title: Transcription factor myocyte enhancer factor 2D regulates interleukin-10 production in microglia to protect neuronal cells from inflammation-induced death.
abstract: BACKGROUND
  Neuroinflammatory responses have been recognized as an important aspect in the pathogenesis of Parkinson's disease (PD). Transcriptional regulation plays a critical role in the process of inflammation. Transcription factor myocyte enhancer factor 2D (MEF2D) is identified as a central factor in transmission of extracellular signals and activation of the genetic programs in response to a wide range of stimuli in several cell types, including neurons. But its presence and function in microglia have not been reported. We therefore investigated the effect of MEF2D in activated microglia on the progress of neuroinflammation and the survival of neurons.
  
  
  METHODS
  BV2 cells and primary cultured glial cells were stimulated with lipopolysaccharide (LPS). Samples from cells were examined for MEF2D expression, interleukin-10 (IL-10), and tumor necrosis factor alpha (TNF-α) by immunoblotting, quantitative real-time PCR (qPCR) or enzyme-linked immunosorbent assay (ELISA). The activity of MEF2D was examined by electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation assay (ChIP). Recombinant lentivirus expressing shRNA specific to MEF2D was used to silence MEF2D expression in BV2 cells. The role of IL-10 transcriptionally induced by MEF2D on neuronal survival was assessed by anti-IL-10 neutralizing antibody. The survival of neurons was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. Male C57bl/6 mice were used to establish an acute PD model. Brain sections and cell slides were tested by immunofluorescence.
  
  
  RESULTS
  We demonstrated that MEF2D was present in microglia. Activation of microglia was associated with an increase in MEF2D level and activity in response to different stimuli in vivo and in vitro. MEF2D bound to a MEF2 consensus site in the promoter region of IL-10 gene and stimulated IL-10 transcription. Silencing MEF2D decreased the level of IL-10, increased the TNF-α mRNA, and promoted inflammation-induced cytotoxicity, consistent with the result of inhibiting IL-10 activity with an anti-IL-10 neutralizing antibody.
  
  
  CONCLUSIONS
  Our study identifies MEF2D as a critical regulator of IL-10 gene expression that negatively controls microglia inflammation response and prevents inflammation-mediated cytotoxicity.
authors.affiliation: Department of Neurosurgery, Tangdu Hospital, The Fourth Military Medical University, Xi'an, 710038, China. yangshaosong@aliyun.com.
authors.forename: Shaosong
authors.initials: S
authors.lastname: Yang
authors.affiliation: Department of Neurosurgery, Tangdu Hospital, The Fourth Military Medical University, Xi'an, 710038, China. gaoli089@yeah.net.
authors.forename: Li
authors.initials: L
authors.lastname: Gao
authors.affiliation: Department of Neurosurgery, Tangdu Hospital, The Fourth Military Medical University, Xi'an, 710038, China. misslff@163.com.
authors.forename: Fangfang
authors.initials: F
authors.lastname: Lu
authors.affiliation: Department of Neurosurgery, Tangdu Hospital, The Fourth Military Medical University, Xi'an, 710038, China. bob.kane@163.com.
authors.forename: Bao
authors.initials: B
authors.lastname: Wang
authors.affiliation: Department of Neurosurgery, Tangdu Hospital, The Fourth Military Medical University, Xi'an, 710038, China. 18092662863@163.com.
authors.forename: Fei
authors.initials: F
authors.lastname: Gao
authors.affiliation: Department of Neurosurgery, Tangdu Hospital, The Fourth Military Medical University, Xi'an, 710038, China. zhugang.0308@163.com.
authors.forename: Gang
authors.initials: G
authors.lastname: Zhu
authors.affiliation: Department of Neurosurgery, Tangdu Hospital, The Fourth Military Medical University, Xi'an, 710038, China. zhibiaocai@163.com.
authors.forename: Zhibiao
authors.initials: Z
authors.lastname: Cai
authors.affiliation: Department of Neurosurgery, Tangdu Hospital, The Fourth Military Medical University, Xi'an, 710038, China. laijuan_1988@163.com.
authors.forename: Juan
authors.initials: J
authors.lastname: Lai
authors.affiliation: Department of Neurosurgery, Tangdu Hospital, The Fourth Military Medical University, Xi'an, 710038, China. qianyang@fmmu.edu.cn.
authors.forename: Qian
authors.initials: Q
authors.lastname: Yang
mesh_terms: D015632:1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine / Q000494:pharmacology; D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D001921:Brain / Q000166:cytology; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002478:Cells, Cultured; D047369:Chromatin Immunoprecipitation; D024202:Electrophoretic Mobility Shift Assay; D016753:Interleukin-10 / Q000235:genetics / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000633:toxicity; D064326:MEF2 Transcription Factors / Q000235:genetics / Q000378:metabolism*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008840:Microfilament Proteins / Q000378:metabolism; D017628:Microglia / Q000187:drug effects / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D013997:Time Factors; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism
chemical_list: C491049:Aif1 protein, mouse; D002135:Calcium-Binding Proteins; D008070:Lipopolysaccharides; D064326:MEF2 Transcription Factors; D008840:Microfilament Proteins; D012333:RNA, Messenger; D016753:Interleukin-10; D015632:1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; D014446:Tyrosine 3-Monooxygenase