Amyloid beta acts synergistically as a pro-inflammatory cytokine

Abstract

The amyloid beta (Aβ) peptide is believed to play a central role in Alzheimer's disease (AD), the most common age-related neurodegenerative disorder. However, the natural, evolutionarily selected functions of Aβ are incompletely understood. Here, we report that nanomolar concentrations of Aβ act synergistically with known cytokines to promote pro-inflammatory activation in primary human astrocytes (a cell type increasingly implicated in brain aging and AD). Using transcriptomics (RNA-seq), we show that Aβ can directly substitute for the complement component C1q in a cytokine cocktail previously shown to induce astrocyte immune activation. Furthermore, we show that astrocytes synergistically activated by Aβ have a transcriptional signature similar to neurotoxic "A1" astrocytes known to accumulate with age and in AD. Interestingly, we find that this biological action of Aβ at low concentrations is distinct from the transcriptome changes induced by the high/supraphysiological doses of Aβ often used in in vitro studies. Collectively, our results suggest an important, cytokine-like function for Aβ and a novel mechanism by which it may directly contribute to the neuroinflammation associated with brain aging and AD.

Keywords: Alzheimer's disease; Amyloid beta; Astrocytes; Inflammation; Transcriptomics.

Fig. 1.. Transcriptome analysis of human astrocytes treated with high and low concentration Aβ.

(A) MA plot showing Log2 fold change and mean gene expression levels in primary human astrocytes treated with 1 μM Aβ for 24 h. Note >5000 significantly increased/decreased genes shown in red (FDR < 0.1). (B) Top transcriptional modules increased/decreased in gene ontology (GO) analysis of 1 μM Aβ-treated astrocytes. (C) MA plot showing Log2 fold change and mean gene expression (no significant changes) in astrocytes treated with 10 nM Aβ for 24 h. All experiments performed in triplicate with cells from one donor.

Fig. 2.. Astrocyte immune activation induced by cytokines and Aβ/cytokine cocktails.

(A) Representative immunofluorescence images of the astrocyte activation marker CD44 in cells treated with TNFα, IL-1α and C1q, as well as Aβ (all alone or in combination). Note greater staining with all-cytokine cocktail and when Aβ is substituted for IL-1α or C1q (relative signal quantified at right). (B) Western blots showing that Aβ monomers (m) and oligomers (o) can substitute for IL-1α or C1q to induce immune activation, marked by increased expression of the pro-inflammatory cytokine IL-1β and the astrocyte activation marker GFAP. Note somewhat greater activation when Aβ is substituted for C1q. (C) Dose-response experiments showing that nanomolar concentrations of Aβ cause astrocyte activation (increased expression of IL-1β and the reactive astrocyte marker ICAM-1) when combined with TNF-α and IL-1α. All experiments performed 3–5 times in primary human astrocytes from one donor. *P < 0.05 vs. control, unpaired two-tailed t-test.

Fig. 3.. Similar transcriptional changes with cytokines and Aβ/cytokine cocktails.

(A-E) MA plots showing Log2 fold change and mean gene expression in astrocytes treated with low-dose (10 nM) Aβ and/or combinations of TNF-α, IL-1α or C1q. Note no transcriptome changes with 10 nM Aβ alone and modest changes with TNF-α + IL-1α, but major and similar differences with both TNF-α + IL-1α + C1q and TNF-α + IL-1α + Aβ. Significantly increased/decreased genes in red (FDR < 0.1). (F) Correlation between gene expression changes induced by cytokine cocktail vs. Aβ/cytokine cocktail. (G) Comparison of gene ontology (GO) terms significantly increased/decreased by cytokine-only and Aβ/cytokine cocktail. (H) Weak correlation between gene expression changes induced by Aβ/cytokine cocktail vs. high concentration (1 μM) Aβ alone; transcripts most different with Aβ/cytokine cocktail vs. Aβ alone in blue. All experiments performed in primary human astrocytes from three different donors.

Fig. 4.. Upregulation of immune response and reactive astrocyte genes by Aβ/cytokine cocktail.

(A) MA plot showing Log2 fold change and mean gene expression in astrocytes treated with TNF-α + IL-1α + Aβ vs. TNF-α + IL-1α alone (significantly increased/decreased genes in red, FDR < 0.1; data on primary human astrocytes from three different donors, as in Fig. 3). (B) Enhanced immune activation/cytokine signaling indicated by gene ontology analysis of genes differentially expressed when Aβ is added to TNF-α + IL-1α (based on significantly modulated transcripts in A). (C–D) Western blot and quantitative RT-PCR conifrmation of reactive astrocyte markers and innate immune mediators identified as increased/decreased with Aβ/cytokine cocktail treatment in RNA-seq data. Data collected using RNA and protein lysate from the same cells (one donor in triplicate) analyzed in Fig. 1 and Supplementary Fig. 1. *P < 0.05 vs. control, unpaired two-tailed t-test. (E) Heat map showing relative expression of reactive astrocyte markers in response to Aβ and/or cytokines based on RNA-seq data from primary human astrocytes (one donor in triplicate as in Fig. 1 and Supplementary Fig. 2).