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Cited by 13Alzheimer's disease research and treatmentsSphingolipid Metabolism and SignalingNeuroinflammation and Neurodegeneration Mechanisms

Nuclear SphK2/S1P signaling is a key regulator of ApoE production and Aβ uptake in astrocytes

Open access
Komai, Masato, Noda, Yuka, Ikeda, Atsuya, Kaneshiro, Nanaka, Kamikubo, Yuji, Sakurai, Takashi, Uehara, Takashi, Takasugi, Nobumasa·2024·Journal of Lipid Research
DOI 10.1016/j.jlr.2024.100510PMC PMC10907773
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Abstract

The link between changes in astrocyte function and the pathological progression of Alzheimer's disease (AD) has attracted considerable attention. Interestingly, activated astrocytes in AD show abnormalities in their lipid content and metabolism. In particular, the expression of apolipoprotein E (ApoE), a lipid transporter, is decreased. Because ApoE has anti-inflammatory and amyloid β (Aβ)-metabolizing effects, the nuclear receptors, retinoid X receptor (RXR) and LXR, which are involved in ApoE expression, are considered promising therapeutic targets for AD. However, the therapeutic effects of agents targeting these receptors are limited or vary considerably among groups, indicating the involvement of an unknown pathological factor that modifies astrocyte and ApoE function. Here, we focused on the signaling lipid, sphingosine-1-phosphate (S1P), which is mainly produced by sphingosine kinase 2 (SphK2) in the brain. Using astrocyte models, we found that upregulation of SphK2/S1P signaling suppressed ApoE induction by both RXR and LXR agonists. We also found that SphK2 activation reduced RXR binding to the APOE promoter region in the nucleus, suggesting the nuclear function of SphK2/S1P. Intriguingly, suppression of SphK2 activity by RNA knockdown or specific inhibitors upregulated lipidated ApoE induction. Furthermore, the induced ApoE facilitates Aβ uptake in astrocytes. Together with our previous findings that SphK2 activity is upregulated in AD brain and promotes Aβ production in neurons, these results indicate that SphK2/S1P signaling is a promising multifunctional therapeutic target for AD that can modulate astrocyte function by stabilizing the effects of RXR and LXR agonists, and simultaneously regulate neuronal pathogenesis.

AI snapshot

Written from the paper’s full text.

TL;DR

In astrocyte models, SphK2/S1P signaling suppresses RXR/LXR agonist-induced ApoE production by reducing nuclear RXRα binding to the APOE promoter, while SphK2 inhibition restores lipidated ApoE and enhances Aβ uptake. The authors propose SphK2/S1P as a multifunctional AD target acting on both astrocytic ApoE metabolism and neuronal Aβ production.

Key findings

  • SphK2 overexpression in U87 astrocytoma cells suppressed ApoE induction by the LXR agonist T0901317 and RXR agonist bexarotene at protein and mRNA levels, while ABCA1 induction was unaffected; SphK2 activity was increased in U87-SphK2 cells.
  • SphK2 knockdown (~60% reduction) enhanced bexarotene-induced secreted ApoE and increased APOE mRNA 1.9-fold; the SphK2 inhibitor ABC294640 increased secreted ApoE 2.4-fold at 1 μM and 4.1-fold at 3 μM, intracellular ApoE 1.5-fold and 1.9-fold, and APOE mRNA 1.8-fold, without affecting ABCA1.
  • In human primary astrocytes, SphK2 overexpression decreased basal ApoE secretion to 0.26-fold, and ABC294640 increased ApoE secretion 2.3-fold; in hippocampal slices, SphK2 overexpression showed a non-significant trend toward reduced bexarotene-induced ApoE.
  • Nuclear SphK2/S1P reduced RXRα binding to the APOE promoter: bexarotene induced RXRα occupancy 1.35-fold in control cells but failed to do so in U87-SphK2 cells; cotreatment with bexarotene plus SphK2 inhibitor enhanced ApoE uptake 1.71-fold and Aβ uptake 2.13-fold in LRP4-expressing U87 cells.

Methods

  • Cell and tissue models: U87 MG control and SphK2-overexpressing cells (ApoE ε3/ε3), human primary fetal astrocytes (19 weeks gestation), and mouse hippocampal slice cultures from C57BL/6J pups (postnatal days 7–10); AAV vectors, siRNA knockdown, and SphK2 inhibitors (ABC294640, SLM6031434) were used.
  • Main assays: immunoblotting, RT-PCR/RT-qPCR, in vitro SphK2 activity, nuclear extraction, biotin-based ChIP for BLRP-hRXRα binding to the APOE promoter, native-PAGE, coimmunoprecipitation with anti-Aβ 4G8, and Aβ/ApoE uptake in LRP4-expressing U87 cells.
  • Statistics: experiments independently repeated at least three times; data shown as mean ± SEM; two-tailed Student’s t-test or one-way ANOVA with Dunnett’s or Bonferroni’s post hoc tests; significance set at P < 0.05.

Limitations

  • The paper states that it cannot fully exclude non-nuclear SphK2/S1P functions, that SphK2 was highly cytoplasmic in some models, that overall sphingolipid changes were not analyzed, and that the mechanism linking SphK2/S1P to ApoE regulation was not fully elucidated.
  • The paper states that ApoE4 was not studied because the work focused on ApoE3, that chloroquine was used in the uptake assay and does not fully reflect physiological Aβ degradation, and that hippocampal slice results were not statistically significant.
  • Evident from the text: most mechanistic data come from U87 cells and primary astrocytes/slice cultures rather than in vivo AD models; several findings rely on supplemental figures; many experiments use n = 3; and no direct S1P measurement or nuclear S1P-binding protein is identified.

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

Integrity signals

Registry

Crossref · 10.1016/j.jlr.2024.100510

Cites

74 of 74 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

81 of 107 cited sources checked · 46 supported · 1 flagged · accuracy not yet measured

  • Premisecitation

    Aβ aggregation in AD model mice was reduced upon SphK2 KO.

    “Aβ aggregation in AD model mice was reduced upon SphK2 KO (19)”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [19] Lei, 2019, Sphingosine kinase 2 potentiates amyloid deposition but protects against hippocampal volume loss and demyelination in a mouse model of Alzheimer’s disease, J. Neurosci. source
  • Premisecitation

    The therapeutic effects of RXR agonists, especially bexarotene, varied widely in follow-up studies.

    “the therapeutic effects of RXR agonists, especially bexarotene, varied widely in follow-up studies (13, 14, 15, 16)”
    Quote in the paperSubstantial claim1 of 3 sources checked
    • cites [13] Fitz, 2013, Comment on “ApoE-directed therapeutics rapidly clear β-amyloid and reverse deficits in AD mouse models.”, Science source
    • cites [14] Tesseur, 2013, Comment on “ApoE-directed therapeutics rapidly clear β-amyloid and reverse deficits in AD mouse models.”, Science source
    • cites [15] Price, 2013, Comment on “ApoE-directed therapeutics rapidly clear β-amyloid and reverse deficits in AD mouse models.”, Science source
  • Interpretationcitation

    Elevated levels of S1P are considered an important activator of astrocyte.

    “elevated levels of S1P are considered an important activator of astrocyte”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [18] Alam, 2023, S1P lyase deficiency in the brain promotes astrogliosis and NLRP3 inflammasome activation via purinergic signaling, Cells source
  • Premisecitation

    Expression of ApoE is downregulated in astrocytes in AD.

    “Interestingly, the expression of ApoE, which regulates lipid homeostasis, is downregulated in astrocytes in AD (9, 10)”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [9] Mathys, 2019, Single-cell transcriptomic analysis of Alzheimer’s disease, Nature source
    • cites [10] Grubman, 2019, A single-cell atlas of entorhinal cortex from individuals with Alzheimer’s disease reveals cell-type-specific gene expression regulation, Nat. Neurosci. source
  • Findingcitation

    Another SphK2 specific inhibitor, SLM6031434, also enhanced the induction of ApoE by bexarotene.

    “In concordance with our finding, another SphK2 specific inhibitor, SLM6031434 (28), also enhanced the induction of ApoE by bexarotene (+0.1 μM SLM: 1.1-fold, +0.3 μM SLM: 1.2-fold, supplemental Fig. S2A–C).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [28] Kharel, 2015, Sphingosine kinase 2 inhibition and blood sphingosine 1-phosphate levels, J. Pharmacol. Exp. Ther. source
    • Rests on an assumption: The human astrocytoma U87 cell line is a valid model for human astrocytes.
  • Classified as probably not a substantial claim. 73
  • May combine two claims. 35

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

    Premisecitation

    Cytoplasmic SphK2-derived S1P has cytoprotective effects, such as promoting cell survival.

    “Cytoplasmic SphK2-derived S1P has cytoprotective effects, such as promoting cell survival”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [53] Igarashi, 2003, Sphingosine kinase 2 is a nuclear protein and inhibits DNA synthesis, J. Biol. Chem. source

      The introductory passage labeled Abstract says sphingosine 1-phosphate regulates survival, and the Abstract reports that SPHK2 can localize in the cytosol. Neither passage establishes that S1P produced by cytoplasmic SPHK2 promotes cell survival; the paper’s reported SPHK2 finding concerns nuclear localization and inhibition of DNA synthesis. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
    • cites [67] Okada, 2005, Involvement of N-terminal-extended form of sphingosine kinase 2 in serum-dependent regulation of cell proliferation and apoptosis, J. Biol. Chem. source

      The Results (page unavailable) report that suppressing SPHK2 protected HEK293 cells from serum-deprivation- and drug-induced apoptosis—the opposite of attributing a survival-promoting effect to SPHK2. The paper observes cytosolic SPHK2-L under high-serum conditions but does not establish that cytoplasmic SPHK2-derived S1P is cytoprotective. (LLM, conf=0.85)

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured

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

    Premisecitation

    S1P is involved in Aβ production in neurons.

    “we have previously shown to be involved in Aβ production in neurons (17)”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [17] Takasugi, 2011, BACE1 activity is modulated by cell-associated sphingosine-1-phosphate, J. Neurosci. source

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

    Premisecitation

    U87-SphK2 cells were established based on the human astrocytoma U87 cell line, which has the most common ApoE genotype, ε3/ε3.

    “we established SphK2 stably overexpressing cells based on human astrocytoma U87 cell line (U87-SphK2 cells), which have the most common genotype of ApoE, ε3/ε3 (26).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [26] Riddell, 2008, Impact of apolipoprotein E (ApoE) polymorphism on brain ApoE levels, J. Neurosci. source

      The astrocytoma cell-culture methods identify U87 as a human astrocytoma cell line, and the Results say the authors genotyped human astrocytoma lines. Our copy does not state that U87 has the ε3/ε3 genotype or that it is the most common ApoE genotype. Establishing U87-SphK2 cells is the citing authors’ own work, not a finding attributed to this source. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
    • Rests on an assumption: The human astrocytoma U87 cell line is a valid model for human astrocytes.

  • Classified as probably not a substantial claim.

    Premisecitation

    S1P is associated with transcriptional regulation to alter cellular function.

    “S1P is associated with transcriptional regulation to alter cellular function (20)”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [20] Hait, 2020, Regulation of hypoxia-inducible factor functions in the nucleus by sphingosine-1-phosphate, FASEB J. source

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

    Premisecitation

    Neuroinflammation has attracted attention as the third hallmark of AD pathology.

    “neuroinflammation has attracted attention as the third hallmark of AD pathology (4)”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [4] Guzman-Martinez, 2019, Neuroinflammation as a common feature of neurodegenerative disorders, Front. Pharmacol. source

      In “Cross-Talks Between Glial Cells and Neurons and Origins of Alzheimer’s Disease” (page unavailable), the paper identifies tau tangles and β-amyloid as major AD hallmarks and calls neuroinflammation a key element alongside them. It supports neuroinflammation’s importance in AD, but does not designate it the third hallmark. (LLM, conf=0.83)

      Experimental — accuracy not yet measured

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

    Premisecitation

    Astrocytes are the most abundant cell type in the brain.

    “Astrocytes are the most abundant cell type in the brain”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [6] Barber, 2019, Lipid metabolism crosstalk in the brain: Glia and neurons, Front. Cell. Neurosci. source

      The Abstract says glial cells are the predominant cell type in the mammalian brain and identifies astrocytes as one of three main glial types. It does not say astrocytes themselves are the most abundant cell type. (LLM, conf=0.82)

      Experimental — accuracy not yet measured

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

    Premisecitation

    Administration of RXR or LXR agonists reduces Aβ aggregation and restores cognitive function in AD mouse model.

    “the administration of RXR or LXR agonists reduces Aβ aggregation and restores cognitive function in AD mouse model (11, 12)”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [11] Cramer, 2012, ApoE-directed therapeutics rapidly clear β-amyloid and reverse deficits in AD mouse models, Science source
    • cites [12] Jiang, 2008, ApoE promotes the proteolytic degradation of Aβ, Neuron source

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

    Premisecitation

    Astrocytes are responsible for maintaining lipid homeostasis.

    “are responsible for maintaining lipid homeostasis (6)”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [6] Barber, 2019, Lipid metabolism crosstalk in the brain: Glia and neurons, Front. Cell. Neurosci. source

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

    Premisecitation

    Reactive astrocytes induced by microglial activation induce cell toxicity via the secretion of saturated lipids.

    “reactive astrocytes induced by microglial activation induce cell toxicity via the secretion of saturated lipids (5, 8)”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [5] Liddelow, 2017, Neurotoxic reactive astrocytes are induced by activated microglia, Nature source

      The Concluding Remarks report that activated microglia induce neurotoxic A1 astrocytes, which secrete a toxin that kills neurons and oligodendrocytes. The paper does not identify that toxin as saturated lipids; Extended Data Figure 8 instead reports that heat inactivation or protease treatment removed the conditioned medium’s toxicity. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [8] Guttenplan, 2021, Neurotoxic reactive astrocytes induce cell death via saturated lipids, Nature source

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

    Premisecitation

    FTY720 is phosphorylated by SphK2 to produce the S1P analog, FTY720-P.

    “The sphingosine analog, FTY720, is phosphorylated by SphK2 to produce the S1P analog, FTY720-P.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [33] Billich, 2003, Phosphorylation of the immunomodulatory drug FTY720 by sphingosine kinases, J. Biol. Chem. source

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

    Premisecitation

    S1P mainly functions as an extracellular ligand for G protein-coupled receptors and as a transcriptional regulator in the nucleus.

    “S1P, a product of SphK2, mainly functions as an extracellular ligand for G protein-coupled receptors and as a transcriptional regulator in the nucleus (49).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [49] Spiegel, 2011, The outs and the ins of sphingosine-1-phosphate in immunity, Nat. Rev. Immunol. source

      The Abstract describes S1P as an agonist of G protein-coupled S1P receptors, and “Regulation of histone deacetylases by S1P” describes nuclear S1P produced by SPHK2 influencing transcription through HDAC1 and HDAC2. The paper also describes other intracellular actions, including regulation of TNFR signalling, so it does not establish that the two functions named are S1P’s main functions. Page numbers were not provided in our copy. (LLM, conf=0.82)

      Experimental — accuracy not yet measured

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

    Premisecitation

    S1P is involved in HDL function and neuroinflammation through regulation of gene expression in the nucleus and alteration in the functioning of various immune cells.

    “The signaling lipid S1P is involved in high-density lipoprotein (HDL) function (44) and neuroinflammation through the regulation of gene expression in the nucleus and alteration in the functioning of various immune cells (45).”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [44] Sato, 2007, Critical role of ABCA1 transporter in sphingosine 1-phosphate release from astrocytes, J. Neurochem. source
    • cites [45] Pérez-Jeldres, 2021, Targeting sphingosine-1-phosphate signaling in immune-mediated diseases: beyond multiple sclerosis, Drugs source

      The Intracellular Targets of S1P section (page unavailable) describes S1P’s effects on nuclear gene transcription, and the S1P Receptors section describes its effects on several immune-cell types. The cited paper does not clearly establish that these mechanisms specifically drive neuroinflammation. HDL function is attached to citation 44, not this source’s citation 45. (LLM, conf=0.82)

      Experimental — accuracy not yet measured

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

    Premisecitation

    Neuroinflammation is characterized by the activation of glial cells, such as microglia and astrocytes.

    “Neuroinflammation is characterized by the activation of glial cells, such as microglia and astrocytes”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [4] Guzman-Martinez, 2019, Neuroinflammation as a common feature of neurodegenerative disorders, Front. Pharmacol. source

  • May combine two claims.

    Findingcitation

    SphK2 overexpression resulted in the downregulation of endogenous RXRα, but not of LXRα/β.

    “Interestingly, as previously reported in all-trans retinoic acid-resistant colon cancer cells, SphK2 overexpression resulted in the downregulation of endogenous RXRα (39), but not of LXRα/β (supplemental Fig. S8A–C).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [39] Shi, 2017, Overexpression of SphK2 contributes to ATRA resistance in colon cancer through rapid degradation of cytoplasmic RXRα by K48/K63-linked polyubiquitination, Oncotarget source
    • Rests on an assumption: The human astrocytoma U87 cell line is a valid model for human astrocytes.

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

    Premisecitation

    Astrocyte activation leading to a chronic inflammatory response has been proposed as a mechanism linking Aβ accumulation and tau pathology.

    “Astrocyte activation leading to a chronic inflammatory response has been proposed as a mechanism linking Aβ accumulation and tau pathology (7)”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [7] Bellaver, 2023, Astrocyte reactivity influences amyloid-β effects on tau pathology in preclinical Alzheimer’s disease, Nat. Med. source

      The Discussion reports that astrocyte reactivity plays a role in the association between Aβ and early tau phosphorylation in preclinical Alzheimer’s disease. It does not establish that astrocyte activation leads to a chronic inflammatory response or identify that response as the linking mechanism. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing).

    Premisecitation

    Astrocyte activity is an executing factor.

    “astrocyte activity is an executing factor (5)”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [5] Liddelow, 2017, Neurotoxic reactive astrocytes are induced by activated microglia, Nature source

  • May combine two claims.

    Premisecitation

    SphK2 activity is upregulated in AD brain and promotes Aβ production in neurons.

    “Considering our previous findings that SphK2 activity is upregulated in AD brain and promotes Aβ production in neurons”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [17] Takasugi, 2011, BACE1 activity is modulated by cell-associated sphingosine-1-phosphate, J. Neurosci. source

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

    Premisecitation

    Astrocyte activation leading to a chronic inflammatory response is considered to be one of the important pathological processes in AD.

    “is considered to be one of the important pathological processes in AD”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [7] Bellaver, 2023, Astrocyte reactivity influences amyloid-β effects on tau pathology in preclinical Alzheimer’s disease, Nat. Med. source

      The Main section describes astrocyte reactivity as one of the earliest abnormalities in AD, and the Discussion links it to early tau pathology in preclinical AD (page numbers unavailable). The paper does not establish that astrocyte activation leads to a chronic inflammatory response; searches for chronic inflammation and neuroinflammation did not find that link. (LLM, conf=0.84)

      Experimental — accuracy not yet measured

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

    Premisecitation

    Neuroinflammation impairs brain network function through the abnormal production of inflammatory cytokines.

    “impairs brain network function through the abnormal production of inflammatory cytokines”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [4] Guzman-Martinez, 2019, Neuroinflammation as a common feature of neurodegenerative disorders, Front. Pharmacol. source

      The sections on neuroinflammation in AD and on tauopathies describe abnormal proinflammatory cytokine release, neuronal dysfunction, and reduced synaptic markers. They do not specifically establish impaired brain network function; the claim extends the reported cellular and synaptic effects to a network-level outcome. No page numbers were available in our copy. (LLM, conf=0.82)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    S1P accumulation induces neurodegeneration, tau pathology, and astrogliosis.

    “S1P accumulation induces neurodegeneration, tau pathology, and astrogliosis (18, 64)”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [18] Alam, 2023, S1P lyase deficiency in the brain promotes astrogliosis and NLRP3 inflammasome activation via purinergic signaling, Cells source
    • cites [64] Alam, 2020, Neurodegeneration caused by S1P-lyase deficiency involves calcium-dependent tau pathology and abnormal histone acetylation, Cells source

      The Introduction reports that increased S1P can induce neuronal death and that S1P accumulation following SGPL1 deficiency increases tau phosphorylation. Results §3.1 confirms increased tau phosphorylation in neurons of SGPL1-deficient mice. The paper reports altered histone acetylation in astrocytes, not astrogliosis; its Discussion describes microglial activation, which is distinct. (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

    These particles regulate various physiological functions, such as Aβ uptake via ApoE receptors and cell migration via S1PRs.

    “These particles regulate various physiological functions, such as Aβ uptake via ApoE receptors and cell migration via S1PRs (68, 69)”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [68] Malchinkhuu, 2003, Assessment of the role of sphingosine 1-phosphate and its receptors in high-density lipoprotein-induced stimulation of astroglial cell function, Biochem. J. source

      The Introduction reports that HDL stimulates astroglial-cell functions and that some of these effects may involve S1P receptors. The passages do not establish cell migration via those receptors or Aβ uptake via ApoE receptors; the latter may be supported by the other citation in the citing sentence. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
    • cites [69] Sato, 2007, HDL-like lipoproteins in cerebrospinal fluid affect neural cell activity through lipoprotein-associated sphingosine 1-phosphate, Biochem. Biophys. Res. Commun. source

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

    Premisecitation

    The nuclear function of SphK2 has been verified in other studies.

    “The nuclear function of SphK2 has been verified in other studies”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [53] Igarashi, 2003, Sphingosine kinase 2 is a nuclear protein and inhibits DNA synthesis, J. Biol. Chem. source
    • cites [54] Ding, 2007, Protein kinase D-mediated phosphorylation and nuclear export of sphingosine kinase 2, J. Biol. Chem. source

  • Classified as probably not a substantial claim.

    Premisecitation

    Amyloid pathology is exacerbated in 5×FAD mice lacking LRP4.

    “amyloid pathology is exacerbated in 5×FAD mice lacking LRP4”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [43] Zhang, 2020, A role of low-density lipoprotein receptor-related protein 4 (LRP4) in astrocytic Aβ clearance, J. Neurosci. source

  • Classified as probably not a substantial claim.

    Premisecitation

    LRP4 is the major ApoE receptor in astrocytes to promote Aβ clearance.

    “we focused on LRP4, the major ApoE receptor in astrocytes, to promote Aβ clearance (43).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [43] Zhang, 2020, A role of low-density lipoprotein receptor-related protein 4 (LRP4) in astrocytic Aβ clearance, J. Neurosci. source

      The Results section says LRP4 in astrocytes promotes Aβ uptake by serving as an ApoE receptor. The Discussion says LRP4 levels are 4–10 times higher than those of LDLR and LRP1 and proposes that it may play a key role in uptake; it does not establish that LRP4 is the major ApoE receptor. Page numbers are unavailable in the indexed passages. (LLM, conf=0.85)

      Experimental — accuracy not yet measured

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

    Premisecitation

    ApoE knockout activates the NF-κB pathway.

    “activation of the NF-κB pathway by ApoE knockout has also been reported (56)”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [56] Yang, 2018, Apolipoprotein E deficiency exacerbates spinal cord injury in mice: inflammatory response and oxidative stress mediated by NF-κB signaling pathway, Front. Cell. Neurosci. source

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

    Premisecitation

    Clinical trials have shown reduction in cortical amyloid levels with bexarotene.

    “clinical trials have shown reduction in cortical amyloid levels”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [71] Cummings, 2016, Double-blind, placebo-controlled, proof-of-concept trial of bexarotene in moderate Alzheimer’s disease, Alzheimers. Res. Ther. source

      The Discussion reports reduced cortical amyloid after bexarotene treatment in ApoE4 noncarriers. But it also states that the trial’s primary outcome was negative, with no difference from placebo when all patients were analyzed. The claim omits that important subgroup limitation. (LLM, conf=0.90)

      Experimental — accuracy not yet measured

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

    Premisecitation

    Bexarotene has been shown to be less effective against ApoE4 carriers than noncarriers.

    “bexarotene has been shown to be less effective against ApoE4 carriers than noncarriers (71)”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [71] Cummings, 2016, Double-blind, placebo-controlled, proof-of-concept trial of bexarotene in moderate Alzheimer’s disease, Alzheimers. Res. Ther. source

      The Results (Outcomes) report reduced brain amyloid in ApoE4 noncarriers after four weeks of bexarotene, but no significant reduction in carriers. The claim overstates this biomarker finding as general effectiveness: the Discussion says the trial’s primary outcome was negative, there were no significant cognitive changes, and the genotype subgroups were small. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

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

    Premisecitation

    Some groups have shown a reduction in SphK2 activity or S1P levels in AD.

    “some groups have shown a reduction in SphK2 activity or S1P levels in AD (65, 66)”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [65] Couttas, 2014, Loss of the neuroprotective factor sphingosine 1-phosphate early in Alzheimer’s disease pathogenesis, Acta Neuropathol. Commun. source
    • cites [66] He, 2010, Deregulation of sphingolipid metabolism in Alzheimer’s disease, Neurobiol. Aging source

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

    Premisecitation

    SphK2 expression and nuclear translocation of p65 were increased in regorafenib-resistant hepatocellular carcinoma-derived cells.

    “SphK2 expression and nuclear translocation of p65 were increased in these cells.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [55] Shi, 2020, Targeting SphK2 reverses acquired resistance of Regorafenib in hepatocellular carcinoma, Front. Oncol. source

      The Results section on SphK2 expression reports that SphK2 was significantly upregulated in regorafenib-resistant HCC cells compared with parental cells. The Results section on NF-κB and STAT3 reports increased phosphorylation of p65, but does not report increased nuclear translocation of p65. Phosphorylation does not establish the claimed change in cellular location. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

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

    Premisecitation

    SphK2 overexpression downregulates endogenous RXRα in all-trans retinoic acid-resistant colon cancer cells.

    “Interestingly, as previously reported in all-trans retinoic acid-resistant colon cancer cells, SphK2 overexpression resulted in the downregulation of endogenous RXRα (39), but not of LXRα/β (supplemental Fig. S8A–C).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [39] Shi, 2017, Overexpression of SphK2 contributes to ATRA resistance in colon cancer through rapid degradation of cytoplasmic RXRα by K48/K63-linked polyubiquitination, Oncotarget source

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

    Premisecitation

    S1P and FTY720-P function as inhibitors of histone deacetylases (HDAC).

    “Because S1P and FTY720-P have been reported to function as inhibitors of histone deacetylases (HDAC) (34, 35)”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [34] Hait, 2009, Regulation of histone acetylation in the nucleus by sphingosine-1-phosphate, Science source

      The paper reports that S1P inhibits HDAC activity, including the activity of HDAC1 and HDAC2 (passage [1], section labeled Abstract; page unavailable). Our copy does not report that FTY720-P inhibits HDACs; searches for FTY720-P and fingolimod phosphate found no supporting passage. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [35] Hait, 2014, Active, phosphorylated fingolimod inhibits histone deacetylases and facilitates fear extinction memory, Nat. Neurosci. source

  • May combine two claims.

    Premisecitation

    FTY720 localizes to the cytoplasm and nucleus after permeabilization of the plasma membrane, and is converted to its S1P analog, FTY720 phosphate (FTY720-P), upon phosphorylation by SphK2.

    “FTY720 localizes to the cytoplasm and nucleus after permeabilization of the plasma membrane, and is converted to its S1P analog, FTY720 phosphate (FTY720-P), upon phosphorylation by SphK2 (33).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [33] Billich, 2003, Phosphorylation of the immunomodulatory drug FTY720 by sphingosine kinases, J. Biol. Chem. source

      The Abstract and Conclusions state that SphK2 phosphorylates FTY720 to form FTY720 phosphate. Our copy does not establish the claimed localization to the cytoplasm and nucleus after plasma-membrane permeabilization; searches for permeabilization and intracellular localization found no supporting passage. Page numbers were not available in the indexed copy. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

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

    Premisecitation

    S1P has been reported to repress HDAC activity.

    “S1P has been reported to repress HDAC activity (34)”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [34] Hait, 2009, Regulation of histone acetylation in the nucleus by sphingosine-1-phosphate, Science source

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

    Premisecitation

    Promotion of ApoE4 lipidation suppresses ApoE4-driven pathology.

    “the promotion of ApoE4 lipidation suppresses ApoE4-driven pathology (42, 73)”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [42] Rawat, 2019, ApoE4 alters ABCA1 membrane trafficking in astrocytes, J. Neurosci. source
    • cites [73] Boehm-Cagan, 2016, Differential effects of apoE4 and activation of ABCA1 on brain and plasma lipoproteins, PLoS One source

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

    Premisecitation

    The effects of bexarotene have been variable in most follow-up studies.

    “the effects of bexarotene have been variable in most follow-up studies (13, 14, 15, 16)”
    Quote in the paperNot a substantial claim1 of 4 sources checked
    • cites [13] Fitz, 2013, Comment on “ApoE-directed therapeutics rapidly clear β-amyloid and reverse deficits in AD mouse models.”, Science source

      In the Abstract, this follow-up study reports mixed effects: bexarotene improved memory and reduced interstitial-fluid Aβ, but did not reduce amyloid deposition. It supports variability in this study’s results, but does not establish what happened in most follow-up studies. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [14] Tesseur, 2013, Comment on “ApoE-directed therapeutics rapidly clear β-amyloid and reverse deficits in AD mouse models.”, Science source
    • cites [15] Price, 2013, Comment on “ApoE-directed therapeutics rapidly clear β-amyloid and reverse deficits in AD mouse models.”, Science source
    • cites [16] Veeraraghavalu, 2013, Comment on “ApoE-directed therapeutics rapidly clear β-amyloid and reverse deficits in AD mouse models.”, Science source

  • May combine two claims.

    Interpretationcitation

    SphK2/S1P signaling is a potential target for multitargeted therapy, that is, Aβ production in neurons and Aβ metabolism via the regulation of ApoE expression in astrocytes.

    “SphK2/S1P signaling is a potential target for multitargeted therapy, that is, Aβ production in neurons and Aβ metabolism via the regulation of ApoE expression in astrocytes”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [17] Takasugi, 2011, BACE1 activity is modulated by cell-associated sphingosine-1-phosphate, J. Neurosci. source

      The Results and Discussion support SphK2/S1P as a potential Alzheimer’s therapeutic target through its effect on neuronal Aβ production. They do not address Aβ metabolism through regulation of ApoE expression in astrocytes, so the proposed multitargeted mechanism is not fully supported. Page numbers are unavailable in our copy. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

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

    Premisecitation

    SphK2 inhibitor has anti-inflammatory activity in several diseases.

    “and SphK2 inhibitor has anti-inflammatory activity in several diseases (40).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [40] Pyne, 2017, Sphingosine kinase 2 in autoimmune/inflammatory disease and the development of sphingosine kinase 2 inhibitors, Trends Pharmacol. Sci. source

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

    Premisecitation

    APOE expression is induced by complex formation with RXR and several transcription factors, not only on the promoter but also on the enhancer region.

    “APOE expression is induced by complex formation with RXR and several transcription factors, not only on the promoter but also on the enhancer region (59, 60)”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [59] Shih, 2000, Duplicated downstream enhancers control expression of the human apolipoprotein E gene in macrophages and adipose tissue, J. Biol. Chem. source

      The Results and Discussion (page unavailable) show that downstream enhancers are needed for apoE expression in macrophages and identify binding motifs for GR and C/EBP in those enhancers; they also describe a previously reported promoter element that binds AP-1. The paper does not identify RXR as part of an apoE-regulating complex or demonstrate that complex formation at the enhancers induces expression. It says the proposed transcription-factor roles require functional tests. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [60] Trusca, 2011, Macrophage-specific up-regulation of apolipoprotein E gene expression by STAT1 is achieved via long range genomic interactions, J. Biol. Chem. source

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

    Premisecitation

    Reducing serum concentration reduces the reactivity of astrocytes and better simulates in vivo conditions.

    “In addition, the serum concentration was gradually reduced and replaced with B-27-supplemented Neurobasal-A medium from DIV4 to DIV6 to reduce the reactivity of astrocytes and better simulate in vivo conditions (30).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [30] Foo, 2011, Development of a method for the purification and culture of rodent astrocytes, Neuron source

      The Results sections on gene expression and calcium imaging report that serum-free, purified astrocyte cultures more closely resemble astrocytes in vivo than traditional cultures. But the paper does not show that gradually reducing serum from DIV4 to DIV6 reduces astrocyte reactivity; its Results section on serum effects says many serum-induced gene-expression changes persisted after serum withdrawal. (LLM, conf=0.84)

      Experimental — accuracy not yet measured

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

    Premisecitation

    S1P3 receptor is involved in the induction of inflammation in reactive astrocytes.

    “this receptor has been reported to be involved in the induction of inflammation in reactive astrocytes (52)”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [52] Dusaban, 2017, Sphingosine 1-phosphate receptor 3 and RhoA signaling mediate inflammatory gene expression in astrocytes, J. Neuroinflammation. source

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

    Premisecitation

    Regulation of the expression of ABCA1 is simpler than that of APOE.

    “regulation of the expression of ABCA1 is simpler than that of APOE (61)”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [61] von Eckardstein, 2015, High density lipoproteins source

      The available indexed passages are the book overview and table of contents, which list a chapter on regulation of HDL genes (pp. 113–179) but do not provide its text. Searches for ABCA1, APOE, and their comparative regulation found no passage stating whether one is simpler to regulate than the other. (LLM, conf=0.70)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    SphK2 KO J20 mice showed hippocampal volume loss, myelin loss, and cognitive impairment.

    “SphK2 KO J20 mice showed hippocampal volume loss, myelin loss, and cognitive impairment (19)”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [19] Lei, 2019, Sphingosine kinase 2 potentiates amyloid deposition but protects against hippocampal volume loss and demyelination in a mouse model of Alzheimer’s disease, J. Neurosci. source

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

    Premisecitation

    ApoE4 promotes AD pathology through toxic gain of function.

    “ApoE4 promotes AD pathology through toxic gain of function (74)”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [74] Fernández-Calle, 2022, APOE in the bullseye of neurodegenerative diseases: impact of the APOE genotype in Alzheimer’s disease pathology and brain diseases, Mol. Neurodegener. source

      The sections on amyloid-β load and tau pathology report that ApoE4 increases amyloid plaque burden and exacerbates tau-mediated pathology. However, the paper’s explicit description of a “toxic gain of function” refers to tau aggregation, not ApoE4, so it does not establish that specific mechanism for ApoE4. (LLM, conf=0.85)

      Experimental — accuracy not yet measured

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

    Premisecitation

    Bexarotene has shown therapeutic effects in AD model mice.

    “Bexarotene has shown therapeutic effects in AD model mice (11)”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [11] Cramer, 2012, ApoE-directed therapeutics rapidly clear β-amyloid and reverse deficits in AD mouse models, Science source

  • Classified as probably not a substantial claim.

    Premisecitation

    HDAC inhibition has been reported to increase ApoE expression.

    “HDAC inhibition has been reported to increase ApoE expression (57)”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [57] Dresselhaus, 2018, Class I HDAC inhibition is a novel pathway for regulating astrocytic apoE secretion, PLoS One source

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

    Premisecitation

    Side effects, particularly triglyceride accumulation, have been observed with bexarotene.

    “side effects, particularly triglyceride accumulation, have been observed (71)”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [71] Cummings, 2016, Double-blind, placebo-controlled, proof-of-concept trial of bexarotene in moderate Alzheimer’s disease, Alzheimers. Res. Ther. source

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

    Premisecitation

    SphK2 KO J20 mice have decreased Aβ aggregation.

    “Lei et al. reported that SphK2 KO J20 mice have decreased Aβ aggregation (19)”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [19] Lei, 2019, Sphingosine kinase 2 potentiates amyloid deposition but protects against hippocampal volume loss and demyelination in a mouse model of Alzheimer’s disease, J. Neurosci. source

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

    Premisecitation

    FTY720 increases Aβ42 levels in AD model mice.

    “we have previously shown that FTY720 increases Aβ42 levels in AD model mice (51)”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [51] Takasugi, 2013, FTY720/fingolimod, a sphingosine analogue, reduces amyloid-β production in neurons, PLoS One source

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

    Premisecitation

    The products of ApoE gene variants differ by only one or two amino acid residues, and these small differences have profound effects on binding to Aβ, ApoE receptors, and lipids.

    “the products of these gene variants differ by only one or two amino acid residues, these small differences have profound effects on binding to Aβ, ApoE receptors, and lipids (46)”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [46] Lanfranco, 2020, ApoE lipidation as a therapeutic target in Alzheimer’s disease, Int. J. Mol. Sci. source

      The Introduction (section 2; page unavailable) says the three main apoE isoforms differ at residues 112 and 158 and that these substitutions confer different receptor- and lipid-binding abilities. Section 7 discusses ApoE’s role in Aβ clearance and degradation, but the passages found do not establish that the isoforms differ in binding to Aβ. (LLM, conf=0.85)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingown data

    Hippocampal slices overexpressing SphK2 showed a trend toward reduced ApoE induction by bexarotene compared to control slices, though no statistically significant differences were obtained.

    “Although no statistically significant differences were obtained, hippocampal slices overexpressing SphK2 showed a trend toward reduced ApoE induction by bexarotene compared to control slices (Fig. 5B, C).”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: The hippocampal slice culture system maintains neural circuits and has coexisting neurons and glial cells.

  • May combine two claims.

    Findingown data

    In U87-SphK2 cells, both LXR and RXR agonists induced ABCA1, but failed to induce APOE transcription.

    “However, in U87-SphK2 cells, both agonists induced ABCA1, but failed to induce APOE transcription (Fig. 1E–I).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The human astrocytoma U87 cell line is a valid model for human astrocytes.

  • May combine two claims.

    Findingown data

    Both LXR agonist T0901317 and RXR agonist bexarotene induced the expression of ApoE and ABCA1 in U87-control cells.

    “Both agonists induced the expression of ApoE and ABCA1, which mediate the lipidation and secretion of ApoE as a lipoprotein.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The human astrocytoma U87 cell line is a valid model for human astrocytes.

  • May combine two claims.

    Findingown data

    Both LXR and RXR agonists upregulated the transcription of APOE and ABCA1 in U87-control cells.

    “We also found that both agonists upregulated the transcription of APOE and ABCA1 in the U87-control cells.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The human astrocytoma U87 cell line is a valid model for human astrocytes.

  • May combine two claims.

    Findingown data

    Suppression of SphK2 activity by knockdown or inhibitors upregulated the induction of ApoE and facilitated Aβ uptake in astrocytes.

    “Intriguingly, the suppression of SphK2 activity by knockdown or inhibitors upregulated the induction of ApoE and facilitated Aβ uptake in astrocytes.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The human astrocytoma U87 cell line is a valid model for human astrocytes.

  • May combine two claims.

    Findingown data

    APOE mRNA expression was increased (+ABC: 1.8-fold) but not ABCA1 by ABC294640.

    “APOE mRNA expression was also increased (+ABC: 1.8-fold) but not ABCA1 by ABC294640 (Fig. 3D, E).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The human astrocytoma U87 cell line is a valid model for human astrocytes.

  • May combine two claims.

    Findingown data

    SphK2 knockdown enhanced APOE expression (1.9-fold), but not ABCA1 at the transcriptional level.

    “In addition, SphK2 knockdown enhanced APOE expression (1.9-fold), but not ABCA1 at the transcriptional level (Fig. 2E, F).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The human astrocytoma U87 cell line is a valid model for human astrocytes.

  • May combine two claims.

    Findingown data

    The NF-κB subunit p65 was upregulated in U87-SphK2 cells and its translocation to the nucleus was increased.

    “The subunit p65 of NF-κB was upregulated in U87-SphK2 cells and its translocation to the nucleus was increased (supplemental Fig. S5A).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The human astrocytoma U87 cell line is a valid model for human astrocytes.

  • May combine two claims.

    Findingown data

    In U87-SphK2 cells, both LXR and RXR agonists induced ABCA1 expression similar to U87-control cells, whereas ApoE induction was strongly suppressed.

    “Interestingly, in U87-SphK2 cells, both agonists induced ABCA1 expression, similar to that in U87-control cells, whereas ApoE induction was strongly suppressed (Fig. 1A–D).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The human astrocytoma U87 cell line is a valid model for human astrocytes.

  • May combine two claims.

    Findingown data

    SphK2 knockdown resulted in enhanced induction of secreted ApoE by bexarotene, and of secreted and intracellular ApoE by T0901317.

    “In contrast to SphK2 overexpression, SphK2 knockdown resulted in enhanced induction of secreted ApoE by bexarotene (Fig. 2A, C, D), and of secreted and intracellular ApoE by T0901317 (supplemental Fig. S1A–C).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The human astrocytoma U87 cell line is a valid model for human astrocytes.

  • May combine two claims.

    Findingown data

    IL6 and IL1β mRNA were upregulated in U87-SphK2 cells and SphK2 knockdown decreased their expression.

    “Moreover, interleukin 6 (IL6) and IL1β mRNA were upregulated in U87-SphK2 cells and SphK2 knockdown decreased their expression (supplemental Fig. S5B–E).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The human astrocytoma U87 cell line is a valid model for human astrocytes.

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

    Findingown data

    SphK2 expression level is increased in U87-SphK2 cells.

    “its expression level is increased in U87-SphK2 cells (Fig. 6A, B)”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    The imbalance between Aβ production and clearance is considered to be a cause of AD.

    “the imbalance between Aβ production and clearance is considered to be a cause of AD (1)”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [1] Hardy, 2002, The amyloid hypothesis of Alzheimer’s disease: progress and problems on the road to therapeutics, Science source

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

    Premisecitation

    Aβ accumulates in the brain prior to tau protein.

    “Aβ accumulates in the brain prior to tau protein”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [1] Hardy, 2002, The amyloid hypothesis of Alzheimer’s disease: progress and problems on the road to therapeutics, Science source

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

    Premisecitation

    SphK2 is predominant in the brain.

    “Of the two SphK isozymes, SphK2 is predominant in the brain (47).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [47] Lei, 2017, Contextual fear conditioning is enhanced in mice lacking functional sphingosine kinase 2, Behav. Brain Res. source

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

    Study design and conductnone given

    All animal experiments were performed in strict accordance with institutional guidelines and the protocol was approved by the Animal Experimentation Committee of Okayama University (approval number OKU-2022622).

    “All animal experiments were performed in strict accordance with the institutional guidelines for animal experiments of Okayama University. The protocol was approved by the Animal Experimentation Committee of Okayama University (approval number: OKU-2022622).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S2 (checkable).

    Interpretationreasoning

    There is an unknown pathological factor causing variation in RXR agonist therapeutic effects.

    “suggesting that there is an unknown pathological factor causing this variation”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    ApoJ is one of the main apolipoproteins in the brain and its expression is reported to be upregulated in AD.

    “ApoJ is one of the main apolipoproteins in the brain and its expression is reported to be upregulated in AD (48).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [48] Moezzi, 2020, Apolipoprotein J in Alzheimer’s disease: shedding light on its role with cell signaling pathway perspective and possible therapeutic approaches, ACS Chem. Neurosci. source

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

    Study design and conductnone given

    The 2−ΔΔCt relative quantification method, using ACTB for normalization, was used to estimate target gene expression.

    “The 2−ΔΔCt relative quantification method, using ACTB for normalization, was used to estimate target gene expression.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    The sequence specificity of the ChIP primers was confirmed by GGGenome.

    “The sequence specificity was confirmed by GGGenome”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    The densitometric intensity of each blot was measured and normalized by the loading control indicated in the figure legends and used for statistical analysis.

    “The densitometric intensity of each blot was measured and normalized by the loading control indicated in the figure legends and used for statistical analysis.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    SphK2/S1P signaling regulates ApoE expression in astrocytes and this mechanism is conserved across species.

    “These results suggest that SphK2/S1P signaling regulates ApoE expression in astrocytes and that this mechanism is conserved across species.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The hippocampal slice culture system maintains neural circuits and has coexisting neurons and glial cells.

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

    Study design and conductown data

    All qPCRs amplified single products, as confirmed by the melting curve and electrophoresis.

    “All qPCRs amplified single products, as confirmed by the melting curve and electrophoresis using 2% agarose gels with 0.5 ng/ml ethidium bromide.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Interpretationreasoning

    Increasing ApoE levels may have therapeutic effects on AD.

    “increasing ApoE levels may have therapeutic effects on AD.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S4 (based).

    Premisecitation

    SphK2 promotes Aβ production in neurons.

    “promotes Aβ production in neurons”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    ApoE has anti-inflammatory and Aβ-metabolizing effects.

    “Lipidated ApoE has anti-inflammatory effects and promotes Aβ metabolism (12)”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [12] Jiang, 2008, ApoE promotes the proteolytic degradation of Aβ, Neuron source

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

    Study design and conductnone given

    All constructed plasmids and inserted sequences were verified by Sanger sequencing.

    “All constructed plasmids and inserted sequences were verified by Sanger sequencing”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    FTY720-P modulates S1P receptors (S1PRs) to reduce their cell surface expression and signaling.

    “In contrast to S1P, FTY720-P modulates S1P receptors (S1PRs) to reduce their cell surface expression and signaling (50).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [50] Matloubian, 2004, Lymphocyte egress from thymus and peripheral lymphoid organs is dependent on S1P receptor 1, Nature source

  • Probably not a substantial claim: may not pass S4 (based).

    Premisecitation

    SphK2 activity is upregulated in AD brain.

    “our previous findings that SphK2 activity is upregulated in AD brain”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    The same amount of protein was applied to each sample.

    “The same amount of protein was applied to each sample.”
    Quote in the paperSubstantial claim

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

    Interpretationown data

    This study cannot fully deny the non-nuclear function of SphK2/S1P signaling.

    “this study cannot fully deny the non-nuclear function of SphK2/S1P signaling”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    The overall sphingolipid changes were not analyzed.

    “the overall sphingolipid changes were not analyzed”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    S1P has low cell permeability and is highly unstable.

    “Considering that S1P has low cell permeability (31) and is highly unstable (32)”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [31] Kihara, 2014, Sphingosine 1-phosphate is a key metabolite linking sphingolipids to glycerophospholipids, Biochim. Biophys. Acta Mol. Cell Biol. Lipids source
    • cites [32] Bradley, 2011, The in vitro metabolism of sphingosine-1-phosphate: identification; inhibition and pharmacological implications, Eur. J. Pharmacol. source

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

    Study design and conductnone given

    Chloroquine was used to evaluate uptake, which does not fully reflect physiological conditions because Aβ is inherently degraded.

    “chloroquine was used to evaluate uptake, which does not fully reflect physiological conditions because Aβ is inherently degraded”
    Quote in the paperNot a substantial claim

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

    Interpretationreasoning

    This study leaves open the possibility that S1P acts in the cytoplasm.

    “this study leaves open the possibility that S1P acts in the cytoplasm”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: S1P shuttles between the cytoplasm and the nucleus.

  • May combine two claims.

    Premisecitation

    Lipidated ApoE has anti-inflammatory effects and promotes Aβ metabolism.

    “Lipidated ApoE has anti-inflammatory effects and promotes Aβ metabolism (12)”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [12] Jiang, 2008, ApoE promotes the proteolytic degradation of Aβ, Neuron source

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

    Study design and conductnone given

    All experiments were independently repeated at least three times.

    “All experiments were independently repeated at least three times.”
    Quote in the paperNot a substantial claim

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

    Study design and conductown data

    The pull-down efficiency of the modified ChIP analysis was confirmed.

    “The pull-down efficiency of this analysis was confirmed (supplemental Fig. S6).”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: The human astrocytoma U87 cell line is a valid model for human astrocytes.

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

    Premisecitation

    RXRα expression is downregulated in AD model mice.

    “RXRα expression is downregulated in AD model mice (58)”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [58] Dheer, 2018, Bexarotene modulates retinoid-X-receptor expression and is protective against neurotoxic endoplasmic reticulum stress response and apoptotic pathway activation, Mol. Neurobiol. source

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

    Premisecitation

    The number of ε4 genes affects the risk and accelerates the onset of AD.

    “the number of ε4 genes affects the risk and accelerates the onset of AD (72)”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [72] Corder, 1993, Gene dose of apolipoprotein E type 4 allele and the risk of Alzheimer’s disease in late onset families, Science source

  • Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing).

    Premisecitation

    The effects of S1P on nuclear receptors are gradually being revealed.

    “the effects of S1P on nuclear receptors, such as peroxisome proliferator-activated receptor γ (63), are gradually being revealed”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [63] Parham, 2015, Sphingosine 1-phosphate is a ligand for peroxisome proliferator-activated receptor-γ that regulates neoangiogenesis, FASEB J. source

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

    Premisecitation

    S1P is incorporated into HDL-like particles in an ABCA1-dependent manner and coexists with ApoE.

    “S1P is incorporated into HDL-like particles in an ABCA1-dependent manner and coexists with ApoE (44)”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [44] Sato, 2007, Critical role of ABCA1 transporter in sphingosine 1-phosphate release from astrocytes, J. Neurochem. source

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

    Premisecitation

    ApoE4 is the strongest genetic risk factor for sporadic AD.

    “ApoE4 is known to be the strongest genetic risk factor for sporadic AD”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [72] Corder, 1993, Gene dose of apolipoprotein E type 4 allele and the risk of Alzheimer’s disease in late onset families, Science source

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    Our study focused on ApoE3.

    “As our study focused on ApoE3”
    Quote in the paperSubstantial claim

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

    Study design and conductown data

    The mechanism underlying the regulation of ApoE expression by SphK2/S1P signaling was not fully elucidated in this study.

    “The mechanism underlying the regulation of ApoE expression by SphK2/S1P signaling was not fully elucidated in this study”
    Quote in the paperNot a substantial claim
94 of 94

  • The paper relies on this without support.

    ApoE is mainly produced in astrocytes.

    “Because ApoE is mainly produced in astrocytes and has anti-inflammatory and Aβ-metabolizing effects, its induction is a promising therapeutic target for AD.”

  • The paper relies on this without support.

    ApoE has anti-inflammatory and Aβ-metabolizing effects.

    “Because ApoE is mainly produced in astrocytes and has anti-inflammatory and Aβ-metabolizing effects, its induction is a promising therapeutic target for AD.”

  • The paper relies on this without support.

    ApoE expression is coordinately regulated by the ligand-activated nuclear receptors RXR and LXR.

    “ApoE expression is coordinately regulated by the ligand-activated nuclear receptors, retinoid X receptor (RXR) and LXR.”

  • The paper relies on this without support.

    In brain, S1P is mainly generated by phosphorylation of sphingosine by SphK2.

    “In brain, S1P is mainly generated by phosphorylation of sphingosine by sphingosine kinase 2 (SphK2).”

  • The paper relies on this without support.

    The human astrocytoma U87 cell line is a valid model for human astrocytes.

    “we established SphK2 stably overexpressing cells based on human astrocytoma U87 cell line (U87-SphK2 cells), which have the most common genotype of ApoE, ε3/ε3 (26).”

  • The paper relies on this without support.

    SphK2/S1P signaling is involved in inflammatory responses in multiple cell types.

    “As SphK2/S1P signaling involves in inflammatory responses in multiple cell types, we analyzed the changes in the activity of the NF-κB pathway and the expression of inflammatory cytokines.”

  • The paper relies on this without support.

    The hippocampal slice culture system maintains neural circuits and has coexisting neurons and glial cells.

    “We examined whether the phenomena observed in U87 cells could be reproduced in a hippocampal slice culture system in which neural circuits are maintained and neurons and glial cells coexist.”

  • The paper relies on this without support.

    S1P shuttles between the cytoplasm and the nucleus.

    “S1P is known to shuttle between the cytoplasm and the nucleus”
8 of 8

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.

  • Conflicting descriptions of SphK2 localization in U87-SphK2 cells

    Major

    The Results state that expressed SphK2 is predominantly nuclear, but the Discussion says SphK2 in U87-SphK2 cells is highly localized in the cytoplasm. These descriptions conflict for the same cell model.

1 of 1

Cites

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1 of the 75 references couldn’t be matched to a known paper.

Cited by

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Related

Authors
Komai, M., Noda, Y., Ikeda, A., Kaneshiro, N., Kamikubo, Y., Sakurai, T., Uehara, T., & Takasugi, N.
Affiliations
  • Okayama University (JP)
  • Juntendo University (JP)
Corresponding author
Takasugi, Nobumasa
ORCID
Venue
Journal of Lipid Research
Publisher
Elsevier BV
Published
2024-03
Volume
65
Issue
3
Pages
100510
ISSN
0022-2275 (ISSN-L)
DOI
10.1016/j.jlr.2024.100510
PMC
PMC10907773
OpenAlex
W4391257819
Cited by
13
References
75
Citations by year
2026: 6 · 2025: 3 · 2024: 4
FWCI
3.67
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of Lipid Research (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Okayama University Scientific Achievement Repository (Okayama University) (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Institutional Repositories DataBase (IRDB) (submittedVersion, cc-by)
Funders
  • Japan Society for the Promotion of Science
  • JST
Topics
  • Health Sciences › Medicine › Physiology › Alzheimer's disease research and treatments
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Sphingolipid Metabolism and Signaling
  • Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
Keywords
sphingosine kinase 2, sphingosine-1-phosphate, apolipoprotein E, amyloid-beta uptake, astrocytes, Alzheimer's disease, retinoid X receptor, liver X receptor, nuclear receptor agonists, lipid metabolism, therapeutic target
MeSH terms
Alzheimer Disease, Apolipoproteins E, Astrocytes, Brain, Humans, Amyloid beta-Peptides

About these checks

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

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-11-02.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-11-02.

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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