A novel long intergenic non-coding RNA, Nostrill, regulates iNOS gene transcription and neurotoxicity in microglia

Abstract

Background Microglia are resident immunocompetent and phagocytic cells in the CNS. Pro-inflammatory microglia, stimulated by microbial signals such as bacterial lipopolysaccharide (LPS), viral RNAs, or inflammatory cytokines, are neurotoxic and associated with pathogenesis of several neurodegenerative diseases. Long non-coding RNAs (lncRNA) are emerging as important tissue-specific regulatory molecules directing cell differentiation and functional states and may help direct proinflammatory responses of microglia. Characterization of lncRNAs upregulated in proinflammatory microglia, such as NR_126553 or 2500002B13Rik, now termed Nostrill (iNOS Transcriptional Regulatory Intergenic LncRNA Locus) increases our understanding of molecular mechanisms in CNS innate immunity. Methods Microglial gene expression array analyses and qRT-PCR were used to identify a novel long intergenic non-coding RNA, Nostrill, upregulated in LPS-stimulated microglial cell lines, LPS-stimulated primary microglia, and LPS-injected mouse cortical tissue. Silencing and overexpression studies, RNA immunoprecipitation, chromatin immunoprecipitation, chromatin isolation by RNA purification assays, and qRT-PCR were used to study the function of this long non-coding RNA in microglia. In vitro assays were used to examine the effects of silencing the novel long non-coding RNA in LPS-stimulated microglia on neurotoxicity. Results We report here characterization of intergenic lncRNA, NR_126553, or 2500002B13Rik now termed Nostrill (iNOS Transcriptional Regulatory Intergenic LncRNA Locus). Nostrill is induced by LPS stimulation in BV2 cells, primary murine microglia, and in cortical tissue of LPS-injected mice. Induction of Nostrill is NF-κB dependent and silencing of Nostrill decreased inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production in BV2 and primary microglial cells. Overexpression of Nostrill increased iNOS expression and NO production. RNA immunoprecipitation assays demonstrated that Nostrill is physically associated with NF-κB subunit p65 following LPS stimulation. Silencing of Nostrill significantly reduced NF-κB p65 and RNA polymerase II recruitment to the iNOS promoter and decreased H3K4me3 activating histone modifications at iNOS gene loci. In vitro studies demonstrated that silencing of Nostrill in microglia reduced LPS-stimulated microglial neurotoxicity. Conclusions Our data indicate a new regulatory role of the NF-κB-induced Nostrill and suggest that Nostrill acts as a co-activator of transcription of iNOS resulting in the production of nitric oxide by microglia through modulation of epigenetic chromatin remodeling. Nostrill may be a target for reducing the neurotoxicity associated with iNOS-mediated inflammatory processes in microglia during neurodegeneration.

Piceatannol Protects Brain Endothelial Cell Line (bEnd.3) against Lipopolysaccharide-Induced Inflammation and Oxidative Stress

“…As an endotoxin, LPS enhances the release of pro-inflammatory mediators, leading to the destruction of brain tissue and BBB dysfunction [24]. iNOS, the most familiar pro-inflammatory cytokine, induces an inflammatory response by inducing the excessive production of NO [25]. LPS has also been reported to cause oxidative stress by increasing ROS generation [24].…”

Epigenetic Changes and Functions in Pneumoconiosis

“…For example, the combination of lncRNA and the chromatin remodeling complex promotes the formation of a dense chromatin structure, thereby generating gene-silencing regions [ 119 ]. lncRNA can also regulate histone modification [ 120 ], promote DNA methylation modification [ 121 ], inhibit transcription factors [ 122 ], and inhibit RNA polymerase II [ 123 ] to regulate mRNA expression, or NATs can directly interfere with mRNA expression, posttranscriptional processing, and transportation [ 112 , 124 , 125 ]. lncRNAs located in the cytoplasm are mainly involved in the regulation and transportation of protein translation.…”

Epigenetic Modulation of Microglia Function and Phenotypes in Neurodegenerative Diseases

“…A novel lncRNA termed Nostrill is highly expressed in LPS-stimulated microglia cells with concomitant increased expression of iNOS. Silencing of Nostrill markedly decreased the expression of NF- κ B p65 and reduced activation of H3K4me3 at the iNOS site, as well as decreased neurotoxicity in mice and BV-2 cells [ 163 ]. LncRNA GAS5 acts as a regulator of microglia polarization, possibly by recruiting polycomb repressive complex 2 (PRC2), which represses transcription of the key factor TRF4 and thus promotes the conversion of microglia to M2 phenotype [ 164 ].…”