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Cited by 247Pluripotent Stem Cells ResearchAlzheimer's disease research and treatmentsNeurogenesis and neuroplasticity mechanisms

Age-dependent instability of mature neuronal fate in induced neurons from Alzheimer’s patients

Open access
Mertens, Jerome, Herdy, Joseph R., Traxler, Larissa, Schafer, Simon T., Schlachetzki, Johannes C.M., Böhnke, Lena, Reid, Dylan A., Lee, Hyungjun +17 more·2021·Cell Stem Cell
DOI 10.1016/j.stem.2021.04.004PMC PMC8423435
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Abstract

Sporadic Alzheimer's disease (AD) exclusively affects elderly people. Using direct conversion of AD patient fibroblasts into induced neurons (iNs), we generated an age-equivalent neuronal model. AD patient-derived iNs exhibit strong neuronal transcriptome signatures characterized by downregulation of mature neuronal properties and upregulation of immature and progenitor-like signaling pathways. Mapping iNs to longitudinal neuronal differentiation trajectory data demonstrated that AD iNs reflect a hypo-mature neuronal identity characterized by markers of stress, cell cycle, and de-differentiation. Epigenetic landscape profiling revealed an underlying aberrant neuronal state that shares similarities with malignant transformation and age-dependent epigenetic erosion. To probe for the involvement of aging, we generated rejuvenated iPSC-derived neurons that showed no significant disease-related transcriptome signatures, a feature that is consistent with epigenetic clock and brain ontogenesis mapping, which indicate that fibroblast-derived iNs more closely reflect old adult brain stages. Our findings identify AD-related neuronal changes as age-dependent cellular programs that impair neuronal identity.

AI snapshot

Written from the paper’s full text.

TL;DR

Directly converted induced neurons (iNs) from Alzheimer’s disease patients retain age-equivalent neuronal identity and show a hypo-mature, de-differentiated state with loss of mature neuronal gene expression and functional deficits. This AD-related instability is age-dependent: rejuvenated iPSC-derived neurons from the same patients show no significant disease transcriptome signature.

Key findings

  • AD iNs had 778 highly significant DE genes (FDR < 0.01), while parental fibroblasts showed no significant AD-specific DE genes; AD iN transcriptomes overlapped with post-mortem AD brain and neuronal single-nucleus AD signatures.
  • Downregulated genes in AD iNs were enriched for mature neuronal categories including synaptic transmission, oxidative phosphorylation, neuronal action potential, ion transport, and mitochondria; AD iNs showed reduced dendritic complexity, fewer synapsin and synapsin/PSD-95 puncta, and reduced spontaneous Ca²⁺ event frequency.
  • Upregulated genes in AD iNs were enriched for cell cycle, stem/progenitor maintenance, stress, apoptosis, senescence, and cancer-related pathways; ATAC-seq showed on average 36% more open chromatin peaks that were 7% wider, with 7,188 differentially accessible peaks, 97% gained in AD.
  • Rejuvenated iPSC-derived neurons showed no significant DE genes between control and AD subjects; iN DNA methylation ages reflected donor adult/old ages, whereas iPSC derivatives reflected pre-natal ages, and AD iNs did not show accelerated epigenetic aging over controls.

Methods

  • Fibroblasts from 16 AD subjects (13 sporadic, 3 familial) and 19 age- and sex-matched non-demented controls were directly converted into iNs using Ngn2/Ascl1-based, small-molecule-enhanced conversion and PSA-NCAM FACS isolation.
  • Assays included whole-genome RNA-seq, ATAC-seq, DNA methylation EPIC arrays, immunofluorescence, ELISA for Aβ42/40, ROS measurement, live-cell Ca²⁺ imaging, and morphological reconstruction.
  • A subset (n = 20) was reprogrammed to iPSCs and converted to iPSC-iNs as rejuvenated isogenic controls; transcriptomes were compared with post-mortem bulk and single-nucleus AD data, BrainSpan fetal/adult brain samples, and published neuronal differentiation trajectories.

Limitations

  • Stated in the paper: fibroblasts carry skin-specific, age-related changes that might interfere with AD-related iN changes; some brain-specific changes might not be captured; Ngn2/Ascl1 overexpression might override endogenous differences; other iPSC differentiation models might recapitulate AD phenotypes more prominently; and the cohort might not fully represent all AD patients.
  • Evident from the text: the model is in vitro and fibroblast-derived, and although AD iNs showed cell cycle re-entry markers and increased DNA content above diploid, no completed neuronal DNA replication was detected, so conclusions about cell cycle re-entry rely on markers and aneuploidy rather than completed division.

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

Integrity signals

Registry

Crossref · 10.1016/j.stem.2021.04.004

Cites

109 of 109 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 57 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    AD iNs reflect a hypo-mature neuronal identity characterized by markers of stress, cell cycle, and de-differentiation.

    “Mapping iNs to longitudinal neuronal differentiation trajectory data demonstrated that AD iNs reflect a hypo-mature neuronal identity characterized by markers of stress, cell cycle, and de-differentiation.”
    Quote in the paperSubstantial claim
  • Findingown data

    AD iNs showed a reduced frequency of Ca2+ neuronal activity events compared with control iNs.

    “We used GcAMP6m-based live-cell Ca2+ imaging to assess spontaneous neuronal activity, and we detected a reduced frequency of Ca2+ neuronal activity events in AD iNs (Figures 3I and 3J).”
    Quote in the paperSubstantial claim
  • Findingnone given

    These GO and KEGG categories were also enriched within upregulated genes in post-mortem AD data.

    “These GO and KEGG categories were also enriched within upregulated genes in post-mortem AD data.”
    Quote in the paperSubstantial claim
  • Findingown data

    These top-ranked downregulated categories were also enriched within downregulated genes in post-mortem AD, indicating a loss of genes promoting neuronal differentiation.

    “These top-ranked categories were also enriched within downregulated genes in post mortem AD, further indicating a loss of genes promoting the differentiation of neurons (Figure 3B; Table S5).”
    Quote in the paperSubstantial claim
  • Findingown data

    GSEA of the most significantly upregulated AD iN DE genes indicated enrichment for cell cycle control, stem/progenitor cell maintenance, signal transduction pathways, apoptosis, and cancer-related pathways.

    “GSEA of the most significantly (padj < 0.01) upregulated AD iN DE genes indicated that these genes were enriched for a broad semantic spectrum around cell cycle control, stem/progenitor cell maintenance, signal transduction pathways, apoptosis, and cancer-related pathways (Figure 4A; Table S5).”
    Quote in the paperSubstantial claim
  • May combine two claims. 40
  • Classified as probably not a substantial claim. 13

  • May combine two claims.

    Findingown data

    AD patient-derived iNs exhibit strong neuronal transcriptome signatures characterized by downregulation of mature neuronal properties and upregulation of immature and progenitor-like signaling pathways.

    “AD patient-derived iNs exhibit strong neuronal transcriptome signatures characterized by downregulation of mature neuronal properties and upregulation of immature and progenitor-like signaling pathways.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Fibroblasts did not display any significant AD-specific DE genes, whereas 778 highly significant (FDR < 0.01) DE genes were identified in iNs.

    “Unsurprisingly, fibroblasts did not display any significant AD-specific DE genes, whereas 778 highly significant (false discovery rate [FDR] < 0.01) DE genes were identified in iNs (Figure 2A; Table S3).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    GSEA of overlapping AD genes showed enrichment for mature neuronal categories such as synaptic transmission and oxidative phosphorylation (downregulated) and response to stress, regulation of cell proliferation, and pathways in cancer (upregulated).

    “Gene set enrichment analysis (GSEA) of overlapping AD genes showed that they were enriched for mature neuronal categories such as synaptic transmission and oxidative phosphorylation (downregulated; Figure 2E) and response to stress, regulation of cell proliferation, and pathways in cancer (upregulated; Figure 2F).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Control and AD iNs separated clearly by tSNE based on DE genes, confirming an AD-specific transcriptome signature that unified most patient iN samples, despite heterogeneity from four outlier samples.

    “The validity of the DE genes was evidenced by a clear separation of control and AD iNs by t-distributed stochastic neighbor embedding (tSNE) that largely confirmed the presence of an AD-specific transcriptome signature that unified most patient iN samples, despite some heterogeneity driven by four outlier samples (Figure 2C).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    AD iNs consistently showed the highest overlap with, and the best correlation to, neuronal cell-type-specific AD signatures from single-nucleus RNA-seq data.

    “AD iNs consistently showed the highest overlap with, and the best correlation to, the neuronal cell-type-specific AD signatures (Figure 2H), with downregulated synapse-related and upregulated stress-related gene categories commonly visible in both systems (Figure 2I).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Top GO terms for upregulated AD iN genes identified overrepresentation of genes involved in cellular stress response, stem cell-related signal transduction, aging, and cell death competence; top KEGG pathways were related to cancer and senescence.

    “The top significantly ranked Gene Ontology (GO) terms also identified an overrepresentation of genes involved in cellular stress response, stem cell-related signal transduction, aging, and cell death competence; the top significantly ranked Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were related to pathways involved in cancer and senescence (Figures 4B and 4C).”
    Quote in the paperSubstantial claim

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

    Findingown data

    iNs express both glutamatergic and GABAergic marker genes.

    “Consistent with the immunocytochemistry and results from previous studies (Kim et al., 2018; Vadodaria et al., 2016), we detected expression of both glutamatergic and GABAergic marker genes in the iNs (Figure 1H).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Gene sets previously found downregulated in AD brains were typically also downregulated in AD iNs, and upregulated gene sets were also upregulated in AD iNs.

    “Indeed, the gene sets that were previously found to be downregulated in AD brains were typically also downregulated in AD iNs, and upregulated gene sets were also upregulated in AD iNs (Figure 2D; Table S4).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Ngn2/Ascl1-based, small molecule-enhanced iN conversion and PSA-NCAM FACS isolation resulted in cultures of ∼90% βIII-tubulin-positive neuronal cells that readily expressed NeuN.

    “Ngn2/Ascl1-based, small molecule-enhanced iN conversion and subsequent polysialylated neural cell adhesion molecule (PSA-NCAM) fluorescence-activated cell sorting (FACS) isolation resulted in cultures of ∼90% βIII-tubulin-positive neuronal cells that readily expressed NeuN (Figures 1C–1E).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Within the AD iN group, there were slightly more neurons with both lower and higher DNA content, with the increase of AD iNs over the diploid level being significant.

    “Within the AD iN group, we observed slightly more neurons with both lower and higher DNA content, with the increase of AD iNs over the diploid level being significant (Figure S2), but we could not detect a completed neuronal DNA replication in iNs (Figures 5E and 5F; Figure S2).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    AD iNs showed changes in DNA damage repair-related gene expression and increased numbers of nuclear H2A.X-positive foci.

    “Indeed, AD iNs displayed increased levels of total ROS (Figure 5A), and we also detected changes in DNA damage repair-related gene expression and increased numbers of nuclear H2A.X-positive foci in AD iN nuclei (Figure S2).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Genes both most upregulated during differentiation and decreased in AD iNs relate to synaptic functions, whereas genes downregulated during differentiation and increased in AD iNs hint toward changes in cell cycle control.

    “The genes that are both the most upregulated during differentiation and also decreased in AD iNs relate to synaptic functions, whereas the genes that are downregulated during differentiation and increased in AD iNs hint toward changes in cell cycle control (Figure 4G).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    ATAC-seq revealed a widespread increase in the number and width of open chromatin regions in AD iNs, which on average displayed 36% more peaks that were 7% wider.

    “ATAC-seq revealed a widespread increase in the number and width of open chromatin regions in AD iNs, which on average displayed 36% more peaks that were 7% wider (Figures 6B and 6C).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Control iNs showed a clear increase in similarity with progressive differentiation, whereas AD iNs showed little increase of similarity with progressive differentiation and higher similarity to immature stages.

    “Control iNs showed a clear increase in similarity with progressive differentiation, whereas AD iNs showed little increase of similarity with progressive differentiation and higher similarity to immature stages (Figure 4F).”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Findingown data

    Cell cycle markers CCNB1 (cyclin B1) and CDK2 become silenced during neuronal differentiation.

    “ROS and DNA damage can trigger neuronal aneuploidy and expression of cell cycle re-entry markers in AD brains (Mosch et al., 2007), and the cell cycle markers CCNB1 (cyclin B1) and CDK2 become silenced during neuronal differentiation (Figure 5B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Both iNs and iPSC-iNs show the highest transcriptome similarity to cortical excitatory neurons from single-cell RNA-seq data.

    “Similar to iNs, iPSC-iN cultures contained vGlut1+ and GABA+ neurons and expressed marker genes for both subtypes, and both showed the highest similarity to cortical excitatory neurons from single-cell RNA-seq (scRNA-seq) data (Figure 7D; Figure S5; Mathys et al., 2019).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Mathys et al., 2019 source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    Differential peak analysis revealed 7,188 highly significantly differentially accessible peaks, 97% of which were gained in AD.

    “Differential peak analysis revealed 7,188 highly significantly differentially accessible peaks, 97% of which were gained in AD (Figure 6E).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    iPSC-iN cultures contain vGlut1+ and GABA+ neurons and express marker genes for both subtypes.

    “Similar to iNs, iPSC-iN cultures contained vGlut1+ and GABA+ neurons and expressed marker genes for both subtypes, and both showed the highest similarity to cortical excitatory neurons from single-cell RNA-seq (scRNA-seq) data (Figure 7D; Figure S5; Mathys et al., 2019).”
    Quote in the paperNot a substantial claim

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

    Findingcitation

    Conversion of iPSCs to iPSC-iNs is highly efficient, with virtually all EGFP-positive iPSCs converting into neurons that predominantly express NeuN and vGlut1.

    “As shown previously, conversion of iPSCs to iPSC-iNs is highly efficient, as virtually all EGFP-positive iPSCs convert into neurons that also predominantly express NeuN and vGlut1 (Figures S5; Schafer et al., 2019).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Schafer et al., 2019

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    iPSC-iNs show more gene expression differences than iPSCs or fibroblasts, but these differences do not reach statistical significance.

    “iPSC-iNs showed more gene expression differences than iPSCs or fibroblasts, which did not reach statistical significance (Figure 7F).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    iPSC-iNs show relatively high transcriptome similarity to pre-natal brain samples but poor correlation with adult brain stages.

    “As expected, we found that iPSC-iNs showed a relatively high degree of similarity with the transcriptomes of the pre-natal brain samples but exhibited relatively poor correlation with adult brain stages (Figure 7L; Figure S6).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Plotting AD iN DE genes across healthy aging iNs does not indicate their regulation by age; rather, a slightly inverse trend is detected.

    “Consistently, aging genes in iNs previously identified by RNA-seq from a healthy aging cohort did not show extended changes in AD iNs, and plotting AD iN DE genes across healthy aging iNs did not indicate their regulation by age; rather, a slightly inverse trend was detected (Figure 7J; Figure S6; Mertens et al., 2015a).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Mertens et al., 2015a

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    Single-nucleus RNA-seq integration shows a similar pattern for excitatory and inhibitory neurons and indicates that adult transcriptome identity is encoded primarily within excitatory neuronal profiles.

    “Integration of single-nucleus RNA-seq data showed a similar pattern for excitatory and inhibitory neurons and further indicated that adult transcriptome identity is encoded primarily within excitatory neuronal profiles (Figure S6).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Along a PCA-based pre-natal versus adult variance dimension, iNs map close to adult brain samples, whereas iPSC-iNs map close to pre-natal samples.

    “Furthermore, along a PCA-based pre-natal versus adult variance dimension, iNs also mapped close to adult brain samples, whereas iPSC-iNs mapped close to pre-natal samples (Figure S6).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    iNs display the highest transcriptome similarity to adult brain samples but not to pre-natal samples.

    “Conversely, iNs displayed the highest similarity to the adult brain samples but not to pre-natal samples, and no similarity differences between control and AD iNs were detected (Figure 7L; Figure S6).”
    Quote in the paperSubstantial claim

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

    Premisecitation

    Sporadic AD cases develop without clear genetic etiology and represent the vast majority of AD cases, with disease risk increasing exponentially with age.

    “However, familial AD is comparatively rare, and sporadic AD cases that develop without clear genetic etiology represent the vast majority of cases, wherein disease risk increases exponentially with age (Querfurth and LaFerla, 2010).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Querfurth and LaFerla, 2010 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Direct conversion of human fibroblasts into iNs bypasses the embryo-like state and directly results in postmitotic neurons with mature marker expression and functionality.

    “In contrast, direct conversion of human fibroblasts into induced neurons (iNs) bypasses the embryo-like state and directly results in postmitotic neurons with mature marker expression and functionality (Liu et al., 2013; Pang et al., 2011).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites Liu et al., 2013 source

      Not checked in the free run.

    • cites Pang et al., 2011

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  • May combine two claims.

    Premisecitation

    Reprogramming into pluripotency leaves old donor-derived iPSC-derived neurons with practically no traces of their donors' ages, and iPSC-associated cellular rejuvenation represents a major challenge when attempting to model age-dependent diseases.

    “However, reprogramming into pluripotency leaves old, donor-derived iPSC-derived neurons with practically no traces of their donors’ ages, and iPSC-associated cellular rejuvenation represents a major challenge when attempting to model age-dependent diseases (Böhnke et al., 2018; Kang et al., 2016; Mertens et al., 2015a; Miller et al., 2013; Tang et al., 2017).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Böhnke et al., 2018

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    • cites Kang et al., 2016

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    • cites Mertens et al., 2015a

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  • May combine two claims.

    Study design and conductown data

    Gene expression differences between control and AD groups largely aligned with clinical diagnosis, whereas other classifiers did not appear to bias the detected DE genes.

    “Gene expression differences between the groups largely aligned with clinical diagnosis, whereas other classifiers did not appear to bias the detected DE genes (Figure 2B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The hypo-mature state of AD iNs partially resembles both malignant transformation-like signatures and immature neuronal stages.

    “This hypo-mature state partially resembles both malignant transformation-like signatures and immature neuronal stages.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    AD iNs gained accessibility at normally silenced LDHA promoter regions, where gained accessibility leads to leaky expression and thus promotion of an immature neuronal state, stem cell- and cancer-associated signaling pathways, and precursor-like metabolic properties.

    “Furthermore, the chromatin landscape signatures at key genes such as LDHA showed that AD iNs gained accessibility at normally silenced promoter regions, where gained accessibility leads to leaky expression and thus promotion of an immature neuronal state, stem cell- and cancer-associated signaling pathways, and precursor-like metabolic properties (Figure 6G).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    AD iN-specific chromatin landscape changes accommodate a persistent lack of a differentiated identity and suggest that epigenetic changes might underlie the transcriptional and functional lack of the differentiated state in AD neurons.

    “Overall, the AD iN-specific chromatin landscape changes accommodate a persistent lack of a differentiated identity and suggest that epigenetic changes might underlie the transcriptional and functional lack of the differentiated state in AD neurons.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Stem and progenitor cells, as well as transformed cells, typically reflect a globally open chromatin state, whereas differentiated cells possess a more defined and closed chromatin landscape.

  • May combine two claims.

    Interpretationown data

    AD iNs successfully completed iN conversion but showed a lack of mature neuronal identity gene expression.

    “AD iNs thus successfully completed iN conversion but showed a lack of mature neuronal identity gene expression.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    AD iNs show signs of elevated ROS, DNA damage, and aneuploid DNA contents, which may trigger neuronal de-differentiation and lead to an aberrant hypo-mature cellular state that partially mirrors a stem/progenitor-associated transcriptional program and expression of non-neuronal proteins.

    “Thus, AD iNs show signs of elevated ROS, DNA damage, and aneuploid DNA contents, which may trigger neuronal de-differentiation and lead to an aberrant hypo-mature cellular state that partially mirrors a stem/progenitor-associated transcriptional program and expression of non-neuronal proteins, including markers of cell cycle and non-neuronal energy metabolism.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: LDHA is inversely associated with mature neuronal identity.

  • May combine two claims.

    Study design and conductown data

    Both control and AD iNs showed equally increasing similarity paralleling progressive iN conversion, and they exhibited equivalent conversion efficiencies, loss of fibroblast signatures, and iN identity marked by NeuN.

    “Both control and AD iNs showed an equally increasing similarity, paralleling progressive iN conversion, and they exhibited equivalent conversion efficiencies, loss of fibroblast signatures, and iN identity marked by NeuN (Figures 1D and 1E; Figures S1 and S3).”
    Quote in the paperSubstantial claim

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

    Premisecitation

    Erosion of epigenetic control, transcriptional drift, and the incidence of malignant transformation are strongly associated with human aging.

    “Erosion of epigenetic control and transcriptional drift and the incidence of malignant transformation are strongly associated with human aging (Veitia et al., 2017; Zheng et al., 2016a).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Veitia et al., 2017

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    • cites Zheng et al., 2016a

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  • May combine two claims.

    Premisecitation

    Neuronal de-differentiation, loss of mature neuronal fate, and markers suggesting re-entry of neurons into the cell cycle are pathological hallmarks of AD.

    “Neuronal de-differentiation, loss of mature neuronal fate, and markers that suggest a re-entry of neurons into the cell cycle are pathological hallmarks of AD (Arendt, 2012; Arendt et al., 2000; Busser et al., 1998; Currais et al., 2009; Herrup and Yang, 2007; McShea et al., 1999; Nagy et al., 1997; Yang et al., 2001).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Arendt, 2012

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    • cites Arendt et al., 2000

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    • cites Busser et al., 1998

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

    Premisecitation

    Outside the nervous system, epigenetic erosion and loss of cell identity are established key features of aging.

    “Outside of the nervous system, epigenetic erosion and loss of cell identity are established key features of aging (Ma et al., 2018; Teschendorff et al., 2013; Veitia et al., 2017; Wang et al., 2008; Zheng et al., 2016a).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Ma et al., 2018

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    • cites Teschendorff et al., 2013

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    • cites Veitia et al., 2017

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  • May combine two claims.

    Interpretationcitation

    Our data are consistent with the observation that oxidative stress and DNA damage can induce cell cycle re-entry markers of neurons, leading to neuronal hypo-maturity and functional defects.

    “Our data are consistent with the observation that oxidative stress and DNA damage can induce cell cycle re-entry markers of neurons (Walton et al., 2019; Yang et al., 2001), leading to neuronal hypo-maturity and functional defects (Kruman et al., 2004; Sharma et al., 2017), that might further lead to metabolic reprogramming, activation of cell death competence (Benn and Woolf, 2004; Kole et al., 2”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Walton et al., 2019

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    • cites Yang et al., 2001

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    • cites Kruman et al., 2004

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  • May combine two claims.

    Interpretationown data

    Features of AD relate to, but are distinct from, features of normal aging.

    “These data further support the view that features of AD relate to, but are distinct from, the features of normal aging, and their emergence in human cell culture models depends on age and adult-specific neuronal features.”
    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

    Subjects were followed for at least 2 years after biopsy with no evidence of cognitive decline in controls and with evidence of progressive impairment in AD.

    “They were followed for at least 2 years after biopsy with no evidence of cognitive decline (controls) and with evidence of progressive impairment (AD).”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    Injury is a well-established trigger of cellular de-differentiation.

    “Injury is a well-established trigger of cellular de-differentiation, and the changes we observed are likely a consequence of human neurons adhering to this principle (Gu et al., 2020; Jopling et al., 2010; Poplawski et al., 2020; Renthal et al., 2020; Tzahor and Poss, 2017; Wagner et al., 2017).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Gu et al., 2020

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    • cites Jopling et al., 2010 source

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    • cites Poplawski et al., 2020 source

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  • May combine two claims.

    Interpretationcitation

    For studying age-related vulnerability and mechanisms relevant to AD, iNs have advantages over iPSC models and complement isogenic iPSC strategies.

    “Collectively, for studying age-related vulnerability and mechanisms relevant to AD, iNs have advantages over iPSC models and thus complement isogenic iPSC strategies (van der Kant et al., 2019; Meyer et al., 2019; Woodruff et al., 2013).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites van der Kant et al., 2019 source

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    • cites Meyer et al., 2019 source

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    • cites Woodruff et al., 2013 source

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

    Study design and conductnone given

    All data for one experiment were acquired from cells cultured and processed in parallel on the same microscope with the exact same setting.

    “All data for one experiment were acquired from cells cultured and processed in parallel on the same microscope with the exact same setting used.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    Statistical values were corrected for false discovery rates using the Benjamini-Hochberg method.

    “Statistical values were corrected for false discovery rates (FDR) using the Benjamini-Hochberg method implemented in R.”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    Previous studies on Huntington's disease have consistently detected signs of neuronal epigenetic instability.

    “Previous studies on Huntington’s disease have consistently detected signs of neuronal epigenetic instability (Ayata et al., 2018; von Schimmelmann et al., 2016).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Ayata et al., 2018 source

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    • cites von Schimmelmann et al., 2016 source

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

    Study design and conductown data

    Both AD and control subjects underwent rigorous clinical assessment including detailed neuropsychological testing and brain imaging, and subjects selected for biopsies were stratified to show either a clear non-demented clinical picture or a clear phenotype of AD.

    “Both AD and control subjects underwent rigorous clinical assessment as part of the ADRC study, including detailed neuropsychological testing and brain imaging (MRI), and the subjects selected for biopsies were stratified to show either a clear non-demented clinical picture or clear phenotype of AD (classified as probable AD until pathological confirmation).”
    Quote in the paperSubstantial claim

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

    Premisecitation

    The 'skin and blood clock' best predicts chronological ages of cell culture samples.

    “the ‘in vitro clock,’ also known as the ‘skin and blood clock,’ was used as it best predicts chronological ages of cell culture samples (Horvath et al., 2019).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Horvath et al., 2019

      Not checked in the free run.

48 of 48

  • The paper relies on this without support.

    Aging is crucial for age-related neurodegenerative diseases to unfold.

    “Because aging is likely crucial for age-related neurodegenerative diseases to unfold, we generated age-equivalent fibroblast-derived iNs from a cohort of primarily sporadic AD patients and cognitively normal controls.”

  • The paper relies on this without support.

    Stem and progenitor cells, as well as transformed cells, typically reflect a globally open chromatin state, whereas differentiated cells possess a more defined and closed chromatin landscape.

    “Stem and progenitor cells, as well as transformed cells, typically reflect a globally open chromatin state, whereas differentiated cells possess a more defined and closed chromatin landscape (Figure 6A).”

  • The paper relies on this without support.

    LDHA is inversely associated with mature neuronal identity.

    “We assessed lactate dehydrogenase A (LDHA), a canonical glycolysis gene that is inversely associated with mature neuronal identity and is downregulated during neuronal differentiation (Figure 5G).”

  • The paper relies on this without support.

    Transcriptome similarity between cultured neurons and BrainSpan cortical samples reflects their ontogenic (developmental) identity.

    “To classify the iNs and iPSC-iNs on an ontogenic identity scale, we compared our transcriptome data with samples from the entire range of human cortical brain development (Figure 7K).”
4 of 4

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • Neurons directly induced from individuals with AD through NGN2/ASCL1 overexpression exhibited age-associated dysregulations such as instability of mature neuronal fate, increased cellular senescence, and metabolic shifts.

    “These neurons exhibited several age-associated dysregulations such as instability of mature neuronal fate, increased cellular senescence, and metabolic shifts (11, 24, 25)”

    From Modeling late-onset Alzheimer’s disease neuropathology via direct neuronal reprogramming (2024) · cites this paper as [11]

    The Results sections on direct iN conversion and stress-related de-differentiation report Ngn2/Ascl1 conversion of AD patient fibroblasts, loss of mature neuronal identity, and changes in genes associated with energy metabolism. The Results section on immature transcriptional programs finds enrichment of senescence-related pathways, but does not establish increased cellular senescence in the neurons themselves; the epigenetic-aging analysis also reports no accelerated aging relative to controls. Page numbers are unavailable in our copy. (LLM, conf=0.86)

    Experimental — accuracy not yet measured

  • No AD hallmark neuropathology was shown in neurons directly induced from individuals with AD through NGN2/ASCL1 overexpression.

    “but no AD hallmark neuropathology was shown.”

    From Modeling late-onset Alzheimer’s disease neuropathology via direct neuronal reprogramming (2024) · cites this paper as [11]

    The Results section, “Direct iN conversion of fibroblasts from AD patients and controls” (page unavailable), confirms that the authors generated neurons from AD donors using Ngn2/Ascl1. But it also reports an increased Aβ42/40 ratio in neurons from familial AD patients, so the unqualified statement that no AD hallmark pathology was shown overstates the findings. The cited passages do not establish the absence of other hallmark neuropathology. (LLM, conf=0.78)

    Experimental — accuracy not yet measured

  • Recent studies have directly induced neurons from individuals with AD through NGN2/ASCL1 overexpression.

    “Recent studies have directly induced neurons from individuals with AD through NGN2/ASCL1 overexpression.”

    From Modeling late-onset Alzheimer’s disease neuropathology via direct neuronal reprogramming (2024) · cites this paper as [11]

  • Increased ROS and DSBs are documented in neurons derived from sporadic AD patient fibroblasts, indicating that age-dependent features of genomic instability are recapitulated in human in vitro models of disease.

    “More recently, increased ROS and DSBs have also been documented in neurons derived directly from sporadic AD patient fibroblasts, indicating that age‐dependent features of genomic instability are recapitulated in human in vitro models of disease (Mertens et al, 2021).”

    From Mechanisms of DNA damage-mediated neurotoxicity in neurodegenerative disease (2022) · cites this paper as Mertens et al, 2021

Self-check

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

Each contradiction between two passages listed here was confirmed by an independent check.

  • Addgene identifiers conflict between resource sections

    Minor

    The key resources table assigns #127289 to the Ascl1 construct and #84777 to the EGFP construct. Materials availability instead assigns #127288 to Ascl1 and #127289 to EGFP.

  • Calcium-imaging cell counts exceed the stated sampling plan

    Minor

    The methods specify 10 donors, three fields per donor, and 10 cells per field, which gives 300 cells total, or 150 per group given the five donors per group in the caption. Figure 3 instead reports 160 control cells and 184 AD cells.

  • Mature-neuron results point to incompatible Figure 4 panels

    Minor

    The text cites Figures 4C and 4D for downregulated mature-neuronal gene sets and markers. The Figure 4 caption identifies those panels as upregulated KEGG pathways and dysregulated gene sets; Figure 3C and 3D are captioned as the mature-neuronal results.

3 of 3

Cites

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1 of the 110 references couldn’t be matched to a known paper.

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Related

Authors
Mertens, J., Herdy, J. R., Traxler, L., Schafer, S. T., Schlachetzki, J. C. M., Böhnke, L., Reid, D. A., Lee, H., Zangwill, D., Fernandes, D. P., Agarwal, R. K., Lucciola, R., Zhou-Yang, L., Karbacher, L., Edenhofer, F., Stern, S., Horvath, S., Paquola, A. C. M., Glass, C. K., Yuan, S. H., Ku, M., Szücs, A., Goldstein, L. S. B., Galasko, D., & Gage, F. H.
Affiliations
  • Salk Institute for Biological Studies (US)
  • University of California San Diego (US)
  • Universität Innsbruck (AT)
  • University of California, Los Angeles (US)
  • University of Minnesota (US)
  • University of Freiburg (DE)
Corresponding author
Gage, Fred H.
ORCID
Venue
Cell Stem Cell
Publisher
Elsevier BV
Published
2021-09
Volume
28
Issue
9
Pages
1533-1548.e6
ISSN
  • 1875-9777 (ISSN-L)
  • 1934-5909
DOI
10.1016/j.stem.2021.04.004
PMC
PMC8423435
OpenAlex
W3157334032
Semantic Scholar
8048718b1b52a70eeaec2916188b07119caa46f5
Cited by
240
References
110
Citations by year
2026: 34 · 2025: 45 · 2024: 54 · 2023: 54 · 2022: 42 · 2021: 11
FWCI
13.19
Type
journal article
Language
en
Open access
Licence
Locations
  • Cell stem cell (publishedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • ELTE Digital Institutional Repository (EDIT) (Eötvös Loránd University) (submittedVersion, other-oa)
Funders
  • Leona M. and Harry B. Helmsley Charitable Trust
  • University of California, San Diego
  • Marshallplan-Jubiläumsstiftung
  • Deutsche Forschungsgemeinschaft
  • Austrian Science Fund (ANN-135291)
  • BrightFocus Foundation
  • Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie
  • Alzheimer&apos;s Disease Research Center, Emory University
  • Alzheimer Nederland
  • European Commission (ERC-STG-2019-852086, H2020-MSCA-IF-2017-797205)
  • Alzheimer&apos;s Association
  • Deutscher Akademischer Austauschdienst
  • American Heart Association
  • G. Harold and Leila Y. Mathers Charitable Foundation
  • Allen Foundation (19PABH134610000)
  • National Institutes of Health (AG056306, AG056511, AG057706, AG062429)
  • European Research Council
  • National Institute on Aging (K99-AG056679)
  • Horizon 2020 Marie Skłodowska-Curie Actions
  • EU Joint Programme – Neurodegenerative Disease Research
  • California Institute for Regenerative Medicine (RT2-01927)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Pluripotent Stem Cells Research
  • Health Sciences › Medicine › Physiology › Alzheimer's disease research and treatments
  • Life Sciences › Neuroscience › Developmental Neuroscience › Neurogenesis and neuroplasticity mechanisms
Keywords
Alzheimer's disease, induced neurons, neuronal identity, neuronal maturation, fibroblast-to-neuron conversion, iPSC-derived neurons, epigenetic clocks, dedifferentiation, neuronal transcriptome
MeSH terms
Aged, Aging, Alzheimer Disease, Fibroblasts, Humans, Neurons, Induced Pluripotent Stem Cells

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 2026-10-10.

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

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.

What counts as a claim ›How we measure confidence ›How we check this ›

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