Investigate The Role Of Mitochondrial Function In SOD1-Related Pathogenesis Of Amyotrophic Lateral Sclerosis In NSC-34 Cell Line

Bckground:Amyotrophic lateral sclerosis(ALS)is an adult-onset progressive fatal neurological disease characterized by motor neuron(MN)loss.It is divided into two types: familial and sporadic.Sporadic cases take up around 95% of all cases.The familial cases follow an autosomal dominant trait.It is revealed in formal studies that ALS is a disease with strong relationship with oxidant interference.It is shown that ALS is closely related to oxidative stress and around 20 percent of familiar cases are associated with mutations in SOD1,among which G93 A mutation is an intensively investigated one.So it is necessary to look deeper into the role of mutant SOD1 in pathogenesis of ALS.It has been clearly shown by amyotrophic lateral sclerosis cell and mouse model that mitochondrial health plays a very important role in the pathology of ALS on molecular level and cellular level.We have been using i PSCs as an in vitro model of ALS and we did not find the difference in mitochondrial function until when cells are differentiated into motor neurons.But due to the significant difficulty of manipulating this cell line and the time-consuming process of differentiation,it is necessary to carry out a new ALS model which mimics the charteristics of motor neurons in vitro.In this model we plan to investigate the influence SOD1 has on mitochondrial functions and to reveal the pathogenesis of ALS.NSC-34 cell line is a hybrid cell line derived from mouse spinal cell and neuroblastoma,studies have shown that NSC-34 cell line can not only keep proliferating actively but also show the features of motor neuron in some aspects(like conduction of action potential).First of all,we established ALS cell model with undifferentiated and differentiated NSC-34 cell line,then we compared the influence SOD1 has on mitochondrial function from three aspects:(1)ROS and ETC,(2)MMP and mitochondrial import,(3)mitophagy protein and c GAS-STING-IRF3.We also try to explore the possible pathogenesis of ALS on the basis of the impact SOD1 brings to mitochondrial.Purpose:First of all,ALS cell model with undifferentiated and differentiated NSC-34 cell line will be established.Then we need to compare mitochondrial function from three aspects: ROS and ETC;MMP and mitochondrial import;mitophagy protein and c GAS-STING-IRF3.At the same time we will evaluate those two cell line as an in vitro model of ALS.Methods:1.Transfect NSC-34 cell line with plasmids(h SODG93A-Ac GFP,h SODWT-Ac GFP)and establish a stable transfected NSC-34-ALS model.There are three groups in this study: non-transfected group(NTX),group transfected with h SODWT-Ac GFP(WT),group transfected with h SODG93A-Ac GFP(G93A).2.Investigating ROS level with DCFDA kit.3.Investigating MMP level with JC-2 kit.4.Investigating ETC,mitophagy,c GAS-STING-IRF3 level with Western Blot.5.Investigating mitochondrial import with mitochondrial import assay.Results:1.Two ALS cell models are successfully established(differentiated and undifferentiated cell lines).Correct gene and protein expressions are observed in lines transfected with either WT plasmid and G93 A plasmid.NSC-34 cell line consists of two group of cells: one group that show a few neurites and one group that exhibit many neurites.When the differentiation is going on,the number and lenth of neurites increase significantly.2.In undifferentiated NSC-34 cell line:1)Overexpression of SOD1 decreased ROS level.2)SOD1G93A increased the level of ETCI,Overexpression of SOD1 and SOD1G93 A increased the level of ETCV.Overexpression of SOD1 increased level of ETCII and ETCIII.3)Overexpression of SOD1 increased level of OPTN in mitochondrial fraction in NSC-34 cell line.4)Neither SOD1G93 A nor overexpression of SOD1 can alter mitochondrial import ability.5)SOD1G93A increased the level of c GAS,STING(P<0.05),but did not influence IRF3.Overexpression of SOD1 increased level of STING,IRF3,but did not influence c GAS.3.In differentiated NSC-34 cell line:1)Overexpression of SOD1 and SOD1G93 A decreased level of MMP.2)Overexpression of SOD1 decreased level of OPTN in whole cell lysate.3)SOD1G93A increased the level of c GAS and decreased level of IRF3.4)Neither SOD1G93 A nor overexpression of SOD1 can alter mitochondrial import ability.Conclusion:1.In undifferentiated NSC-34 cell line,overexpression of SOD1 is beneficial to mitochondrial health in terms of ROS.In both undifferentiated and differentiated NSC-34 cell lines SOD1G93 A leads to an increase in c GAS,indicating that SOD1G93 A causes ALS via activating cell apoptosis.2.The influence of SOD1G93 A and overexpression of SOD1 on ROS,ETC,MMP is dependent of the differentiation status of NSC-34 cell line.But the influence on c GAS is independent of the differentiation status of NSC-34 cell line.We must take a closer look at the differentiation status of cell line if we are trying to investigate ALS in cell model.