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PreprintCited by 8Neuroinflammation and Neurodegeneration MechanismsNeuroscience and Neuropharmacology ResearchSingle-cell and spatial transcriptomics

Single-cell resolution analysis of the crosstalk between chemogenically activated astrocytes and microglia

Open access
Philtjens, Stéphanie, Turnbull, Marion T., Thedy, Brian P., Moon, Younghye, Kim, Jungsu·2020·bioRxiv (Cold Spring Harbor Laboratory)
DOI 10.1101/2020.04.27.064881
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Abstract

Summary: Astrocytes are the most common glial cell type in the brain, yet, it is unclear how their activation affects the transcriptome of neighboring cells. Engineered G protein-coupled receptors (GPCRs) called Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) enable selective activation of specific cell types, such as astrocytes. Here, we combine activation of astrocytes in the hippocampus and cortex of healthy mice with single-cell RNA sequencing. Our data show that long-term activation of astrocytes dramatically alters the transcriptome of astrocytes and microglia. Genes that were differentially expressed in Gq-DREADD-activated astrocytes are involved in neurogenesis and low-density lipoprotein particle biology, while those in the microglia were involved in lipoprotein handling, purinergic receptor activity, and immune cell migration and chemotaxis. Furthermore, network analysis showed that Gq-DREADD-mediated activation in astrocytes resulted in an upregulation of genes involved in the GPCR signaling pathways and calcium ion homeostasis, confirming astrocyte activation. This dataset will serve as a resource for the broader neuroscience community, and our findings highlight the importance of studying transcriptomic alterations in microglia after astrocyte activation in vivo .

AI snapshot

Written from the paper’s full text.

TL;DR

Chronic Gq-DREADD activation of astrocytes in healthy mouse hippocampus and cortex altered the transcriptome of astrocytes and neighboring microglia at single-cell resolution. The authors report 34,736 cells passing QC and identify transcriptionally distinct astrocyte, microglial, and mixed glia subclusters after 8 weeks of CNO treatment.

Key findings

  • In astrocytes, 396 genes were differentially expressed (p < 0.05) between CNO and saccharin groups; upregulated genes were enriched for adenylate cyclase activator activity and cell motility/migration, while downregulated genes related to gene expression and translation. Network analysis linked 16 of 50 genes in a CMKLR1/SPARCL1/Rich1/Transcobalamin II/OLFML3 network to GPCR signaling and calcium ion homeostasis.
  • Astrocyte subclustering showed condition-specific shifts: cluster 0 was 53.26% of saccharin-group astrocytes vs. 3.96% of CNO-group astrocytes, while cluster 1 was 0.87% vs. 66.55%, respectively.
  • Despite astrocyte-restricted Gq-DREADD expression, microglial clusters changed: microglia I increased 35% and microglia II decreased 42% after CNO; microglia I subclusters 4 and 6 were observed only in the CNO group.
  • Mixed glia I increased 48% and mixed glia II decreased 50% after CNO; in mixed glia II, cluster 2 went from 2.69% in saccharin to 66.26% in CNO, and cluster 3 from 0.05% to 20.45%. Gq-DREADD-activated astrocytes did not match PAN-reactive, A1, A2, disease-associated, or other previously described astrocyte transcriptomic phenotypes.

Methods

  • Male C57BL/6J mice (n = 4; n = 2/group) received bilateral stereotaxic injections of AAV5-GFAP-hM3D(Gq)-mCherry into hippocampus and cortex at 8 weeks of age, then CNO (0.25 mg/mL in drinking water, approximating 5 mg/kg/day) or saccharin control for 8 weeks.
  • Single cells from hippocampus and cortex were processed with 10x Genomics Chromium Single Cell 3′ Library & Gel Bead Kit v2 and sequenced on Illumina NovaSeq 6000; Cell Ranger v3.0.2 aligned to mm10, and Seurat v3.0.2 was used for QC, clustering, and differential expression.
  • After QC, 34,736 cells remained (16,107 CNO; 18,629 saccharin); clustering used the top 3,000 variable genes and first 21 PCs, yielding 15 clusters at resolution 0.5. Differential expression used a likelihood test with Bonferroni-corrected p < 0.05; enrichment used g:Profiler and network analysis used MetaCore.

Limitations

  • Stated: the single-cell dissociation protocol enriched glial cells and depleted neurons, with less than 1% of the total cell population consisting of neuronal cells, so the authors could not confirm neuronal activation at the transcriptomic level.
  • Stated: about 60% of cells were annotated as “Mixed Glia,” and the authors note difficulty in manual annotation, regional heterogeneity from pooling cortex and hippocampus, and the need for reliable automatic annotation tools.
  • Evident: the study used only n = 2 mice per group, only male C57BL/6J mice, and healthy animals rather than a disease model; the authors also state that further research is needed to determine the biological relevance of the activated astrocyte phenotype and the induction of DAM genes.

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

Integrity signals

Registry

Crossref · 10.1101/2020.04.27.064881

Cites

74 of 74 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

38 of 58 cited sources checked · 16 supported · 0 flagged · accuracy not yet measured

  • Findingown data

    Gq-DREADD expression remained restricted to astrocytes based on co-localization with GFAP and morphology consistent with astrocytic immunoreactivity patterns.

    “Histological visualization with the fluorescent reporter mCherry confirmed that Gq-DREADD expression remained restricted to astrocytes based on their co-localization with the astrocyte marker GFAP (Figure 1C) and morphology consistent with astrocytic immunoreactivity patterns (Benediktsson et al., 2005;Bushong et al., 2002;Scofield et al., 2015).”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites Benediktsson et al., 2005

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

    • cites Bushong et al., 2002 source
    • cites Scofield et al., 2015 source

      The Results section ('Astrocyte-specific Expression of Gq-DREADD') describes astrocyte-like morphology and apparent co-localization of viral HA with GFAP, supporting the basis for identifying astrocytic expression. It calls astrocyte-specific expression 'very likely,' however, and the Discussion says a minor neuronal contribution cannot be ruled out. The source therefore does not establish that expression was strictly restricted to astrocytes. (LLM, conf=0.87)

      Experimental — accuracy not yet measured
  • Premisecitation

    Expression of DREADDs in astrocytes allows for activation of this cell type through selective binding of its ligand.

    “Expression of engineered G protein-coupled receptors (GPCRs), called Designer Receptors Exclusively Activated by Designer Drugs (DREADDs), in astrocytes allows for activation of this cell type through selective binding of its ligand (Lee et al., 2014;Roth, 2016;Urban and Roth, 2015).”
    Quote in the paperSubstantial claim1 of 3 sources checked
    • cites Lee et al., 2014

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

    • cites Roth, 2016 source
    • cites Urban and Roth, 2015

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

  • Findingown data

    Our astrocyte population in the CNO group did not show the transcriptional profile of reactive astrogliosis typically seen in neurodegenerative diseases.

    “We also observed that our astrocyte population in the CNO group did not show the transcriptional profile of reactive astrogliosis that is typically seen in neurodegenerative diseases (Supplementary Figure 2D) (Kraft et al., 2013).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Kraft et al., 2013 source

      The introductory passage (labeled Abstract in our copy; page unavailable) describes reactive astrocytes as showing increased GFAP and vimentin, and the Results describe their characteristic process hypertrophy in APP/PS1 mice. These passages support a general description of reactive astrogliosis, but do not establish a broader transcriptional profile typical of neurodegenerative diseases. The CNO-group observation is the citing authors’ own result, not a finding attributed to Kraft et al. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
  • Premisecitation

    None of these previous studies looked at the effect of long-term Gq-DREADD-mediated activation on the transcriptome of astrocytes or other neighboring brain cells.

    “However, none of these studies looked at the effect of long-term Gq-DREADD-mediated activation on the transcriptome of astrocytes or other neighboring brain cells.”
    Quote in the paperSubstantial claim3 of 3 sources checked
    • cites Agulhon et al., 2013 source
    • cites Bull et al., 2014 source

      The sections on stimulating NAcore astrocytes report calcium responses, reward-related behavior, and ethanol self-administration after Gq-DREADD activation, not transcriptomic effects. The Discussion says downstream mechanisms remain unknown. This supports the characterization of this study, but the paper does not establish that none of the other previous studies examined transcriptomes after long-term activation. (LLM, conf=0.76)

      Experimental — accuracy not yet measured
    • cites Scofield et al., 2015 source
  • Interpretationnone given

    This is the first study to show transcriptional changes in glia that occur after long-term activation of astrocytes with the Gq-DREADD in an in vivo model.

    “To the best of our knowledge, this is the first study to show transcriptional changes in glia that occur after long-term activation of astrocytes with the Gq-DREADD in an in vivo model.”
    Quote in the paperSubstantial claim3 of 3 sources checked
    • cites Agulhon et al., 2013 source
    • cites Bull et al., 2014 source

      The section “Stimulating NAcore Astrocytes Decreased the Motivation to Self-Administer Ethanol after 3 Weeks Abstinence” reports Gq-DREADD activation in rat astrocytes and a behavioral effect. The cited paper does not report transcriptional changes after long-term activation or establish a first study to show them. (LLM, conf=0.84)

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

      The Results sections on astrocyte-specific expression and Gq-DREADD activation describe an in vivo model in which Gq-DREADD was expressed and activated in astrocytes. They report glutamate release, not transcriptional changes after long-term activation. The 'first study' wording is the citing authors’ characterization of their own study, not a finding established by this source. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
  • May combine two claims. 34
  • Classified as probably not a substantial claim. 24

  • May combine two claims.

    Premisecitation

    AAV5 is astrocyte specific and its injection into the striatum and substantia nigra has previously demonstrated astrocyte-specific expression with no expression in neurons or microglia.

    “AAV5 is astrocyte specific and its injection into the striatum and substantia nigra has previously demonstrated astrocyte-specific expression with no expression in neurons or microglia (Drinkut et al., 2012;Merienne et al., 2013).”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites Drinkut et al., 2012

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

    • cites Merienne et al., 2013 source

      The ‘Targeting astrocytes with transcription regulatory elements’ section reports astrocyte-specific expression, with no residual expression in neurons or microglia, after high-titer AAV5-gfa2-eGFP injection into the striatum or substantia nigra. But the broader statement that AAV5 itself is astrocyte-specific omits the gfa2 promoter and conflicts with the ‘Altering the entry of viral vectors’ section, which says AAV5 can also infect neurons and other cells. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Activation of Gq-coupled hM3Dq results in Ca2+ mobilization and ERK1/2 phosphorylation.

    “In this study we used the Gq-coupled human muscarinic type 3 receptor (hM3Dq) whose activation results in Ca 2+ mobilization and ERK1/2 phosphorylation (Armbruster et al., 2007).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Armbruster et al., 2007 source

  • May combine two claims.

    Premisecitation

    Mutated muscarinic DREADD receptors respond exclusively to CNO and are unresponsive to endogenous ligand acetylcholine.

    “These mutated muscarinic receptors respond exclusively to clozapine-N-oxide (CNO) and are unresponsive to their endogenous ligand acetylcholine (Armbruster et al., 2007;Rogan and Roth, 2011).”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites Armbruster et al., 2007

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

    • cites Rogan and Roth, 2011 source

  • Classified as probably not a substantial claim.

    Findingcitation

    Cell clusters were annotated manually using marker genes conserved between both conditions as astrocytes, microglia, neurons, endothelial cells, ependymal cells, and mixed glia.

    “The cell clusters were annotated manually using marker genes that were conserved between both conditions as astrocytes, microglia, neurons, endothelial cells, ependymal cells, and mixed glia (Supplementary Data 1) (Arneson et al., 2018;McKenzie et al., 2018).”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites Arneson et al., 2018 source

      The Methods section, “Resolving cell identities of the cell clusters” (page unavailable), says the authors assigned cluster identities using known cell-type markers and marker-gene overlap. The Results section, “Unbiased identification of cell identities in hippocampus” (page unavailable), identifies astrocytes, microglia, neurons, endothelial cells, and ependymal cells. Our copy does not say the markers were selected for being conserved between conditions or identify a cluster as “mixed glia.” (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites McKenzie et al., 2018 source

      The Discussion (page unavailable) says the paper annotated marker genes for astrocytes, endothelial cells, microglia, neurons, oligodendrocytes, and OPCs. It therefore supports using its markers for four of the six cell labels in the citing sentence, but does not supply markers for ependymal cells or mixed glia. The manual annotation and conservation between conditions describe the citing authors’ procedure, not a finding established by this source. (LLM, conf=0.84)

      Experimental — accuracy not yet measured

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

    Premisecitation

    In vivo functionality of DREADD-mediated astrocyte activation has previously been demonstrated in mice, with excitation of hM3D-expressing astrocytes in CA1 resulting in increased neuronal activity and enhanced memory.

    “The in vivo functionality of DREADD-mediated astrocyte activation has previously been demonstrated in mice, with excitation of hM3Dexpressing astrocytes in the CA1 region of the hippocampus resulting in increased neuronal activity and enhanced memory (Adamsky et al., 2018).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Adamsky et al., 2018 source

      The Results (Figures 4 and 6; page numbers not supplied) show that activating hM3Dq-expressing CA1 astrocytes in mice enhanced memory and increased neuronal activity. The neuronal increase was observed only when activation was coupled with learning, not in home-caged mice; the citing sentence omits that condition. (LLM, conf=0.85)

      Experimental — accuracy not yet measured

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

    Premisecitation

    Previous studies have expressed the Gq-DREADD specifically in astrocytes using the GFAP promotor and showed an increase in intracellular Ca2+ after activation with CNO.

    “Previous studies have expressed the Gq-DREADD specifically in astrocytes using the GFAP promotor and showed an increase in intracellular Ca 2+ after activation with CNO (Agulhon et al., 2013;Bull et al., 2014;Scofield et al., 2015).”
    Quote in the paperNot a substantial claim3 of 3 sources checked
    • cites Agulhon et al., 2013 source

      The Discussion says hM3Dq DREADD was expressed under the GFAP promoter and restricted to astrocytes within the CNS, but also expressed in non-myelinating Schwann cells and satellite cells in the PNS. The Results report CNO-induced Ca2+ increases in 95.51% of hippocampal astrocytes. Thus the activation finding is supported, but expression was not specific to astrocytes throughout the mouse. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites Bull et al., 2014 source
    • cites Scofield et al., 2015 source

      The Results section, “Astrocyte-specific Expression of Gq-DREADD” (page unavailable), reports GFAP-driven Gq-DREADD expression in astrocytes. The paper measured CNO-induced glutamate release, not an increase in intracellular Ca2+; its calcium discussion cites a separate report and infers a role for internal calcium stores. Thus, this study does not itself show the claimed calcium increase. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

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

    Premisecitation

    This subgroup of microglia (DAM) were identified while studying the immune cells at the single cell level in a mouse model for AD.

    “This subgroup of microglia were identified while studying the immune cells at the single cell level in a mouse model for AD (Deczkowska et al., 2018;Keren-Shaul et al., 2017).”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites Deczkowska et al., 2018 source
    • cites Keren-Shaul et al., 2017 source

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

    Premisecitation

    Activation of astrocytes in the mouse hippocampus using the Gq-DREADD influences the excitability of neurons.

    “Previous research also showed that activation of astrocytes in the mouse hippocampus using the Gq-DREADD influences the excitability of neurons.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Durkee et al., 2019 source

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

    Premisecitation

    Enzymatic dissociation of brain tissue at 37°C results in an upregulation of inflammatory genes in microglia.

    “Research has also shown that the enzymatic dissociation of brain tissue at 37°C results in an upregulation of inflammatory genes in microglia.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Bennett et al., 2016 source

      The section “RNAseq Profiles Quality and Purity: Lower Expression of Activation Marker Genes” reports lower expression of inflammatory activation markers in microglia isolated by the authors’ procedure than in published datasets that used enzymatic digestion or Percoll. It does not specify 37°C or establish that enzymatic dissociation itself caused the higher expression; the procedures differed in other ways too. The “Other Analyses: Microglial Activation” section attributes inflammatory-gene upregulation to LPS, not dissociation. Page numbers are unavailable in our copy. (LLM, conf=0.82)

      Experimental — accuracy not yet measured

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

    Premisecitation

    One drawback of an ice-cold dissociation is the decrease in efficiency of dissociation with certain tissues such as the brain.

    “However, one drawback of an ice-cold dissociation is the decrease in efficiency of dissociation with certain tissues such as the brain (Adam et al., 2017;Denisenko et al., 2019;van den Brink et al., 2017).”
    Quote in the paperNot a substantial claim1 of 3 sources checked
    • cites Adam et al., 2017 source
    • cites Denisenko et al., 2019 source

      The Discussion (page not provided) describes lower efficiency in releasing hard-to-dissociate cell types as a possible drawback of cold dissociation, using kidney loop-of-Henle cells as a tentative example. It does not establish reduced dissociation efficiency in brain tissue; the study used mouse kidneys. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites van den Brink et al., 2017 source

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

    Premisecitation

    These astrocytes (Batiuk et al.'s cluster 5) seem to be enriched in cortical layers 2/3 and 5, and in the dentate gyrus of the hippocampus.

    “These astrocytes seem to be enriched in cortical layers 2/3 and 5, and in the dentate gyrus of the hippocampus (Batiuk et al., 2020).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Batiuk et al., 2020 source

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

    Premisecitation

    A recent study (Batiuk et al., 2020) determined the transcriptomic profile of astrocytes in the hippocampus and cortex of C57BL/6J mice at post-natal day 56 and showed the existence of five transcriptionally different astrocytic clusters.

    “Furthermore, a recent study (Batiuk et al., 2020) determined the transcriptomic profile of astrocytes in the hippocampus and cortex of C57BL/6J mice at post-natal day 56 and showed the existence of five transcriptionally different astrocytic clusters.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Batiuk et al., 2020 source

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

    Premisecitation

    Previous studies mostly perform scRNA seq in younger mice, while single nucleus RNA sequencing is more common in adult mice.

    “Previous studies mostly perform scRNA seq in younger mice, while single nucleus RNA sequencing is more common in adult mice (Hook et al., 2018;Loo et al., 2019;Zeisel et al., 2015;Zhou et al., 2020).”
    Quote in the paperNot a substantial claim1 of 3 sources checked
    • cites Hook et al., 2018

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

    • cites Loo et al., 2019 source

      The Results section, “Single-cell transcriptomics of the E14.5 and P0 cortex” (page unavailable), confirms that this study used single-cell RNA sequencing in embryonic and newborn mice. The paper does not establish that previous studies mostly used younger mice or that single-nucleus sequencing was more common in adults; its Introduction also describes single-cell RNA-seq being used to catalog adult nervous-system cells. (LLM, conf=0.84)

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

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

    Premisecitation

    Previous research has shown that the transcriptomic profile of astrocytes varies in response to different insults, e.g. long-term activation.

    “Previous research has shown that the transcriptomic profile of astrocytes varies in response to different”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites Anderson et al., 2014

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

    • cites Hamby et al., 2012 source

      The Results section ('Treatment-dependent global changes in gene expression profiles'; page unavailable) reports different astrocyte gene-expression changes under different inflammatory treatments. The Methods section ('Cytokine and endotoxin exposure'; page unavailable) describes exposures lasting hours to about two days, not long-term activation, so that example is not supported. (LLM, conf=0.85)

      Experimental — accuracy not yet measured

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

    Premisecitation

    It is well established that both astrocytes and microglia show regional heterogeneity at the transcriptome level.

    “A possible explanation for this phenomenon is that we analyzed the cortex and hippocampus together, while it is well established that both astrocytes and microglia show regional heterogeneity at the transcriptome level (Batiuk et al., 2020;Tan et al., 2020).”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites Batiuk et al., 2020 source

      The Results (Single-cell mRNA sequencing reveals astrocyte heterogeneity) report transcriptomic differences between cortical and hippocampal astrocyte subtypes, supporting the astrocyte part of the jointly cited claim. Our copy does not establish regional transcriptomic heterogeneity in microglia. (LLM, conf=0.85)

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

      The Introduction describes spatial patterns of astrocyte-specific genes, and “Heterogeneity in microglial molecular signature across CNS regions” reports transcriptomic studies showing regional differences in microglial gene expression. But that section also describes conflicting single-cell results, including adult mouse microglia with remarkably similar transcriptomes across regions. Thus the source supports regional transcriptomic heterogeneity in both cell types, but not the unqualified assertion that it is well established for microglia. (LLM, conf=0.85)

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim.

    Study design and conductcitation

    All cell clusters were annotated manually based on marker genes that were conserved between the saccharin group and the CNO group and gene lists present in literature.

    “All cell clusters were annotated manually based on marker genes that were conserved between the saccharin group and the CNO group and gene lists present in literature (Arneson et al., 2018;McKenzie et al., 2018).”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites Arneson et al., 2018 source

      The Methods section, “Resolving cell identities of the cell clusters” (page unavailable), describes assigning cluster identities using curated markers from previous studies and checking their expression in the clusters. It does not describe markers conserved between saccharin and CNO groups, so the cited paper supports the literature-marker approach but not that group-specific detail. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
    • cites McKenzie et al., 2018 source

  • May combine two claims.

    Premisecitation

    The central nervous system of older mice is more complex than that of younger mice, making it harder to dissociate it into single cells, and as a result some cell types can get over- or underrepresented.

    “The central nervous system of older mice is more complex than that of younger mice, making it harder to dissociate it into single cells, and as a result some cell types can get over-or underrepresented (Darmanis et al., 2015;Grindberg et al., 2013;Krishnaswami et al., 2016;Lake et al., 2016;Lake et al., 2017;Tasic et al., 2018).”
    Quote in the paperSubstantial claim3 of 3 sources checked
    • cites Darmanis et al., 2015 source

      The Materials and Methods section says that human brain tissue was dissociated into single-cell suspensions, but does not compare older and younger mice. The Abstract and Results discuss human developmental stages and differences between humans and mice, not age-related difficulty of dissociating mouse CNS tissue or resulting over- or underrepresentation of cell types. Searches for dissociation difficulty and cell-type sampling bias found no passage addressing the claim; page numbers were not available in our copy. (LLM, conf=0.75)

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

      The Discussion (page unavailable) says that intact single cells are difficult to isolate from complex CNS tissue, supporting the dissociation difficulty. It does not compare older and younger mice or say that dissociation over- or underrepresents particular cell types. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites Krishnaswami et al., 2016 source

      The Introduction (page unavailable) says that CNS cells are difficult to isolate intact because neurons are highly interconnected. It does not compare older and younger mice or say that dissociation causes cell types to be over- or underrepresented. (LLM, conf=0.85)

      Experimental — accuracy not yet measured

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

    Premisecitation

    Increased intracellular Ca2+ levels in astrocytes stimulate glutamate release, which results in activation of neuronal NMDA receptors.

    “The increased intracellular Ca 2+ levels stimulated glutamate release which resulted in activation of neuronal NMDA receptors (Durkee et al., 2019).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Durkee et al., 2019 source

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

    Findingown data

    A total of 34,736 cells passed quality control filters, with 16,107 cells in CNO treated group and 18,629 cells in saccharin group.

    “A total of 34,736 cells passed the quality control filters, 16,107 cells in the CNO treated group and 18,629 cells in the saccharin group.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    In microglia I, clusters 0, 3 and 5 were more common in the saccharin group, while clusters 1 and 2 were more pronounced in the CNO group.

    “In addition, clusters 0, 3 and 5 were more common in the saccharin group, while clusters 1 and 2 were more pronounced in the CNO group (Figure 3E and Supplementary Table 3).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Gene expression analysis showed a relative upregulation of homeostatic microglial genes in clusters 1, 4 and 5 of microglia I, while they were downregulated in clusters 0, 2, 3 and 6.

    “Gene expression analysis showed a relative upregulation of homeostatic microglial genes in clusters 1, 4 and 5, while they were downregulated in clusters 0, 2, 3 and 6 (Figure 3F).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    mCherry did not show co-localization with the neuronal marker NeuN or the microglial marker Iba1.

    “Moreover, mCherry did not show co-localization with the neuronal marker, NeuN, or the microglial marker, Iba1 (Supplementary Figure 1A and1B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    After astrocyte activation with CNO, there was a 35% increase in number of cells in microglia I cluster and a 42% decrease in microglia II cluster.

    “Even though Gq-DREADD was only expressed in astrocytes, we observed a 35% increase and a 42% decrease in the number of cells in the microglia I and II clusters, respectively, after astrocyte activation with CNO (Figure 1E and Supplementary Table 1).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Re-clustering of microglia I cluster resulted in seven sub-clusters of which clusters 4 and 6 were only observed in the CNO group.

    “Re-clustering of the microglia I cluster resulted in seven sub-clusters of which clusters 4 and 6 were only observed in the CNO group (Figure 3D, 3E and Supplementary Table 3).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In microglia II, three sub-clusters (0, 2 and 3) were mainly observed in saccharin group, two (1 and 4) were more common in CNO group, and two (5 and 6) were present in both groups.

    “Three sub-clusters were mainly observed in the saccharin group (clusters 0, 2 and 3), two were more common in the CNO group (clusters 1 and 4), and two others (clusters 5 and 6) were present in both groups (Figure 3G, 3H and Supplementary Table 3).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Gene enrichment of cluster 0 of microglia V showed these cells were responsive to cytokines, while cluster 2 showed involvement in MHC binding.

    “The gene enrichment and functional annotation of the differentially expressed genes for cluster 0 showed that these cells were responsive to cytokines, while cluster 2 showed an involvement in major histocompatibility complex (MHC) binding.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In microglia III cluster, differentially expressed genes showed an enrichment for transcripts involved in the immune system and cell killing, while a downregulation of transcripts involved in monocyte differentiation was observed.

    “In the microglia III cluster, gene enrichment and functional analysis of the differentially expressed genes showed an enrichment for transcripts involved in the immune system and cell killing, while a downregulation of transcripts involved in monocyte differentiation was observed (Supplementary Figure 3A and Supplementary Data 1).”
    Quote in the paperSubstantial claim

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

    Findingown data

    In mixed glia II, clusters 1 and 2 had only one differentially expressed gene each.

    “No gene ontology analysis was performed on clusters 1 and 2 since there was only one differentially expressed gene in each cluster (Supplementary Data 9).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    In microglia III, cluster 0 was six times more prominent in saccharin group (56.08%) compared to CNO group (8.95%), and cluster 2 was nine times more pronounced in CNO group (36.24%) than in saccharin group (3.97%).

    “Although all of them were observed in both groups, cluster 0 was six times more prominent in the saccharin group (56.08%) compared to the CNO group (8.95%), and cluster 2 was nine times more pronounced in the CNO group (36.24%) than in the saccharin group (3.97%, Figure 4A, 4B and Supplementary Table 3).”
    Quote in the paperSubstantial claim

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

    Findingown data

    Microglia IV cluster showed five sub-clusters after re-clustering.

    “The microglia IV cluster showed five sub-clusters after re-clustering (Figure 4C).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    In microglia V, clusters 0 and 2 showed a 1.7-fold increase and a 50% decrease in cell numbers between saccharin and CNO groups, respectively.

    “Clusters 0 and 2, showed a 1.7-fold increase and a 50% decrease in cell numbers between the saccharin and the CNO groups, respectively (Supplementary Figure 3E and Supplementary Table 3).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Although the up- or downregulation of differentially expressed genes was relatively low, transcriptionally different astrocytic sub-clusters were clearly observed.

    “Although the up-or downregulation of differentially expressed genes was relatively low, we could clearly observe transcriptionally different astrocytic sub-clusters (Figure 2F).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Activation of astrocytes using CNO led to a 48% increase in cell numbers in mixed glia I cluster and a 50% decrease in mixed glia II cluster.

    “Activation of astrocytes using CNO led to a 48% increase in cell numbers in the mixed glia I cluster and a 50% decrease in the mixed glia II cluster (Figure 1E and Supplementary Table 1).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In microglia V, clusters 0, 2 and 3 showed relatively low expression of homeostatic genes, while cluster 1 showed increased expression compared to the other three clusters.

    “Cluster 0, 2 and 3 showed relatively low expression of homeostatic genes, while cluster 1 showed an increased expression of these genes compared to the other three clusters (Supplementary Figure 2F).”
    Quote in the paperNot a substantial claim

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

    Findingown data

    Two cell clusters were annotated as 'Mixed Glia' because it was not clear from conserved marker genes whether these cells should be annotated as astrocytes, microglia or oligodendrocytes.

    “We annotated two cell clusters as 'Mixed Glia' since it was not clear from their conserved marker genes whether these cells should be annotated as astrocytes (e.g.Cpe and Apoe), microglia (e.g.Actb) or oligodendrocytes (e.g.Dpysl2; Figure 5A and5B).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    A strong downregulation of homeostatic microglial genes was observed in cluster 6 of microglia II, while an upregulation was observed in cluster 5.

    “A strong downregulation of homeostatic microglial genes was observed in cluster 6, while an upregulation was observed in cluster 5 (Figure 3I).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    We observed microglial clusters that were absent in the control group and dominating in the CNO group.

    “We observed microglial clusters that were absent in the control group and dominating in the CNO group (Figures 3D,3G,4A and 4C).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    We observed a 48% increase and 50% decrease in cell numbers in the two largest cell clusters, mixed glia I and mixed glia II respectively, after Gq-DREADD-mediated activation of astrocytes.

    “We further observed a 48% increase and 50% decrease in cell numbers in the two largest cell clusters, mixed glia I and mixed glia II respectively, after Gq-DREADD-mediated activation of astrocytes.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Our single cell dissociation protocol shows a dramatic enrichment of glial cells and a relative depletion of neurons.

    “Lastly, our single cell dissociation protocol shows a dramatic enrichment of glial cells and a relative depletion of neurons.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Certain microglial sub-clusters showed an increased expression of genes that are known to characterize homeostatic microglia, while other subclusters showed a decreased expression of these genes.

    “Additionally, we observed that certain microglial sub-clusters showed an increased expression of genes that are known to characterize homeostatic microglia, while other subclusters showed a decreased expression of these genes (Figure 3F, 3I, 4E and Supplementary Figure 3F).”
    Quote in the paperSubstantial claim

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

    Premisecitation

    Activated astrocytes release ATP that can activate purinergic receptors on neighboring microglia, resulting in microglial activation.

    “Previous research has shown that activated astrocytes release adenosine triphosphate (ATP) that can activate the purinergic receptors on neighboring microglia, resulting in microglial activation (Figure 3A) (Davalos et al., 2005;Liu et al., 2011;Schipke et al., 2002;Verderio and Matteoli, 2001).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Davalos et al., 2005 source
    • cites Liu et al., 2011 source
    • cites Schipke et al., 2002

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

  • May combine two claims.

    Study design and conductown data

    Less than 1% of our total cell population consists of neuronal cells, so we were unable to confirm neuronal activation at the transcriptomic level.

    “Since less than 1% of our total cell population subsists of neuronal cells, we were unable to confirm this neuronal activation at the transcriptomic level.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S1 (asserted).

    Interpretationown data

    DAM genes are induced following astrocyte activation.

    “Our data hypothesizes that DAM genes are induced following astrocyte activation.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S1 (asserted).

    Interpretationreasoning

    Long-term activation of astrocytes changes their transcriptional profile to a thus far transcriptionally uncharacterized phenotype.

    “Therefore, we hypothesize that long-term activation of astrocytes changes their transcriptional profile to a, thus far transcriptionally uncharacterized phenotype.”
    Quote in the paperNot a substantial claim

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

    Study design and conductreasoning

    To arrest ongoing gene expression and to decrease the likelihood of inducing heat shock proteins or immediate-early response genes, we performed our dissociation protocol at 4°C instead of 37°C.

    “To arrest ongoing gene expression and to decrease the likelihood of inducing heat shock proteins or immediate-early response genes, we performed our dissociation protocol at 4°C instead of 37°C.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Activated astrocytes release ATP which activates the purinergic receptors on microglia, resulting in microglial activation.

    “As mentioned earlier, activated astrocytes release ATP which activates the purinergic receptors on microglia, resulting in microglial activation (Figure 3A) (Davalos et al., 2005;Liu et al., 2011;Schipke et al., 2002;Verderio and Matteoli, 2001).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Davalos et al., 2005 source
    • cites Liu et al., 2011 source
    • cites Schipke et al., 2002

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

  • Probably not a substantial claim: may not pass S1 (asserted). May combine two claims.

    Interpretationown data

    DAM cells may not always be linked to amyloid pathology and thus are not always disease-associated.

    “We observed an upregulation of DAM genes in some of our microglial sub-clusters (Supplementary Figure 4), resulting in the hypothesis that DAM cells may not always be linked to amyloid pathology and thus are not always disease-associated.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S1 (asserted).

    Interpretationreasoning

    The microglial response is secondary to the astrocytic response in AD.

    “This would implicate that the microglial response is secondary to the astrocytic response in AD, which can have major implications for AD research.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: Gq-DREADD-mediated astrocyte activation in healthy mice models the astrocytic activation that occurs in AD.

  • May combine two claims.

    Interpretationown data

    After long-term activation of astrocytes, the microglial transcriptome suggests a role in lipoprotein particle processes, and migration and chemotaxis of immune cells.

    “After long-term activation of astrocytes, the microglial transcriptome suggests a role in lipoprotein particle processes, and migration and chemotaxis of immune cells.”
    Quote in the paperSubstantial claim
49 of 49

  • The authors state this as an assumption.

    The effect of astrocyte activation on the transcriptome of neighboring cells had not been investigated before this study.

    “However, the effect of astrocyte activation on the transcriptome of neighboring cells was not investigated.”

  • The paper relies on this without support.

    Increase of intracellular Ca2+ after astrocytic activation results in calcium waves that can activate neighboring astrocytes and microglia.

    “In addition, it is known that the increase of intracellular Ca 2+ after astrocytic activation results in calcium waves that can activate neighboring astrocytes and microglia (Figure 3A).”

  • The authors state this as an assumption.

    This study is the first to investigate the effect of Gq-DREADD activated astrocytes on surrounding brain cells at the single cell level using scRNA seq.

    “To the best of our knowledge, our study is the first to investigate the effect of Gq-DREADD activated astrocytes on surrounding brain cells at the single cell level using scRNA seq.”

  • The paper relies on this without support.

    Gq-DREADD-mediated astrocyte activation in healthy mice models the astrocytic activation that occurs in AD.

    “This would implicate that the microglial response is secondary to the astrocytic response in AD, which can have major implications for AD research.”
4 of 4

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.

  • Microglial cell-count changes are calculated from percentages, not counts

    Minor

    The reported 35% increase and 42% decrease match changes in the clusters' shares of each group, not changes in cell numbers. Using the stated group totals of 18,629 saccharin cells and 16,107 CNO cells, the implied microglia I count increases by about 17%, while the microglia II count decreases by about 50%.

1 of 1

Cites

Flagged first, then by citations

Research works printed but not linked: 2

  1. 10.1038/nbt.4096 · DOI · Paper page
  2. 10.1038/s41380-019-0609- · DOI

Cited by

Sorted by citations

Related

Authors
Philtjens, S., Turnbull, M. T., Thedy, B. P., Moon, Y., & Kim, J.
Affiliations
  • Indiana University School of Medicine
  • Mayo Clinic (US)
Corresponding author
Kim, Jungsu
ORCID
Venue
bioRxiv (Cold Spring Harbor Laboratory)
Publisher
openRxiv
Published
2020-04-28
DOI
10.1101/2020.04.27.064881
OpenAlex
W3165839118
Cited by
7
References
64
Citations by year
2024: 2 · 2022: 2 · 2021: 3
Type
posted content
Language
en
Open access
Licence
Locations
bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by)
Funders
  • National Science Foundation (UL1TR002529, 0521433)
  • Eli Lilly and Company
  • Indiana Clinical and Translational Sciences Institute (UL1TR002529)
  • Lilly Endowment
  • Servier
  • National Institutes of Health (3ul1tr002529-03s1, R01AG053500, UL1 TR002529, R01AG053242, R21AG050804)
  • School of Medicine, Indiana University (UL1TR002529)
  • National Institute on Aging (R01AG054102)
  • National Center for Advancing Translational Sciences (UL1TR002529)
Topics
  • Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Neuroscience and Neuropharmacology Research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Single-cell and spatial transcriptomics
Keywords
astrocyte activation, microglia, astrocyte-microglia crosstalk, DREADD, single-cell transcriptomics, hippocampus, cortex, lipoprotein metabolism, purinergic receptors, leukocyte migration, calcium homeostasis, G protein-coupled receptor signaling, neurogenesis

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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.

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Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-17.

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