| TDP43is encodede by human TARDBP gene. Human TARDBP gene islocaded on chromosome1p36and encodes a414-residue protein of43kDa.Recently, the ubiquitin-positive conclusion was discovered in pathology ofsome ALS and FTLD-U patient and TDP43is the primary component of theconclusion. TDP43is primary in nucleus and shuttles between nucleus andcytoplasm.TDP43participates in the transport, translation and degradation ofRNA. TDP43also maintains the stability of RNA. But TDP43transfers tocytoplasm, and aggregates in cytoplasm in the form of ubiquitin,phosphorylation and traction in central nervous system of some ALS andFTLD-U patients. TDP43gene is likely to be the high mutation genefollowing SOD1gene which causes ALS. Hyperexcitability of SOD1mice hasalready developed presymptomatic. The hyperexcitability of neurons maypromote energy metabolism, increase ATP consumption, aggravate theburden of mitochondria, lead to energy depletion, ultimately promote thedevelopment of clinical symptoms. However, the excitability of motorneuron-like cells that stably expressed mutant and wild-type TDP43protein isunclear. At present, there are more and more study on the relation of ALS andTDP43. In NSC34cell line, normal and mutational TDP43gene result inabnormal morphology of mitochondrial, the downgrade of mitochondrialcomplex1, the absence potential of mitochondrial, elevation of uncouplingprotein-2(UCP2). In some study, analogues of curcumin double methoxylcurcumin (Dimethoxy Curcumin, DMC), improves mitochondrialtransmembrane potential, ameliorates mitochondrial complex1activity andmitochondrial morphology in NSC34cells stably transfected with mutatedTDP43. In addition, previous research in our lab shows that DMC can improve the fusion barrier between the autophagosomes and lysosomes.Furthermore, it can enhance the ability of autophagic to eliminate toxicprotein. But after administration of DMC, whether the excitability ofmotorneuron-like cells that expressed mutant and wild-type TDP43proteinchanged, we still don’t know.Objective: Studying the characteristics of action potention inmotorneuron-like cells that stabled transfected mutational TDP43(Q331K) andwide type TDP43to speculate the level of excitatory in these cells. Whethercharacteristics of action potential of these cells change after giving DMC.Analyze the role of protein TDP43in the pathogenesis of ALS.Methods: We used three different NSC34cell lines that stabledtransfected three different stable TDP43plasmid which are empty vector,TDP43wide type, TDP43Q331K. These three cell lines constructed by ourlaboratory. We consult the method of incubating NSC34cell line to incubateour cells. We identify the express of TDP43with immunohistochemicalmethod. We study the action potential of the three cells using the whole-cellpatch clamp technique. We observe the delay, threshold, amplitude andfrequency of action potential of the three cell and the changes of them aftergiving DMC.Results:First: Comparation of characteristics of action potential in the threegroup between the groups before giving DMCCompared with the cell lines that stably transfected empty vector(Emptygroup),the delay of the cells that stably transfected wide type TDP43(WTgroup) was significantly shorter. When giving all of the depolarizing current,the difference was significant(P<0.05). For20pA Empty group205.6942.35msWT group152.8724.28ms. For30pA Empty group164.5443.55ms WTgroup130.6412.12ms. For40pA Empty group138.2128.86ms WT group112.259.25ms. For60pA Empty group111.5419.83ms WT group92.859.13ms. For80pA Empty group98.6413.94ms WT group80.796.40ms. The other index of action potential was similar to Empty group (P>0.05).Compared with the empty group, the frequency of the cell that stablytransfected mutated Q331K(Q331K group) was significantly faster. Whengiving depolarizing current of30,40,60,80pA the different issignificant(P<0.05). For30pA Q331K group13.352.04Hz Empty group11.151.90Hz. For40pA Q331K group15.241.77Hz Empty group11.932.36Hz. For60pA Q331K group17.441.65Hz Empty group13.632.60Hz. For80pA Q331K group18.371.75Hz Empty group14.331.73Hz. The other index of action potential was similar to Emptygroup (P>0.05).Compared with WT group, the frequency of Q331K group wassignificantly faster. When giving depolarizing current of30,60,80pA thedifferent is significant(P<0.05). For30pA Q331K group13.352.04Hz WTgroup11.572.44Hz. For60pA Q331K group17.441.65Hz WTgroup13.622.53Hz. For80pA Q331K group18.371.75Hz WTgroup14.682.24Hz The other index of action potential was similar to WTgroup (P>0.05).Second: Comparison of before and after the administration of DMCAfter giving DMC, the amplitude of empty group was significantlyreduced. When giving all of the depolarizing current, the difference wassignificant(P<0.05). For20pA, before giving DMC63.869.92mv aftergiving DMC52.5710.78mv. For30pA, before giving DMC69.299.47mvafter giving DMC55.4311.14mv. For40pA, before giving DMC72.607.06mv after giving DMC59.4710.00mv. For60pA, beforegiving DMC77.286.27mv after giving DMC63.5110.80mv. For80pA,before giving DMC81.144.77mv after giving DMC66.7510.86mv.Theother index of action potential was similar to the cells that before giving DMC(P>0.05).After giving DMC, the amplitude of WT group was significantlyreduced. When giving20,30pA depolarizing current, the difference wassignicicnat. For20pA before giving DMC67.3514.66mv after giving DMC 53.4610.08mv. For30pA, before giving DMC70.8313.91mv after givingDMC56.5910.16mv. The other index of action potential was similar to thecells that before giving DMC (P>0.05).After giving DMC, the amplitude of Q331K group was significantlyreduced. When giving all of the depolarizing current, the difference wassignificant(P<0.05). For20pA, before giving DMC63.4010.34mv aftergiving DMC51.607.67mv. For30pA, before giving DMC65.739.40mvafter giving DMC55.277.85mv. For40pA,before giving DMC72.048.58mv after giving DMC after giving DMC55.958.40mv. For60pA, before giving DMC75.509.83mv after giving DMC59.165.51mv.For80pA, before giving DMC76.547.79mv after giving DMC61.766.14mv.The frequency of action potential in Q331K was significantly slowerafter giving DMC. When giving40,60,80pA depolarizing current thedifference was significant(P<0.05). For40pA before giving DMC15.241.77Hz after giving DMC12.082.25Hz. For60pA before giving DMC17.441.65Hz after giving DMC12.342.37Hz. For80pA before givingDMC18.371.75Hz after giving DMC13.363.02Hz.The threshold of action potential was significantly rised after givingDMC. When giving all of the depolarizing current，the difference wassignificant(P<0.05). For20pA before giving DMC-28.055.00mv aftergiving DMC-19.485.49mv. For30pA before giving DMC-28.055.00mvafter giving DMC-19.485.49mv. For40pA before giving DMC-30.134.28mv after giving DMC-19.485.49mv. For60pA before givingDMC-30.134.28mv after giving DMC-20.345.67mv. For80pA beforegiving DMC-30.134.28mv，after giving DMC-20.036.49mv. The otherindex of action potential was similar to the cells that before giving DMC(P>0.05).Thrid: After administration of DMC, the comparison of the three groupbetween the groups, no difference in the indicators of action potential(P>0.05)。 Conclusion: Before giving DMC, the excitability of WT group andQ331K group are higher than Empty group. Both Wild type TDP43andmutant Q331K may participate in the pathogenesis of ALS. The excitability ofthe mutant Q331K cells is higher than wide type, so the nerve damage may bemore serious.After giving DMC, the threshold was increased significantly, frequencyof action potential wass significantly slower and excitability of Q331K wassignificantly decreased, so DMC may be involved in the protect of neuronaldamage relevant with the TDP43gene. The amplitude of action potential in allof the three cell group were reduced. This proclaim that DMC may influencethe sodium channel of cells that stabled transfected mutational TDP43(Q331K),wide type TDP43and empty vector.After administration of DMC, the comparison of the three group between thegroups, there is no difference in the indicators of action potential. |
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