Regulation Of C9orf72 On ALS Related Proteins TDP-43 And FUS

Objective: To study the regulation of C9orf72 on Amyotrophic lateral sclerosis(ALS)related proteinstransactive response DNA-binding protein of 43 kDa(TDP-43)and fused in sarcoma protein(FUS).Methods: The plasmids vector with target gene(plasmids of C9orf72 gene related proteins,TDP-43 and FUS,etc.)and the competent cells(DH-5 alpha competent Escherichia coli)were mixed,and bacterial transformation;Inoculation and culture the Escherichia coli which containing the corresponding plasmid;The plasmidswere extracted by Sanprep column plasmid DNA mini-preps kit,and different plasmids were obtained,which would be kept in-20 ℃and reserved;HEK293 cells(human embryonic kidney epithelial cells)were transfected with the corresponding plasmids;Western blotting was used to detect the proteins expression of TDP-43 and FUS;The localization and aggregation of C9orf72 related proteins,TDP-43 and FUS were detected by immunoprecipitation and immunofluorescence.Results: In this study,we found that the two FUS mutants were more likely to accumulate in the cytoplasm than the wild type,and the cytoplasm of the mutantswere evenly distributed,andthe two FUS mutants showed obvious aggregation after Arsenite induction.TDP-43 protein was transported to cytoplasm after Arsenite induction,but not like FUS easy to be formed aggregates.Normally,Poly-GA/GP/PA promoted TDP-43 to transport to the cytoplasm,while Poly-GR/PR did not have the effect.Under the condition of Arsenite stress,Poly-GR/PR could interdict the cytoplasmic transport of TDP-43.After co-transfection of Poly-GR/PR and FUS mutants,the protein aggregates,which formed by FUS under the pressure of Arsenite,could be depolymerized.Conclusion:FUS and TDP-43 nucleocytoplasmic transport and abnormal protein aggregation was a classical phenomenon in the pathological process of ALS,this study showed that Poly-GR/PR can significantly regulate the nucleocytoplasmic transport and protein aggregation of TDP-43 and FUS,were suggested that abnormal encoding repeat proteins of C9orf72 and FUS/TDP-43 can be the same function in the same pathway.