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Cited by 578Neuroinflammation and Neurodegeneration MechanismsAlzheimer's disease research and treatmentsNeuroscience and Neuropharmacology Research

Neurotoxic reactive astrocytes induce cell death via saturated lipids

Open access
Guttenplan, Kevin A., Weigel, Maya K., Prakash, Priya, Wijewardhane, Prageeth R., Hasel, Philip, Rufen-Blanchette, Uriel, Münch, Alexandra E., Blum, Jacob A. +7 more·2021·Nature
DOI 10.1038/s41586-021-03960-yPMC PMC12054010
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Abstract

Astrocytes regulate the response of the central nervous system to disease and injury and have been hypothesized to actively kill neurons in neurodegenerative disease1–6. Here we report an approach to isolate one component of the long-sought astrocyte-derived toxic factor5,6. Notably, instead of a protein, saturated lipids contained in APOE and APOJ lipoparticles mediate astrocyte-induced toxicity. Eliminating the formation of long-chain saturated lipids by astrocyte-specific knockout of the saturated lipid synthesis enzyme ELOVL1 mitigates astrocyte-mediated toxicity in vitro as well as in a model of acute axonal injury in vivo. These results suggest a mechanism by which astrocytes kill cells in the central nervous system. Astrocytes can respond to diseases and injuries of the central nervous system by driving the death of neurons and mature oligodendrocytes through the delivery of long-chain saturated fatty acids contained in lipoparticles.

AI snapshot

Written from the paper’s full text.

TL;DR

Reactive astrocytes kill neurons and oligodendrocytes by secreting saturated lipids—long-chain saturated free fatty acids and very-long-chain phosphatidylcholines—carried in ApoE/ApoJ lipoparticles, rather than by a protein toxin. Astrocyte-specific knockout of the saturated lipid synthesis enzyme ELOVL1 reduces long-chain saturated lipid production and mitigates astrocyte-mediated toxicity in vitro and partially in an optic nerve crush model in vivo.

Key findings

  • Reactive astrocyte conditioned media (ACM) toxicity was not attributable to proteins: ApoE/ApoJ immunodepletion reduced toxicity, while ACM from Apoe−/−, Clu−/−, and Apoe−/−Clu−/− astrocytes was as toxic as C57Bl/6 control ACM. Lipid removal with a Lipidex 3000 column eliminated toxicity, and the eluted lipids were toxic.
  • Lipidomics/metabolomics of 1501 lipids from 10 classes and 717 metabolites showed reactive astrocytes upregulate very-long-chain phosphatidylcholines (VLCPCs) in membranes and long-chain saturated free fatty acids (FFAs) in ACM; both were toxic to cultured oligodendrocytes, with longer chain lengths causing greater toxicity.
  • The death mechanism resembled lipoapoptosis rather than ferroptosis: Ferrostatin-1 and ethoxyquin did not affect toxicity; reactive ACM induced FOXO3a dephosphorylation, PUMA upregulation, and Caspase 3 cleavage; PUMA−/− oligodendrocytes were resistant, whereas CHOP−/− oligodendrocytes were not.
  • Astrocyte-specific Elovl1 conditional knockout reduced long-chain saturated FFAs and made reactive ACM significantly less toxic to oligodendrocytes in vitro; after optic nerve crush, Elovl1 cKO mice still showed retinal ganglion cell death but less than WT mice.

Methods

  • Primary mouse and rat astrocytes, oligodendrocytes, and retinal ganglion cells were purified by immunopanning; reactive astrocytes were induced with IL1α (3 ng/ml), TNF (30 ng/ml), and C1q (400 ng/ml) for 24 h, and ACM was concentrated ~10× and used at 50 μg/ml unless otherwise noted.
  • The study combined protein mass spectrometry, unbiased lipidomics/metabolomics, biochemical fractionation, ELISAs, size-exclusion HPLC, Lipidex lipid depletion, reconstituted ApoE/ApoJ lipoparticles, western blotting, siRNA knockdown, and an astrocyte-specific Gfap-Cre–driven Elovl1 cKO mouse.
  • Toxicity was measured by Calcein AM/ethidium homodimer live/dead analysis, generally 24 h after treatment, with at least 3 replicates from separate primary cell preparations; optic nerve crush was performed on P30–P50 mice and retinas collected 14 days after crush.

Limitations

  • The paper states that reducing saturated lipids does not completely eliminate neurotoxicity; future work is needed to identify other astrocyte-derived toxins or determine whether remaining toxicity reflects incomplete knockdown of saturated lipids or contributions from proposed toxic proteins, miRNAs, or other lipids.
  • The paper notes that Elovl1 cKO mice still showed neuronal death after optic nerve crush, unlike Il1a−/−Tnf−/−C1qa−/− mice, indicating that long-chain FFAs and PCs only partially mediate RGC toxicity in vivo.
  • Evident from the text: some validation is limited—e.g., the ApoE ELISA on HDL fractions analyzed only one control and one reactive sample—and the main in vitro toxicity assays focus on oligodendrocytes and RGCs, so broader CNS cell-type and disease-context generality remains to be established.

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

Integrity signals

Registry

Crossref · 10.1038/s41586-021-03960-y

Cites

38 of 38 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 28 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    Reactive astrocyte membrane fractions were more toxic than the membranes of quiescent astrocytes and HEK cells.

    “reactive astrocyte membrane fractions were more toxic than the membranes of quiescent astrocytes and HEK cells (Figure 2d).”
    Quote in the paperSubstantial claim
  • Findingown data

    ACM depleted of ApoE and ApoJ, but not with IgG control antibodies, showed decreased toxicity.

    “ACM depleted of ApoE and ApoJ, but not with IgG control antibodies, showed decreased toxicity”
    Quote in the paperSubstantial claim
  • Findingown data

    The most prominent class of proteins upregulated in purified reactive media, other than known markers such as C3, were lipoparticle proteins such as ApoE and ApoJ.

    “The most prominent class of proteins upregulated in purified reactive media, other than known markers such as C3, were lipoparticle proteins such as ApoE and ApoJ”
    Quote in the paperSubstantial claim
  • Findingown data

    Toxicity was most prominent in the flowthrough of anion and cation exchange columns as well as the final elutions of a hydrophobic interaction chromatography column.

    “Toxicity was most prominent in the flowthrough of anion and cation exchange columns as well as the final elutions of a hydrophobic interaction chromatography column (Figure 1f).”
    Quote in the paperSubstantial claim
  • Findingown data

    ApoE and ApoJ lipoparticle proteins are abundant in reactive ACM.

    “We also noted a surprising abundance of lipoparticle proteins (lipoproteins) ApoE and ApoJ in reactive ACM”
    Quote in the paperSubstantial claim
  • Classified as probably not a substantial claim. 31
  • May combine two claims. 24

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

    Findingown data

    176 of 3660 detected proteins changed in abundance within reactive astrocytes.

    “While 176 of 3660 detected proteins changed in abundance within reactive astrocytes (Extended Data Figure 1, Methods)”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Both long-chain, saturated FFAs and VLCPCs were toxic to cultured oligodendrocytes, with greater toxicity caused by longer chain lengths.

    “Both long-chain, saturated FFAs and VLCPCs were toxic to cultured oligodendrocytes, with greater toxicity caused by longer chain lengths (Extended Data Figure 7).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    There were significant changes in lipid metabolism and, to a lesser extent, the metabolome in reactive compared to quiescent astrocytes.

    “We observed significant changes in lipid metabolism and, to a lesser extent, the metabolome in reactive compared to quiescent astrocytes (Figure 2h, Extended Data Figure 6a-c).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Most of the changes in protein abundance within reactive astrocytes correspond to known reactivity signature genes, with complement component C3 showing the largest increase.

    “most of these changes correspond to known reactivity signature genes, with the inflammatory astrocyte protein complement component C3 showing the largest increase (Extended Data Figure 1d)2.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [2] Liddelow, S. A. et al. Neurotoxic reactive astrocytes are induced by activated microglia. Nature 541, 481–487 (2017). source

      Not checked in the free run.

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

    Findingown data

    Reactive ACM showed an expected increase in abundance of C3, lipocalin-2, and other reactivity markers.

    “we found the expected increase in abundance of C3, lipocalin-2, and other reactivity markers in reactive ACM (Figure 1e)10.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [10] Bi, F. et al. Reactive astrocytes secrete lcn2 to promote neuron death. Proc. Natl Acad. Sci. USA 110, 4069–4074 (2013). source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    Uptake of fluorescently labeled rHDL into treated oligodendrocytes was dependent on cell type and on the presence of lipoparticle proteins.

    “uptake was dependent on cell type and on the presence of lipoparticle proteins (Figure 2e, Extended Data Figure 5).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    There was a substantial upregulation of phosphatidylcholines with very-long chain fatty acid acyl chains (VLCPCs) in astrocyte cell membranes and long-chain saturated free fatty acids (FFAs) in reactive astrocyte ACM.

    “there was a substantial upregulation of phosphatidylcholines with very-long chain fatty acid acyl chains (VLCPCs) in astrocyte cell membranes and long-chain saturated free fatty acids (FFAs) in reactive astrocyte ACM”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    ApoE and ApoJ are found in HDL-like fractions, with an increase in ApoJ concentration within HDL-like fractions.

    “we found ApoE and ApoJ in HDL-like fractions, with an increase in ApoJ concentration within HDL-like fractions (Extended Data Figure 4)11.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [11] Fagan, A. et al. Unique lipoproteins secreted by primary astrocytes from wild type, apoE−/−, and human apoE transgenic mice. J. Biol. Chem. 274, 30001–30007 (1999). source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    Treating oligodendrocytes with quiescent ACM-lipid-containing rHDL was non-toxic, but treatment with reactive ACM-lipid-containing rHDL was highly toxic.

    “Treating oligodendrocytes with quiescent ACM-lipid-containing rHDL was non-toxic, but treatment with reactive ACM-lipid-containing rHDL was highly toxic”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Both reactive ACM and reconstituted lipoparticles bearing reactive ACM lipids are toxic to RGCs in vitro.

    “both reactive ACM and reconstituted lipoparticles bearing reactive ACM lipids are toxic to RGCs in vitro (Figure 4e)”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingcitation

    Elovl1 cKO mice still showed neuronal death following ONC, unlike Il1a−/−Tnf−/−C1qa−/− mice, which show no RGC death following axon crush, but the amount of death was reduced.

    “While Elovl1 cKO mice still showed neuronal death following ONC (unlike in Il1a−/−Tnf−/−C1qa−/− mice, which show no RGC death following axon crush2,3,20), the amount of death was reduced”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [2] Liddelow, S. A. et al. Neurotoxic reactive astrocytes are induced by activated microglia. Nature 541, 481–487 (2017). source

      Not checked in the free run.

    • cites [3] Guttenplan, K. A. et al. Neurotoxic reactive astrocytes drive neuronal death after retinal injury. Cell Rep. 31, 107776 (2020). source

      Not checked in the free run.

    • cites [20] Sterling, J. K. et al. GLP-1 receptor agonist NLY01 reduces retinal inflammation and neuron death secondary to ocular hypertension. Cell Rep. 33, 108271 (2020). source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    Concentrating reactive ACM 10-fold, Elovl1 cKO reactive ACM became toxic to oligodendrocytes, but identically concentrated WT reactive ACM proved much more toxic.

    “Concentrating this reactive ACM 10-fold, Elovl1 cKO reactive ACM became toxic to oligodendrocytes, but identically concentrated WT reactive ACM proved much more toxic.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Reactive ACM, but not Lcn2, Lgals1, or complement component C3 family members, is toxic to retinal ganglion cell neuron cultures.

    “Reactive ACM, but not Lcn2, Lgals1, or complement component C3 family members, is toxic to retinal ganglion cell (RGC) neuron cultures.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Reactive versus control astrocytes and ACM are somewhat separable in PCA space based on their metabolome, but less so than by their lipidome.

    “Reactive (red) vs control (gray) astrocytes and ACM are somewhat separable in PCA space based on their metabolome, but less so than by their lipidome (Figure 2).”
    Quote in the paperNot a substantial claim

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

    Findingown data

    3660 total unique proteins were detected in astrocytes and 183 total unique proteins were detected in ACM.

    “3660 total unique proteins detected in astrocytes and 183 total unique proteins detected in ACM.”
    Quote in the paperNot a substantial claim

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

    Findingown data

    All tested cell types incorporate reconstituted lipoparticles except for endothelial cells.

    “Note that all cells incorporate reconstituted lipoparticles except for endothelial cells.”
    Quote in the paperNot a substantial claim

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

    Findingown data

    PCA plots of cellular and ACM protein mass spectrometry show clear separation of the proteome and secretome of reactive versus control astrocytes.

    “PCA plots of cellular and ACM protein mass spectrometry of all proteins detected in at least 4 of 10 astrocytes samples see shows clear separation of the proteome and secretome of reactive versus control astrocytes.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    Astrocytes activated by IL-1α, TNF, and C1q in vitro secrete factors that are toxic to neurons and oligodendrocytes, but not to other CNS cells.

    “Astrocytes activated by these cytokines in vitro secrete factors that are toxic to neurons and oligodendrocytes, but not to other CNS cells2.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [2] Liddelow, S. A. et al. Neurotoxic reactive astrocytes are induced by activated microglia. Nature 541, 481–487 (2017). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Mature oligodendrocytes are highly viable in serum-free conditions and do not require astrocyte trophic support.

    “We screened for ACM toxicity using mature oligodendrocytes because they are highly viable in serum-free conditions and do not require astrocyte trophic support (Figure 1a-b)7”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [7] Barres, B., Schmid, R., Sendnter, M. & Raff, M. Multiple extracellular signals are required for long-term oligodendrocyte survival. Development 118, 283–295 (1993). source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    Previous evidence suggested the toxic activity of reactive astrocytes is mediated by a secreted protein.

    “Previous evidence suggested the toxic activity of reactive astrocytes is mediated by a secreted protein5,6”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [5] Nagai, M. et al. Astrocytes expressing ALS-linked mutated SOD1 release factors selectively toxic to motor neurons. Nat. Neurosci. 10, 615–622 (2007). source

      Not checked in the free run.

    • cites [6] Giorgio, F., Carrasco, M. A., Siao, M. C., Maniatis, T. & Eggan, K. Non-cell autonomous effect of glia on motor neurons in an embryonic stem cell–based ALS model. Nat. Neurosci. 10, 608–614 (2007). source

      Not checked in the free run.

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

    Interpretationown data

    The abundance of ApoE and ApoJ in reactive ACM suggests a potential change in astrocytic lipid efflux.

    “suggesting a potential change in astrocytic lipid efflux (Figure 1e).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    Lipids themselves are both necessary and sufficient components of the ACM toxicity.

    “These results provide evidence that lipids themselves are both necessary and sufficient components of the ACM toxicity (Figure 2c).”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given

    The injection solvent containing a quantitative mass spectrometry internal standard was used as the quality control sample to monitor peaks over time and confirm proper instrument working.

    “The injection solvent containing the quantitative mass spectrometry internal standard consisting of a mixture of 13 deuterated lipid internal standards at a concentration of 100 μg/mL each (Avanti Polar Lipids, #330731) was used as the “quality control” sample to monitor their peaks over time to confirm the proper working of the instrument.”
    Quote in the paperNot a substantial claim

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

    Study design and conductcitation

    Protein mass spectrometry data were validated using the standard reverse-decoy technique at a 1% false discovery rate.

    “Data were validated using the standard reverse-decoy technique at a 1% false discovery rate as described previously37.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [37] Elias, J. E. & Gygi, S. P. Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry. Nat. Methods 4, 207–214 (2007). 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 conductnone given

    RNA quality and integrity was evaluated and only samples with RIN > 9.0 were used for downstream analysis.

    “RNA quality and integrity was evaluated using an Agilent RNA 6000 Pico assay (Agilent 2100 Bioanalyzer) and only samples with RIN > 9.0 were used for downstream analysis.”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    RGCs are known to be sensitive to lipotoxicity.

    “aligning with the known sensitivity of RGCs to lipotoxicity19.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [19] Yan, P. et al. Palmitic acid triggers cell apoptosis in RGC-5 retinal ganglion cells through the Akt/FoxO1 signaling pathway. Metab. Brain Dis. 32, 453–460 (2017). source

      Not checked in the free run.

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

    Premisecitation

    A peripheral mechanism to inactivate lipopolysaccharide (LPS) is the loading of its lipid component onto lipoparticles.

    “Because a peripheral mechanism to inactivate lipopolysaccharide (LPS) is the loading of its lipid component onto lipoparticles26”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [26] Wurfel, M. M., Kunitake, S. T., Lichenstein, H., Kane, J. P. & Wright, S. D. Lipopolysaccharide (LPS)-binding protein is carried on lipoproteins and acts as a cofactor in the neutralization of LPS. J. Exp. Med. 180, 1025–1035 (1994). source

      Not checked in the free run.

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

    Study design and conductnone given

    All primers used had 90-105% efficiency.

    “All primers had 90–105% efficiency.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    Subsequent experiments were performed blind to age and genotype.

    “subsequent experiments performed blind to age and genotype.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    Efficiency of siRNA knockdown was validated using qPCR.

    “Efficiency of siRNA knockdown validated using qPCR as outlined in the following methods section.”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given

    Mice were randomly selected within genotype for assignment to different experiments.

    “Mice were randomly selected within genotype for assignment to different experiments.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    Primer pairs were designed to amplify products spanning exon-exon junctions to avoid amplification of genomic DNA.

    “We designed primer pairs to amplify products that spanned exon–exon junctions to avoid amplification of genomic DNA.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    Reactive astrocytes drive neuron cell death by secreting toxic lipids both in vitro and in vivo, identifying at least one long-sought astrocyte-secreted neurotoxic class of molecules.

    “These results demonstrate that reactive astrocytes drive neuron cell death by secreting toxic lipids both in vitro and in vivo, identifying at least one long-sought astrocyte-secreted neurotoxic class of molecules.”
    Quote in the paperSubstantial claim

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

    Premisecitation

    Astrocytes and microglia secrete and degrade a huge array and quantity of lipids.

    “these results and those of others demonstrate that astrocytes and microglia secrete and degrade a huge array and quantity of lipids”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [21] Ioannou, M. S. et al. Neuron-astrocyte metabolic coupling protects against activity-induced fatty acid toxicity. Cell 177, 1522–1535 (2019). source

      Not checked in the free run.

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

    Study design and conductnone given

    For all experiments, all attempts at replication were successful.

    “For all experiments, all attempts at replication were successful.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    All in vitro experiments were performed at least 3 times from separate individual primary cell preparations.

    “All in vitro experiments were performed at least 3 times from separate individual primary cell preparations.”
    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 conductcitation

    Sample sizes were determined by reference to previous literature, with sample sizes for optic nerve crush and in vitro experiments determined according to Liddelow et al.

    “Sample sizes were determined by reference to previous literature, with samples sizes for optic nerve crush and in vitro experiments determined according to Liddelow et al.2”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [2] Liddelow, S. A. et al. Neurotoxic reactive astrocytes are induced by activated microglia. Nature 541, 481–487 (2017). source

      Not checked in the free run.

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

    Premisecitation

    Upregulation of this lipid class is a common phenomenon in TLR4-mediated immune cell activation.

    “upregulation of this lipid class is a common phenomenon in TLR4-mediated immune cell activation25.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [25] Oishi, Y. et al. SREBP1 contributes to resolution of pro-inflammatory TLR4 signaling by reprogramming fatty acid metabolism. Cell Metab. 25, 412–427 (2017). source

      Not checked in the free run.

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

    Premisecitation

    Microglia change their lipid metabolism in response to the lipid flux that occurs when neurons and oligodendrocytes die in neurodegenerative contexts.

    “microglia change their lipid metabolism in response to the lipid flux that occurs when neurons and oligodendrocytes die in neurodegenerative contexts27”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [27] Nugent, A. A. et al. TREM2 regulates microglial cholesterol metabolism upon chronic phagocytic challenge. Neuron 105, 837–854 (2020). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    In lipoapoptosis, saturated lipids activate the PERK endoplasmic reticulum stress response pathway likely due to changes in lipid membrane structure, leading to cell death via PUMA or CHOP following dephosphorylation of FOXO3a.

    “In lipoapoptosis, saturated lipids activate the PERK (Protein Kinase R-like ER Kinase) endoplasmic reticulum stress response pathway likely due to changes in lipid membrane structure, leading to cell death via PUMA (p53 upregulated modulator of apoptosis, Bbc3) or CHOP (C/EBP homologous protein, Ddit3) following dephosphorylation of FOXO3a (Figure 3a)15.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [15] Cunha, D. A. et al. Death protein 5 and p53-upregulated modulator of apoptosis mediate the endoplasmic reticulum stress–mitochondrial dialog triggering lipotoxic rodent and human β-cell apoptosis. Diabetes 61, 2763–2775 (2012). source

      Not checked in the free run.

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

    Study design and conductown data

    Primer specificity was tested by PCR with mouse whole-brain cDNA and examination of PCR products by agarose gel electrophoresis.

    “We tested the specificity of the primer pairs by PCR with mouse whole-brain cDNA (prepared fresh) and examined PCR products by agarose gel electrophoresis.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    In all instances, 3 separate replicates were performed on 3 separate primary cell culturing events to ensure reproducibility.

    “In all instances, 3 separate replicates were performed on 3 separate primary cell culturing events to ensure reproducibility.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    One optic nerve crush was performed on each animal and the retina of the uncrushed eye was used as a within-animal control.

    “One ONC was performed on each animal and the retina of the uncrushed eye used as a within-animal control.”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given

    Three regions of interest were selected randomly throughout the retina, ensuring multiple eccentricities per retina, blind to genotype and condition, and the average number of RBPMS+ cells in the three images was calculated.

    “Three regions of interest were selected randomly throughout the retina (ensuring multiple eccentricities selected for each retina) blind to genotype and condition and the average number of RBPMS+ cells in the three images calculated.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Saturated lipids mediate the toxic activity of reactive astrocytes and Elovl1 cKO reduces the production of these toxic lipids.

    “Together, these results suggest that saturated lipids mediate the toxic activity of reactive astrocyte and Elovl1 cKO reduces the production of these toxic lipids.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Study design and conductown data

    The p-values for lipid and metabolite comparisons were adjusted for multiple testing using the BH method to FDR.

    “These p-values were then adjusted for multiple testing using the BH method to FDR.”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given

    All animal studies were performed on animals from 2 different litters over many months.

    “All animal studies were performed on animals from different 2 different litters over many months.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    After surgery, retinal blood flow was validated by checking the eye fundi.

    “After surgery, retinal blood flow was validated by checking the eye fundi.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    Unbound media was assessed by Bradford assay to ensure final relative protein concentrations were identical between control and reactive ACM.

    “Unbound media was assessed by Bradford Assay to ensure final relative protein concentrations were identical between control and reactive ACM.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The free radical scavenger ethoxyquin did not impact oligodendrocyte survival when treated in isolation and did not impact the toxicity of reactive ACM.

    “suggesting that the free radical scavenger did not impact cell survival when treated in isolation and did not impact the toxicity of reactive ACM.”
    Quote in the paperSubstantial claim
50 of 50

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?

  • Reactive astrocytes secrete long-chain saturated FFAs that kill injured neurons and oligodendrocytes in vitro and in vivo.

    “Reactive astrocytes secrete long-chain saturated FFAs that kill injured neurons and oligodendrocytes in vitro and in vivo41.”

    From Amyloid-β induces lipid droplet-mediated microglial dysfunction via the enzyme DGAT2 in Alzheimer’s disease (2025) · cites this paper as [41]

    The Discussion reports that reactive astrocytes drive neuronal and mature oligodendrocyte death through delivery of FFAs and very-long-chain phosphatidylcholines. Extended Data Figure 7 shows saturated FFA toxicity to cultured oligodendrocytes, while the optic-nerve-crush results concern injured retinal neurons in vivo. The paper does not establish oligodendrocyte death in vivo, and it describes the in-vivo contribution of FFAs and phosphatidylcholines as partial rather than attributing it to FFAs alone. (LLM, conf=0.88)

    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 [186]

    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

  • An increased number of C3-positive reactive astrocytes is observed in tissues of patients with neurodegenerative diseases.

    “An increased number of C3-positive reactive astrocytes has been observed in the tissues of patients with neurodegenerative diseases”

    From Astrocyte-specific inhibition of the primary cilium suppresses C3 expression in reactive astrocyte (2024) · cites this paper as Guttenplan et al. 2021

    The paper reports increased C3 protein in reactive astrocytes and their conditioned media (Abstract; no page given). It does not report an increased number of C3-positive astrocytes in tissues from patients with neurodegenerative diseases; the reported comparison is in astrocyte cultures. (LLM, conf=0.84)

    Experimental — accuracy not yet measured

  • Recent work shows deleterious effects of conditioned media on neurons from astrocytes and microglia.

    “This approach takes inspiration from recent work showing the deleterious effects of conditioned media on neurons from astrocytes30 and microglia4.”

    From APOE4/4 is linked to damaging lipid droplets in Alzheimer’s disease microglia (2024) · cites this paper as [30]

  • Reactive astrocytes also induce adverse outcomes.

    “Although the purpose of astrocyte activation is to limit tissue damage, growing evidence shows that reactive astrocytes also induce adverse outcomes (Guttenplan et al., 2021; Liddelow and Barres, 2017; Liddelow et al., 2017).”

    From Sterol dysregulation in Smith–Lemli–Opitz syndrome causes astrocyte immune reactivity through microglia crosstalk (2022) · cites this paper as Guttenplan et al., 2021

Self-check

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

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

  • Figure 1g is assigned to two different experiments

    Minor

    The results identify Figure 1g as the purified-ACM protein analysis, whereas the reconstituted-lipoparticle method identifies Figure 1g as cell-survival data from lipoparticle treatments.

  • Universal in-vitro replication claim conflicts with a single-sample analysis

    Minor

    The methods state that all in-vitro experiments used at least three separate primary-cell preparations, but the HDL-fraction ApoE analysis reports only one control and one reactive sample.

  • Lipid-depletion resin has two different names

    Minor

    The results say toxicity was eliminated using a Lipidex 3000 column, while the corresponding lipid-depletion method specifies Lipidex 1000 resin.

3 of 3

Cites

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Authors
Guttenplan, K. A., Weigel, M. K., Prakash, P., Wijewardhane, P. R., Hasel, P., Rufen-Blanchette, U., Münch, A. E., Blum, J. A., Fine, J., Neal, M. C., Bruce, K. D., Gitler, A. D., Chopra, G., Liddelow, S. A., & Barres, B. A.
Affiliations
  • Stanford Medicine (US)
  • Purdue University West Lafayette (US)
  • NYU Langone Health (US)
  • Stanford University (US)
  • University of Colorado Anschutz (US)
Corresponding author
Liddelow, Shane A.
ORCID
Venue
Nature
Publisher
Springer Science and Business Media LLC
Published
2021-10-06
Volume
599
Issue
7883
Pages
102-107
ISSN
  • 0028-0836 (ISSN-L)
  • 1476-4687
DOI
10.1038/s41586-021-03960-y
PMC
PMC12054010
OpenAlex
W3201931145
Semantic Scholar
004c2001b806c6c2b3507f6de48e2871f3b72be8
Cited by
578
References
38
Citations by year
2026: 75 · 2025: 112 · 2024: 135 · 2023: 137 · 2022: 106 · 2021: 12
FWCI
39.44
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature (publishedVersion)
  • PubMed (publishedVersion)
  • Europe PMC (PubMed Central) (acceptedVersion)
  • PubMed Central (acceptedVersion)
  • RePEc: Research Papers in Economics (submittedVersion)
Funders
  • National Institutes of Health (UL1TR002529)
  • National Institute on Aging (R21AG061549)
  • National Cancer Institute (P30CA023168, P30CA124435)
  • National Institute of Neurological Disorders and Stroke (P30NS069375)
  • National Center for Research Resources (S10RR027425)
  • National Center for Advancing Translational Sciences
Topics
  • Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
  • Health Sciences › Medicine › Physiology › Alzheimer's disease research and treatments
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Neuroscience and Neuropharmacology Research
Keywords
reactive astrocytes, saturated fatty acids, ELOVL1, axonal injury, oligodendrocyte death, neuronal cell death, neurodegeneration
MeSH terms
Fatty Acid Elongases, Animals, Astrocytes, Female, Lipids, Male, Neurotoxins, Cell Death, Culture Media, Conditioned, Mice, Knockout, Neurodegenerative Diseases, Mice, Gene Knockout Techniques

About these checks

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

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

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

Statistics and arithmetic are recomputed, not judged.

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

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