Roles Of Potassium Channels In β-Amyloid Peptide 25-35 Induced Neurotoxicity And Antagonism Of HN: Electrophysiological And Apoptic Study

Alzheimer's disease (AD) is a primary irreversible neurodegenerative disorder characterized by the presence of extensive extracellular amyloid plaques, intracellular neurofibrillary tangles and neuronal death in cerebral cortex and hippocampus, along with progressive impairment of learning, memory and unrelenting cognitive decline.β-amyloid (Aβ) is a polypeptide of 39-43 amino acids and major protein component of senile plaques. As reported widely, the full-length of Aβmolecules, no matter in vivo or in vitro experiments, is neurotoxic. In addition, it is generally accepted that Aβ25-35, a shorter fragment of Aβpeptide, exerts a similar effect as that of the full molecule in different experimental models and thus widely used for exploring the neurotoxicity of Aβ. Several lines of evidence indicates that an abnormal accumulation of Aβis the leading cause and pathological characteristic of AD. Therefore, it is critical to study the mechanisms of Aβ-induced neurotoxicity and find a neuroprotective agence for inhibiting the Aβ's toxicity.Multiple evidence has been reported to show that the disruption of ion homeostasis is one of molecular mechanisms of the neurotoxicity of Aβ. Recent reports suggested that changes in ionic content, primarily K+currents, played a pivotal role in the progression of AD. There are several types of K+ currents in hippocampal neurons which co-exist and apparently contribute specifically to various aspects of neuronal electrophysical properties, such as the resting potential, spike repolarization, spike-frequency adaptation, and delayed excitation. In addition, K+ currents also regulate Ca2+ influx and affect the Ca2+ homeostasis. Accordingly, functional alterations of K+ channels would lead to profound changes in neuronal excitability and intracellular Ca2+ concentration, and finally result in subsequent neuronal dysfunction and even cell death. It was reported that alternations of potassium channel, played an important role in learning and memory. In central nervous system and periphery tissue of AD, there exists abnormal function of potassium channel. Very recently, there was evidence showing that disfunction of K+ channels were involved in apoptosis, in which increase of K+ efflux contributed to apoptosis. However, the relationship between potassium channel dysfunction and pathophysiology of AD is still unclear. Although there were multiple evidence showing the Aβ-induced alternations of potassium currents, there is definitely debate concerning the effects of Aβon potassium channels, especially in different experimental conditions, alternation of potassium currents subjected to Aβwere even opposite.Calcium/phospholipid-regulated protein kinase C (PKC) signaling is known to be involved in cellular functions relevant to brain health and disease, including ion channel modulations, receptor regulations, neurotransmitter release, synaptic plasticity, and neuronal survival. The modulation of K+ channel activities via protein phosphorylation is crucial with regard to the regulation of both neuronal excitability and cellular signaling. The activation of PKC has been shown to modulate neuronal K+ currents in vitro and the expression of KV (voltage-dependent K+ current) family. Furthermore, convincing evidences indicate a close link between Aβand PKC. For example, PKC mediates Aβ31-35-induced suppression of hippocampal late-phase long-term potentiation in vivo. Thus, whether PKC is involved in the modulation of Aβon Ca2+-independent K+ currents?Studies have demonstrated that apoptosis is a major form of neuronal death in AD which could be induced by Aβ31-35. Massive evidence supported that apoptosis occured while K+ efflux is increasing. Thus, whether potassium channels involve in Aβ-induced cell death? Whether K+ channel blockers antagonize neuronal apoptosis induced by Aβ25-35?Humanin (HN) is a 24 amino peptide encoded by a newly identified gene cloned from an apparently normal brain region from patients with AD. Initial studies showed that HN peptide was demonstrated as a selectively neuroprotective factor rescuing neurons from Alzheimer's disease-related insults. Recently, evidence has revealed that HN appeared possessing a broader spectrum of protective activity other than AD-related insults. Thus, HN may play an important protective role in neurodegenerative disease including AD and other pathological insults. Although investigations had paid attention to the mechanisms underlying the neuroprotection of HN, no evidence showed its electrophysiological effects on the neurons.We have testified that HN can antagonize Aβinduced cell death. And whether K+ channel involved in HN against Aβinduced toxicity? Whether HN can protect neurons from K+ efflux induced cell death?Previous experiments showed that HN against Aβ-induced inhibition of Ca2+-dependent K+ currents. Therefore, the purposes of the present study are as follows: (1) clarifying the effects of Aβon the Ca2+-independent K+ currents including a fast transient current (IA) and a delayed rectifier-like current (IK) in hippocampal CA1 neurons and the effects of HN on Aβ-mediated change of Ca2+-independent K+ currents by using whole cell patch clamp technique and calcium fluorescent image;(2) evaluating the potential involvement of PKC in Aβ-mediated the alterations of Ca2+-independent K+ currents and the possible mechanism of HN against Aβ-mediated change of Ca2+-independent K+ currents by using whole cell patch clamp and Western blot techniques; (3) investigating the involvement of K+ channel in Aβ-induced cell death (including apoposis) and antagonistic effects of HN by using cell toxicity assay (cck-8 cell viability assay, LDH release and Caicein-AM staining), apoptosis assay (the quantitative analysis of sub-summit of PI, the measurement of caspase-3 and TUNEL staining ).PartⅠ:Humanin suppressed Aβ25-35-mediated inhibition of Ca2+-independent K+ currentsIn order to explore the electrophysiological mechanisms underlying the Aβ-induced neurotoxicity and the neuroprotection of HN, we designed the experimemts to investigate the effects of HN on Aβ25-35 mediated inhibition of the Ca2+-independent K+ currents including IA and IK in hippocampal CA1 neurons by using whole cell patch clamp technique and calcium fluorescent image. Command potential -80 mV, total K+ currents stimulated with 200 ms depolarizing pulse from -40 mV to +70 mV in 10 mV steps following a hyperpolarizing prepulse of 150 ms to -110 mV. IK stimulated with similar protocol as total K+ currents, except for a 150 ms prepulse to -50 mV. The amplitude of total K+ currents, IA was measured at the peak of the current, and the amplitude of IK was measured at 158 ms of the current.The results showed that: (1) After application of Aβ25-35 (5 mol/L), the amplitude of total K+ currents were significantly decreased throughout the entire voltage-clamp step. The relative amplitude of total K+ currents, IK and IA were 66.23±10.29% (n=11, p<0.05), 71.94±11.20% (n=11, p<0.05) and 47.95±19.25% (n=11, p<0.05), respectively; (2) Pretreatment with HN (5 mol/L), prevented Aβ25-35-mediated inhibition of K+ currents in hippocampal CA1 neurons. The amplitude of total K+ currents were unchanged after application of Aβ25-35 in the presence of HN (103.70±6.64% and 80.16±9.78%, n=8, p>0.05); (3) Post-treatment with HN, reversed Aβ25-35-mediated inhibition of K+ currents in hippocampal CA1 neurons. The relative amplitude of total K+ currents, IK and IA were 80.50±10.05% (n=11, p<0.05 vs Aβ25-35 group), 87.49±13.50% (n=11, p<0.05 vs Aβ25-35 group) and 59.71±22.75% (n=11, p<0.05 vs Aβ25-35 group);(4) In the presence of mixture of Aβ25-35 and HN simultaneously, HN antagonized Aβ25-35-mediated inhibition of K+ currents in hippocampal CA1 neurons. Compared with control, the relative current amplitude of total K+, IK and IA current were 122.82±18.89% (n=5, p>0.05), 116.46± 17.40% (n=5, p>0.05) and 99.71±30.42% (n=5, p>0.05), respectively. Aβ25-35 did not elicit the inhibition of K+ currents; (5) the [Ca2+]i was significantly increased by application of K+ channel blocker as well as Aβ25-35; (6) Aβ25-35-induced elevation in [Ca2+]i was suppressed by HN.These results indicated that: (1) the average amplitude of total K+ currents (including IA and IK) were significantly decreased after acute application of Aβ25-35 in isolated hippocampal CA1 neurons which in turn produce the toxic events including increasing the cellular excitability or enhancing Ca2+ influx (Ca2+ overloading); (2) HN suppressed Aβ25-35-induced inhibition of Ca2+-independent K+ currents and Aβ25-35-induced elevation of [Ca2+]i in hippocampal CA1 neurons; (3) voltage-dependent potassium channel might be one of the targets for HN against Aβ25-35-induced neurotoxicit; (4) combind the previous study and the present results, we hypothesis that HN might suppress the Aβ-mediated responses including electrophysiologial activaties (Aβ-mediated inhibition of IA and IK) by competently occupying the same receptor, no matter the applying ways (before/after Aβ, or coapplied HN and Aβ) of HN and HN is more potent than Aβ.PartⅡ:Roles of PKC in Aβ25-35-induced inhibition of Ca2+-independent K+ currents and Antagonism of HNThe activation of PKC has been shown to modulate neuronal K+ currents and convincing evidence indicates a close link between PKC and activites of Aβ. In the first part of experiment, we have showed that Aβ25-35 suppressed the Ca2+-independent K+ currents including IA and IK. Thus, in the present study, PKC agonist PDBu and antagonist chelerythrine chloride were used to explore if PKC signaling pathway involved in Aβ25–35-induced suppression of IK and further elucidate the mechanism of how HN antagonizes Aβ's effects, in which whole-cell patch clamp and westerm blot were performed in acutely dissociated rat hippacampal neurons.The results showed that: (1) Application of PDBu (1 mol/L, 10 mol/L, 100 mol/L), an activator of PKC resulted in a dose-dependent depression of IK. The relative currents amplitude were 85.00±5.75% (n=5, p<0.05), 67.13±6.83% (n=5, p<0.05), 54.25±8.90% (n=5, p<0.05), respectively. While IA had no significant change after application of PDBU; (2) Application of chelerythrine (20 mol/L), an inhibitor of PKC, antagonized Aβ-induced inhibition of IK in hippocampal CA1 neurons. The relative current amplitude of total K+ currents and IK were 107.08±6.54% (n=12, p>0.05), 106.90±20.54% (n=12, p>0.05). In other words, Aβ25-35-induced inhibition of total K+ currents and IK were prevented; (3) HN (5 mol/L) reversed PDBu (5 mol/L)-induced inhibition of IK in hippocampal CA1 neurons. The inhibition of IK was reduced to 15.4 % (n=5, p<0.05 vs PDBu 5 mol/L group); (4) Aβ25-35 (5 mol/L) activated PKC or phosphorytated PKC. Coadministration of Aβand HN (5 mol/L, each) suppressed Aβ-induced phosphorylation of PKC in hippocampal neurons (n=4, p<0.05).These results indicated that: (1) PKC activator, PDBu resulted in a dose-dependent depression of IK which mimiced the effects of Aβon K+ currents; (2) Aβactivated PKC (phosphorylation of PKC) and in turn mediate Aβ-induced inhibition of K+ currents, while PKC inhibitor chelerythrine antagonized Aβ-induced inhibition of IK; (3) HN suppressed Aβ25-35-induced inhibition of K+ currents by antagonizing Aβ-induced phosphorylation of PKC and PKC-mediated effection on K+ currents. Therefore, PKC is involved in, the depressive effects of Aβon IK which may be one of mechanisms of Aβinduced neurotoxicity; inhibition of PKC-mediated Aβneurotoxicity may be one of mechanisms of HN against Aβinduced neurotoxicity.Part : Roles of K+ channels in Aβ-induced Neurotoxicity and Antagonism of HNIt is well known that suppression of IA and IK channels caused by Aβcould lead to increase duration of depolarization during an action potential, which in turn increase Ca2+ influx, or Ca2+ overloading, and Ca2+-dependent insults. However, the results concerning the effects of Aβon membrane K+ channels were definitely different. It was reported recently that enhanced K+ efflux had been shown to be an essential process of early apoptotic cell shrinkage and also of downstream caspase activation and DNA fragmentation leading neuronal apoptosis.In order to identify whether the K+ channel is involved in Aβ-induced neurotoxicity and antagonism of HN, potassium channel blocker (including TEA for IK, TPeA, analog of TEA, 4-AP for IA) and elevated extracellular [K+] (25 mmol/L KCl) were used to observe if inhibition of K+ currents could suppress Aβ-induced cell death. Additionally, to observe whether HN can antagonize K+ efflux-induced apoptosis, K+ ionophores, valinomycin, a potassium ionophore that allows K+ efflux based on the K+ electrochemical gradient, and can induce apoptosis in many cell types, was used for observations.The results showed that: (1) Aβ25–35 (25 mol/L) produced neurotoxic damage in cortical cell cultures and pretreated with potassium channel blocker TEA (5 mmol/L), TPeA (1 nmol/L) or elevated extracellular K+ (25 mmol/L KCl) 30 min earlier than Aβ25-35, attenuated Aβ25–35-induced neuronal insults measured by cell viability and apoptosis assay; while 4-AP (5 mmol/L) had no significant protection against Aβ25-35-induced neuronal death; (2) K+ ionophore, valinomycin (10 nmol/L, 100 nmol/L, 1000 nmol/L) inhibited neuronal cell viability and increased apoptotic rate in a dose-dependent manner by K+ efflux; (3) Pretreatment of HN (5 mol/L) for 16 h, inhibited valinomycin (100 nmol/L)-induced neuronal insults; while HN (15 mol/L and 25 mol/L) had no effects on them; (4) Pretreated with HN (25 mol/L) for 16 h antagonized Aβ25–35 (25 mol/L)-induced neuronal insults.These results demonstrated that: (1) Chronic exposing of Aβinduced cultured neuronal apoptosis; (2) Cultured neuronal apoptosis induced by Aβwas related with K+ channels open and subsequently massive K+ efflux; (3) IK might play an important role in certain form of cell toxicity and programmed cell death induced by Aβ; (4) HN, at least partly, protected neuron from K+ channels open-mediated cell apoptosis. Therefore, HN protected neuron from K+ channels opening-mediated cell apoptosis, which might be one of mechanism of HN against Aβinduced neuronal apoptosis.Conclusions:(1) Aβ25-35 significantly decreased the amplitude of total K+ currents (including IA and IK) and subsequently induced elevation of [Ca2+]i; HN suppresses Aβ25-35-induced inhibition of Ca2+-independent K+ currents and elevation of [Ca2+]i;(2) Aβ25-35 activate PKC and in turn mediate the inhibition of K+ currents;(3) HN suppressed Aβ25-35-induced inhibition of K+ currents by antagonizing Aβ-induced phosphorylation of PKC and PKC-mediated inhibition of K+ currents;(4) Chronic exposing of Aβinduced cultured neuronal apoptosis; massive K+ channels open might be one of mechanisms of Aβinduced neuronal apoptosis;(5) HN protected neuron from K+ channels opening-mediated cell apoptosis, which might be one of mechanism of HN against Aβinduced neuronal apoptosis;(6) Aβ25-35-induced alternations in K+ ionic homeostasis although it exerted different effects on K+ flux, acute application of Aβ25-35 suppressed K+ currents which in turn increase intracellular Ca2+ (Ca2+ overloading) or excitability; long exposure of Aβenhanced K+ currents which in turn induce apoptosis. However, these different effects of Aβ25-35 definitely produced the same events or toxic results including over excitibility (Ca2+ overloading) or apoptosis which might provide the molecular mechanisms of Aβunderlying the neurodegeneration occurred in AD. (7) The different application of Aβon K+ currents in neurons induced different results by disparate mechanisms. Aβsuppresses K+ channels activity is because acute application of Aβresulted from direct interaction with K+ channels, while Aβmediate the excessive K+ efflux owing to chronic exposure to Aβresulted from the increased expression of potassium channel protein which in turn enhance the K+ efflux.