Cited by 151Neuroinflammation and Neurodegeneration MechanismsPrion Diseases and Protein MisfoldingMicroRNA in disease regulation

The DOI matches its Crossref record; 57 of the 57 references it cites were found in a registry or an index; 39 of the 55 sources its claims cite were checked for support.

MicroRNA 146a (miR-146a) Is Over-Expressed during Prion Disease and Modulates the Innate Immune Response and the Microglial Activation State

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Abstract

Increasing evidence supports the involvement of microRNAs (miRNAs) in inflammatory and immune processes in prion neuropathogenesis. MiRNAs are small, non-coding RNA molecules which are emerging as key regulators of numerous cellular processes. We established miR-146a over-expression in prion-infected mouse brain tissues concurrent with the onset of prion deposition and appearance of activated microglia. Expression profiling of a variety of central nervous system derived cell-lines revealed that miR-146a is preferentially expressed in cells of microglial lineage. Prominent up-regulation of miR-146a was evident in the microglial cell lines BV-2 following TLR2 or TLR4 activation and also EOC 13.31 via TLR2 that reached a maximum 24-48 hours post-stimulation, concomitant with the return to basal levels of transcription of induced cytokines. Gain- and loss-of-function studies with miR-146a revealed a substantial deregulation of inflammatory response pathways in response to TLR2 stimulation. Significant transcriptional alterations in response to miR-146a perturbation included downstream mediators of the pro-inflammatory transcription factor, nuclear factor-kappa B (NF-κB) and the JAK-STAT signaling pathway. Microarray analysis also predicts a role for miR-146a regulation of morphological changes in microglial activation states as well as phagocytic mediators of the oxidative burst such as CYBA and NOS3. Based on our results, we propose a role for miR-146a as a potent modulator of microglial function by regulating the activation state during prion induced neurodegeneration.

Overview

AI snapshot

Written from the paper’s full text.

TL;DR

miR-146a is over-expressed in prion-infected mouse brain and is enriched in microglial cells, where TLR2/TLR4 stimulation induces it with delayed kinetics relative to cytokines. Gain- and loss-of-function studies indicate miR-146a broadly dampens microglial inflammatory and phagocytosis-related gene programs, suggesting it modulates the microglial activation state during prion disease.

Key findings

  • miR-146a was consistently up-regulated in prion-infected mouse brain: 3.3-fold in 22A whole brain, 8.3-fold in RML hippocampus, 16.3-fold in RML cerebellum, and 10.0-fold in BSE cerebellum versus age-matched mock-infected controls (Table 1).
  • miR-146a expression was enriched in microglial cell lines and induced by TLR2 or TLR4 agonists in BV-2 and EOC 13.31 cells, reaching a maximum at 24–48 hours and remaining elevated at 72 hours, while IL-6, CSF2/GM-CSF, and TNF transcripts peaked before 24 hours and returned to basal levels by 48 hours.
  • In EOC 13.31 cells, miR-146a over-expression altered 1,668 genes by ≥1.5-fold (FDR ≤2%) in resting cells; after TLR2/LPS stimulation, 977 genes were deregulated at 24 hours (778 down, 199 up), with enrichment in NF-κB, JNK, and MAPK signaling.
  • miR-146a over-expression significantly reduced IL-6 and GM-CSF protein after TLR2 stimulation (p<0.001), while anti-miR knock-down caused a small but significant increase in IL-6; predicted targets included IL1B, SMAD3, NOS2, QK1, and BCL2L1.

Methods

  • CD-1 mice were inoculated intracerebrally with 20 µl of 1% RML scrapie brain homogenate; at clinical symptoms, 6 mice per group were sacrificed and hippocampus or cerebellar tissue was used for RNA extraction, with equal numbers of age-matched mock-infected controls.
  • Murine microglial cell lines BV-2 and EOC 13.31 were stimulated with LPS or heat-killed Listeria monocytogenes (HKLM), with anti-TLR2 neutralization; miR-146a was over-expressed with 30 nM pre-miR mimic or knocked down with 50 nM anti-miR, followed by qRT-PCR (normalized to U6, 2−ΔΔCt) and IL-6/GM-CSF ELISA.
  • miRNA profiling used an in-house microarray; mRNA expression used Agilent whole mouse genome 4×44K arrays, analyzed with Feature Extraction, SAM (p=0.01), Ingenuity Pathway Analysis, and TargetScan 5.1; raw data deposited under GEO accession GSE17759.

Limitations

  • The paper states that in situ hybridization for miR-146a was inconclusive, that miR-146a may be expressed at low levels in other cell types including neurons, and that attempts to induce miR-146a in cultured microglia with recombinant PrP or PrP106–126 were unsuccessful.
  • The paper states that in vitro culture lacks multiple cell types, cell-to-cell signaling, normal cellular functions, and the long disease time course, and that TLR4 transcripts were below detection in their prion model; it also notes that MyD88 knockout data imply TLR2 signaling may not be essential in disease and that their model may not fully mimic disease processes.
  • Evident from the text: findings rely on mouse models and microglial cell lines rather than human tissue; direct miR-146a targets are largely inferred from microarray intersections and bioinformatic prediction, with limited validation; some in vivo miR-146a increases were not statistically significant (22A p=9.4×10−2; BSE p=7.6×10−2), and IL-10 up-regulation was not detectable by ELISA.

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Integrity signals

Registry

Crossref · 10.1371/journal.pone.0030832

Cites

57 of 57 references verified0 citation errors · 0 not checkable

Statistics

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Verification

Claims the paper makes

39 of 55 cited sources checked · 14 supported · 1 flagged · accuracy not yet measured

Show all 83 claims

  • Probably not a substantial claim. 31
  • It may state two separate claims as one. 42
  • The quote only partly states this claim. 4
64 of 64
  • The paper relies on this without support.

    PrPSc induces inflammatory responses by the brain's glial cells (astrocytes and microglia) by activating innate immunity pathways.

    “PrPSc induces inflammatory responses by the brain's glial cells (astrocytes and microglia) by activating innate immunity pathways.”
  • The paper relies on this without support.

    The microglial surface receptor that is engaged leading to activation during prion pathogenesis is unknown.

    “MiR-146a is reported to be up-regulated in LPS stimulated human macrophages, however, the microglial surface receptor that is engaged leading to activation during prion pathogenesis is unknown [16].”
  • The paper relies on this without support.

    The specific stimuli and signaling pathways that lead to modulations of microglial functions, and the mechanisms by which microglial activation influences chronic neurodegeneration, are as yet unknown.

    “The specific stimuli and signaling pathways that lead to these modulations of functions, and the mechanisms by which microglial activation influences chronic neurodegeneration, are as yet unknown.”
  • The paper relies on this without support.

    Whether activation of the innate immune response is beneficial or harmful in prion-like pathologies is still unknown.

    “Such an inflammatory milieu has been recognized in the prion infected brain for some time; however, whether the activation of this innate immune response is beneficial or harmful in prion-like pathologies is still unknown.”
  • The authors state this as an assumption.

    Most translational repression is accompanied by miRNA-mediated mRNA degradation.

    “It has been previously reported that most translational repression is accompanied by miRNA-mediated mRNA degradation, we therefore used this assumption to predict potential direct targets of miR-146a in this system [34], [35].”
  • The paper relies on this without support.

    Direct targets of miR-146a important in immune activation are likely to be those found at the intersection of genes up-regulated following knock-down and down-regulated after over-expression, provided they exhibit altered expression detectable at the mRNA level.

    “Direct targets of miR-146a important in immune activation are likely to be those found at the intersection of genes that are up-regulated following knock-down and down-regulated after over-expression, provided that they exhibit altered expression that is detectable at the mRNA level.”
  • The paper relies on this without support.

    Microglial cells respond to prion deposition by converting from a resting to an activated phenotype, but synthesize low levels of pro-inflammatory cytokines.

    “Microglial cells, the brain's resident immune effector cells, respond to prion deposition by the conversion of a resting phenotype into one that is ‘activated’. However, although they take on an activated phenotype they synthesize fairly low levels of pro-inflammatory cytokines”
  • The paper relies on this without support.

    Phenotypic changes in microglia are one of the earliest apparent alterations in tissue pathology detectable during prion disease.

    “However, it is clear that phenotypic changes in microglia are one of the earliest apparent alterations in tissue pathology detectable during disease.”
  • The paper relies on this without support.

    The in vivo transcriptional response likely represents the host's response to the process of neurodegeneration as much, if not more than, the response specifically to prions.

    “It is often speculated that the in vivo transcriptional response likely represents the host's response to the process of neurodegeneration as much, if not more than, the response specifically to prions, which may account for our inability to detect a robust miR-146a induction in the cultured microglia.”
  • The paper relies on this without support.

    Effector molecules downstream of multiple cell surface receptors, including TLR2 and TLR4 signals, are similar.

    “However, effector molecules downstream of multiple cell surface receptors, including TLR2 (and TLR4) signals, are similar”
  • The paper relies on this without support.

    A reduction in TLR2 expression is linked with an increase in phagocytosis, implying an inverse relation between TLR2 expression and phagocytic activity.

    “Studies also link a reduction in TLR2 expression with an increase in phagocytosis, implying an inverse relation between TLR2 expression and phagocytic activity.”
  • The paper relies on this without support.

    MyD88 is the main adaptor protein implicated in TLR signaling and is considered essential for the induction of inflammatory cytokines triggered by most TLRs, including TLR2.

    “MyD88 is the main adaptor protein implicated in TLR signaling and is considered essential for the induction of inflammatory cytokines triggered by most TLRs, including TLR2.”
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Claims other papers make about this one

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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 4B is cited for pathways it does not depict

    Minor

    The discussion directs readers to Figure 4B for alterations in NF-κB and JAK-STAT pathway mediators, but Figure 4B is described as a measurement of miR-146a knock-down over time.

  • An 8-hour stimulated result is dated from transfection rather than stimulation

    Minor

    The paper says LPS stimulation occurred 24 hours after mimic transfection, then describes a stimulated result at 8 hours post transfection. A result eight hours after transfection would precede the stated LPS stimulation.

  • TLR1 is called significantly up-regulated despite its reported p-value

    Minor

    The text names TLR1 as significantly up-regulated, but Table 2 reports p = 0.081 for TLR1. That exceeds the conventional 0.05 threshold, as well as the paper's stated microarray differential-expression threshold of 0.01.

  • The named up-regulated Fc receptor differs from Table 2

    Minor

    The results name FCGR3 as an over-expressed Fc receptor, whereas Table 2 lists FCGR1 and FCGR2, not FCGR3.

4 of 4

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Metadata

Authors
Saba, R., Gushue, S., Huzarewich, R. L. C. H., Manguiat, K., Medina, S., Robertson, C., & Booth, S. A.
Affiliations
Public Health Agency of Canada (CA)
Corresponding author
Booth, Stephanie A.
Venue
PLoS ONE
Published
2012-02-17
Volume
7
Issue
2
Pages
e30832
ISSN
1932-6203 (ISSN-L)
OpenAlex
W2084713640
Cited by
151
References
57
Citations by year
2026: 4 · 2025: 5 · 2024: 4 · 2023: 4 · 2022: 10 · 2021: 10 · 2020: 13 · 2019: 9 · 2018: 13 · 2017: 15 · 2016: 11 · 2015: 16 · 2014: 14 · 2013: 16 · 2012: 7
FWCI
8.04
Type
journal article
Language
en
Open access
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
Funders
  • Public Health Agency of Canada
  • Public Health Agency
Topics
  • Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Prion Diseases and Protein Misfolding
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › MicroRNA in disease regulation
Keywords
miR-146a, prion disease, microglial activation, innate immune response, TLR2, TLR4, NF-κB signaling, JAK/STAT signaling, neuroinflammation, respiratory burst
MeSH terms
Animals, Brain, Cell Line, Cell Movement, Immunity, Innate, Kinetics, Lipopolysaccharides, Oxidative Phosphorylation, Prions, Time Factors, Transcription, Genetic, Protein Biosynthesis, Signal Transduction, Up-Regulation, Cluster Analysis, Cytokines, Prion Diseases, Microglia, Inflammation Mediators, Gene Expression Profiling, MicroRNAs, Toll-Like Receptor 2, Toll-Like Receptor 4, Mice

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