Abstract
Delivery of exogenous heat shock protein 90α (Hsp90α) and/or its induced expression in neural tissues has been suggested as a potential strategy to combat neurodegenerative disease. However, within a neurodegenerative context, a pro-inflammatory response to extracellular Hsp90α (eHsp90α) could undermine strategies to use it for therapeutic intervention. The aim of this study was to investigate the biological effects of eHsp90α on microglial cells, the primary mediators of inflammatory responses in the brain. Transcriptomic profiling by RNA-seq of primary microglia and the cultured EOC2 microglial cell line treated with eHsp90α showed the chaperone to stimulate activation of innate immune responses in microglia that were characterized by an increase in NF-kB-regulated genes. Further characterization showed this response to be substantially lower in amplitude than the effects of other inflammatory stimuli such as fibrillar amyloid-β (fAβ) or lipopolysaccharide (LPS). Additionally, the toxicity of conditioned media obtained from microglia treated with fAβ was attenuated by addition of eHsp90α. Using a co-culture system of microglia and hippocampal neuronal cell line HT22 cells separated by a chamber insert, the neurotoxicity of medium conditioned by microglia treated with fAβ was reduced when eHsp90α was also added. Mechanistically, eHsp90α was shown to activate Nrf2, a response which attenuated fAβ-induced nitric oxide production. The data thus suggested that eHsp90α protects against fAβ-induced oxidative stress. We also report eHsp90α to induce expression of macrophage receptor with collagenous structure (Marco), which would permit receptor-mediated endocytosis of fAβ.
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13 July 2022
A Correction to this paper has been published: https://doi.org/10.1007/s12192-022-01285-x
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Acknowledgements
We wish to thank the Department of Radiation Oncology for their continued support. Suraya Yasmine, Reeham Choudhury and Lay-Hong Ang are thanked for fine technical contributions. We thank Harvard Biopolymers Facility and Harvard Research Computing for their technical support. Figures 2A and 4A were made with Biorender.com.
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The original version of this article was revised: due to a technical error Figures 1 and 2 were incomplete. Figs 1F, 1G and Figs 2G, 2H have been added.
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12192_2022_1279_Fig7_ESM.png
Supplementary Fig. 1 (PNG 705 KB) (A) Gene set enrichment GO analysis of DEG list of cultured primary microglia treated with or without 10 µg/ml Hsp90α for 12 h. B Relative levels of mRNAs in EOC2 cells and primary microglia treated with or without 10 µg/ml Hsp90α for 4 h or 12 h, respectively, that were found to be differentially expressed and that have been categorized under GO:1901701 ‘cellular response to oxygen-containing compound’. Gene expression levels were quantified by RNA-seq and the relative levels shown are z-scores of TMM-normalized counts.
12192_2022_1279_Fig8_ESM.png
Supplementary Fig. 2 (PNG 543 KB) (A) Relative levels of Marco mRNA in EOC2 cells treated with or without 10 µg/ml Hsp90α for 4 h, quantified by RNA-seq. Relative levels shown are z-scores of TMM-normalized counts. Fold change was determined using the edgeR statistical package. B RT-qPCR quantitation of Marco normalized to Rpl32 in BV2 cells after 12 h. * p < 0.05. n=4. C Quantitation of Immunofluorescence staining for Marco in paraformaldehyde fixed EOC2 cells treated with 10 µg/ml Hsp90α for 24 h, scale bar =100 µm. n=3. D BV2 cells were treated with Nrf2 inhibitor ML385 (ML) or vehicle control for the indicated periods at which time levels of Nrf2-encoding mRNA (Nfe2l2) were assessed.
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Okusha, Y., Lang, B.J., Murshid, A. et al. Extracellular Hsp90α stimulates a unique innate gene profile in microglial cells with simultaneous activation of Nrf2 and protection from oxidative stress. Cell Stress and Chaperones 27, 461–478 (2022). https://doi.org/10.1007/s12192-022-01279-9
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DOI: https://doi.org/10.1007/s12192-022-01279-9