2 atoms — 0 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 1 unverifiable. Expand the claim to see per-atom verdicts.

      Experimental — accuracy not yet measured
    • cites Sumi et al., 1992 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Disruption of the autophagy-lysosomal pathway is a pathological hallmark in human tauopathy brain.

    “Disruption of the autophagy-lysosomal pathway (ALP) is another pathological hallmark in human tauopathy brain (Bain et al., 2019; Piras et al., 2016; Wang et al., 2009).”
    Quote in the paperNot a substantial claim3 of 3 sources checked
    • cites Bain et al., 2019 source

      The Discussion reports lower LAMP-1 levels in human FTLD-tau cases and suggests that deficient lysosomes or defective vesicular transport may impair tau clearance. This supports disruption of the pathway in the FTLD-tau cases studied, but the paper does not establish it as a pathological hallmark across human tauopathies; its mechanistic conclusion is hedged. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
    • cites Piras et al., 2016 source
    • cites Wang et al., 2009 source

      The Discussion (page unavailable) states that the autophagy–lysosomal system is disturbed in Alzheimer disease brain, and the Introduction says lysosomal alterations in Alzheimer disease are well established. The paper does not establish that this disruption is a pathological hallmark across human tauopathies; its own tauopathy experiments use a cell model. (LLM, conf=0.84)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    ELAVL4 regulates glutamate levels and neuronal excitability.

    “glutamate levels, and neuronal excitability (Ince-Dunn et al., 2012; Jung and Lee, 2021)”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites Ince-Dunn et al., 2012 source

      The Results section, “A new role for nElavl in regulating glutamate in the brain” (page not provided), reports reduced glutamate in mice lacking both Elavl3 and Elavl4. The Results section, “Seizures in Elavl3-/- Mice” (page not provided), links abnormal cortical activity to loss of Elavl3. These findings support a role for the neuronal Elav-like proteins collectively, but do not establish that ELAVL4 alone regulates both glutamate levels and neuronal excitability. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites Jung and Lee, 2021 source

      The Introduction identifies HuD as ELAVL4. Section 4.1 reports that mice lacking neuronal ELAV proteins had impaired splicing of genes regulating cellular glutamate levels and spontaneous epileptic seizures. That supports a link to glutamate regulation and excitability, but the passage concerns the neuronal ELAV group, not ELAVL4 individually. (LLM, conf=0.85)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    ELAVL4 expression decreases self-renewal of neural progenitor cells and promotes deep-layer glutamatergic neuronal differentiation.

    “ELAVL4 expression decreases self-renewal of neural progenitor cells and promotes deep-layer glutamatergic neuronal differentiation (Akamatsu et al., 2005; DeBoer et al., 2014; Popovitchenko et al., 2020).”
    Quote in the paperSubstantial claim3 of 3 sources checked
    • cites Akamatsu et al., 2005 source

      The Results (page not provided) show increased self-renewal and reduced neuronal differentiation in neural stem/progenitor cells from HuD-deficient mice, supporting a role for HuD in limiting self-renewal and promoting neuronal differentiation. The paper does not establish that the neurons are specifically deep-layer or glutamatergic, and it tests HuD loss rather than experimentally increasing its expression. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites DeBoer et al., 2014 source

      The Results section, “HuD is critical for the balanced expression of Tle4 in lower layer neocortical neurons” (page unavailable), reports fewer Tle4-positive lower-layer neurons after HuD deletion and suggests a role in their specification and/or maintenance. That supports a connection to deep-layer neuronal identity, but does not establish that ELAVL4 expression promotes differentiation specifically. Our copy does not report that ELAVL4 decreases neural progenitor self-renewal. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites Popovitchenko et al., 2020 source

      The Results section, “Elavl4-v3 promotes lower layer (LL) identity when not repressed in RG” (page unavailable), reports that premature Elavl4-v3 expression in radial glia increased a lower-layer neuronal identity marker, supporting the differentiation part of the claim. It does not establish that ELAVL4 expression decreases neural progenitor self-renewal; the Results section, “Celf1 regulates the balance of glutamatergic neurons” (page unavailable), instead reports greater progenitor retention in a condition with increased Elavl4 protein. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    ELAVL4 binds to the glutamatergic receptor GRM5 and other genes within the long-term potentiation (LTP) pathway.

    “binds to the glutamatergic receptor GRM5 and other genes within the long-term potentiation (LTP) pathway (Bolognani et al., 2010)”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Bolognani et al., 2010 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    ELAVL4 is a target of FUS activity.

    “ELAVL4 is a target of FUS activity”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites Fallini et al., 2012 source

      The Discussion describes FUS as a regulator of neuronal mRNA transport and stability, and separately discusses HuD and other ELAV-family proteins in relation to TDP-43 aggregates. Searches for ELAVL4 as a FUS target and for FUS regulation of HuD found no passage connecting them in our copy. (LLM, conf=0.75)

      Experimental — accuracy not yet measured
    • cites De Santis et al., 2019 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Tau-TIA1 interaction can lead to formation and stabilization of toxic tau oligomers.

    “tau-TIA1 interaction can lead to formation and stabilization of toxic tau oligomers (Ash et al., 2021).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Ash et al., 2021 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    ELAVL4 binds the 3′ UTR of MAPT RNA in rat primary neurons and in mouse P19 cells.

    “ELAVL4 binds the 3′ UTR of MAPT RNA in rat primary neurons and in mouse P19 cells”
    Quote in the paperNot a substantial claim1 of 3 sources checked
    • cites Atlas et al., 2007 source
    • cites Behar et al., 1995 source
    • cites Jung and Lee, 2021 source

      The Introduction identifies HuD as ELAVL4, and §3.1 says HuD regulates tau expression. A reference listed in the paper concerns tau mRNA and HuD protein in differentiated P19 neuronal cells, but our copy does not establish direct binding to the MAPT 3′ UTR in either P19 cells or rat primary neurons. (LLM, conf=0.84)

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim.

    Premisecitation

    ELAVL4 regulates RNA splicing.

    “ELAVL4 regulates RNA splicing (Scheckel et al., 2016)”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Scheckel et al., 2016 source

      The Results section, “nELAVL regulates splicing of human brain targets” (page unavailable), reports altered exon splicing after nELAVL depletion. But the depletion targeted ELAVL2, ELAVL3, and ELAVL4 together, as specified in “nELAVL proteins regulate mRNA abundance of human brain targets” (page unavailable). The paper therefore supports a role for the neuronal ELAVL proteins in splicing, not an independently established role for ELAVL4 alone. (LLM, conf=0.85)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    ELAVL4 is present in TAR DNA-binding protein 43 (TDP-43) inclusions.

    “is present in TAR DNA-binding protein 43 (TDP-43) inclusions (Fallini et al., 2012; De Santis et al., 2019).”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites Fallini et al., 2012 source
    • cites De Santis et al., 2019 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Tau influences stress granule formation in neurons.

    “Tau influences stress granule formation in neurons”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Ash et al., 2021 source

      The Discussion (page unavailable) says tau selectively associates with TIA1-containing stress granules. The paper examines TIA1’s effects on tau phase separation and tests tau toxicity in cultured neurons, but the passages do not show that tau influences stress granule formation in neurons. (LLM, conf=0.82)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    PIKFYVE inhibition increases the number of EEA1-positive endosomes and LAMP1-positive lysosomes.

    “PIKFYVE inhibition increases the number of EEA1-positive endosomes and LAMP1-positive lysosomes”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites Shi et al., 2018 source

      The section on small-molecule regulators of endosomal trafficking (page not provided) reports that PIKFYVE inhibition increased the size of EEA1-positive endosomes, not their number. The lysosomal-biogenesis section attributes restored numbers of LAMP1-positive lysosomes to forced C9ORF72 expression; our copy does not report that PIKFYVE inhibition increased their number. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites Staats et al., 2019 source

  • May combine two claims.

    Premisecitation

    Humans and mice haplodeficient for PIKFYVE are normal.

    “humans and mice haplodeficient for PIKFYVE are normal”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites Ikonomov et al., 2011 source

      The Results section (page unavailable) reports that heterozygous PIKfyve WT/KO mice developed without ostensible defects. But the Discussion (page unavailable) says humans with heterozygous PIKFYVE mutations can have fleck corneal dystrophy, a pathology not seen in the mice. The claim that affected humans are normal is therefore not supported. (LLM, conf=0.90)

      Experimental — accuracy not yet measured
    • cites Sands et al., 2010 source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Microglia contribute to tau pathology, spread, and synaptic degeneration.

    “It would be of interest to include microglia in the model, given their contribution to tau pathology, spread, and synaptic degeneration (Vogels et al., 2019).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Vogels et al., 2019 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductcitation

    The false discovery rate of the differential expression test was estimated using the Benjamini-Hochberg method.

    “The false discovery rate (FDR) of the differential expression test was estimated using the Benjamini-Hochberg (BH) method (Reiner et al., 2003).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Reiner et al., 2003 source

  • Classified as probably not a substantial claim.

    Study design and conductcitation

    Data from different sequencing runs, batches and ages were integrated and scaled using SCTransform while regressing out covariates including percentage of mitochondrial genes, number of cells per parent organoid, number of genes per cell, and number of reads per cell.

    “Data from different sequencing runs, batches and ages were integrated and scaled using SCTransform (Hafemeister and Satija, 2019), while regressing out the following covariates; percentage of mitochondrial genes, the number of cells collected per parent organoid, the number of genes per cell and the number of reads per cell.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Hafemeister and Satija, 2019 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductcitation

    Differential gene expression between MAPT mutation organoids and isogenic controls was predicted by linear mixed model analysis accounting for sample correlations within isogenic lines and differentiation batches.

    “Differential gene expression between MAPT mutation organoids and isogenic controls was predicted by linear mixed model analysis using the variancePartition package (v1.14.1) in the R programming environment (Hoffman and Roussos, 2021) to account for sample correlations within isogenic lines and differentiation batches.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Hoffman and Roussos, 2021 source

  • May combine two claims.

    Findingown data

    Over time, the proportion of upper cortical layer-enriched (ExM-U) neurons increased, as did inhibitory neurons, astrocytes, and oligodendrocytes.

    “Overtime, the proportion of upper cortical layer-enriched (ExM-U) neurons increased, as did inhibitory neurons, astrocytes, and oligodendrocytes (all p < 2.2 × 10−16; Figures 1B–1D).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    IHC analysis showed a reduction in NeuN+ neurons at 4 and 6 months and in MAP2+ neurons at 6 months in tau-V337M organoids.

    “These differences were validated by immunohistochemistry (IHC) analysis, which showed a reduction in neuronal nuclear protein+ (NeuN+) neurons at 4 and 6 months (Figures 2B and 2C) and in MAP2+ neurons at 6 months (Figures S2A and S2B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Lysosome-related multi-membrane lamellar bodies were rare in healthy neurons but more frequent in tau-V337M neurons, including those showing early signs of apoptosis.

    “Lysosome-related multi-membrane lamellar bodies were rare in healthy neurons (Figure 2E) but more frequent in tau-V337M neurons, including those showing early signs of apoptosis (Figure 2F), suggesting ALP dysfunction.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Pro-CTSD was increased significantly at 6 months in the absence of increased active CTSD, whereas p62 and LC3-II were reduced significantly at the same time point.

    “In contrast, pro-CTSD was increased significantly at 6 months in the absence of increased active CTSD, whereas sequestosome 1 (p62) and microtubule-associated protein 1A/1B-light chain 3 (LC3-II) were reduced significantly at the same time point (Figures 2H and 2I), suggesting defects in proteolytic cleavage and activity.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Tau-V337M organoids showed no genotype-dependent differences at 2 months, but a significant increase in total tau and the P-tau S396/total tau ratio emerged between 4 and 6 months compared with isogenic controls.

    “Tau-V337M organoids showed no genotype-dependent differences at 2 months (Figure 2J), but a significant increase in total tau and the P-tau S396/total tau ratio emerged between 4 and 6 months compared with isogenic controls (Figures 2J–2L).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    In Figure 7K, the log-rank test for neuronal survival between isogenic control organoids treated with negative control and those treated with ASO yielded n=120 each, chi-sq=0.3814, p=0.5369.

    “Figure 7K: nisogenic+nc = 120, nisogenic+ASO = 120, Chi-sq = 0.3814, p = 0.5369.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Findingown data

    In Figure 7B, the log-rank test for neuronal survival between mutant and isogenic control organoids yielded n=80 each, chi-sq=3.723, p=0.0537.

    “for Figures 7B: nisogenic = 80, nmutant = 80, Chi-sq = 3.723, p = 0.0537;”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Study design and conductown data

    There was no batch-specific effect on differentiation over three replicates and no clustering because of mutation at 2 or 4 months.

    “There was no batch-specific effect on differentiation over three replicates assessed by uniform manifold approximation and projection (UMAP) (Figure S1B) and no clustering because of mutation at 2 or 4 months (Figure S1C).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Transcriptional signatures in the human frontal cortex at gestational week 17-18 correlate highly with gene expression profiles of organoids.

    “Transcriptional signatures in the human frontal cortex at this age correlate highly with gene expression profiles of organoids (Qian et al., 2016).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Qian et al., 2016 source

  • May combine two claims.

    Interpretationreasoning

    PIKFYVE inhibition may rescue glutamate-induced neurodegeneration by correcting ALP dysfunction, increasing turnover of misfolded tau, and favoring elimination of internalized NMDA and AMPA receptors over recycling back to the synapse.

    “Therefore, PIKFYVE inhibition may rescue glutamate-induced neurodegeneration by correcting ALP dysfunction, increasing turnover of misfolded tau, and favoring elimination of internalized NMDA and AMPA receptors over recycling back to the synapse.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    iPSC-tau-V337M organoids phenocopy aspects of ALP dysfunction seen in the FTD brain, which may be triggered by expression of mutant tau protein in neurons early in disease development.

    “These findings demonstrate that iPSC-tau-V337M organoids phenocopy aspects of ALP dysfunction seen in the FTD brain, which may be triggered by expression of mutant tau protein in neurons early in disease development.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Increased HMW ubiquitinated species in tau-V337M organoids at 2 months suggests disrupted degradation and accumulation of poly-ubiquitinated proteins and increased proteostasis stress.

    “High-molecular-weight (HMW) ubiquitinated species were increased significantly in tau-V337M organoids at 2 months, suggesting disrupted degradation and accumulation of poly-ubiquitinated proteins and, thus, increased proteostasis stress in mutant organoids.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Interpretationnone given

    Dysfunctional proteostasis may result in increased levels of tau and ELAVL4 because of impaired protein clearance, promoting their cytosolic accumulation.

    “Additionally, dysfunctional proteostasis may result in increased levels of tau and ELAVL4 because of impaired protein clearance, promoting their cytosolic accumulation.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    Perturbed ELAVL4 expression and localization could contribute to several phenotypes observed in tau-V337M organoids, including aberrant MAPT/tau expression, early neuronal maturation, aberrant glutamate signaling, and dysregulated splicing.

    “Given its pleiotropic functions, our observation of perturbed ELAVL4 expression and localization could contribute to several phenotypes observed in tau-V337M organoids, including aberrant MAPT/tau expression, early neuronal maturation, aberrant glutamate signaling, and dysregulated splicing.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    All presented differential expression, GSEA, pathway enrichment and splicing analyses p values were adjusted for multiple test correction.

    “All presented differential expression, GSEA, pathway enrichment and splicing analyses p values were adjusted for multiple test correction.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    Two organoids were excluded from further bulk RNA-sequencing analysis due to inconsistent positioning away from the remaining samples by principal components analysis.

    “Two organoids were excluded from further analysis due to inconsistent positioning away from the remaining samples by principal components analysis”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationreasoning

    Additional effects of the mutant 4R protein may be missed due to low 4R tau expression in these organoid models.

    “Given the low 4R tau expression in these organoid models, additional effects of the mutant 4R protein may be missed.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: The organoid models have low 4R tau expression.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    The resulting single-cell RNA-sequencing dataset for analysis included 379,064 cells (V337M n=161,706, V337V n=217,358).

    “The resulting dataset for analysis included 379,064 cells (V337M n = 161,706, V337V n = 217,358).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Protein bands' densitometry was normalized to the respective internal control (GAPDH) band.

    “Protein bands’ densitometry (pixel mean intensity in arbitrary units, a.u.) was measured with ImageJ and normalized to the respective internal control (GAPDH) band.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Data was also corrected for differentiation batch.

    “Data was also corrected for differentiation batch.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    The final bulk RNA-sequencing dataset consisted of 239 organoids (V337M n=99, V337V n=140).

    “resulting in a final dataset consisting of 239 organoids (V337M n = 99, V337V n = 140).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S4 (based).

    Interpretationnone given

    The MAPT mutation results in accumulation of mutant tau.

    “In our working model, the MAPT mutation results in accumulation of mutant tau”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Study design and conductnone given

    All reported p values were adjusted for multiple test correction, with significance defined as adjusted p < 0.05.

    “All reported p values were adjusted for multiple test correction; Significance was defined as adjusted p < 0.05.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    For every set of slides stained, a neuropathologically confirmed severe AD case and healthy control case was included as a batch control.

    “For every set of slides stained, a neuropathologically confirmed severe AD case and healthy control case was included as a batch control.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductown data

    Pathological changes assessed using semiquantitative analysis were scored by two researchers blinded to mutational and control conditions.

    “Pathological changes assessed using semiquantitative analysis were scored by two researchers (JFC and KW) blinded to mutational and control conditions using a semiquantitative scoring system”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    Of the single-cell RNA-sequencing organoids, 339 passed quality control, determined by samples containing detectable cells with clear HTO identities.

    “of which 339 passed quality control, determined by samples containing detectable cells with clear HTO identities.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing).

    Study design and conductnone given

    The data characterizing glutamatergic signaling in tau-V337M organoids require additional validation by comprehensive electrophysiological assessment to confirm the hypothesized mechanism.

    “Our data characterizing glutamatergic signaling in tau-V337M organoids require additional validation by comprehensive electrophysiological assessment to confirm our hypothesized mechanism.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    These changes are initially accommodated by plasticity, but over time they result in glutamatergic neuron death.

    “Although these changes are initially accommodated by plasticity, over time they result in glutamatergic neuron death.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    Band intensity was normalized against total protein by Ponceau S staining and expressed relative to the normalized control within each blot.

    “Band intensity was analyzed using GeneTools, normalized against total protein by Ponceau S staining of each blot, and band intensity was expressed relative to the normalized control within each blot.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    Accumulation of mutant tau alters glutamatergic synaptic signaling via impaired proteostasis, preventing recycling of receptors away from the synapse and promoting further tau accumulation.

    “and second by impaired proteostasis, preventing recycling of receptors away from the synapse and promoting further tau accumulation.”
    Quote in the paperSubstantial claim
53 of 53

  • The authors state this as an assumption.

    Organoid models do not accurately reflect adult aging.

    “Given that organoid models do not accurately reflect adult aging, it is interesting that we observed neurodegeneration.”

  • The paper relies on this without support.

    MAPT is alternatively spliced into 3R and 4R tau isoforms by exclusion or inclusion of exon 10.

    “MAPT is alternatively spliced, resulting in two major isoforms, 3R and 4R tau, defined by exclusion or inclusion of exon 10, respectively.”

  • The paper relies on this without support.

    Temporal analysis of human organoid models has revealed early pathological events in neurodegeneration.

    “Temporal analysis of human organoid models has revealed early pathological events in neurodegeneration.”

  • The paper relies on this without support.

    The organoid models have low 4R tau expression.

    “Given the low 4R tau expression in these organoid models”
4 of 4

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

  • Single-cell count exceeds the stated per-organoid range

    Minor

    The reported final dataset contains 379,064 cells from 339 organoids, or about 1,118 cells per organoid-above the stated approximate range of 800-1,000.

    Reported 1000 · recomputed 1118.182891

1 of 1

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Authors
Bowles, K. R., Silva, M. C., Whitney, K., Bertucci, T., Berlind, J. E., Lai, J. D., Garza, J. C., Boles, N. C., Mahali, S., Strang, K. H., Marsh, J. A., Chen, C., Pugh, D. A., Liu, Y., Gordon, R. E., Goderie, S. K., Chowdhury, R., Lotz, S., Lane, K., Crary, J. F., Haggarty, S. J., Karch, C. M., Ichida, J. K., Goate, A. M., & Temple, S.
Affiliations
  • Icahn School of Medicine at Mount Sinai (US)
  • Harvard University (US)
  • Neural Stem Cell Institute (US)
  • University of Southern California (US)
  • Washington University in St. Louis (US)
Corresponding author
  • Goate, Alison M.
  • Temple, Sally
ORCID
Venue
Cell
Publisher
Elsevier BV
Published
2021-08
Volume
184
Issue
17
Pages
4547-4563.e17
ISSN
0092-8674 (ISSN-L)
DOI
10.1016/j.cell.2021.07.003
PMC
PMC8635409
OpenAlex
W3183726003
Cited by
175
References
112
Citations by year
2026: 32 · 2025: 35 · 2024: 37 · 2023: 29 · 2022: 37 · 2021: 5
FWCI
14.05
Type
journal article
Language
en
Open access
Licence
Locations
  • Cell (publishedVersion)
  • PubMed (publishedVersion)
  • PubMed Central (submittedVersion)
Funders
  • National Institutes of Health (AG046374)
  • New York State Stem Cell Science (C029158)
  • National Institute of Neurological Disorders and Stroke (R35 NS097277, F31NS117075)
  • Harrington Discovery Institute, University Hospitals
  • Hope Center for Neurological Disorders
  • Eisai
  • Rainwater Charitable Foundation
  • New York Genome Center (U54NS092089, U01AG045390)
  • U.S. Department of Defense (W81XWH-21-1-0168, W81XWH-21-1-0131, W81XWH-20-1-0424)
  • National Institute on Aging (R01 AG056293, 2R01NS097850)
  • John Douglas French Alzheimer&apos;s Foundation
  • Biogen
  • ALS Association
  • Association for Frontotemporal Degeneration
  • AbbVie
  • BrightFocus Foundation
  • Genentech
  • Alzheimer&apos;s Drug Discovery Foundation
  • Denali Therapeutics
  • Tau Consortium
  • California Institute for Regenerative Medicine
  • New York Stem Cell Foundation
  • GlaxoSmithKline
  • Sächsische Aufbaubank
Topics
  • Health Sciences › Medicine › Physiology › Alzheimer's disease research and treatments
  • Health Sciences › Medicine › Neurology › Amyotrophic Lateral Sclerosis Research
  • Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
Keywords
frontotemporal dementia, MAPT mutations, ELAVL4, RNA-binding proteins, alternative splicing, glutamatergic neurons, glutamate excitotoxicity, stress granules, autophagy, tau accumulation, brain organoids, iPSC-derived neurons, apilimod, neurodegeneration
MeSH terms
ELAV-Like Protein 4, Stress Granules, Autophagy, Cell Line, Humans, Hydrazones, Lysosomes, Morpholines, Mutation, Neurons, Organoids, Phosphorylation, Pyrimidines, RNA Splicing, Synapses, Signal Transduction, Biomarkers, Up-Regulation, tau Proteins, Cell Death, Glutamic Acid, Body Patterning, Cerebrum

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-23.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-23.

Cited sources are checked by gpt-6-sol.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

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