Effect Of The Human SOD1-G93A Gene On TBK1 In NSC34 Cell

Objective: Amyotrophic lateral sclerosis(ALS)is a lethal and progressive neurodegenerative disease characterized by the loss of motor neurons.The hypothesis of mutations in superoxide dismutase 1(SOD1)has an important role in the pathogenesis of ALS,especially the SOD1-G93 A mutations gene.In our experiment,we use NSC34 cells stably tranfected with the human SOD1-G93 A gene,exploring Effect of the human SOD1-G93 A gene on TBK1,laying a basis for the future.TBK1 is a ubiquitously distributed protein gene and has an influential role in many cytological features.TBK1 is a uncanonicala inhibitor of nuclear factor ?Bkinase(IKK)–related protein kinase.TBK1 is a 729 amino acid protein which has four functionally distinct domains;a kinase domain(KD)at the N-terminus,two putative coiled-coil-containing regions in the C-terminal region,including a C-terminal leucine zipper(LZ)and a helix-loop-helix(HLH)motif;a ubiquitin-likedomain(ULD).TBK1 has an important role in autophagy.In addition,TBK1 participates in innate immunity,clearance of pathogens and inhibition the growth and proliferation of cells.Methods:1)the group of experimentNSC34 cells stably tranfected with the human SOD1-G93 A gene(mutant group),empty and human SOD1-Wide Type(control groups)2)the intervention of drugs Three stable tranfected cells were seeded in 96-wells in a similar density.And then the autophagy blockers Bafilomycin A and theproteasome blockers MG132 were injected.Cell vability was measured by cell counting kit(CCK-8).The confocal was used to observe the quantitative variance of pTBK1.In addition,we examined protein levels of SOD1?TBK1 ?pTBK1 and autophagy related proteins P62?LC3B-II in the stable cell line by western blot.Results1 Bafilomycin A and MG132 decrease three stable tranfected cells vability,but the mutant group decreased more obviously than the control group(p < 0.05).2 In confocal we observed with the intervention of Bafilomycin A,the quantitative variance of pTBK1 in the mutant group decreased more obviously than the empty group.3 Western Blot shows Bafilomycin A increased the level of autophagy related proteins P62?LC3B-II.In addition the level of pTBK1 in the mutant group was less than the empty group with the Bafilomycin A intervention(p < 0.05);the total leval of TBK1 increased in empty group compared with untreated goup,but the mutant group had no obvious variance.MG132 increased the level of autophagy related proteins P62?LC3B-II.The level of pTBK1 in the mutant group was less than the empty group with the MG132 intervention(p < 0.05).Besides,the total leval of TBK1 had no variance.Conclusions:1 The mutant SOD1 was degraded by the autophagy pathways and the proteasome pathway.2 The level of pTBK1 in the mutant group was less than the empty group with the Bafilomycin A intervention,so we estimate that the mutant SOD1 inhibited the activation of TBK1.However,the total level of TBK1 in empty group increased compared with untreated goup,but the mutant group had no obvious variance.We infer mutant SOD1 restain the degradation of TBK1 through autophagy pathway.3 MG132 inhibited proteasome pathway and indirectly activated the autophagy pathway.Then induced the activation of TBK1.The level of pTBK1 in the mutant group was less than the empty group,which prompted mutant SOD1 restain the activitation of TBK1.