Noncanonical Contribution of Microglial Transcription Factor NR4A1 to Post-stroke Recovery Through TNF mRNA Destabilization
Pinyi Liu, Yan Chen, Zhi Zhang, Zengqiang Yuan, Jian-Guang Sun, Shengnan Xia, Xiang Cao, Jian Chen, Cun-Jin Zhang, Yanting Chen, Hui Zhan, Yuexinzi Jin, Xinyu Bao, Yue Gu, Meijuan Zhang, Yun Xu
Abstract
Microglia-mediated neuroinflammation is involved in various neurological diseases, including ischemic stroke, but the endogenous mechanisms preventing unstrained inflammation is still unclear. The anti-inflammatory role of transcription factor nuclear receptor subfamily 4 group A member 1 (NR4A1) in macrophages and microglia has previously been identified. However, the endogenous mechanisms that how NR4A1 restricts unstrained inflammation remain elusive. Here, we observed that NR4A1 is up-regulated in the cytoplasm of activated microglia and localizes to processing bodies (P-bodies). In addition, we found that cytoplasmic NR4A1 functions as an RNA-binding protein (RBP) that directly binds and destabilizes Tnf mRNA in an N6-methyladenosine (m6A)-dependent manner. Remarkably, conditional microglial deletion of Nr4a1 elevates Tnf expression and worsens outcomes in a mouse model of ischemic stroke, in which case NR4A1 expression is significantly induced in the cytoplasm of microglia. Thus, our study illustrates a novel mechanism that NR4A1 posttranscriptionally regulates Tnf expression in microglia and determines stroke outcomes.
Introduction
Microglia are the resident macrophages in the brain, which exert critical physiological functions during development and homeostasis, such as scanning of the brain microenvironment, remodeling of synapses, and support for the development of oligodendrocytes [1,2]. In most neurological diseases, however, microglia respond rapidly to brain injuries and undergo distinct morphological and transcriptional changes [3,4]. In ischemic stroke, microglia are first activated after ischemia, releasing proinflammatory cytokines and chemokines to initiate early proinflammatory response and trigger the subsequent infiltration of peripheral immune cells to damage the brain [5–7]. Thus, a stronger mechanistic understanding of the regulation of neuroinflammation by microglia during ischemic stroke could pave the way for a novel therapy that would reduce ischemic brain injury.
Materials and methods
Ethics statement
All animal procedures were approved by the Animal Care and Use Committee of the Model Animal Research Center, Nanjing University (approval number: 2018010025) and performed according to the Guidelines for the Ethical Review of Laboratory Animal Welfare (GB/T 35892–2018) and the General Requirements for Laboratory Animal Experiments (GB/T 35823–2018) of the People’s Republic of China.
Results
NR4A1 localizes to P-bodies in activated microglia
NR4A1 is expressed in microglia and exhibits anti-inflammatory effect under inflammatory stimuli [12]. However, the underlying mechanism is still unclear. Injured neurons release various damage-associated molecular patterns (DAMPs), which activates microglia through Toll-like receptors and purinergic receptors [15,16], we therefore used ATP and LPS to activate primary microglia and confirmed that NR4A1 was significantly up-regulated after ATP treatment and ATP+LPS co-stimulation (Figs 1A and S1A). Notably, we found that ATP+LPS stimulation induced an upper band of NR4A1 as compared to ATP alone (S1A Fig). Since NR4A1 is a canonical transcription factor with its nuclear localization, and previous studies also have demonstrated that NR4A1 is present in the cytoplasm of neurons and PC12 cells in its phosphorylated form [17,18], we further examined the subcellular localization of NR4A1 in microglia after ATP+LPS treatment. Western blot analysis of compartmentalized proteins showed that ATP+LPS stimulation resulted in an up-regulation of an upper band of NR4A1 in the cytoplasm but not the nucleus (Fig 1B).
Discussion
In the present study, we found that NR4A1 was significantly up-regulated in the cytoplasm of activated microglia, which was inconsistent with the role of NR4A1 as a transcription factor, indicating microglial NR4A1 might also function as a non-transcriptional regulator. We then discovered that microglial NR4A1 localized to P-bodies, where it destabilized Tnf mRNA in an m6A-dependent manner. Global and conditional microglial knockout of Nr4a1 up-regulated Tnf expression and worsened stroke outcomes. Therefore, we uncovered a previously unidentified role of NR4A1 as an RBP, highlighting that microglial NR4A1 is a posttranscriptional brake that suppresses Tnf expression.
Acknowledgments
We thank Chinese Brain Bank Center for providing human post-mortem specimens. We also thank Prof. Jie Zhang (Xiamen University), Dr. Shiji Deng (Nanjing University), and Dr. Huiya Li (Nanjing University) for their intellectual and technical support during the revision.
Citation: Liu P, Chen Y, Zhang Z, Yuan Z, Sun J-G, Xia S, et al. (2023) Noncanonical contribution of microglial transcription factor NR4A1 to post-stroke recovery through TNF mRNA destabilization. PLoS Biol 21(7): e3002199. https://doi.org/10.1371/journal.pbio.3002199
Academic Editor: Richard Daneman, UCSD, UNITED STATES
Received: May 23, 2022; Accepted: June 14, 2023; Published: July 24, 2023
Copyright: © 2023 Liu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: This work was supported by the National Natural Science Foundation of China (81630028, 81920108017, to Y.X.) and the Key Research and Development Program of Jiangsu Province of China (BE2020620, to Y.X.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Abbreviations: AGC, automatic gain control; Co-IP, coimmunoprecipitation; DAMP, damage-associated molecular pattern; DBD, DNA-binding domain; DBP, DNA-binding protein; EAE, experimental autoimmune encephalomyelitis; GCR, glucocorticoid receptor; LBD, ligand-binding domain; MCA, middle cerebral artery; MCAO, middle cerebral artery occlusion; mNSS, modified neurological severity score; miRNA, microRNA; NCM, neuron-conditioned media; OD, optical density; OGD, oxygen-glucose deprivation; P-bodies, processing bodies; PI, propidium iodide; RBP, RNA-binding protein; REMSA, RNA electrophoretic mobility shift assay; RIP-seq, RNA-binding protein immunoprecipitation sequencing; TSA, trichostatin A; WT, wild-type
https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002199#abstract0
