Mechanism Of Abnormal Lipid Metabolism In ALS Skeletal Muscle Induced By FUS Gene Mutation

Objective:To investigate the correlation between clinical phenotypes and lipid droplets accumulation in muscle of ALS patients with different genotypes.To observe the changes in mitochondrial morphology and dynamics after FUS mutation,as well as the changes in lipid metabolism,transport and related ultrastructure,and to explore the possible pathogenesis.Methods:1.The clinical data of 41 patients with diagnosed ALS and 53 patients without ALS and the pathological sections of muscle biopsy were collected to analyze the differences and correlations between clinical phenotypes and pathological changes.2.C2C12 cells were infected with adenovirus to construct the cell model of over-expressed FUS-K510Q gene mutation.The expression level of FUS was detected by Western blot.The intracellular FUS protein localization was detected by confocal immunofluorescence,and lipid droplet deposition and mitochondrial damage were observed.3.The levels of ROS and mitochondrial membrane potential were detected by fluorescent dye labeling combined with fluorescence inverted microscope,and the level of ATP was detected by ATP detection kit.The deposition of lipid droplets was observed by oil red O staining and electron microscopy.4.The expression levels of FUS,CPT1A,PLLIN2 and ATGL were detected by Western blotting.5.By verifying the phenotypes of lipid droplet deposition and exercise ability in the muscle of Drosophila and normal Drosophila expressing FUS-K510Q mutant protein,Objective to explore the role of mitochondrial damage leading to abnormal lipid metabolism in the pathogenesis of ALS by exploring the changes of protein related to the lipid transport pathway of FUS in cell model.Results:1.In the oil red O staining of muscle tissue,ALS patients were more likely to find more fat droplets than normal controls,and in different genotypes of ALS,ALS patients with FUS gene mutation were more likely to find more fat droplets than other ALS patients,suggesting that ALS patients with Fus mutation are more likely to have fat metabolism disorders.2.FUS-K510Q mutant myoblast cells showed abnormal accumulation of FUS protein in cytoplasm compared with the no-load group.3.Compared with the no-load group,the myoblasts with FUS-K510Q mutation showed significantly higher levels of reactive oxygen species(ROS),lower mitochondrial membrane potential and ATP levels.4.Compared with normal myoblasts,the myoblasts with FUS-K510Q mutation were more likely to cause abnormal mitochondrial morphology under electron microscopy.5.The FUS-K510Q mutant myoblasts were more likely to cause the accumulation of lipid droplets than the normal group.6.The muscle specific expression of FUS mutant protein in Drosophila is more likely to lead to the decline of motor ability than the normal group.7.The level of triglyceride in muscle tissue of Drosophila which specifically expressed FUS mutant protein was higher than that in normal group.8.No matter whether there is oleic acid load or not,FUS-K510Q mutation reduces the expression level of ATGL and CPT1A,which leads to the decline of lipolysis ability and fatty acid transport ability;FUS-K510Q mutation increases the expression level of PLIN2,leading to the accumulation of lipid droplets.Conclusion:Mitochondrial dysfunction and abnormal lipid metabolism are stored in FUS mutant myoblasts.This study provides a theoretical basis for the involvement of FUS mutation in lipid metabolism disorders in the pathogenesis of ALS,and alleviating muscle pathology or providing therapeutic drugs to motor neurons may become a new treatment strategy for ALS.