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Cited by 226Neurogenesis and neuroplasticity mechanismsMultiple Sclerosis Research StudiesSphingolipid Metabolism and Signaling

Accumulation of 8,9-unsaturated sterols drives oligodendrocyte formation and remyelination

Open access
Hubler, Zita, Allimuthu, Dharmaraja, Bederman, Ilya, Elitt, Matthew S., Madhavan, Mayur, Allan, Kevin C., Shick, H. Elizabeth, Garrison, Eric +13 more·2018·Nature
DOI 10.1038/s41586-018-0360-3PMC PMC6423962
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Abstract

Regeneration of myelin is mediated by oligodendrocyte progenitor cells-an abundant stem cell population in the central nervous system (CNS) and the principal source of new myelinating oligodendrocytes. Loss of myelin-producing oligodendrocytes in the CNS underlies a number of neurological diseases, including multiple sclerosis and diverse genetic diseases 1-3. High-throughput chemical screening approaches have been used to identify small molecules that stimulate the formation of oligodendrocytes from oligodendrocyte progenitor cells and functionally enhance remyelination in vivo 4-10. Here we show that a wide range of these pro-myelinating small molecules function not through their canonical targets but by directly inhibiting CYP51, TM7SF2, or EBP, a narrow range of enzymes within the cholesterol biosynthesis pathway. Subsequent accumulation of the 8,9-unsaturated sterol substrates of these enzymes is a key mechanistic node that promotes oligodendrocyte formation, as 8,9-unsaturated sterols are effective when supplied to oligodendrocyte progenitor cells in purified form whereas analogous sterols that lack this structural feature have no effect. Collectively, our results define a unifying sterol-based mechanism of action for most known small-molecule enhancers of oligodendrocyte formation and highlight specific targets to propel the development of optimal remyelinating therapeutics.

AI snapshot

Written from the paper’s full text.

TL;DR

Many small molecules that promote oligodendrocyte formation and remyelination act by inhibiting CYP51, TM7SF2, or EBP, causing accumulation of 8,9-unsaturated sterols. These sterols are sufficient to enhance oligodendrocyte formation, and pathway inhibitors improve remyelination in mice and human spheroid models.

Key findings

  • Pro-myelinating small molecules from diverse structural classes act not through their canonical targets but by inhibiting a narrow window of cholesterol biosynthesis enzymes—CYP51, TM7SF2, or EBP—leading to accumulation of 8,9-unsaturated sterol intermediates.
  • Nine natural and unnatural 8,9-unsaturated sterols enhanced MBP+ oligodendrocyte formation when supplied directly to OPCs, whereas sterols lacking the 8,9-unsaturation, including cholesterol, were ineffective; 2,2-dimethyl-zymosterol was optimally potent among sterols evaluated.
  • In a screen of over 3,000 bioactive small molecules at 2 μM, all 10 top novel enhancers led to 8,9-unsaturated sterol accumulation; benztropine, clemastine, tamoxifen, and U50488 inhibited EBP and enhanced oligodendrocyte formation, while liothyronine and bexarotene showed minimal sterol effects.
  • Ifenprodil (TM7SF2) and tamoxifen (EBP) enhanced remyelination in a mouse lysolecithin focal demyelination model after 8 days, and miconazole, ifenprodil, and tamoxifen led to brain accumulation of 8,9-unsaturated sterols; miconazole and ifenprodil also enhanced human oligodendrocyte generation in cortical spheroids. In total, 27 small molecules were characterized as enhancing myelination and elevating 8,9-unsaturated sterol intermediates.

Methods

  • High-content imaging of mouse epiblast stem cell-derived OPCs (OPC-5 and OPC-1 lines) and mouse primary OPCs, using MBP or PLP1 immunostaining to quantify oligodendrocyte formation; treatments included small molecules, purified sterols, siRNA against CYP51, and CRISPR/Cas9 targeting of EBP.
  • GC/MS- and LC/MS-based sterol profiling with cholesterol-d7 as internal standard, plus enzymatic assays for CYP51 and EBP, qPCR, nuclear receptor reporter assays, electrospun microfiber ensheathment assays, LPC-induced focal demyelination in C57BL/6 mice, and human glioma cells and human pluripotent stem cell-derived cortical spheroids.
  • Statistics: mean ± standard deviation; unpaired two-tailed Student’s t-test for pairwise comparisons except Figure 4b, which used an unpaired two-tailed Mann-Whitney test; biological replicate numbers are reported per figure.

Limitations

  • The paper states that no statistical methods were used to predetermine sample size, and that the direct cellular targets of 8,9-unsaturated “meiosis-activating sterols” remain poorly understood; NHRs and SREBP2 were discounted, suggesting mechanisms beyond these.
  • The paper notes that in vitro OPC assays model only the initial differentiation event, although it then tests maturation and remyelination in vitro and in vivo.
  • Evident from the text: most mechanistic screening used EpiSC-derived OPC lines, with key results validated in primary OPCs and human spheroids; in vivo remyelination was tested in a single focal LPC mouse model with n = 4–6 per group, and no formal limitations section is included.

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

Integrity signals

Registry

Crossref · 10.1038/s41586-018-0360-3

Cites

40 of 40 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

10 of 26 cited sources checked · 3 supported · 0 flagged · accuracy not yet measured

  • Premisecitation

    Among the top 10 novel enhancers of oligodendrocyte formation, four molecules had previously been shown to inhibit TM7SF2 or EBP in CNS-derived cells.

    “Among the top 10 novel enhancers of oligodendrocyte formation, we noted four molecules had previously been shown to inhibit TM7SF2 or EBP in CNS-derived cells11,21”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [11] Korade, Z. et al. The effect of small molecules on sterol homeostasis: measuring 7-dehydrocholesterol in DHCR7-deficient Neuro2a cells and human fibroblasts. J. Med. Chem. 59, 1102–1115 (2016). source
    • cites [21] Canfrán-Duque, A. et al. Atypical antipsychotics alter cholesterol and fatty acid metabolism in vitro. J. Lipid Res. 54, 310–324 (2013). source
  • Findingcitation

    Twenty-seven small molecules have been characterized as both enhancing myelination and elevating 8,9-unsaturated sterol intermediate levels.

    “In all we have characterized twenty-seven small molecules as both enhancing myelination and elevating 8,9-unsaturated sterol intermediate levels11,21,23”
    Quote in the paperSubstantial claim1 of 3 sources checked
    • cites [11] Korade, Z. et al. The effect of small molecules on sterol homeostasis: measuring 7-dehydrocholesterol in DHCR7-deficient Neuro2a cells and human fibroblasts. J. Med. Chem. 59, 1102–1115 (2016). source

      The Results (Validation of HTS Hit Compounds; page unavailable) report that several small molecules increased zymostenol or zymosterol levels, supporting the sterol-intermediate part of the claim. The paper does not characterize twenty-seven such molecules or report that they enhance myelination; its experiments examine sterol profiles in Neuro2a cells and human fibroblasts. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [21] Canfrán-Duque, A. et al. Atypical antipsychotics alter cholesterol and fatty acid metabolism in vitro. J. Lipid Res. 54, 310–324 (2013). source
    • cites [23] Gylling, H. et al. Tamoxifen and toremifene lower serum cholesterol by inhibition of delta 8-cholesterol conversion to lathosterol in women with breast cancer. J. Clin. Oncol. 13, 2900–2905 (1995). source
  • Findingown data

    Following 8 days of treatment with ifenprodil or tamoxifen, remyelination was widespread throughout the lesion.

    “By contrast, following 8 days of treatment with ifenprodil or tamoxifen, remyelination was widespread throughout the lesion”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [9] Gonzalez, G. A. et al. Tamoxifen accelerates the repair of demyelinated lesions in the central nervous system. Sci. Rep. 6, 31599 (2016). source

      The Results (‘Tamoxifen promotes CNS remyelination’; page unavailable) report increased remyelinated axons at 14 days post-lesion with tamoxifen. The Methods (‘Induction of focal demyelination’; page unavailable) specify treatment from day 3 through day 14, not 8 days. Our copy does not establish widespread remyelination throughout the lesion or an effect of ifenprodil. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
  • Findingown data

    Across a panel of nine azole-containing molecules, the ability to inhibit CYP51 in vitro and in OPCs predicted enhanced formation of MBP+ oligodendrocytes from mouse epiblast stem cell-derived OPCs.

    “We noted that across a panel of nine azole-containing molecules, the ability to inhibit CYP51 in vitro and in OPCs predicted enhanced formation of myelin basic protein-positive (MBP+) oligodendrocytes from mouse epiblast stem cell-derived OPCs”
    Quote in the paperSubstantial claim
  • Findingown data

    Treatment of OPCs with 8,9-unsaturated sterols, including 14-dehydrozymostenol and zymostenol, enhanced the formation of MBP+ oligodendrocytes.

    “Treatment of OPCs with 8,9-unsaturated sterols, including 14-dehydrozymostenol (accumulates following TM7SF2 inhibition) and zymostenol (accumulates following EBP inhibition), enhanced the formation of MBP+ oligodendrocytes”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Exogenously supplied 8,9-unsaturated sterols enter OPCs and reach the relevant intracellular compartment to enhance oligodendrocyte formation.
    • Rests on an assumption: The enhanced oligodendrocyte formation observed with purified 8,9-unsaturated sterols is due to the supplied sterols themselves rather than to their metabolites.
  • May combine two claims. 28
  • Classified as probably not a substantial claim. 32
  • The quote only partly states this claim. 1

  • May combine two claims.

    Premisecitation

    The imidazole antifungal miconazole, which targets CYP51, penetrates the mouse blood brain barrier and enhances remyelination in mouse models of demyelination.

    “Previously we established that the imidazole antifungal miconazole, which targets CYP51, penetrates the mouse blood brain barrier and enhances remyelination in mouse models of demyelination 4”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [4] Najm, F. J. et al. Drug-based modulation of endogenous stem cells promotes functional remyelination in vivo. Nature 522, 216–220 (2015). source

      The paper’s main text (listed under Abstract in our copy; no page given) identifies miconazole as an imidazole antifungal, says it crosses the mouse blood–brain barrier, and reports enhanced remyelination in a mouse demyelination model. It describes miconazole as inhibiting cytochrome P450 but does not identify CYP51 specifically, so that part of the claim is not supported by our copy. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingown data

    Only molecules targeting CYP51 (ketoconazole), TM7SF2 (amorolfine), and EBP (TASIN-1) enhanced formation of MBP+ oligodendrocytes, whereas inhibitors of five other pathway enzymes were ineffective.

    “Only molecules targeting CYP51 (ketoconazole), TM7SF2 (amorolfine15), and EBP (TASIN-116) enhanced formation of MBP+ oligodendrocytes, whereas inhibitors of the five other pathway enzymes were ineffective”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [15] Jachak, G. R. et al. Silicon incorporated morpholine antifungals: design, synthesis, and biological evaluation. ACS Med. Chem. Lett. 6, 1111–1116 (2015). source
    • cites [16] Zhang, L. et al. Selective targeting of mutant adenomatous polyposis coli (APC) in colorectal cancer. Sci. Transl. Med. 8, 361ra140 (2016). source

  • May combine two claims.

    Premisecitation

    Previously identified structurally-diverse imidazole antifungal drugs stimulate generation of new mouse and human oligodendrocytes and enhance remyelination in mouse disease models.

    “Previously we identified structurally-diverse imidazole antifungal drugs as a robust class of small molecules that stimulate the generation of new mouse and human oligodendrocytes and enhance remyelination in mouse disease models 4.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [4] Najm, F. J. et al. Drug-based modulation of endogenous stem cells promotes functional remyelination in vivo. Nature 522, 216–220 (2015). source

      The paper reports that miconazole, an imidazole antifungal, promotes human oligodendrocyte generation in vitro (Abstract; Methods, “Generation and screening of human OPCs”) and remyelination in mouse models (Abstract; Extended Data Fig. 4). Its structure–activity analysis identifies an active imidazole class, but the reported human-cell and mouse-model tests concern miconazole, not multiple structurally diverse imidazole antifungal drugs. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

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

    Premisecitation

    Myelin is cholesterol-enriched, and past work has established that genetic or pharmacological treatments that inhibit early enzymes in cholesterol biosynthesis lead to hypomyelination in vivo.

    “Myelin is cholesterol-enriched, and past work has established that genetic or pharmacological treatments that inhibit early enzymes in cholesterol biosynthesis lead to hypomyelination in vivo 28–30”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites [28] Miron, V. E. et al. Statin therapy inhibits remyelination in the central nervous system. Am. J. Pathol. 174, 1880–1890 (2009). source

      The section on long-term simvastatin treatment says cholesterol is concentrated in myelin, reports reduced myelin load after treatment in animals, and describes abnormal myelination after genetic manipulation of oligodendrocytes in vivo. It does not establish that the genetic manipulation inhibited an early cholesterol-biosynthesis enzyme, and its own drug result concerns myelin maintenance rather than developmental hypomyelination. Page numbers were not available for these passages. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
    • cites [29] Klopfleisch, S. et al. Negative impact of statins on oligodendrocytes and myelin formation in vitro and in vivo. J. Neurosci. 28, 13609–13614 (2008). source

      The Introduction identifies statins as HMG-CoA reductase inhibitors, and ‘Intrinsic remyelination is delayed by statins in vivo’ reports impaired remyelination in simvastatin-treated mice after cuprizone-induced demyelination (page unavailable). The paper does not establish that genetic inhibition causes hypomyelination, and its finding concerns remyelination after injury rather than initial myelination. The retrieved text also does not state that myelin is cholesterol-enriched. (LLM, conf=0.87)

      Experimental — accuracy not yet measured
    • cites [30] Saher, G. et al. High cholesterol level is essential for myelin membrane growth. Nat. Neurosci. 8, 468–475 (2005). source

  • May combine two claims.

    Findingcitation

    Various small molecules caused accumulation of the expected 8,9-unsaturated sterol intermediates both in a human glioma cell line and in human pluripotent stem cell-derived cortical spheroids, confirming that these molecules similarly engage the sterol synthesis pathway in mouse and human cells and CNS tissue.

    “Various small molecules caused accumulation of the expected 8,9-unsaturated sterol intermediates both in a human glioma cell line and in human pluripotent stem cell-derived cortical spheroids27, confirming that these molecules similarly engage the sterol synthesis pathway in mouse and human cells and CNS tissue”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [27] Madhavan, M. et al. Induction of myelinating oligodendrocytes in human cortical spheroids. Nat. Methods https://doi.org/10.1038/s41592-018-0081-4 (2018). source

      Split into 2 atoms — 0 confirmed, 0 partially supported, 0 disputed, 0 misattributed, 2 unverifiable. Expand the claim to see per-atom verdicts.

      Experimental — accuracy not yet measured

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

    Study design and conductcitation

    GBM528 cells were validated as unique by STR profiling.

    “These cells were validated as unique by STR profiling (Miller et al., Nature, 2017 547 355–359).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Miller et al., Nature, 2017 547 355–359 source

  • May combine two claims.

    Findingown data

    Analysis of three additional structurally-related EZH2 inhibitors revealed that EPZ005687 uniquely inhibited EBP and enhanced oligodendrocyte formation.

    “However, analysis of three additional structurally-related EZH2 inhibitors revealed that EPZ005687 uniquely inhibited EBP and enhanced oligodendrocyte formation”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Ketoconazole (CYP51), amorolfine (TM7SF2), and TASIN-1 (EBP) all significantly enhanced MBP+ oligodendrocyte tracking along and wrapping of electrospun microfibers.

    “Ketoconazole (CYP51), amorolfine (TM7SF2), and TASIN-1 (EBP) all significantly enhanced MBP+ oligodendrocyte tracking along and wrapping of the microfibers”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    4-hydroxy-tamoxifen showed 100-fold enhanced cellular antiestrogen activity relative to tamoxifen, but both molecules have comparable potency for inhibition of EBP and comparable potency for enhancing oligodendrocyte formation.

    “Conversely, while 4-hydroxy-tamoxifen as expected showed 100-fold enhanced cellular antiestrogen activity relative to tamoxifen, both molecules have comparable potency for inhibition of EBP and comparable potency for enhancing oligodendrocyte formation”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In vehicle treated animals, remyelinating axons were sparsely distributed mainly at the periphery of lysolecithin lesions, and ultrastructural analyses revealed unmyelinated axons or axons with a single wrap of myelin.

    “In vehicle treated animals, profiles of sparsely distributed remyelinating axons characterized by thin myelin sheaths were detected mainly at the periphery of the lesion, while ultrastructural analyses revealed unmyelinated axons or axons with a single wrap of myelin”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    A total of nine natural and unnatural 8,9-unsaturated sterols enhanced oligodendrocyte formation from OPCs, with 2,2-Dimethyl-zymosterol optimally potent among the sterols evaluated to date.

    “A total of nine natural and unnatural 8,9-unsaturated sterols enhanced oligodendrocyte formation from OPCs, with 2,2-Dimethyl-zymosterol optimally potent among the sterols evaluated to date”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Co-treating OPCs with ketoconazole and MAS-412 provided no further benefit over ketoconazole alone, confirming a redundant mechanism.

    “Finally, co-treating OPCs with ketoconazole and MAS-412 provided no further benefit over ketoconazole alone, confirming a redundant mechanism”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    OPCs expressing Cas9 and guide RNA targeting EBP demonstrated reduced EBP transcript levels, robust accumulation of the expected intermediate zymostenol, and enhanced formation of oligodendrocytes under differentiation-permissive conditions.

    “OPCs expressing Cas9 and guide RNA targeting EBP demonstrated reduced EBP transcript levels, robust accumulation of the expected intermediate zymostenol, and enhanced formation of oligodendrocytes under differentiation-permissive conditions”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Cell-permeable siRNA reagents depleted CYP51 transcript levels in OPCs by 80%, led to significant accumulation of lanosterol, and enhanced formation of MBP+ oligodendrocytes.

    “Cell-permeable siRNA reagents depleted CYP51 transcript levels in OPCs by 80% 14, led to significant accumulation of lanosterol, and enhanced formation of MBP+ oligodendrocytes”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [14] Mir, F. & Le Breton, G. C. A novel nuclear signaling pathway for thromboxane A2 receptors in oligodendrocytes: evidence for signaling compartmentalization during differentiation. Mol. Cell. Biol. 28, 6329–6341 (2008). source

  • May combine two claims.

    Findingown data

    GCMS-based sterol profiling revealed that all 10 top hits led to 8,9-unsaturated sterol accumulation at the screening dose, while randomly selected library members had no effect on sterol levels or oligodendrocyte formation.

    “In fact, GCMS-based sterol profiling revealed that all 10 top hits led to 8,9-unsaturated sterol accumulation at the screening dose, while randomly selected library members had no effect on sterol levels or oligodendrocyte formation”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Therapeutic dosing regimens of miconazole, ifenprodil, and tamoxifen all led to significant accumulation of 8,9-unsaturated sterols within the mouse brain, indicating inhibition of CYP51, TM7SF2, and EBP.

    “Critically, we used GCMS-based sterol profiling of brain tissue from mice treated with miconazole, ifenprodil, and tamoxifen to demonstrate that these therapeutic dosing regimens all led to significant accumulation of 8,9-unsaturated sterols within the mouse brain, indicating inhibition of CYP51, TM7SF2, and EBP”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Among selective estrogen receptor modulators, toremifine and ospemifine are structurally near-identical and show comparable cellular antiestrogen activity, but only toremifine inhibited EBP in OPCs and only toremifine enhanced oligodendrocyte formation.

    “Likewise, among selective estrogen receptor modulators, toremifine and ospemifine are structurally near-identical and show comparable cellular antiestrogen activity. However, only toremifine inhibited EBP in OPCs, and only toremifine enhanced oligodendrocyte formation”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Among the six muscarinic receptor antagonists, only clemastine and benztropine inhibited EBP in OPCs, and only clemastine and benztropine enhanced oligodendrocyte formation.

    “Among these molecules, only clemastine and benztropine inhibited EBP in OPCs, and only clemastine and benztropine enhanced oligodendrocyte formation”
    Quote in the paperSubstantial claim

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

    Findingown data

    In a screen of over 3,000 bioactive small molecules and approved drugs at 2 μM, many confirmed hits had known targets that did not cluster into easily discernible categories.

    “we also identified many confirmed hits whose known targets did not cluster into easily discernible categories”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    At concentrations that promoted oligodendrocyte formation, benztropine, clemastine, tamoxifen, and U50488 induced accumulation of zymostenol and zymosterol and decreased basal sterol levels, indicative of inhibition of EBP in OPCs.

    “At concentrations that promoted oligodendrocyte formation, benztropine, clemastine, tamoxifen, and U50488 induced accumulation of zymostenol and zymosterol and decreased basal sterol levels, indicative of inhibition of EBP in OPCs”
    Quote in the paperSubstantial claim

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

    Findingown data

    In confocal cross-sections, green fluorescence appears to encircle several microfibers in OPCs seeded onto aligned microfibers and treated 14 days with ketoconazole (2.5 μM).

    “the cross-section, in which green fluorescence appears to encircle several microfibers, is shown in the bottom panel”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Findingown data

    A two-tailed Student's t-test yielded P = 0.0009 for the comparison of MBP+ oligodendrocyte percentage in OPC-5 infected with lentivirus expressing Cas9 and an independent guide RNA targeting EBP.

    “Two-tailed Student’s t-test, * P = 0.0009.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Findingnone given

    Azole molecules have varying degrees of potency for mammalian CYP51 inhibition.

    “Azole molecules with varying degrees of potency for mammalian CYP51 inhibition.”
    Quote in the paperSubstantial claim

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

    Findingown data

    Metabolites highlighted in green accumulate after treatments that enhance oligodendrocyte formation.

    “Green text highlights metabolites that accumulate after treatments that enhance oligodendrocyte formation (Fig. 2e, Extended Data Fig. 3c).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Fig. 2e

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

    • cites Extended Data Fig. 3c

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

  • May combine two claims.

    Interpretationown data

    Several lines of evidence support a central signaling role for 8,9-unsaturated sterols in the observed enhanced oligodendrocyte formation, including the ability of nine independent 8,9-unsaturated sterols to enhance the formation of oligodendrocytes when supplied to OPCs.

    “Mechanistically, several lines of evidence support a central signaling role for 8,9-unsaturated sterols in the observed enhanced oligodendrocyte formation, including the ability of nine independent 8,9-unsaturated sterols to enhance the formation of oligodendrocytes when supplied to OPCs”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Exogenously supplied 8,9-unsaturated sterols enter OPCs and reach the relevant intracellular compartment to enhance oligodendrocyte formation.
    • Rests on an assumption: The enhanced oligodendrocyte formation observed with purified 8,9-unsaturated sterols is due to the supplied sterols themselves rather than to their metabolites.

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

    Study design and conductown data

    The effects of azole molecules were confirmed using an orthogonal image quantitation approach, a second oligodendrocyte marker, and an LC/MS method for detecting cellular sterols.

    “Additionally, the effects of azole molecules were confirmed using an orthogonal image quantitation approach, a second oligodendrocyte marker, and an LC/MS method for detecting cellular sterols”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Additional experiments discounted a role for SREBP2, suggesting these sterols act by mechanisms beyond NHRs or SREBP2.

    “Additional experiments discounted a role for SREBP2, which transcriptionally regulates cholesterol homeostasis, suggesting these sterols act by mechanisms beyond NHRs or SREBP2”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Small molecule inhibitors of CYP51, TM7SF2, and EBP can engage their sterol pathway targets and significantly enhance remyelination in mice.

    “Collectively, these data show that small molecule inhibitors of CYP51, TM7SF2, and EBP can engage their sterol pathway targets and significantly enhance remyelination in mice”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Combining liothyronine with a range of sterol-modulating OPC differentiation-inducing treatments produced additive effects on oligodendrocyte formation, indicating these molecules likely function by mechanisms other than thyroid hormone receptor signaling.

    “Combining the thyroid hormone agonist liothyronine with a range of sterol-modulating OPC differentiation-inducing treatments produced additive effects on oligodendrocyte formation, indicating that these molecules likely function by mechanisms other than thyroid hormone receptor signaling to enhance oligodendrocyte generation”
    Quote in the paperSubstantial claim

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

    Premisecitation

    Past work suggested nuclear hormone receptors (NHRs) may play a role in the direct cellular targets of 8,9-unsaturated 'meiosis-activating sterols'.

    “While the direct cellular targets of 8,9-unsaturated ‘meiosis-activating sterols’ remain poorly understood, past work suggested nuclear hormone receptors (NHRs) may play a role19”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [19] Byskov, A. G., Andersen, C. Y. & Leonardsen, L. Role of meiosis activating sterols, MAS, in induced oocyte maturation. Mol. Cell. Endocrinol. 187, 189–196 (2002). source

  • May combine two claims.

    Interpretationown data

    Miconazole and ifenprodil significantly enhanced the generation of human oligodendrocytes in a three-dimensional human pluripotent stem cell-derived cortical spheroid model, indicating conservation of function across species.

    “Critically, miconazole and ifenprodil also significantly enhanced the generation human oligodendrocytes in a three-dimensional human pluripotent stem cell-derived cortical spheroid model, indicating conservation of function across species”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Study design and conductown data

    All effects of small molecules on oligodendrocyte formation and sterol levels were confirmed using a second, independently isolated batch of OPCs, and key results were validated using mouse primary OPCs.

    “Notably, we confirmed all effects of small molecules on oligodendrocyte formation and sterol levels using a second, independently isolated batch of OPCs, and key results were also validated using mouse primary OPCs”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    Panels c, d, h, k-o, and r are representative of two independent experiments.

    “panels c, d, h, k-o, and r are representative of two independent experiments”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    All treatments were assayed in two batches of epiblast stem cell-derived OPCs, and key results were confirmed using mouse primary OPCs.

    “To rigorously assess effects of small molecule and genetic treatments on OPCs, all treatments were assayed in two batches of epiblast stem cell-derived OPCs, and key results were confirmed using mouse primary OPCs”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Study design and conductown data

    All findings have been confirmed in a second independent derivation of OPCs (Fig. 1).

    “all findings have been confirmed in a second independent derivation of OPCs (Fig. 1).”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given

    All plates for the primary screen were processed and analyzed simultaneously to minimize variability.

    “All plates for the primary screen were processed and analyzed simultaneously to minimize variability.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    Panels a, c, e-h are representative of two independent experiments.

    “All bar graphs indicate mean +/− standard deviation, and panels a, c, e-h are representative of two independent experiments.”
    Quote in the paperNot a substantial claim

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

    Study design and conductown data

    Intermediates in blue have been confirmed in our GC/MS-based sterol profiling assay using authentic standards.

    “Intermediates in blue have been confirmed in our GC/MS-based sterol profiling assay using authentic standards.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    Selective estrogen receptor modulators and EZH2 inhibitors affect cellular EBP function and oligodendrocyte formation.

    “Effect of selective estrogen receptor modulators and EZH2 inhibitors on cellular EBP function and oligodendrocyte formation”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    No statistical methods were used to predetermine sample size.

    “No statistical methods were used to predetermine sample size”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    Two animals were excluded due to surgical complications.

    “(2 animals were excluded due to surgical complications)”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    Panels b, d, h, i, k, l, o and p are representative of two independent experiments.

    “panels b, d, h, i, k, l, o and p are representative of two independent experiments”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    T47D cells were used without further authentication beyond the observed estrogen-dependent cell proliferation.

    “T47D cells were provided by the Translational Research Shared Resource of the Case Comprehensive Cancer Center and used without further authentication beyond the observed estrogen-dependent cell proliferation.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    EpiSC-derived OPCs were sorted to purity by fluorescence activated cell sorting at passage five with CD140a-APC and NG2-AF488 antibodies.

    “To ensure uniformity throughout all in vitro screening experiments, EpiSC-derived OPCs were sorted to purity by fluorescence activated cell sorting at passage five with conjugated CD 140a-APC (eBioscience, 17–1401; 1:80) and NG2-AF488 (Millipore, AB5320A4; 1:100) antibodies”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given

    Animals were randomized into treatment groups prior to treatment.

    “At day 4, animals were randomized into treatment groups prior to treatment”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    Inhibition of sterol 14-reductase activity can lead to accumulation of the expected upstream intermediate (FF-MAS) or 14-dehydrozymostenol, also known as cholesta-8,14-dien-3-β-ol.

    “Consistent with past reports (ref. 21), inhibition of sterol 14-reductase activity can lead to accumulation of the expected upstream intermediate (FF-MAS) or 14-dehydrozymostenol, also known as cholesta-8,14-dien-3-β-ol.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites ref. 21

      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).

    Study design and conductnone given

    The experiment was done in a blinded manner: compounds were coded to ensure the researchers performing the experiments were unaware of the treatment being administered to each animal.

    “This experiment was done in a blinded manner: compounds were coded to ensure the researchers performing the experiments were unaware of the treatment being administered to each animal.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    Cultures were regularly tested and shown to be mycoplasma free.

    “Cultures were regularly tested and shown to be mycoplasma free”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). The quote only partly states this claim.

    Study design and conductown data

    The RT-qPCR results for EBP mRNA levels in OPCs infected with lentivirus expressing Cas9 and either of two guide RNAs targeting EBP were validated in an independent experiment.

    “One well per condition, with results validated in an independent experiment.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    Panels a-n, and t-v are representative of two independent experiments.

    “All bar graphs indicate mean +/− standard deviation, and panels a-n, and t-v are representative of two independent experiments.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    Panels b, c, e, i, j, k, n and q are representative of two independent experiments.

    “panels b, c, e, i, j, k, n and q are representative of two independent experiments”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    Panels a, d, g, i and k are representative of two independent experiments.

    “All bar graphs indicate mean +/− standard deviation, and panels a, d, g, i and k are representative of two independent experiments.”
    Quote in the paperNot a substantial claim

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

    Study design and conductown data

    CRISPR-based targeting of EBP with a second sgRNA was functionally validated using GC/MS-based quantitation of zymostenol levels.

    “Functional validation of CRISPR-based targeting of EBP with a second sgRNA using GC/MS-based quantitation of zymostenol levels.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    The identity and purity of small molecules were authenticated by LC/MS before use.

    “The identity and purity of small molecules were authenticated by LC/MS before use”
    Quote in the paperNot a substantial claim

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

    Study design and conductown data

    The sterol measurements in human cortical spheroids treated with the indicated small molecules are representative of two independent experiments.

    “n = 3 spheroids per condition; representative of two independent experiments.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    Panels a-d are representative of two independent experiments.

    “panels a-d are representative of two independent experiments”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    Multiple molecules now annotated by us as enhancing 8,9-unsaturated sterol intermediate levels can regenerate functional myelin in vivo, as evidenced by reversal of paralysis in mice with MS-like disease.

    “Importantly, we and others have independently shown that multiple molecules now annotated by us as enhancing 8,9-unsaturated sterol intermediate levels can regenerate functional myelin in vivo, as evidenced by reversal of paralysis in mice with MS-like disease 465”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [465]

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

57 of 57

  • The paper relies on this without support.

    Exogenously supplied 8,9-unsaturated sterols enter OPCs and reach the relevant intracellular compartment to enhance oligodendrocyte formation.

    “Treatment of OPCs with 8,9-unsaturated sterols, including 14-dehydrozymostenol (accumulates following TM7SF2 inhibition) and zymostenol (accumulates following EBP inhibition), enhanced the formation of MBP+ oligodendrocytes”

  • The paper relies on this without support.

    The enhanced oligodendrocyte formation observed with purified 8,9-unsaturated sterols is due to the supplied sterols themselves rather than to their metabolites.

    “as 8,9-unsaturated sterols are effective when supplied to OPCs in purified form”

  • The paper relies on this without support.

    Sterol 14-reductase activity in mouse is shared by two genes, TM7SF2 and LBR.

    “Sterol 14-reductase activity in mouse is shared by two genes, TM7SF2 and LBR.”
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.

  • Nuclear receptor assay target is listed as both PPARα and PPARδ

    Minor

    The nuclear receptor profiling method lists PPARα among the receptors tested, while its assay-design table lists PPARδ instead.

  • CYP51 siRNA treatment duration differs between caption and method

    Minor

    The figure caption says CYP51 mRNA was measured after 96 hours of siRNA treatment, but the qPCR method says siRNA was added 24 hours after plating and RNA was isolated after three days of treatment (72 hours).

2 of 2

Cites

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Authors
Hubler, Z., Allimuthu, D., Bederman, I., Elitt, M. S., Madhavan, M., Allan, K. C., Shick, H. E., Garrison, E., T. Karl, M., Factor, D. C., Nevin, Z. S., Sax, J. L., Thompson, M. A., Fedorov, Y., Jin, J., Wilson, W. K., Giera, M., Bracher, F., Miller, R. H., Tesar, P. J., & Adams, D. J.
Affiliations
  • Case Western Reserve University (US)
  • George Washington University (US)
  • Rice University (US)
  • Leiden University Medical Center (NL)
  • Ludwig-Maximilians-Universität München (DE)
Corresponding author
Adams, Drew J.
ORCID
Venue
Nature
Publisher
Springer Science and Business Media LLC
Published
2018-07-25
Volume
560
Issue
7718
Pages
372-376
ISSN
  • 0028-0836 (ISSN-L)
  • 1476-4687
DOI
10.1038/s41586-018-0360-3
PMC
PMC6423962
OpenAlex
W2884521265
Cited by
226
References
40
Citations by year
2026: 11 · 2025: 18 · 2024: 28 · 2023: 27 · 2022: 31 · 2021: 38 · 2020: 37 · 2019: 31 · 2018: 5
FWCI
9.04
Type
journal article
Language
en
Open access
Licence
www.springer.com/tdm (tdm)
Locations
  • Nature (publishedVersion)
  • PubMed (publishedVersion)
  • Europe PMC (PubMed Central) (submittedVersion)
  • PubMed Central (submittedVersion)
  • Data Archiving and Networked Services (DANS) (submittedVersion)
  • RePEc: Research Papers in Economics (submittedVersion)
  • Leiden Repository (Leiden University) (submittedVersion)
Funders
  • National Institutes of Health (1s10od016164-01)
  • National Institute of Mental Health (F30 MH116581)
  • National Cancer Institute (P30CA043703)
  • National Institute of General Medical Sciences (T32GM007250)
  • National Institute of Neurological Disorders and Stroke (R01 NS095280)
  • National Center for Advancing Translational Sciences (TL1 TR002549)
  • Eunice Kennedy Shriver National Institute of Child Health and Human Development (U54-HD090257)
Topics
  • Life Sciences › Neuroscience › Developmental Neuroscience › Neurogenesis and neuroplasticity mechanisms
  • Health Sciences › Medicine › Pathology and Forensic Medicine › Multiple Sclerosis Research Studies
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Sphingolipid Metabolism and Signaling
Keywords
remyelination, oligodendrocyte precursor cells, cholesterol biosynthesis, CYP51, evidence-based practice, small molecule enhancers, multiple sclerosis
MeSH terms
Remyelination, Animals, Cholesterol, Humans, Imidazoles, Male, Membrane Proteins, Mice, Inbred C57BL, Multiple Sclerosis, Myelin Sheath, Oligodendroglia, Spinal Cord, Steroid Isomerases, Sterols, Substrate Specificity, Spheroids, Cellular, Oxidoreductases Acting on CH-CH Group Donors, Mice, High-Throughput Screening Assays, HEK293 Cells, Sterol 14-Demethylase, 14-alpha Demethylase Inhibitors

About these checks

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

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-11.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-11.

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.

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