Protective effects of apigenin on the brain transcriptome with aging

Abstract

Brain aging is associated with reduced cognitive function that increases the risk for dementia. Apigenin is a bioactive plant compound that inhibits cellular aging processes and could protect against age-related cognitive dysfunction, but its mechanisms of action in the brain have not been comprehensively studied. We characterized brain transcriptome changes in young and old mice treated with apigenin in drinking water. We observed improved learning/memory in old treated mice, and our transcriptome analyses indicated that differentially expressed genes with aging and apigenin were primarily related to immune responses, inflammation, and cytokine regulation. Moreover, we found that genes/transcripts that were increased in old vs. young mice but downregulated with apigenin treatment in old animals were associated with immune activation/inflammation, whereas transcripts that were reduced with aging but increased with apigenin were related neuronal function and signaling. We also found that these transcriptome differences with aging and apigenin treatment were driven in part by glial cells. To follow up on these in vivo transcriptome findings, we studied aged astrocytes in vitro, and we found that apigenin reduced markers of inflammation and cellular senescence in these cells. Collectively, our data suggest that apigenin may protect against age-related cognitive dysfunction by suppressing neuro-inflammatory processes.

Keywords: Astrocytes; Brain aging; Cognitive decline; Neuroinflammation; Transcriptomics.

Figure 1.. Apigenin improves learning/memory in old mice and modulates transcriptomic signatures of inflammation/immune activation.

(A-E) Cognitive-behavioral data on young and old mice treated with apigenin in drinking water for 6 weeks (YA and OA), as well as vehicle-treated young and old controls (YC and OC). (A) Total distance traveled in meters during open field testing; (B) time spent immobile during open field testing; (C) Average movement speed during open field testing; (D) Recognition index in NOR testing; (E) Discrimination ratio in NOR testing; Cognitive-behavioral data are means ± SD; n=8–12/group; * p<0.05 vs. YC, # p<0.05 vs, YA, † p<0.05 vs. AC. (F-I) Brain RNA-seq data on young and old apigenin-treated mice. (F) Volcano plot showing gene expression differences in OC vs. YC (n=4–5/group, red/green points=p<0.01); (G) Top biological processes/pathways related to gene expression differences in OC vs. YC (FDR<0.05; (H) Volcano plot showing gene expression differences in OA vs. OC (n=4–5/group, red/green points p<0.01); (I) Top significant pathways related to OA vs. OC (FDR<0.05). Pathway terms simplified for presentation; full list provided in supplement.

Figure 2.. Apigenin reverses gene expression signatures related to inflammation and immune activation in the aging mouse brain.

(A) Log2 fold changes of genes with the greatest increase in expression in old control (OC) vs. young control (YC) mice and the same genes in old apigenin-treated mice (OA) vs. OC (top 100 genes shown). (B) Top 20 significant KEGG and Reactome pathways (FDR<0.05) related to the most increased genes/transcripts in OC vs. YC that were also decreased in OA vs. OC. (C) Log2 fold changes of genes with the greatest decrease in expression in OC vs. YC and those same genes in OA vs. OC (top 100 genes shown). (D) biological processes/pathways (FDR<0.05) related to the most decreased genes/transcripts in OC vs. YC that were also increased in OA vs. OC. Pathway terms simplified for presentation, full list provided in supplement.

Figure 3.. Non-neuronal and glial cells contribute strongly to gene expression changes with aging and apigenin treatment.

(A) Principal component analyses of the top genes that increased (left) and decreased (right) in expression in old vs. young control mice. (B) Principal component analyses of the top ~150 genes that increased (left) and decreased (right) in old apigenin-treated mice vs. old controls. All data based on deconvolved bulk RNA-seq data from the same mice in Figures 1–2; n=4–5/group.

Figure 4.. Apigenin reduces markers of senescence and inflammation in aging-like primary human astrocytes.

(A) Representative immunoblots for markers of senescence and inflammation [all immunoblots shown in supplement fig. S2]. (B-C) Relative levels of the cellular senescence markers p16 and phosphorylated H2ax in fetal/young and aging-like (replicatively aged) astrocytes treated with or without 25 μM apigenin for 24 h. (D-E) Relative levels of the innate immune activation/inflammatory markers phosphorylated NFκB and interferon gamma (IFNγ) in the same cells. YC=Young control; YA=Young apigenin; AC=Aged control; AA=Aged apigenin. Data are means ± SD, normalized to GAPDH and YC; n=6–9/condition; * p<0.05 vs. YC, # p<0.05 vs, YA, † p<0.05 vs. AC.