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Cited by 90Extracellular vesicles in diseaseMicroRNA in disease regulationAmyotrophic Lateral Sclerosis Research

Distinct repertoires of microRNAs present in mouse astrocytes compared to astrocyte-secreted exosomes

Open access
Jovičić, Ana, Gitler, Aaron D.·2017·PLOS ONE
DOI 10.1371/journal.pone.0171418PMC PMC5289606
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Abstract

BACKGROUND: Astrocytes are the most abundant cell type in the central nervous system (CNS) and secrete various factors that regulate neuron development, function and connectivity. microRNAs (miRNAs) are small regulatory RNAs involved in posttranslational gene regulation. Recent findings showed that miRNAs are exchanged between cells via nanovesicles called exosomes. In this study, we sought to define which miRNAs are contained within exosomes secreted by astrocytes. We also explored whether astroglial miRNA secretion via exosomes is perturbed in a mouse model of amyotrophic lateral sclerosis (ALS), a neurodegenerative disease where astrocytes play a crucial role in driving disease progression. METHODOLOGY/PRINCIPAL FINDINGS: By isolating and profiling the expression of miRNAs from primary mouse astrocytes and from the exosomes that astrocytes secrete, we compared miRNA expression in the cells and secreted vesicles. We established that miRNA expression profiles of astrocytes and their exosomes are vastly different. In addition, we determined that exosomal miRNA expression in astrocytes is not significantly perturbed in a mouse model of ALS. CONCLUSIONS: Astrocytes secrete numerous miRNAs via exosomes and miRNA species contained in exosomes are considerably different from miRNAs detectable in astrocytes, suggesting the existence of a mechanism to select certain miRNAs for inclusion or exclusion from exosomes. The exosomal miRNA profiling dataset we have generated will provide a resource to aid in the investigation of this selection mechanism. Finally, the miRNA expression profile in astrocyte-secreted exosomes is not perturbed by expression of mutant SOD1-G93A.

AI snapshot

Written from the paper’s full text.

TL;DR

Mouse astrocytes and their secreted exosomes have vastly different miRNA repertoires, with exosomes containing a more restricted set and some miRNAs highly enriched or exclusive to exosomes. Exosomal miRNA profiles were not significantly altered in SOD1-G93A ALS astrocytes, although SOD1-G93A astrocyte-conditioned media was toxic to motor neurons.

Key findings

  • Profiling 752 mature rodent miRNAs in 4 independent primary astrocyte and exosome RNA samples showed that astroglial exosomes have substantially different miRNA content from astrocytes; hierarchical clustering separated the 4 exosomal samples from the 4 astroglial samples.
  • 217 miRNAs were exclusively detected in astrocytes, and 61 miRNAs were more than 2-fold enriched in astrocytes compared to exosomes. Exosomal miRNA expression was more restrictive: 12 miRNAs were exclusively present in exosomes, and 42 miRNAs were more than 2-fold enriched in exosomes compared to astrocytes; among these, 6 were >100-fold and 26 were >1000-fold enriched.
  • qPCR validated exosome-specific miRNAs: miR-10b had cycle threshold 21.92 in exosomes and was undetermined in astrocytes; miR-208 had cycle threshold 25.43 in exosomes and was undetermined in astrocytes; astrocyte-specific miR-188-5p had cycle threshold 15.99 in astrocytes and was undetermined in exosomes.
  • Exosomal miRNA expression in SOD1-G93A ALS mouse astrocytes was not significantly perturbed compared with wild-type astrocytes, while conditioned media from SOD1-G93A astrocytes significantly decreased motor neuron viability compared with wild-type astrocyte media (Student’s t-test, p<0.01). Several array features—miR-1274a, miR-1937b, miR-1937c, miR-2134, and miR-2182—were actually tRNA fragments and were highly enriched in exosomes.

Methods

  • Primary mouse astrocytes were prepared from P1 cortex, cultured, and exosomes were purified from astrocyte-conditioned media by differential centrifugation, filtration, and ultracentrifugation; vesicles were validated by transmission electron microscopy (50–100 nm double membrane-bound vesicles) and Western blot for CD63 and Tsg101.
  • miRNA expression was profiled using TaqMan rodent miRNA arrays for 752 mature rodent miRNAs in 4 independently prepared primary astrocyte and exosome RNA samples, with hierarchical clustering and qPCR validation for selected miRNAs.
  • For ALS experiments, primary astrocytes from SOD1-G93A mice and wild-type littermates were used; exosomal miRNAs were profiled, and astrocyte-conditioned media was applied to primary motor neurons to assess viability.

Limitations

  • Stated: the study profiled only the 752 mature miRNAs present on commercial TaqMan arrays, so miRNAs not represented on the arrays cannot be excluded from contributing to astrocyte toxicity.
  • Stated: whether exosomes, through their RNA, DNA, protein, or lipid content, contribute to SOD1-G93A astrocyte-mediated toxicity remains to be examined.
  • Evident from the text: miRNA profiling used n=4 per group, qPCR validation covered only a small number of selected miRNAs, and the mechanism selecting specific miRNAs for exosomal inclusion or exclusion was not determined.

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

Integrity signals

Registry

Crossref · 10.1371/journal.pone.0171418

Cites

36 of 36 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

5 of 16 cited sources checked · 3 supported · 0 flagged · accuracy not yet measured

  • Findingown data

    Conditioned media from SOD1-G93A astrocytes is toxic to motor neurons.

    “Consistent with previous studies [23–26], we validated that the conditioned media from these cultures is toxic to motor neurons.”
    • Also stated in: § The profile of exosomal miRNAs is not changed in mouse SOD1-G93A ALS astrocytes · plain
      “Treating primary spinal cord neurons with SOD1-G93A astrocyte-conditioned media significantly decreased neuronal viability after 3 days compared to the media from wild-type astrocytes (Fig 3).”
    Quote in the paperSubstantial claim2 of 4 sources checked
    • cites [23] M Nagai, 2007, Astrocytes expressing ALS-linked mutated SOD1 release factors selectively toxic to motor neurons, Nat Neurosci source
    • cites [24] MCN Marchetto, 2008, Non-Cell-Autonomous Effect of Human SOD1G37R Astrocytes on Motor Neurons Derived from Human Embryonic Stem Cells, Cell Stem Cell source

      Checked before — its earlier verdict is held back until a new check runs.

    • cites [25] AM Haidet-Phillips, 2011, Astrocytes from familial and sporadic ALS patients are toxic to motor neurons, Nat Biotech source

      The paper reports that conditioned media from patient-derived familial and sporadic ALS astrocytes accelerated motor-neuron death (results describing Fig. 2e–f; page unavailable). It also reports motor-neuron toxicity when SOD1-G93A was overexpressed in astrocytes, but that result came from co-culture experiments, not a reported test of conditioned media from those astrocytes. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [26] K Meyer, 2014, Direct conversion of patient fibroblasts demonstrates non-cell autonomous toxicity of astrocytes to motor neurons in familial and sporadic ALS, Proceedings of the National Academy of Sciences source

      Checked before — its earlier verdict is held back until a new check runs.

    • Rests on an assumption: The primary astrocyte cultures used were free of contaminating microglia and oligodendrocyte precursor cells.
  • Findingown data

    qPCR detected miR-188-5p in astrocytes but not in exosomes.

    “On the other hand, miR-188-5p which is astrocyte-specific was detected in astrocytes, but not in exosomes (Table 3).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The primary astrocyte cultures used were free of contaminating microglia and oligodendrocyte precursor cells.
    • Rests on an assumption: The exosome preparations were not contaminated by serum-derived exosomes.
  • Findingcitation

    Primary mouse astrocytes secrete exosomes containing miRNAs.

    “Astrocytes secrete numerous miRNAs via exosomes”
    • Also stated in: § Discussion · boosted
      “In the present study we have confirmed that primary astrocytes secrete miRNA-containing exosomes [11].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [11] L Zhang, 2015, Microenvironment-induced PTEN loss by exosomal microRNA primes brain metastasis outgrowth, Nature source

      Checked before — its earlier verdict is held back until a new check runs.

    • Rests on an assumption: The primary astrocyte cultures used were free of contaminating microglia and oligodendrocyte precursor cells.
    • Rests on an assumption: The exosome preparations were not contaminated by serum-derived exosomes.
  • Findingown data

    qPCR detected miR-10b and miR-208 in exosomes but not in astrocytes.

    “While we were unable to detect them in astrocytes, we readily detected them in exosomes.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The primary astrocyte cultures used were free of contaminating microglia and oligodendrocyte precursor cells.
    • Rests on an assumption: The exosome preparations were not contaminated by serum-derived exosomes.
  • Findingown data

    42 miRNAs had higher than 2-fold enrichment in exosomes compared to astrocytes.

    “only 12 miRNAs were exclusively present in exosomes, and 42 miRNAs had higher than 2-fold enrichment in exosomes compared to astrocytes (Table 2).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The primary astrocyte cultures used were free of contaminating microglia and oligodendrocyte precursor cells.
    • Rests on an assumption: The exosome preparations were not contaminated by serum-derived exosomes.
  • Classified as probably not a substantial claim. 7
  • May combine two claims. 5

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

    Premisecitation

    ALS is a progressive neurodegenerative disease with a strong non-cell autonomous component contributing to motor neuron death.

    “ALS is a progressive neurodegenerative disease with a strong non-cell autonomous component contributing to motor neuron death [17].”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [17] H Ilieva, 2009, Non–cell autonomous toxicity in neurodegenerative disorders: ALS and beyond, The Journal of Cell Biology source

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

    Premisecitation

    miR-302 regulates neural progenitor proliferation, differentiation timing, and survival during neurulation; knockout results in early embryonic lethality and open neural tube defects.

    “For example, miR-302, which we have found to be highly enriched in exosomes, has been shown to regulate neural progenitor proliferation, differentiation timing, and survival during neurulation, and knockout or miR-302 results in early embryonic lethality and open neural tube defects [28].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [28] S-L Yang, 2015, MiR-302/367 regulate neural progenitor proliferation, differentiation timing, and survival in neurulation, Developmental Biology source

      The Abstract reports that deletion of the miR-302/367 cluster caused early embryonic lethality and open neural tube defects, with altered neural progenitor proliferation, differentiation, and survival. The Discussion clarifies that this study deleted both miR-302 and miR-367, so its knockout and cell-survival findings cannot be attributed to miR-302 alone. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim.

    Premisecitation

    Selectively deleting mutant SOD1 transgene expression in astrocytes sharply slows disease progression in the SOD1 mouse model of ALS.

    “Selectively deleting mutant SOD1 transgene expression in astrocytes, sharply slows the disease progression in the SOD1 mouse model of ALS [22].”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [22] K Yamanaka, 2008, Astrocytes as determinants of disease progression in inherited amyotrophic lateral sclerosis, Nat Neurosci source

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

    Premisecitation

    tRNA fragments are not random by-products of tRNA degradation or biogenesis and have biological regulatory roles.

    “These tRNA fragments are not random by-products of tRNA degradation or biogenesis and have biological regulatory roles [21].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [21] YS Lee, 2009, A novel class of small RNAs: tRNA-derived RNA fragments (tRFs), Genes & Development source

      Checked before — its earlier verdict is held back until a new check runs.

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

    Premisecitation

    Increased exocytosis has been observed in SOD1-G93A astrocytes and it contributes to astrocyte-mediated toxicity.

    “Interestingly, increased exocytosis has been observed in SOD1-G93A astrocytes and it contributes to the astrocyte-mediated toxicity [27].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [27] H Kawamata, 2014, Abnormal Intracellular Calcium Signaling and SNARE-Dependent Exocytosis Contributes to SOD1G93A Astrocyte-Mediated Toxicity in Amyotrophic Lateral Sclerosis, The Journal of Neuroscience source

  • Classified as probably not a substantial claim.

    Premisecitation

    Astrocytes seem to secrete toxic factor(s) that are detrimental to motor neuron health.

    “Moreover, astrocytes seem to secrete toxic factor(s) that are detrimental to motor neuron health [23–26].”
    Quote in the paperSubstantial claim0 of 4 sources checked
    • cites [23] M Nagai, 2007, Astrocytes expressing ALS-linked mutated SOD1 release factors selectively toxic to motor neurons, Nat Neurosci source

      Queued — the background check reaches it, usually within a day.

    • cites [24] MCN Marchetto, 2008, Non-Cell-Autonomous Effect of Human SOD1G37R Astrocytes on Motor Neurons Derived from Human Embryonic Stem Cells, Cell Stem Cell source

      Queued — the background check reaches it, usually within a day.

    • cites [25] AM Haidet-Phillips, 2011, Astrocytes from familial and sporadic ALS patients are toxic to motor neurons, Nat Biotech source

      Queued — the background check reaches it, usually within a day.

    • cites [26] K Meyer, 2014, Direct conversion of patient fibroblasts demonstrates non-cell autonomous toxicity of astrocytes to motor neurons in familial and sporadic ALS, Proceedings of the National Academy of Sciences source

      Queued — the background check reaches it, usually within a day.

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

    Premisecitation

    Astrocytes secrete exosomes containing miR-19a, which regulates PTEN expression in brain tumor cells, thus influencing the outgrowth of brain metastases.

    “Recently it has been demonstrated that astrocytes secrete exosomes containing miR-19a, which regulates PTEN expression in brain tumor cell, thus influencing the outgrowth of brain metastases [11].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [11] L Zhang, 2015, Microenvironment-induced PTEN loss by exosomal microRNA primes brain metastasis outgrowth, Nature source

      Checked before — its earlier verdict is held back until a new check runs.

7 of 7

  • The paper relies on this without support.

    The primary astrocyte cultures used were free of contaminating microglia and oligodendrocyte precursor cells.

    “After 10–14 d in vitro, microglial cells and oligodendrocyte precursors were eliminated by shaking on a rotary shaker at 300 rpm for 3 h, after which the medium was replaced.”

  • The paper relies on this without support.

    The exosome preparations were not contaminated by serum-derived exosomes.

    “In order to eliminate serum-derived exosome contamination in the media, before use fetal bovine serum was ultracentrifuged at 120,000g for 90 min.”

  • The paper relies on this without support.

    The study had sufficient statistical power to detect differences in exosomal miRNA expression between SOD1-G93A and wild-type astrocytes.

    “expression profiling of exosomal miRNAs in SOD1-G93A and wild type astrocytes did not show any significant differences.”
3 of 3

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.

  • miR-547 is listed as exclusive to both astrocytes and exosomes

    Major

    Table 1 lists miR-547 among species detected in astrocytes but not exosomes, while Table 2 lists it among species detected in exosomes but not astrocytes.

  • miR-190b is both absent from and highly enriched in exosomes

    Major

    Table 1 places miR-190b in the astrocyte-only row, whereas Table 2 reports it in the row enriched more than 1,000-fold in exosomes.

  • Motor-neuron viability is reported at both three and seven days

    Minor

    The Results say viability decreased after three days of treatment, while the caption for the cited Fig 3 says viability was assessed after seven days.

  • Table 1 assigns miR-543 to incompatible enrichment categories

    Minor

    Table 1 includes miR-543 in both its astrocyte-only row and its row for species with a measured greater-than-twofold astrocyte/exosome enrichment.

  • The tRNA-fragment results cite the wrong enrichment table

    Minor

    The text directs readers to Table 1 for tRNA fragments highly enriched in exosomes, but Table 1 is labeled as enrichment in astrocytes; the named fragments appear in Table 2.

  • Table 2 lists miR-685 and miR-715 as both exosome-only and fold-enriched

    Minor

    The exosome-only row says these species were not detected in astrocytes and gives their fold enrichment as N/A, but the next row assigns both a greater-than-twofold enrichment.

6 of 6

Cites

Flagged first, then by citations

1 of the 37 references couldn’t be matched to a known paper.

Cited by

Sorted by citations

Related

Authors
Jovičić, A., & Gitler, A. D.
Affiliations
Stanford Medicine (US)
Corresponding author
Jovičić, Ana
ORCID
Venue
PLOS ONE
Publisher
Public Library of Science (PLoS)
Published
2017-02-02
Volume
12
Issue
2
Pages
e0171418
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0171418
PMC
PMC5289606
OpenAlex
W2583401467
Cited by
86
References
37
Citations by year
2026: 2 · 2025: 4 · 2024: 5 · 2023: 10 · 2022: 14 · 2021: 15 · 2020: 14 · 2019: 13 · 2018: 7 · 2017: 2
FWCI
3.57
Type
journal article
Language
en
Open access
Licence
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, cc-by-sa)
  • Figshare (submittedVersion, cc-by)
Funders
  • National Institutes of Health
  • National Institute of Neurological Disorders and Stroke (R01NS073660, R01NS093865, R01NS065317)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Extracellular vesicles in disease
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › MicroRNA in disease regulation
  • Health Sciences › Medicine › Neurology › Amyotrophic Lateral Sclerosis Research
Keywords
microRNA, astrocytes, exosomes, amyotrophic lateral sclerosis, SOD1 G93A, mouse model, extracellular vesicles, miRNA expression profiling
MeSH terms
Animals, Astrocytes, Cells, Cultured, Female, Gene Expression Regulation, Male, Motor Neurons, Blotting, Western, Polymerase Chain Reaction, MicroRNAs, Mice, Exosomes, Transcriptome

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