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Cited by 476Alzheimer's disease research and treatmentsNeuroinflammation and Neurodegeneration MechanismsSingle-cell and spatial transcriptomics

Single-nucleus transcriptome analysis reveals dysregulation of angiogenic endothelial cells and neuroprotective glia in Alzheimer’s disease

Open access
Lau, Shun-Fat, Cao, Han, Fu, Amy K. Y., Ip, Nancy Y.·2020·Proceedings of the National Academy of Sciences
DOI 10.1073/pnas.2008762117PMC PMC7568283
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Abstract

Significance: Alzheimer’s disease (AD) has complex pathological mechanisms. Our limited understanding of the molecular and cellular changes in AD hindered the identification of therapeutic targets. In this study, we profiled 169,496 nuclei from the prefrontal cortical samples of AD patients and healthy controls by single-nucleus RNA sequencing. The results indicated that there is a loss of neuroprotective glial cells in AD, which may contribute to impaired neurotransmitter recycling in astrocytes, demyelination in oligodendrocytes, and perturbed synaptic pruning in microglia. Moreover, the analysis revealed a role of antigen presentation by angiogenic endothelial cells in AD. Together, our findings offer important insights into the therapeutic potential of targeting glial- and endothelial-specific pathways to restore brain homeostasis in AD.

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Written from the paper’s full text.

TL;DR

Single-nucleus RNA sequencing of 169,496 nuclei from AD and control prefrontal cortex identifies cell type-specific dysregulation, including loss of neuroprotective glial subpopulations and expansion of angiogenic endothelial cells with antigen-presentation signatures. The findings implicate glial and endothelial pathways in AD pathogenesis and therapeutic targeting.

Key findings

  • Profiling 21 prefrontal cortex samples (AD n = 12; NC n = 9) yielded 169,496 nuclei (90,713 AD; 78,783 NC) across six major cell types: astrocytes 11.9 ± 1.4%, endothelial cells 2.3 ± 0.5%, excitatory neurons 45.2 ± 1.7%, inhibitory neurons 14.1 ± 0.9%, microglia 4.7 ± 0.6%, and oligodendrocytes 21.8 ± 2.5%. Endothelial cell proportion was higher in AD than NC (3.0 ± 0.9% vs. 1.2 ± 0.3%).
  • AD-associated transcriptomic changes were largely cell type-specific: 2,190 DEGs total, with 551 in astrocytes, 299 in endothelial cells, 347 in excitatory neurons, 125 in inhibitory neurons, 340 in microglia, and 528 in oligodendrocytes; only 11 DEGs were shared across all six cell types.
  • Subcluster analysis showed reduced neuroprotective/homeostatic subpopulations: astrocyte subpopulation a3 was 23.5% smaller in AD, while a1 and a6 were 9.9% and 10.2% larger; oligodendrocyte subpopulations o3 and o5 (myelination genes MAG, MOBP, OPALIN) were reduced; microglial m6 (C1QA/B/C, IL4R, IL1RAP) was smaller. Endothelial subpopulations e1, e3, and e4 were AD-up-regulated and enriched for angiogenesis genes (CLDN5, ERG, FLT1, VWF) and MHC-I antigen presentation (HLA-E).
  • Cross-study validation found 1,113 and 764 DEGs significantly differentially expressed in prefrontal and temporal cortex microarray data, respectively; 86.7% of replicable prefrontal DEGs showed concordant changes. Comparison with Mathys et al. snRNA-seq identified 273 shared cell type-specific DEGs, with >90% concordant changes for astrocyte- and neuron-specific DEGs.

Methods

  • Postmortem prefrontal cortex tissue (BA6, BA8, BA9) from the South West Dementia Brain Bank; AD classified by Braak stage ≥4 and NC ≤2; 21 subjects (AD mean age 74.6 y; NC 85.4 y; 8 male/4 female AD, 6 male/3 female NC).
  • Nuclei isolated from frozen tissue, snRNA-seq libraries prepared with Chromium Single Cell 3′ Library Kit v3 (10× Genomics), sequenced on NovaSeq 6000; reads aligned to GRCh38 pre-mRNA reference with Cell Ranger v3.0.1; QC and downstream analysis in Seurat v3.0.
  • Cell clustering by UMAP and K-nearest neighbor clustering; DEGs identified by Wilcoxon rank-sum test (adjusted P < 0.1, log2 fold change ≥ 0.1 or ≤ −0.1); subcluster analysis used enrichment scores >0.5 for AD-associated subpopulations; validation used bulk microarray datasets (GSE33000, GSE15222), prior snRNA-seq data, and mouse model transcriptome data.

Limitations

  • No formal limitations section is included. The paper states that postmortem delay varied among samples, that bulk microarray data cannot distinguish cell type-specific changes, and that a previous snRNA-seq dataset had too few endothelial cells to validate endothelial findings; it also notes unresolved questions about how endothelial dysregulation leads to blood–brain barrier abnormalities and why remyelinating oligodendrocytes fail to overcome demyelination.
  • Evident from the text: the sample size is modest (21 subjects), AD and NC groups differ in mean age (74.6 vs. 85.4 y), and the design is cross-sectional and associational, with no functional or causal validation of the identified pathways.

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Registry

Crossref · 10.1073/pnas.2008762117

Cites

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Statistics

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Claims the paper makes

0 of 59 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    The proportion of endothelial cells was higher in AD samples than NC samples (3.0 ± 0.9% vs. 1.2 ± 0.3%).

    “However, the proportion of endothelial cells was higher in the AD samples than the NC samples (3.0 ± 0.9% vs. 1.2 ± 0.3%, respectively; Fig. 2 A and B).”
    Quote in the paperSubstantial claim
  • Findingown data

    Only three of the seven identified endothelial cell subpopulations-e1, e3, and e4-contributed to the transcriptomic changes observed in endothelial cells in AD.

    “Our subcluster analysis further revealed that only three of the seven identified endothelial cell subpopulations—e1, e3, and e4—contributed to the transcriptomic changes observed in endothelial cells in AD (Fig. 5A and SI Appendix, Fig. S7 A and B and Table S5).”
    Quote in the paperSubstantial claim
  • Findingown data

    The transcriptome profiles of the AD-up-regulated oligodendrocyte subpopulations o1 and o2 were characterized by enriched expression of HSPA1A, NEAT1, and PDE1A.

    “Notably, the transcriptome profiles of the AD-up-regulated oligodendrocyte subpopulations o1 and o2 were characterized by enriched expression of HSPA1A (a heat shock protein), NEAT1 (a nuclear noncoding RNA), and PDE1A (which encodes phosphodiesterase 1A) (SI Appendix, Fig. S5 I and J); this expression pattern resembles that of remyelinating oligodendrocytes in patients with multiple sclerosis (22”
    Quote in the paperSubstantial claim
  • Findingown data

    273 cell type-specific DEGs were in common between our dataset and Mathys et al. snRNA-seq data: 53 in astrocytes, 97 in excitatory neurons, 24 in inhibitory neurons, 13 in microglia, and 86 in oligodendrocytes.

    “We found 273 cell type-specific DEGs in common between the two datasets, including 53 in astrocytes, 97 in excitatory neurons, 24 in inhibitory neurons, 13 in microglia, and 86 in oligodendrocytes (SI Appendix, Fig. S4 A, B, D, E, G, H, J, K, M, and N).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [12] Mathys H.., Single-cell transcriptomic analysis of Alzheimer’s disease. Nature 570, 332– 337 (2019). 31042697 PMC6865822 source

      Not checked in the free run.

  • Findingown data

    Genes associated with angiogenesis (CLDN5, ERG, FLT1, and VWF) and antigen presentation-especially MHC-I machinery (HLA-E)-were enriched in the three AD-up-regulated endothelial subpopulations.

    “Transcriptome profiling showed that genes associated with angiogenesis (i.e., CLDN5, ERG, FLT1, and VWF) and antigen presentation—especially MHC-I (major histocompatibility complex class I) machinery (i.e., HLA-E)—were enriched in these three AD-up-regulated subpopulations (Fig. 5C and SI Appendix, Table S5).”
    Quote in the paperSubstantial claim
  • May combine two claims. 30
  • Classified as probably not a substantial claim. 27

  • May combine two claims.

    Findingown data

    The proportions of astrocytes, excitatory neurons, inhibitory neurons, microglia, and oligodendrocytes were similar between AD and NC brain samples.

    “UMAP cluster analysis revealed that the proportions of astrocytes, excitatory neurons, inhibitory neurons, microglia, and oligodendrocytes were similar between the AD and NC brain samples.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    There were 2,190 differentially expressed genes (DEGs) between AD and NC brain samples: 551 in astrocytes, 299 in endothelial cells, 347 in excitatory neurons, 125 in inhibitory neurons, 340 in microglia, and 528 in oligodendrocytes.

    “We identified the following 2,190 differentially expressed genes (DEGs) between the AD and NC brain samples: 551 in astrocytes, 299 in endothelial cells, 347 in excitatory neurons, 125 in inhibitory neurons, 340 in microglia, and 528 in oligodendrocytes (Fig. 2C and SI Appendix, Table S3).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The 169,496 nuclei from 21 prefrontal cortex samples clustered into six major cell types: astrocytes (11.9 ± 1.4%), endothelial cells (2.3 ± 0.5%), excitatory neurons (45.2 ± 1.7%), inhibitory neurons (14.1 ± 0.9%), microglia (4.7 ± 0.6%), and oligodendrocytes (21.8 ± 2.5%).

    “This analysis yielded 43 unique cell clusters (SI Appendix, Fig. S1B), which we subsequently categorized into the following six major cell types according to their individual transcriptome profiles and previously reported cell-type markers: astrocytes (AQP4+, 11.9 ± 1.4% of total nuclei), endothelial cells (CLDN5+, 2.3 ± 0.5%), excitatory neurons (CAMK2A+, 45.2 ± 1.7%), inhibitory neurons (GAD1+,”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The six major cell types expressed unique signature genes, including ADGRV1, GPC5, and RYR3 in astrocytes; ABCB1 and EBF1 in endothelial cells; CBLN2 and LDB2 in excitatory neurons; LHFPL3 and PCDH15 in inhibitory neurons; LRMDA and DOCK8 in microglia; and PLP1 and ST18 in oligodendrocytes.

    “In addition to the previously reported cell-type markers, these six major cell types expressed the following unique signature genes, which can serve as novel cell-type markers: ADGRV1, GPC5, and RYR3 were expressed by astrocytes; ABCB1 and EBF1 by endothelial cells; CBLN2 and LDB2 by excitatory neurons; LHFPL3 and PCDH15 by inhibitory neurons; LRMDA and DOCK8 by microglia; and PLP1 and ST18 by”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Compared to NC samples, the relative proportions of astrocyte subpopulations a1 and a6 were 9.9% and 10.2% larger in AD samples, respectively, while that of a3 was 23.5% smaller.

    “However, compared to the NC samples, the relative proportions of a1 and a6 were 9.9% and 10.2% larger in the AD samples, respectively, while that of a3 was 23.5% smaller (Fig. 4A and SI Appendix, Fig. S5A).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The overlapping DEGs were associated with transsynaptic signaling in astrocytes, synaptic signaling in excitatory neurons, mitochondrial functions in inhibitory neurons, secretion in microglia, and axonogenesis in oligodendrocytes.

    “Moreover, pathway analysis showed that these overlapping DEGs were associated with transsynaptic signaling in astrocytes (SI Appendix, Fig. S4C), synaptic signaling in excitatory neurons (SI Appendix, Fig. S4F), mitochondrial functions in inhibitory neurons (SI Appendix, Fig. S4I), secretion in microglia (SI Appendix, Fig. S4L), and axonogenesis in oligodendrocytes (SI Appendix, Fig. S4O).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Subcluster analysis of microglia identified 13 subpopulations; only m1, m6, and m7 contributed to the transcriptomic changes in microglia in AD.

    “Finally, subcluster analysis of microglia identified 13 subpopulations; only 3 of them—m1, m6, and m7—contributed to the transcriptomic changes in microglia in AD (SI Appendix, Fig. S6 A and B and Table S5).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Only astrocyte subpopulations a1, a3, and a6 expressed DEGs; a1 and a6 were enriched with up-regulated signature genes, whereas a3 was enriched in down-regulated signature genes.

    “Only subpopulations a1, a3, and a6 expressed DEGs; specifically, subpopulations a1 and a6 were enriched with up-regulated signature genes, whereas subpopulation a3 was enriched in down-regulated signature genes (SI Appendix, Fig. S5B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The AD brain contains a reduced proportion of neuroprotective astrocytes, which are associated with glutamate recycling and synaptic signaling.

    “Indeed, we showed that the AD brain contains a reduced proportion of neuroprotective astrocytes, which are associated with glutamate recycling and synaptic signaling.”
    Quote in the paperSubstantial claim

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

    Premisecitation

    Previous studies using transgenic mouse models of AD have identified various mechanisms that lead to synaptic impairment in AD, including the accumulation of soluble amyloid-beta, Tau hyperphosphorylation, loss of VGF signaling, and glial state dysregulation.

    “For example, previous studies using transgenic mouse models of AD have identified various mechanisms that lead to synaptic impairment in AD, including the accumulation of soluble amyloid-beta, Tau hyperphosphorylation, loss of VGF signaling, and glial state dysregulation (e.g., the induction of disease-associated microglia and disease-associated astrocytes) (9, 23–30).”
    Quote in the paperNot a substantial claim0 of 9 sources checked
    • cites [9] Liddelow S. A.., Neurotoxic reactive astrocytes are induced by activated microglia. Nature 541, 481– 487 (2017). 28099414 PMC5404890 source

      Not checked in the free run.

    • cites [23] Hong S.., Complement and microglia mediate early synapse loss in Alzheimer mouse models. Science 352, 712– 716 (2016). 27033548 PMC5094372 source

      Not checked in the free run.

    • cites [24] Sheng M., Sabatini B. L., Südhof T. C., Synapses and Alzheimer’s disease. Cold Spring Harb. Perspect. Biol. 4, 10 (2012). PMC3331702 22491782 source

      Not checked in the free run.

    • cites [25] Tremblay M. È., Sierra A., Microglia in Health and Disease, (Springer, 2014). source

      Not checked in the free run.

    • cites [26] El Gaamouch F.., VGF-derived peptide TLQP-21 modulates microglial function through C3aR1 signaling pathways and reduces neuropathology in 5xFAD mice. Mol. Neurodegener. 15, 4 (2020). 31924226 PMC6954537 source

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    • cites [27] Butovsky O.., Identification of a unique TGF-β-dependent molecular and functional signature in microglia. Nat. Neurosci. 17, 131– 143 (2014). 24316888 PMC4066672 source

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    • cites [29] Keren-Shaul H.., A unique microglia type Associated with restricting development of Alzheimer’s disease. Cell 169, 1276– 1290.e17 (2017). 28602351 source

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    • cites [30] Beckmann N. D.., Multiscale causal networks identify VGF as a key regulator of Alzheimer’s disease. Nat. Commun. 11, 3942 (2020). 32770063 PMC7414858 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    The cell type-specific transcriptomic changes in AD are associated with four molecular pathways: angiogenesis in endothelial cells, immune response in endothelial cells and microglia, myelination in oligodendrocytes, and synaptic signaling in astrocytes and neurons.

    “Together, our results reveal that the cell type-specific transcriptomic changes in AD are associated with four molecular pathways: angiogenesis in endothelial cells, immune response in endothelial cells and microglia, myelination in oligodendrocytes, and synaptic signaling in astrocytes and neurons (Fig. 2E).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The dysregulation of these pathways in AD is due to changes in the subpopulation heterogeneity of respective cell types, including the reduced proportions of neuroprotective astrocytes and oligodendrocytes as well as the increased proportions of endothelial cells, which are associated with enhanced angiogenesis and antigen presentation.

    “Moreover, we showed that the dysregulation of these pathways in AD is due to changes in the subpopulation heterogeneity of respective cell types, including the reduced proportions of neuroprotective astrocytes and oligodendrocytes as well as the increased proportions of endothelial cells, which are associated with enhanced angiogenesis and antigen presentation.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    This could lead to synaptic loss due to excitotoxicity and reduced synaptogenesis due to the destabilization of nascent synapses, which would result in a net loss of synapses during disease progression.

    “This could lead to synaptic loss due to excitotoxicity (6, 33, 34) and reduced synaptogenesis due to the destabilization of nascent synapses (16, 17), which would result in a net loss of synapses during disease progression.”
    Quote in the paperSubstantial claim0 of 5 sources checked
    • cites [6] Hynd M. R., Scott H. L., Dodd P. R., Glutamate-mediated excitotoxicity and neurodegeneration in Alzheimer’s disease. Neurochem. Int. 45, 583– 595 (2004). 15234100 source

      Not checked in the free run.

    • cites [33] Eid T., Tu N., Lee T. S. W., Lai J. C. K., Regulation of astrocyte glutamine synthetase in epilepsy. Neurochem. Int. 63, 670– 681 (2013). 23791709 PMC3825815 source

      Not checked in the free run.

    • cites [34] Petr G. T.., Conditional deletion of the glutamate transporter GLT-1 reveals that astrocytic GLT-1 protects against fatal epilepsy while neuronal GLT-1 contributes significantly to glutamate uptake into synaptosomes. J. Neurosci. 35, 5187– 5201 (2015). 25834045 PMC4380995 source

      Not checked in the free run.

    • cites [16] Stogsdill J. A.., Astrocytic neuroligins control astrocyte morphogenesis and synaptogenesis. Nature 551, 192– 197 (2017). 29120426 PMC5796651 source

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    • cites [17] Südhof T. C., Synaptic neurexin complexes: A molecular code for the logic of neural circuits. Cell 171, 745– 769 (2017). 29100073 PMC5694349 source

      Not checked in the free run.

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

    Premisecitation

    Impaired recycling of neurotransmitters, especially glutamate, results in excitotoxicity and leads to neuronal death.

    “Indeed, impaired recycling of neurotransmitters, especially glutamate, results in excitotoxicity and leads to neuronal death (6).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [6] Hynd M. R., Scott H. L., Dodd P. R., Glutamate-mediated excitotoxicity and neurodegeneration in Alzheimer’s disease. Neurochem. Int. 45, 583– 595 (2004). 15234100 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    In AD, three specific astrocyte subpopulations contribute to the transcriptomic changes in astrocytes; the subpopulation of homeostatic astrocytes (a3), crucial for neurotransmitter recycling, is reduced; and alarmin-expressing astrocytes (a1 and a6) are induced.

    “Thus, our results show that in AD, (i) three specific astrocyte subpopulations contribute to the transcriptomic changes in astrocytes, (ii) the subpopulation of homeostatic astrocytes (a3), which are crucial for neurotransmitter recycling, is reduced, and (iii) alarmin-expressing astrocytes are induced (a1 and a6).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Single-nucleus transcriptome analysis identified four major molecular pathways dysregulated in specific neural cell types in AD: impaired synaptic signaling in astrocytes and neurons, reduced myelination in oligodendrocytes, aberrant immune activation in microglia and endothelial cells, and enhanced angiogenesis in endothelial cells.

    “Accordingly, our single-nucleus transcriptome analysis identified the four major molecular pathways that are dysregulated in specific neural cell types in patients with AD: impaired synaptic signaling in astrocytes and neurons, reduced myelination in oligodendrocytes, aberrant immune activation in microglia and endothelial cells, and enhanced angiogenesis in endothelial cells.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    Such changes were not observed in previous snRNA-seq studies, possibly due to a lower proportion of endothelial nuclei being sampled.

    “However, such changes were not observed in previous snRNA-seq studies (12, 13), possibly due to a lower proportion of endothelial nuclei being sampled.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [12] Mathys H.., Single-cell transcriptomic analysis of Alzheimer’s disease. Nature 570, 332– 337 (2019). 31042697 PMC6865822 source

      Not checked in the free run.

    • cites [13] Grubman A.., A single-cell atlas of entorhinal cortex from individuals with Alzheimer’s disease reveals cell-type-specific gene expression regulation. Nat. Neurosci. 22, 2087– 2097 (2019). 31768052 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationcitation

    The dysregulated pathways in neurons and oligodendrocytes are associated with synaptic signaling and myelination, respectively; these findings are similar to those in previous studies despite variations in study cohorts, brain regions, sample preparation, and sequencing protocols.

    “For example, we showed that the dysregulated pathways in neurons and oligodendrocytes are associated with synaptic signaling and myelination, respectively; these findings are similar to those in previous studies despite variations in study cohorts, brain regions, sample preparation, and sequencing protocols (12, 13, 23).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [12] Mathys H.., Single-cell transcriptomic analysis of Alzheimer’s disease. Nature 570, 332– 337 (2019). 31042697 PMC6865822 source

      Not checked in the free run.

    • cites [13] Grubman A.., A single-cell atlas of entorhinal cortex from individuals with Alzheimer’s disease reveals cell-type-specific gene expression regulation. Nat. Neurosci. 22, 2087– 2097 (2019). 31768052 source

      Not checked in the free run.

    • cites [23] Hong S.., Complement and microglia mediate early synapse loss in Alzheimer mouse models. Science 352, 712– 716 (2016). 27033548 PMC5094372 source

      Not checked in the free run.

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

    Study design and conductcitation

    The mean numbers of transcripts and genes detected per nucleus were comparable to those reported in a previous snRNA-seq study, confirming the quality of our samples prior to downstream analysis.

    “In addition, these parameters were comparable to those reported in a previous snRNA-seq study (15), confirming the quality of our samples prior to downstream analysis.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [15] Habib N.., Massively parallel single-nucleus RNA-seq with DroNc-seq. Nat. Methods 14, 955– 958 (2017). 28846088 PMC5623139 source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    Aberrant complement signaling mediates nonselective synaptic pruning via phagocytosis.

    “Given that aberrant complement signaling mediates nonselective synaptic pruning via phagocytosis (23), our results suggest that the loss of this typical microglial subpopulation can contribute to the imbalanced complement signaling and synaptic pruning in AD.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [23] Hong S.., Complement and microglia mediate early synapse loss in Alzheimer mouse models. Science 352, 712– 716 (2016). 27033548 PMC5094372 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    In AD, there is a decrease in the number of mature myelinated oligodendrocytes and an increase in the number of remyelinating oligodendrocytes.

    “Thus, our results show that in AD, there is a decrease in the number of mature myelinated oligodendrocytes and an increase in the number of remyelinating oligodendrocytes.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    Dysregulated communication with neurons can impair synaptic plasticity.

    “For astrocytes in particular, dysregulated communication with neurons can impair synaptic plasticity (31, 32).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [31] Hillen A. E. J., Burbach J. P. H., Hol E. M., Cell adhesion and matricellular support by astrocytes of the tripartite synapse. Prog. Neurobiol. 165-167, 66– 86 (2018). 29444459 source

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    • cites [32] Perea G., Navarrete M., Araque A., Tripartite synapses: Astrocytes process and control synaptic information. Trends Neurosci. 32, 421– 431 (2009). 19615761 source

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    Study design and conductown data

    Differential expression analysis of the Narayanan dataset was fitted by linear regression, adjusting for age and sex.

    “For differential expression analysis, we fitted the gene expression profiles by linear regression, adjusting for age and sex.”
    Quote in the paperNot a substantial claim

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

    Study design and conductown data

    APOE genotype was not intentionally used to include/exclude samples prior to snRNA-seq library preparation, but APOE genotypes were confirmed by TaqMan assay.

    “We did not intentionally include/exclude samples according to APOE genotype prior to snRNA-seq library preparation but confirmed APOE genotypes by TaqMan assay with a probe from Thermo Scientific (assay ID: C_3084793_20 and C_904973_10) following the manufacturer’s instructions.”
    Quote in the paperNot a substantial claim

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    Premisecitation

    AD is associated with viral infection.

    “Interestingly, several recent studies suggest that AD is associated with viral infection (47, 48).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [47] Eimer W. A.., Alzheimer’s disease-associated β-Amyloid is rapidly seeded by herpesviridae to protect against brain infection. Neuron 99, 56– 63.e3 (2018). 30001512 PMC6075814 source

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      Not checked in the free run.

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

    Study design and conductown data

    The final filtered matrix contained 169,496 nuclei and 29,171 genes.

    “The final filtered matrix contained 169,496 nuclei and 29,171 genes.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Study design and conductown data

    The variation in the number of nuclei that passed quality control among samples was independent of age, sex, postmortem delay, and pathology, and there was no batch effect.

    “During sample preparation, we targeted ∼16,000 nuclei per sample and found that while the number of nuclei that passed quality control varied among samples, the variation was independent of age, sex, postmortem delay, and pathology, and there was no batch effect (SI Appendix, Table S1).”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    Aging-related myelin loss is associated with cognitive impairment in AD.

    “Indeed, aging-related myelin loss is associated with cognitive impairment in AD (35).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [35] Bartzokis G., Age-related myelin breakdown: A developmental model of cognitive decline and Alzheimer’s disease. Neurobiol. Aging 25, 5– 18, author reply 49–62 (2004). 14675724 source

      Not checked in the free run.

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    Premisecitation

    Once activated CD8+ T cells have infiltrated the brain, they can release IFN-γ to stimulate excessive microglial synaptic pruning or inhibit adult neural stem cell proliferation, impairing neuronal homeostasis.

    “Once activated CD8+ T cells have infiltrated the brain, they can release IFN-γ to stimulate excessive microglial synaptic pruning or inhibit adult neural stem cell proliferation (44, 49), impairing neuronal homeostasis.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [44] Di Liberto G.., Neurons under T Cell attack coordinate phagocyte-mediated synaptic stripping. Cell 175, 458– 471.e19 (2018). 30173917 source

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      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Study design and conductcitation

    Nuclei with ≤200 genes, ≥20,000 unique molecular identifiers, or ≥20% mitochondrial genes were filtered out to exclude potential dead cells and cell debris.

    “To exclude potential dead cells and cell debris from the dataset, we filtered out nuclei with ≤200 genes, ≥20,000 unique molecular identifiers, or ≥20% mitochondrial genes as described in several previous snRNA-seq studies (12, 13, 22, 53).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [12] Mathys H.., Single-cell transcriptomic analysis of Alzheimer’s disease. Nature 570, 332– 337 (2019). 31042697 PMC6865822 source

      Not checked in the free run.

    • cites [13] Grubman A.., A single-cell atlas of entorhinal cortex from individuals with Alzheimer’s disease reveals cell-type-specific gene expression regulation. Nat. Neurosci. 22, 2087– 2097 (2019). 31768052 source

      Not checked in the free run.

    • cites [22] Jäkel S.., Altered human oligodendrocyte heterogeneity in multiple sclerosis. Nature 566, 543– 547 (2019). 30747918 PMC6544546 source

      Not checked in the free run.

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

    Study design and conductown data

    Data validation was performed by obtaining datasets from published studies and comparing them with our findings.

    “For data validation, we obtained datasets from published studies and compared them with our findings.”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    Upon viral infection, endothelial MHC-I presents endogenous viral products and facilitates CD8+ T cell activation.

    “Upon viral infection, endothelial MHC-I presents endogenous viral products and facilitates CD8+ T cell activation (44–46).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [44] Di Liberto G.., Neurons under T Cell attack coordinate phagocyte-mediated synaptic stripping. Cell 175, 458– 471.e19 (2018). 30173917 source

      Not checked in the free run.

    • cites [45] Razakandrainibe R., Pelleau S., Grau G. E., Jambou R., Antigen presentation by endothelial cells: What role in the pathophysiology of malaria? Trends Parasitol. 28, 151– 160 (2012). 22365903 source

      Not checked in the free run.

    • cites [46] Pober J. S., Merola J., Liu R., Manes T. D., Antigen presentation by vascular cells. Front. Immunol. 8, 1907 (2017). 29312357 PMC5744398 source

      Not checked in the free run.

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

    Study design and conductown data

    Subjects were selected according to age and postmortem delay, and a balanced sex ratio was ensured.

    “For snRNA-seq library preparation, we subsequently selected subjects according to age and postmortem delay and ensured a balanced sex ratio.”
    Quote in the paperNot a substantial claim

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

    Interpretationreasoning

    Another possible role of this MHC-I induction is the triggering of CD8+ T cell activation and subsequent clonal expansion.

    “Another possible role of this MHC-I induction is the triggering of CD8+ T cell activation and subsequent clonal expansion.”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    In animal models, demyelination can be triggered by the accumulation of amyloid-beta peptides, phosphorylated Tau, or proinflammatory cytokines.

    “In animal models, demyelination can be triggered by the accumulation of amyloid-beta peptides, phosphorylated Tau, or proinflammatory cytokines (35–37).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [35] Bartzokis G., Age-related myelin breakdown: A developmental model of cognitive decline and Alzheimer’s disease. Neurobiol. Aging 25, 5– 18, author reply 49–62 (2004). 14675724 source

      Not checked in the free run.

    • cites [36] Anderson J. M.., Abnormally phosphorylated tau is associated with neuronal and axonal loss in experimental autoimmune encephalomyelitis and multiple sclerosis. Brain 131, 1736– 1748 (2008). 18567922 source

      Not checked in the free run.

    • cites [37] Stadelmann C., Wegner C., Brück W., Inflammation, demyelination, and degeneration - recent insights from MS pathology. Biochim. Biophys. Acta 1812, 275– 282 (2011). 20637864 source

      Not checked in the free run.

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

    Premisecitation

    Transcriptome data for mouse models of amyloid-beta deposition and Tau hyperphosphorylation were obtained from MOUSEAC.

    “We obtained transcriptome data for mouse models of amyloid-beta deposition and Tau hyperphosphorylation from MOUSEAC (55).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [55] Matarin M.., A genome-wide gene-expression analysis and database in transgenic mice during development of amyloid or tau pathology. Cell Rep. 10, 633– 644 (2015). 25620700 source

      Not checked in the free run.

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

    Interpretationreasoning

    The induction of MHC-I in endothelial cells in AD might reflect response to viral infection.

    “Therefore, the induction of MHC-I in endothelial cells in AD might reflect response to viral infection.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    Increased clonal expansion and CD8+ T cell infiltration in the brain are observed both in AD patients and during aging.

    “Indeed, increased clonal expansion and CD8+ T cell infiltration in the brain are observed both in AD patients and during aging (48, 49).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [48] Gate D.., Clonally expanded CD8 T cells patrol the cerebrospinal fluid in Alzheimer’s disease. Nature 577, 399– 404 (2020). 31915375 PMC7445078 source

      Not checked in the free run.

    • cites [49] Dulken B. W.., Single-cell analysis reveals T cell infiltration in old neurogenic niches. Nature 571, 205– 210 (2019). 31270459 PMC7111535 source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    Subjects with other neurodegenerative diseases, vascular diseases, intoxicated state, infection, prions, inflammatory diseases, structural brain disorders, metabolic/nutritional diseases, trauma, delirium, genetic disorders, or other systemic diseases were excluded from initial sample selection.

    “For initial sample selection from the SWDBB, we excluded subjects with other neurodegenerative diseases, vascular diseases, an intoxicated state, infection, prions, inflammatory diseases, structural brain disorders, metabolic/nutritional diseases, trauma, delirium, genetic disorders (e.g., Down syndrome), or other systemic diseases (SI Appendix, Table S1).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Both patients with AD and AD animal models exhibit neurovascular system impairment including abnormalities in the number, diameter, and density of blood vessels, which lead to decreased brain perfusion and blood-brain barrier disruption.

    “Both patients with AD and AD animal models exhibit neurovascular system impairment including abnormalities in the number, diameter, and density of blood vessels, which lead to decreased brain perfusion and blood–brain barrier disruption (10, 39, 40).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [10] Zlokovic B. V., Neurovascular pathways to neurodegeneration in Alzheimer’s disease and other disorders. Nat. Rev. Neurosci. 12, 723– 738 (2011). 22048062 PMC4036520 source

      Not checked in the free run.

    • cites [39] Sweeney M. D., Sagare A. P., Zlokovic B. V., Blood-brain barrier breakdown in Alzheimer disease and other neurodegenerative disorders. Nat. Rev. Neurol. 14, 133– 150 (2018). 29377008 PMC5829048 source

      Not checked in the free run.

    • cites [40] Bennett R. E.., Tau induces blood vessel abnormalities and angiogenesis-related gene expression in P301L transgenic mice and human Alzheimer’s disease. Proc. Natl. Acad. Sci. U.S.A. 115, E1289– E1298 (2018). 29358399 PMC5819390 source

      Not checked in the free run.

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

    Study design and conductcitation

    Comparison with Jäkel et al. confirmed the expression patterns of cell-type markers in the different cell types in our study.

    “To further confirm the cell-type specificity of these markers, we compared our data with those of Jäkel et al. (22), which confirmed the expression patterns of these markers in the different cell types in our study.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [22] Jäkel S.., Altered human oligodendrocyte heterogeneity in multiple sclerosis. Nature 566, 543– 547 (2019). 30747918 PMC6544546 source

      Not checked in the free run.

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

    Premisecitation

    Proper remyelination can restore the impaired saltatory conduction in neurons.

    “While proper remyelination can restore the impaired saltatory conduction in neurons (38), it remains unclear why remyelinating oligodendrocytes fail to overcome the demyelination observed in AD.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [38] Franklin R. J. M., Ffrench-Constant C., Remyelination in the CNS: From biology to therapy. Nat. Rev. Neurosci. 9, 839– 855 (2008). 18931697 source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Study design and conductcitation

    AD diagnosis was based on DSM-5 criteria.

    “The clinical diagnosis of AD was based on the Diagnostic and Statistical Manual of Mental Disorders-5 criteria for AD (SI Appendix, Table S1).”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    Samples were categorized according to Braak stage (AD: ≥4, NC: ≤2).

    “We categorized samples according to Braak stage (i.e., AD: ≥4 and NC: ≤2).”
    Quote in the paperSubstantial claim

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

    Premisecitation

    MHC-I mostly presents endogenous proteins that originate in the cytoplasm, making it essential in the response to viral infection.

    “MHC-I mostly presents endogenous proteins that originate in the cytoplasm, making it essential in the response to viral infection (43).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [43] Neefjes J., Jongsma M. L. M., Paul P., Bakke O., Towards a systems understanding of MHC class I and MHC class II antigen presentation. Nat. Rev. Immunol. 11, 823– 836 (2011). 22076556 source

      Not checked in the free run.

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

    Study design and conductown data

    The study included 21 subjects (mean age: AD, 74.6 y; NC, 85.4 y) including 8 males and 4 females with AD as well as 6 male and 3 female NC subjects.

    “Thus, we included 21 subjects (mean age: AD, 74.6 y; NC, 85.4 y) including 8 males and 4 females with AD as well as 6 male and 3 female NC subjects.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    The same pipeline was used to process the microarray dataset of AD temporal cortical samples from Webster et al. (GEO GSE15222).

    “We also used the same pipeline to process the microarray dataset of AD temporal cortical samples from Webster et al. (GEO accession no. GSE15222) (19).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [19] Webster J. A..; NACC-Neuropathology Group, Genetic control of human brain transcript expression in Alzheimer disease. Am. J. Hum. Genet. 84, 445– 458 (2009). 19361613 PMC2667989 source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    A subpopulation with an enrichment score > 0.5 was designated as an AD-associated subpopulation.

    “A subpopulation with an enrichment score > 0.5 was designated as an AD-associated subpopulation.”
    Quote in the paperSubstantial claim
48 of 48

  • The paper relies on this without support.

    Endothelial cells are the primary component of the neurovascular system, which maintains blood–brain barrier integrity.

    “Endothelial cells are the primary component of the neurovascular system, which maintains blood–brain barrier integrity.”
1 of 1

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?

  • The snRNA-seq dataset contains over 100,000 astrocyte nuclei in each brain region with ≈6,000 average total reads and ≈2,500 average total genes detected, which is orders of magnitude higher than previous studies.

    “resulting in over 100,000 astrocyte nuclei in each brain region with ≈6,000 average total number of reads and ≈2,500 average total number of genes detected, which is orders of magnitude higher than previous studies9–15”

    From Astrocyte transcriptomic changes along the spatiotemporal progression of Alzheimer’s disease (2024) · cites this paper as [13]

    In Results, “Single-Nucleus Transcriptome Profiling of the Prefrontal Cortex in AD” (page unavailable), the paper reports 169,496 nuclei in total from prefrontal cortex samples, with astrocytes comprising 11.9% of those nuclei—not over 100,000 astrocyte nuclei in each brain region. It also says the mean numbers of transcripts and genes detected per nucleus were comparable to a previous study, contrary to the claimed orders-of-magnitude increase. The quoted averages of approximately 6,000 reads and 2,500 genes are not established by these passages. (LLM, conf=0.90)

    Checked by a reasoning model — may be wrong.

    Experimental — accuracy not yet measured

  • The exhausted phase may have confounded previous studies that compared only control and severe AD donors or grouped late- and end-stage donors (Braak stage V/VI) together.

    “may have confounded previous studies that compared only control and severe AD donors or grouped late- and end-stage donors (Braak stage V/VI) together9–15”

    From Astrocyte transcriptomic changes along the spatiotemporal progression of Alzheimer’s disease (2024) · cites this paper as [13]

    The Methods (Selection Criteria for Brain Tissue Collection; page unavailable) classify donors as AD at Braak stage ≥4 or controls at ≤2, and the snRNA-seq Analysis Methods compare those two groups. This supports the claim that the study grouped AD stages for comparison with controls, but the copy does not identify a distinct V/VI grouping or say that an exhausted phase confounded the results. (LLM, conf=0.82)

    Experimental — accuracy not yet measured

  • Transcriptomic analysis suggested that several subpopulations of endothelial cells in AD are associated with immune response characterized by increased expression of antigen presentation genes, especially MHC-I machinery.

    “Transcriptomic analysis suggested that several subpopulations of endothelial cells in AD are associated with immune response which was characterized by increased expression of genes associated with antigen presentation—especially major histocompatibility complex class I (MHC-I) machinery.473”

    From Role of neuroinflammation in neurodegeneration development (2023) · cites this paper as [473]

  • Previous studies described AD-associated molecular and cellular changes; however, cross-study comparisons are challenging without common cell annotations.

    “Previous studies described AD-associated molecular and cellular changes5–15; however, cross-study comparisons are challenging without common cell annotations.”

    From Integrated multimodal cell atlas of Alzheimer’s disease (2024) · cites this paper as [6]

  • The study harmonized snRNA-seq data and associated donor metadata from the PFC from ten additional AD studies spanning 707 donors.

    “To corroborate the results, we harmonized snRNA-seq data and associated donor metadata from the PFC from ten additional AD studies spanning 707 donors5–14.”

    From Integrated multimodal cell atlas of Alzheimer’s disease (2024) · cites this paper as [6]

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.

  • Total DEG count treats shared genes as distinct

    Minor

    The six cell-type DEG counts add to exactly 2,190, but 11 genes are reported as differentially expressed in all six cell types. Counting those shared genes once would make the number of distinct DEGs at least 55 lower than 2,190.

1 of 1

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Authors
Lau, S.-F., Cao, H., Fu, A. K. Y., & Ip, N. Y.
Affiliations
Hong Kong University of Science and Technology (HK)
Corresponding author
Ip, Nancy Y.
ORCID
Venue
Proceedings of the National Academy of Sciences
Publisher
National Academy of Sciences
Published
2020-09-28
Volume
117
Issue
41
Pages
25800-25809
ISSN
  • 0027-8424 (ISSN-L)
  • 1091-6490
DOI
10.1073/pnas.2008762117
PMC
PMC7568283
OpenAlex
W3087821509
Semantic Scholar
f58e0c665760e51542f8ab62fc65854fc60cbdbc
Cited by
476
References
55
Citations by year
2026: 65 · 2025: 100 · 2024: 96 · 2023: 96 · 2022: 79 · 2021: 39 · 2020: 1
FWCI
32.47
Type
journal article
Language
en
Open access
Licence
Locations
  • Proceedings of the National Academy of Sciences (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Rare & Special e-Zone (The Hong Kong University of Science and Technology) (submittedVersion)
Funders
  • Research Grants Council, University Grants Committee (C6027-19GF, T13-607/12R, HKUST16149616, HKUST16103017, HKUST16100418, HKUST16102019)
  • National Key R&D Program of China (2017YFE0190000, 2018YFE0203600)
  • University Grants Committee (AoE/M-604/16)
  • Innovation and Technology Commission (ITCPD/17-9)
  • Guangdong Provincial Key S&T Program (2018B030336001)
  • Shenzhen Knowledge Innovation Program (JCYJ20180507183642005)
  • Shenzhen Knowledge Innovation Progra (JCYJ20170413173717055)
Topics
  • Health Sciences › Medicine › Physiology › Alzheimer's disease research and treatments
  • Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Single-cell and spatial transcriptomics
Keywords
Alzheimer's disease, single-nucleus transcriptomics, prefrontal cortex, endothelial cells, glial cells, astrocytes, oligodendrocytes, microglia, demyelination, synaptic pruning, neurotransmitter recycling, antigen presentation, angiogenesis, neuroinflammation, therapeutic target
MeSH terms
Aged, Aged, 80 and over, Alzheimer Disease, Cell Nucleus, Female, Humans, Male, Neuroglia, Prefrontal Cortex, Gene Expression Profiling, Endothelial Cells, Single-Cell Analysis, Transcriptome

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

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-06.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-06.

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