The Influence Of Stably Transfected TDP-25 And The Regulating Effect Of N-acetylcysteine On The Voltage-gated Sodium Channels Of Motor Neurons

Amyotrophic lateral sclerosis(ALS) is a chronic,progressive,fatalneurodegenerative disease.The spinal cord anterior horn cells,the brainstem,cortex pyramidal cells and pyramidal tract are selectively invaded.Theclinical feature of the disease is both upper and lower motor neuron aredamaged.The pathogenesis is still not known,perhaps many factors includinggenetics,oxidative stress,excitatory toxicity,metabolism and so on are allinvolved in this disease.TDP-43(TAR DNA-binding protein) positiveinclusions are found in the cytoplasm of both neurons and gliocytes in mostpatients of ALS.Pathologic protein aggregates,identified as ubiquitinatedinclusions,are the main feature of ALS.TDP-43 inclusions are found in bothFALS and SALS patients.TDP-25,a 25 k Da C-terminal fragment of TDP-43,isdiscovered in patients of ALS.The recent studies show that TDP-25 ispoisonous to neurons and can promote the genesis of TDP-43,causing injuryof the cells,eventually leading to neurodegenerative diseases. However,thedefinite pathogenesis of TDP-25 to ALS is not yet confirmed.Many present understandings of neuron excitability come from studiesabout mutant SOD1 gene related ALS.The previous research suggests thatboth patients of FALS and of SALS all show high corticalexcitability.Therefore we predict that a similar pathogenesis exists in the twotypes.A series of reactive oxygen species(ROS) are generated in the process ofmetabolism of the cells.Normally,the generation and removal of ROS are in abalanced state and the cells will not be damaged by ROS.Studies indicate thatmutant TDP-43 induced oxidative stress,which leading to an unbalancebetween the generation and removal of ROS and mitochondrialdysfunction.Different types of ROS could increase the activity ofvoltage-gated sodium channel(VGSC),then the excitability of the neuronscould be enhanced.As is demonstrated by our laboratory,in TDP-43 cellmodels,oxidative stress and mitochondrial dysfunction existedsimultaneously,which could be improved by a safe,effective antioxidant calleddimethoxycurcumin(DMC).Voltage-gated sodium channels(VGSCs) play an important role in theinitiation and propagation of action potentials,which is the basis of cellexcitability.Therefore the primary goal of our experiment is to investigate theeffect of TDP-25 to VGSCs of motor neuron cell lines,by which to analysiswhether there are changes in terms of excitability of the cells.N-acetylcysteine(NAC), a derivative of cysteine,is an effective expectorant totreat respiratory infection clinically.This kind of sulfydryl-containing drug canbe served as hydrogen donor to play a role of antioxidant directly.In addition,itcan compound glutathione that has biological activity to play an indirect roleof antioxidant.Some sduties demonstrate that NAC has multiple effects asfollows:to interfere the generation of free radicals,clear the generated ROSaway,anti-apoptosis,modulate genetic expression and signal conductionsystem.So the second objective of the research is to study whether theabberant sodium electrophysiology and cell excitability induced by mutantTDP-25 can be ameliorated by the protective NAC.Objective:To detect whether the properties of sodium channels of motorneuron-like cell lines that were stably transfected with mutant TDP-25 change,in order to analysis the possible mechanisms that mutant TDP-25 results in motor neuron degeneration.And next we will discuss how NACmodulates VGSCs.Methods:Two different types of cell lines including stable transfectedwith mutant TDP-25 and empty vector were used in this experiment.The celllines,constructed by our neurological laboratory and cultured in accordancewith the method of NSC34 cell strain,were cultured and passaged.Next we putthe petri dish that contained cells into an incubator,which could provide athirty-seven centigrade degree and five percent carbon dioxideenvironment.Twenty-four hours later,the petri dish was withdrawn to roomtemperature.With the whole-cell patch clamp technique,in voltage clampmode,we used five different stimulation protocols, which are voltagedependence of activation,fast inactivation and recovery from fastinactivation,entry into slow inactivation,voltage dependence of slowinactivation,to record sodium currents of the two cells and analysis theirproperties.The second step of the experiment was as follows:TDP-25 cellswere dealt with three different kinds of NAC concentration,20μM 、 50μM 、80μM.As the above,twenty four hours later,the cells were taken out.We gavethe same protocols and recorded sodium currents.Results:1 Voltage dependence of activation:Nav channels were all activated at-35 m V,and the sodium currents reached the maximum at voltages between-15and-5m V(P > 0.05).There was no obvious statistical significance in terms ofV1/2.The slope factor of TDP-25 group was smaller than Empty group(P <0.05)and than NAC50 group(P<0.01).2 Voltage dependence of steady-state fast inactivation:Voltages of halfinactivation(V1/2) and the slope factors(k)were similar among the fivegroups,in other words,there was no remarkable difference(P>0.05).3 Recovery from fast inactivation:The τ value of TDP-25 group(n=15 τ=5.252±1.574)was significantly decreased compared with NAC50 group(n=13 τ=6.863±1.827,P<0.05) and Empty group(n=18 τ=7.238±2.104,P<0.01).There were no significant statistical differences between TDP-25 group and low(n=12 τ=5.567±1.756,P>0.05) and high(n=18 τ=5.826±2.215,P>0.05) concentration NAC groups.4 Onset of slow inactivation:Fraction available of Nav channels ofNAC50 group(n=10 0.355±0.178)was clearly less than the other four groups,with TDP-25 group(n=13 0.576±0.157,P<0.05),Empty group(n=9 0.529±0.187,P<0.05),NAC20 group(n=12 0.522±0.200,P<0.05) and NAC80 group(n=10 0.513±0.159,P<0.05).The proportion of Nav channels thatentering into slow inactivation state of the posterior four groups was similar and of no apparent discrepancy(P>0.1).5 Voltage dependence of slow inactivation:The course of slow inactivation of the five groups was evidently dependent on the prepulses.Availabilities of NAC50 group(n=12)at potentials from-50 to-10 m V weremuch smaller than other four groups(P<0.05).In the case of V1/2,therewere significant differences between TDP-25 group(n=11 V1/2=-16.580±0.658) and Empty group(n=9 V1/2=-24.113±0.668,P<0.05),and NAC50group(n=8 V1/2=-40.763±1.081,P<0.001).Voltages of half inactivation ofthe latter two groups reached earlier than the former.The slope factor kvalue of NAC80 group(n=10 k=15.466±2.235)was larger than the other four groups(P<0.05).Conclusions:1 The course of recovery from fast inactivation of Nav channels inTDP-25 group is faster than Empty group,meaning that Nav channels inTDP-25 group can enter the resting state faster.Thus the time of absoluterefractory is shortened and the firing frequency of action potentialincreases,which lead to higher excitability of motor neurons.2 50 μM NAC can effectively eliminate free radicals.In our experiment,the recovery time constant from fast inactivation and the proportionof Nav channels that entering into slow inactivation are both obviouslymore than TDP-25 group.These suggest that the function of fast and slow inactivation of Nav channels can be improved effectively after oxygenspecies are cleared away appropriately,which help to prolong the time ofabsolute refractory and reduce the frequency of action potential.3 We infer that oxidative stress is one of the most importantpathogenesis of motor neuronal degeneration due to the outcome that thedysfunction of Nav channels caused by mutant TDP-25 can be ameliorated bythe proper elimination(50μM NAC)of reactive oxygen species.4 Appropriate reactive oxygen species are needed to maintain normalfunction of motor neurons because removing reactive oxygen species toolittle(20μM NAC) or too much(8μM NAC) can not recover the abnormalitiesof Nav channels caused by mutant TDP-25.