N-acetylcysteine Can Recover The Dysfunction Of The Persistent Sodium Channel On TDP-25 Transfection Motor Neuron-like Cells

Amyotrophic lateral sclerosis(ALS) is a progressive neurodegenerative disease characterized by selective damage of motor neurons. Most of the cases are sporadic(s ALS), but 10% are familial(f ALS) inherited in an autosomal dominant manner.Mutant TDP-43 could be involved in 3%familial ALS and 2.9% sporadic ALS,suggesting that TDP-43 may play a significant role in ALS.TDP-43 is a nuclear protein which regulates transcription and RNA splicing, and participates in m RNA transport and stability.Many studies have shown that TDP-43 in tissue taken from cases of FTLD-TDP or ALS is often hyperphosphorylated, cleaved, ubiquitinated, mislocalized,and poorly soluble。 These features suggest various loss of function and gain of toxic function mechanisms for TDP-43 in disease pathogenesis. Pathological TDP-43 is proteolytically cleaved to form a 25 k Da pathological C-terminal fragment, the TDP-25 protein is charactered by aggregation and cytotoxicity. Recently,we research the mitochondrial dysfunction and oxidation damage causing by TDP-25,which may play an important role in the pathogenesis of ALS.Compared with full-length TDP-43, C-terminal fragments 164 were more likely to form compact cytoplasmic and nuclear inclu-165 sionsinduced apoptosis and mislocalization of TDP-43, and caused shorter motor neuronal axons. Previous results found that the present of mutant TDP-43 induces high excitability, including high firing frequency and low threshold of APs.Mutant TDP-43 results in mitochondia dysfunction,oxidative injury and induce an imbalance between the generation and removal of reactive oxygen species(ROS).Correspondingly, TDP-43-expressing models indicated by accumulation of reactive oxygen species(ROS), loss of mitochondrial membrane potential and impairment of the respiratory chain. Mitophagy involved in the modulation of Na channel function in neurons. We suppose that high excitability and oxidative stress exist simultaneously in motor neurons cell lines transfected with mutant TDP-25.N-acetyl cysteine(NAC) is a kind of small molecular substances which be easy through the cell membrane and enter the cells out of acetyl and promote the production of glutathione and maintain stable redox potential in the cell, thereby eliminating intracellular ROS, become an effective antioxidant.So is also known as the reduced glutathione precursors, and ROS scavenger directly, can through the cell membrane and mitochondrial membrane.This experiment by using NAC antioxidant protection, record the persistent sodium current again, further analysis of persistent sodium current recorded in the TDP-25 after the treatment of NAC, to verify whether there were positive effects.Objective: To analysys the difference of the persistent sodium current between the TDP-25(transgenic cells with stably expressing the TDP-25) and the empty vector motor-like neurons in our experiment.Then we verified whether there were toxic effects in the TDP-25 cells.Investigate the influence of the NAC drug concentration on the TDP-25 cells. Also we discussed whether the protein TDP-25 paly an important role in the pathogenesis of ALS.Methods: In this study, we used two different NSC34 cell lines with empty vector(Empty) and TDP-25. The cell lines were constructed by our laboratory and were cultured as the same method as NSC cell lines. We used high saturation antibiotics to select monoclonal cells. The cell lines were identificated by inmmunocytochemistry. Whole cell configuration of the patch-clamp technique was used to record persistent sodium currents in voltage clamp in the resting state, and we would record it again after influence of various drug concentration of NAC, including 20umol/L、50umol/L、80umol/L.Then we observed the protection of NAC.Results: The amplitude and density of the persistent sodium current of the TDP-25 group were larger than the E group.We recorded the persistent sodium current after 24 hours,as the 20umol/L、50umol/L、80umol/L NAC influence on the TDP-25 cell.The results were that the amplitude and density of the persistent sodium current of 50umol/L NAC group were reduced obviously;the current of the 20umol/L NAC group were decreased slightly;there were no significant changes in the 80umol/LNAC groups.The results are as following: TDP-25 group:n=25 current amplitude:-74.69±17.64 p A;current density:-3.18±1.723 p A/pf Empty group: n=20 current amplitude:-30.60±6.72 p A;current density: 0.52±0.024 p A/pf The results of drug groups,as follows: 20μmol/L NAC group:n=10 current amplitude:-62.62±11.63 p A;current density: 0.52±0.024 p A/pf 50μmol/L NAC group:n=10 current amplitude:-34.56±10.07 p A;current density:-1.73±0.42 p A/pf 80μmol/L NAC group:n=10 current amplitude:-66.30±18.23 p A;current density:-3.06±0.955 p A/pf Non-linear index equation was fitted according to the results above. Two indexes equation and graph were presented, the fitted equation was as following:Conclusions: In our experiment study,we recorded the persistent sodium current successfully.Comparing with the Empty group,the TDP-25 group increased the amplitude and density of the persistent sodium current,which attach great importance in the firing frequency and the excitability of the nerve cell.We also summarized the advantageous function of the different NAC concentration.The middle drug concentration influenced the TDP-25 cells in a great extent. There were no significant changes in the 20umol/L NAC groups and 80umol/L NAC groups.As the verification of conclusions,we kown middle drug concentration NAC could alleviate high excitability and oxidative stress,which palyed an important role in the pathogenesis of ALS.