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Cited by 83Amyotrophic Lateral Sclerosis ResearchMicroRNA in disease regulationMuscle Physiology and Disorders

MicroRNA Profiling Reveals Marker of Motor Neuron Disease in ALS Models

Open access
Hoye, Mariah L., Koval, Erica D., Wegener, Amy J., Hyman, Theodore S., Yang, Chengran, O'Brien, David R., Miller, Rebecca L., Cole, Tracy +9 more·2017·The Journal of Neuroscience
DOI 10.1523/jneurosci.3582-16.2017PMC PMC5452343
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Abstract

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder marked by the loss of motor neurons (MNs) in the brain and spinal cord, leading to fatally debilitating weakness. Because this disease predominantly affects MNs, we aimed to characterize the distinct expression profile of that cell type to elucidate underlying disease mechanisms and to identify novel targets that inform on MN health during ALS disease time course. microRNAs (miRNAs) are short, noncoding RNAs that can shape the expression profile of a cell and thus often exhibit cell-type-enriched expression. To determine MN-enriched miRNA expression, we used Cre recombinase-dependent miRNA tagging and affinity purification in mice. By defining the in vivo miRNA expression of MNs, all neurons, astrocytes, and microglia, we then focused on MN-enriched miRNAs via a comparative analysis and found that they may functionally distinguish MNs postnatally from other spinal neurons. Characterizing the levels of the MN-enriched miRNAs in CSF harvested from ALS models of MN disease demonstrated that one miRNA (miR-218) tracked with MN loss and was responsive to an ALS therapy in rodent models. Therefore, we have used cellular expression profiling tools to define the distinct miRNA expression of MNs, which is likely to enrich future studies of MN disease. This approach enabled the development of a novel, drug-responsive marker of MN disease in ALS rodents. SIGNIFICANCE STATEMENT Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease in which motor neurons (MNs) in the brain and spinal cord are selectively lost. To develop tools to aid in our understanding of the distinct expression profiles of MNs and, ultimately, to monitor MN disease progression, we identified small regulatory microRNAs (miRNAs) that were highly enriched or exclusive in MNs. The signal for one of these MN-enriched miRNAs is detectable in spinal tap biofluid from an ALS rat model, where its levels change as disease progresses, suggesting that it may be a clinically useful marker of disease status. Furthermore, rats treated with ALS therapy have restored expression of this MN RNA marker, making it an MN-specific and drug-responsive marker for ALS rodents.

AI snapshot

Written from the paper’s full text.

TL;DR

Using Cre-dependent miRNA tagging and affinity purification (miRAP) in adult mice, the authors defined motor neuron (MN)-enriched miRNAs and identified miR-218 as a marker of MN disease in ALS models. miR-218 was depleted in ALS spinal cord due to MN loss, increased in CSF of SOD1 G93A rats, and reduced by SOD1-lowering antisense oligonucleotide therapy.

Key findings

  • miRAP profiling of adult mouse brainstem and spinal cord across ChAT (MNs), Syn (all neurons), GFAP (astrocytes), and Lyz2 (microglia/myeloid) populations identified cell-type-enriched miRNA profiles; five of the top six MN-enriched miRNAs were confirmed by RT-qPCR, with miR-218-5p showing the strongest enrichment in adult MNs compared with all other spinal neurons. miR-218 was detected predominantly in ChAT+ anterior horn MNs by in situ hybridization, and all five MN-enriched miRNAs were significantly enriched in ChAT+ MNs versus Cnp1+ oligodendrocytes; miR-338-3p was ~25-fold more abundant in oligodendrocytes than MNs.
  • In SOD1 G93A mouse spinal cord, MN-enriched miR-218 and miR-138 were significantly depleted temporally beginning as early as 126 d and maximized at end stage, whereas pan-neuronal miR-382 and miR-672 decreased but were not significantly depleted even at end stage. miR-218 was also significantly depleted in SOD1 G93A rat spinal cord and in human ALS autopsy spinal cord compared with non-neurodegenerative controls.
  • miR-218 levels were maintained in surviving ALS MNs: miRAP showed no significant change in ALS MNs from presymptomatic 70 d to symptomatic 140 d, and miR-218 in situ fluorescence was not significantly different between control and SOD1 G93A 140 d mice, while ChAT+ MN number per unit area was significantly reduced.
  • In ALS SOD1 G93A rat CSF, miR-218 increased temporally, was not increased in hSOD1 WT overexpressing rats, and neuronal miR-132/miR-124 were not significantly changed. hSOD1 ASO treatment reduced lumbar spinal cord hSOD1 mRNA by ~75%, preserved hindlimb grip strength, and reduced CSF miR-218; miR-218 CSF levels correlated with SMI-32+ MN loss and hSOD1 mRNA levels. miR-218 was also released from injured primary Hb9-GFP MNs and human iPSC-derived MNs.

Methods

  • miRAP: LSL-tAgo2 mice were crossed to Syn-Cre, ChAT-Cre, GFAP-Cre, or Lyz2-Cre lines; brainstem and spinal cord from 63-d-old adult mice were processed for myc or Ago2 immunoprecipitation, TaqMan miRNA microarrays, and individual RT-qPCR validation. Analyses included LoessM normalization, empirical Bayes, Benjamini-Hochberg correction, and specificity index (pSI).
  • ALS models and CSF: SOD1 G93A mice were harvested at 56, 84, 105, 126, and 147 d and at end stage; SOD1 G93A rats were used for CSF collection via cisterna magna puncture. Rats at 65 d received intrathecal 1000 µg hSOD1 ASO, scrambled ASO, or aCSF; CSF and spinal cord were harvested at 115 d, with SMI-32+ MN counts and hindlimb grip strength assessed.
  • Human and cell culture: cervical spinal cord autopsy tissue from 10 ALS patients and 4 non-ALS controls was used for miRNA analysis; human iPSC-derived MN cultures and primary Hb9-GFP mouse MN cultures were treated with puromycin or sodium arsenite, respectively, with miR-218 and LDH measured in media.

Limitations

  • Stated: miR-218 is below the limit of detection in some human CSF samples, so a valid comparison between ALS and control human CSF is not yet achievable; the authors state miR-218 needs further validation, especially in human ALS CSF.
  • Stated: the mechanism underlying increased miR-218 in ALS rat CSF is unknown; some miR-218 is released before significant MN loss, so release may partly reflect an active response independent of MN loss. It also remains possible that miR-218 is downregulated acutely in individual MNs shortly before death.
  • Stated/evident: one of the top six MN-enriched miRNAs, miR-380-5p, could not be confirmed due to nonspecific TaqMan primers. The human autopsy comparison used a small sample (10 ALS, 4 non-ALS controls), and the main disease-marker findings come from SOD1 rodent models, so generalization to sporadic ALS or other MN diseases is not established in this paper.

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

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Crossref · 10.1523/jneurosci.3582-16.2017

Cites

69 of 69 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

6 of 31 cited sources checked · 3 supported · 0 flagged · accuracy not yet measured

  • Findingcitation

    miR-338-3p was approximately 25-fold more abundant in oligodendrocytes than MNs.

    “As expected, miR-338-3p, a miRNA known to be enriched in oligodendrocytes, was ϳ25-fold more abundant in oligodendrocytes than MNs (Zhao et al., 2010;de Faria et al., 2012) (Fig. 2D).”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites Zhao et al., 2010 source

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

    • cites de Faria et al., 2012 source

      The Results (section “Regulation of the miR-338 cluster during OPC differentiation,” Figure 3E) report that miR-338-3p is enriched in white matter, and the Discussion (“miR regulation in human OLs”) reports its expression in human oligodendrocytes. The source does not compare oligodendrocytes with motor neurons or report a 25-fold difference between them; the citing sentence points to its own Figure 2D for that measurement. (LLM, conf=0.87)

      Experimental — accuracy not yet measured
  • Premisecitation

    Affinity purification via antibodies against Ago2 serves to isolate active miRNAs from tissue lysates.

    “affinity purification via antibodies against Ago2 serves to isolate active miRNAs from tissue lysates (Chi et al., 2009).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Chi et al., 2009 source

      The ‘Ago RNA targets in the mouse brain’ section (page unavailable) reports immunoprecipitating Ago from mouse neocortex and recovering Ago-bound miRNAs. The Methods summary names two monoclonal antibodies, but our copy does not identify them as Ago2-specific or establish that every recovered miRNA was active; the method also includes UV crosslinking and further purification. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
  • Premisecitation

    Inhibiting miR-155 significantly delays disease progression in SOD1G93A mice.

    “Inhibiting miR-155 significantly delays disease progression in SOD1 G93A mice (Koval et al., 2013;Butovsky et al., 2015).”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites Koval et al., 2013 source
    • cites Butovsky et al., 2015 source
  • Premisecitation

    miR-155 is upregulated in the spinal cords of end-stage ALS model SOD1G93A mice and human autopsy.

    “Work from our lab and others found that miR-155, a glialenriched miRNA, is upregulated in the spinal cords of end-stage ALS model (SOD1 G93A ) mice and human autopsy (Koval et al., 2013;Butovsky et al., 2015).”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites Koval et al., 2013 source
    • cites Butovsky et al., 2015 source

      The Discussion (page unavailable) says miR-155 was the most upregulated miRNA in microglia and spinal cord tissue of SOD1G93A mice and ALS subjects. The Results report increased miR-155 in human ALS spinal-cord specimens, and the Methods identify those specimens as post-mortem. The passages do not establish that the reported miR-155 increase in mice was specifically measured at end-stage. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
  • Findingown data

    This depletion was maximized in the end-stage ALS mouse model spinal cord.

    “This depletion was maximized in the end-stage ALS mouse model spinal cord.”
    Quote in the paperNot a substantial claim
  • May combine two claims. 20
  • Classified as probably not a substantial claim. 19

  • May combine two claims.

    Findingown data

    miRNA expression profiles from IP of tagged and untagged Ago2 from each transgenic mouse clustered together in PCA, indicating global miRNA expression in these mice is comparable.

    “Furthermore, miRNA expression profiles from IP of tagged and untagged Ago2 from each transgenic mouse clustered together in PCA, indicating that the global miRNA expression in these mice is comparable (Fig. 1E).”
    Quote in the paperNot a substantial claim

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

    Findingnone given

    In ALS model rats, disease onset similarly occurs between 120 and 140 days.

    “In ALS model rats, disease onset similarly occurs between 120 and 140 d.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The number of ChAT+ MNs per unit area was significantly different, confirming ChAT+ MNs were lost in ALS mouse model spinal cords.

    “but the number of ChAT ϩ MNs per unit area were, confirming that ChAT ϩ MNs were lost in ALS mouse model spinal cords (Fig. 5C,D).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Using a heuristic approach, six miRNAs were enriched in either brainstem or spinal MNs compared with all other neurons (≥1.75-fold, adjusted p≤0.1) and abundantly expressed (CT≤30).

    “Using the heuristic approach, we identified six miRNAs that were enriched in either brainstem or spinal MNs compared with all other neurons (Ն1.75-fold, adjusted p Յ 0.1) and abundantly expressed (CT Յ30) with the arrays.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    CSF levels of neuronal miR-132 and 124 were not affected by SOD1 ASO treatment, demonstrating miR-218 CSF levels are selectively responsive to MN-function-sparing therapy.

    “However, the CSF levels of neuronal miR-132 and 124 were not affected by SOD1 ASO treatment, demonstrating that miR-218 CSF levels are selectively responsive to MNfunction-sparing therapy (Fig. 7E).”
    Quote in the paperSubstantial claim

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

    Findingcitation

    In the congenic ALS mouse model, disease onset typically occurs approximately 130 days, as marked by time to peak weight.

    “In our congenic ALS mouse model [B6.Cg-Tg(SOD1 G93A )1Gur/J] colony (Wooley et al., 2005), disease onset typically occurs ϳ130 d, as marked by time to peak weight.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Wooley et al., 2005 source

  • May combine two claims.

    Findingown data

    MN-enriched miR-218 is significantly depleted in ALS rat model spinal cord and more so than the pan-neuronal miRNAs miR-382 and miR-672.

    “MN-enriched miR-218 is significantly depleted in ALS rat model spinal cord and more so than the pan-neuronal miRNAs miR-382 and miR-672.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    miR-218 is a marker of motor neuron loss and/or injury in ALS rat model CSF.

    “Our data also demonstrate that miR-218 is a marker of MN loss and/or injury in ALS rat model CSF.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The MN-enriched miRNAs miR-218 and miR-138 are significantly depleted in ALS mouse SOD1 G93A model spinal cord, temporally, maximized at end stage.

    “The MN-enriched miRNAs miR-218 and miR-138 are significantly depleted in ALS mouse (SOD1 G93A ) model spinal cord.This depletion occurs temporally and is maximized at end stage.”
    Quote in the paperSubstantial claim

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

    Findingown data

    miR-380-5p could not be confirmed due to nonspecificity of TaqMan primers.

    “(miR-380-5p could not be confirmed due to nonspecificity of TaqMan primers: repeated amplification in no-template control).”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    miR-218 is indispensable for embryonic motor neurons.

    “and is indispensable for embryonic MNs (Amin et al., 2015).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Amin et al., 2015 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

    ALS is a fatal, adult-onset neurodegenerative disease in which motor neurons are selectively lost.

    “Amyotrophic lateral sclerosis (ALS) is a fatal, adult-onset neurodegenerative disease in which motor neurons (MNs) are selec-tively lost.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Pasinelli and Brown, 2006 source
    • cites Rothstein, 2009 source

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

  • May combine two claims.

    Interpretationown data

    miR-218 release is related to motor neuron dysfunction and death and occurs in both animal and human models of motor neuron loss and disease.

    “These data suggest that miR-218 release is related to MN dysfunction and death and occurs in both animal and human models of MN loss and disease.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Study design and conductown data

    At present, using available methods, miR-218 is below the limit of detection in some human samples, so a valid comparison between ALS and controls is not yet achievable.

    “at the present time using available methods, miR-218, although quantifiable in some samples, is below the limit of detection in others and thus a valid comparison between ALS and controls is not yet achievable.”
    Quote in the paperSubstantial claim

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

    Premisecitation

    A single miRNA can potentially regulate hundreds of mRNAs.

    “Because only partial complementarity is required for miRNA-mRNA interactions, a single miRNA can potentially regulate hundreds of mRNAs (Selbach et al., 2008).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Selbach et al., 2008 source

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

    Interpretationreasoning

    There may be a physiological level of miR-218 release outside the context of MN disease.

    “Although increased miR-218 CSF levels are associated with MN loss, because miR-218 can be measured readily in the CSF of nontransgenic rats, there may be a physiological level of miR-218 release outside of the context of MN disease.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    Grip strength assessors were blinded to treatment groups.

    “Assessors were blinded to treatment groups.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    All animal protocols were approved by the Institutional Animal Care and Use Committee of Washington University at St. Louis and adhered to NIH guidelines.

    “All animal protocols were approved by the Institutional Animal Care and Use Committee of Washington University at St. Louis and adhered to National Institutes of Health (NIH) guidelines.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    miR-218 retains its MN specificity in adults and may be essential in maintaining MN identity distinct from other spinal neurons in addition to interneurons.

    “Importantly, our work suggests miR-218 retains its MN specificity in adults and may be essential in maintaining MN identity distinct from other spinal neurons in addition to interneurons.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Both ALS rodent models and human patient autopsy spinal cord tissue indicate a robust depletion of MN-enriched miRNA expression, consistent with MN loss throughout disease progression.

    “Both ALS rodent models and human patient autopsy spinal cord tissue indicate a robust depletion of MN-enriched miRNA expression, consistent with MN loss throughout disease progression.”
    Quote in the paperSubstantial claim

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

    Study design and conductown data

    MN counts were performed by a blinded reviewer.

    “The counting was performed by a blinded reviewer.”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    miRNAs direct translational repression by partial binding to the 3' UTR of mRNAs after incorporation into an RNA-induced silencing complex containing Argonaute-2 (Ago2).

    “miRNAs direct translational repression by partial binding to the 3Ј UTR of mRNAs after first being incorporated into an RNA-induced silencing complex containing Argonaute-2 (Ago2) (Gregory et al., 2005;Chekulaeva and Filipowicz, 2009;Ha and Kim, 2014).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Gregory et al., 2005 source

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

    • cites Chekulaeva and Filipowicz, 2009 source
    • cites Ha and Kim, 2014 source

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

    Premisecitation

    miR-218 is enriched in motor neurons of other organisms.

    “recent work indicates that miR-218 is en-riched in MNs of other organisms (Kapsimali et al., 2007;Punnamoottil et al., 2015)”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Kapsimali et al., 2007 source

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

    • cites Punnamoottil et al., 2015 source

  • May combine two claims.

    Premisecitation

    Laser capture microdissection is lower throughput and overlapping processes from neighboring cells may contaminate the motor neuron sample.

    “Likewise, LCM is lower throughput and overlapping processes from neighboring cells may contaminate the MN sample (Kummari et al., 2015).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Kummari et al., 2015 source

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

  • Classified as probably not a substantial claim.

    Premisecitation

    Tissue-enriched miRNAs are increased in serum after injury to those tissues.

    “It has been demonstrated previously that tissue-enriched miRNAs are increased in serum after injury to those tissues (Corsten et al., 2010;Zhang et al., 2010).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites Corsten et al., 2010 source

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

    • cites Zhang et al., 2010 source

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

    Premisecitation

    miR-218 may be important for motor neuron differentiation.

    “may be important for MN differentiation (Thiebes et al., 2015)”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Thiebes et al., 2015 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).

    Study design and conductnone given

    All surgeries and downstream analyses were done by personnel blinded to treatment groups.

    “All surgeries and downstream analyses were done by personnel blinded to treatment groups.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    Increased miR-218 CSF levels are directly responsive to MN-function-sparing therapy and may be a relevant proxy for MN health during MN disease.

    “indicating that the increased miR-218 CSF levels are directly responsive to MNfunction-sparing therapy and may be a relevant proxy for MN health during MN disease (Fig. 7F).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Study design and conductreasoning

    Inclusion of a pan-neuronal line allowed the study to define which miRNAs distinguish motor neurons specifically from neurons in general, and comparison with microglia and astrocyte profiles provided controls and baseline data.

    “Inclusion of a pan-neuronal line allowed us to define which miRNAs distinguish MNs specifically from neurons in general, whereas comparison with profiles containing microglia and astrocytes provided key controls and baseline data”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisenone given

    Motor neurons have long processes that would be difficult to maintain during tissue dissociation for FACS, and dissociation is particularly challenging from adult animals.

    “MNs have long processes that would be difficult to maintain during tissue dissociation for FACS and dissociation is particularly challenging from adult animals”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    miR-218 loss from spinal cord tissue is likely subsequent to motor neuron loss in ALS and thus may not be a cause of motor neuron degeneration in ALS rodent models.

    “These data indicate that miR-218 loss from spinal cord tissue is likely subsequent to MN loss in ALS and thus may not be a cause of MN degeneration in ALS rodent models.”
    Quote in the paperSubstantial claim

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

    Interpretationreasoning

    The study's approach highlights a pipeline for hypothesis-driven development of specific miRNA markers of particular cell types and a means of predicting their functionality on mRNA expression in relevant cell types.

    “Our approach highlights a pipeline for hypothesis-driven development of specific miRNA markers of particular cell types and a means of predicting their functionality on mRNA expression in relevant cell types.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: There are a limited number of reliable biomarkers for ALS.
    • Rests on an assumption: None of the existing ALS biomarkers has been shown to change longitudinally within individual patients as disease progresses.

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

    Study design and conductnone given

    Image analyses were conducted by a blinded reviewer.

    “Image analyses were conducted by a blinded reviewer.”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    The MN-enrichment of miR-218 has been established previously in developing MNs and was found to be important for MN function.

    “The MN-enrichment of one of these miRNAs (miR-218) has been established previously in developing MNs and was found to be important for MN function (Amin et al., 2015;Thiebes et al., 2015).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Amin et al., 2015 source

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

    • cites Thiebes et al., 2015 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).

    Premisecitation

    Gliosis and glial dysfunction may precede neuronal loss in many neurodegenerative diseases.

    “there is a growing body of work showing that gliosis and glial dysfunction may precede neuronal loss in many neurodegenerative diseases (Hong et al., 2016; Clayton et al., 2017)”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Hong et al., 2016 source

      Not checked in the free run.

    • cites Clayton et al., 2017 source

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

35 of 35

  • The paper relies on this without support.

    Mice and rats containing mutated human SOD1 recapitulate ALS disease pathology faithfully.

    “Currently, mice and rats containing mutated human SOD1 (hSOD1) are the most widely used models and recapitulate the disease pathology faithfully.”

  • The paper relies on this without support.

    MN loss in ALS model rats is due to expression of mutant hSOD1.

    “Because MN loss in ALS model rats is due to expression of mutant hSOD1, we hypothesized that decreasing the levels of the toxic transgene SOD1 G93A would spare MNs and their function”

  • The paper relies on this without support.

    SMI-32 immunostaining specifically marks spinal MNs in the anterior horn of the lumbar spinal cord.

    “To mark MNs in the anterior horns of the lumbar spinal cord, we used SMI-32 (which is also known as neurofilament heavy).”

  • The paper relies on this without support.

    There are a limited number of reliable biomarkers for ALS.

    “Despite many efforts, there are a limited number of reliable biomarkers for ALS.”

  • The paper relies on this without support.

    None of the existing ALS biomarkers has been shown to change longitudinally within individual patients as disease progresses.

    “However, none of these markers has been shown to change longitudinally within individual patients as disease progresses.”
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?

  • MiR-155-5p and miR-146a-5p expression is likely driven by microglia, as both showed an over 10-fold enrichment in microglia compared to brainstem.

    “MiR-155-5p and miR-146a-5p expression is likely driven by microglia, as both showed an over 10-fold enrichment in microglia compared to brainstem38,39 (Fig. 5a, Supplementary Fig. 9a).”

    From A spatio-temporal brain miRNA expression atlas identifies sex-independent age-related microglial driven miR-155-5p increase (2025) · cites this paper as [38]

    The Significance Statement (page unavailable) describes miR-155 as glial-enriched, and the Discussion says the authors identified microglia-enriched miRNAs. The passages in our copy do not identify miR-146a-5p as microglia-enriched or report over 10-fold enrichment for either miRNA compared with brainstem. (LLM, conf=0.82)

    Experimental — accuracy not yet measured

Self-check

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

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

  • Butovsky citation year differs from reference list

    Minor

    The text cites Butovsky et al. as a 2015 publication, but the corresponding reference lists it as 2014.

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Authors
Hoye, M. L., Koval, E. D., Wegener, A. J., Hyman, T. S., Yang, C., O'Brien, D. R., Miller, R. L., Cole, T., Schoch, K. M., Shen, T., Kunikata, T., Richard, J.-P., Gutmann, D. H., Maragakis, N. J., Kordasiewicz, H. B., Dougherty, J. D., & Miller, T. M.
Affiliations
  • Washington University in St. Louis (US)
  • Ionis Pharmaceuticals (United States) (US)
  • Johns Hopkins University (US)
ORCID
Venue
The Journal of Neuroscience
Publisher
Society for Neuroscience
Published
2017-04-17
Volume
37
Issue
22
Pages
5574-5586
ISSN
  • 0270-6474 (ISSN-L)
  • 1529-2401
DOI
10.1523/jneurosci.3582-16.2017
PMC
PMC5452343
OpenAlex
W2605681290
Semantic Scholar
a48ff2726dcfae44a40552a1df02311b8d578d8f
Cited by
82
References
69
Citations by year
2026: 7 · 2025: 7 · 2024: 6 · 2023: 8 · 2022: 10 · 2021: 7 · 2020: 11 · 2019: 9 · 2018: 16 · 2017: 1
FWCI
7.00
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of Neuroscience (publishedVersion, cc-by-nc-sa)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
  • Digital Commons@Becker (Washington University School of Medicine) (submittedVersion)
Funders
  • Brain and Behavior Research Foundation
  • Foundation for Barnes-Jewish Hospital
  • Children's Discovery Institute
  • National Alliance for Research on Schizophrenia and Depression
  • Hope Center for Neurological Disorders
  • National Institutes of Health
  • Center for Cellular Imaging, Washington University
  • National Institute of Neurological Disorders and Stroke (U01NS084970, F31NS077781, R24NS086741, K08NS074194, F31NS092340, R01NS078398, R01NS102272)
Topics
  • Health Sciences › Medicine › Neurology › Amyotrophic Lateral Sclerosis Research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › MicroRNA in disease regulation
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Muscle Physiology and Disorders
Keywords
amyotrophic lateral sclerosis, motor neurons, miRNA expression profiling, miR-218, cerebrospinal fluid, spinal cord neurons, neurodegeneration
MeSH terms
Amyotrophic Lateral Sclerosis, Animals, Female, Male, Mice, Inbred C57BL, Motor Neurons, Sensitivity and Specificity, Reproducibility of Results, Biomarkers, Gene Expression Profiling, MicroRNAs, Mice, Transcriptome

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 2026-11-06.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-11-06.

Cited sources are checked by gpt-6-sol.

Statistics and arithmetic are recomputed, not judged.

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

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

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