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Cited by 8Genetics and Neurodevelopmental DisordersCholesterol and Lipid MetabolismLysosomal Storage Disorders Research

An iPSC-derived astrocyte model of fragile X syndrome exhibits dysregulated cholesterol homeostasis

Open access
Talvio, Karo, Wagner, Victoria A., Minkeviciene, Rimante, Kirkwood, Jay S., Kulinich, Anna O., Umemori, Juzoh, Bhatia, Anil, Hur, Manhoi +3 more·2023·Communications Biology
DOI 10.1038/s42003-023-05147-9PMC PMC10387075
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Abstract

Cholesterol is an essential membrane structural component and steroid hormone precursor, and is involved in numerous signaling processes. Astrocytes regulate brain cholesterol homeostasis and they supply cholesterol to the needs of neurons. ATP-binding cassette transporter A1 (ABCA1) is the main cholesterol efflux transporter in astrocytes. Here we show dysregulated cholesterol homeostasis in astrocytes generated from human induced pluripotent stem cells (iPSCs) derived from males with fragile X syndrome (FXS), which is the most common cause of inherited intellectual disability. ABCA1 levels are reduced in FXS human and mouse astrocytes when compared with controls. Accumulation of cholesterol associates with increased desmosterol and polyunsaturated phospholipids in the lipidome of FXS mouse astrocytes. Abnormal astrocytic responses to cytokine exposure together with altered anti-inflammatory and cytokine profiles of human FXS astrocyte secretome suggest contribution of inflammatory factors to altered cholesterol homeostasis. Our results demonstrate changes of astrocytic lipid metabolism, which can critically regulate membrane properties and affect cholesterol transport in FXS astrocytes, providing target for therapy in FXS.

AI snapshot

Written from the paper’s full text.

TL;DR

This study reports dysregulated cholesterol homeostasis in fragile X syndrome (FXS) astrocytes, with reduced ABCA1 expression in human and mouse FMRP-deficient astrocytes and cholesterol accumulation in Fmr1 KO mouse astrocytes. The findings link altered astrocytic lipid metabolism and cytokine responses to FXS pathology and suggest ABCA1-related pathways as potential therapeutic targets.

Key findings

  • ABCA1 was reduced in human FXS iPSC-derived astrocytes at mRNA level (P = 0.011) and protein level (P = 0.013), and in FMR1 KO hESC-derived astrocytes (log2 −6.41-fold change, P = 0.0004, P adjust = 0.125); ABCA1 immunoreactivity was also reduced in Fmr1 KO mouse cortical astrocytes (P = 0.034).
  • Human FXS astrocyte-conditioned medium showed a higher cholesterol/cholesteryl ester ratio than controls (P = 0.029), while total cholesterol and cholesteryl esters did not differ significantly; Fmr1 KO mouse astrocytes had increased cholesterol (P = 0.038) and desmosterol (P = 0.036), with decreased desmosterol in conditioned medium (P = 0.030).
  • Fmr1 KO mouse astrocyte lipidomes showed increased polyunsaturated phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine species, decreased sphingomyelin, and a higher phosphatidylserine/phosphatidylinositol ratio.
  • Human FXS astrocyte secretomes had reduced anti-inflammatory cytokines IL-13 and IL-10 (both 54.3% of control) and reduced CCL1 (36.2% of control); FXS astrocytes showed abnormal CCL5 and GFAP responses to IL-1β and TNF-α (sixfold increase and twofold decrease, respectively, P < 0.0001).

Methods

  • Human forebrain astrocytes were differentiated from male FXS and control iPSC lines, plus an FMR1 KO hESC line and isogenic control; primary cortical astrocytes were cultured from P1 Fmr1 KO and wild-type mice.
  • Main analyses included immunocytochemistry, RT-qPCR, RNA-seq, cholesterol assay, cytokine antibody array, and untargeted LC-MS lipidomics of cells and astrocyte-conditioned medium.
  • Statistics used Student’s t test, ANOVA for cytokine-response gene expression, and PCA/SIMCA for lipidomic group comparisons; significance was set at P < 0.05.

Limitations

  • The paper does not state a limitations section.
  • Evident limitations include small biological sample sizes (typically 3–4 cell lines or 3 mice per group), pooled samples for the cytokine/chemokine array, and the RNA-seq ABCA1 change losing significance after adjustment (P adjust = 0.125).
  • Evident limitations also include differences between human serum-free and mouse serum-containing culture systems, and the absence of functional rescue or direct therapeutic testing of ABCA1 or lipid pathways in this study.

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

Integrity signals

Registry

Crossref · 10.1038/s42003-023-05147-9

Cites

87 of 87 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

43 of 58 cited sources checked · 17 supported · 2 flagged · accuracy not yet measured

  • Interpretationcitation

    Reduced IL-10 (54.3% of control) may be a consequence of low ABCA1 expression.

    “On the other hand, reduced IL-10 (54.3% of control) may be the consequence of the low ABCA1 expression36.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [36] Ma, L., Dong, F., Zaid, M., Kumar, A. & Zha, X. ABCA1 protein enhances Toll-like receptor 4 (TLR4)-stimulated interleukin-10 (IL-10) secretion through protein kinase A (PKA) activation. J. Biol. Chem. 287, 40502–40512 (2012). source
  • Premisecitation

    FMRP deficiency has an impact on lipid homeostasis.

    “Furthermore, FMRP deficiency has an impact on lipid homeostasis23 and n-3 polyunsaturated fatty acid (n-3 PUFA) supplementation has beneficial effects on the behavioral phenotype of the Fmr1 KO mice24,25.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [23] Leboucher, A. et al. The translational regulator FMRP controls lipid and glucose metabolism in mice and humans. Mol. Metab. 21, 22–35 (2019). source
  • Findingown data

    Desmosterol was substantially decreased in Fmr1 KO astrocyte-conditioned medium compared to wild type (P = 0.030).

    “Unexpectedly, a substantial decrease in desmosterol (367.3357 m/z; P = 0.030) (Fig. 2d), the last precursor of cholesterol in the Bloch unsaturated sterol side chain pathway30, was seen in the positive ion mode in Fmr1 KO ACM when compared to that in WT ACM.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [30] Allen, L. B., Genaro-Mattos, T. C., Porter, N. A., Mirnics, K. & Korade, Z. Desmosterolosis and desmosterol homeostasis in the developing mouse brain. J. Inherit. Metab. Dis. 42, 934–943 (2019). source
    • Rests on an assumption: Fmr1 KO mouse astrocytes are a valid model of FXS astrocyte cholesterol dysregulation.
  • Premisecitation

    Cholesterol homeostasis is dysregulated in many syndromes associated with autism spectrum disorder, including fragile X syndrome.

    “Cholesterol homeostasis is dysregulated in many syndromes associated with autism spectrum disorder (ASD), including fragile X syndrome (FXS)11.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [11] Esposito, C. M., Buoli, M., Ciappolino, V., Agostoni, C. & Brambilla, P. The role of cholesterol and fatty acids in the etiology and diagnosis of autism spectrum disorders. Int. J. Mol. Sci. 22, 3550 (2021). source

      The Introduction and Results §3.1 link disrupted cholesterol metabolism to ASD and identify Smith–Lemli–Opitz syndrome as an associated condition with impaired cholesterol biosynthesis. They do not establish the claim for many ASD-associated syndromes or for fragile X syndrome specifically; a search for fragile X/FXS found no supporting passage. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
  • Findingcitation

    hESC-derived FMR1 KO astrocytes expressed less ABCA1 than their isogenic controls (log2 −6.41-fold change, P = 0.0004, P adjust = 0.125) in RNA-Seq analysis.

    “Similarly, human embryonic stem cell (hESC)-derived FMR1 KO astrocytes expressed less ABCA1 than their isogenic controls (log2 −6.41-fold change, P = 0.0004, P adjust = 0.125) in RNA-Seq analysis26.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [26] Peteri, U. K. et al. Urokinase plasminogen activator mediates changes in human astrocytes modeling fragile X syndrome. Glia 69, 2947–2962 (2021). source

      Sections 2.3 and 3.2 describe RNA-seq comparing isogenic FMR1-knockout and control astrocytes. However, the passages retrieved by searching for ABCA1 and the reported values do not identify an ABCA1 result or verify its fold change and P values; the claim cannot be checked against our copy. (LLM, conf=0.72)

      Experimental — accuracy not yet measured
    • Rests on an assumption: Human iPSC-derived astrocytes are a valid model of human FXS astrocytes.
  • May combine two claims. 31
  • Classified as probably not a substantial claim. 30

  • May combine two claims.

    Premisecitation

    Individuals with FXS have reduced peripheral cholesterol and triacylglycerol, and an abnormal profile of fatty acids.

    “Individuals with FXS have reduced peripheral cholesterol and triacylglycerol (TAG), and an abnormal profile of fatty acids (FA)19–21, which questions the appropriateness of the lovastatin treatment.”
    Quote in the paperSubstantial claim3 of 3 sources checked
    • cites [19] Berry-Kravis, E. et al. Cholesterol levels in fragile X syndrome. Am. J. Med. Genet. A 167A, 379–384 (2015). source

      The Discussion (page not provided) reports lower total, HDL and LDL cholesterol in males with FXS; findings for females were inconclusive. It calls for a future study measuring fasting triglycerides and does not report reduced triacylglycerol or an abnormal fatty-acid profile. (LLM, conf=0.90)

      Experimental — accuracy not yet measured
    • cites [20] Lisik, M. Z., Gutmajster, E. & Sieroń, A. L. Low levels of HDL in fragile X syndrome patients. Lipids 51, 189–192 (2016). source

      The Results report significantly higher, not reduced, serum triacylglycerols in FXS patients compared with controls. Total cholesterol was numerically lower but did not differ statistically; the paper reports lower HDL-C and does not report a fatty-acid profile. (LLM, conf=0.90)

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured
    • cites [21] Abolghasemi, A. et al. Alteration of fatty acid profile in fragile X syndrome. Int. J. Mol. Sci. 23, 10815 (2022). source

      The Results (section 2.2) report lower plasma fatty-acid levels in FXS, and the Discussion reports previously observed lower plasma cholesterol. The paper does not report reduced triacylglycerol: its Methods (section 4.3) mention triglycerides only as part of an analytical standard. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

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

    Study design and conductcitation

    Features with a CV >30% across QC injections were removed.

    “Features with a CV >30% across QC injections were removed79,80.”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [79] Barupal, D. K. et al. Generation and quality control of lipidomics data for the alzheimer’s disease neuroimaging initiative cohort. Sci. Data 5, 180263 (2018). source

      The Technical validation passage says lipids with technical errors greater than 25% RSD in QC samples were removed, not those exceeding 30% CV. The supplied copy does not give a page number for this passage. (LLM, conf=0.88)

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured
    • cites [80] Dunn, W. B. et al. Procedures for large-scale metabolic profiling of serum and plasma using gas chromatography and liquid chromatography coupled to mass spectrometry. Nat. Protoc. 6, 1060–1083 (2011). source

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

    Premisecitation

    ABCA1 is the main cholesterol efflux transporter in astrocytes and transfers cellular cholesterol and phospholipids onto lipid-poor apolipoproteins.

    “The main cholesterol efflux transporter in astrocytes is ATP-binding cassette transporter A1 (ABCA1), which transfers cellular cholesterol and PLs onto lipid-poor apolipoproteins that shuttle cholesterol8–10.”
    Quote in the paperSubstantial claim3 of 3 sources checked
    • cites [8] Rawat, V. et al. ApoE4 alters ABCA1 membrane trafficking in astrocytes. J. Neurosci. 39, 9611–9622 (2019). source

      The section on ABCA1-mediated efflux says that a primary function of ABCA1 is to transport cholesterol and phospholipids to apolipoprotein acceptor particles. The Introduction describes cholesterol transfer into nascent ApoE, and the paper studies ABCA1-mediated efflux in astrocytes. Our copy does not establish the comparative claim that ABCA1 is the main cholesterol efflux transporter in astrocytes. (LLM, conf=0.83)

      Experimental — accuracy not yet measured
    • cites [9] Steck, T. L. & Lange, Y. Cell cholesterol homeostasis: mediation by active cholesterol. Trends Cell Biol. 20, 680–687 (2010). source

      The “Reverse cholesterol transport” section says ABCA1 appears to transfer cholesterol and phospholipids to lipid-poor apolipoprotein A-I. It does not identify ABCA1 as the main cholesterol efflux transporter in astrocytes or discuss astrocytes in this context; no page number is available in our copy. (LLM, conf=0.87)

      Experimental — accuracy not yet measured
    • cites [10] Chen, L., Zhao, Z. W., Zeng, P. H., Zhou, Y. J. & Yin, W. J. Molecular mechanisms for ABCA1-mediated cholesterol efflux. Cell Cycle 21, 1121–1139 (2022). source

      The Introduction states that ABCA1 transports free cholesterol and phospholipids to apoA-I, and §3 calls it the most important transporter of intracellular cholesterol efflux. The paper notes ABCA1 expression in the brain but does not establish that it is the main efflux transporter specifically in astrocytes. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

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

    Premisecitation

    Astrocytes produce excess cholesterol and secrete most of the produced cholesterol for use by neurons.

    “Astrocytes produce excess cholesterol and secrete most of the produced cholesterol for use by neurons7.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [7] Genaro-Mattos, T. C., Anderson, A., Allen, L. B., Korade, Z. & Mirnics, K. Cholesterol biosynthesis and uptake in developing neurons. ACS Chem. Neurosci. 10, 3671–3681 (2019). source

      The Results and Discussion section reports that, in cholesterol-free cultures, astrocytes secreted most of their newly synthesized cholesterol. The neuronal-uptake section shows that neurons can take up ApoE-bound cholesterol, but it does not show that they used the cholesterol secreted by those astrocytes; it also says astrocytic support is unlikely during embryonic development. Page numbers are unavailable in our copy. (LLM, conf=0.84)

      Experimental — accuracy not yet measured

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

    Premisecitation

    FXS is caused by the absence of FMRP, which is essential for normal synapse formation and plasticity.

    “FXS is caused by the absence of Fragile X messenger ribonucleoprotein (FMRP) which is essential for normal synapse formation and plasticity13.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [13] Richter, J. D. & Zhao, X. The molecular biology of FMRP: new insights into fragile X syndrome. Nat. Rev. Neurosci. 22, 209–222 (2021). source

      The Introduction (page unavailable) says that nearly all FXS cases involve FMR1 silencing and loss of FMRP, and that FMR1 inactivation impairs dendrite morphogenesis and circuit integration. A section on translation initiation also links FMRP-related processes to dendritic spine morphogenesis. Those non-abstract passages support the cause and synapse-formation aspects, but do not establish the claim about normal synaptic plasticity. (LLM, conf=0.76)

      Experimental — accuracy not yet measured

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

    Premisecitation

    Early brief treatment with lovastatin prevented associative learning deficits in Fmr1 KO rats.

    “Early brief treatment with lovastatin also prevented associative learning deficits in Fmr1 KO rats15, but cholesterol was not measured in relation to lovastatin treatment responses in the rodent FXS models.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [15] Asiminas, A. et al. Sustained correction of associative learning deficits after brief, early treatment in a rat model of Fragile X Syndrome. Sci. Transl. Med. 11, eaao0498 (2019). source

  • May combine two claims.

    Premisecitation

    Treatment with lovastatin dampens neuronal hyperexcitability in the brain of Fmr1 KO mice and rescues part of the mouse FXS phenotype.

    “Treatment with lovastatin, an inhibitor of 3-hydroxy-3-methylglutarylcoenzyme A (HMG-CoA) reductase which regulates the early irreversible and rate-limiting step in the biosynthesis of cholesterol, dampens neuronal hyperexcitability in the brain of FXS mouse model, Fmr1 KO mice, and rescues part of the mouse FXS phenotype14.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [14] Osterweil, E. K. et al. Lovastatin corrects excess protein synthesis and prevents epileptogenesis in a mouse model of fragile X syndrome. Neuron 77, 243–250 (2013). source

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

    Premisecitation

    Lovastatin did not enhance the treatment response of parent-implemented language intervention in a randomized, double-blind trial.

    “However, lovastatin did not enhance the treatment response of parent-implemented language intervention in a randomized, double-blind trial18, and it remained unclear whether lovastatin has less effect on speech-related mechanisms than mechanisms underlying other symptoms such as hyperactivity and stereotypy in FXS.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [18] Thurman, A. J. et al. Controlled trial of lovastatin combined with an open-label treatment of a parent-implemented language intervention in youth with fragile X syndrome. J. Neurodev. Disord. 12, 12 (2020). source

  • May combine two claims.

    Interpretationcitation

    In KO astrocytes, the membrane phospholipid profile favored highly unsaturated phosphatidylcholine species and contained less sphingomyelin, rendering the membrane structurally incompatible with cholesterol.

    “In KO astrocytes, the membrane PL profile favored the highly unsaturated PC species and contained less SM, rendering the membrane structurally incompatible with cholesterol44.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [44] Wassall, S. R. & Stillwell, W. Polyunsaturated fatty acid-cholesterol interactions: domain formation in membranes. Biochim. Biophys. Acta 1788, 24–32 (2009). source

      Section 5 (‘DHA has an aversion for cholesterol’; page unavailable) reports reduced cholesterol solubility in highly polyunsaturated PC membranes, and Section 6 describes cholesterol-poor domains formed by such lipids. The paper does not report a phospholipid profile for KO astrocytes or show that their membranes contain less sphingomyelin; its model-membrane findings do not establish that those astrocyte membranes are structurally incompatible with cholesterol. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Interpretationcitation

    Decrease in ABCA1 led to accumulation of astrocytic cholesterol, suggesting activation of a non-ABCA1-mediated efflux or increase in diffusion, perhaps due to a larger cholesterol gradient between medium lipoproteins and cellular plasma membrane.

    “Decrease in ABCA1 led to the accumulation of astrocytic cholesterol, suggesting activation of a non-ABCA1-mediated efflux or increase in diffusion, perhaps due to a larger cholesterol gradient between medium lipoproteins and cellular plasma membrane42.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [42] Juhl, A. D. & Wüstner, D. Pathways and mechanisms of cellular cholesterol efflux-insight from imaging. Front. Cell Dev. Biol. 10, 834408 (2022). source

      Split into 2 atoms — 0 confirmed, 2 partially supported, 0 disputed, 0 misattributed, 0 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).

    Premisecitation

    n-3 polyunsaturated fatty acid supplementation has beneficial effects on the behavioral phenotype of Fmr1 KO mice.

    “n-3 polyunsaturated fatty acid (n-3 PUFA) supplementation has beneficial effects on the behavioral phenotype of the Fmr1 KO mice24,25.”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [24] Pietropaolo, S. et al. Dietary supplementation of omega-3 fatty acids rescues fragile X phenotypes in Fmr1-KO mice. Psychoneuroendocrinology 49, 119–129 (2014). source
    • cites [25] Nolan, S. O., Hodges, S. L., Okoh, J. T., Binder, M. S. & Lugo, J. N. Prenatal high-fat diet rescues communication deficits in Fmr1 mutant mice in a sex-specific manner. Dev. Neurosci. 42, 94–104 (2020). source

      The Results (page unavailable) report that prenatal omega-3-enriched diet ameliorated reduced vocalization spectral purity in female Fmr1 knockout pups. The claim is broader than this finding: the same section says a control high-fat diet also ameliorated spectral purity, while omega-3 exposure decreased the number of calls in female knockouts. The male Results section reports no genotype-by-diet interaction for the vocalization measures tested. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Clinical studies have shown that peripheral cholesterol levels are reduced in FXS males and that an abnormal plasma lipid profile including decreased n-6 PUFAs and n-3 PUFAs occurs in FXS.

    “Clinical studies have shown that peripheral cholesterol levels are reduced in FXS males and the recent report of Abolghasemi et al. showed an abnormal plasma lipid profile including decreased n-6 PUFAs and n-3 PUFAs in FXS21.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [21] Abolghasemi, A. et al. Alteration of fatty acid profile in fragile X syndrome. Int. J. Mol. Sci. 23, 10815 (2022). source

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

    Study design and conductcitation

    An extension of the metabolomics standard initiative guidelines was used to assign annotation level confidence.

    “An extension of the metabolomics standard initiative guidelines was used to assign annotation level confidence82,83.”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [82] Sumner, L. W. et al. Proposed minimum reporting standards for chemical analysis Chemical Analysis Working Group (CAWG) Metabolomics Standards Initiative (MSI). Metabolomics 3, 211–221 (2007). source
    • cites [83] Schymanski, E. L. et al. Identifying small molecules via high resolution mass spectrometry: communicating confidence. Environ. Sci. Technol. 48, 2097–2098 (2014). source

  • May combine two claims.

    Interpretationcitation

    In addition to inhibiting ABCA1 function in cholesterol efflux, the biased membrane lipid composition potentially interferes with functions of several channels on the membrane directly or by altering bilayer stiffness and hydrophobic mismatch.

    “In addition to inhibiting ABCA1 function in cholesterol efflux, the biased membrane lipid composition potentially interferes with functions of several channels on the membrane directly or by altering bilayer stiffness and hydrophobic mismatch between transmembrane domains and the lipid bilayer69.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [69] Levitan, I., Fang, Y., Rosenhouse-Dantsker, A. & Romanenko, V. Cholesterol and ion channels. Subcell. Biochem. 51, 509–549 (2010). source

      Split into 2 atoms — 1 confirmed, 0 partially supported, 0 disputed, 0 misattributed, 1 unverifiable. Expand the claim to see per-atom verdicts.

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Protein kinase C activation promotes apoA-I-induced stabilization of ABCA1 protein, which is downregulated in KO astrocytes.

    “Protein kinase C activation promotes apoA-I-induced stabilization of ABCA1 protein57, downregulated in the KO astrocytes.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [57] Yamauchi, Y., Hayashi, M., Abe-Dohmae, S. & Yokoyama, S. Apolipoprotein A-I activates protein kinase Cα signaling to phosphorylate and stabilize ATP binding cassette transporter A1 for the high density lipoprotein assembly. J. Biol. Chem. 278, 47890–47897 (2003). source

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

    Premisecitation

    Several clinical trials have shown beneficial effects of n-3 PUFA supplementation in ASD and ADHD, but not all results are consistent.

    “Several clinical trials have shown beneficial effects of n-3 PUFA supplementation in ASD and ADHD, but not all results are consistent and the ratio of EPA and DHA in n-3 PUFA supplements and the overall dietary intake of FAs may affect the clinical outcome62–65.”
    Quote in the paperNot a substantial claim3 of 4 sources checked
    • cites [62] De Crescenzo, F. et al. Impact of polyunsaturated fatty acids on patient-important outcomes in children and adolescents with autism spectrum disorder: a systematic review. Health Qual. Life Outcomes 18, 28 (2020). source

      The Results report reduced anxiety in children and adolescents with ASD, while the Discussion describes mixed trial findings and low to very low certainty of evidence. The Introduction says EPA and DHA have been studied for ADHD, but our copy does not report beneficial ADHD trial results. (LLM, conf=0.87)

      Experimental — accuracy not yet measured
    • cites [63] Widenhorn-Müller, K. et al. Effect of supplementation with long-chain ω-3 polyunsaturated fatty acids on behavior and cognition in children with attention deficit/hyperactivity disorder (ADHD): a randomized placebo-controlled intervention trial. Prostaglandins Leukot. Essent. Fat. Acids 91, 49–60 (2014). source
    • cites [64] Bos, D. J. et al. Reduced symptoms of inattention after dietary omega-3 fatty acid supplementation in boys with and without attention deficit/hyperactivity disorder. Neuropsychopharmacology 40, 2298–2306 (2015). source

      The Introduction and Discussion report beneficial but inconsistent results for omega-3 supplementation in ADHD. The Discussion also suggests that the DHA-to-EPA ratio may help explain differing outcomes. The paper does not address ASD, and its dietary controls do not establish that overall fatty-acid intake affects clinical outcomes. Page numbers are unavailable in our copy. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [65] Czumaj, A. & Śledziński, T. Biological role of unsaturated fatty acid desaturases in health and disease. Nutrients 12, 356 (2020). source

      Section 3.3 links FADS gene variants to ADHD but does not report n-3 PUFA supplementation trials for ADHD or ASD. Its discussion of inconsistent supplementation results concerns schizophrenia, not either condition in the claim. Searches for ASD and ADHD supplementation trials found no passage addressing the claimed clinical outcomes. (LLM, conf=0.73)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Interpretationcitation

    The study demonstrated that altered cholesterol homeostasis in FXS astrocytes was associated with broader changes in lipidome and inflammatory factors, which are potentially directly modulated by statins.

    “Our study demonstrated that altered cholesterol homeostasis in FXS astrocytes was associated with broader changes in lipidome and inflammatory factors, which are potentially directly modulated by statins61.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [61] Fu, H. et al. The differential statin effect on cytokine production of monocytes or macrophages is mediated by differential geranylgeranylation-dependent Rac1 activation. Cell Death Dis. 10, 880 (2019). source

      The Discussion (page not provided) reports that statins affect inflammatory cytokine production in macrophages, supporting the possibility that statins modulate inflammatory factors. The Results (page not provided) do not examine lipidome changes, and the paper studies monocytes and macrophages rather than FXS astrocytes. The cited portion therefore supports only the inflammatory-factor aspect of the proposed modulation. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Interpretationcitation

    Reduced ABCA1 in FXS astrocytes may reflect defective LXR/RXR signaling potentially caused by dysregulated RXRA and RXRB by the absence of FMRP.

    “LXR/retinoic X receptor (RXR) ligands are known to stimulate ABCA1 expression and reduced ABCA1 in FXS astrocytes may reflect defective LXR/RXR signaling potentially caused by dysregulated RXRA and RXRB by the absence of FMRP48.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [48] Ascano, M. Jr. et al. FMRP targets distinct mRNA sequence elements to regulate protein expression. Nature 49, 382–6 (2012). source

      The Abstract and Results and Discussion (pages unavailable in our copy) state that FXS typically involves loss of FMRP and that FMRP affects the protein levels of its mRNA targets. They do not address RXRA, RXRB, LXR/RXR signaling, ABCA1, or FXS astrocytes; searches for those terms found no supporting passage. Thus the proposed pathway from FMRP loss to reduced astrocyte ABCA1 is not supported by this source. (LLM, conf=0.77)

      Experimental — accuracy not yet measured

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

    Study design and conductcitation

    Features belonging to the same metabolite were assigned a cluster-ID using RAMClust.

    “To aid in the identification of features that belong to the same metabolite, features were assigned a cluster-ID using RAMClust79,81.”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [79] Barupal, D. K. et al. Generation and quality control of lipidomics data for the alzheimer’s disease neuroimaging initiative cohort. Sci. Data 5, 180263 (2018). source

      The Methods > LC-MS data processing passages describe targeted ion extraction and selecting one adduct per lipid using quality-control variance. Searches for RAMClust and cluster-ID found no passage saying that features were assigned cluster IDs using RAMClust. (LLM, conf=0.74)

      Experimental — accuracy not yet measured
    • cites [81] Broeckling, C. D., Afsar, F. A., Neumann, S., Ben-Hur, A. & Prenni, J. E. RAMClust: a novel feature clustering method enables spectral-matching-based annotation for metabolomics data. Anal. Chem. 86, 6812–6817 (2014). source

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

    Premisecitation

    Potential detrimental effects of altering the optimal ratio of EPA to DHA on cognitive functions have been reported.

    “Potential detrimental effects of altering the optimal ratio of EPA to DHA on cognitive functions have been reported68.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [68] Liu, J. H. Acute EPA-induced learning and memory impairment in mice is prevented by DHA. Nat. Commun. 11, 5465 (2020). source

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

    Premisecitation

    High membrane phosphatidylserine activates many membrane-bound kinases, which have been studied as targets of therapy in FXS.

    “High membrane PS activates many membrane-bound kinases, which have been studied as targets of therapy in FXS55.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [55] D'Incal, C., Broos, J., Torfs, T., Kooy, R. F. & Vanden Berghe, W. Towards kinase inhibitor therapies for fragile X syndrome: tweaking twists in the autism spectrum kinase signaling network. Cells 11, 1325 (2022). source

      Section 4.15 (page unavailable) says protein kinase C signals in a lipid-dependent manner, including through phosphatidylserine binding, and discusses PKC inhibitors as potential FXS treatments. Section 4 more broadly reviews kinases involved in FXS. The paper does not establish that high membrane phosphatidylserine activates many membrane-bound kinases, or that this is why those kinases have been studied as therapeutic targets. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

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

    Premisecitation

    Statin treatment might improve behavioral outcomes in FXS children and lovastatin has beneficial effects on the phenotype of Fmr1 KO mice.

    “Statin treatment might improve behavioral outcomes in FXS children16,17 and lovastatin has beneficial effects on the phenotype of Fmr1 KO mice14.”
    Quote in the paperNot a substantial claim2 of 3 sources checked
    • cites [16] Çaku, A., Pellerin, D., Bouvier, P., Riou, E. & Corbin, F. Effect of lovastatin on behavior in children and adults with fragile X syndrome: an open-label study. Am. J. Med. Genet. A 164A, 2834–2842 (2014). source
    • cites [17] Champigny, C. et al. Combining lovastatin and minocycline for the treatment of fragile X syndrome: Results from the lovaMiX clinical trial. Front. Psychiatry 12, 762967 (2022). source
    • cites [14] Osterweil, E. K. et al. Lovastatin corrects excess protein synthesis and prevents epileptogenesis in a mouse model of fragile X syndrome. Neuron 77, 243–250 (2013). source

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

    Interpretationcitation

    Low levels of IL-13 contributed to the reduced ABCA1 expression in FXS astrocytes.

    “It is possible that low levels of IL-13 contributed to the reduced ABCA1 expression in FXS astrocytes35.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [35] Cardilo-Reis, L. et al. Interleukin-13 protects from atherosclerosis and modulates plaque composition by skewing the macrophage phenotype. EMBO Mol. Med. 4, 1072–1086 (2012). source

      The Results section reports higher ABCA1 expression in IL-13-stimulated macrophage foam cells and in lesions of IL-13-treated mice (Fig. 5C–F). That supports the proposed IL-13–ABCA1 link, but the paper does not establish that low IL-13 contributes to reduced ABCA1 expression in FXS astrocytes. (LLM, conf=0.84)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingown data

    CCL5 expression was sixfold higher (P < 0.0001) in FXS astrocytes activated with IL-1β (10 ng/ml) and 2-fold lower (P < 0.0001) after treatment with TNF-α (50 ng/ml) compared to controls.

    “However, the expression of CCL5 was sixfold higher (P < 0.0001) in FXS astrocytes activated with IL-1β (10 ng/ml) and 2-fold lower (P < 0.0001) after the treatment with TNF-α (50 ng/ml) compared to controls (Fig. 4c), indicating abnormalities in responses to pro-inflammatory cytokines40 in FXS astrocytes.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [40] Meeuwsen, S., Persoon-Deen, C., Bsibsi, M., Ravid, R. & van Noort, J. M. Cytokine, chemokine and growth factor gene profiling of cultured human astrocytes after exposure to proinflammatory stimuli. Glia 43, 243–253 (2003). source
    • Rests on an assumption: Human iPSC-derived astrocytes are a valid model of human FXS astrocytes.
    • Rests on an assumption: The control iPSC lines are appropriate controls for FXS iPSC lines.

  • May combine two claims.

    Findingown data

    Treatment with IL-1β resulted in sixfold increase (P < 0.0001) in GFAP expression, whereas TNF-α treatment decreased (twofold) GFAP expression in FXS astrocytes compared with controls.

    “Analogously, treatment with IL-1β resulted in sixfold increase (P < 0.0001) in GFAP expression, whereas TNF-α treatment decreased (twofold) GFAP expression in FXS astrocytes compared with controls (Fig. 4d).”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: Human iPSC-derived astrocytes are a valid model of human FXS astrocytes.
    • Rests on an assumption: The control iPSC lines are appropriate controls for FXS iPSC lines.

  • May combine two claims.

    Findingown data

    The phosphatidylcholine species profile was different between control and Fmr1 KO astrocytes, and polyunsaturated phosphatidylcholine species were elevated both in Fmr1 KO astrocyte-conditioned medium and in lysates from KO astrocytes compared to controls.

    “The PC species profile was different between the control and Fmr1 KO astrocytes, and polyunsaturated PC species were elevated both in Fmr1 KO ACM and in lysates from KO astrocytes compared to the controls.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Levels of monounsaturated phosphatidylcholine species were lower and there were slight elevations of polyunsaturated phosphatidylcholine species in Fmr1 KO astrocyte-conditioned medium compared with controls.

    “Levels of monounsaturated PC species (e.g., 32:1 and 34:1) were lower and there were slight elevations of polyunsaturated PC species in Fmr1 KO ACM compared with controls (Fig. 2f, Supplementary Figure 1).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Fmr1 KO mouse astrocytes are a valid model of FXS astrocyte cholesterol dysregulation.

  • May combine two claims.

    Findingown data

    KO astrocytes had increased levels of polyunsaturated phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine species and some species with the shortest chains, and contained less phosphatidylcholine and sphingomyelin harboring one or two monounsaturated acyl chains.

    “Comparison of the PL species composition of Fmr1 KO and WT astrocytes using mass spectrometry revealed that KO astrocytes had increased levels of polyunsaturated PC, phosphatidylethanolamine (PE), and phosphatidylserine (PS) species and some species with the shortest chains, and contained less PC and SM harboring one or two monounsaturated acyl chains (Fig. 3d–f).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Fmr1 KO mouse astrocytes are a valid model of FXS astrocyte cholesterol dysregulation.
    • Rests on an assumption: Lipidomic changes in cultured astrocytes reflect in vivo astrocyte lipid metabolism.

  • May combine two claims.

    Findingown data

    Cholesterol levels were not significantly different between FXS and control astrocyte-conditioned medium (P = 0.093), although there was a trend suggesting an increase.

    “Utilizing mass spectrometry (MS), we found that cholesterol levels were not significantly different between FXS and control ACM (P = 0.093), although there was a trend suggesting an increase (Fig. 1e).”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: Human iPSC-derived astrocytes are a valid model of human FXS astrocytes.
    • Rests on an assumption: The control iPSC lines are appropriate controls for FXS iPSC lines.

  • May combine two claims.

    Findingown data

    Despite increased phosphatidylserine in KO astrocytes, phosphatidylinositol levels remained constant.

    “Despite increased PS in KO astrocytes, the PI levels remained constant.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Fmr1 KO astrocytes had high phosphatidylserine content and low relative amounts of saturated and monounsaturated sphingomyelin species.

    “Our finding that Fmr1 KO astrocytes had high PS content and low relative amounts of saturated and monounsaturated SM species is especially interesting.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    In mouse Fmr1 KO astrocytes cultured with serum, the ABCA1 defect caused accumulation of cholesterol and desmosterol connected with changes in inflammatory properties and lipidome.

    “In human FXS astrocytes, the ABCA1 defect was associated with changes in cytokine secretion, while in mouse Fmr1 KO astrocytes cultured with serum, the ABCA1 defect caused accumulation of cholesterol and desmosterol connected with changes in inflammatory properties and lipidome (Fig. 5).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Increased desmosterol in KO astrocytes concomitant with reduced desmosterol abundance in astrocyte-conditioned medium suggested higher uptake of desmosterol by Fmr1 KO mouse astrocytes in serum-containing medium.

    “However, increased desmosterol in KO astrocytes (P = 0.036; Fig. 3b) concomitant with reduced desmosterol abundance in ACM suggested higher uptake of desmosterol by Fmr1 KO mouse astrocytes in the serum-containing medium.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Changes in desmosterol abundance in astrocyte-conditioned medium and astrocytes reflect astrocytic uptake of desmosterol from serum-containing medium.

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

    Interpretationcitation

    Desmosterol accumulation could be regarded as an attempt to activate LXR signaling and thereby increase sterol secretion from astrocytes.

    “Desmosterol accumulation could also be regarded as an attempt to activate LXR signaling and thereby increase sterol secretion from astrocytes32,33.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [32] Wang, N. et al. ATP-binding cassette transporters G1 and G4 mediate cholesterol and desmosterol efflux to HDL and regulate sterol accumulation in the brain. FASEB J. 22, 1073–1082 (2008). source
    • cites [33] Jansen, M. et al. What dictates the accumulation of desmosterol in the developing brain? FASEB J. 27, 865–870 (2013). source

  • May combine two claims.

    Interpretationreasoning

    Because total cholesterol in human and mouse astrocyte medium was not affected by FMRP deficiency, the data suggested accumulation of cholesterol in astrocytes.

    “Since total cholesterol in human and mouse astrocyte medium was not affected by FMRP deficiency, the data suggested accumulation of cholesterol in astrocytes.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Polyunsaturated species showed higher levels in KO astrocytes, likely affecting vesiculation, membrane fluidity, and lipid-derived mediator signaling.

    “Consistent with lipidomic findings in ACM, the polyunsaturated species showed higher levels in KO astrocytes, likely affecting vesiculation, membrane fluidity, and lipid-derived mediator signaling that is involved in a variety of biological processes in concert with cytokines.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The findings suggested accumulation of cholesterol and increased polyunsaturated membrane phospholipids in Fmr1 KO astrocytes.

    “Our findings suggested accumulation of cholesterol and increased polyunsaturated membrane PLs in Fmr1 KO astrocytes.”
    Quote in the paperSubstantial claim

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

    Premisecitation

    n-3 PUFA supplementation of Fmr1 KO mice from weaning to adulthood rescues many behavioral symptoms and neuroinflammatory parameters.

    “Furthermore, n-3 PUFA supplementation of Fmr1 KO mice from weaning to adulthood rescues many behavioral symptoms and neuroinflammatory parameters24.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [24] Pietropaolo, S. et al. Dietary supplementation of omega-3 fatty acids rescues fragile X phenotypes in Fmr1-KO mice. Psychoneuroendocrinology 49, 119–129 (2014). source

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Study design and conductnone given

    The absence of FMRP in FXS iPSC-derived astrocytes was confirmed in the authors' previous studies.

    “The absence of FMRP in FXS iPSC-derived astrocytes was confirmed in our previous studies.”
    Quote in the paperNot a substantial claim

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

    Study design and conductown data

    The cytokine/chemokine profile of FXS and control astrocytes was analyzed using pooled media from four iPSC-derived astrocyte lines.

    “For the analysis, media from four iPSC-derived astrocyte lines were pooled and array performed with undiluted medium according to the manufacturer’s instructions.”
    • Also stated in: § Statistics and reproducibility · plain
      “The chemokine and cytokine profile of FXS and control astrocytes was shown in pooled samples.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductown data

    RNA-seq data were generated using triplicate samples of isogenic hPSC (H1 control and FMR1 KO cell lines)-derived cells at 95 DIV.

    “RNA sequencing (RNA-seq) data were generated using triplicate samples of isogenic hPSC (H1 control and FMR1 KO cell lines)-derived cells at 95 DIV and NextSeq500 platform.”
    Quote in the paperSubstantial claim

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

    Premisecitation

    Increased availability of PUFA ligand for nuclear receptor LXR alters LXR-mediated gene expression.

    “In addition, increased availability of PUFA ligand for nuclear receptor LXR alters LXR-mediated gene expression47.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [47] Lengqvist, J. et al. Polyunsaturated fatty acids including docosahexaenoic and arachidonic acid bind to the retinoid X receptor alpha ligand-binding domain. Mol. Cell. Proteom. 3, 692–703 (2004). source

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

    Interpretationreasoning

    The abundant phosphatidylinositol precursor supported intracellular cholesterol transport in FXS astrocytes.

    “Apparently, the abundant PI precursor supported such transport in FXS astrocytes.”
    Quote in the paperNot a substantial claim

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

    Interpretationcitation

    Because desmosterol impairs membrane lipid packing compared to cholesterol, it could contribute to membrane fluidity control in cells with excess cholesterol.

    “Since desmosterol impairs membrane lipid packing compared to cholesterol, it could contribute to membrane fluidity control in cells with excess cholesterol51.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [51] Hąc-Wydro, K., Węder, K., Mach, M., Flasiński, M. & Wydro, P. The influence of cholesterol precursor – desmosterol – on artificial lipid membranes. Biochim. Biophys. Acta 1848, 1639–1645 (2015). source

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

    Study design and conductnone given

    A quality control sample, generated by pooling equal aliquots of each sample, was analyzed periodically to monitor system stability and performance in LC-MS.

    “A quality control sample, generated by pooling equal aliquots of each sample, was analyzed periodically to monitor system stability and performance.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    For ABCA1 analysis, at least 10 images were captured per culture (100-200 astrocytes) with 4-5 cultures per group.

    “For ABCA1 analysis, at least 10 images were captured per culture (100-200 astrocytes) with 4–5 cultures per group.”
    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 conductown data

    LC-MS analysis used cultures derived from three Fmr1 KO and three WT mice, and ABCA1 expression studies used 4-5 cultures of both genetic groups.

    “We used cultures derived from three Fmr1 KO and WT mice in LC-MS analysis and 4–5 cultures of both genetic groups for ABCA1 expression studies.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    FMRP deficiency in astrocytes affects the balance of membrane lipids, decreasing the proportion of phosphatidylcholine and sphingomyelin species with a low degree of unsaturation and increasing the proportion of polyunsaturated phospholipid species.

    “The present study demonstrated that FMRP-deficiency in astrocytes affects the balance of the membrane lipids, decreasing the proportion of PC and SM species with a low degree of unsaturation, which is structurally compatible with cholesterol, and increasing the proportion of polyunsaturated PL species, incompatible with cholesterol but having crucial signaling roles.”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given

    All cell cultures were tested regularly to be free from mycoplasma contamination using a Mycoplasma detection kit.

    “All cell cultures were tested regularly to be free from mycoplasma contamination using a Mycoplasma detection kit (MycoAlert, Lonza Group Ltd.)”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    Experiments using human astrocytes were performed with cells derived from at least three different FXS human iPSC lines representing biological patient-specific replicates and controls, except cytokine response studies which used three separate cultures of both HEL11.4 control and HEL69.5 cell lines.

    “The experiments utilizing human astrocytes were performed with cells derived from at least three different FXS human iPSC lines representing biological patient-specific replicates and controls, except studies of cytokine responses where three separate cultures of both HEL11.4 control and HEL69.5 cell lines were used.”
    Quote in the paperSubstantial claim

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

    Study design and conductown data

    RT-qPCR experiments were performed with three technical replicates.

    “Experiments were performed with three technical replicates.”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    Excess EPA may functionally inhibit ABCA1-mediated cholesterol efflux.

    “For example, excess EPA may functionally inhibit ABCA1-mediated cholesterol efflux66.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [66] Fournier, N. et al. Eicosapentaenoic acid membrane incorporation impairs ABCA1-dependent cholesterol efflux via a protein kinase A signaling pathway in primary human macrophages. Biochim. Biophys. Acta 1861, 331–341 (2016). source

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

    Study design and conductnone given

    All samples and standards in the cholesterol assay were run in duplicate.

    “All samples and standards were run in duplicate.”
    Quote in the paperNot a substantial claim
53 of 53

  • The paper relies on this without support.

    Human iPSC-derived astrocytes are a valid model of human FXS astrocytes.

    “Here we show dysregulated cholesterol homeostasis in astrocytes generated from human induced pluripotent stem cells (iPSCs) derived from males with fragile X syndrome (FXS)”

  • The paper relies on this without support.

    Fmr1 KO mouse astrocytes are a valid model of FXS astrocyte cholesterol dysregulation.

    “The FXS-specificity of the reduced ABCA1 expression in human forebrain FXS astrocyte model was confirmed by the observation of reduced ABCA1 immunoreactivity in cortical astrocytes of Fmr1 KO mice compared with wild type (WT) controls”

  • The paper relies on this without support.

    The control iPSC lines are appropriate controls for FXS iPSC lines.

    “human forebrain astrocytes generated from three FXS patient-specific male iPSC lines compared with controls”

  • The paper relies on this without support.

    The cytokine antibody array reliably quantifies cytokine and chemokine levels in astrocyte-conditioned medium.

    “Using a commercial cytokine antibody array, we found that several anti-inflammatory cytokines were reduced in ACM of human FXS astrocytes.”

  • The paper relies on this without support.

    Lipidomic changes in cultured astrocytes reflect in vivo astrocyte lipid metabolism.

    “Our results demonstrate changes of astrocytic lipid metabolism, which can critically regulate membrane properties and affect cholesterol transport in FXS astrocytes”

  • The paper relies on this without support.

    Changes in desmosterol abundance in astrocyte-conditioned medium and astrocytes reflect astrocytic uptake of desmosterol from serum-containing medium.

    “concomitant with reduced desmosterol abundance in ACM suggested higher uptake of desmosterol by Fmr1 KO mouse astrocytes in the serum-containing medium.”
6 of 6

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?

  • PC species were the most differently regulated membrane lipid class in the Fmr1 KO astrocyte lipidomics analysis.

    “PC species, which are the most common membrane phospholipids, were the most differently regulated membrane lipid class in the Fmr1 KO astrocyte lipidomics analysis (Talvio et al., 2023).”

    From Astrocytes in fragile X syndrome (2024) · cites this paper as Talvio et al., 2023

    The Results section on Fmr1 KO mouse astrocytes reports a significantly different PC species profile, but does not rank PC as the most differently regulated membrane lipid class in the cells. The Results section on astrocyte-conditioned medium says differences were most pronounced in PC species profiles; that comparison concerns the medium, not the astrocyte membrane lipidome. The Discussion describes PC as the most abundant phospholipid class in eukaryotic cells. (LLM, conf=0.87)

    Experimental — accuracy not yet measured

  • Cholesterol levels did not differ between human or mouse FMRP-deficient and control astrocyte conditioned medium, suggesting that astrocytes maintained extracellular cholesterol homeostasis to avoid cholesterol toxicity.

    “Here, cholesterol levels did not differ between human or mouse FMRP-deficient and control astrocyte conditioned medium, suggesting that astrocytes maintained extracellular cholesterol homeostasis to avoid cholesterol toxicity (Adachi et al., 2022; Talvio et al., 2023).”

    From Astrocytes in fragile X syndrome (2024) · cites this paper as Talvio et al., 2023

  • In Fmr1 KO mouse astrocytes, cholesterol and desmosterol accumulated in astrocytes.

    “In studies of Fmr1 KO mouse astrocytes, cholesterol, and desmosterol (the last precursor of cholesterol in the Bloch pathway) accumulated in astrocytes (Talvio et al., 2023).”

    From Astrocytes in fragile X syndrome (2024) · cites this paper as Talvio et al., 2023

  • Fmr1 KO astrocytes contained more of the most unsaturated PC species.

    “Fmr1 KO astrocytes contained more of the most unsaturated PC species (Talvio et al., 2023).”

    From Astrocytes in fragile X syndrome (2024) · cites this paper as Talvio et al., 2023

  • The capacity of Fmr1 KO astrocyte membranes to buffer cholesterol overload and prevent cholesterol toxicity was compromised due to a phospholipid profile containing highly unsaturated PC species and less sphingomyelin.

    “The capacity of the Fmr1 KO astrocyte membranes to buffer cholesterol overload and prevent cholesterol toxicity was compromised due to the phospholipid profile containing highly unsaturated PC species and less sphingomyelin (Talvio et al., 2023).”

    From Astrocytes in fragile X syndrome (2024) · cites this paper as Talvio et al., 2023

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.

  • Two different injection volumes are specified for the lipidomics analysis

    Major

    The LC-MS methods specify both 2 μl and 6 μl as the injection volume for data acquisition, without distinguishing separate analyses.

1 of 1

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Authors
Talvio, K., Wagner, V. A., Minkeviciene, R., Kirkwood, J. S., Kulinich, A. O., Umemori, J., Bhatia, A., Hur, M., Käkelä, R., Ethell, I. M., & Castrén, M. L.
Affiliations
  • University of Helsinki (FI)
  • University of California, Riverside (US)
  • University of Eastern Finland (FI)
Corresponding author
Castrén, Maija L.
ORCID
Venue
Communications Biology
Publisher
Springer Science and Business Media LLC
Published
2023-07-29
Volume
6
Issue
1
Pages
789-789
ISSN
2399-3642 (ISSN-L)
DOI
10.1038/s42003-023-05147-9
PMC
PMC10387075
OpenAlex
W4385380125
Semantic Scholar
4581b504806e4d890b3a4fbc5be345258556ef71
Cited by
8
References
87
Citations by year
2026: 2 · 2025: 3 · 2024: 3
FWCI
1.16
Type
journal article
Language
en
Open access
Licence
Locations
  • Communications Biology (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • eScholarship (California Digital Library) (submittedVersion, cc-by)
  • PubMed Central (submittedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Työväentutkimus Vuosikirja (submittedVersion)
Funders
  • FRAXA Research Foundation
  • California Institute of Regenerative Medicine (EDUC4-12752)
  • Academy of Finland
  • Lastentautien Tutkimussäätiö
  • Biocenter Finland
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Genetics and Neurodevelopmental Disorders
  • Health Sciences › Medicine › Surgery › Cholesterol and Lipid Metabolism
  • Health Sciences › Medicine › Physiology › Lysosomal Storage Disorders Research
Keywords
fragile X syndrome, astrocytes, cholesterol homeostasis, ABCA1, induced pluripotent stem cells, desmosterol, lipidome, polyunsaturated phospholipids, astrocyte secretome, neuroinflammation, cholesterol efflux, intellectual disability, iPSC-derived astrocytes
MeSH terms
Animals, Astrocytes, Fragile X Syndrome, Homeostasis, Humans, Male, Cytokines, Lipid Metabolism, Mice, Induced Pluripotent Stem Cells

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 2026-10-27.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-27.

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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