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Cited by 55Alzheimer's disease research and treatmentsMitochondrial Function and PathologyDementia and Cognitive Impairment Research

APOE4 is Associated with Differential Regional Vulnerability to Bioenergetic Deficits in Aged APOE Mice

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Area-Gomez, Estela, Larrea, Delfina, Pera, Marta, Agrawal, Rishi R., Guilfoyle, David N., Pirhaji, Leila, Shannon, Kathleen, Arain, Hirra A. +9 more·2020·Scientific Reports
DOI 10.1038/s41598-020-61142-8PMC PMC7062695

Published version of a preprint

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Abstract

The ε4 allele of apolipoprotein E ( APOE ) is the dominant genetic risk factor for late-onset Alzheimer’s disease (AD). However, the reason for the association between APOE4 and AD remains unclear. While much of the research has focused on the ability of the apoE4 protein to increase the aggregation and decrease the clearance of Aβ, there is also an abundance of data showing that APOE4 negatively impacts many additional processes in the brain, including bioenergetics. In order to gain a more comprehensive understanding of APOE4 ′s role in AD pathogenesis, we performed a transcriptomics analysis of APOE4 vs. APOE3 expression in the entorhinal cortex (EC) and primary visual cortex (PVC) of aged APOE mice. This study revealed EC-specific upregulation of genes related to oxidative phosphorylation (OxPhos). Follow-up analysis utilizing the Seahorse platform showed decreased mitochondrial respiration with age in the hippocampus and cortex of APOE4 vs. APOE3 mice, but not in the EC of these mice. Additional studies, as well as the original transcriptomics data, suggest that multiple bioenergetic pathways are differentially regulated by APOE4 expression in the EC of aged APOE mice in order to increase the mitochondrial coupling efficiency in this region. Given the importance of the EC as one of the first regions to be affected by AD pathology in humans, the observation that the EC is susceptible to differential bioenergetic regulation in response to a metabolic stressor such as APOE4 may point to a causative factor in the pathogenesis of AD.

AI snapshot

Written from the paper’s full text.

TL;DR

In aged APOE targeted-replacement mice, APOE4 expression is associated with region-specific bioenergetic changes: hippocampus and cortex show reduced mitochondrial respiration, while entorhinal cortex (EC) shows upregulated oxidative phosphorylation genes and increased mitochondrial coupling efficiency. The authors propose that EC-specific compensatory bioenergetic mechanisms may contribute to APOE4-related Alzheimer’s disease pathogenesis.

Key findings

  • RNA-sequencing of EC and primary visual cortex (PVC) from 14–15-month-old male APOE3/3 (10 mice) vs. APOE3/4 (19 mice) revealed enrichment of “Oxidative Phosphorylation” KEGG pathway, driven by upregulated ETC complex I–V genes in the EC of APOE3/4 mice.
  • Seahorse analysis in 20-month-old male APOE4/4 vs. APOE3/3 mice (2 per genotype, pooled) showed significant reductions in Complex I- and Complex II-mediated respiration in hippocampus and cortex, but not in EC; EC mitochondria showed significant increases in State 3 Complex I-mediated respiration and increased respiratory control ratio (RCR; state3u/state4o), indicating higher coupling efficiency.
  • Untargeted metabolomics in EC of aged male APOE4/4 vs. APOE3/3 mice showed increased energy-related metabolites including malate, citrate/isocitrate, fructose-6-phosphate, carnitine, and ATP, plus a higher ATP:ADP ratio; free fatty acids were decreased in both EC and PVC. PIUMet analysis of 304 differentially altered EC features inferred 32 metabolite identities and enriched ketone catabolic process, among other GO terms.
  • 1H NMR spectroscopy of EC in 18–19-month-old male APOE3/3 and APOE4/4 mice (7 per genotype) showed increased phosphocreatine (PCr; 0.84 ± 0.27 mM to 2.55 ± 0.18 mM) and PCr:Cr ratio, with decreased creatine, glutamate, GABA, taurine, and glycerophosphocholine in APOE4/4 mice.

Methods

  • Transcriptomics: RNA-sequencing of EC and PVC from 14–15-month-old male APOE3/3 (10) and APOE3/4 (19) targeted-replacement mice; DESeq2 differential expression and Cytoscape/ClueGo KEGG pathway analysis.
  • Seahorse respirometry: mitochondria isolated from EC, PVC, hippocampus, and cortex of APOE3/3 vs. APOE4/4 mice at 6, 15, and 20 months (male; 20-month females in supplement), with samples pooled by region and genotype; OCR measured under Complex I- and Complex II-mediated conditions using XF24e analyzer.
  • Metabolomics and NMR: untargeted LC-TOF MS metabolomics on EC/PVC from 14–15-month-old male APOE3/3 (8), APOE3/4 (9), and APOE4/4 (7) mice, followed by PIUMet network analysis; in vivo 1H NMR spectroscopy at 7T on EC of 18–19-month-old male APOE3/3 (7) and APOE4/4 (7) mice.

Limitations

  • The paper states more work is needed to understand specific cell-type contributions and whether the EC-specific compensatory mechanisms observed in aged APOE4 mice exist in human APOE4 carriers; direct comparison to human data is difficult due to limitations of human imaging and lack of published omics data on human EC from APOE4 vs. APOE3 carriers.
  • The paper states 1H NMR limitations: inherently low signal-to-noise, small EC requiring a small voxel, spectral overlap for some metabolites, and chemical shift displacement; they report shim values of 8–15 Hz and short echo time helped achieve reliable measures.
  • Evident from the text: several Seahorse comparisons use very small numbers and pooled samples (e.g., 2 APOE3/3 and 2 APOE4/4 per genotype/region at 20 months; 3 and 3 at 6 months; 4 and 4 at 15 months), and the main transcriptomics, metabolomics, and NMR cohorts are male only, with female Seahorse data limited to supplementary 2-per-group comparisons.

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

Integrity signals

Registry

Crossref · 10.1038/s41598-020-61142-8

Cites

101 of 101 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

24 of 36 cited sources checked · 13 supported · 0 flagged · accuracy not yet measured

  • Premisecitation

    APOE4 expression causes impaired insulin signaling.

    “impaired insulin signaling19,20”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [19] Ong, Q. R., Chan, E. S., Lim, M. L., Cole, G. M. & Wong, B. S. Reduced phosphorylation of brain insulin receptor substrate and Akt proteins in apolipoprotein-E4 targeted replacement mice. Sci. Rep. 4, 3754 (2014). source
    • cites [20] Zhao, N. et al. Apolipoprotein E4 Impairs Neuronal Insulin Signaling by Trapping Insulin Receptor in the Endosomes. Neuron 96, 115–129 e115 (2017). source
  • Premisecitation

    The endosomal-lysosomal dysregulation observed in the brains of aged APOE4 mice was not restricted to the EC.

    “As the endosomal-lysosomal dysregulation that we observed in the brains of aged APOE4 mice was not restricted to the EC30”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [30] Nuriel, T. et al. The Endosomal-Lysosomal Pathway Is Dysregulated by APOE4 Expression in Vivo. Front. Neurosci. 11, 702 (2017). source
  • Premisecitation

    The neuronal hyperactivity observed appears to be primarily localized to the EC within the hippocampal formation.

    “the neuronal hyperactivity that we observed appears to be primarily localized to the EC within the hippocampal formation33.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [33] Nuriel, T. et al. Neuronal hyperactivity due to loss of inhibitory tone in APOE4 mice lacking Alzheimer’s disease-like pathology. Nat. Commun. 8, 1464 (2017). source
  • Premisecitation

    The brains of APOE4 carriers display decreased glucose utilization in numerous AD-relevant brain regions, as observed by FDG-PET.

    “As shown in previous studies, the brains of APOE4 carriers display decreased glucose utilization in numerous AD-relevant brain regions, as observed by FDG-PET16–18.”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites [16] Reiman, E. M. et al. Preclinical evidence of Alzheimer’s disease in persons homozygous for the epsilon 4 allele for apolipoprotein E. N. Engl. J. Med. 334, 752–758 (1996). source

      The PET and Discussion passages report reduced glucose metabolism in posterior cingulate, parietal, temporal, and prefrontal regions associated with Alzheimer's disease. The study's comparison, however, was of ε4 homozygotes against noncarriers; it does not establish the same pattern for APOE4 carriers generally. Page numbers are not available in the supplied passages. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [17] Reiman, E. M. et al. Functional brain abnormalities in young adults at genetic risk for late-onset Alzheimer’s dementia. Proc. Natl Acad. Sci. U S Am. 101, 284–289 (2004). source
    • cites [18] Reiman, E. M. et al. Correlations between apolipoprotein E epsilon4 gene dose and brain-imaging measurements of regional hypometabolism. Proc. Natl Acad. Sci. U S Am. 102, 8299–8302 (2005). source
  • Interpretationcitation

    The decline in mitochondrial functionality observed in the cortex and hippocampus is likely below the pathological threshold.

    “although it is important to note that the decline in mitochondrial functionality that we observed in the cortex and hippocampus, while significant, is likely below the pathological threshold50.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [50] Rossignol, R., Malgat, M., Mazat, J. P. & Letellier, T. Threshold effect and tissue specificity. Implic. mitochondrial cytopathies. J. Biol. Chem. 274, 33426–33432 (1999). source

      The Results report biochemical thresholds above 50% in the rat tissues studied, supporting the idea that a mitochondrial defect can remain below a functional threshold. The Discussion says thresholds vary by tissue and cautions that results from isolated mitochondria cannot be directly applied to mitochondrial pathology. The paper therefore does not establish that the citing authors’ observed declines in cortex and hippocampus were below a pathological threshold. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
  • Classified as probably not a substantial claim. 29
  • May combine two claims. 23

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

    Premisecitation

    APOE4 expression has deleterious effects on numerous Aβ-independent pathways, including lipid metabolism, tau pathology, bioenergetics, neuronal development, synaptic plasticity, the neuro-vasculature, and neuro-inflammation.

    “APOE4 expression has also been shown to have deleterious effects on numerous Aβ-independent pathways, including lipid metabolism, tau pathology, bioenergetics, neuronal development, synaptic plasticity, the neuro-vasculature, and neuro-inflammation”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [14] Huang, Y. Abeta-independent roles of apolipoprotein E4 in the pathogenesis of Alzheimer’s disease. Trends Mol. Med. 16, 287–294 (2010). source
    • cites [15] Wolf, A. B. et al. Apolipoprotein E as a beta-amyloid-independent factor in alzheimer’s disease. Alzheimers Res. Ther. 5, 38 (2013). source

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

    Premisecitation

    Glutamate and GABA can be utilized as energy metabolites.

    “Although glutamate and GABA are mostly known for their roles as neurotransmitters in excitatory and inhibitory neurons, respectively, they can also be utilized as energy metabolites46–49.”
    Quote in the paperNot a substantial claim3 of 4 sources checked
    • cites [46] Schousboe, A., Bak, L. K. & Waagepetersen, H. S. Astrocytic Control of Biosynthesis and Turnover of the Neurotransmitters Glutamate and GABA. Front. Endocrinol. 4, 102 (2013). source
    • cites [47] Kreft, M., Bak, L. K., Waagepetersen, H. S. & Schousboe, A. Aspects of astrocyte energy metabolism, amino acid neurotransmitter homoeostasis and metabolic compartmentation. ASN neuro 4 (2012). source
    • cites [48] Falkowska, A. et al. Energy Metabolism of the Brain, Including the Cooperation between Astrocytes and Neurons, Especially in the Context of Glycogen Metabolism. Int. J. Mol. Sci. 16, 25959–25981 (2015). source

      Section 6 (Figure 5; page unavailable) says glutamate can be converted to α-ketoglutarate for further metabolism in the TCA cycle, supporting its use in energy metabolism. The supplied passages and searches for GABA and gamma-aminobutyric acid found no corresponding support for GABA as an energy metabolite. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [49] Patel, A. B. et al. The contribution of GABA to glutamate/glutamine cycling and energy metabolism in the rat cortex in vivo. Proc. Natl Acad. Sci. USA 102, 5588–5593 (2005). source

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

    Premisecitation

    Ketone bodies readily cross the blood brain barrier and can act as a potent fuel for the brain.

    “These ketone bodies readily cross the blood brain barrier and can act as a potent fuel for the brain35–37.”
    Quote in the paperNot a substantial claim2 of 3 sources checked
    • cites [35] Robinson, A. M. & Williamson, D. H. Physiological roles of ketone bodies as substrates and signals in mammalian tissues. Physiological Rev. 60, 143–187 (1980). source
    • cites [36] Zhang, Y. et al. Ketosis proportionately spares glucose utilization in brain. J. Cereb. blood flow. metabolism: Off. J. Int. Soc. Cereb. Blood Flow. Metab. 33, 1307–1311 (2013). source

      The Introduction identifies ketone bodies as alternative brain energy substrates, and the Discussion says they can supply up to 70% of brain energy needs after maximal metabolic adaptation. The Discussion also describes blood–brain barrier transporters upregulating during ketosis, but does not establish that ketone bodies cross the barrier readily without that adaptation. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
    • cites [37] Lin, A. L., Zhang, W., Gao, X. & Watts, L. Caloric restriction increases ketone bodies metabolism and preserves blood flow in aging brain. Neurobiol. aging 36, 2296–2303 (2015). source

      The Discussion states that ketone bodies are an alternative fuel for brain cells and can support most, if not all, basal neuronal oxidative metabolism. It does not state that they readily cross the blood–brain barrier; it says brain ketones in the studied rats appeared to come primarily from astrocytes rather than blood. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

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

    Premisecitation

    APOE4 expression causes altered genetic expression of glucose-regulating enzymes and transporters.

    “altered genetic expression of glucose-regulating enzymes and transporters23,24”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [23] Keeney, J. T., Ibrahimi, S. & Zhao, L. Human ApoE Isoforms Differentially Modulate Glucose and Amyloid Metabolic Pathways in Female. Brain: Evid. Mechanism Neuroprotection ApoE2 Implic. Alzheimer’s Dis. Prev. Early Intervention. J. Alzheimer’s disease: JAD. 48, 411–424 (2015). source
    • cites [24] Wu, L., Zhang, X. & Zhao, L. Human ApoE Isoforms Differentially Modulate Brain Glucose and Ketone Body Metabolism: Implications for Alzheimer’s Disease Risk Reduction and Early Intervention. J. neuroscience: Off. J. Soc. Neurosci. 38, 6665–6681 (2018). source

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

    Premisecitation

    Both young and old APOE4 carriers display decreased glucose utilization, measured by FDG-PET, in brain regions similar to those seen with AD patients.

    “early reports by Reiman and colleagues demonstrated that both young and old APOE4 carriers display decreased glucose utilization, as measured by fluorodeoxyglucose positron emission tomography (FDG-PET), in brain regions similar to those seen with AD patients16–18.”
    Quote in the paperNot a substantial claim2 of 3 sources checked
    • cites [16] Reiman, E. M. et al. Preclinical evidence of Alzheimer’s disease in persons homozygous for the epsilon 4 allele for apolipoprotein E. N. Engl. J. Med. 334, 752–758 (1996). source

      The PET results and Discussion report reduced glucose metabolism in APOE ε4 homozygotes in the same regions affected in patients with probable Alzheimer’s disease; the Brain Imaging section specifies that the PET scans used fluorodeoxyglucose. But this study examined carriers aged 50–65, not both young and old carriers, and its findings concern people with two ε4 alleles rather than APOE4 carriers generally. Page numbers are unavailable in our copy. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [17] Reiman, E. M. et al. Functional brain abnormalities in young adults at genetic risk for late-onset Alzheimer’s dementia. Proc. Natl Acad. Sci. U S Am. 101, 284–289 (2004). source
    • cites [18] Reiman, E. M. et al. Correlations between apolipoprotein E epsilon4 gene dose and brain-imaging measurements of regional hypometabolism. Proc. Natl Acad. Sci. U S Am. 102, 8299–8302 (2005). source

      The Results report lower FDG-PET measurements of glucose metabolism in APOE ε4 carriers in regions also implicated in Alzheimer’s dementia. The study examined cognitively normal people aged 47–68 (study summary and Methods; page unavailable), so it does not establish the claim for both young and old carriers. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim.

    Premisecitation

    PIUMet discovers dysregulated molecular pathways and components associated with changes in untargeted metabolomics data by analyzing a network built from curated protein-protein and protein-metabolite interactions (PPMI).

    “PIUMet discovers dysregulated molecular pathways and components associated with changes in untargeted metabolomics data by analyzing a network built from curated protein-protein and protein-metabolite interactions (PPMI).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [34] Pirhaji, L. et al. Revealing disease-associated pathways by network integration of untargeted metabolomics. Nat. methods 13, 770–776 (2016). source

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

    Premisecitation

    Weighted averaging improves statistical power in NMR spectroscopic studies.

    “To improve statistical significance, Miller et al. recently demonstrated the use of weighted averaging in NMR spectroscopic studies83.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [83] Miller, J. J., Cochlin, L., Clarke, K. & Tyler, D. J. Weighted averaging in spectroscopic studies improves statistical power. Magnetic Reson. Med. 78, 2082–2094 (2017). source

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

    Premisecitation

    APOE4 expression causes reduced cerebral blood volume and cognitive function in response to a high fat diet.

    “reduced cerebral blood volume and cognitive function in response to a high fat diet (HFD)21,22”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [21] Johnson, L. A. et al. Apolipoprotein E4 mediates insulin resistance-associated cerebrovascular dysfunction and the post-prandial response. Journal of cerebral blood flow and metabolism: official journal of the International Society of Cerebral Blood Flow and Metabolism, 271678X17746186 (2017). source
    • cites [22] Johnson, L. A., Torres, E. R., Impey, S., Stevens, J. F. & Raber, J. Apolipoprotein E4 and Insulin Resistance Interact to Impair Cognition and Alter the Epigenome and Metabolome. Sci. Rep. 7, 43701 (2017). source

      The Results (page unavailable) report that high-fat-diet-induced deficits in spatial learning and memory were more pronounced in E4 mice than E3 mice. The paper does not report reduced cerebral blood volume in response to the diet; its Discussion raises vascular dysfunction only as a possibility. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

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

    Premisecitation

    APOE4 expression generates a toxic C-terminal fragment of apoE4 that can directly target electron transport chain (ETC) complexes in mitochondria.

    “the generation of a toxic C-terminal fragment of apoE4 that can directly target electron transport chain (ETC) complexes in the mitochondria25–27”
    Quote in the paperNot a substantial claim2 of 3 sources checked
    • cites [25] Chang, S. et al. Lipid- and receptor-binding regions of apolipoprotein E4 fragments act in concert to cause mitochondrial dysfunction and neurotoxicity. Proc. Natl Acad. Sci. U S Am. 102, 18694–18699 (2005). source

      The Results report that apoE4(1-272) fragments localize to mitochondria in Neuro-2a cells and impair mitochondrial function, as measured by MitoTracker fluorescence (Figs. 2 and 5). They do not show direct targeting of electron transport chain complexes. The paper describes the toxic fragment as C-terminal-truncated apoE4, not an isolated C-terminal fragment. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [26] Huang, Y. et al. Apolipoprotein E fragments present in Alzheimer’s disease brains induce neurofibrillary tangle-like intracellular inclusions in neurons. Proc. Natl Acad. Sci. U S Am. 98, 8838–8843 (2001). source
    • cites [27] Nakamura, T., Watanabe, A., Fujino, T., Hosono, T. & Michikawa, M. Apolipoprotein E4 (1-272) fragment is associated with mitochondrial proteins and affects mitochondrial function in neuronal cells. Mol. Neurodegener. 4, 35 (2009). source

      The Conclusion (page not provided) reports that apoE4(1–272) associates with proteins in mitochondrial respiratory complexes III and IV and that its overexpression reduces their activities. The Background (page not provided) describes this as a C-terminal-truncated fragment—residues 1–272 remain—not a fragment consisting of apoE4’s C terminus. The paper also treats its direct binding to the complexes as a suggested mechanism. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim.

    Premisecitation

    Neuronal activity and glucose utilization have previously been shown to exist in a near 1:1 stoichiometric relationship.

    “neuronal activity and glucose utilization have previously been shown to exist in a near 1:1 stoichiometric relationship72”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [72] Yellen, G. Fueling thought: Management of glycolysis and oxidative phosphorylation in neuronal metabolism. J. Cell Biol. 217, 2235–2246 (2018). source

      In “The source of neuronal energy during activation” (page unavailable), the review reports that neuronal stimulation increased glucose metabolism more than twofold, supporting a link between activity and glucose use. It does not report a near 1:1 stoichiometric relationship between them. Its separate discussion of stoichiometry concerns oxygen and glucose use in the resting brain, not neuronal activity and glucose use. (LLM, conf=0.87)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingown data

    Seahorse analysis showed decreased mitochondrial respiration with age in the hippocampus and cortex of APOE4 vs. APOE3 mice, but not in the EC.

    “Follow-up analysis utilizing the Seahorse platform showed decreased mitochondrial respiration with age in the hippocampus and cortex of APOE4 vs. APOE3 mice, but not in the EC of these mice.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The resulting PIUMet network is enriched in 18 putative GO biological processes and 124 total metabolites.

    “The resulting network is enriched in 18 putative GO biological processes (Table 3) and 124 total metabolites (Supplementary Fig. S4).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    PIUMet inferred the identity of 32 metabolite features and revealed a network of protein-protein and protein-metabolite interactions associated with their dysregulation.

    “PIUMet was able to infer the identity of 32 metabolite features and revealed a network of protein-protein and protein-metabolite interactions associated with their dysregulation.”
    Quote in the paperSubstantial claim

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

    Findingown data

    Among the putative metabolite identifications were acetone, adenylosuccinate, alpha-tocotrienol, and oleamide.

    “while the most interesting putative metabolite identifications were acetone, adenylosuccinate, alpha-tocotrienol, and oleamide.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The metabolomics data from both the EC and PVC of aged male APOE4/4 vs. APOE3/3 mice show decreased fatty acid levels and increased carnitine levels.

    “This is represented by the decreased fatty acid levels and increased carnitine levels observed in the metabolomics data from both the EC and PVC of the aged male APOE4/4 vs. APOE3/3 mice.”
    Quote in the paperSubstantial claim

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

    Findingown data

    79% of the resulting PIUMet nodes have specificity scores over 60%.

    “79% of the resulting nodes have specificity scores of over 60%.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Among the enriched GO biological processes were fatty acid metabolic process, IMP metabolic process, ketone catabolic process, steroid metabolic process, and vitamin metabolic process.

    “Among these, the most intriguing GO biological processes were fatty acid metabolic process, inosine 5′-monophosphate (IMP) metabolic process, ketone catabolic process, steroid metabolic process, and vitamin metabolic process”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    In 6-month-old male APOE4/4 vs. APOE3/3 mice, there was a reduction in State 3 respiration of mitochondria from the Ctx in the presence of pyruvate-malate (Complex I-mediated activity), and very slight reductions in State 3 respiration of mitochondria from the Hip and EC.

    “we observed a reduction in State 3 respiration of mitochondria from the Ctx of 6-month-old male APOE4/4 vs. APOE3/3 mice, and very slight reductions in State 3 respiration of mitochnodria from the Hip and EC of these mice.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In the EC of aged male APOE4 mice, Seahorse data show no changes in proton leak or mitochondrial mass.

    “mitochondria in the EC of aged male APOE4 mice show increased ADP-stimulated oxygen consumption and increased maximal respiration rate, without changes in proton leak (Fig. 3) or mitochondrial mass (Supplementary Fig. S2).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In 15-month-old APOE mice, there was a significant reduction in both Complex I-mediated and Complex II-mediated respiration in mitochondria from the PVC of APOE4/4 vs. APOE3/3 mice, but no difference in respiration between genotypes in mitochondria from the EC.

    “we observed a significant reduction in both the Complex I-mediated and Complex II-mediated respiration in mitochondria from the PVC of these APOE4/4 vs. APOE3/3 mice, but no difference in the respiration between genotypes in the mitochondria from the EC region.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Untargeted metabolomics study uncovered increases in numerous energy-related metabolites, including TCA cycle metabolites (malate, citrate, isocitrate), fructose-6-phosphate, carnitine, and ATP, each higher in the EC of aged APOE4/4 vs. APOE3/3 mice.

    “this metabolomics study uncovered increases in numerous energy-related metabolites, including several TCA cycle metabolites (malate, citrate and isocitrate), as well as fructose-6-phosphate, carnitine, and ATP, each of which had higher levels in the EC of the aged APOE4/4 vs. APOE3/3 mice (Fig. 4A).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Metabolomics data revealed an APOE4-associated decrease in levels of numerous free fatty acids in aged male APOE4/4 vs. APOE3/3 mice, which were differentially altered in both the EC and the PVC.

    “our metabolomics data also revealed an APOE4-associated decrease in the levels of numerous free fatty acids in aged male APOE4/4 vs. APOE3/3 mice (Fig. 4C), which, unlike each of the previously mentioned energy-related metabolites (with the exception of carnitine), were differentially altered in both the EC and the PVC.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In the EC of aged male APOE4 mice, Seahorse data show increased ADP-stimulated oxygen consumption and increased maximal respiration rate.

    “mitochondria in the EC of aged male APOE4 mice show increased ADP-stimulated oxygen consumption and increased maximal respiration rate, without changes in proton leak (Fig. 3) or mitochondrial mass (Supplementary Fig. S2).”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given

    Metabolites identified as differentially expressed were further validated for chromatographic and biological accuracy, and non-validated metabolites were removed.

    “Following differential expression analysis, metabolites identified as differentially expressed were further validated for chromatographic and biological accuracy, and non-validated metabolites were removed.”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given

    The paper corrected for multiple testing using the Benjamini-Hochberg procedure and selected all genes that possessed a corrected p-value of less than 0.05.

    “We corrected for multiple testing using the Benjamini-Hochberg procedure and selected all genes that possessed a corrected p-value of less than 0.05.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    EC mitochondria from 20-month-old APOE4/4 mice are highly coupled, resulting in increased production of ATP per unit of oxygen.

    “This analysis indicates that EC mitochondria from the 20-month-old APOE4/4 mice are highly coupled, resulting in increased production of ATP per unit of oxygen.”
    Quote in the paperSubstantial claim

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

    Study design and conductown data

    The paper achieved full width half maximums of 8-15 Hz using a customized second and third order shim coil.

    “we were able to achieve full width half maximums of 8–15 Hz using a customized second and third order shim coil.”
    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 conductreasoning

    The chemical shift displacement error was relatively small and still allowed for sufficient specificity.

    “However, this displacement was relatively small and still allowed for sufficient specificity.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    Nodes with robustness scores less than 60% were removed from the PIUMet results.

    “Nodes with robustness scores less than 60% were removed from the results.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Study design and conductown data

    At 7T with shim values in the range 8-15 Hz and a short echo time of 8 ms, the spectral peaks were reasonably separated, allowing for direct and reliable measures of metabolite concentrations.

    “However, at 7T with shim values in the range 8–15 Hz and a short echo time of 8 ms, the spectral peaks were reasonably separated, allowing for direct and reliable measures of metabolite concentrations.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    APOE targeted replacement mice express human APOE in place of their mouse Apoe gene and do not develop overt AD pathology.

    “APOE targeted replacement mice, which express human APOE in place of their mouse Apoe gene and which do not develop overt AD pathology28,29”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [28] Sullivan, P. M., Mace, B. E., Maeda, N. & Schmechel, D. E. Marked regional differences of brain human apolipoprotein E expression in targeted replacement mice. Neurosci. 124, 725–733 (2004). source
    • cites [29] Sullivan, P. M. et al. Targeted replacement of the mouse apolipoprotein E gene with the common human APOE3 allele enhances diet-induced hypercholesterolemia and atherosclerosis. J. Biol. Chem. 272, 17972–17980 (1997). source

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

    Study design and conductnone given

    Each feature was manually validated following extraction with investigators blinded to genotype assignments.

    “Each feature was also manually validated following extraction (with investigators blinded to genotype assignments during validation), and re-integration was performed when necessary.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    All data used in the final NMR analysis had Cramer-Rao lower bounds of 20% or less.

    “All data used in the final analysis had Cramer-Rao lower bounds of 20% or less.”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given

    Every biological replicate consisted of three technical replicates.

    “Every biological replicate consisted of three technical replicates.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    The ε4 allele of apolipoprotein E (APOE) is the major genetic risk factor for late-onset Alzheimer's disease (AD).

    “The ε4 allele of apolipoprotein E (APOE) is the dominant genetic risk factor for late-onset Alzheimer’s disease (AD).”
    • Also stated in: § Introduction · plain
      “Possession of the ε4 allele of apolipoprotein E (APOE) is the major genetic risk factor for late-onset Alzheimer’s disease (AD).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [5] Farrer, L. A. et al. Effects of age, sex, and ethnicity on the association between apolipoprotein E genotype and Alzheimer disease. A meta-analysis. APOE and Alzheimer Disease Meta Analysis Consortium. JAMA 278, 1349–1356 (1997). source
    • cites [6] Rubinsztein, D. C. & Easton, D. F. Apolipoprotein E genetic variation and Alzheimer’s disease. a meta-analysis. Dement. Geriatr. Cogn. Disord. 10, 199–209 (1999). source

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    All results are averages of five or more biological replicates.

    “All results are averages of five or more biological replicates.”
    Quote in the paperSubstantial claim

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

    Interpretationreasoning

    The bioenergetic counterbalancing observed in the EC of aged APOE4 mice may be directly correlated to the observed neuronal hyperactivity in these mice.

    “suggesting that the bioenergetic counterbalancing that we observe in the EC of aged APOE4 mice may be directly correlated to the observed neuronal hyperactivity in these mice33.”
    Quote in the paperNot a substantial claim

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

    Study design and conductreasoning

    The study achieved high quality spectra in the EC region with high quantification accuracy.

    “Despite the challenges of in vivo mouse brain 1H NMR, we were able to achieve high quality spectra in this difficult region with high quantification accuracy.”
    Quote in the paperSubstantial claim

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

    Premisecitation

    The EC is known to have higher energy demands and to be more metabolically active than many other brain regions.

    “As such, it is known to have higher energy demands and to be more metabolically active that many other brain regions75.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [75] Egorov, A. V., Hamam, B. N., Fransen, E., Hasselmo, M. E. & Alonso, A. A. Graded persistent activity in entorhinal cortex neurons. Nat. 420, 173–178 (2002). source

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

    Study design and conductown data

    One APOE3/4 PVC sample possessed a chromatographic error and was removed from all future analyses.

    “Prior to analysis, it was observed that one APOE3/4 PVC sample possessed a chromatographic error and was removed from all future analyses.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    All mice used in this study were treated in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Columbia University Medical Center and the Nathan Kline Institute's Institutional Animal Care and Use Committee (IACUC).

    “All mice used in this study were treated in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Columbia University Medical Center and the Nathan Kline Institute’s Institutional Animal Care and Use Committee (IACUC).”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    During AD pathogenesis, the EC is one of the first regions to develop tauopathy.

    “During AD pathogenesis, the EC is one of the first regions to develop tauopathy, which eventually develops in the hippocampus and other cortical regions as the disease progresses76.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [76] Braak, H. & Braak, E. Neuropathological stageing of Alzheimer-related changes. Acta Neuropathol. 82, 239–259 (1991). source

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

    Study design and conductown data

    All RNA samples possessed a RIN of 8 or higher.

    “All RNA samples possessed a RIN of 8 or higher.”
    Quote in the paperNot a substantial claim
43 of 43

  • The paper relies on this without support.

    The EC is one of the first regions to be affected by AD pathology in humans.

    “Given the importance of the EC as one of the first regions to be affected by AD pathology in humans”

  • The paper relies on this without support.

    The EC is acutely vulnerable to AD pathology and the PVC is less vulnerable.

    “a brain region that is acutely vulnerable to AD pathology (the EC) vs. a less vulnerable brain region (the PVC)”

  • The paper relies on this without support.

    APOE4 expression acts as a metabolic stressor.

    “in response to a metabolic stressor such as APOE4”

  • The paper relies on this without support.

    Possession of the APOE4 allele greatly increases an individual’s risk of developing AD.

    “Possession of the APOE4 allele greatly increases an individual’s risk of developing AD.”

  • The paper relies on this without support.

    The precise cause of the association between APOE4 and AD remains unknown.

    “Although numerous theories have been proposed, the precise cause of this association remains unknown.”

  • The paper relies on this without support.

    To the authors' knowledge, this is the first in vivo 1H NMR study of the EC performed in mice.

    “To our knowledge, this is the first in vivo 1H NMR study of the EC performed in mice.”

  • The authors state this as an assumption.

    The internal reference method assumes known values of water concentrations of gray and white matter.

    “This internal reference method assumes known values of water concentrations of gray and white matter5–7.”

  • The paper relies on this without support.

    It is often difficult to resolve Glu from Gln, NAA from NAAG, and PCh from GPC, particularly when spectral quality is poor due to inadequate shim.

    “It is often quite difficult to resolve Glu from Gln, NAA from NAAG and PCh from GPC, particularly if the spectral quality is poor due to an inadequate shim.”

  • The paper relies on this without support.

    Total concentrations of Glu and Gln, NAA and NAAG, and PCh and GPC are a more reliable metric than individual metabolites.

    “These total concentrations are thought to be a more reliable metric.”

  • The paper relies on this without support.

    1H NMR metabolite measures are inherently low in signal to noise.

    “One such limitation is that is that 1H NMR metabolite measures are inherently low in signal to noise.”

  • The paper relies on this without support.

    The metabolite signals measured are approximately 10,000-fold less than the proton signal used in imaging.

    “The metabolite signals measured are approximately 10,000-fold less than the proton signal used in imaging.”
11 of 11

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?

  • APOE4 humanized mice have altered brain levels of palmitate, succinate, lactate, and malate.

    “For example, APOE4 humanized mice have altered brain levels of palmitate, succinate, lactate, and malate 21 .”

    From APOE4-drives brain bioenergetic shifts independent of Alzheimer’s disease pathology (2026) · cites this paper as [21]

    In the Results metabolomics table (Table 1; page unavailable), palmitic acid (palmitate) is lower and malic acid (malate) is higher in the entorhinal cortex of aged male APOE4/4 mice than in APOE3/3 mice. The NMR Results list lactate as measured but not among the metabolites with significant differences (Table 4); the Methods mention succinate as an assay substrate, not as a metabolite with altered brain levels. (LLM, conf=0.88)

    Experimental — accuracy not yet measured

  • Pathways and proteins associated with oxidative phosphorylation, glycolysis, and lipid metabolism have altered brain expression profiles in APOE4 humanized mice.

    “Pathways and proteins associated with oxidative phosphorylation, glycolysis, and lipid metabolism demonstrate altered brain expression profiles in APOE4 humanized mice 21,22,24 .”

    From APOE4-drives brain bioenergetic shifts independent of Alzheimer’s disease pathology (2026) · cites this paper as [21]

    The Results (page unavailable) report altered expression of oxidative-phosphorylation genes in the entorhinal cortex of aged human-APOE targeted-replacement mice. The Discussion reports altered expression of genes for a shuttle that links glycolysis to mitochondrial metabolism, while the lipid-related Results chiefly report changes in fatty-acid metabolites. The paper does not establish altered protein expression across all three named pathways. (LLM, conf=0.84)

    Experimental — accuracy not yet measured

  • APOE4-TR mice exhibit multiple alterations of energy metabolism compared with APOE3-TR mice, including impaired mitochondrial respiration in the cortex and hippocampus, upregulation of oxidative phosphorylation genes in the entorhinal cortex and differential levels of fatty acids and other metabolites.

    “APOE4-TR mice exhibit multiple alterations of energy metabolism compared with APOE3-TR mice, including impaired mitochondrial respiration in the cortex and hippocampus, upregulation of oxidative phosphorylation genes in the entorhinal cortex and differential levels of fatty acids and other metabolites174.”

    From Multifaceted roles of APOE in Alzheimer disease (2024) · cites this paper as [174]

  • Prior lipidomic, metabolomic, and magnetic resonance spectroscopy studies in mouse models show that APOE genetic variation modifies brain metabolite levels.

    “Prior lipidomic, metabolomic, and magnetic resonance spectroscopy (MRS) studies in mouse models show that genetic variation in APOE modify brain metabolite levels [20][21][22][23] .”

    From APOE4-drives brain bioenergetic shifts independent of Alzheimer’s disease pathology (2026) · cites this paper as [21]

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.

  • Seahorse methods count three experiments after listing four

    Minor

    The methods explicitly enumerate experiments 1 through 4, then describe normalization for each of three experiments without identifying an exclusion.

  • DHA finding is attributed to a Seahorse figure

    Minor

    The discussion cites Fig. 2B for the EC fatty-acid finding, but Figure 2 presents Seahorse mitochondrial-respiration measurements, not metabolite levels.

  • APOE3/3 total creatine does not equal its components

    Minor

    Table 4 reports 3.73 mM creatine and 0.84 mM phosphocreatine for APOE3/3 mice, which sum to 4.57 mM, not the reported 5.58 mM total.

    Reported 5.58 · recomputed 4.57

3 of 3

Cites

Flagged first, then by citations

2 of the 103 references couldn’t be matched to a known paper.

Cited by

Sorted by citations

Related

Authors
Area-Gomez, E., Larrea, D., Pera, M., Agrawal, R. R., Guilfoyle, D. N., Pirhaji, L., Shannon, K., Arain, H. A., Ashok, A., Chen, Q., Dillman, A. A., Figueroa, H. Y., Cookson, M. R., Gross, S. S., Fraenkel, E., Duff, K. E., & Nuriel, T.
Affiliations
  • Columbia University (US)
  • Universitat Internacional de Catalunya (ES)
  • Nathan Kline Institute for Psychiatric Research (US)
  • Massachusetts Institute of Technology (US)
  • Weill Cornell Medicine (US)
  • National Institutes of Health (US)
  • Broad Institute (US)
  • UK Dementia Research Institute (GB)
  • Columbia University Irving Medical Center (US)
Corresponding author
Nuriel, Tal
ORCID
Venue
Scientific Reports
Publisher
Springer Science and Business Media LLC
Published
2020-03-09
Volume
10
Issue
1
Pages
4277-4277
ISSN
2045-2322 (ISSN-L)
DOI
10.1038/s41598-020-61142-8
PMC
PMC7062695
OpenAlex
W3010213203
Cited by
55
References
103
Citations by year
2026: 6 · 2025: 9 · 2024: 9 · 2023: 5 · 2022: 12 · 2021: 11 · 2020: 3
FWCI
4.66
Type
journal article
Language
en
Open access
Licence
Locations
  • Scientific Reports (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nd)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DIGITAL.CSIC (Spanish National Research Council (CSIC)) (acceptedVersion)
  • DSpace@MIT (Massachusetts Institute of Technology) (submittedVersion, cc-by-nc)
  • UCL Discovery (University College London) (submittedVersion)
Funders
  • U.S. Department of Health & Human Services | NIH | National Institute on Aging (AG045335, AG056387, AG057331, AG047797, K01AG061264)
  • U.S. Department of Defense (W911NF-12-1-9159, W911F-15-1-0169)
  • U.S. Department of Health & Human Services | NIH | Eunice Kennedy Shriver National Institute of Child Health and Human Development (HD67244)
  • U.S. Department of Health & Human Services | NIH | National Heart, Lung, and Blood Institute (HL87062)
  • U.S. Department of Health & Human Services | NIH | National Institute of Neurological Disorders and Stroke (NS089076)
  • Cure Alzheimer's Fund
  • BrightFocus Foundation
Topics
  • Health Sciences › Medicine › Physiology › Alzheimer's disease research and treatments
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Mitochondrial Function and Pathology
  • Health Sciences › Medicine › Psychiatry and Mental health › Dementia and Cognitive Impairment Research
Keywords
APOE ε4, apolipoprotein E, Alzheimer's disease, bioenergetics, mitochondrial respiration, oxidative phosphorylation, entorhinal cortex, primary visual cortex, hippocampus, APOE3, aging, transcriptomics, mitochondrial coupling efficiency, metabolic stress, ApoE-deficient mice
MeSH terms
Animals, Brain, Energy Metabolism, Male, Mitochondria, Mice, Apolipoprotein E4, Metabolome, Transcriptome

About these checks

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

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

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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