The Effect Of TDP-25on The Motoneuronal Electrophysical Propeties Of Delayed Rectifier Potassium Current And The Protective Effect Of Dimethoxy Curcumin

Amyotrophic Lateral Sclerosis(ALS)，a fatal neurodegerative disease,ischaracterized by the progressive loss of motor neurons.TDP-43（TARDNA bingding protein of43kDa）was identified as the major component ofthe ubiquitin-positive inclusions found in the brain of ALS patentiens. It maybe involved in specific pre-mRNA splicing and transcription, mRNA stabilityand the biosynthesis of microRNAs.Up to now, over40dominant mutations inthe TDP-43gene have been linked to sporadic and familial ALS, whichprovided strong evidence for a direct link between TDP-43dysfunction andneurodegeneration.TDP-25are25kDa C-terminal fragments of TDP-43whichare recovered in the affected brain regions of ALS patients.It has been foundthat TDP-25serve to the process of TDP-43aggregation and is toxic tomotorneurons,resulting in the degeneration of neurons,so play an importantpart in the ALS pathogenisis，but up to now,the mechanism is still unclear. Asthe further investigations to ALS，the relationship between ALS and potassiumchannels has attracted extensive attention.Delayed rectifier potassium current（IKdr） is the major component contributing to the action potentialrepolarization.But few studies were carried out involving the potassiumcurrent on cultured cells of ALS models. Meanwhile, DimethoxyCurcumin(DMC) is the analog of curcumin which is highlighted in recentyears because of its anti-oxidant, anti-carcinogen, anti-mutagenic,immune-modulatory and anti-inflammatory effect.The previous data in ourlaboratory illustrated its protective effect to the mitochondria of themotoneurons-like cells stably transfected with wide type and mutantTDP-43,and indicated that it could enhance the ability of mitophagy,inaddition,curcurmin can also decrease the impairments caused by oxidant effect by upregulating the Nrf2and LCII.Objective： In this study we aimed to investigate whether thephysiological properties of the delayed rectifier potassium channels weremodified in the neurons stably transfected with TDP-25and to explore thepotential pathway in which TDP-25exerts its toxic effect and to furtherexplore the modulatory effect to voltage gated potassium channels of DMC.Method: NSC-34cell lines stably transfected with Empty,TDP-25wereused in our work. The cell lines were constructed by our laboratory and werecultured with the same method as the NSC34cell line. We used highsaturation antibiotics to select monoclonal cells. The stably transfected cellswere seeded on a glass cover in six-well plates, then medium was changedafter12hours and we treated the two cell lines with10μM DMC for24hoursand then observed changes of the biophysical properties of delayed rectifierpotassium current.Whole cell configuration of the patch-clamp technique wasused to record currents in voltage clamp.In mode of current-clamp,we gavethe two cells with four ramping current stimulations of20pA,40pA,60pA and80pA.Then we made a record of threshold potential,APA.APD50and delay.Result：1Compared with the Empty group，the current density ofTDP-25group was smaller， there was statistical difference at60mV（10.65±3.24for TDP-25group and13.26±6.51for Empty group，P<0.05）.2The steady-state activation curve was shifed to more negativepotential，the voltages of the half-activation (V1/2) decreased by3.73mV（P＜0.05，n=14），11.91±4.34mV and8.18±4.95mV for TDP-25group andEmpty group respectively, indicating that the potassium channels of TDP-25group cells are more prone to be activated in more negative potentials,andthere was no significant difference for the slope factor between TDP-25group（11.46±2.41）and Empty group（12.82±1.89），P>0.05.3As for the actionpotential,the threshold potential of the two groups(n=14for TDP-25group andn=11Empty group) at each injection current were similar.The delay time ofTDP-25group was longer than that of Empty group,and was statisticallysignificant at40pA(38.99±9.48ms and30.13±6.96ms, P<0.05）. The APA was larger in TDP-25group than in Empty group(42.96±7.29mV and47.10±8.69mV, P<0.05）.The APD50of TDP-25group was longer than that ofEmpty group,with significant statistical difference at40pA,60pA,80pA(80.41±25.51ms,64.03±23.35ms,55.16±18.97ms and53.18±7.65ms,44.13±5.58ms,36.56±9.24ms respectively）.This difference may be correlated withthe alteration of activation kinetics of TDP-25group cells.4After treating theTDP-25group cells with10uM DMC for24hours,the current density waslarger in the drug group than that of control group,indicating that DMC has thetendency of increasing the IKdr,but there was no statistical difference betweenthe two groups.5Finally,the steady-state activation curve shifted to morepositive potential,the half activation potential moved to hyperpolarlizationdirection for about8.93mV，V1/2=8.18±4.96mV for TDP-25group and17.11±7.99mV for drug group（P＜0.05)，indicating that DMC changed theactivation kenetic behavior of IKdr,promting the channels opening.The slopefactor increased from11.46±2.4to16.12±2.40（P＜0.01),indicating that DMCenhanced the sensitivity of the potassium channel to voltage changing.Conclusion: In this study,we detected the delayed rectifier potassiumsuccessfully.TDP-25inhibited IKdr activities and decreased the currentdensity by altering the activation kinetics,prolonging the action potentialrepolarization phase,which resulted in the increase of firing frequency andthus increased the excitbiliy,according to which the inhibitory effect to IKdr ofTDP-25may be one of the mechanisms resulting in the neuronsdegeneration.DMC,as an opener of IKdr,can change the activation kineticsbehavior of IKdr by which decrease the neurons’excitbility,and thus exert itsprotective effect to motoneurons.