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Cited by 246Neuroinflammation and Neurodegeneration MechanismsMicroRNA in disease regulationTraumatic Brain Injury and Neurovascular Disturbances

Evidence for miR‐181 involvement in neuroinflammatory responses of astrocytes

Open access
Hutchison, Emmette R., Kawamoto, Elisa M., Taub, Dennis D., Lal, Ashish, Abdelmohsen, Kotb, Zhang, Yongqing, Wood, William H., Lehrmann, Elin +4 more·2013·Glia
DOI 10.1002/glia.22483PMC PMC4624280
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Abstract

Inflammation is a common component of acute injuries of the central nervous system (CNS) such as ischemia, and degenerative disorders such as Alzheimer's disease. Glial cells play important roles in local CNS inflammation, and an understanding of the roles for microRNAs in glial reactivity in injury and disease settings may therefore lead to the development of novel therapeutic interventions. Here, we show that the miR‐181 family is developmentally regulated and present in high amounts in astrocytes compared to neurons. Overexpression of miR‐181c in cultured astrocytes results in increased cell death when exposed to lipopolysaccharide (LPS). We show that miR‐181 expression is altered by exposure to LPS, a model of inflammation, in both wild‐type and transgenic mice lacking both receptors for the inflammatory cytokine TNF‐α. Knockdown of miR‐181 enhanced LPS‐induced production of pro‐inflammatory cytokines (TNF‐α, IL‐6, IL‐1β, IL‐8) and HMGB1, while overexpression of miR‐181 resulted in a significant increase in the expression of the anti‐inflammatory cytokine IL‐10. To assess the effects of miR‐181 on the astrocyte transcriptome, we performed gene array and pathway analysis on astrocytes with reduced levels of miR‐181b/c. To examine the pool of potential miR‐181 targets, we employed a biotin pull‐down of miR‐181c and gene array analysis. We validated the mRNAs encoding MeCP2 and X‐linked inhibitor of apoptosis as targets of miR‐181. These findings suggest that miR‐181 plays important roles in the molecular responses of astrocytes in inflammatory settings. Further understanding of the role of miR‐181 in inflammatory events and CNS injury could lead to novel approaches for the treatment of CNS disorders with an inflammatory component.

AI snapshot

Written from the paper’s full text.

TL;DR

This study reports that miR-181 family members are developmentally regulated, enriched in astrocytes, and altered by LPS-induced neuroinflammation. Reducing miR-181 promotes pro-inflammatory cytokine production, while over-expressing miR-181c increases IL-10 and cell death, and MeCP2 and XIAP are validated as direct miR-181 targets.

Key findings

  • miR-181b, miR-181c, and miR-181d were higher in adult cortex than embryonic telencephalon and enriched in cultured astrocytes relative to BDNF-differentiated neurons. In vivo, LPS decreased miR-181b/c/d in wild-type mouse cortex at 4 h; in TNFR1/TNFR2 double knockout mice, LPS did not significantly reduce miR-181b/c, but miR-181d decreased (p<0.04).
  • Over-expression of miR-181c increased astrocyte cell death by LDH release regardless of LPS concentration, and caused a small but significant increase in proliferation by MTS. Knockdown of miR-181b/c enhanced LPS-induced production of pro-inflammatory cytokines TNF-α, IL-6, IL-1β, IL-8, and HMGB1; over-expression of miR-181c enhanced LPS-induced IL-10.
  • MeCP2 and XIAP mRNAs were validated as direct miR-181 targets by reporter assays: MeCP2 reporter showed a 72% reduction in light units, and XIAP reporter showed roughly a 63% decrease in relative light units. SIRT1 reporter showed no repression by miR-181c, and HMGA1 was identified as a non-canonical potential target.

Methods

  • Primary mouse cortical astrocyte cultures were transfected with miR-181c over-expression, miR-181b/c hairpin inhibitors, or control C. elegans miR-67 at 20 nM, then treated with LPS; cell death was measured by LDH, proliferation by MTS, and cytokines by Bio-Plex multiplex assays.
  • In vivo, adult male wild-type C57BL/6J and TNFR1/TNFR2 double knockout mice received a single intraperitoneal LPS injection at 250 μg/kg and were euthanized 4 h later for cortex miRNA analysis.
  • Transcriptome and target analyses used Illumina BeadChips after miR-181b/c knockdown and biotinylated miR-181c pull-down, with RT-qPCR validation and luciferase reporter assays for MeCP2, SIRT1, and XIAP.

Limitations

  • The paper does not state a limitations section. Evident limitations include reliance on mouse primary astrocyte cultures and mouse in vivo models, with no human tissue or clinical data reported.
  • Some findings are trends or context-dependent: FGF2 was only a trend in control astrocytes, SIRT1 was not repressed by miR-181c, and HMGA1 was not predicted by Targetscan and is described as a non-canonical potential target.
  • Multiplex assays for IL-5, IL-4, IL-13, IFN-γ, IL-12p70, IL-7, IL-2, and SAA were below the detection threshold, limiting cytokine conclusions to the measured factors.

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

Integrity signals

Registry

Crossref · 10.1002/glia.22483

Cites

67 of 67 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 26 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    Levels of FGF2 were increased as a result of knocking down miR-181 and exposing astrocytes to LPS.

    “We also found that levels of FGF2, a potent mitogen for astrocytes (Joy et al. 1997), were also increased as a result of knocking down miR-181 and exposing astrocytes to LPS.”
    Quote in the paperSubstantial claim
  • Findingown data

    Knockdown of miR-181b/c in primary astrocytes alters major pathways related to cell death, cancer, and inflammatory responses.

    “Pathway and signaling pathway analysis identified alterations of several major pathways related to cell death, cancer and the inflammatory responses (Figure 4B, 4C and Supplemental Table 4).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The miR-181b/c hairpin inhibitors specifically knock down miR-181b and miR-181c without off-target effects.
  • Findingown data

    Reduction of miR-181 causes a dramatic increase in the production of pro-inflammatory cytokines including LIF, IL-6, TNF-α, IL-1β, IL-8 and HMGB1.

    “The most striking phenotype that we observed as a consequence of miR-181 reduction was a dramatic increase in the production of pro-inflammatory cytokines including LIF, IL-6, TNF-α, IL-1β, IL-8 and HMGB1.”
    Quote in the paperSubstantial claim
  • Findingown data

    Knockdown of miR-181s results in significant elevation of FGF2 levels in LPS-treated astrocytes.

    “Knockdown of miR-181s resulted in a trend towards elevated FGF2 levels in control astrocytes, and a significant elevation of FGF2 levels in LPS-treated astrocytes.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The miR-181b/c hairpin inhibitors specifically knock down miR-181b and miR-181c without off-target effects.
  • Findingown data

    Knockdown of miR-181s results in a trend towards elevated FGF2 levels in control astrocytes.

    “Knockdown of miR-181s resulted in a trend towards elevated FGF2 levels in control astrocytes, and a significant elevation of FGF2 levels in LPS-treated astrocytes.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: The miR-181b/c hairpin inhibitors specifically knock down miR-181b and miR-181c without off-target effects.
  • May combine two claims. 13
  • Classified as probably not a substantial claim. 12

  • May combine two claims.

    Findingown data

    Bcl-2 and NAMPT mRNA were enriched in the miR-181c pulldown.

    “Bcl-2, a previously validated miR-181 target (Ouyang et al., 2012a) and the predicted target NAMPT mRNA were also enriched in the pull-down (Figure 7A).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    MeCP2 mRNA is a direct target of miR-181, validated by a Psi-CHECK2 MeCP2 reporter assay showing a 72% reduction in light units.

    “The predicted MeCP2 binding site was validated as a miR-181 target, as the Psi-CHECK2 MeCP2 reporter showed a 72% reduction in light units (Figure 7C).”
    • Also stated in: § Discussion · plain
      “In the present study we validated MeCP2 mRNA as a direct target of miR-181 by reporter assay.”
    • Also stated in: § Discussion · plain
      “We have identified two novel targets of miR-181, MeCP2 and XIAP mRNAs, as well as a non-canonical potential target, HMGA1.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    XIAP mRNA is a direct target of miR-181, validated by a Psi-CHECK2 XIAP reporter assay showing a roughly 63% decrease in relative light units.

    “The XIAP predicted binding site was also validated as a miR-181 target, with a roughly 63% decrease in relative light units for the Psi-CHECK2 XIAP reporter (Figure 7D).”
    • Also stated in: § Discussion · plain
      “The mRNA encoding X-linked inhibitor of apoptosis (XIAP) is another miR-181 target that we validated by reporter assay.”
    • Also stated in: § Discussion · boosted
      “We have identified two novel targets of miR-181, MeCP2 and XIAP mRNAs, as well as a non-canonical potential target, HMGA1.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    miR-181b, c and d are present at high levels in the mature CNS, but not early in development prior to astrogenesis.

    “Taken together with our observation that miR-181b, c and d are present at high levels in the mature CNS, but not early in development prior to astrogenesis, these data support a possible role for miR-181 in developmental specification of cell fate in the CNS and warrant further investigation.”
    Quote in the paperSubstantial claim

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

    Premisecitation

    miR-181 regulates other transcripts involved in cell survival and responses to stress, including hsp-70, MCL-1 and Bim.

    “It should be noted that miR-181 regulates other transcripts involved in cell survival and responses to stress including hsp-70, MCL-1 and Bim (Ouyang et al., 2011a, 2011b).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Ouyang et al., 2011a, 2011b

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

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

    Interpretationcitation

    HMGA1 activation of STAT3, which results in an increase of miR-181 expression, could represent an important feedback loop to regulate expression of HMGA1 in adult tissues.

    “HMGA1 activation of STAT3, which results in an increase of miR-181 expression, could represent an important feedback loop to regulate expression of HMGA1 in adult tissues (Hillion et al., 2008).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Hillion et al., 2008 source

  • Classified as probably not a substantial claim.

    Premisecitation

    TNFα is produced by astrocytes exposed to LPS.

    “TNFα is produced by astrocytes exposed to LPS (Chung et al., 1990);”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Chung et al., 1990 source

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

    Premisecitation

    Bcl-2 is a previously validated miR-181 target.

    “Bcl-2, a previously validated miR-181 target (Ouyang et al., 2012a) and the predicted target NAMPT mRNA were also enriched in the pull-down (Figure 7A).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Ouyang et al., 2012a

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

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

    Premisecitation

    HMGA1 enhances NF-κB binding, as in the case of the IFN1-γ promoter.

    “HMGA1 has been observed to enhance NF-κB binding, as in the case of the IFN1-γ promoter (Thanos et al., 1992).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Thanos et al., 1992 source

  • May combine two claims.

    Interpretationown data

    miR-181s negatively regulate the production of multiple cytokines and FGF2 in astrocytes, effectively suppressing their production in an inflammatory environment.

    “These results suggest that miR-181s negatively regulate the production of multiple cytokines and FGF2 in astrocytes, effectively suppressing their production in an inflammatory environment.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The miR-181b/c hairpin inhibitors specifically knock down miR-181b and miR-181c without off-target effects.
    • Rests on an assumption: Transfection with miR-181c specifically over-expresses miR-181c without off-target effects.

  • Classified as probably not a substantial claim.

    Premisecitation

    Pro-inflammatory stimuli such as LPS engage an innate immune response through TLR4 receptors, resulting in activation of astrocytes.

    “Pro-inflammatory stimuli such as lipopolysaccharide (LPS), an endotoxin component of gram-negative bacterial cell membranes, engage an innate immune response through TLR4 receptors, resulting in activation of astrocytes (Sofroniew et al., 2009b; Tang et al., 2007; Okun et al., 2012).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Sofroniew et al., 2009b

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

    • cites Tang et al., 2007 source
    • cites Okun et al., 2012

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

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

    Premisecitation

    Activation of Stat3, an important component of the NF-κB signaling pathway, increases miR-181 expression.

    “Activation of Stat3, an important component of the NF-κB signaling pathway, has also been shown to increase miR-181 expression (Iliopoulos et al., 2010).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Iliopoulos et al., 2010 source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    HMGA1 promotes an inflammatory transcriptional profile, at least in part by increasing Cox-2 and STAT-3 levels.

    “Particularly relevant to the role of miR-181 repression of HMGA1 in the inflammatory response, HMGA1 has been shown to promote an inflammatory transcriptional profile, at least in part by increasing Cox-2 and STAT-3 levels (Resar, 2010 for a review; Schuldenfrei et al., 2011).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites Resar, 2010 source
    • cites Schuldenfrei et al., 2011 source

  • May combine two claims.

    Premisecitation

    miR-181 directly targets the mRNA encoding Bcl-2 and other mRNAs encoding proteins involved in responses to stress including heat-shock protein 70.

    “miR-181 directly targets the mRNA encoding Bcl-2, a mitochondrial membrane-associated protein that inhibits apoptosis, and other mRNAs encoding proteins involved in responses to stress including heat-shock protein 70 (Chen et al., 2010; Ouyang et al., 2011a, b).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Chen et al., 2010

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

    • cites Ouyang et al., 2011a, b

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

  • May combine two claims.

    Interpretationreasoning

    miR-181s are involved in a general cell stress response and cell survival-related changes in transcriptional profiles.

    “Our results therefore suggest that miR-181s are involved in a general cell stress response and cell survival-related changes in transcriptional profiles.”
    Quote in the paperSubstantial claim

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

    Premisecitation

    Reactive gliosis is broadly characterized by an increase in expression of GFAP, activation of NF-κB, and the production of nitric oxide and inflammatory cytokines such as TNFα.

    “Reactive gliosis is broadly characterized by an increase in expression of the cytoskeletal protein GFAP, activation of the transcription factor NF-κB, and the production of nitric oxide and inflammatory cytokines such as TNFα (Pekny et al., 2005; Kang et al., 2011).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Pekny et al., 2005 source
    • cites Kang et al., 2011 source

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

    Premisecitation

    XIAP inhibits TNF-α-induced cell death and its expression is regulated by NF-κB.

    “XIAP inhibits TNF-α-induced cell death and its expression is regulated by NF-κB (Chu et al., 1997; Rothe et al., 1995; Stehlik et al., 1998).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Chu et al., 1997 source
    • cites Rothe et al., 1995 source
    • cites Stehlik et al., 1998 source

  • May combine two claims.

    Interpretationreasoning

    By enabling the production of GDNF and VEGF, a reduction in miR-181s might reduce neuronal vulnerability and enhance neurogenesis.

    “By enabling the production of GDNF and VEGF, a reduction in miR-181s might therefore be expected to reduce neuronal vulnerability and enhance neurogenesis.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    miR-181s are fundamentally involved in major programs of posttranscriptional regulation of cell differentiation and stress responses in astrocytes, with increasing miR-181 expression resulting in increased differentiation and vulnerability to environmental stress.

    “Taken together with the other confirmed targets of the miR-181 family, our findings argue that miR-181s are fundamentally involved in major programs of posttranscriptional regulation of cell differentiation and stress responses in astrocytes, with increasing miR-181 expression resulting in increased differentiation and vulnerability to environmental stress.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    GDNF can protect dopaminergic neurons against oxidative and metabolic stress in experimental models of Parkinson's disease.

    “GDNF can protect dopaminergic neurons against oxidative and metabolic stress in experimental models of Parkinson’s disease (Ramaswamy et al., 2009).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Ramaswamy et al., 2009 source

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

    Premisecitation

    VEGF stimulates cerebral angiogenesis and neurogenesis.

    “VEGF stimulates cerebral angiogenesis and neurogenesis (MacKenzie and Ruhrberg, 2012).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites MacKenzie and Ruhrberg, 2012 source

  • May combine two claims.

    Interpretationown data

    miR-181 repression of MeCP2 in astrocytes could play a physiological role in reactive gliosis, the developmental differentiation of astrocytes, and possibly Rett Syndrome.

    “miR-181 repression of MeCP2 in astrocytes could therefore play a physiological role in reactive gliosis, the developmental differentiation of astrocytes, and possibly Rett Syndrome.”
    Quote in the paperSubstantial claim
22 of 22

  • The paper relies on this without support.

    The C. elegans miR-67 mimic used as a control has no known mammalian targets.

    “For all miRNA experiments a C. elegans miR-67 mimic (Dharmacon), a miRNA with no known mammalian targets, was used.”

  • The paper relies on this without support.

    The miR-181b/c hairpin inhibitors specifically knock down miR-181b and miR-181c without off-target effects.

    “or to knock down (KD) miR-181 using miR-181c and miR-181b microRNA hairpin inhibitors (Dharmacon).”

  • The paper relies on this without support.

    Cultured primary astrocytes are representative of astrocytes in vivo for inferring miR-181 function in neuroinflammation.

    “For mouse primary astrocyte cultures, the cerebral cortex was removed from the brains of newborn mice and placed in cold Hank’s balanced saline solution (HBSS).”

  • The paper relies on this without support.

    Transfection with miR-181c specifically over-expresses miR-181c without off-target effects.

    “Lipofectamine LTX (Invitrogen) was used to transfect cells to over-express (OE) either miR-181c or a control miRNA (the C. Elegans miR-67; CTRL), or to knock down (KD) miR-181 using miR-181c and miR-181b microRNA hairpin inhibitors (Dharmacon).”

  • The paper relies on this without support.

    The observed changes in miR-181 expression in cortex after LPS occur in astrocytes.

    “We show that miR-181 expression is altered in brain cells in vivo in response to LPS, a model of inflammation, in both wild-type mice and transgenic mice lacking both receptors for the inflammatory cytokine TNF-α.”
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?

  • In AD-related contexts, miR-181a is downregulated in astrocytes, where it normally prevents excessive GFAP/S100B reactivity and supports balanced survival signaling.

    “In AD-related contexts, miR-181a is downregulated in astrocytes, where it normally prevents excessive GFAP/S100B reactivity and supports balanced survival signaling (Hutchison et al., 2013; Stary et al., 2017).”

    From Cell-specific MicroRNA networks orchestrate the pathogenesis of Alzheimer’s disease (2026) · cites this paper as Hutchison et al., 2013

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Authors
Hutchison, E. R., Kawamoto, E. M., Taub, D. D., Lal, A., Abdelmohsen, K., Zhang, Y., Wood, W. H., Lehrmann, E., Camandola, S., Becker, K. G., Gorospe, M., & Mattson, M. P.
Affiliations
National Institutes of Health (US)
Corresponding author
Mattson, Mark P.
ORCID
Venue
Glia
Publisher
Wiley
Published
2013-05-07
Volume
61
Issue
7
Pages
1018-1028
ISSN
  • 0894-1491 (ISSN-L)
  • 1098-1136
DOI
10.1002/glia.22483
PMC
PMC4624280
OpenAlex
W2159268524
Semantic Scholar
761fecbed94be3b582dc7ef8b7f8144246032b5c
Cited by
246
References
67
Citations by year
2026: 11 · 2025: 8 · 2024: 14 · 2023: 12 · 2022: 16 · 2021: 20 · 2020: 17 · 2019: 23 · 2018: 26 · 2017: 23 · 2016: 29 · 2015: 16 · 2014: 20 · 2013: 10
FWCI
12.47
Type
journal article
Language
en
Open access
Licence
onlinelibrary.wiley.com/termsAndConditions#vor (vor)
Locations
  • Glia (publishedVersion)
  • PubMed (publishedVersion)
  • Europe PMC (PubMed Central) (acceptedVersion)
  • PubMed Central (acceptedVersion)
Funders
  • National Institutes of Health
  • National Institute on Aging (ZIAAG000314, ZIAAG000312)
Topics
  • Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › MicroRNA in disease regulation
  • Health Sciences › Medicine › Neurology › Traumatic Brain Injury and Neurovascular Disturbances
Keywords
miR-181, microRNA, astrocytes, neuroinflammation, lipopolysaccharide, inflammatory cytokines, HMGB1, IL-10, MeCP2, XIAP, miRNA target genes, central nervous system injury, astrocyte transcriptome
MeSH terms
Animals, Astrocytes, Cells, Cultured, Cerebral Cortex, L-Lactate Dehydrogenase, Lipopolysaccharides, Male, Neurons, Transfection, Neuroimmunomodulation, Cytokines, Mice, Knockout, Brain-Derived Neurotrophic Factor, Biotinylation, MicroRNAs, Receptors, Tumor Necrosis Factor, Type I, Receptors, Tumor Necrosis Factor, Type II, Mice, X-Linked Inhibitor of Apoptosis Protein, Methyl-CpG-Binding Protein 2

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