| BackgroundAmyotrophic Lateral Sclerosis(ALS)is the most common type of motor neuron diseases and clinically manifests as severe dysfunction of both upper and lower motor neurons.Short survival,lack of therapeutic drugs,high cost,and poor outcomes are the main dilemmas of the treatment of ALS.Studying the pathogenesis of ALS may help us to find potential treatments.Optineurin,the protein encoded by OPTN,one of the ALS-associated genes,is implicated in several cellular processes including autophagy,mitochondrial autophagy,vesicle transport,activation of the NF-κB signaling pathway,and so on.In our previous study,we found that microglia cells transfected with ALS-associated-mutants of OPTN,which may cause the dysfunction of optineurin,induced motor neuron loss.Microglia cells are one of the important members that mediate neuron-inflammation in the central nervous system(CNS).At present,microglia cells are considered to participate in the occurrence and development of ALS,and play a dual role of both neuroprotection and cytotoxicity.Pro-inflammatory M1 microglia cells are predominant in the very early stage and neuroprotective M2 microglia cells are predominant in the early stage of the disease.M1 microglia cells gradually dominate in the CNS during the development of the disease and are predominant in the advanced stage.Therefore,we hypothesis that silencing OPTN,the ALS-associated gene,can activate the transition of microglia cells to the pro-inflammatory M1 phenotype and lead to motor neuron(MN)loss.Our study aims to verify the hypothesis and provide new ideas and therapeutic targets for the treatments of OPTN-ALS.In addition,other studies suggest that activation of the NF-κB in microglia cells can induce the M1 microglia cells polarization while optineurin participate in the activation of the NF-κB.Therefore we explored the the role of NF-κB signaling pathway in our further study.Materials and Methods1.To study the role of the microglia cells in OPTN-ALS,BV-2 cells(microglia-like cells)transfected with WT,Q398X or E478G mutant of OPTN and OPTN siRNA were cocultured with normal NSC-34 cells,and NSC-34 cell viability was measured.OPTN siRNA was transfected into BV2 microglia cells to silence the expression of OPTN in microglia cells and followed experiments were performed:2.To study the effect of dysfunction of optineurin on microglia polarization,real Time Quantitative PCR,ELISA,and Western Blot were used to detect the expression levels of M1/M2 phenotype biomarkers in microglia cells.3.For further confirmation that dysfunction of OPTN may activate M1 phenotype microglia cells and induce the apoptosis of motor neuron,BV-2 microglia cells transfected with OPTN siRNA were cocultured with NSC-34 cells at a ratio as 1:1 by using Transwell chamber.Alexa Fluor? 488 annexin V/Dead Cell Apoptosis Kit was used to detect the apoptosis rates of NSC-34 cells.4.To explore the mediation role of NF-κB signaling pathway,the location of phosphorylated p65,a subunit of the NF-κB transcription factor complex,was detected by Immunofluorescent Staining;the expression of phosphorylated p65 was measured by Western Blot;the transcriptional activation of NF-κB was determined by using the Dual-Luciferase Reporter Assay and the expression of mRNA of TNF-α,the product of the NF-κB signaling pathway,was measured by Real Time Quantitative PCR to verify that dysfunction of optineurin may activate the NF-κB signaling pathway.NF-κB regulators were added,and the expression levels of the biomarkers of phenotypes and apoptosis rates of cocultured NSC-34 cells were detected later.Results1.Severe cell death was seen in neurons cocultured with microglia cells transfected with Q398X or E478G mutant(ALS-associated)of OPTN or OPTN siRNA,while increase in survival rate of NSC-34 cells was found in WT group.Results indicated the negative role of microglia in OPTN-ALS.2.The expression levels of MHC-II and NOS2(M1 phenotype biomarkers)increased while the expression levels of ARG-1 and FIZZ1(M2 phenotype biomarkers)decreased in OPTN siRNA-BV-2 microglia cells.In addition,the secretion of pro-inflammatory cytokines including IL-1β,IL-6,TNF-α and NO were promoted and anti-inflammatory cytokines including TGF-B and IL-10 were decreased in OPTN siRNA-BV-2 microglia cells.Results indicated that silencing the OPTN gene may alternatively activate the M1 phenotype microglia cells.3.The apoptosis rate of cocultured NSC-34 cells were increased in OPTN siRNA group compared to control group which may indicated that the dysfunction of OPTN may activate M1 phenotype microglia cells and induce the apoptosis of motor neuron.4.P65 nuclear translocation,the expression levels of phosphorylated p65,the transcriptional activation of NF-κB,and the expression levels of TNF-α mRNA increased compared to that in normal BV-2 microglia cells,which suggested the activation of NF-κB in OPTN siRNA-BV-2 microglia cells.Withaferin A,the NF-κB inhibitor,may abolish the effect of dysfunction of optineurin on microglia polarization.In addition,NF-κB inhibitor can abolish the negative effect of OPTN siRNA-BV-2 microglia cells on NSC-34 cells while NF-κB activator can aggravate the effect.ConclusionOptineurin,the protein encoded by one of the pathogenic genes of ALS,may participate in the activation of transition of microglia phenotypes,and thus affect the apoptosis of motor neurons.This mechanism may be associated with the negative effect of optineurin on the activation of NF-κB.Results may provide insights into the pathogenesis of OPTN-ALS. |
