The Influence Of Human TDP-43-M337V On ROCK/PTEN Signaling Pathway In NSC-34 Cells

Objective: We observe the impact of h TDP-43-M337 V on ROCK/PTEN signaling pathway in NSC-34 cells.Methods: 1. cell culture: The three cell lines including empty transformation, wild type and mutant type were removed from liquid nitrogen and thawed quickly in 37 0C water. Then the cells were seeded into 25 cm2 glass culture bottles. The culture bottles were maintained at 37 0C in a 5 % CO2 humidified atmosphere. 2. We use the immunohistochemical method to observe morphological changes in stably transfected cells; 3. We detect content of intracellular Malondialdehyde(MDA) which reflects the degree of cell oxidative damage; 4. We detect the influence of transfected genes on the NSC-34 cells' activity by detecting the MTT content in cells; 5. We detect the different protein expression levels of ROCK, PTEN and Akt in these three cell lines by using Western blots method; 6. We detect ROCK activity through Rho-kinase Assay kit, so as to observe changes of ROCK activity in the signaling pathway.Result: 1. In our study,we found that the soma became round and the number of neurite decreased in the NSC-34 cells transfected with p DEST30-EGFP-TDP-43-M337 V plasmid, and the neurites were shorter compared with the other two cell lines; After intracellular MDA content test,we found that content of intracellular MDA in mutant group was significantly higher than the other two groups(P<0.05); At the same time, after determined by MTT test,we found inhibition rate of transfected p DEST30-EGFPTDP-43-M337 V cells increased significantly(P<0.05).2. The Western blots showed that ROCK1, ROCK2, PTEN, Akt had no difference in the total protein expression levels in different groups of ALS transfected cells(P>0.05); Compared with empty plasmid transfected group of cells, protein expression levels of pPTEN transfected p DEST30-EGFP-TDP-43-M337 V plasmid groups significantly increased, while the levels of p-Akt decreased obviously(P<0.05). While in the wild type of plasmid transfected cells, there was no significant difference between protein expression levels of p-PTEN and p-Akt, compared to empty plasmid transfected groups(P>0.05). 3. By using ROCK activity detection techenology,We also found that the activity of Rho-kinase had no obvious change among TDP-43-M337 V mutant cells,compared with the other groups of cells(P>0.05).Conclusion: Transfected TDP-43-M337 V reduces the NSC-34 cell activity and ability to resist oxidative damage, and increases the vulnerability of motor neurons in stress induced toxic injury. TDP-43-M337 V on the NSC-34 cells had no obvious influence on the activity and protein expression of ROCK, but it can change the phosphorylation level of PTEN and Akt. This may indicate that transfected TDP-43 mutant gene activates the cell apoptosis signaling pathways, and then induce cell apoptosis through different mechanisms or different ways.