钾通道介导Aβ的神经毒及HN的拮抗作用：电生理和凋亡的实验观察
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摘要
阿尔茨海默病(Alzheimer’s disease,AD),又称老年痴呆,是以记忆、认知功能受损为主要特征的神经退行性疾病,其典型的病理学特征为老年斑(senile plaque, SP)、神经纤维缠结(neurofibrillary tangle, NFT)和大量的神经元丧失。β-淀粉样蛋白(β-amyloid protein,Aβ)是老年斑的主要成分,由39～43个氨基酸组成。有关Aβ1-40和Aβ1-42完整肽链的神经毒作用已有广泛的报道。研究发现Aβ25-35,一个短的分子片断,具有与Aβ完整肽链相同的神经毒作用,因此被广泛应用于AD的实验研究。由于Aβ被普遍认为是AD的关键性致病因素,在AD发病过程中发挥重要作用,因而深入研究其毒性作用机制,从而抑制其毒性将是防治AD的重要环节。
有关Aβ的毒性机制众说纷纭,近来研究表明,Aβ通过扰乱细胞内离子稳态,影响膜电生理特性而发挥重要作用。大量研究证实,钾离子通道的功能紊乱在AD的发病过程中发挥重要作用。钾离子通道能够影响胞内离子稳态、细胞容积,并通过调节膜电位调控递质释放、激素分泌等生理功能。在几乎所有兴奋性细胞和绝大多数非兴奋性细胞静息膜电位的维持过程中钾通道发挥主要作用,同时还可影响动作电位的频率和时程,由此调控细胞的兴奋性以及参与Ca2+稳态的调节。此外,研究表明,钾通道在学习记忆中发挥重要作用,钾通道与Cp20 (一种分子量为20 kDa的GTP结合蛋白)、胞内钙离子、蛋白激酶C (PKC)共同参与学习和记忆的过程。病理条件下,如发生AD时中枢神经系统和外周组织中均存在钾通道功能异常。近来研究还发现,钾通道除影响膜电生理特性、兴奋性和钙稳态及一些生理及病理过程,还参与神经元凋亡的诱导,促进凋亡发生。
然而钾通道功能异常产生的神经损伤作用与AD的病理变化关系十分混乱。虽有相关文献报道,但存在很大争议,特别是在不同的实验条件(如电生理记录和细胞培养)观察时,甚至有结果相反的报道。一方面,钾通道功能异常造成的毒性作用报道不一。既有钾通道开放抑制、钾电流减少引起毒性作用(兴奋性提高、钙超载)的报道,也有钾通道开放增加、钾电流增大引起的毒性作用(诱导凋亡)的报道。另一方面,Aβ对钾通道功能的毒性作用结果也报道不一,既有抑制通道开放使钾电流减少的结果,也有促使通道开放使钾外流增加的报道。因此有必要做进一步研究并分析其机制及原因,明确Aβ在产生毒性作用时对钾通道功能的影响,明确钾通道功能异常产生的毒性作用,从而为解释钾通道的病理生理作用及Aβ的毒性作用机制提供依据。
蛋白激酶C (PKC)作为细胞内重要的信号物质,参与许多生理功能的调节。在对钾通道的调节过程中,PKC尤为重要。研究显示,PKC激活可使钾电流抑制。而且有证据显示,Aβ的毒性作用与PKC密切相关。本研究室以往的研究也证实PKC介导了Aβ31-35引起的海马长持续-长时程(L-LTP)压抑。那么,PKC是否也参与了Aβ所致的钾通道功能的异常改变呢?此外,本室及其他的研究者以往的工作表明,Aβ能引起神经元细胞活力降低、促进凋亡发生,那么钾通道是否也参与Aβ引起的细胞凋亡过程?
Humanin (HN)是2001年首次由日本学者在AD患者脑内发现的由24个氨基酸组成的线性多肽,能够有效抑制多种FAD (Family Alzheimer’s disease)基因突变和Aβ衍生物诱发的神经毒作用,初始被认为是AD特异或AD相关毒性的神经保护肽。然而有关HN拮抗Aβ的神经毒作用机制还不清楚,尤其是对神经元电生理特性的影响未见报道。而且,我们前期的实验观察到HN能够拮抗Aβ引起的凋亡而发挥保护作用,那么钾通道是否在HN拮抗Aβ引起的毒性作用中发挥重要作用?
基于上述的研究背景,为进一步明确Aβ对钾通道功能的影响及钾通道功能异常产生的毒性作用;为明确HN拮抗Aβ神经毒的电生理(钾通道)机制;明确PKC在Aβ引起钾通道功能异常时的作用及HN的拮抗作用;明确钾通道在Aβ所致凋亡中的作用以及HN的神经保护作用,我们设计了以下三部分实验:(1)采用全细胞膜片钳技术观察Aβ对钙非依赖性的钾电流(包括快速失活钾电流和延迟整流钾电流)的影响,以及HN对Aβ引起的钾通道功能改变的拮抗作用;(2)采用全细胞膜片钳技术和免疫蛋白印迹技术观察PKC在Aβ引起钾通道功能改变过程中的作用和HN拮抗作用;(3)采用细胞毒性检测方法和凋亡检测手段,观察钾通道在Aβ诱导的神经毒尤其是诱导凋亡产生中的作用,以及HN的保护作用。为深入理解钾通道功能异常、Aβ神经毒性作用及AD的病理机制提供理论依据。
第一部分: Aβ25-35介导的钙非依赖性钾电流的抑制及HN的拮抗作用
为了深入理解Aβ的神经毒作用的离子机制及探讨HN发挥神经保护作用的电生理机制,本实验利用全细胞膜片钳技术观察Aβ25-35对急性分离的海马神经元钙非依赖性的钾电流(包括快速失活钾电流(fast transient current,IA)和延迟整流钾电流(delayed rectifier-like current,IK))的影响以及HN的拮抗作用。实验过程中,我们采用三种不同的给药顺序进行观察,即在灌流Aβ之前、同时或之后给予同等浓度的HN。在全细胞电压钳记录的条件下,给予细胞由-40～+70mV(阶跃10mV)的去极化脉冲,分别记录钙非依赖性的总的钾电流和IK电流,再经公式换算得出IA。依据文献报道,IK取其158ms处的稳态电流,而IA取其峰值电流进行分析。在同一海马神经元上,比较HN不同的给药方式对Aβ引起的钾电流作用的影响。
结果显示:(1)单独应用Aβ25-35 (5 mol/L),使钙非依赖性的总的钾电流、IK和IA都受到明显的抑制,其相对电流幅度分别为66.23±10.29% (n=11, p<0.05)、71.94±11.20% (n=11, p<0.05)和47.95±19.25% (n=11, p<0.05);(2)HN (5 mol/L)预处理取消了Aβ25-35 (5 mol/L)对钙非依赖性的钾电流的抑制作用。在HN预处理条件下,给予Aβ25-35后,其电流幅度基本不变(103.70±6.64%和80.16±9.78%, n=8, p>0.05),即HN取消了Aβ对钾电流的抑制;(3)HN (5 mol/L)后处理逆转了Aβ25-35 (5 mol/L)引起的对钙非依赖性钾电流的抑制。总的钾电流、IK和IA电流幅度分别为80.50±10.05% (n=11)、87.49±13.50% (n=11)和59.71±22.75% (n=11),同Aβ25-35处理组相比,均有统计学差异(n=11, p<0.05);(4)HN (5 mol/L)与Aβ25-35 (5 mol/L)同时作用于神经元时,HN同样拮抗了Aβ25-35对钾通道的抑制作用。总的钾电流,IK,IA相对电流幅度分别为122.82±18.89% (n=5), 116.46±17.40% (n=5), 99.71±30.42% (n=5),同Aβ25-35处理组相比,均具有统计学差异(n=5, p <0.05);(5)Aβ25-35以及使用钾通道的阻断剂TEA和4-AP或提高细胞外钾离子浓度能够显著的增高细胞内钙离子水平;(6) HN与Aβ25-35同时作用于神经元时,HN能够拮抗Aβ25-35引起的胞内钙升高。
以上结果表明:(1)急性给予Aβ25-35可使分离神经元的钙非依赖性的总的钾电流包括IK、IA抑制,这种作用可能通过提高兴奋性影响钙稳态,如形成钙超载而发挥毒性作用;(2)HN拮抗Aβ25-35介导的对钙非依赖性钾电流的抑制以及胞内钙升高;(3)电压依赖性钾通道可能是HN拮抗Aβ发挥神经保护作用的靶点之一;(4)结合以往实验和本实验结果推测,HN和Aβ可能作用于细胞膜同一靶点而发挥作用,HN比Aβ可能更具有亲和力。
第二部分: PKC介导的Aβ25-35对钾通道的抑制及HN的拮抗作用
鉴于PKC在钾通道调节过程中的重要作用,即PKC激活可抑制钾电流以及广泛报道的PKC介导Aβ的毒性作用。在第一部分研究结果的基础上(Aβ抑制钙非依赖性钾电流),通过使用PKC的激动剂PDBu和阻断剂chelerythrine,应用全细胞膜片钳技术观察PKC是否介导了Aβ对钙非依赖性钾电流的抑制和HN的拮抗作用。同时,采用蛋白免疫印迹技术进一步观察Aβ对PKC的活化以及HN的拮抗作用。
结果显示:(1)不同浓度的PKC激动剂PDBu (1 mol/L, 10 mol/L, 100 mol/L)能够剂量依赖性抑制延迟整流钾电流(IK),相对于对照组,IK的电流幅度分别为85.00±5.75% (n=5, p <0.05), 67.13±6.83% (n=5, p <0.05)和54.25±8.90% (n=5, p <0.05)。而快速失活钾电流(IA)则不受PDBu的影响;(2)PKC阻断剂chelerythrine (20 mol/L)能够拮抗Aβ引起的对IK的抑制。钙非依赖性的总的钾电流和IK相对于正常电流幅度分别为107.08±6.54% (n=12)和106.90±20.54% (n=12),而同单独应用Aβ25-35 (5 mol/L)时两种电流的相对值分别为66.23±10.29% (n=11)和71.94±11.20% (n=11),均有统计学差异(p<0.05)(;3)HN拮抗PKC激动剂PDBu引起的对IK的抑制。第一部分的实验结果证实,HN能拮抗Aβ25-35介导的对钙非依赖性钾电流的抑制,且前述的实验证实Aβ通过PKC介导其对IK的抑制。因此,我们观察了HN对PKC激动剂PDBu引起IK抑制的影响。结果发现,在PDBu (5 mol/L)之后给予HN (5 mol/L),IK相对电流幅度升高为84.60±6.79% (n=8,p <0.05 vs PDBu组),即HN能够拮抗由PDBu引起的对IK的抑制作用;(4)Aβ25-35促进神经元PKC的活化。Aβ25-35 (5 mol/L)作用30 min后能够激活PKC,即磷酸化的PKC明显增多,而Aβ(5 mol/L)和HN (5 mol/L)共同作用30 min后,同单独应用Aβ组相比,PKC的磷酸化水平不再增加(n=4, p<0.05)。
以上结果表明:(1)PKC激动剂剂量依赖性的抑制IK,即激活PKC模拟了Aβ对钾通道的作用;(2)Aβ能够活化(磷酸化)神经元PKC,活化的PKC介导了对钾通道的抑制,而PKC阻断剂拮抗Aβ引起的IK电流的抑制;(3)HN抑制Aβ诱导的PKC的活化(磷酸化);(4)HN拮抗PKC介导的Aβ对IK的抑制。综上,Aβ通过活化PKC介导其对钾通道的作用,这可能是Aβ毒性作用的重要机制之一,抑制Aβ对PKC的活化及抑制PKC介导的毒性作用可能是HN拮抗Aβ神经毒的机制之一。
第三部分:钾通道开放介导的Aβ25-35致凋亡及HN的拮抗作用
我们前一部分的实验及其他一些电生理实验观察显示,Aβ能够抑制钾通道开放及钾电流,使膜电位负值变小,由此导致膜容易去极化,动作电位的时程和有效不应期延长,胞内钙内流增多,继发钙超载,从而导致细胞毒性。新近的文献报道,钾通道开放增加、钾外流增多亦可产生毒性作用,但不是影响兴奋性和钙信号而是诱导凋亡。在培养的神经元发现慢性孵育Aβ通过引起钾通道开放增加,大量钾外流诱导凋亡发生。上述Aβ对钾通道的不同作用(通道抑制,钾电流减少或通道开放,钾外流增加)是在不同的实验条件下观察的结果,前者是在电生理记录条件下通过急性给药观察的结果,后者是在细胞培养条件下慢性孵育药物观察的结果。我们在完成了电生理条件下进行的Aβ对钾通道功能影响的观察后,设计了本部分实验,即使用原代培养的皮层神经元,观察钾通道在Aβ引起的神经元凋亡中的作用及HN的拮抗作用。
实验中采用钾通道的阻断剂,包括对IK敏感的TEA和其同源类似物TPeA以及对IA敏感的4-AP以及使用高浓度的KCl提高细胞外钾离子浓度来抑制钾离子外流,观察阻止钾离子过渡外流能否拮抗Aβ引起的神经元死亡;同时应用钾离子的载体valinomycin(相当于开放剂)诱导神经元凋亡(这是目前研究钾外流增多引起凋亡的通用模型),在此基础上观察HN对钾外流增多引起凋亡的拮抗作用,明确HN拮抗Aβ引起神经元死亡的可能机制。实验过程采用原代培养的皮层神经元,通过细胞毒性检测方法(cck-8试剂盒,乳酸脱氢酶试剂盒,Calcein-AM染色)和凋亡检测方法(流式细胞术,caspase-3试剂盒,TUNEL染色)分析钾通道在Aβ诱导的细胞凋亡中的作用。
结果显示:(1)Aβ25-35 (25 mol/L)孵育24 h引起皮层神经元的凋亡,使用钾通道(IK)的阻断剂TEA (5 mmol/L)、TPeA (1 nmol/L )或提高细胞外的钾离子浓度(KCl 25 mmol/L)预处理30分钟,抑制了Aβ25-35诱导的细胞凋亡,而IA的阻断剂4-AP (5 mmol/L)预孵育则对Aβ25-35引起的神经元凋亡没有保护作用;(2)钾离子载体valinomycin (10 nmol/L, 100 nmol/L, 1000 nmol/L)通过钾通道的开放,钾离子的外流剂量依赖性的降低神经元的细胞活力,并导致凋亡发生;(3) HN (5 mol/L)预处理16 h能够拮抗由valinomycin(100 nmol/L)诱导的细胞凋亡,而15 mol/L和25 mol/L HN却未能发挥保护作用;(4)HN (25 mol/L)预处理16 h能够拮抗Aβ25-35 (25 mol/L)引起的皮层神经元凋亡。
以上结果表明:(1)在离体神经细胞培养的条件下,Aβ导致培养神经元产生凋亡;(2)Aβ引起的神经元凋亡与慢性孵育Aβ引起钾通道开放导致钾离子大量外流有关;(3)HN可拮抗钾通道开放诱导的神经元凋亡。综上,HN可通过拮抗钾通道开放抑制Aβ诱导的神经元凋亡,这可能是HN拮抗Aβ致凋亡作用的机制之一
综上结果,可得出如下结论:
(1)急性给予Aβ25-35可使分离的神经元钙非依赖的钾通道(钾电流)抑制,包括IA和IK,进而引起细胞内钙离子浓度的升高; HN可拮抗由Aβ25-35诱导的钙非依赖性的钾电流的抑制以及胞内钙升高;
(2)Aβ25-35可能通过活化PKC系统介导对钾电流的抑制;
(3)HN通过拮抗Aβ25-35诱导的PKC活化(磷酸化)从而拮抗Aβ25-35诱导的对钾电流的抑制;
(4)慢性孵育Aβ25-35导致培养的神经元凋亡,钾通道的大量开放可能是介导Aβ25-35致凋亡的机制之一;
(5)HN可通过拮抗钾通道开放抑制Aβ诱导的神经元凋亡,这可能是HN拮抗Aβ致凋亡作用的机制之一;
(6)钾通道功能异常无论是开放抑制(钾电流减小)或开放增加(钾外流增加)均可造成毒性作用,前者通过提高神经兴奋性或引起胞内钙超载而产生毒性作用,而后者则通过诱导凋亡发挥毒性作用;
(7)不同的实验条件及给药方式可能通过不同的机制使Aβ产生对钾通道的不同效应。电生理记录过程的急性给药使通道抑制,钾电流减小;而细胞培养过程的慢性孵育可使通道开放增加,钾外流增加。急性给药产生的钾通道抑制可能是Aβ对膜通道及膜电流产生的直接效应,而慢性孵育可能是由于上调了钾通道蛋白表达,使其数量增加,开放增加。
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+ currents
In 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 HN
The 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 HN
It 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.
有关Aβ的毒性机制众说纷纭,近来研究表明,Aβ通过扰乱细胞内离子稳态,影响膜电生理特性而发挥重要作用。大量研究证实,钾离子通道的功能紊乱在AD的发病过程中发挥重要作用。钾离子通道能够影响胞内离子稳态、细胞容积,并通过调节膜电位调控递质释放、激素分泌等生理功能。在几乎所有兴奋性细胞和绝大多数非兴奋性细胞静息膜电位的维持过程中钾通道发挥主要作用,同时还可影响动作电位的频率和时程,由此调控细胞的兴奋性以及参与Ca2+稳态的调节。此外,研究表明,钾通道在学习记忆中发挥重要作用,钾通道与Cp20 (一种分子量为20 kDa的GTP结合蛋白)、胞内钙离子、蛋白激酶C (PKC)共同参与学习和记忆的过程。病理条件下,如发生AD时中枢神经系统和外周组织中均存在钾通道功能异常。近来研究还发现,钾通道除影响膜电生理特性、兴奋性和钙稳态及一些生理及病理过程,还参与神经元凋亡的诱导,促进凋亡发生。
然而钾通道功能异常产生的神经损伤作用与AD的病理变化关系十分混乱。虽有相关文献报道,但存在很大争议,特别是在不同的实验条件(如电生理记录和细胞培养)观察时,甚至有结果相反的报道。一方面,钾通道功能异常造成的毒性作用报道不一。既有钾通道开放抑制、钾电流减少引起毒性作用(兴奋性提高、钙超载)的报道,也有钾通道开放增加、钾电流增大引起的毒性作用(诱导凋亡)的报道。另一方面,Aβ对钾通道功能的毒性作用结果也报道不一,既有抑制通道开放使钾电流减少的结果,也有促使通道开放使钾外流增加的报道。因此有必要做进一步研究并分析其机制及原因,明确Aβ在产生毒性作用时对钾通道功能的影响,明确钾通道功能异常产生的毒性作用,从而为解释钾通道的病理生理作用及Aβ的毒性作用机制提供依据。
蛋白激酶C (PKC)作为细胞内重要的信号物质,参与许多生理功能的调节。在对钾通道的调节过程中,PKC尤为重要。研究显示,PKC激活可使钾电流抑制。而且有证据显示,Aβ的毒性作用与PKC密切相关。本研究室以往的研究也证实PKC介导了Aβ31-35引起的海马长持续-长时程(L-LTP)压抑。那么,PKC是否也参与了Aβ所致的钾通道功能的异常改变呢?此外,本室及其他的研究者以往的工作表明,Aβ能引起神经元细胞活力降低、促进凋亡发生,那么钾通道是否也参与Aβ引起的细胞凋亡过程?
Humanin (HN)是2001年首次由日本学者在AD患者脑内发现的由24个氨基酸组成的线性多肽,能够有效抑制多种FAD (Family Alzheimer’s disease)基因突变和Aβ衍生物诱发的神经毒作用,初始被认为是AD特异或AD相关毒性的神经保护肽。然而有关HN拮抗Aβ的神经毒作用机制还不清楚,尤其是对神经元电生理特性的影响未见报道。而且,我们前期的实验观察到HN能够拮抗Aβ引起的凋亡而发挥保护作用,那么钾通道是否在HN拮抗Aβ引起的毒性作用中发挥重要作用?
基于上述的研究背景,为进一步明确Aβ对钾通道功能的影响及钾通道功能异常产生的毒性作用;为明确HN拮抗Aβ神经毒的电生理(钾通道)机制;明确PKC在Aβ引起钾通道功能异常时的作用及HN的拮抗作用;明确钾通道在Aβ所致凋亡中的作用以及HN的神经保护作用,我们设计了以下三部分实验:(1)采用全细胞膜片钳技术观察Aβ对钙非依赖性的钾电流(包括快速失活钾电流和延迟整流钾电流)的影响,以及HN对Aβ引起的钾通道功能改变的拮抗作用;(2)采用全细胞膜片钳技术和免疫蛋白印迹技术观察PKC在Aβ引起钾通道功能改变过程中的作用和HN拮抗作用;(3)采用细胞毒性检测方法和凋亡检测手段,观察钾通道在Aβ诱导的神经毒尤其是诱导凋亡产生中的作用,以及HN的保护作用。为深入理解钾通道功能异常、Aβ神经毒性作用及AD的病理机制提供理论依据。
第一部分: Aβ25-35介导的钙非依赖性钾电流的抑制及HN的拮抗作用
为了深入理解Aβ的神经毒作用的离子机制及探讨HN发挥神经保护作用的电生理机制,本实验利用全细胞膜片钳技术观察Aβ25-35对急性分离的海马神经元钙非依赖性的钾电流(包括快速失活钾电流(fast transient current,IA)和延迟整流钾电流(delayed rectifier-like current,IK))的影响以及HN的拮抗作用。实验过程中,我们采用三种不同的给药顺序进行观察,即在灌流Aβ之前、同时或之后给予同等浓度的HN。在全细胞电压钳记录的条件下,给予细胞由-40～+70mV(阶跃10mV)的去极化脉冲,分别记录钙非依赖性的总的钾电流和IK电流,再经公式换算得出IA。依据文献报道,IK取其158ms处的稳态电流,而IA取其峰值电流进行分析。在同一海马神经元上,比较HN不同的给药方式对Aβ引起的钾电流作用的影响。
结果显示:(1)单独应用Aβ25-35 (5 mol/L),使钙非依赖性的总的钾电流、IK和IA都受到明显的抑制,其相对电流幅度分别为66.23±10.29% (n=11, p<0.05)、71.94±11.20% (n=11, p<0.05)和47.95±19.25% (n=11, p<0.05);(2)HN (5 mol/L)预处理取消了Aβ25-35 (5 mol/L)对钙非依赖性的钾电流的抑制作用。在HN预处理条件下,给予Aβ25-35后,其电流幅度基本不变(103.70±6.64%和80.16±9.78%, n=8, p>0.05),即HN取消了Aβ对钾电流的抑制;(3)HN (5 mol/L)后处理逆转了Aβ25-35 (5 mol/L)引起的对钙非依赖性钾电流的抑制。总的钾电流、IK和IA电流幅度分别为80.50±10.05% (n=11)、87.49±13.50% (n=11)和59.71±22.75% (n=11),同Aβ25-35处理组相比,均有统计学差异(n=11, p<0.05);(4)HN (5 mol/L)与Aβ25-35 (5 mol/L)同时作用于神经元时,HN同样拮抗了Aβ25-35对钾通道的抑制作用。总的钾电流,IK,IA相对电流幅度分别为122.82±18.89% (n=5), 116.46±17.40% (n=5), 99.71±30.42% (n=5),同Aβ25-35处理组相比,均具有统计学差异(n=5, p <0.05);(5)Aβ25-35以及使用钾通道的阻断剂TEA和4-AP或提高细胞外钾离子浓度能够显著的增高细胞内钙离子水平;(6) HN与Aβ25-35同时作用于神经元时,HN能够拮抗Aβ25-35引起的胞内钙升高。
以上结果表明:(1)急性给予Aβ25-35可使分离神经元的钙非依赖性的总的钾电流包括IK、IA抑制,这种作用可能通过提高兴奋性影响钙稳态,如形成钙超载而发挥毒性作用;(2)HN拮抗Aβ25-35介导的对钙非依赖性钾电流的抑制以及胞内钙升高;(3)电压依赖性钾通道可能是HN拮抗Aβ发挥神经保护作用的靶点之一;(4)结合以往实验和本实验结果推测,HN和Aβ可能作用于细胞膜同一靶点而发挥作用,HN比Aβ可能更具有亲和力。
第二部分: PKC介导的Aβ25-35对钾通道的抑制及HN的拮抗作用
鉴于PKC在钾通道调节过程中的重要作用,即PKC激活可抑制钾电流以及广泛报道的PKC介导Aβ的毒性作用。在第一部分研究结果的基础上(Aβ抑制钙非依赖性钾电流),通过使用PKC的激动剂PDBu和阻断剂chelerythrine,应用全细胞膜片钳技术观察PKC是否介导了Aβ对钙非依赖性钾电流的抑制和HN的拮抗作用。同时,采用蛋白免疫印迹技术进一步观察Aβ对PKC的活化以及HN的拮抗作用。
结果显示:(1)不同浓度的PKC激动剂PDBu (1 mol/L, 10 mol/L, 100 mol/L)能够剂量依赖性抑制延迟整流钾电流(IK),相对于对照组,IK的电流幅度分别为85.00±5.75% (n=5, p <0.05), 67.13±6.83% (n=5, p <0.05)和54.25±8.90% (n=5, p <0.05)。而快速失活钾电流(IA)则不受PDBu的影响;(2)PKC阻断剂chelerythrine (20 mol/L)能够拮抗Aβ引起的对IK的抑制。钙非依赖性的总的钾电流和IK相对于正常电流幅度分别为107.08±6.54% (n=12)和106.90±20.54% (n=12),而同单独应用Aβ25-35 (5 mol/L)时两种电流的相对值分别为66.23±10.29% (n=11)和71.94±11.20% (n=11),均有统计学差异(p<0.05)(;3)HN拮抗PKC激动剂PDBu引起的对IK的抑制。第一部分的实验结果证实,HN能拮抗Aβ25-35介导的对钙非依赖性钾电流的抑制,且前述的实验证实Aβ通过PKC介导其对IK的抑制。因此,我们观察了HN对PKC激动剂PDBu引起IK抑制的影响。结果发现,在PDBu (5 mol/L)之后给予HN (5 mol/L),IK相对电流幅度升高为84.60±6.79% (n=8,p <0.05 vs PDBu组),即HN能够拮抗由PDBu引起的对IK的抑制作用;(4)Aβ25-35促进神经元PKC的活化。Aβ25-35 (5 mol/L)作用30 min后能够激活PKC,即磷酸化的PKC明显增多,而Aβ(5 mol/L)和HN (5 mol/L)共同作用30 min后,同单独应用Aβ组相比,PKC的磷酸化水平不再增加(n=4, p<0.05)。
以上结果表明:(1)PKC激动剂剂量依赖性的抑制IK,即激活PKC模拟了Aβ对钾通道的作用;(2)Aβ能够活化(磷酸化)神经元PKC,活化的PKC介导了对钾通道的抑制,而PKC阻断剂拮抗Aβ引起的IK电流的抑制;(3)HN抑制Aβ诱导的PKC的活化(磷酸化);(4)HN拮抗PKC介导的Aβ对IK的抑制。综上,Aβ通过活化PKC介导其对钾通道的作用,这可能是Aβ毒性作用的重要机制之一,抑制Aβ对PKC的活化及抑制PKC介导的毒性作用可能是HN拮抗Aβ神经毒的机制之一。
第三部分:钾通道开放介导的Aβ25-35致凋亡及HN的拮抗作用
我们前一部分的实验及其他一些电生理实验观察显示,Aβ能够抑制钾通道开放及钾电流,使膜电位负值变小,由此导致膜容易去极化,动作电位的时程和有效不应期延长,胞内钙内流增多,继发钙超载,从而导致细胞毒性。新近的文献报道,钾通道开放增加、钾外流增多亦可产生毒性作用,但不是影响兴奋性和钙信号而是诱导凋亡。在培养的神经元发现慢性孵育Aβ通过引起钾通道开放增加,大量钾外流诱导凋亡发生。上述Aβ对钾通道的不同作用(通道抑制,钾电流减少或通道开放,钾外流增加)是在不同的实验条件下观察的结果,前者是在电生理记录条件下通过急性给药观察的结果,后者是在细胞培养条件下慢性孵育药物观察的结果。我们在完成了电生理条件下进行的Aβ对钾通道功能影响的观察后,设计了本部分实验,即使用原代培养的皮层神经元,观察钾通道在Aβ引起的神经元凋亡中的作用及HN的拮抗作用。
实验中采用钾通道的阻断剂,包括对IK敏感的TEA和其同源类似物TPeA以及对IA敏感的4-AP以及使用高浓度的KCl提高细胞外钾离子浓度来抑制钾离子外流,观察阻止钾离子过渡外流能否拮抗Aβ引起的神经元死亡;同时应用钾离子的载体valinomycin(相当于开放剂)诱导神经元凋亡(这是目前研究钾外流增多引起凋亡的通用模型),在此基础上观察HN对钾外流增多引起凋亡的拮抗作用,明确HN拮抗Aβ引起神经元死亡的可能机制。实验过程采用原代培养的皮层神经元,通过细胞毒性检测方法(cck-8试剂盒,乳酸脱氢酶试剂盒,Calcein-AM染色)和凋亡检测方法(流式细胞术,caspase-3试剂盒,TUNEL染色)分析钾通道在Aβ诱导的细胞凋亡中的作用。
结果显示:(1)Aβ25-35 (25 mol/L)孵育24 h引起皮层神经元的凋亡,使用钾通道(IK)的阻断剂TEA (5 mmol/L)、TPeA (1 nmol/L )或提高细胞外的钾离子浓度(KCl 25 mmol/L)预处理30分钟,抑制了Aβ25-35诱导的细胞凋亡,而IA的阻断剂4-AP (5 mmol/L)预孵育则对Aβ25-35引起的神经元凋亡没有保护作用;(2)钾离子载体valinomycin (10 nmol/L, 100 nmol/L, 1000 nmol/L)通过钾通道的开放,钾离子的外流剂量依赖性的降低神经元的细胞活力,并导致凋亡发生;(3) HN (5 mol/L)预处理16 h能够拮抗由valinomycin(100 nmol/L)诱导的细胞凋亡,而15 mol/L和25 mol/L HN却未能发挥保护作用;(4)HN (25 mol/L)预处理16 h能够拮抗Aβ25-35 (25 mol/L)引起的皮层神经元凋亡。
以上结果表明:(1)在离体神经细胞培养的条件下,Aβ导致培养神经元产生凋亡;(2)Aβ引起的神经元凋亡与慢性孵育Aβ引起钾通道开放导致钾离子大量外流有关;(3)HN可拮抗钾通道开放诱导的神经元凋亡。综上,HN可通过拮抗钾通道开放抑制Aβ诱导的神经元凋亡,这可能是HN拮抗Aβ致凋亡作用的机制之一
综上结果,可得出如下结论:
(1)急性给予Aβ25-35可使分离的神经元钙非依赖的钾通道(钾电流)抑制,包括IA和IK,进而引起细胞内钙离子浓度的升高; HN可拮抗由Aβ25-35诱导的钙非依赖性的钾电流的抑制以及胞内钙升高;
(2)Aβ25-35可能通过活化PKC系统介导对钾电流的抑制;
(3)HN通过拮抗Aβ25-35诱导的PKC活化(磷酸化)从而拮抗Aβ25-35诱导的对钾电流的抑制;
(4)慢性孵育Aβ25-35导致培养的神经元凋亡,钾通道的大量开放可能是介导Aβ25-35致凋亡的机制之一;
(5)HN可通过拮抗钾通道开放抑制Aβ诱导的神经元凋亡,这可能是HN拮抗Aβ致凋亡作用的机制之一;
(6)钾通道功能异常无论是开放抑制(钾电流减小)或开放增加(钾外流增加)均可造成毒性作用,前者通过提高神经兴奋性或引起胞内钙超载而产生毒性作用,而后者则通过诱导凋亡发挥毒性作用;
(7)不同的实验条件及给药方式可能通过不同的机制使Aβ产生对钾通道的不同效应。电生理记录过程的急性给药使通道抑制,钾电流减小;而细胞培养过程的慢性孵育可使通道开放增加,钾外流增加。急性给药产生的钾通道抑制可能是Aβ对膜通道及膜电流产生的直接效应,而慢性孵育可能是由于上调了钾通道蛋白表达,使其数量增加,开放增加。
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+ currents
In 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 HN
The 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 HN
It 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.
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