Mitochondrial dynamics modulate the expression of pro‐inflammatory mediators in microglial cells

Abstract

Over-activation of microglia cells in the brain contributes to neurodegenerative processes promoted by the production of various neurotoxic factors including pro-inflammatory cytokines and nitric oxide. Recently, accumulating evidence has suggested that mitochondrial dynamics are an important constituent of cellular quality control and function. However, the role of mitochondrial dynamics in microglial activation is still largely unknown. In this study, we determined whether mitochondrial dynamics are associated with the production of pro-inflammatory mediators in lipopolysaccharide (LPS)-stimulated immortalization of murine microglial cells (BV-2) by a v-raf/v-myc carrying retrovirus (J2). Excessive mitochondrial fission was observed in lentivirus-transfected BV-2 cells stably expressing DsRed2-mito following LPS stimulation. Furthermore, mitochondrial localization of dynamin-related protein 1 (Drp1) (a key regulator of mitochondrial fission) was increased and accompanied by de-phosphorylation of Ser637 in Drp1.Interestingly, inhibition of LPS-induced mitochondrial fission and reactive oxygen species (ROS) generation by Mdivi-1 and Drp1 knock-down attenuated the production of pro-inflammatory mediators via reduced nuclear factor kappa-light-chainenhancer of activated B cells (NF-jB) and mitogen-activated protein kinase (MAPK) signaling. Our results demonstrated for the first time that mitochondrial fission regulates mitochondrial ROS production in activated microglial cells and influences the expression of pro-inflammatory mediators through the activation of NF-jB and MAPK. We therefore suggest that mitochondrial dynamics may be essential for understanding pro-inflammatory mediator expression in activated microglial cells. This could represent a new therapeutic approach for preventing neurodegenerative diseases. Keywords: Drp1, lipopolysaccharide, Mdivi-1, microglia, mitochondrial dynamics, neuroinflammation. Address correspondence and reprint requests to Dong-Seok Lee, College of Natural Sciences, Kyungpook National University, Daegu, Republic of Korea. E-mail: lee1@knu.ac.kr 1 These authors contributed equally to this work. Abbreviations used: CMVie, cytomegalovirus immediate early; Cox-2, cyclooxygenase-2; COXIV, cytochrome c oxidase; cPPT, central polypurine tract; DMEM, Dulbecco's modified Eagle's medium; Drp1, dynamin-related protein 1; DsRed2, discosoma sp. red fluorescent; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; Fis1, mitochondrial fission 1; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; iNOS, inducible nitric oxide synthase; JNK, c-jun N-terminal kinase; LPS, lipopolysaccharide; LTR, long terminal repeat; MAPK, mitogen-activated protein kinase; Mdivi-1, 3-(2,4-dichloro-5-methoxyphenyl)-2,3-dihydro-2-thioxo-4(1H)-quinazolinone3-(2,4-dichloro-5-methoxyphenyl)-2-sulfanyl-4(3H)-quinazolinone; Mfn1, mitofusin 1; Mfn2, mitofusin 2; MOI, multiplicity of infection; MTS, mitochondrial targeting sequence; NF-jB, nuclear factor kappa-light-chainenhancer of activated B cells...

Metabolic Alterations Contribute to Enhanced Inflammatory Cytokine Production in Irgm1-deficient Macrophages

“…Mitochondrial dynamics have generally been associated with different metabolic states; cells relying on fatty acid oxidation or oxidative phosphorylation often display fused networks of mitochondria, whereas those relying on glycolysis tend to have more punctate mitochondria (71)(72)(73)(74)(75). Further, promoting mitochondrial fission and/or blocking fusion can stimulate pro-inflammatory cytokine production (55,59,76,77). Nevertheless, our results suggest that the punctate mitochondrial morphology seen in Irgm1-deficient cells is secondary to accumulation of LC-AC in those cells, because treatment of the cells with fatty acid synthase inhibitors to reduce fatty acid concentrations not only blunted RANTES production in IFN-␥-primed, Irgm1-deficient macrophages, it also eliminated the punctate mitochondrial phenotype.…”

“…increases mitochondrial fragmentation (27,55,56), with the fragmentation resulting in part from the high production of reactive oxygen species that occurs with activation (56 -59). We thus reasoned that the fragmented mitochondrial morphology in Irgm1-deficient macrophages may be a result of the metabolic changes in those cells in the context of high ROS levels.…”

The mitochondrial dynamics in cancer and immune-surveillance

“…The inflammasome requirement of mitochondrial elongation has been recently demonstrated also in Drp1-silenced macrophages [184] and is correlated with reduced Opa-1 levels in rat [185]. Additionally, receptor-interacting protein kinase -1 and -3 (RIPK1/3)-dependent and lipopolysaccharide (LPS)-dependent inflammasome activation, and cytokine production upon viral infection, requires Drp1-dependent mitochondrial fission to induce mitochondrial damage and generate ROS [186,187]. The mitochondrial phosphatase phosphoglycerate mutase 5 (PGAM5) is the mediator of such RIPK-dependent Drp1 activation [188].…”

“…The mitochondrial phosphatase phosphoglycerate mutase 5 (PGAM5) is the mediator of such RIPK-dependent Drp1 activation [188]. Similarly, microglial cells (MCs) in the central nervous system require Drp1-driven mitochondrial fragmentation and ROS signalling for correct cytokine production [189] ( Fig. 2A).…”

The Drosophila retinoblastoma protein, Rbf1, induces a debcl and drp1-dependent mitochondrial apoptosis.

“…Rbf1 induces mitochondrial fragmentation which is required for ROS production and cell death Mitochondrial fission, which can occur during apoptosis in mammalians and Drosophila cells (Desagher and Martinou, 2000;Goyal et al, 2007), can regulate ROS generation (Park et al, 2013;Qi et al, 2013). Interestingly, apoptosis can be blocked with a dominant-negative mutation or specific RNAi-mediated knockdown of the pro-fission gene Drp1 (Abdelwahid et al, 2007;Arnoult et al, 2005;Breckenridge et al, 2003;Frank et al, 2001).…”