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...

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Endoplasmic reticulum (ER) stress generally occurs in secretory cell types. It has been reported that Leydig cells, which produce testosterone in response to human chorionic gonadotropin (hCG), express key steroidogenic enzymes for the regulation of testosterone synthesis. In this study, we analyzed whether hCG induces ER stress via three unfolded protein response (UPR) pathways in mouse Leydig tumor (mLTC-1) cells and the testis. Treatment with hCG induced ER stress in mLTC-1 cells via the ATF6, IRE1a/XBP1, and eIF2α/GADD34/ATF4 UPR pathways, and transient expression of 50 kDa protein activating transcription factor 6 (p50ATF6) reduced the expression level of steroidogenic 3β-hydroxy-steroid dehydrogenase Δ5-Δ4-isomerase (3β-HSD) enzyme. In an in vivo model, high-level hCG treatment induced expression of p50ATF6 while that of steroidogenic enzymes, especially 3β-HSD, 17α-hydroxylase/C17–20 lyase (CYP17), and 17β-hydrozysteroid dehydrogenase (17β-HSD), was reduced. Expression levels of steroidogenic enzymes were restored by the ER stress inhibitor tauroursodeoxycholic acid (TUDCA). Furthermore, lentivirus-mediated transient expression of p50ATF6 reduced the expression level of 3β-HSD in the testis. Protein expression levels of phospho-JNK, CHOP, and cleaved caspases-12 and -3 as markers of ER stress-mediated apoptosis markedly increased in response to high-level hCG treatment in mLTC-1 cells and the testis. Based on transmission electron microscopy and H&E staining of the testis, it was shown that abnormal ER morphology and destruction of testicular histology induced by high-level hCG treatment were reversed by the addition of TUDCA. These findings suggest that hCG-induced ER stress plays important roles in steroidogenic enzyme expression via modulation of the ATF6 pathway as well as ER stress-mediated apoptosis in Leydig cells.

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