Na~+, K~+-ATPase In TDP-25Transfection Motor Neuron-like Cells

Amyotrophic lateral sclerosis(ALS) is a neurodegenerative diseasecharacterized by degeneration of both upper and lower motor neurons, locatedin cortex, brain stem and spinal cord. ALS is sporadic in90%of all the cases,and the remaining10%are familial heredity. Among the ensured familialheredity cases, mutations in the copper-zinc superoxide dismutase(SOD1)gene on chromosome21was the fisrt to be recognized to cause autosomaldominant ALS, as well as the most common one. It has been reported that thespecific mutations in the gene encoding TDP-43is linked to the pathogenesisof ALS, while the probability of the mutation clearly increases as ageing,indicating that other genetic or environmental factors may promote thedevelopment of ALS.TDP-43is DNA-RNA binding protein encoded by the TARDBP gene,which exits in nuclear normally. In abnormal brains of ALS or frontotemporallobar degeneration with ubiquitin-positive incluxions(FTLD-U), pathologicalTDP-43translocates from nuclear to cytoplasm and prones to aggregation incytoplasm, which forms insoluble ubiquitin-positive inclusions. PathologicalTDP-43is proteolytically cleaved to form a25kDa pathological C-terminalfragment, which is charactered by aggregation and cytotoxicity. Thegeneration and aggregation of this fragment, which are detected in patientswho are suffering from ALS or FTLD-U, could be the cause of the underlyingneurodegeneration. Eventhough TDP-25plays an important role in thepathogenesis of the neurodegeneration disease, we still have knew little aboutthis peptide.Na+, K+-ATPase is critical to neuron survival, which plays a significantrole in physiological activity, such as keeping the homeostasis of iron,maintaining the capacity of cell and the membrane excitability of the muscleand neurocyte and so on. While the inhibition of Na+, K+-ATPase would give rise to the less excretion of Na+and the more intracellular aggregation of Na+,which could then provoke the transportation of Na+-Ca2+exchangers. All ofthese would lead to calcium overload in cytoplasm and end up with neuroninjury or even apoptosis ultimately.It is reported that transgenic mice that express an ALS-associated SOD1mutation(G93A), in the probably presymptomatic phase, have alreadyelevated the excitability of the spinal cord neuron. The hyperexcitability ofneurons may increase the consumption of ATP as well as the burden ofmitochondria and facilitate the progression of the clinical symptoms.Otherwise, it has been detected that the depressed of Na+,K+-ATPase in spinalcord neurons from ALS mice. However, up to now, there are few reports aboutthe activity of Na+,K+-ATPase in transgenic motor-like neurons with stableexpression of the TDP-25.Objective: In this study we aimed to determine whether there is adifference in the activity of Na+,K+-ATPase between the TDP-25(transgeniccells with stably expressing the TDP-25) and the empty vector motor-likeneurons. Then we verified whether there were two subunits in Na+,K+-ATPaseand detected changes of the two subunits in TDP-25cells. Also we discussedthe protein TDP-25in the pathogenesis of ALS.Methods: In this study, we used two different NSC34cell lines withempty vector(Empty) and TDP-25. The cell lines were constructed by ourlaboratory and were cultured as the same method as NSC cell lines. We usedhigh saturation antibiotics to select monoclonal cells. The cell lines wereidentificated by inmmunocytochemistry. Whole cell configuration of thepatch-clamp technique was applied to record the membrane current in theresting state, and we would record it again after perfusion of variousconcentration of ouabain, including10-9,10-8,10-7,10-6,10-5,10-4,10-3mol/L.Thenwe observed the axtivity of Na+,K+-ATPase.Results: Membrane current was recorded after perfusion of variousconcentration of ouabain, as following:TDP-25group：10-9mol/L (n=6):0.10538±0.07058, 10-8mol/L(n=6):0.10122±0.07452,10-/mol/L(n=6):0.23377±0.0683,10-6mol/L(n=6):0.48538±0.1471,10-5mol/L(n=6):0.47068±0.08315,10-4mol/L(n=6):0.64007±0.04886Empty group:10-9mol/L (n=9):0.10662±0.08123,10-8mol/L(n=6):0.15378±0.04578,10-/mol/L(n=7):0.32086±0.09789,10-6mol/L(n=6):0.28407±0.12265,10-5mol/L(n=7):0.60686±0.11757,10-4mol/L(n=8):0.69882±0.1077Non-linear index equation was fitted according to the results above. Two indexes equation and graph were presented, the fitted equation was as following:IP means currents of Na+, K+-ATPase, k means currents which is recorded when one single molecule of Na+, K+-ATPase conjugates with ouabain, K-high and K-low means the index which is the capacity when high or low affinity Na+, K+-ATPase conjugates with ouabain, fh and fl means the percentage of currents which are recorded when high and low affinity Na K+-ATPase conjugates with ouabain,[Oua] means the concentration of ouabain.Na+, K+-ATPase can be classified as high and low affinity according to the differences between K-high and K-lovThe results are as following:Empty group:K-high=58.79nmol/L,K-low=197.67μmol/L, fh=21.8%,f l=78.2%.TDP25group:K-high=32.86nmol/L,K-low=168.95μmol/L,fh=32.6%,f l=67.4%.Conclusions: Na+,K+-ATPase was composed of two subunits. There weretwo kinds of sites conjugated to ouabain in αsubunit, the high affinity siteand the low affinity site. The sensitivity of two sites to ouabain could besignificant improved in the existence of ouabain, and TDP25may promote theexpression of high affinity Na+, K+-ATPase.