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Cited by 7,971Neuroinflammation and Neurodegeneration MechanismsNeuroscience and Neuropharmacology ResearchTraumatic Brain Injury and Neurovascular Disturbances

Neurotoxic reactive astrocytes are induced by activated microglia

Open access
Liddelow, Shane A., Guttenplan, Kevin A., Clarke, Laura E., Bennett, Frederick C., Bohlen, Christopher J., Schirmer, Lucas, Bennett, Mariko L., Münch, Alexandra E. +13 more·2017·Nature
DOI 10.1038/nature21029PMC PMC5404890
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Abstract

Reactive astrocytes are strongly induced by central nervous system (CNS) injury and disease, but their role is poorly understood. Here we show that a subtype of reactive astrocytes, which we termed A1, is induced by classically activated neuroinflammatory microglia. We show that activated microglia induce A1 astrocytes by secreting Il-1α, TNF and C1q, and that these cytokines together are necessary and sufficient to induce A1 astrocytes. A1 astrocytes lose the ability to promote neuronal survival, outgrowth, synaptogenesis and phagocytosis, and induce the death of neurons and oligodendrocytes. Death of axotomized CNS neurons in vivo is prevented when the formation of A1 astrocytes is blocked. Finally, we show that A1 astrocytes are abundant in various human neurodegenerative diseases including Alzheimer's, Huntington's and Parkinson's disease, amyotrophic lateral sclerosis and multiple sclerosis. Taken together these findings help to explain why CNS neurons die after axotomy, strongly suggest that A1 astrocytes contribute to the death of neurons and oligodendrocytes in neurodegenerative disorders, and provide opportunities for the development of new treatments for these diseases.

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Crossref · 10.1038/nature21029

Cites

48 of 48 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

8 of 11 cited sources checked · 4 supported · 1 flagged · accuracy not yet measured

  • Premisecitation

    Il-1α, TNFα, and C1q are highly expressed specifically by microglia.

    “All three of these cytokines are highly expressed specifically by microglia13,15, again suggesting a critical role for microglia in inducing A1 reactive astrocytes.”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [13] Zhang, Y. et al. An RNA-sequencing transcriptome and splicing database of glia, neurons, and vascular cells of the cerebral cortex. J. Neurosci. 34, 11929–11947 (2014) source

      Table 2 (Additional information; page unavailable) lists C1qa, C1qb, and C1qc among microglia-enriched ligands, but places Tnf and Il1a in the myelinating-oligodendrocyte column. The source therefore contradicts the claim that all three are specifically highly expressed by microglia. (LLM, conf=0.87)

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured
    • cites [15] Bennett, M. L. et al. New tools for studying microglia in the mouse and human CNS. Proc. Natl Acad. Sci. USA 113, E1738–E1746 (2016) source

      In “Tmem119 mRNA Expression Is Highly Enriched in the CNS and Specific,” Il1a is listed among highly expressed microglia-enriched candidates. The RNA-seq section reports much higher C1q gene expression in microglia than in other myeloid cells, but also identifies C1q-positive macrophages. The activation section reports Tnf upregulation after LPS, not that TNFα is highly expressed specifically by microglia. No page numbers are supplied for these passages. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
  • Findingcitation

    Wild-type littermate controls had a normal response to LPS injection with robust induction of an A1 response.

    “As expected, wild-type littermate controls had a normal response to LPS injection5,17, with robust induction of an A1 response (Fig. 1a)”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [5] Zamanian, J. L. et al. Genomic analysis of reactive astrogliosis. J. Neurosci. 32, 6391–6410 (2012) source
    • cites [17] Cartmell, T., Luheshi, G. N. & Rothwell, N. J. Brain sites of action of endogenous interleukin-1 in the febrile response to localized inflammation in the rat. J. Physiol. (Lond.) 518, 585–594 (1999) source
  • Findingown data

    The phagocytic deficit corresponded with a 90% decrease in Mertk and 60% decrease in Megf10 mRNA.

    “This phagocytic deficit corresponded with a 90% decrease in Mertk and 60% decrease in Megf10 mRNA, phagocytic receptors we have previously found mediate synaptic phagocytosis3 (Fig. 3f).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [3] Chung, W.-S. S. et al. Astrocytes mediate synapse elimination through MEGF10 and MERTK pathways. Nature 504, 394–400 (2013) source
  • Findingown data

    Death of axotomized CNS neurons in vivo is prevented when A1 formation is blocked.

    “Death of axotomized CNS neurons in vivo is prevented when A1 formation is blocked.”
    Quote in the paperSubstantial claim
  • Findingown data

    A1s induce death of oligodendrocytes.

    “A1s lose the ability to promote neuronal survival, outgrowth, synaptogenesis and phagocytosis, and induce death of neurons and oligodendrocytes.”
    Quote in the paperSubstantial claim
  • Classified as probably not a substantial claim. 16
  • May combine two claims. 37

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

    Premisecitation

    Reactive astrocytes induced by ischemia promote CNS recovery and repair.

    “previous studies have provided evidence that reactive astrocytes induced by ischemia promote CNS recovery and repair1,10,11.”
    Quote in the paperNot a substantial claim2 of 3 sources checked
    • cites [1] Sofroniew, M. V. & Vinters, H. V. Astrocytes: biology and pathology. Acta Neuropathol. 119, 7–35 (2010) source
    • cites [10] Bush, T. G. et al. Leukocyte infiltration, neuronal degeneration, and neurite outgrowth after ablation of scar-forming, reactive astrocytes in adult transgenic mice. Neuron 23, 297–308 (1999) source
    • cites [11] Zador, Z., Stiver, S., Wang, V. & Manley, G. T. Role of aquaporin-4 in cerebral edema and stroke. Handb. Exp. Pharmacol. 190, 159–170 (2009) source

      Section 3.2 discusses ischemic edema and tissue damage, but does not say that reactive astrocytes promote recovery or repair. Searches for reactive astrocytes, recovery, repair, and glial scar formation found no passage making that claim; the glial-scar reference alone does not establish it. (LLM, conf=0.73)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Neuroinflammation and ischemia induced two different types of reactive astrocytes termed A1 and A2.

    “We previously purified and gene profiled reactive astrocytes from mice treated either with a systemic injection of lipopolysaccharide (LPS), or received middle cerebral artery occlusion to induce ischemia5. We found neuroinflammation and ischemia induced two different types of reactive astrocytes that we termed “A1” and “A2” respectively”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [5] Zamanian, J. L. et al. Genomic analysis of reactive astrogliosis. J. Neurosci. 32, 6391–6410 (2012) source

      The Discussion section on stimulus-dependent phenotypes reports distinct LPS- and MCAO-induced reactive astrocyte subtypes, supporting the core finding. Our copy calls them the LPS and MCAO subtypes, but does not show that this paper termed them “A1” and “A2”; searches for those labels returned no matching passage. Page numbers were unavailable. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

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

    Study design and conductcitation

    Quantitative RT-PCR was conducted following MIQE guidelines.

    “Quantitative RT-PCR was conducted following the MIQE (minimum information for publication of quantitative real-time PCR experiments) guidelines38.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [38] Bustin, S. A. et al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin. Chem. 55, 611–622 (2009) source

  • May combine two claims.

    Findingown data

    Il-1α, TNFα, and C1q together are necessary and sufficient to induce A1s.

    “We show that activated microglia induce A1s by secreting Il-1α, TNFα, and C1q, and that these cytokines together are necessary and sufficient to induce A1s.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Weekly injection of neutralizing antibodies to Il-1α, TNFα, and C1q together into the vitreous from the time of injury inhibited A1 formation and prevented death of RGCs at least as long as 14 days post-ONC.

    “Weekly injection of neutralizing antibodies to Il-1α, TNFα, and C1q together into the vitreous of the eye from the time of injury inhibited A1 formation and prevented death of RGCs at least as long as 14 days post-ONC (Fig. 4g–j).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    LPS-activated MCM, but not resting MCM, strongly induced A1 reactive astrocytes.

    “LPS-activated MCM, but not resting MCM, strongly induced A1 reactive astrocytes (Fig. 1a).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Microglia from these knockout mice still upregulate inflammatory mediators in response to LPS but fail to release A1 initiators.

    “Microglia from these same knock-out mice still upregulate inflammatory mediators in response to LPS injection, but simply fail to release A1 initiators (Extended Data Fig. 4).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Levels of Il-1α, TNFα and C1q were all significantly elevated after microglial activation.

    “Levels of Il-1α, TNFα and C1q were all significantly elevated after microglial activation (Fig. 1b, c).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Il-1β secretion increased in LPS-activated MCM but was unable to induce expression of A1 transcripts.

    “Il-1β secretion also increased in LPS-activated MCM, but was unable to induce expression of A1 transcripts (Fig. 1a).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    A1s were not toxic to oligodendrocyte precursor cells but were able to slow their differentiation and division.

    “Although not toxic to oligodendrocyte precursor cells, A1s were able to slow their differentiation and division (Extended Data Fig. 8).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    ONC rapidly induced robust A1 generation that was temporally paired with death of RGCs.

    “ONC rapidly induced robust A1 generation that was temporally paired with death of RGCs (Fig. 4i–o).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Control astrocytes are able to robustly phagocytose myelin debris, but upon conversion to an A1 reactive phenotype almost completely lose this capacity.

    “Similarly, we found control astrocytes are able to robustly phagocytose myelin debris, but upon conversion to an A1 reactive phenotype almost completely lose this capacity (Fig. 3a,c).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Quantitative PCR showed decreased Gpc6 and Sparcl1, while simultaneously showing increased expression of Thbs1/2.

    “Quantitative PCR showed decreased Gpc6 and Sparcl1, while simultaneously showing increased expression of Thbs1/2 (Fig. 2c).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Double and triple knockouts showed decreases in A1 reactivity, with triple knockouts having no response following systemic LPS injection.

    “Additionally, we looked at double (Il-1α−/−TNFα−/−) and triple knock-out mice (Il-1α−/−TNFα−/−C1q−/−) and saw decreases in A1 reactivity, with triple knock-out animals having no response following systemic LPS injection (Fig. 1e).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Death could not be attributed to Il-1α, TNFα, and C1q alone, which did not cause death of cells in purified cultures.

    “This death could not be attributed to Il-1α, TNFα, and C1q alone, which did not cause death of cells in purified cultures.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Death was blocked by caspase-2/-3 inhibitors but not by necrostatin or glutamate blockers and thus appeared to be due to apoptosis.

    “Death was blocked by caspase-2/-3 inhibitors but not by necrostatin or glutamate blockers and thus appeared to be due to apoptosis (Extended Data Fig. 9).”
    Quote in the paperSubstantial claim

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

    Findingown data

    S100A10 positive, GFAP colabelled astrocytes were only faintly labelled, and total numbers were very low.

    “S100A10 positive, GFAP colabelled astrocytes (red arrows) were only faintly labelled, and total numbers were very low.”
    Quote in the paperNot a substantial claim

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

    Findingown data

    A2-specific GFAP+ reactive astrocytes (S100A10) are present in low CD68+ density areas.

    “Note presence of A2-specific GFAP+ reactive astrocytes (S100A10; red arrows) in low CD68+ density areas (phagocytes; yellow arrowheads).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Both CFB and MX1 co-localised 100% with C3, and were found in close association with CD68+ reactive microglia.

    “Both CFB and MX1 co-localised 100% with C3, and were found in close association with CD68+ reactive microglia (yellow arrowheads).”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: CD68+ cells in MS lesions are reactive microglia.

  • May combine two claims.

    Findingown data

    PLX-3397 treatment dramatically reduces microglia number in adult mice, with around a 95% decrease in P21 wildtype mice after 7 days.

    “Pexinartinib (PLX-3397)- treated adult mice have dramatic reduction in number of microglia and no increase in myeloid cell infiltration after LPS compared to vehicle control treatment”
    • Also stated in: § Depletion of microglia using Pexidartinib (PLX-3397) · plain
      “At this stage flow cytometry showed around 95% decrease in microglia cell number (Extended Data Fig. 1).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In OPCs treated with A1 ACM, there is no increase in mature oligodendrocyte marker Mbp transcript, and no change in OPC markers Pdgfra and Cspg4 expression.

    “f–h, Representative RT-PCR ethidium bromide gel showing no increase in mature OL marker Mbp transcript in OPCs treated with A1 ACM, with no change in OPC marker Pdgfra and Cspg4 expression – evidence of a lack of differentiation into mature oligodendrocytes.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In saline-injected control mice, AQP4 protein localized robustly to astrocytic endfeet on blood vessels; after LPS injection, AQP4 immunoreactivity lost polarization, bled away from endfeet, and increased in other astrocyte regions.

    “Saline injected (control) mice had robust AQP4 protein localization to astrocytic endfeet on blood vessels (red stain, white arrows), while LPS injected mice had loss of polarization of AQP4 immunoreactivity, with bleeding of immunoreactivity away from endfeet (white arrows) and increased staining in other regions of the astrocyte (yellow arrowheads).”
    Quote in the paperNot a substantial claim

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

    Findingnone given

    A1s are not directly toxic to bacteria.

    “Although A1s are not directly toxic to bacteria”
    Quote in the paperNot a substantial claim

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

    Findingown data

    The expression levels of S100a10, determined by qPCR, was not significantly altered at different stages of MS.

    “The expression levels of S100a10, determined by qPCR, was not significantly altered at different stages of MS.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    24 hours following LPS-induced systemic neuroinflammation, astrocytes were either non-reactive or fell into three forms of reactivity, all with A1 reactive cassette genes upregulated.

    “24 hours following LPS-induced systemic neuroinflammation, astrocytes were either non-reactive (no reactive genes upregulated), or fell into three forms of reactivity – all with A1 reactive cassette genes upregulated.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Conditioned media from control astrocytes contained less chondroitin sulphate proteoglycans Brevican, Ng2, Neurocan and Versican, while having higher levels of heparan sulphate proteoglycans Syndecan and Glypican.

    “Conditioned media from control astrocytes contained less chondroitin sulphate proteoglycans Brevican, Ng2, Neurocan and Versican, while simultaneously having higher levels of heparan sulphate proteoglycans Syndecan and Glypican.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: Cultured immunopanned astrocytes retain their non-activated in vivo gene profiles.

  • May combine two claims.

    Findingown data

    Triple knockout mice lacking Il1α, TNFα, and C1q retained AQP4 immunoreactivity in endfeet following LPS-induced neuroinflammation, though some low-level ectopic immunoreactivity remained.

    “Triple knock-out mice (Il1α−/−TNFα−/−C1q−/−) did retained AQP4 immunoreactivity in endfeet following LPS-induced neuroinflammation (white arrows), though some low-level ectopic immunoreactivty was still seen (yellow arrowheads).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Individual knockout animals (Il1α−/−, TNFα−/−, or C1q−/−) had far fewer TUNEL+ cells in the dentate gyrus than wildtype animals after systemic LPS injection; no TUNEL+ cells were seen in Il1α−/− or TNFα−/− animals.

    “Individual knock-out animals had far less TUNEL+ cells in the dentate gyrus (no cells in Il1α−/− or TNFα−/− animals) than wild type animals, suggesting A1-induced toxicity may be apoptosis.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The number of C3+GFAP+ colabelled cells was highest in acute active demyelinating lesions, but they were still present in chronic active and inactive lesions.

    “Number of C3+GFAP+ colabelled cells is quantified and was highest in acute active demyelinating lesions, however they were still present in chronic active and inactive lesions.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    A1 reactive astrocytes are harmful, contributing to neuron death after acute CNS injury.

    “Thus A1 reactive astrocytes are harmful, contributing to neuron death after acute CNS injury.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    A1s secrete a soluble toxin that rapidly kills a subset of CNS neurons and mature oligodendrocytes, but not other CNS cell types.

    “Thus, A1s secrete a soluble toxin that rapidly kills a subset of CNS neurons and mature oligodendrocytes, but not other CNS cell types.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    A1s are either unable to maintain synapses or actively disassemble them.

    “suggesting that A1s are either unable to maintain these synapses or actively disassemble them.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    A1 reactive astrocytes induce formation of fewer synapses, and the few synapses they do induce are significantly weaker when compared to those produced by healthy resting astrocytes.

    “Taken together these results show A1 reactive astrocytes induce formation of fewer synapses, and the few synapses they do induce are significantly weaker when compared to those produced by healthy resting astrocytes.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Death of RGCs after axotomy is not due to trophic deprivation but is instead due to release of a toxic signal from nearby neurotoxic A1 reactive astrocytes.

    “These data provide strong evidence that death of RGCs after axotomy is not due to trophic deprivation but is instead due to release of a toxic signal from nearby neurotoxic A1 reactive astrocytes.”
    Quote in the paperSubstantial claim

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

    Interpretationreasoning

    A1s may lose the capacity to clear myelin debris in vivo.

    “and suggest that A1s may well lose the capacity to clear myelin debris in vivo, an important area for future investigation.”
    Quote in the paperNot a substantial claim

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

    Interpretationnone given

    A1s secrete many classical complement cascade components that are expected to greatly enhance clearance of bacteria by the immune system.

    “they do secrete many classical complement cascade components that are expected to greatly enhance clearance of bacteria by the immune system.”
    Quote in the paperNot a substantial claim

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

    Study design and conductown data

    Cell-type specific transcripts were detected for microglia, oligodendrocyte lineage cells, and neurons, with any astrocyte samples containing measurable levels of other cell types removed from further analysis.

    “Cell-type specific transcripts were also detected for microglia, oligodendrocyte lineage cells, and neurons, with any astrocyte samples containing measurable levels of other cell types removed from further analysis.”
    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

    All procedures were conducted in accordance with animal care standards of the National Institute of Health and approved by Stanford University's Administrative Panel on Laboratory Animal Care.

    “All Procedure were conducted in accordance with the animal care standards of the National Institute of Health and approved by Stanford University’s Administrative Panel on Laboratory Animal Care.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationcitation

    Activated microglia strongly induce A1s, which could drive neurodegeneration by secreting a neurotoxin and by releasing multiple complement components that help drive synapse degeneration.

    “they strongly induce A1s, which could drive neurodegeneration not only by secreting a neurotoxin but by releasing multiple complement components that help to drive synapse degeneration33.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [33] Stevens, B. et al. The classical complement cascade mediates CNS synapse elimination. Cell 131, 1164–1178 (2007) source

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

    Study design and conductnone given

    Negative control sections without primary antibodies were processed in parallel for human immunohistochemistry.

    “Negative control sections without primary antibodies were processed in parallel.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    All experiments with mutants were performed blindly without knowledge of their genotype.

    “All experiments with mutants were performed blindly without knowledge of their genotype.”
    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

    Animals were randomly assigned numbers and evaluated thereafter blind to both experimental condition and genotype.

    “Animals were randomly assigned numbers and evaluated thereafter blind (to both experimental condition and genotype).”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductcitation

    Retinal ganglion cells were purified from postnatal day 5 rats by sequential immunopanning to greater than 99% purity.

    “We purified retinal ganglion cells from postnatal day 5 rats by sequential immunopanning to greater than 99% purity and cultured them in serum-free medium as previously described41.”
    Quote in the paperSubstantial claim

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

    Study design and conductown data

    Group sizes provided at least 80% calculable power with Type I error 0.05 and conservative effect size 0.25.

    “Group sizes were used to provide at least 80% calculable power with the following parameters: probability of Type I error (0.05), conservative effect size (0.25).”
    Quote in the paperNot a substantial claim

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

    Study design and conductown data

    At least 6 independent experiments were conducted for each condition in the survival/cell toxicity assays.

    “At least 6 independent experiments were conducted for each condition.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: Cultured immunopanned astrocytes retain their non-activated in vivo gene profiles.

  • May combine two claims.

    Interpretationown data

    A1 neurotoxic reactive astrocyte formation is a fundamental pathological response of the CNS to LPS-induced neuroinflammation, acute CNS injury, and most or all neurodegenerative diseases.

    “Here we have identified A1 neurotoxic reactive astrocyte formation as a fundamental pathological response of the CNS to LPS-induced neuroinflammation, acute CNS injury, and most or all neurodegenerative diseases.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationnone given

    A1s are highly present in neurodegenerative diseases where their presence may well contribute to neurodegeneration and help to drive disease progression.

    “A1s are also highly present in neurodegenerative diseases where their presence may well contribute to neurodegeneration and help to drive disease progression.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    A1s are rapidly induced after acute CNS injury and responsible for the death of axotomized RGCs.

    “A1s are rapidly induced after acute CNS injury and responsible for the death of axotomized RGCs.”
    Quote in the paperSubstantial claim
48 of 48

  • The paper relies on this without support.

    The role of reactive astrocytes in CNS injury and disease is poorly understood.

    “Reactive astrocytes are strongly induced by central nervous system (CNS) injury and disease but their role is poorly understood.”

  • The paper relies on this without support.

    It has been a longstanding mystery why CNS neurons die after axotomy.

    “It has been a longstanding mystery why CNS neurons die after axotomy.”

  • The paper relies on this without support.

    Reactive microglia are found in neurodegenerative diseases.

    “because reactive microglia are found in neurodegenerative diseases”

  • The paper relies on this without support.

    Cultured immunopanned astrocytes retain their non-activated in vivo gene profiles.

    “These astrocytes retain their non-activated in vivo gene profiles.”

  • The paper relies on this without support.

    CD68+ cells in MS lesions are reactive microglia.

    “CD68+ reactive microglia (yellow arrowheads)”
5 of 5

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?

  • Aging-related cell states exhibited upregulation of genes (e.g., B2m and Trem2 in Micro-3, Xdh in Endo-3, Gfap and C4b in Astro-2) previously shown to be enriched in activated microglia, endothelial cells, and astrocytes.

    “These aging-related cell states exhibit upregulation of genes (e.g., B2m and Trem2 in Micro-3, Xdh in Endo-3, and Gfap and C4b in Astro-2) (Figure 2C) that have been previously shown to be enriched in microglia, endothelial cells, and astrocytes activated by inflammation and/or aging.”

    From Molecular and spatial signatures of mouse brain aging at single-cell resolution (2023) · cites this paper as [40]

    The section ‘A1 reactive astrocytes in human disease’ and Extended Data Fig. 13 identify GFAP-positive reactive astrocytes in neuroinflammatory disease, supporting the astrocyte part of the comparison. Our copy does not establish enrichment of B2m or Trem2 in activated microglia, Xdh in endothelial cells, or C4b in activated astrocytes; it discusses C3 rather than C4b. The citing paper’s own aging-related cell-state results are not findings attributed to this source. (LLM, conf=0.82)

    Experimental — accuracy not yet measured

  • For microglia and astrocytes, genes highly upregulated with age overlapped substantially with genes previously reported as upregulated in activated (reactive) states (e.g., Gfap and C4b for astrocytes; B2m and Lyz2 for microglia).

    “For microglia and astrocytes, we observed that the genes highly upregulated with age overlapped substantially with genes that have been previously reported as being upregulated in the activated (or “reactive”) state of these cell types (e.g., Gfap and C4b for astrocytes; B2m and Lyz2 for microglia)”

    From Molecular and spatial signatures of mouse brain aging at single-cell resolution (2023) · cites this paper as [40]

    The Introduction reports that reactive A1 astrocytes upregulate complement-cascade genes, and Extended Data Figure 13 identifies GFAP-positive reactive astrocytes. The cited paper does not establish C4b as an upregulated reactive-astrocyte gene or B2m and Lyz2 as upregulated activated-microglia genes. The age-related overlap is presented as the citing authors’ own observation, not a finding attributed to this source. (LLM, conf=0.83)

    Experimental — accuracy not yet measured

  • Two major astrocyte activation states have been described in neurodegenerative diseases: A1 neurotoxic astrocytes and A2 neuroprotective astrocytes.

    “Two major astrocyte activation states have been described in neurodegenerative diseases: A1 neurotoxic astrocytes and A2 neuroprotective astrocytes 23 .”

    From APOE2 attenuates APOE4-associated amyloid pathology and related toxicity in a mouse model of Alzheimer’s disease (2026) · cites this paper as [23]

    The Introduction describes two reactive astrocyte types, A1 and A2, and the Concluding Remarks characterize A1 as neurotoxic and A2 as promoting neuronal survival and tissue repair. But the paper reports A1 in neurodegenerative diseases while describing A2 as induced by ischemia; it does not establish both states in neurodegenerative diseases. (LLM, conf=0.86)

    Experimental — accuracy not yet measured

  • Activated microglia secrete chemokines and cytokines that promote astrocyte activation.

    “activated microglia secrete chemokines and cytokines that promote astrocyte activation [42]”

    From The protective PLCγ2-P522R variant mitigates Alzheimer’s disease-associated pathologies by enhancing beneficial microglial functions (2025) · cites this paper as [42]

    Section 2 reports that activated microglia secrete IL-1α, TNFα and C1q, and that these signals induce A1 reactive astrocytes. The cited passages do not establish that secreted chemokines promote astrocyte activation, so that part of the claim is unsupported. (LLM, conf=0.85)

    Experimental — accuracy not yet measured

  • Neurotoxic reactive astrocytes associated with AD release pro-inflammatory cytokines and can no longer support neuronal synapse formation, facilitate synaptic pruning and clearance of myelin debris and may even induce cell death in neighboring neurons, thereby promoting further neurodegeneration and inflammation.

    “It is thought that the “neurotoxic” reactive astrocytes associated with AD release pro-inflammatory cytokines and can no longer support neuronal synapse formation, facilitate synaptic pruning and clearance of myelin debris and may even induce cell death in neighboring neurons, thereby promoting further neurodegeneration and inflammation [17, 186].”

    From Microglial function, INPP5D/SHIP1 signaling, and NLRP3 inflammasome activation: implications for Alzheimer’s disease (2023) · cites this paper as [17]

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

    Experimental — accuracy not yet measured

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.

  • C3 marker evidence cites the OPC figure

    Minor

    The cited Extended Data Figure 10 addresses OPC proliferation and differentiation. The caption reporting that A2-cassette-expressing cells were not C3-positive is Extended Data Figure 12.

  • LGN experiment cites the synapse-formation figure

    Minor

    The cited Extended Data Figure 6 is captioned as a synapse-formation and neurite-outgrowth experiment. The legend describing LGN astrocytes after LPS injection is numbered Figure 7.

  • Motor-neuron subtype result cites the LGN figure

    Minor

    The motor-neuron subtype analysis is identified as panel m of Extended Data Figure 8, not Figure 7; the Figure 7 caption concerns LGN astrocytes.

  • LPS response in cultured astrocytes points to an unrelated extended-data figure

    Minor

    The cited Extended Data Figure 2 concerns microglia depletion in PLX-3397-treated mice, not the response of cultured astrocytes to LPS.

  • Synapse assay cites the astrocyte-morphology figure

    Minor

    The RGC synapse assay cites Extended Data Figure 5, whose caption concerns astrocyte morphology. Synapse formation is the subject of Extended Data Figure 6.

  • Knockout microglial-response citation points to a different experiment

    Minor

    The text cites Extended Data Figure 4 for microglial activation in knockout mice, but that experiment is captioned Extended Data Figure 11. Figure 4 instead describes astrocyte treatments with candidate mediators.

6 of 6

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Authors
Liddelow, S. A., Guttenplan, K. A., Clarke, L. E., Bennett, F. C., Bohlen, C. J., Schirmer, L., Bennett, M. L., Münch, A. E., Chung, W.-S., Peterson, T. C., Wilton, D. K., Frouin, A., Napier, B. A., Panicker, N., Kumar, M., Buckwalter, M. S., Rowitch, D. H., Dawson, V. L., Dawson, T. M., Stevens, B., & Barres, B. A.
Affiliations
  • The University of Melbourne (AU)
  • Stanford Medicine (US)
  • University of California, San Francisco (US)
  • Korea Advanced Institute of Science and Technology (KR)
  • Boston Children's Hospital (US)
  • Johns Hopkins University (US)
Corresponding author
Liddelow, Shane A.
ORCID
Venue
Nature
Publisher
Springer Science and Business Media LLC
Published
2017-01
Volume
541
Issue
7638
Pages
481-487
ISSN
  • 0028-0836 (ISSN-L)
  • 1476-4687
DOI
10.1038/nature21029
PMC
PMC5404890
OpenAlex
W2572710398
Cited by
7,971
References
48
Citations by year
2026: 917 · 2025: 978 · 2024: 1067 · 2023: 1007 · 2022: 1043 · 2021: 1015 · 2020: 754 · 2019: 553 · 2018: 428 · 2017: 194 · 2013: 1 · 2012: 2
FWCI
261.80
Type
journal article
Language
en
Open access
Licence
www.springer.com/tdm (tdm)
Locations
  • Nature (publishedVersion)
  • PubMed (publishedVersion)
  • PubMed Central (acceptedVersion)
  • RePEc: Research Papers in Economics (submittedVersion)
  • Apollo (University of Cambridge) (submittedVersion)
  • Apollo
Funders
  • Parkinson's UK (J-1402)
  • National Institute on Aging (R01AG048814)
  • National Institute on Drug Abuse (R01DA015043, R37DA015043)
  • National Institute of Neurological Disorders and Stroke (R01NS084298, R37NS067525, P50NS038377)
  • Eunice Kennedy Shriver National Institute of Child Health and Human Development (U54HD090255)
Topics
  • Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Neuroscience and Neuropharmacology Research
  • Health Sciences › Medicine › Neurology › Traumatic Brain Injury and Neurovascular Disturbances
Keywords
reactive astrocytes, microglial activation, neuroinflammation, interleukin-1 alpha, TNF-α, C1q, neuronal cell death, oligodendrocyte death, axotomy, neurodegenerative diseases, Alzheimer's disease, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, synaptogenesis, phagocytosis
MeSH terms
Animals, Astrocytes, Cell Survival, Central Nervous System, Humans, Inflammation, Mice, Inbred C57BL, Neurons, Oligodendroglia, Phagocytosis, Phenotype, Synapses, Toxins, Biological, Tumor Necrosis Factor-alpha, Complement C1q, Cell Death, Rats, Sprague-Dawley, Microglia, Disease Progression, Cell Culture Techniques, Neurodegenerative Diseases, Axotomy, Mice, Rats, Interleukin-1alpha

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 2026-10-18.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-18.

Cited sources are checked by gpt-6-sol.

Statistics and arithmetic are recomputed, not judged.

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

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

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