| Amyotrophic lateral sclerosis (ALS) is characterized by the degenerationof corticomotoneurons in both the cerebral cortex and the spinal cord.However the mechanisms leading to selective degeneration of motor neuronsare far from being understood. Previous results have established that corticalhyperexcitability develops similarly both in familial ALS and in sporadic ALSpatients, suggesting a common pathophysiological process. In addition it hasalso been shown that intracortical inhibition (ICI) and excitability of thestructures along the corticomotoneuronal pathways are impaired in ALSpatients and correlate with disease duration and clinical stage. Moreover,pre-symptomatic carriers of the SOD1mutation have already developed anintrinsic hyperexcitability, cortical hyperexcitability may contribute tochanges in oxidative stress, as well as altered mitochondrial and energymetabolism, and perhaps a chronic state of energy source depletion and thusprecede the development of clinical symptoms. The importance ofvoltage-gated sodium channels in the action potential initiation andpropagation is well established. They are responsible for the rising phase ofthe action potential in the membranes of neurons and most electricallyexcitable cells. So the orchestrated activation and inactivation gating ofsodium channels is vital to normal neuronal signaling. Even smallsyncopations in this normal gating rhythm may alter cellular excitability,leading to many different disorders. It is reported that G93A mice, in an earlyand probably presymptomatic phase, have already developed an alteration ofthe fast transient voltage-gated Na+channel functions. But in the recentlyfound pathogenic genes model [mutant TAR-DNA-binding protein of43kDa(TDP-43)], there was no record about the properties of voltage-gated Na+ channel.Curcumin(1,7-bis[4-hydroxy-3-methoxyphenyl]-1,6-hep-tadiene-3,5-dione), a member of the curcuminoid family, has a number of biological andpharmacological activities such as anti-carcinogen, anti-angiogenic,anti-malarial, anti-oxidant, anti-mutagenic, anti-bacterial, immune-modulatory,and anti-inflammatory. Dimethoxy Curcumin (DMC) is an analog of curcuminobtained by methylation of both free phenolic groups in the parent compound.It has been reported that DMC has antiamyloido-genic properties in Alzheimerdisease, the previous research in our lab showed protective effect onmitochondria of TDP-43transfected NSC34cell lines. But it is unknown whatwould happen to voltage-gated Na+channel in the condition of giving DMC.So, we put forward four questions:1.Would the neurons stabletransfected with Wide type and Q331K mutant TDP-43in consistent with theones transfected with mutant SOD1show hyperexcitability?2. Does mutant orwide type TDP-43affect the properties of voltage-gated sodium channelwhich is vital in initiating and propagating action potential?3. After theintracellular mutant and wild-type TDP-43level have been regulated by DMC,will the properties of voltage-gated sodium channel change?4. ALS is aneuronal degeneration disease, although the mechanism is still unknown,abnormal in ion channels was not consideded as the fundamental cause, thenwhat effect does voltage-gated sodium channel have on the production andprogress of the disease.To solve the above problems, this research is divided into two parts:Comparing the voltage-gated sodium channel properties of NSC-34cellstransfected human normal and mutated TDP-43to analysis the excitability ofthe cells. Meanwhile observe the changes of the above propetries after havingbeen treated with DMC, then analysis the role of voltage-gated sodiumchannels in ALS pathogenesis.Part ⅠThe Effect of Mutant or Wide-type TDP-43on the Propeties ofNeuronal Voltage-gated Sodium ChannelsObjective: In this study we aimed to determine whether the physiological properties of the voltage-dependent Na+channels were modified in theneurons stably transfected with mutant or wide-type TDP-43.Methods: NSC-34cell lines stable transfected with Empty, WT andQ331K mutant TDP-43were used in our work. The cell lines were constructedby our laboratory and were cultured with the same method as NSC34cell line.High saturation antibiotics were used to select monoclonal cells. Theexpression of TDP-43was identificated by Immunocytochemistry. Whole cellconfiguration of the patch-clamp technique was used to record currents involtage clamp.Results: The properties of voltage-dependent Na+channels were similarin NSC34and Empty groups. While compared with the other three cell lines,in TDP-43Q331K cells, the Na+current density significantly increased(Q331K group-30mv-105.516±43.949;-20mv-123.379±45.219; WT group-30mv-55.062±49.624P<0.01;-20mv-71.088±44.544P<0.01; Empty group-30mv-34.041±45.892P<0.001;-20mv-79.986±40.790P<0.05; NSC34group-30mv-53.048±46.427P<0.05), the voltages of the half-activation (V1/2)increased toward more negative values(Q331K group-37.121±3.991; WTgroup-30.141±7.356, P<0.01; Empty group-26.956±5.609, P<0.001; NSC34group-30.686±2.147, P<0.05), the recovery from fast inactivation wasaccelerated significantly (Q331K group τ=4.066±1.422; WT groupτ=6.838±2.493P<0.01; Empty group τ=5.695±1.856P<0.05), the rate ofNa+channels entry into slow inactivation decreased significantly（Q331Kgroup0.709±0.154; NSC34group0.528±0.116, P<0.01), the rate of Na+channels entry into slow inactivation1s after the depolarizing conditioningpulse increased significantly (when the depolarizing conditioning potentialwas-20，-10,0，10,20mV, the rate of Na+channels entry into slowinactivation decreased significantly in Q331K group than in NSC34group(P<0.05); when the depolarizing conditioning potential was-60and-20mV, the rate of Na+channels entry into slow inactivation decreasedsignificantly in Q331K group than in Empty group(P<0.05)). TDP-43widetype cells showed some similar properties with TDP-43Q331K cells at slow inactivation entry and voltage dependency. Compared with Empty group(0.606±0.161, P<0.05) and NSC34group (0.528±0.116, P<0.01), in WTgroup (0.721±0.124), the rate of Na+channels entry into slow inactivationdecreased significantly. When the depolarizing conditioning potential was-20and-10mV, the rate of Na+channels entry into slow inactivation decreasedsignificantly in WT group than in NSC34group(P<0.05); when thedepolarizing conditioning potential was-50and-10mV, the rate of Na+channels entry into slow inactivation decreased significantly in WT group thanin Empty group(P<0.05). Whereas the slope factors of activation, the timeconstant of fast Inactivation, Na+current steady-state inactivation were similarin the four cell types.Conclusions:1.The properties of voltage-dependent Na+channels weresimilar in NSC34and Empty groups which indicated that the vector did notaffect voltage-dependent Na+currents. The Q331K mutation TDP-43improved the voltage-gated sodium channel activity by changing theproperties of activation and fast and slow inactivation and may increase thefrequency of action potentials and neuronal excitability.3. Wild-type TDP-43improved voltage-gated sodium channel activity mainly by changing thenature of the slow inactivation and may then increase the excitability ofneurons, leading to neuronal degeneration.Part Ⅱ The effects of DMC on voltage-dependent Na+currents inamyotrophic lateral sclerosis cell modelsObjective: This study was to evaluate the effect of DMC onvoltage-dependent Na+currents in Q331K, WT, Empty groups, and theprotective effects of curcumin on the cells and its underlying mechanism.Methods: The stably transfected cells were seeded in six-well plates on aglass cover, medium was changed after12hours and we treated the three celllines with10μM DMC for24hours and then observed changes of thebiophysical properties of voltage-activated sodium channels. The samestimulation protocol as in control group was applied.Results: The sodium currents in WT group（-30mv-7.555±17.681; -20mv-36.178±27.921）were significantly larger than in Q331K group (-30mv-4.255±8.174P<0.001;-20mv-28.122±24.506P<0.05), while it wassignificantly larger in Q331K group (0mv-38.521±21.932) than in Emptygroup(0mv-28.347±10.479P<0.05). The sodium current in WT group wassignificantly larger than in Empty group at the potential of-20、40mv（P<0.01），-10、0、10mv(P<0.001)，20、30mv (P<0.01). The voltages ofthe half-activation (V1/2) in Empty group(-20.286±3.526) were significantlylarger than in Q331K group (-13.200±3.575P<0.01） and WT group（-13.855±4.123P<0.01）. Slope factors of activation, steady-stateinactivation, recovery from fast inactivation, the rate of Na+channels entryinto slow inactivation and recovery from slow inactivation were notsignificantly different.After Q331K group having been treated with10μM DMC for24hours,the sodium current decreased significantly (-40-+40mv P<0.001), the voltagesof the half-activation (V1/2) decreased significantly (Drug group-13.200±3.575; Control group-37.121±3.991P<0.001), the slope factors (a)increased significantly (Drug group2.578±1.075, Control group0.303±0.101P<0.001), V1/2of Na+current steady-state inactivation decreased significantly(Drug group-32.740±6.916; Control group-46.366±11.625P<0.01), recoveryfrom fast inactivation slowed down significantly (Drug group18.279±6.706;Control group4.066±1.422P<0.001). After a long time stimulation the rate ofentry into slow inactivation increased significantly (Drug group0.475±0.179;Control group0.709±0.154P<0.01) and the rate of Na+channels entry intoslow inactivation1s after different depolarizing conditioning pulse was alsosignificantly increased(-70-+20mV P<0.05). The recovery from slowinactivation slowed significantly (4s, P<0.01;7s, P<0.05;10s, P<0.05).Conclusions:1. DMC downregulate the activity of voltage-gated sodiumchannel by changing the properties of activation and fast and slow inactivation.2. Mis-aggregation of TDP-43may be the main reason for changes ofvoltage-gated sodium channel properties.3. DMC is a promising therapeuticcandidate for TDP-43proteinopathies and aggregation-related diseases. |
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