CoChat
Sign Up
Cited by 736Single-cell and spatial transcriptomicsGenetics, Aging, and Longevity in Model OrganismsNeuroinflammation and Neurodegeneration Mechanisms

Single-cell transcriptomic profiling of the aging mouse brain

Open access
Ximerakis, Methodios, Lipnick, Scott L., Innes, Brendan T., Simmons, Sean K., Adiconis, Xian, Dionne, Danielle, Mayweather, Brittany A., Nguyen, Lan +10 more·2019·Nature Neuroscience
DOI 10.1038/s41593-019-0491-3

Published version of a preprint

Open this paper in CoChat — free accountOpen PDF

Abstract

The mammalian brain is complex, with multiple cell types performing a variety of diverse functions, but exactly how each cell type is affected in aging remains largely unknown. Here we performed a single-cell transcriptomic analysis of young and old mouse brains. We provide comprehensive datasets of aging-related genes, pathways and ligand-receptor interactions in nearly all brain cell types. Our analysis identified gene signatures that vary in a coordinated manner across cell types and gene sets that are regulated in a cell-type specific manner, even at times in opposite directions. These data reveal that aging, rather than inducing a universal program, drives a distinct transcriptional course in each cell population, and they highlight key molecular processes, including ribosome biogenesis, underlying brain aging. Overall, these large-scale datasets (accessible online at https://portals.broadinstitute.org/single_cell/study/aging-mouse-brain ) provide a resource for the neuroscience community that will facilitate additional discoveries directed towards understanding and modifying the aging process.

AI snapshot

Written from a shortened version of the paper’s full text (it is longer than we read).

TL;DR

Single-cell RNA-seq of young (2–3 months) and old (21–23 months) mouse brains profiled 37,089 cells across 25 cell populations, revealing that aging does not induce a universal transcriptional program but instead drives shared, cell-type-specific, and even oppositely directed changes. The authors provide a public resource of aging-related genes, pathways, and ligand–receptor interactions, highlighting ribosomal biogenesis as a pathway regulated in opposite directions between differentiated cells and progenitors.

Key findings

  • Of 14,699 detected genes, 3,897 were significantly affected by aging in at least one cell type (FDR<0.05); 1,117 passed a 10% fold-change threshold, of which 1,034 changed in the same direction across cell types (494 upregulated, 540 downregulated) and 83 changed in different directions depending on cell type.
  • Cell identity and overall composition were largely preserved with age, though the previously reported decline of neuronal-restricted precursors (NRP) was confirmed; other subtype shifts (OPC, OLG, ASC, MG) were not statistically significant.
  • GSEA identified 451 aging-related pathways (1,142 terms; p<0.05, q<0.25): 234 shared by ≥2 cell types and 217 unique; 339 changed in the same direction (195 up, 144 down) and 112 varied in direction across cell types.
  • Ribosomal genes showed bidirectional regulation: most differentiated cell types upregulated ribosomal genes with aging, while most progenitor cells (e.g., OPC, ASC) downregulated them — validated by bulk RNA-seq in FACS-purified ASC, EC, and MG.
  • Common aging-upregulated genes included ribosomal genes (e.g., Rpl6), lncRNAs (e.g., Malat1), and immunoregulatory genes (e.g., B2m); common downregulated genes included mitochondrial respiratory chain genes (e.g., mt-Nd1), glycolysis genes (e.g., Aldoc), selenoproteins (e.g., Sepw1), and tetraspanins (e.g., Tspan7).

Methods

  • Unbiased 10x Genomics Chromium scRNA-seq of dissociated whole brains from 8 young (2–3 months) and 8 old (21–23 months) male mice, yielding 50,212 cells initially and 37,089 cells after QC, representing 25 cell populations; a modified dissociation protocol was developed for both ages.
  • Clustering and QC in Seurat (graph-based clustering, PCA); differential gene expression with MAST (hurdle model, normalized UMI covariate); gene set enrichment analysis (GSEA, 5 reference datasets, 1,000 permutations); ligand–receptor network analysis using curated receptor/ligand/ECM gene lists and iRefIndex, Pathway Commons, and BioGRID interactions.
  • Validation by bulk RNA-seq (4 young, 4 old) and qRT-PCR (5 young, 6 old) on FACS-purified EC, MG, and ASC, plus RNAscope in situ hybridization and immunohistochemistry.

Limitations

  • Stated by the authors: potential sampling problems from enzymatic dissociation (possibly addressable with single-nuclei approaches); potential age-associated biases in dissociation, cell encapsulation, and other procedures; relatively shallow sequencing depth limiting analysis to highly transcribed genes; relatively small number of cells sequenced relative to total brain tissue, restricting comparative analyses to more abundant populations; and inability to assess sex-specific changes because only male mouse brains were analyzed.
  • Evident from the text: estimated cell-type percentages do not necessarily reflect actual brain proportions due to differential dissociation sensitivity; DGE power depends on cell number, transcription level, and algorithm, and MAST's Bayesian shrinkage underestimates fold change while the approach may underrepresent changes in lowly transcribed genes.

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

Integrity signals

Registry

Crossref · 10.1038/s41593-019-0491-3

Cites

140 of 140 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 55 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    Differences in transcriptional variability between young and old cells were observed in many cell types.

    “We observed differences in the variability of transcription between young and old cells in many cell types.”
    Quote in the paperSubstantial claim
  • Findingown data

    The most commonly aging-downregulated genes were mitochondrial respiratory chain complex genes (such as mt-Nd1), glycolysis-related genes (such as Aldoc), genes encoding selenoproteins (such as Sepw1) and tetraspanins (such as Tspan7).

    “The most commonly aging-downregulated genes were mitochondrial respiratory chain complex genes (such as mt-Nd1), glycolysis-related genes (such as Aldoc), genes encoding selenoproteins (such as Sepw1) and tetraspanins (such as Tspan7)”
    Quote in the paperSubstantial claim
  • Findingown data

    Specific markers distinguish each cell type regardless of age.

    “This also permitted us to identify specific markers that distinguish each type regardless of age (Table S3).”
    Quote in the paperNot a substantial claim
  • Findingown data

    Aggregated whole-brain profile verified previously identified top aging-related genes (Apod, B2m, C1qa, C4b, Ctss, Il33, Rpl8).

    “As expected, this analysis verified previously identified top aging-related genes (such as Apod, B2m, C1qa, C4b, Ctss, Il33, Rpl8).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [5] Zahn, J. M. et al. AGEMAP: a gene expression database for aging in mice. PLoS Genet. 3, e201 (2007). source

      Not checked in the free run.

    • cites [7] Clarke, L. E. et al. Normal aging induces A1-like astrocyte reactivity. Proc. Natl Acad. Sci. USA 115, E1896–E1905 (2018).

      Not checked in the free run.

    • cites [37] Holtman, I. R. et al. Induction of a common microglia gene expression signature by aging and neurodegenerative conditions: a co-expression meta-analysis. Acta Neuropathol. Commun. 3, 31 (2015). source

      Not checked in the free run.

  • Findingown data

    The study identified novel aging-related genes not reported previously (Apoc1, Caly, Cxcl12, Nell2, Ybx1).

    “we were able to identify a set of novel aging-related genes not reported previously (such as Apoc1, Caly, Cxcl12, Nell2, Ybx1; presented in Table S5).”
    Quote in the paperSubstantial claim
  • May combine two claims. 34
  • Classified as probably not a substantial claim. 20

  • May combine two claims.

    Findingown data

    The analysis identified 37,089 cells representing 25 cell populations with distinct expression profiles.

    “Ultimately, our analysis led to the identification of 37,089 cells (Fig. S5A), representing 25 cell populations (Fig. 1B) with distinct expression profiles”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Certain genes traditionally used as cell type specific markers change with aging, such as aging-related downregulation of Mog in OLG and Csf1r in MG, and aging-related upregulation of Cxcl12 in EC.

    “these data revealed that certain genes that are traditionally used as cell type specific markers change with aging, such as the agingrelated downregulation of Mog in OLG and Csf1r in MG, and the aging-related upregulation of Cxcl12 in EC”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Of the 1,117 genes, 1,034 exhibited the same directionality regardless of cell type identity (494 upregulated and 540 downregulated), while 83 genes changed direction across cell populations.

    “of those, 1,034 exhibited the same directionality regardless of the cell type identity (494 upregulated and 540 downregulated), while the direction of change in the expression of 83 genes was different across cell populations”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Nell2 is mostly transcribed in neuronal lineage cells and OEG, but its levels changed with aging only in OEG.

    “Another example is Nell2, which is mostly transcribed in neuronal lineage cells and OEG, but its levels changed with aging only in OEG.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Ctss is highly transcribed in all immune cells (MG, MAC, MNC, DC) but was only significantly changed with aging in MG.

    “For example, Ctss, while highly transcribed in all immune cells (MG, MAC, MNC, DC; see Table S2), was only significantly changed with aging in MG (Table S5).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    GSEA showed that with aging, the vast majority of differentiated brain cell types exhibited upregulation of genes encoding ribosomal subunits, while the majority of progenitor cells exhibited the opposite regulation.

    “this pathway analysis, despite employing stringent significance criteria, clearly showed that, with aging, the vast majority of the differentiated brain cell types exhibited upregulation of genes encoding ribosomal subunits, while the majority of progenitor cells exhibited the opposite regulation”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Aggregating all sequenced single cells and performing DGE comparable to prior bulk sequencing studies validated many previously identified aging-related genes and extended the list.

    “By aggregating all our sequenced single cells and performing DGE analysis comparable to prior bulk sequencing studies, we validated many of the previously identified aging-related genes 5,7,37 and extended the list to include numerous additional gene signatures.”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [5] Zahn, J. M. et al. AGEMAP: a gene expression database for aging in mice. PLoS Genet. 3, e201 (2007). source

      Not checked in the free run.

    • cites [7] Clarke, L. E. et al. Normal aging induces A1-like astrocyte reactivity. Proc. Natl Acad. Sci. USA 115, E1896–E1905 (2018).

      Not checked in the free run.

    • cites [37] Holtman, I. R. et al. Induction of a common microglia gene expression signature by aging and neurodegenerative conditions: a co-expression meta-analysis. Acta Neuropathol. Commun. 3, 31 (2015). source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    DGE and pathway analyses showed that the majority of differentiated brain cell types exhibited an aging-driven upregulation of ribosomal genes, while the majority of progenitor cells exhibited the opposite regulation.

    “Data from both DGE and pathway analyses showed that the majority of differentiated brain cell types exhibited an aging-driven upregulation of ribosomal genes, while the majority of progenitor cells exhibited the opposite regulation.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Both OPC and ASC downregulate a fraction of their ribosomal genes with aging, while other cell types upregulate their expression.

    “both OPC and ASC were found to downregulate a fraction of their ribosomal genes with aging, while the other cell types upregulated their expression.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    EC exhibit aging-regulated pathways including induction of senescence, hypoxia signaling and response to ketone signaling, and reduction of xenobiotic metabolism, lipid metabolism and hormone processing.

    “GSEA showed that EC exhibit numerous aging-regulated cellular pathways and biological processes, such as the aging-related induction of senescence, hypoxia 6 signaling and response to ketone signaling, and the aging-related reduction of xenobiotic metabolism, lipid metabolism and hormone processing”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Cldn5 is downregulated with aging in EC but upregulated in OEG.

    “We found aging-related downregulation of Cldn5 in EC but upregulation in OEG.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In total, 451 pathways (1,142 GSEA terms) changed significantly (p<0.05 & q<0.25); 234 were expressed in at least 2 cell types, while 217 were unique for specific cell populations.

    “In total, 451 pathways (1,142 GSEA terms) changed significantly (p<0.05& q<0.25); 234 were expressed in at least 2 cell types, while the remaining 217 were unique for specific cell populations.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Of the 451 aging-related pathways, 339 exhibited the same directionality regardless of cell type identity (195 upregulated and 144 downregulated), while directionality of changes in 112 varied across cell types.

    “Of the 451 aging-related pathways, 339 exhibited the same directionality regardless of cell type identity (195 were upregulated and 144 downregulated), while the directionality of changes in the remaining 112 varied across cell types”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Single cell type DGE analyses yielded a large number of aging-related genes that are commonly regulated among cell types, specific to certain cell types, and discordant between cell types.

    “More specifically, single cell type DGE analyses yielded a large number of aging-related genes that are: (a) commonly regulated among cell types, (b) specific to certain cell types and (c) discordant between cell types.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Immunohistochemistry verified the aging-related downregulation of SPARC in MG and the broad aging-related upregulation of IL33.

    “we verified the aging-related downregulation of SPARC in MG and the broad aging-related upregulation of IL33”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    RNAscope in situ hybridization in mouse hippocampi shows aging-related upregulation of Rpl6 and of the lncRNAs Malat1 and Meg3.

    “RNAscope in situ hybridization images (middle panels) of mouse hippocampi showing the aging-related upregulation of Rpl6, and of the lncRNAs Malat1 and Meg3.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    IL33, mainly expressed in oligodendrocytes, is upregulated with aging.

    “Violin plots and immunohistochemistry showing the aging-related up-regulation of IL33 (mainly expressed in OLG based on”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    In the vast majority of cell populations, Vcp levels remain unaltered with aging, in contrast to other commonly used genes like Gapdh.

    “Our analysis showed that in the vast majority of cell populations its levels remain unaltered with aging, in contrast to other more commonly used genes, like Gapdh.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The work identified aging-related changes in nearly all mouse brain cell types and revealed different patterns of aging across different cell populations, many of which were validated.

    “Nonetheless, our work identified aging-related changes in nearly all mouse brain cell types and revealed different patterns of aging across different cell populations, many of which we validated in this study.”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given

    The ability to identify genes whose transcription changes significantly with aging and the calculation of fold-change is dependent on several factors, including number of cells within each population, level of transcription, and algorithm for analysis.

    “our ability to identify genes whose transcription changes significantly with aging and the calculation of fold-change is dependent on several factors, including the number of cells within each population, the level of transcription, and the algorithm for analysis.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationcitation

    The directionality of change in transcriptional variability was not consistent among cell types, providing evidence that aging is not broadly associated with increased transcriptional variation.

    “However, the directionality of change was not consistent among cell types, providing evidence that aging is not broadly associated with increased transcriptional variation 34 .”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [34] Angelidis, I. et al. An atlas of the aging lung mapped by single cell transcriptomics and deep tissue proteomics. Nat. Commun. 10, 963 (2019). source

      Not checked in the free run.

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

    Premisecitation

    Lifespan and the integrity of multiple tissues can be regulated by discrete molecular modifications in model organisms.

    “That this might be achievable is supported by a plethora of studies using model organisms demonstrating that not only lifespan, but also the integrity of multiple tissues, can be regulated by discrete molecular modifications [2][3][4] .”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [2][3][4]

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Cells in the brain do not change with aging identically; while overlapping signatures exist, the cellular consequences of aging are not universal.

    “Our computational analysis suggests that cells in the brain do not change with aging identically, indicating that, while overlapping signatures exist, the cellular consequences of aging are not universal.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Prior transcriptomic studies generally use aggregated RNA from mixed cell populations or cell populations purified using known markers, which may themselves change during aging.

    “However, these studies generally utilize aggregated RNA from either mixed cell populations [5][6][7][8][9][10] that may vary in distinct ways with age, or cell populations purified using known markers [11][12][13][14][15] , which themselves may also change during aging.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [5][6][7][8][9][10]

      Not checked in the free run.

    • cites [11][12][13][14][15]

      Not checked in the free run.

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

    Premisecitation

    Aging, the time-dependent functional decline of organs and tissues, is the biggest risk factor for many diseases, including several neurodegenerative and cardiovascular disorders.

    “Aging, the time-dependent functional decline of organs and tissues, is the biggest risk factor for many diseases, including several neurodegenerative and cardiovascular disorders 1 .”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [1] Lopez-Otin, C., Blasco, M. A., Partridge, L., Serrano, M. & Kroemer, G. The hallmarks of aging. Cell 153, 1194–1217 (2013). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    Although estimated percentages for each cell type do not necessarily reflect actual proportions due to dissociation sensitivity, observed changes in cell type ratios appear to reflect a real biological effect.

    “although the estimated percentages for each cell type do not necessarily reflect their actual proportions in the mouse brain, mainly due to differences in their sensitivity to tissue dissociation, the observed changes in cell type ratios appear to reflect a real biological effect.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Differences in sensitivity to tissue dissociation do not confound observed age-related changes in cell type ratios.

  • May combine two claims.

    Premisecitation

    Recently published transcriptomic studies showed aging-related downregulation of ribosomal genes in ASC and NSC, and upregulation in MG of aged and diseased brains.

    “recently published transcriptomic studies showed an agingrelated downregulation of ribosomal genes in both ASC 14 and NSC 61 , and an upregulation in MG of both aged 62,63 and diseased brains 62,64 .”
    Quote in the paperNot a substantial claim0 of 4 sources checked
    • cites [14] Rosenberg, A. B. et al. Single-cell profiling of the developing mouse brain and spinal cord with split-pool barcoding. Science 360, 176–182 (2018). source

      Not checked in the free run.

    • cites [61] Llorens-Bobadilla, E. et al. Single-cell transcriptomics reveals a population of dormant neural stem cells that become activated upon brain injury. Cell Stem Cell. 17, 329–340 (2015). source

      Not checked in the free run.

    • cites [62] Shah, P. T. et al. Single-cell transcriptomics and fate mapping of ependymal cells reveals an absence of neural stem cell function. Cell 173, 1045–1057.e1049 (2018). source

      Not checked in the free run.

    • cites [63] Zywitza, V., Misios, A., Bunatyan, L., Willnow, T. E. & Rajewsky, N. Single-cell transcriptomics characterizes cell types in the subventricular zone and uncovers molecular defects impairing adult. Neurogenesis. Cell Rep. 25, 2457–2469.e2458 (2018). source

      Not checked in the free run.

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

    Premisecitation

    Attenuation of protein synthesis by dietary restriction or genetic manipulation of translation-associated genes increases lifespan of multiple species.

    “Over the past decades, it has been clearly shown that the attenuation of protein synthesis by dietary restriction or genetic manipulation of translationassociated genes, including those encoding ribosomal subunits, increases the lifespan of multiple species 53 .”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [53] Zheng, G. X. et al. Massively parallel digital transcriptional profiling of single cells. Nat. Commun. 8, 14049 (2017). source

      Not checked in the free run.

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

    Premisecitation

    Zahn et al. reported aging-driven upregulation of ribosomal genes in human brain and muscle tissues and later upregulation in mouse neuronal tissues but downregulation in non-neuronal tissues.

    “Zahn et al. reported an aging-driven upregulation of ribosomal genes in human brain and muscle tissues 37 and, in a later study, reported an aging-driven upregulation of ribosomal genes in mouse neuronal tissues 6 but a downregulation of the same genes in non-neuronal tissues 6 .”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [37] Holtman, I. R. et al. Induction of a common microglia gene expression signature by aging and neurodegenerative conditions: a co-expression meta-analysis. Acta Neuropathol. Commun. 3, 31 (2015). source

      Not checked in the free run.

    • cites [6] de Magalhaes, J. P., Curado, J. & Church, G. M. Meta-analysis of age-related gene expression profiles identifies common signatures of aging. Bioinformatics 25, 875–881 (2009). source

      Not checked in the free run.

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

    Premisecitation

    Down-regulation of ribosomal genes and bulk protein synthesis has long been considered a hallmark of aging.

    “Notably, the down-regulation of ribosomal genes and bulk protein synthesis has been long considered as a hallmark of aging 54,55 .”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [54] Satija, R., Farrell, J. A., Gennert, D., Schier, A. F. & Regev, A. Spatial reconstruction of single-cell gene expression data. Nat. Biotechnol. 33, 495–502 (2015). source

      Not checked in the free run.

    • cites [55] Macosko, E. Z. et al. Highly parallel genome-wide expression profiling of individual cells using nanoliter droplets. Cell 161, 1202–1214 (2015). source

      Not checked in the free run.

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

    Premisecitation

    Aging-driven down-regulation of ribosomal genes had been widely accepted, mostly based on transcriptomic studies in yeast.

    “It appears that aging-driven down-regulation of ribosomal genes had been widely accepted, mostly based on transcriptomic studies in yeast 56,57 .”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [56] Butler, A., Hoffman, P., Smibert, P., Papalexi, E. & Satija, R. Integrating single-cell transcriptomic data across different conditions, technologies, and species. Nat. Biotechnol. 36, 411–421 (2018). source

      Not checked in the free run.

    • cites [57] Marques, S. et al. Oligodendrocyte heterogeneity in the mouse juvenile and adult central nervous system. Science 352, 1326–1329 (2016). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    The interferon signaling upregulation in EPC aligns with induction of interferon-stimulated genes (like Ifit1) in DGE analysis and is similar to what has been previously reported for choroid plexus epithelium.

    “This aligns with the induction of certain interferon-stimulated genes (like Ifit1) that was shown in the DGE analysis (Fig. 2E and Table S5) and is similar to what has been previously reported for the choroid plexus epithelium 39 .”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [39] Keren-Shaul, H. et al. A unique microglia type associated with restricting development of Alzheimer’s disease. Cell 169, 1276–1290.e1217 (2017). source

      Not checked in the free run.

  • May combine two claims.

    Study design and conductown data

    The finding on transcriptional variability is in line with Warren et al. but in contrast to other studies that suggested increased transcriptional variability is a common feature of aging.

    “This is in line with Warren et al. 51 , but in contrast to other studies that suggested increased transcriptional variability is a common feature of aging 34,52 .”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [51] Zhang, R., Lahens, N. F., Ballance, H. I., Hughes, M. E. & Hogenesch, J. B. A circadian gene expression atlas in mammals: implications for biology and medicine. Proc. Natl Acad. Sci. USA 111, 16219–16224 (2014). source

      Not checked in the free run.

    • cites [34] Angelidis, I. et al. An atlas of the aging lung mapped by single cell transcriptomics and deep tissue proteomics. Nat. Commun. 10, 963 (2019). source

      Not checked in the free run.

    • cites [52] Saxena, A. et al. Trehalose-enhanced isolation of neuronal sub-types from adult mouse brain. Biotechniques 52, 381–385 (2012). source

      Not checked in the free run.

  • May combine two claims.

    Study design and conductown data

    Bulk RNA-seq recapitulated scRNA-seq data for a subset of key ribosomal genes, verifying divergent aging-driven ribosomal expression patterns among tested cell populations.

    “bulk RNA-seq recapitulated the scRNA-seq data for a subset of key genes, thus verifying the existence of divergent aging-driven ribosomal expression patterns among the tested cell populations”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    Recent findings from our lab and others have shown that certain secreted factors, either derived from brain parenchyma or blood, are able to modulate brain aging, degeneration and rejuvenation.

    “This is also of high importance as recent findings from our lab 66 and others 39,[67][68][69] have shown that certain secreted factors, either derived from brain parenchyma or blood, are able to modulate brain aging, degeneration and rejuvenation.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [66] Tasic, B. et al. Adult mouse cortical cell taxonomy revealed by single cell transcriptomics. Nat. Neurosci. 19, 335–346 (2016). source

      Not checked in the free run.

    • cites 39,[67][68][69]

      Not checked in the free run.

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

    Premisecitation

    A very recent study reported increased ribosomal biogenesis and activity as hallmarks of premature aging in human fibroblasts.

    “Intriguingly, a very recent study reported increased ribosomal biogenesis and activity as hallmarks of premature aging in human fibroblasts 65 .”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [65] Hochgerner, H., Zeisel, A., Lonnerberg, P. & Linnarsson, S. Conserved properties of dentate gyrus neurogenesis across postnatal development revealed by single-cell RNA sequencing. Nat. Neurosci. 21, 290–299 (2018). source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Study design and conductreasoning

    Including antibodies targeting unwanted cell populations is critical because it helps exclude these cells during sorting, minimizing cross-contamination events.

    “This step is critical as it helps to exclude these cells during sorting, thus minimizing crosscontamination events.”
    Quote in the paperSubstantial claim

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

    Study design and conductown data

    Post-sequencing quality control was performed on each library to assess coverage depth, mRNA enrichment, rRNA fraction, and number of detected genes.

    “Post-sequencing quality control on each of the libraries was performed to assess coverage depth, enrichment for messenger RNA (exon/intron and exon/intergenic density ratios), fraction of rRNA reads and number of detected genes using bespoke scripts.”
    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

    A chloroform extraction step and an on-column DNase digestion step were included to remove myelin debris/lipids and genomic/mitochondrial DNA, respectively.

    “Prior to RNA extraction, a chloroform extraction step was included to remove myelin debris/lipids, as well as, an on-column DNase digestion step (Qiagen #79254) to remove genomic and mitochondrial DNA.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    To exclude cellular debris and dead cells, Calcein Blue AM was added to stain live cells before sorting.

    “To exclude cellular debris and dead cells, 15min before sorting, 10uM Calcein Blue AM (BD Biosciences #564060) was added to the FACS tubes to stain live cells.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Study design and conductreasoning

    Due to shrinkage in the Bayes approach leveraged by MAST, the analysis was able to detect significance in very small changes in transcription but also underestimated fold change.

    “It is worth mentioning that due to shrinkage in the Bayes approach leveraged by MAST, we were able to detect significance in very small changes in transcription but there was also an underestimation of fold change.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductown data

    Doublet artifacts arising from co-capture of multiple cells in one droplet were removed.

    “The final pre-processing stage was to remove likely doublet artifacts arising from the co-capture of multiple cells in one droplet.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Study design and conductreasoning

    The ability to establish a baseline level of transcription is proportional to the number of cells measured, and thus more subtle changes in abundant populations can be deemed significant.

    “Finally, our ability to establish a baseline level of transcription is proportional to the number of cells measured and thus more subtle changes in abundant populations can be deemed significant.”
    Quote in the paperNot a substantial claim

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

    Study design and conductown data

    Multiple NextSeq runs achieved over 70% sequencing saturation (median 75%).

    “Multiple NextSeq runs were conducted to achieve over 70% sequencing saturation as determined again by Cell Ranger (median: 75%; Fig. S2F).”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    Vcp is a housekeeping gene that has been proposed for calibration in quantitative experiments.

    “The fold-change (FC) in gene expression was determined by the 2 -DDC T method 100 , and all values were normalized to the endogenous expression of Vcp; a housekeeping gene that has been proposed for calibration in quantitative experiments 101 .”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [101] Mann, H. B. & Whitney, D. R. On a test of whether one of two random variables is stochastically larger than the other. Ann. Math. Stat. 18, 50–60 (1947). source

      Not checked in the free run.

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

    Study design and conductnone given

    Gene lists were manually curated to correct or remove genes that were misclassified.

    “Gene lists were manually curated to correct or remove genes that were misclassified.”
    Quote in the paperNot a substantial claim

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

    Study design and conductown data

    After initial quality control, 38,244 cells and 14,699 genes were maintained.

    “After initial quality control (QC), we maintained a total of 38,244 cells and 14,699 genes.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    While there may be hallmarks of aging that occur in most cell types, such as mitochondrial dysfunction and loss of proteostasis, the data argue against the hypothesis that aging induces a single universal molecular program in all cells and tissues.

    “Thus, while there may be hallmarks of aging that occur in most cell types, such as mitochondrial dysfunction and loss of proteostasis 1,4,54 , our data argue against the hypothesis that aging induces a single universal molecular program in all cells and tissues 16 .”
    Quote in the paperSubstantial claim0 of 4 sources checked
    • cites [1] Lopez-Otin, C., Blasco, M. A., Partridge, L., Serrano, M. & Kroemer, G. The hallmarks of aging. Cell 153, 1194–1217 (2013). source

      Not checked in the free run.

    • cites [4] Lee, C. K., Weindruch, R. & Prolla, T. A. Gene-expression profile of the ageing brain in mice. Nat. Genet. 25, 294–297 (2000). source

      Not checked in the free run.

    • cites [54] Satija, R., Farrell, J. A., Gennert, D., Schier, A. F. & Regev, A. Spatial reconstruction of single-cell gene expression data. Nat. Biotechnol. 33, 495–502 (2015). source

      Not checked in the free run.

    • cites [16] Sim, F. J., Zhao, C., Penderis, J. & Franklin, R. J. The age-related decrease in CNS remyelination efficiency is attributable to an impairment of both oligodendrocyte progenitor recruitment and differentiation. J. Neurosci. 22, 2451–2459 (2002). source

      Not checked in the free run.

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

    Study design and conductnone given

    Before data analysis, melting curves were examined and only reactions with a single peak at the expected melting temperature were included.

    “Before data analysis, we examined the melting curves for each reaction and included only those with a single peak at the expected melting temperature.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Study design and conductreasoning

    The DGE techniques employed have more power to assign significance to subtle changes in highly transcribed genes, and therefore the results may underrepresent changes in lowly transcribed genes.

    “Additionally, the DGE techniques employed here have more power to assign significance of subtle changes in highly transcribed genes and therefore our results may underrepresent changes in lowly transcribed genes.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    Collecting cells directly in RL buffer supplemented with BME minimizes RNA degradation.

    “To minimize RNA degradation, cells were collected directly in RL buffer (Norgen Biotek #48500) supplemented with 10% BME, in a 1:1 final ratio (50% lysis buffer : 50% cells in sheath fluid; the PBS-based solution that is derived from the flow cytometer).”
    Quote in the paperNot a substantial claim
51 of 51

  • The paper relies on this without support.

    Exactly how the brain is affected with aging remains largely unknown.

    “The mammalian brain is complex, with multiple cell types performing a variety of diverse functions, but exactly how the brain is affected with aging remains largely unknown.”

  • The paper relies on this without support.

    This study is the first large-scale transcriptomic analysis of aging for the vast majority of individual brain cell types.

    “This is the first large-scale transcriptomic analysis of aging for the vast majority of individual brain cell types.”

  • The paper relies on this without support.

    Differences in sensitivity to tissue dissociation do not confound observed age-related changes in cell type ratios.

    “although the estimated percentages for each cell type do not necessarily reflect their actual proportions in the mouse brain, mainly due to differences in their sensitivity to tissue dissociation, the observed changes in cell type ratios appear to reflect a real biological effect.”

  • The paper relies on this without support.

    Aging processes observed in the mouse brain are representative of mammalian brain aging.

    “The transcriptomic database reported here is the first to examine the aging process in the mammalian brain at a single-cell level.”

  • The paper relies on this without support.

    Only a small fraction of the genes reported here have been previously associated with brain aging.

    “To the best of our knowledge, only a small fraction of the genes reported here have been previously associated with brain aging.”
5 of 5

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 aging-related Oligo-3 cluster exhibited substantial upregulation of C4b and interleukin 33 (Il33).

    “The aging-related cluster Oligo-3 exhibited substantial upregulation of C4b (Figure 2C), a complement protein of the innate immunity system, and interleukin 33 (Il33) (Figure 2C), a cytokine involved in inflammatory and innate immune response,39 consistent with previous results.17”

    From Molecular and spatial signatures of mouse brain aging at single-cell resolution (2023) · cites this paper as [17]

    The ‘Identification of shared and cell-type specific aging signatures’ section reports aging-related IL33 upregulation, with most of its signal arising from oligodendrocytes (Fig. 2E; page unavailable). Our copy does not identify an Oligo-3 cluster or report C4b upregulation in it, so it does not support those more specific parts of the claim. (LLM, conf=0.85)

    Experimental — accuracy not yet measured

  • Genes upregulated with age in neurons, especially inhibitory neurons, were enriched in neurodegenerative disease, oxidative response, and mitochondria pathways, whereas age-upregulated genes in non-neuronal cells tended to be associated with inflammatory and immune response.

    “genes upregulated with age in neurons, in particular in inhibitory neurons, were enriched in pathways associated with neurodegenerative diseases, oxidative response, and mitochondria function, whereas genes upregulated with age in non-neuronal cells tended to be associated with inflammatory and immune response”

    From Molecular and spatial signatures of mouse brain aging at single-cell resolution (2023) · cites this paper as [17]

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

    Experimental — accuracy not yet measured

  • Aging-related cell states exhibited upregulation of genes (e.g., B2m and Trem2 in Micro-3, Xdh in Endo-3, Gfap and C4b in Astro-2) previously shown to be enriched in activated microglia, endothelial cells, and astrocytes.

    “These aging-related cell states exhibit upregulation of genes (e.g., B2m and Trem2 in Micro-3, Xdh in Endo-3, and Gfap and C4b in Astro-2) (Figure 2C) that have been previously shown to be enriched in microglia, endothelial cells, and astrocytes activated by inflammation and/or aging.”

    From Molecular and spatial signatures of mouse brain aging at single-cell resolution (2023) · cites this paper as [17]

    The ‘Identification of shared and cell-type specific aging signatures’ section reports aging-related upregulation of B2m, identifies C4b as an aging-related gene, and describes Gfap as enriched in the astrocyte lineage. Our copy does not establish the stated Micro-3, Endo-3, or Astro-2 gene assignments, or show that the listed genes are enriched in microglia, endothelial cells, and astrocytes activated by inflammation or aging; searches for Trem2, Xdh, and the cluster names did not resolve those details. No page numbers were available for these passages. (LLM, conf=0.82)

    Experimental — accuracy not yet measured

  • Among genes most differentially expressed in oligodendrocytes are several genes that characterize this cell type during ageing and neurodegeneration.

    “Among the genes that were most differentially expressed in oligodendrocytes were several genes that characterize this cell type during ageing and neurodegeneration, including H2-D1, Ifi27, C4b, Spock3, Trf or Sgk1 (refs. 42,43).”

    From cGAS–STING drives ageing-related inflammation and neurodegeneration (2023) · cites this paper as [43]

    The 'Identification of shared and cell-type specific aging signatures' section reports aging-related downregulation of Mog in oligodendrocytes (OLG), supporting the general point that this cell type has an aging-related expression signature. Our copy does not identify the sentence's listed genes as the most differentially expressed in oligodendrocytes or establish that they characterize oligodendrocytes during neurodegeneration; it mentions C4b only among previously identified aging-related genes in a whole-brain analysis. Page numbers were not available in the indexed passages. (LLM, conf=0.84)

    Experimental — accuracy not yet measured

  • Previous transcriptomic studies have revealed widespread changes in cell state in specific cell types with age, with many highlighting increased inflammation as a key aspect of brain aging.

    “Previous transcriptomic studies have revealed widespread changes in cell state in specific cell types with age, with many of these studies highlighting a role for increased inflammation as a key aspect of brain aging.17–20,25–30”

    From Molecular and spatial signatures of mouse brain aging at single-cell resolution (2023) · cites this paper as [17]

Cites

Flagged first, then by citations

Cited by

Sorted by citations

Related

Authors
Ximerakis, M., Lipnick, S. L., Innes, B. T., Simmons, S. K., Adiconis, X., Dionne, D., Mayweather, B. A., Nguyen, L., Niziolek, Z., Ozek, C., Butty, V. L., Isserlin, R., Buchanan, S. M., Levine, S. S., Regev, A., Bader, G. D., Levin, J. Z., & Rubin, L. L.
Affiliations
  • Broad Institute (US)
  • University of Toronto (CA)
  • Harvard University (US)
  • Massachusetts Institute of Technology (US)
Corresponding author
  • Ximerakis, Methodios
  • Rubin, Lee L.
ORCID
Venue
Nature Neuroscience
Publisher
Springer Science and Business Media LLC
Published
2019-09-24
Volume
22
Issue
10
Pages
1696-1708
ISSN
  • 1097-6256 (ISSN-L)
  • 1546-1726
DOI
10.1038/s41593-019-0491-3
OpenAlex
W2975507539
Semantic Scholar
3c98121a8e1343fb4d5094e185619fa67919a6c0
Cited by
736
References
140
Citations by year
2026: 67 · 2025: 142 · 2024: 130 · 2023: 133 · 2022: 117 · 2021: 82 · 2020: 58 · 2019: 6 · 2018: 1
FWCI
22.18
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Neuroscience (publishedVersion)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
  • PubMed (publishedVersion)
  • Digital Access to Scholarship at Harvard (DASH) (Harvard University) (acceptedVersion)
  • DSpace@MIT (Massachusetts Institute of Technology) (submittedVersion, cc-by-nc)
Funders
The work was supported by Ono Pharmaceutical Co., Ltd (L.L.R.)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Single-cell and spatial transcriptomics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Aging › Genetics, Aging, and Longevity in Model Organisms
  • Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
Keywords
brain aging, mouse brain, single-cell transcriptomics, cell type-specific gene expression, aging-related genes, ligand-receptor interactions, ribosome biogenesis, gene expression signatures
MeSH terms
Aging, Animals, Brain, Cell Communication, Gene Expression Regulation, Male, Mice, Inbred C57BL, Neurons, Ribosomes, Cell Lineage, Gene Expression Profiling, Mice, Single-Cell Analysis, High-Throughput Nucleotide Sequencing, 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 2026-10-10.

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

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.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review