Role Of Voltage-dependent Potassium Channels And C-Jun N-terminal Kinase Pathway In Aβ_1-40-induced Neuronal Apoptosis

Alzheimer's disease (AD) is a progressive neurogenerative condition affectingthe memory and cognitive abilities of 5-10ï¼…of the population over the age of 65.Brains of AD patients are characterized by three diagnostic hallmarks: senile plaques,neurofibrillary tangles and prominent cortical neurons loss. The senile plaques areinsoluble proteinaceous deposits, principally composed of a 38-43 amino acidpolypeptide known as amloidβprotein (Aβ). It is widely believed that the cellularactions of Aβare responsible for the neurodegeneration observed in AD. Studies invitro and in vivo have demonstrated that neuronal death induced by Aβinvolves anapoptotic pathway. Research on the mechanisms by which Aβmediates neurotoxicityhas made great strides over the last decade. Aβcan generate free radicals, causes aloss of Ca²⁺ homeostasis, and induce tau protein phosphorylation, which results inloss of microtubule binding capacity.Substantive advances have been made, including our laboratory that neuronaldeath induced by many insults including Aβis associated with an enhancement ofoutward K⁺ currents, in particular the delayed rectifier potassium current (I_K). At thesame time, JNK signal pathway also takes part in the generation of senile plaques,causing dispersed Aβdeposit. And after large quantity deposit of Aβ, JNK alsoparticipates in the progress of neuronal apoptosis. At the present study, we study therole of voltage dependent potassium channel and c-Jun N-terminal Kinase pathway inAβ_1-40-induced neuronal apoptosis.Partâ… Role of Potassium Channels in Aβ_1-40-induced Neuronal Apoptosis MTT staining and Hoechst33342 dye were used to assess the cell viability andapoptosis. Viability was gradually decreased with cortical neurons exposed to Aβ_1-40 5μM for 12-48 h. And Aβ_1-40 also brought about apoptotic cell death with nuclearchromatin condensation or fragment after Aβ_1-40 incubation for 48 h.Whole-cell patch clamp technique was used to study the effect of Aβ_1-40incubation on I_K and I_A in cultured cortical neurons. Aβ_1-40 increases I_K in time andconcentration dependent manner. After neurons were exposed to Aβ_1-40 5μM for 6-48h, the density of steady state current of I_K (current just before the end of clamp pulse)at+40 mV was significantly enhanced and reached the maximal value at 12 h. It wasmaintained until 48 h. And the increased current is sensitive to TEA 5 mM.Incubation with Aβ_1-40 2μM,5μM and 10μM for 12 h increa ed the current densityof I_K by 27.48ï¼…, 42.32ï¼…and 58.07ï¼…. In addition, the outward rectifiercharacterization of I_K was not affected. But transient outward K⁺ currents (I_A) did notchange significantly after exposure to Aβ_1-40 at different concentration and differenttime period.After pretreatment with potassium channel blocker TEA 5 mM for 30 min, MTTstaining and Hoechst33342 dye were used and Aβ_1-40 neurotoxicity was dramaticallysuppressed.Colorimetric assay was applied to test the Caspase-3 activation after differenttime incubation with Aβ_1-40 5μM. The Ac-DEVD-pNA cleavage activity wassignificantly elevated as early as 12 h after treatment with Aβ_1-405μM and reachedthe maximal level at 24 h. So we selected the time point 24 h to test the effect of TEAon Caspase-3 activation induced by Aβ. After pretreatment with potassium channelblocker TEA 5 mM for 30 min, the activation of Caspase-3 induced by Aβ_1-40 wassignificantly attenuated. Western blot was used to measure the protein expression ofBcl-2, Bax and cytochrome c release. Compared with control group, exposure of cellsto Aβ_1-40 for 24 h resulted in a significant decrease of Bcl-2 protein, an evident increase of Bax protein and cytochrome c release into cytosol from mitochondria.And pretreatment with TEA 5 mM for 30 min significantly prevented thesealterations.Conclusion: Incubation with Aβ_1-405μM induced apoptosis of cortical neurons,and selectively upmodulated I_K of cortical neurons. TEA 5 mM significantlyalleviated the neuronal apoptosis induced by Aβ_1-40incubation. It suggested that theactivation of delayed rectifier potassium channel played a vital role in neuronalapoptosis.TEA may protect cortical neurons against the apoptosis induced by Aβ_1-405μM by affecting those apoptosis-related protein. And the upmodulation of potassiumchannel occured in the early phase.Partâ…¡Role of c-Jun N-terminal Kinase Pathway in Aβ_1-40-induced NeuronalApoptosis and Potassium Channels UpmodulationWestern blot was used to test the phosphorylation of JNK. JNK wasphosphorylated at all time points after cortical neurons were incubated with Aβ_1-40 5μM for 0-48 h. JNK phosphorylation was gradually increased with time and reachedthe maximal value at 24 h. JNK selective blocker SP600125 significantly inhibitedthe phosphorylation.After pretreatment with SP600125 10μM for 30 min, cortical neurons viabilityinduced by Aβ_1-40was studied with MTT staining method. SP600125 dramaticallyalleviated Aβ_1-40-induced neuronal death. And also decreased neuronal apoptosis.Colorimetric assay and western blot methods were applied to observe the effectof SP600125 on the Caspase-3, Bcl-2, Bax and cytochrome c release induced byAβ_1-40in cultured cortical neurons. And SP600125 significantly affected all thoseapoptosis-related protein alterations. Whole-cell patch clamp technique was used to observe the effect of SP600125on delayed rectifier potassium currents induced by Aβ_1-40. After pretreatment withSP600125 10μM for 30 min, I_K activation in cortical neurons incubated with Aβfor6-48 h was obviously altered. I_K was gradually increased during 24 h incubation withAβ, but compared with Aβtreated, goup, I_K activation was inhibited to a certainextent, and at 12 h I_K activation was significantly inhibited. And then I_K wasgradually increased and maintained at a higher level after 24h.After pretreatment with TEA 5 mM, western blot method was used to test theeffect of TEA on JNK phosphorylation induced by Aβ_1-40. The result showed thatTEA 5 mM didn't significantly change JNK phosphorylation after incubation withAβ_1-40 for 24 h.Conclusion: Aβ_1-405μM induced JNK phosphorylation in cortical neurons in atime dependent manner. JNK phosphorylation was increased after 1 h incubation andthen reached the maximal value after 24 h. JNK selective blocker SP600125 10μMprotected cortical neurons against Aβ_1-40-induced death and apotosis occurrence wasdecreased. And SP600125 prevented neurons from death by affectingapoptosis-related protein alterations.TEA has no effect on Aβ-induced JNK phosphorylation. I_K activation wasn'tcompletely inhibited by SP600125 and maintained a higher level after 24 h. Itsuggested that potassium channel upmodulation may coordinate with JNK activationin the Aβ-induced neuronal apoptosis.