Abstract
The emergence of degenerative disease after traumatic brain injury (TBI) is often described as an acceleration of normal age-related processes. Whether similar molecular processes occur after injury and in age is unclear. Here, we identify a functionally dynamic and lasting transcriptional response in glia, mediated by the conserved transcription factor AP1. In the early post-TBI period, glial AP1 is essential for recovery, ensuring brain integrity and animal survival. In sharp contrast, chronic AP1 activation promotes human tau pathology, tissue loss and mortality. We show a similar process activates in healthy fly brains with age. In humans, AP1 activity is detected after moderate TBI and correlates with microglial activation and tau pathology. Our data provide key molecular insight into glia, highlighting that the same molecular process drives dynamic and contradictory glia behavior in TBI and possibly age, first acting to protect but chronically promoting disease.
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Data availability
The differential gene expression lists generated in this study are included in this article (Supplementary Tables 5–7). dTBI sequencing data (fastq files and HTSeq counts) that support the findings of this study have been deposited in the Gene Expression Omnibus (accession code GSE171185). Additional data that support the findings of this study are available from the corresponding author upon reasonable request.
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Acknowledgements
We thank G. Donahue for early guidance on analyzing RNA-seq data; V. Lee and S. Narasimhan for helpful discussions on tau; R. Bonasio, L. Goodman, E. Lee, A. Perlegos and K. Simeonov for manuscript feedback; F. Carranza and Z. Jin for technical assistance; M. Kayser, G. Ming and D. Meany for helpful thesis feedback; and S. Lindquist for continued inspiration. This work was supported by T32-AG000255, F31-NS111868 (to C.N.B.), a predoctoral Howard Hughes Medical Institute fellowship (to J.S.) and R35-NS097275 (to N.M.B.).
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C.N.B., under the supervision of N.M.B., executed the conceptualization, investigation, formal analysis and visualization; J.S. contributed to RNA-seq study conceptualization. C.N.B. wrote and edited the manuscript, with input from N.M.B. Funding acquisition was obtained by C.N.B., J.S. and N.M.B.
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Peer review information Nature Aging thanks David Sharp and the other, anonymous reviewer(s) for their contribution to the peer review of this work.
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Extended data
Extended Data Fig. 1 A lasting AP1 transcriptional response to TBI.
a, Kaplan-Meier survival curve (with Log-rank test) for 10 and 15 dpi RNAseq replicate animals. b, Venn diagram showing the number of common and distinct differentially expressed genes (FDR < 0.05) between post-injury times. Value in parenthesis shows total (up and down) number of DE genes for a given timepoint. c, Results for HOMER de novo motif enrichment among downregulated genes (FDR < 0.05). d, Tile plot showing Reactome pathways enriched among upregulated genes with a predicted AP1 motif (FDR < 0.05). Presence of a colored tile indicates enrichment at a given post-injury time. Tile opacity encodes significance. Color corresponds to the parent process, as defined by the Reactome annotation database (annotated on left). e, Average sham (black) and severe dTBI (red) expression of genes related to the heat shock response at ≤1 dpi (top) and ≥1 dpi (bottom; blue star indicates FDR < 0.05 at a given time). f, Average sham (black) and severe dTBI (red) expression of canonical stress response genes at ≤1 dpi (top) and ≥1 dpi (bottom; blue star indicates FDR < 0.05 at a given time). For full statistical reporting, including exact p-values, see Source Data Extended Data Fig. 1. See Supplementary Table 1 for genotypes.
Extended Data Fig. 2 Sustained and severity-dependent activation of AP1.
a, Representative z-stacked wholemount brains at 1 dpi in flies with dsRed expressed under a mutated TRE-promoter (MREdsRed; representative of n = 9–11 brains per condition from two independent experiments). b, Mean relative expression of select RNAseq predicted AP1 genes by RT-qPCR at 1, 7 and 15 dpi across injury conditions (each point = 1 biological replicate (9 dissected brains); n = 6 biological replicates per condition; Kruskal–Wallis test with Dunn’s multiple comparison test and Holm adjustment for each gene). Black symbols, sham. Pink symbols, mild dTBI. Red symbols, severe dTBI. Statistical annotations are ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, .<p < 0.10. For full statistical reporting, including exact p-values, see Source Data Extended Data Fig. 2. See Supplementary Table 1 for genotypes. All scale bars 100 μm.
Extended Data Fig. 3 Glial AP1 activation is draper and JNK-independent.
a, Z-stack of the first 6 slices (2 μm each) of the antennal lobe with (RU; top) or without (vehicle; bottom) glial draper-RNAi. White arrows highlight hypertrophic glial processes. 1 day after 3rd antennal segment ablation (AL injury; n = 7–9 brains per condition). b, Left, z-stacked whole mount brains at 1 dpi with (RU; top) and without (vehicle; bottom) glial draper-RNAi. Right, quantification relative to left condition (each point = 1 brain; n = 11–14 brains per condition from two independent experiments; p = 2.17e-06, Kruskal–Wallis test with Wilcoxon rank sum test). c, Mean relative expression by RT-qPCR at 1 dpi, with or without glial draper-RNAi (each point = 1 biological replicate (9 dissected brains pooled); n = 6 biological replicates per condition; Kruskal–Wallis test with Dunn’s multiple comparison test). d, Average sham (black) and severe dTBI (red) expression of draper from RNAseq experiment (blue star FDR < 0.05). e, Representative whole mount for lysotracker at 1 dpi (severe) in WT (top; w1118) and draper-/- flies (middle: dTBI; bottom: sham). Right, quantification relative to left condition (each point = 1 brain; n = 7–11 brains per condition from two independent experiments; p = 0.00072, Kruskal–Wallis test with Dunn’s test). f, Western immunoblots for all replicates in Fig. 4a. See Source Data for uncropped gels with molecular weights. g, Quantification of dsRed immunofluorescence for Fig. 4b, relative to left condition (each point = 1 brain; n = 9-10 brains from two independent experiments; p = 0.000238, Kruskal–Wallis test with Wilcoxon test). h, Mean relative expression of basal dsRed and basket by RT-qPCR in 3 d whole flies, with (blue) or without JNK-RNAi (green) under a ubiquitous driver (each point = 1 biological replicate, n = 20 flies per biological replicate; Welch’s t-test). a-g, Black symbols, sham. Red symbols, severe dTBI. Statistical annotations are ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05. All statistical tests were two-sided. For full statistical reporting, see Source Data Extended Data Fig. 3. All scale bars 100 μm. See Supplementary Table 1 for genotypes.
Extended Data Fig. 4 AP1 activation requires ERK.
a, Images from Scope Single Cell Atlas of the Drosophila adult brain61 showing relative expression levels of repo, elav, rolled (ERK), and basket (JNK). b, Western immunoblots for all replicates of Fig. 4c. Samples are biological replicates. c, Quantification of dsRed immunofluorescence fold change for representative whole mount brains shown in Fig. 4d, relative to leftmost condition (each point = 1 brain; n = 9-15 brains per condition pooled from two independent experiments; p = 6.08e-7, one-way ANOVA with Tukey’s test). d, RNAseq data showing average sham (black) and severe dTBI (red) dFos and dJun expression (blue star FDR < 0.05). e, Quantification of dJun-GFP (left; p = 2.07e-5, Student’s t-test) and dFos-GFP (right; p = 2.56e-7, Student’s t-test) immunofluorescence change for Figs. 4e and 4f, relative to sham (each point = 1 brain; n = 7-10 brains per condition from two independent experiments). f, Quantification of dsRed immunofluorescence change for Fig. 4g, relative to left condition (each point = 1 brain; n = 9-15 brains per condition from two independent experiments; p = 0.0040, one-way ANOVA with Tukey’s test). g, pERK/ERK levels at 1 hpi (each point = 1 biological replicate, 8 dissected brains; n = 3 biological replicates per condition/genotype; p = 0.0159, two-way ANOVA). h, Mean relative expression of AP1 target genes by RT-qPCR at 15 dpi, with or without glial ERK RNAi from 12 dpi (each point = 1 biological replicate, 9 dissected brains; n = 6 biological replicates per condition; Kruskal–Wallis test with Dunn’s multiple comparison test and Holm adjustment). i, Western immunoblot for ERK in whole flies with (blue) or without (green) ERK RNAi expressed under an inducible ubiquitous promoter. Right, quantification (each point = 1 biological replicate, 8 whole flies; p = 0.0173, Welch t-test). b-h, Black symbols, sham. Red symbols, severe dTBI. Statistical annotations are ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05. All statistical tests were two-sided where applicable. For full statistical reporting, including exact p-values, see Source Data Extended Data Fig. 4. See Source Data for uncropped gels with molecular weights. See Supplementary Table 1 for genotypes.
Extended Data Fig. 5 Glial AP1 is required for TBI recovery.
a, Representative z-stacked whole mount brains focused on the central brain at 1 dpi in flies expressing a GFP-tagged variant of the AP1 target gene, Ets21c. Ets21c-GFP is detected in a handful of neurons at baseline but is dramatically upregulated in glia by 1 dpi (representative of n = 11-15 brains per condition, two independent experiments). b, Post-injury survival with (RU; dashed line) or without (vehicle; solid line) Ets21c-RNAi expressed in glia (n = 100 per condition, 5 vials of 20 flies; p < 0.0001, Kaplan-Meier analysis with log-rank comparison). c, Representative brain vacuolization at 1 dpi under sham and dTBI conditions, with (RU) or without (vehicle) JNK RNAi expression in glia. Quantification on right, expressed as % of total brain area that is vacuolized (each point = 1 brain; n = 9-10 brains per condition; two-way ANOVA revealed a non-significant interaction but a significant effect of dTBI, p = 6.1e-05). d, Representative brain vacuolization at 1 dpi under sham and dTBI conditions, with (RU) or without (vehicle) puckered expression in neurons. Quantification on right, expressed as % of total brain area that is vacuolized (each point = 1 brain; n = 18-19 brains per condition pooled from two independent experiments; two-way ANOVA revealed a non-significant interaction but a significant effect of dTBI, p = 3.55e-06).e, Post-injury survival with (RU; dashed line) or without (vehicle; solid line) puckered expression in neurons (n = 100 per condition, 5 vials of 20 flies; p < 0.0001, Kaplan-Meier analysis with log-rank comparison). Black symbols, sham. Red symbols, severe dTBI. Statistical annotations are ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05. All statistical tests were two-sided where applicable. For full statistical reporting, including exact p-values, see Source Data Extended Data Fig. 5. All scale bars 100 μm. See Supplementary Table 1 for genotypes.
Extended Data Fig. 6 Sustained glial AP1 activity drives human tau pathology.
a, Representative section of paraffin-embedded head at 10 dpi stained for AT8 when human tau is expressed in neurons (representative of n = 7 animals). b, Membrane immunostained for human tau in phosphatase treated (+) and untreated (-) protein samples isolated from the heads of 5 dpi (repoGS > UAS-hTau) or WT (w1118) flies (n = 30 per sample). c, Pearson correlation (two-sided) between percentage of total brain area vacuolized and number of AT8 + puncta (top) or AT100 + puncta (bottom) at 5 and 10 dpi (combined) in sham, mild or severe dTBI. Data shown is an alternative representation of Fig. 6a. d, Representative sections of paraffin embedded heads immunostained for AT100 (top row) or AT8 (bottom row), highlighting the concentration of phosphorylated tau puncta in neuropil and around vacuoles. Vacuoles are visualized by autofluorescence. e, Representative z-stacked hemisections of paraffin-embedded in WT (left; w1118) or AP1 blocked (right) mild dTBI at 10 dpi, immunostained for AT100. AP1 was blocked by expressing a dominant negative variant of dFos (dFos-DN). f, Quantification of AT00 + puncta (top) and % brain vacuolization (bottom) at 10 dpi with (UAS-dFos-DN) or without AP1 blockade (WT) in glia from 3 dpi (each point = 1 brain, n = 9-10 per condition/genotype pooled from two independent experiments; AT100: p = 0.051, brain vacuolization: p = 0.072, two-way ANOVA with Tukey’s test). g, Post-injury survival with (solid) or without (dashed) UAS-dFos-DN expression in glia in the setting of tau expression (n = 100 per condition, 5 vials of 20 flies; p < 0.0001, Kaplan-Meier analysis with two-sided log-rank comparison). Black symbols, sham. Pink symbols, mild dTBI. Red symbols, severe dTBI. Statistical annotations are ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05. All statistical tests were two-sided where applicable. For full statistical reporting, including exact p-values, see Source Data Extended Data Fig. 6. All scale bars 100 μm. See Supplementary Table 1 for genotypes.
Extended Data Fig. 7 Evidence of AP1 activity after moderate human TBI.
a, TBI exposure history for donors; the same data is visualized two ways. Top shows age at first TBI, with most injuries occurring before age 30. Donors are further separated by duration of loss-of-consciousness. Total lifetime TBI events for each donor are shown by point size. Bottom shows the duration of time between first TBI and death. b, PCA of all samples (left) compared to PCA of samples with outliers removed (right). Each point represents 1 tissue sample. Shape encodes brain region. Black symbols, non-TBI samples. Red symbols, TBI samples. c, Top 5 Molecular Signature Data Base (MSigDb) Hallmark gene sets and top 10 gene ontology (GO) terms enriched among upregulated genes (FDR < 0.10) in TBI donors. d, Violin plots showing expression of all predicted FRA1/JUND target genes in non-TBI (black; n samples = 81) and TBI (red; n samples = 80) exposed donors (FDR < 0.10).
Extended Data Fig. 8 Evidence of a dTBI-specific dFos isoform.
Aligned bigWig files (IGV; aligned to Fly Base d. mel r6.17) for severe dTBI (top row) and sham injury at 24 hpi (bottom row), along with controls (0h; middle row), at the dFos locus (Fly gene name kay or kayak). With dTBI, reads map to dFos-RB, which encodes isoform B.
Supplementary information
Supplementary Information
Supplementary Tables 1–4; Supplementary statistical outputs (detailed statistical results)
Source data
Source Data Fig. 2
Full statistical results, including exact P values.
Source Data Fig. 4
Full statistical results, including exact P values.
Source Data Fig. 4
Unprocessed western blots.
Source Data Fig. 5
Full statistical results, including exact P values.
Source Data Fig. 5
Unprocessed western blots.
Source Data Fig. 6
Full statistical results, including exact P values.
Source Data Extended Data Fig. 1
Full statistical results, including exact P values.
Source Data Extended Data Fig. 2
Full statistical results, including exact P values.
Source Data Extended Data Fig. 3
Full statistical results, including exact P values.
Source Data Extended Data Fig. 4
Full statistical results, including exact P values.
Source Data Extended Data Fig. 4
Unprocessed western blots.
Source Data Extended Data Fig. 5
Full statistical results, including exact P values.
Source Data Extended Data Fig. 6
Full statistical results, including exact P values.
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Byrns, C.N., Saikumar, J. & Bonini, N.M. Glial AP1 is activated with aging and accelerated by traumatic brain injury. Nat Aging 1, 585–597 (2021). https://doi.org/10.1038/s43587-021-00072-0
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DOI: https://doi.org/10.1038/s43587-021-00072-0