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Cited by 110Telomeres, Telomerase, and SenescenceGenetics, Aging, and Longevity in Model OrganismsNeuroinflammation and Neurodegeneration Mechanisms

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Changes in the Transcriptome of Human Astrocytes Accompanying Oxidative Stress-Induced Senescence

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Crowe, Elizabeth P., Tuzer, Ferit, Gregory, Brian D., Donahue, Greg, Gosai, Sager J., Cohen, Justin, Leung, Yuk Y., Yetkin, Emre, Nativio, Raffaella, Wang, Li-San, Sell, Christian, Bonini, Nancy M., Berger, Shelley L., Johnson, F. Brad, Torres, Claudio·2016·Frontiers in Aging Neuroscience
DOI 10.3389/fnagi.2016.00208PMC PMC5005348
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Abstract

Aging is a major risk factor for many neurodegenerative disorders. A key feature of aging biology that may underlie these diseases is cellular senescence. Senescent cells accumulate in tissues with age, undergo widespread changes in gene expression, and typically demonstrate altered, pro-inflammatory profiles. Astrocyte senescence has been implicated in neurodegenerative disease, and to better understand senescence-associated changes in astrocytes, we investigated changes in their transcriptome using RNA sequencing. Senescence was induced in human fetal astrocytes by transient oxidative stress. Brain-expressed genes, including those involved in neuronal development and differentiation, were downregulated in senescent astrocytes. Remarkably, several genes indicative of astrocytic responses to injury were also downregulated, including glial fibrillary acidic protein and genes involved in the processing and presentation of antigens by major histocompatibility complex class II proteins, while pro-inflammatory genes were upregulated. Overall, our findings suggest that senescence-related changes in the function of astrocytes may impact the pathogenesis of age-related brain disorders.

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FIGURE 1 Differential expression analysis of pre-senescent vs. senescent astrocyte transcriptomes. (A) Principle component analysis (PCA) revealed the greatest variability in the RNA-Seq data was due to the treatment condition (PC = principle component). (B) DESeq Plot showing the expression fold changes compared to pre-senescent and the expression levels of 19520 transcripts (gray) out of which 3569 are differentially expressed transcripts with each symbol representing a single transcript (red symbols are statistically significant at p < 0.05 Benjamini–Hochberg adjusted). A positive log2 fold change indicates transcripts that are upregulated in senescent cells, whereas a negative log2 fold change represents transcripts significantly downregulated in pre-senescent astrocytes. “Mean normalized counts” are the raw read counts, normalized by the total library size, and averaged for each group.
FIGURE 1 Differential expression analysis of pre-senescent vs. senescent astrocyte transcriptomes. (A) Principle component analysis (PCA) revealed the greatest variability in the RNA-Seq data was due to the treatment condition (PC = principle component). (B) DESeq Plot showing the expression fold changes compared to pre-senescent and the expression levels of 19520 transcripts (gray) out of which 3569 are differentially expressed transcripts with each symbol representing a single transcript (red symbols are statistically significant at p < 0.05 Benjamini–Hochberg adjusted). A positive log2 fold change indicates transcripts that are upregulated in senescent cells, whereas a negative log2 fold change represents transcripts significantly downregulated in pre-senescent astrocytes. “Mean normalized counts” are the raw read counts, normalized by the total library size, and averaged for each group.CC BY · PubMed Central
FIGURE 2 Gene Ontology (GO) term enrichment analysis for senescent astrocyte down- and upregulated transcripts. (A) The top overrepresented GO biological process classes in astrocyte downregulated transcripts (padj < 0.05 and fold change of 1.5 or greater; Supplementary Table S3) are shown in the pie chart. The weighted number of genes in each category corresponds to the width of the wedges in the pie chart. (B) GO terms that are not cell division related are expanded below in a bar graph. Developmental categories are coded in gray, immune function is in green, sterol metabolism in blue, and development of the nervous system is in black. (C) The top overrepresented GO biological process classes by fold enrichment within astrocyte upregulated transcripts (padj < 0.05 and fold change of 1.5 or greater; Supplementary Table S4). (D) All GO terms overrepresented by the senescence upregulated genes, ranked by fold enrichment, color coded as in (C). *Full names of GO terms: antigen processing and presentation of peptide or polysaccharide antigen via major histocompatibility complex (MHC) class II, regulation of Wnt receptor signaling pathway, morphogenesis of a branching structure, the combined term of “cell projection morphogenesis, cell morphogenesis involved in differentiation, neuron differentiation,” regulation of smooth muscle cell proliferation, embryonic skeletal system development, regulation of I-kappaB kinase/NF-kappaB cascade, positive regulation of cytokine production, positive regulation of protein kinase cascade.
FIGURE 2 Gene Ontology (GO) term enrichment analysis for senescent astrocyte down- and upregulated transcripts. (A) The top overrepresented GO biological process classes in astrocyte downregulated transcripts (padj < 0.05 and fold change of 1.5 or greater; Supplementary Table S3) are shown in the pie chart. The weighted number of genes in each category corresponds to the width of the wedges in the pie chart. (B) GO terms that are not cell division related are expanded below in a bar graph. Developmental categories are coded in gray, immune function is in green, sterol metabolism in blue, and development of the nervous system is in black. (C) The top overrepresented GO biological process classes by fold enrichment within astrocyte upregulated transcripts (padj < 0.05 and fold change of 1.5 or greater; Supplementary Table S4). (D) All GO terms overrepresented by the senescence upregulated genes, ranked by fold enrichment, color coded as in (C). *Full names of GO terms: antigen processing and presentation of peptide or polysaccharide antigen via major histocompatibility complex (MHC) class II, regulation of Wnt receptor signaling pathway, morphogenesis of a branching structure, the combined term of “cell projection morphogenesis, cell morphogenesis involved in differentiation, neuron differentiation,” regulation of smooth muscle cell proliferation, embryonic skeletal system development, regulation of I-kappaB kinase/NF-kappaB cascade, positive regulation of cytokine production, positive regulation of protein kinase cascade.CC BY · PubMed Central
FIGURE 3 Tissue expression of detected transcripts in senescent astrocytes. (A) Tissue annotation expression for astrocyte 1.5-fold or more downregulated transcripts using DAVID (controlling for an FDR at 10%). The width of wedges corresponds to the number of astrocyte downregulated transcripts with tissue expression annotation information that are expressed in the indicated tissues. (B) Tissue expression annotation for all transcripts detected in astrocytes.
FIGURE 3 Tissue expression of detected transcripts in senescent astrocytes. (A) Tissue annotation expression for astrocyte 1.5-fold or more downregulated transcripts using DAVID (controlling for an FDR at 10%). The width of wedges corresponds to the number of astrocyte downregulated transcripts with tissue expression annotation information that are expressed in the indicated tissues. (B) Tissue expression annotation for all transcripts detected in astrocytes.CC BY · PubMed Central
FIGURE 4 Validation of selected genes by quantitative real-time PCR (qRT-PCR) and comparison with RNA-Seq log2 fold change. (A) A scatter plot of pre-senescence to senescence log2-fold changes obtained by RNA-Seq and qRT-PCR for selected transcripts. Each symbol represents a single transcript, n = 17, Pearson r = 0.809, (p < 0.001 95% CI 0.659 to 0.939). (B) Relative levels of astrocyte/brain enriched-genes. Error bars indicate SEM of two independent samples. (C) Relative levels of senescence or SASP-related transcripts. Error bars indicate SEM of two independent samples. ∗indicates p < 0.05, student’s t-test.
FIGURE 4 Validation of selected genes by quantitative real-time PCR (qRT-PCR) and comparison with RNA-Seq log2 fold change. (A) A scatter plot of pre-senescence to senescence log2-fold changes obtained by RNA-Seq and qRT-PCR for selected transcripts. Each symbol represents a single transcript, n = 17, Pearson r = 0.809, (p < 0.001 95% CI 0.659 to 0.939). (B) Relative levels of astrocyte/brain enriched-genes. Error bars indicate SEM of two independent samples. (C) Relative levels of senescence or SASP-related transcripts. Error bars indicate SEM of two independent samples. ∗indicates p < 0.05, student’s t-test.CC BY · PubMed Central
FIGURE 5 Cell cycle analysis of senescent astrocytes. (A) Representative cell cycle profiles of subconfluent pre-senescent astrocytes that were serum-starved, kept in complete growth media, or kept in complete growth media and treated with H2O2 (200 μM) to induce senescence 7 days prior to staining for DNA content and flow cytometric analysis. Arrowheads indicate, left: Diploid (2N), right: tetraploid (4N) DNA content. (B) Graph for percentage of cells in G0/G1 (blue), S (green), or G2/M (red) phases of the cell cycle, that is representative of at least four independent experiments. ∗p < 0.02, ∗∗p < 0.001 vs. pre-senescent in growth medium using one-way ANOVA followed by Bonferroni post hoc testing. (C) Pre-senescent and senescent astrocytes were stained for DNA content and BrdU incorporation. The BrdU-positive population was significantly reduced in senescent astrocytes compared with pre-senescent controls ∗p < 0.01, Student’s t-test. (D) Representative images of immunofluorescence staining for mitosis marker phosphorylated histone H3 (pH3) (red) and DAPI (blue), with percent of cells staining positive for pH3 shown in the bar graph. ∗p < 0.01, student’s t-test.
FIGURE 5 Cell cycle analysis of senescent astrocytes. (A) Representative cell cycle profiles of subconfluent pre-senescent astrocytes that were serum-starved, kept in complete growth media, or kept in complete growth media and treated with H2O2 (200 μM) to induce senescence 7 days prior to staining for DNA content and flow cytometric analysis. Arrowheads indicate, left: Diploid (2N), right: tetraploid (4N) DNA content. (B) Graph for percentage of cells in G0/G1 (blue), S (green), or G2/M (red) phases of the cell cycle, that is representative of at least four independent experiments. ∗p < 0.02, ∗∗p < 0.001 vs. pre-senescent in growth medium using one-way ANOVA followed by Bonferroni post hoc testing. (C) Pre-senescent and senescent astrocytes were stained for DNA content and BrdU incorporation. The BrdU-positive population was significantly reduced in senescent astrocytes compared with pre-senescent controls ∗p < 0.01, Student’s t-test. (D) Representative images of immunofluorescence staining for mitosis marker phosphorylated histone H3 (pH3) (red) and DAPI (blue), with percent of cells staining positive for pH3 shown in the bar graph. ∗p < 0.01, student’s t-test.CC BY · PubMed Central

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Authors
Crowe, E. P., Tuzer, F., Gregory, B. D., Donahue, G., Gosai, S. J., Cohen, J., Leung, Y. Y., Yetkin, E., Nativio, R., Wang, L.-S., Sell, C., Bonini, N. M., Berger, S. L., Johnson, F. B., & Torres, C.
Affiliations
  • Drexel University (US)
  • University of Pennsylvania (US)
Corresponding author
Torres, Claudio
ORCID
Venue
Frontiers in Aging Neuroscience
Publisher
Frontiers Media SA
Published
2016-08-31
Volume
8
Pages
208-208
ISSN
1663-4365 (ISSN-L)
DOI
10.3389/fnagi.2016.00208
PMC
PMC5005348
OpenAlex
W2508551815
Cited by
110
References
74
Citations by year
2026: 8 · 2025: 15 · 2024: 13 · 2023: 14 · 2022: 15 · 2021: 10 · 2020: 13 · 2019: 8 · 2018: 8 · 2017: 6
FWCI
5.86
Type
journal article
Language
en
Open access
Licence
cc-by
Locations
  • Frontiers in Aging Neuroscience (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • ScholarlyCommons (University of Pennsylvania) (submittedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
Funders
  • National Institute of Neurological Disorders and Stroke (RO1NS078283)
  • National Institute on Aging (R21AG046943, F30AG043307)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Telomeres, Telomerase, and Senescence
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Aging › Genetics, Aging, and Longevity in Model Organisms
  • Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
Keywords
astrocyte senescence, oxidative stress, transcriptome, cellular senescence, neurodegenerative diseases, aging, pro-inflammatory genes, glial fibrillary acidic protein, MHC class II antigen presentation

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