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Cited by 439Neuroinflammation and Neurodegeneration MechanismsAlzheimer's disease research and treatmentsSingle-cell and spatial transcriptomics

Alzheimer’s Patient Microglia Exhibit Enhanced Aging and Unique Transcriptional Activation

Open access
Srinivasan, Karpagam, Friedman, Brad A., Etxeberria, Ainhoa, Huntley, Melanie A., van der Brug, Marcel P., Foreman, Oded, Paw, Jonathan S., Modrusan, Zora +3 more·2020·Cell Reports
DOI 10.1016/j.celrep.2020.107843PMC PMC7422733

Published version of a preprint

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Abstract

Damage-associated microglia (DAM) profiles observed in Alzheimer's disease (AD)-related mouse models reflect an activation state that could modulate AD risk or progression. To learn whether human AD microglia (HAM) display a similar profile, we develop a method for purifying cell types from frozen cerebrocortical tissues for RNA-seq analysis, allowing better transcriptome coverage than typical single-nucleus RNA-seq approaches. The HAM profile we observe bears little resemblance to the DAM profile. Instead, HAM display an enhanced human aging profile, in addition to other disease-related changes such as APOE upregulation. Analyses of whole-tissue RNA-seq and single-cell/nucleus RNA-seq datasets corroborate our findings and suggest that the lack of DAM response in human microglia occurs specifically in AD tissues, not other neurodegenerative settings. These results, which can be browsed at http://research-pub.gene.com/BrainMyeloidLandscape, provide a genome-wide picture of microglial activation in human AD and highlight considerable differences between mouse models and human disease.

AI snapshot

Written from the paper’s full text.

TL;DR

Human Alzheimer’s disease microglia (HAM) purified from frozen cortex show a transcriptional profile that is largely distinct from the mouse damage-associated microglia (DAM) profile, with a prominent enhanced aging signature and APOE upregulation. The HAM signature is validated across cortical regions and whole-tissue datasets, while DAM-like induction is stronger in multiple sclerosis lesions and human microglia xenotransplanted into 5xFAD mice than in AD microglia.

Key findings

  • In sorted CD11b+ microglia from superior frontal gyrus, genome-wide differential expression identified 45 genes increased and 21 decreased in AD versus control; only APOE upregulation survived genome-wide correction (fold change = 4.1, adjusted p = 0.0004).
  • The HAM profile bore little resemblance to mouse DAM: the mouse neurodegeneration-related/DAM module was only slightly increased in AD microglia (p = 0.045), and among more than 100 module genes only APOE was significantly increased.
  • AD microglia displayed an enhanced human aging phenotype: age-related DE scores were significantly higher in AD microglia than controls, with older-subject genes such as IL15, MS4A6A, MS4A4A, NME8, and GPR141 trending upward and CECR2 trending downward; this was not explained by age differences between groups.
  • Validation in fusiform gyrus qRT-PCR replicated the direction of effect for nearly every DE gene tested (22 genes), and whole-tissue datasets (GSE95587, GSE125583, ROSMAP) showed significant elevation of the HAM-Up gene set, most apparent in later Braak stages. DAM induction was stronger in MS lesion microglia and human xenografted microglia in 5xFAD mouse brains than in AD microglia.

Methods

  • The authors adapted a method for isolating cell types from frozen post-mortem human brain: brief Accutase treatment at 4°C, mechanical dissociation, ethanol fixation, immunolabeling, and FACS for NeuN+ neurons, GFAP+ astrocytes, CD31+ endothelial cells, and CD11b+ microglia/myeloid cells, followed by RNA-seq or qPCR.
  • Frontal cortex (superior frontal gyrus) samples were processed from 22 AD and 21 control tissues; after QC, 113 cell-type-specific expression profiles were retained, including microglia profiles from 15 control and 10 AD subjects. Temporal cortex (fusiform gyrus) samples totaled 25 AD and 21 control tissues and were analyzed by qRT-PCR.
  • Analyses included DESeq2 differential expression with Cook’s distance filtering, gene set scoring, myeloid balancing for whole-tissue datasets, and pseudobulk analysis of published single-cell/single-nucleus RNA-seq datasets from AD, MS, and xenotransplanted microglia.

Limitations

  • The paper states that RNA quality after purification was poor, with Bioanalyzer RIN scores typically 1–3 due to freeze-thaw, fixation, and post-mortem tissue processing; alternative dissociation and fixation conditions were unsuccessful or counterproductive.
  • The authors state that the sorted-cell approach does not afford single-cell resolution, and the SFG RNA-seq cohort was small (10 AD and 15 control microglia profiles). They also note that snRNA-seq datasets suffer from extensive gene dropout, making downregulated gene sets difficult to detect.
  • The text makes evident additional limitations: APOE4 carrier status was imbalanced between AD and control groups (0 controls versus 9 AD subjects overall; 0 versus 6 among QC-passing myeloid profiles), and the new whole-tissue cohort had imperfect age matching, with AD cases on average about a half decade younger. The authors also present alternative interpretations for aging-related changes, including possible increased peripheral monocyte/macrophage infiltration versus microglial transcriptional modulation toward a monocyte-like state.

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

Integrity signals

Registry

Crossref · 10.1016/j.celrep.2020.107843

Cites

51 of 51 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 11 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    Most HAM genes exhibiting age-related DE in the Galatro et al. (2017) study showed corresponding expression changes in monocytes relative to microglia.

    “we saw that most HAM genes exhibiting age-related DE in the Galatro et al. (2017) study showed corresponding expression changes in monocytes relative to microglia (Figure 6D).”
    Quote in the paperSubstantial claim
  • Findingown data

    The method for purifying cell types from frozen cerebrocortical tissues for RNA-seq analysis allows better transcriptome coverage than typical single-nucleus RNA-seq approaches.

    “we develop a method for purifying cell types from frozen cerebrocortical tissues for RNA-seq analysis, allowing better transcriptome coverage than typical single-nucleus RNA-seq approaches.”
    • Also stated in: § Defining the HAM Gene Expression Profile Using Frozen Tissues · plain
      “our bulk-sorted microglia profiles from frozen tissues displayed coverage of the transcriptome similar to that of bulk-sorted microglia from fresh post-mortem tissues (Galatro et al., 2017; Gosselin et al., 2017) and better coverage than snRNA-seq profiles”
    Quote in the paperSubstantial claim
  • Findingown data

    The HAM-Up gene set was more robustly elevated in AD whole-tissue RNA than either of the mouse-derived gene sets.

    “We observed that the HAM-Up gene set was more robustly elevated in AD whole-tissue RNA than either of the mouse-derived gene sets (Figures 3D and S4C)”
    Quote in the paperSubstantial claim
  • Findingown data

    Genes whose expression changed with age in Galatro et al. (2017)'s dataset showed the same direction of age-related change among our control microglia samples.

    “we observed a clear tendency for genes whose expression changed with age in Galatro et al. (2017)’s dataset to show the same direction of age-related change among our control microglia samples (Figure 5C), validating our SFG RNA-seq profiles.”
    Quote in the paperSubstantial claim
  • Findingown data

    GPNMB upregulation was robust in xMG and MS cells, but in AD microglia, it was meager or absent.

    “For instance, GPNMB upregulation was robust in xMG and MS cells, but in AD microglia, it was meager or absent (Figure S7B).”
    Quote in the paperSubstantial claim
  • May combine two claims. 32
  • Classified as probably not a substantial claim. 30

  • May combine two claims.

    Findingown data

    Expression of the resting microglia module defined in mouse microglia was not reduced in microglia from AD tissues (it increased in the snRNA-seq dataset) or in human xMG from 5xFAD mouse brains.

    “Surprisingly, expression of the resting microglia module defined in mouse microglia was not reduced in microglia from AD tissues (it increased in the snRNA-seq dataset) or even in human xMG from 5xFAD mouse brains.”
    Quote in the paperSubstantial claim

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

    Findingown data

    RNA extracted from whole tissue using QIAGEN miRNeasy mini columns yielded samples with RNA integrity (RIN) scores averaging 6.5.

    “RNA was extracted using QIAGEN miRNeasy mini columns, yielding samples with RNA integrity (RIN) scores averaging 6.5.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The mouse microglia aging profile, the PS2APP profile, and the PVM profile each showed statistically significant enrichment in AD versus control microglia.

    “The mouse microglia aging profile, the PS2APP profile, and the PVM profile each showed statistically significant enrichment in AD versus control microglia (Figure 4B).”
    Quote in the paperSubstantial claim

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

    Findingreasoning

    The RNA purified from collected cell types is of low quality because of unavoidable aspects of preparing fixed cell suspensions from frozen and thawed post-mortem tissue samples.

    “A notable caveat of our method is the low quality of the RNA after its purification from the collected cell types because of unavoidable aspects of preparing fixed cell suspensions from frozen and thawed post-mortem tissue samples.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Findingreasoning

    Only brief dissociation at 4°C and fixation immediately thereafter permitted cell populations to suitably endure the subsequent immunolabeling and FACS procedures.

    “Only brief dissociation at 4°C and fixation immediately thereafter permitted cell populations to suitably endure the subsequent immunolabeling and FACS procedures.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    DAM+ nuclei did not cluster separately from HAM+ nuclei in the snRNA-seq datasets; to whatever extent the DAM signal was induced, it occurred in the same nuclei in which the HAM signal was detected.

    “Third, we did not observe instances of DAM+ nuclei clustering separately from HAM+ nuclei in the snRNA-seq datasets; instead, these datasets revealed that to whatever extent the DAM signal was induced, it occurred in the same nuclei in which the HAM signal was detected (see Data S4, panel 2).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Of more than 100 genes in the DAM module, only APOE was significantly increased in SFG microglia from AD patients (fold change = 4.1, p = 0.0004), and most other genes showed no clear trends.

    “However, of more than 100 genes in the module, only APOE was significantly increased in SFG microglia from AD patients (fold change = 4.1, p = 0.0004), and most other genes showed no clear trends in either direction (Figure S5A; Table S1).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingcitation

    Many HAM genes induced in human AD tissues overlap with those induced by natural aging, though the genes involved are distinct between species.

    “Just as DAM genes induced in neurodegenerative mouse models overlap with those induced by natural aging (Friedman et al., 2018; Holtman et al., 2015), so do many HAM genes induced in human AD tissues (Figure 5A), though the genes involved are distinct between species (Galatro etal.,2017).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Friedman et al., 2018 source

      Queued — the background check reaches it, usually within a day.

    • cites Holtman et al., 2015 source

      Queued — the background check reaches it, usually within a day.

    • cites Galatro et al., 2017 source

      Queued — the background check reaches it, usually within a day.

  • May combine two claims.

    Findingown data

    Human xMG from 5xFAD mouse brains displayed similar extents of induction for HAM-Up and DAM gene sets, although no changes were observed in the Aging-Up and Aging-Down gene sets.

    “In contrast, human xMG from 5xFAD mouse brains displayed similar extents of induction for HAM-Up and DAM gene sets, although no changes were observed in the Aging-Up and Aging-Down gene sets (Figure 7).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In all three whole-tissue datasets, the HAM-Up gene set was significantly increased; this was most apparent in later Braak stages, which also showed decreased expression of the HAM-Down gene set.

    “In all three whole-tissue datasets, the HAM-Up gene set was significantly increased (Figure 3D); this was most apparent in later Braak stages, which also showed decreased expression of our HAM-Down gene set (Figure S4C).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Using 22 genes to assign a DE score to each sample revealed a clear difference in FuG microglia between AD and control groups, reproducing the signal observed in the SFG RNA-seq data.

    “Using these 22 genes to assign a DE score to each sample revealed a clear difference in FuG microglia between AD and control groups, reproducing the signal observed in the SFG RNA-seq data (Figure 3C).”
    Quote in the paperSubstantial claim

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

    Findingown data

    None of the alternative dissociation techniques attempted were as good as the described method in terms of cell yield and RNA recovery.

    “None of these attempts were as good as the method described above in terms of cell yield and RNA recovery.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingnone given

    Sorted mouse microglia from the PS2APP and Tau-P301S models showed strong DAM induction and no changes in the HAM-Up gene set.

    “As a point of reference, sorted mouse microglia from the PS2APP and Tau-P301S models showed strong DAM induction and no changes in the HAM-Up gene set.”
    Quote in the paperNot a substantial claim

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

    Findingown data

    RIN scores for all sorted cell types were typically between 1 and 3.

    “RIN scores for all cell types were typically between 1 and 3.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingcitation

    Most genes with higher expression in microglia from older subjects trended toward elevated expression in AD relative to control microglia; genes with lower expression in older subjects tended to be reduced in AD microglia.

    “Most genes with higher expression in microglia from older subjects, including IL15 and the candidate AD risk genes MS4A6A, MS4A4A, NME8, and GPR141, trended toward elevated expression in AD relative to control microglia; conversely, genes with lower expression in microglia from older subjects, like CECR2, tended to be reduced in AD microglia (Figures 5A and 6A).”
    Quote in the paperSubstantial claim

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

    Findingown data

    The 89 samples from subjects already profiled in GSE95587 showed very similar sample-by-sample DE profiles as the corresponding samples from the same subjects in GSE95587.

    “These samples, which came from new fusiform gyrus tissue blocks, showed very similar sample-by-sample DE profiles as the corresponding samples from the same subjects in GSE95587 (Figure S4A)”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    All components of the HAM profile-elevated HAM-Up and Aging-Up scores and reduced HAM-Down and Aging-Down scores-were clearly represented in microglia from MS lesions, usually with larger effect sizes and lower p values than the DAM scores in the same cells.

    “All components of the HAM profile—elevated HAM-Up and Aging-Up scores and reduced HAM-Down and Aging-Down scores— were clearly represented in microglia from MS lesions, usually with larger effect sizes and lower p values than the DAM scores in the same cells (Figure 7).”
    Quote in the paperNot a substantial claim

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

    Findingown data

    Of 289 total samples, 89 were from subjects that had already been profiled in the previous study GSE95587.

    “Of 289 total samples, 89 were from subjects that had already been profiled in our previous study, GSE95587.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingnone given

    Fixation adversely affects RNA integrity (RIN), but it also permeabilizes the cells and enables labeling of intracellular markers for sorting.

    “Although the fixation adversely affects RNA integrity (RIN), it also permeabilizes the cells and enables labeling of intracellular markers for sorting.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Many genes with higher expression in younger subjects in Galatro et al. (2017)'s dataset were microglia enriched in Gosselin et al. (2017)'s dataset; many genes with elevated expression in older subjects were monocyte enriched.

    “many genes with higher expression in younger subjects in Galatro et al. (2017)’s dataset, like CECR2, were microglia enriched in Gosselin et al. (2017)’s dataset; conversely, many genes with elevated expression in older subjects, like IL15, were monocyte enriched (Figures 6A and 6C).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    APOE, ABCA7, GPR141, PTK2B, SPI1, and ZYX appeared upregulated in AD microglia, whereas MEF2C appeared downregulated (unadjusted p < 0.05).

    “we observed that APOE, ABCA7, GPR141, PTK2B, SPI1, and ZYX appeared upregulated in AD microglia, whereas MEF2C appeared downregulated (unadjusted p < 0.05) (Figure 2B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    CD44 was increased in AD samples in FuG myeloid cell sorts by qPCR (unadjusted p = 0.041), so its DE is considered genuine.

    “We included CD44 in our panel of genes tested by qPCR in FuG myeloid cell sorts, and it was again increased in the AD samples (unadjusted p = 0.041), so we consider its DE to be genuine”
    Quote in the paperNot a substantial claim

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

    Findingown data

    Differential expression analysis revealed that about 80 genes were significantly increased in microglia from subjects with larger post-mortem interval (PMI).

    “Differential expression analysis (Data S4, panel 1) revealed that the expression of about 80 genes was significantly increased in microglia from subjects with larger post-mortem interval (PMI).”
    Quote in the paperNot a substantial claim

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

    Findingown data

    The authors discarded 1/43 neuron, 19/38 astrocyte, 14/41 endothelial, and 18/43 microglia libraries from original frozen tissues, and 16/16 microglia libraries from twice frozen tissues.

    “We discarded 1/43 neuron libraries, 19/38 astrocyte libraries, 14/41 endothelial cell libraries, and 18/43 microglia libraries from original frozen tissues, and 16/16 microglia libraries from twice frozen tissues (which underscored the liabilities of the freeze-thaw process).”
    Quote in the paperNot a substantial claim

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

    Findingown data

    The Cook's distance filter left 4 DE genes in neurons, 66 in myeloid cells, and 135 in endothelial cells.

    “The Cook’s distance filter eliminated 6/10 neuronal DE genes, 9/75 myeloid DE genes, and 382/517 endothelial DE genes from consideration, leaving 4 DE genes in neurons, 66 in myeloid cells, and 135 in endothelial cells.”
    Quote in the paperSubstantial claim

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

    Findingown data

    For APOE genotype, only one DE gene, ACY3, was detected in AD microglia between APOE-e4 carriers versus non-carriers.

    “For APOE genotype, we only detected one DE gene, ACY3, in AD microglia between APOE-e4 carriers versus non-carriers.”
    Quote in the paperNot a substantial claim

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

    Findingown data

    Compared to Pass libraries, Fail libraries generally showed higher percentages of intergenic reads and lower percentages of exonic and intronic reads.

    “Compared to”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Findingnone given

    The sorted cell RNA preparations were highly fragmented.

    “Given the highly fragmented condition of our sorted cell RNA preps”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    voom+limma identified only 12 DE genes (adjusted p ≤ 0.05) in myeloid cells and none in the other sorted cell types.

    “DE between AD and controls for this study’s sorted cell populations was first attempted using voom+limma, which identified only 12 DE genes (adjusted p ≤ 0.05) in myeloid cells and none in the other cell types.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    The relative lack of DAM gene induction in human microglia was a peculiar feature of human AD tissues and may be a specific feature of AD pathogenesis.

    “Thus, the relative lack of DAM gene induction in human microglia was a peculiar feature of human AD tissues and may be a specific feature of AD pathogenesis.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Study design and conductown data

    Linear model testing and visual exploration revealed no significant correlation between PMI and diagnosis, sex, ApoE4 status, age, or Last MMSE.

    “Linear model testing as well as visual exploration revealed no significant correlation between PMI and any of the other variables (diagnosis, see also Figure S2C PMI panel; sex; ApoE4 status; age; or Last MMSE).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    Human microglia are capable of responding in a DAM-like manner, but this response is blunted in AD patients (at least in the disease stages examined).

    “Thus, human microglia are capable of responding in a DAM-like manner, but for some reason this response is blunted in AD patients (at least in the disease stages we examined).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Study design and conductreasoning

    The approach allowed sampling a suitably large number of tissues with known histopathological characteristics while obtaining broad coverage of the transcriptome.

    “our approach allowed us to sample a suitably large number of tissues with known histopathological characteristics while obtaining broad coverage of the transcriptome.”
    Quote in the paperSubstantial claim

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

    Study design and conductcitation

    Whole-tissue polyA-selected Illumina RNA-Seq was performed on samples with RNA integrity (RIN) scores at least 5 and post-mortem intervals (PMIs) no greater than 5 hr.

    “Standard polyA-selected Illumina RNA-Seq analysis was performed as described (Srinivasan et al., 2016) on samples with RNA integrity (RIN) scores at least 5 and post-mortem intervals (PMIs) no greater than 5 hr.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Srinivasan et al., 2016 source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Study design and conductreasoning

    The method provides rich gene expression profiles to enable genome-wide analyses of differential expression.

    “provides rich gene expression profiles to enable genome-wide analyses of differential expression (DE)”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Study design and conductcitation

    All AD subjects were clinically diagnosed with AD in life and brains were neuropathologically confirmed to have frequent CERAD neuritic plaque densities and Braak score V or VI.

    “All AD subjects were clinically diagnosed with AD in life and brains were neuropathologically confirmed to have”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    The method allows collection of desired cell types by the tens (or hundreds) of thousands from each tissue sample.

    “The method we developed allows the collection of desired cell types by the tens (or hundreds) of thousands from each tissue sample”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Study design and conductreasoning

    The 89 duplicated samples were omitted from all other analyses associated with the manuscript to avoid overlap between the two datasets.

    “but were omitted in all other analyses associated with this manuscript to avoid overlap between the two datasets”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Study design and conductown data

    In the new whole-tissue cohort, age was not well controlled, and the direction of the age difference was anti-conservative, with AD cases on average about a half decade younger than controls.

    “Although age was not well controlled in the new cohort, the direction of difference was anti-conservative, with the AD cases on average about a half decade younger.”
    Quote in the paperSubstantial claim

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

    Premisecitation

    Several genes associated with AD incidence (APOE, CLU, ABCA7, SORL1, INPP5D, and PLCG2) function in lipid transport or signaling.

    “Several genesassoci-ated with AD incidence (APOE, CLU, ABCA7, SORL1, INPP5D, and PLCG2)(Jansen et al., 2019; Kunkle et al., 2019; Marioni et al., 2018) also function in lipid transport or signaling.”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Jansen et al., 2019 source

      Not checked in the free run.

    • cites Kunkle et al., 2019 source

      Not checked in the free run.

    • cites Marioni et al., 2018 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    The HAM profile in AD microglia reflected a mixture of an enhanced aging process and an age-independent, disease-related activation process.

    “These data indicated that the HAM profile in AD microglia reflected a mixture of an enhanced aging process and an age-independent, disease-related activation process.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    We completed this process for 22 AD and 21 control SFG tissues.

    “We completed this process for 22 AD and 21 control SFG tissues.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S1 (asserted), S3 (load-bearing).

    Premisecitation

    The DAM activation state in β-amyloid models is a protective response by healthy microglia.

    “A third explanation for the dissimilarity could be that the DAM activation state in b-amyloid models is a protective response by healthy microglia (Keren-Shaul et al., 2017)”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Keren-Shaul et al., 2017 source

      Not checked in the free run.

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

    Study design and conductown data

    For the sorted cell cohort, controls had either zero or sparse CERAD neuritic plaque densities, and mostly had Braak scores ranging from 0 to III (median II).

    “For sorted cell cohort (GSE125050), controls had either”
    Quote in the paperNot a substantial claim

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

    Study design and conductreasoning

    The Braak stage analyses did not include corrective measures for altered cellular makeup of AD tissues.

    “(The Braak stage analyses did not include the corrective measures for altered cellular makeup of AD tissues, because it was impractical to reduce the sample size.)”
    Quote in the paperNot a substantial claim

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

    Study design and conductreasoning

    Labeling nuclei with DAPI helps ensure that only singlet cell bodies are collected.

    “Labeling nuclei with DAPI helps ensure that only singlet cell bodies are collected, because doublets with a higher DAPI signal and cell fragments that lack nuclei are easily excluded.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    The method allows selection of sample cohorts that include a suitably large number of AD and control subjects with desired histopathological or clinical characteristics.

    “allows the selection of sample cohorts that include a suitably large number of AD and control subjects with desired histopathological or clinical characteristics.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    The DAM activation state is generally considered protective in mouse neurodegeneration models.

    “That the DAM activation state—generally considered protective in mouse neurodegeneration models—was more readily observed in microglia from MS lesions and in xMG from 5xFAD mouse brains”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Keren-Shaul et al., 2017 source

      Not checked in the free run.

  • May combine two claims.

    Study design and conductown data

    qRT-PCR for specific cell-type markers validated the specificity of the collected populations and was successful for nearly all RNA samples.

    “qRT-PCR for specific cell-type markers validated the specificity of the collected populations and was successful for nearly all RNA samples (data not shown).”
    Quote in the paperSubstantial claim

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

    Study design and conductown data

    After QC, cells with less than 800 total transcripts or greater than 30% mitochondrial transcripts were discarded, resulting in 1,738 QC-passing cells for analysis.

    “After this step cells with less than 800 total transcripts or greater than 30% mitochondrial transcripts were discarded, resulting in 1,738 QC-passing cells for analysis.”
    Quote in the paperNot a substantial claim

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

    Study design and conductown data

    The authors set aside neurons and endothelial cells and focused on whether changes in myeloid cells could be validated.

    “led us to set these cell types aside (taking a conservative position) and focus on the whether the changes in myeloid cells could be validated.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: There was a lack of other human AD datasets available at the time for cross-comparison.

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

    Interpretationown data

    ACY3 showed variable expression levels in the Controls (all non-carriers), so it may be a false positive.

    “It showed variable expression levels in the Controls (all non-carriers), so it may be a false positive.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    All qPCR assays were performed in duplicate and the average of the two Ct values was kept, except for twelve sample/assay pairs with a difference greater than 2.82, which were discarded.

    “All assays were performed in duplicate and the average of these two Ct values was kept, except for twelve sample/assay pairs for which the difference was more than 2.82 (corresponding to a standard deviation of 2), which were discarded.”
    Quote in the paperNot a substantial claim

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

    Study design and conductown data

    The authors did not use ROSMAP-DLPFC samples from batches 7 and 8 because they saw strong clustering of these two batches separately from the first 6 on PCA.

    “We did not use the samples from batches 7 and 8 since, despite restricting to the batch-normalized values, we still saw very strong clustering of these two batches separately from the first 6 on PCA.”
    Quote in the paperNot a substantial claim
54 of 54

  • The authors state this as an assumption.

    Damage from the freeze-thaw process led to cellular disintegration when cells remained unfixed for too long or were incubated at 37°C.

    “We presume that damage from the freeze-thaw process led to cellular disintegration when cells remained unfixed for too long or were incubated at 37°C.”

  • The paper relies on this without support.

    Gray matter atrophy was pronounced in the fusiform gyrus from AD subjects.

    “since gray matter atrophy was pronounced in FuG from AD subjects”

  • The paper relies on this without support.

    Activated microglia often display elevated CD45 reactivity.

    “since we did not want to exclude activated microglia which often display elevated CD45 reactivity”

  • The paper relies on this without support.

    There was a lack of other human AD datasets available at the time for cross-comparison.

    “the lack of other human AD datasets available at the time for cross-comparison”
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?

  • Transcriptional profiles of microglia isolated from frozen human AD and control cortical tissue suggest an overall species difference in the disease associated signature.

    “Transcriptional profiles of microglia isolated from frozen human AD and control cortical tissue suggest an overall species difference in the disease associated signature, though an increased SPI1 expression was measured in AD samples compared to controls55 validating previous observations by others31.”

    From Modest changes in Spi1 dosage reveal the potential for altered microglial function as seen in Alzheimer’s disease (2021) · cites this paper as [55]

  • Increased SPI1 expression was measured in AD samples compared to controls.

    “Transcriptional profiles of microglia isolated from frozen human AD and control cortical tissue suggest an overall species difference in the disease associated signature, though an increased SPI1 expression was measured in AD samples compared to controls55 validating previous observations by others31.”

    From Modest changes in Spi1 dosage reveal the potential for altered microglial function as seen in Alzheimer’s disease (2021) · cites this paper as [55]

  • Human single nucleus RNA sequencing data from Mathys and colleagues and Lake and colleagues were obtained from The Myeloid Landscape 2 portal.

    “Human single nucleus RNA sequencing data from Mathys and colleagues [5] and Lake and colleagues [12] were obtained from The Myeloid Landscape 2 portal (http://research-pub.gene.com/BrainMyeloidLandscape) [70].”

    From Selective suppression of oligodendrocyte-derived amyloid beta rescues neuronal dysfunction in Alzheimer’s disease (2024) · cites this paper as [70]

  • The 832 LOAD spheroid DEGs overlap with bulk RNA-seq datasets from various brain regions in individuals with AD, GO terms related to apoptosis/neurodegeneration, and AD risk genes from Alzgene database, with overlaps ranging from 8 to 135 genes.

    “We further compared 832 DEGs with bulk RNA-seq datasets from various brain regions in individuals with AD (80–84), gene members in GO terms related to apoptosis/neurodegeneration, and AD risk genes from Alzgene database, revealing distinct sets of DEGs with overlaps ranging from 8 to 135 genes (Table S7, page 2, 3 and 4).”

    From Modeling late-onset Alzheimer’s disease neuropathology via direct neuronal reprogramming (2024) · cites this paper as [84]

  • APOE is greatly upregulated in human AD microglia from human AD brains.

    “APOE is greatly upregulated in human AD microglia (HAM) from human AD brains”

    From ApoE4 reduction: An emerging and promising therapeutic strategy for Alzheimer's disease (2022) · cites this paper as Srinivasan et al 2020

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.

  • Frontal-cortex AD subject count differs between text and table

    Minor

    The Results state that 22 AD and 21 control SFG tissues were processed, but the sorted-cell cohort table lists 21 AD and 21 control subjects under "All Subjects."

1 of 1

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Authors
Srinivasan, K., Friedman, B. A., Etxeberria, A., Huntley, M. A., van der Brug, M. P., Foreman, O., Paw, J. S., Modrusan, Z., Beach, T. G., Serrano, G. E., & Hansen, D. V.
Affiliations
  • Genentech (US)
  • Banner Sun Health Research Institute (US)
Corresponding author
  • Friedman, Brad A.
  • Hansen, David V.
ORCID
Venue
Cell Reports
Publisher
Elsevier BV
Published
2020-06
Volume
31
Issue
13
Pages
107843
ISSN
2211-1247 (ISSN-L)
DOI
10.1016/j.celrep.2020.107843
PMC
PMC7422733
OpenAlex
W3039072393
Semantic Scholar
161193b958b5ad9f3c708d4d10a0ed89678d1d84
Cited by
427
References
52
Citations by year
2026: 55 · 2025: 60 · 2024: 68 · 2023: 61 · 2022: 74 · 2021: 89 · 2020: 18 · 2019: 2
FWCI
24.01
Type
journal article
Language
en
Open access
Licence
Locations
  • Cell Reports (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nd)
  • PubMed (publishedVersion)
  • PubMed Central (acceptedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • Michael J. Fox Foundation for Parkinson&apos;s Research (GSE125050, GSE125583)
  • Genentech
  • Arizona Department of Health Services (211002)
  • Arizona Biomedical Research Commission (0011, 05-901, 1001, 4001)
  • National Institute on Aging (P30 AG19610)
  • National Institute of Neurological Disorders and Stroke (U24 NS072026)
Topics
  • Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
  • Health Sciences › Medicine › Physiology › Alzheimer's disease research and treatments
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Single-cell and spatial transcriptomics
Keywords
Alzheimer's disease, human microglia, microglial activation, aging, apolipoprotein E, transcriptome, single-nucleus RNA sequencing, single-cell RNA sequencing, mouse model, neuroinflammation
MeSH terms
Aged, Aged, 80 and over, Alzheimer Disease, Animals, Female, Frontal Lobe, Frozen Sections, Humans, Male, Monocytes, Multiple Sclerosis, Phenotype, Risk Factors, Temporal Lobe, Reproducibility of Results, Transcriptional Activation, Cellular Senescence, Microglia, Genetic Predisposition to Disease, Gene Expression Profiling, Databases, Genetic, Mice, Heterografts

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 2026-10-07.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-07.

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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