聚腺苷二磷酸核糖多聚酶在无镁致痫大鼠海马神经元的死亡机制研究
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摘要
癫痫是一种多种病因导致的临床综合征,以脑部神经元高度同步化异常放电引起的发作性、短暂性、重复性和刻板性的中枢神经系统功能障碍为特点,是最常见的神经系统疾病之一。全球约有5000万,我国约有900万癫痫患者,患病率约为5‰,其中难治性癫痫约占25%,抗癫痫药物和手术治疗效果不佳,且发病机制目前尚未完全清楚。众多研究证实,胆碱能受体激动剂匹鲁卡品和兴奋性毒性谷氨酸受体激动剂海人酸(kainic acid, KA)诱导的癫痫动物模型,在行为学、神经电生理学及海马神经元损伤的病理学方面与人类难治性癫痫尤其是颞叶癫痫类似,是常用的颞叶癫痫体内动物模型。1995年Sombati等利用无镁细胞外液诱导体外培养的海马神经元产生自发性反复性癫痫样放电(spontaneous recurrent epileptiform discharges, SREDs),建立了癫痫体外细胞模型,具有快捷有效、可靠稳定、便于观察药物效应和电生理变化的特点,并且排除了化学致痫药物本身的毒性干扰,在探索新型抗癫痫神经保护药物方面发挥了重要作用。
聚腺苷二磷酸核糖多聚酶[poly(ADP-ribose) polymerase, PARP]主要存在于真核细胞核中,是一类非组蛋白染色体蛋白质,其主要生物学功能包括：(1)修复DNA损伤并维持基因稳定性；(2)调节相关蛋白的转录水平,影响其表达；(3)影响复制和分化,参与维持端粒长度；(4)清除机体内部损伤细胞,调控细胞死亡。其中PARP-1是PARP家族成员中含量最高(90%以上)、研究最清楚的蛋白。在生理状态下,PARP-1被DNA断裂损伤激活,形成同型二聚体并将烟酰胺腺嘌呤二核甘酸(nicotinamide adenine dinucleotide, NAD)分解为烟酰胺和腺苷二磷酸核糖大分子聚合物,聚腺苷二磷酸核糖[poly(ADP-ribose), PAR]可与组蛋白、转录因子及PARP-1结合,调节其活性和功能,利JDNA修复的进行。而DNA受损严重时,PARP-1被过度激活,大量消耗细胞内NAD+和ATP,激活细胞死亡相关蛋白,引起炎症反应,可见PARP-1的生物学效应是一把双刃剑。
研究发现,PARP-1的药物抑制或基因敲除在神经系统退行性病变、外伤、炎症、缺血再灌注和癫痫等疾病中发挥了明显的神经保护作用。PARP-1过度激活介导细胞损伤的机制很复杂,主要包括：Caspase依赖性细胞凋亡,非Caspase依赖性细胞死亡,炎性介质过度表达,能量代谢障碍和Ca2+/Mg2+依赖性核酸内切酶活性增高等。近期观点认为,PARP介导细胞死亡的形态学与分子生物学特征同经典的凋亡与坏死存在明显的区别,为非Caspase依赖性细胞死亡的一种,并被命名为Parthanatos。其生化特征包括：PARP迅带激活,早期PAR的积累,线粒体膜电位去极化并通透性改变,凋亡诱导因子(apoptosis-inducing factor, AIF)向细胞核转位,细胞内NAD+(?)(?)ATP大量消耗,晚期Caspase激活,细胞萎缩等。近期研究发现沉默信息调节因子2同源蛋白1(silencing information regulator2homolog1,SIRT1)能够通过调节细胞能量代谢、生长分化发挥保护作用,但其生物学效应受NAD+水平制约。因此对PARP-1, NAD+, AIF, SIRT1等潜在靶点的调控可为PARP(?)(?)关性疾病的治疗开辟新的途径。然而,Parthanatos的具体机制尚未完全明确,在癫痫病理生理过程中的作用研究较少。
为此,本课题建立无镁诱导大鼠海马神经元癫痫放电模型,排除体内众多因素的影响,模拟边缘系统受损性癫痫,探讨癫痫体外模型中海马神经元的电生理特性,并应用多种药物干预手段,检测PARP-1介导的细胞死亡信号通路和相关蛋白变化,以此深入研究癫痫的分子生物学机制并为癫痫的治疗提供新的靶点。本研究为三个部分：
第一部分无镁诱导大鼠海马神经元癫痫放电模型的建立及形态学、电生理学观察
目的
建立无镁诱导大鼠海马神经元癫痫放电模型,观察自发性癫痫放电对体外培养大鼠海马神经元细胞形态学及电生理学特征的影响。
方法
采用新生24h内的Sprague-Dawley大鼠断头取脑,显微镜下分离海马神经元进行的原代培养。培养过程中观察神经元的形态学特征,并用培养至14d的细胞用神经元特异性烯醇化酶(neuron-specific enolase, NSE)抗体,采用免疫细胞化学技术鉴定神经元纯度。培养至14d的海马神经元经过无镁细胞外液处理3h后恢复至正常培养基,在不同时间点采用全细胞膜片钳技术记录神经元放电情况,验证无镁诱导大鼠海马神经元癫痫放电模型建立成功。
结果
1.细胞形态学特征：刚接种至六孔板培养基内的海马神经元为小圆形,散在分布。大部分神经元在培养12-24h后贴壁,突起明显增多,长短不一。培养至7d,已具有典型的轴突与树突,细胞结构立体,折光感强,细胞核呈空泡状,神经元间尚未形成突触联系,为单个生长的细胞。10d左右神经元间形成突触联系,呈网状结构。培养10-14d时,细胞体积最大,突起最为清晰,网络最为密集。至25-28d,培养液中有悬浮的细胞碎片。
2.神经元纯度检验：培养至14d的神经元细胞爬片采用免疫细胞化学技术染色后,神经元的胞浆和突起被染成棕色,镜下计数后神经元纯度可达94.7%。
3.电生理学特征：在正常生理外液中,大部分神经元可记录到正常突触后电活动,包括兴奋性突触后电位(excitatory postsynaptic potential, EPSP)、抑制性突触后电位(inhibitory postsynaptic potential, IPSP)和偶发动作电位(action potential, AP)。其静息电位范围为-60--40mV,动作电位幅度为15～30mV。神经元在无镁刺激过程中,放电形式发生改变,表现为持续强直性高频爆发和“楔形”去极化。经无镁处理3h并恢复正常培养基后,表现为阵发性持续棘波样爆发,另外存在有“楔形”去极化及阵发性去极化偏移(paroxysmal depolarizing shifts,PDS)。无镁刺激恢复正常后持续至96h仍可检测到周期性自发放电表现。
结论
1.本研究所用方法培养的大鼠海马神经元在数量、纯度、存活时间方而较好,表面光滑,轮廓清楚,有较强的立体感,具有适于膜片钳研究的细胞结构基础。
2.本模型中海马神经元虽为体外培养,但神经元之间能形成广泛的突触网状联系,具有信息传递的形态学基础。
3.无镁刺激能诱发神经元产生持久的自发性反复性放电,与癫痫发作时电生理活动类似,具有体外癫痫细胞模型研究的电生理基础。
4.无镁诱导海马神经元癫痫放电模型稳定可靠、可控性强,是良好的癫痫细胞模型,为研究癫痫病理生理机制及新型抗癫痫药物的研发提供了良好平台。
第二部分PARP-1调控无镁致痫大鼠海马神经元中AIF表达、分布及PI3K/Akt信号通路的机制研究
目的
评估无镁诱导大鼠海马神经元癫痫放电模型中的细胞生存状态：探讨神经元内AIF的表达、分布及PI3K/Akt信号通路随时间的变化规律；探讨PARP-1及其抑制剂DPQ(3,4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)一isoquinolinone)对上述指标的调节作用及相关机制。
方法
1.将体外培养至14d的大鼠海马神经元随机分为对照组、尤镁组、在镁+Z-VAD-FMK(Caspase抑制剂)组、无镁饮+DPQ组,均选用致痫后1h、24h两个时间点,采用FUNEI及LDH检测法,评估无镁诱导后神经元死亡情况,并探讨DPQ对其影响。
2.将大鼠海马神经元随机分为对照组、无镁组、无镁+DMSO祖、无镁(?)DPQ组、无镁+dpq+WTN(wortmannin,PI3K抑制剂组,提取总蛋白、细胞核及线粒体蛋白,采用Western blot法检测PAR、PARP-1\AIF、(p-)Akt、(p-)GSK3β的表达
3.采用细胞免疫荧光共聚集技术,检测AIF在神经元内的分布。
结果
1.无镁诱导后24h,TUNEL细胞核阳性率可达71.2%：而在镁+FMK组及无镁+DPQ组的该比例分别降至45.25%和26.88%,LDH漏出率分别为在镁24h组的57.35%和36.94%。
2.PAR的蛋白量在无镁诱导3h后显著升高,24h达最高水平。线粒体中AIF表达量随时间降低,诱导后24h达最低水平(p<0.05)；与之对应,细胞核中AIF表达逐渐增高。无镁+DPQ组中PAR表达量下降,线粒体内AIF的含量明显高于无镁+DMSO组(p<0.05)；细胞核内AIF含量则显著降低(p<0.05)。细胞免疫荧光检测结果与上述趋势相一致。
3.无镁诱导后,Akt、GSK3β磷酸化水平在1h显著降低(p<0.05),在12h基本恢复至对照组水平。无镁+DPQ组Akt及GSK3β磷酸化水平显著高于无镁+DMSO组(p<0.05)。而无镁+DPQ+WTN组,Akt、GSK3P磷酸化水平及线粒体内AIF表达显著低于无镁+DPQ组(p<0.05),细胞核内AIF表达则与之相反。
结论
无镁诱导大鼠海马神经元癫痫模型中,PARP-1被过度激活,导致AIF由线粒体向细胞核内转位,神经元大量死亡。而PARP-1抑制剂DPQ通过激活PI3K/Akt/GSK3β信号通路,抑制AIF向细胞核的转位,减少神经元死亡,发挥了细胞保护作用。
第三部分PARP-1调节无镁致痫大鼠海马神经元中NAD+及SIRT1水平的机制研究
目的
探讨无镁诱导大鼠海马神经元癫痫放电模型中,PARP-1介导的细胞死亡与神经元内NAD+含量、SIRT1的表达及去乙酰化活性的关系；探讨细胞内NAD+含量对AIF由线粒体向细胞核转位的作用。
方法
将体外培养至14d的大鼠海马神经元随机分为对照组、无镁组、无镁+DPQ组、无镁+NAD (5,10mM)组、无镁+白藜芦醇(resveratrol, SIRT1激动剂)组及无镁+sirtinol (SIRT1抑制剂)组。检测无镁诱导后NAD+的含量、SIRT1的mRNA和蛋白水平随时间的变化规律：探讨应用DPQ或NAD干预后,TUNEL阳性细胞率与NAD+水平的变化关系、PARP-1的表达与活性状态、AIF的胞内分布以及SIRT1(?)的表达和活性对神经元存活状况的影响。
结果
1.无镁组大鼠海马神经元内NAD+含量随时间逐渐下降。与无镁组相比,无镁+DPQ组的NAD+含量显著增高(p<0.05),TUNEL阳性细胞率显著降低(p<0.05)。
2.细胞免疫荧光和Western blot(?)支术均证实,无镁24h+NAD组神经元细胞核AIF的蛋白表达量显著低于无镁24h组(p<0.05),而线粒体AIF表达量高于无镁组(p<0.05)。
3.SIRT1的mRNA水平随无镁诱导后时间延长而增高,但蛋白水平与对照{组无显著差异；应用DPQ或NAD干预后SIRT1的mRNA水平较在镁明显著降低(p<0.05),但蛋白水平与无镁组相比无显著差异。
4.在镁+DPQ组SIRT1去乙酰化活性是无镁组的3倍,TUNEL阳性率为27.62%。无镁+resveratrol组将SIRT1(?)活性提高至无镁组的3.72倍,TUNEL阳性率为35.73%。无镁+sirtinol组SIRT1活性显著降低,TUNEL(?)(?)性率与无镁组无显著差异(p>0.05)。
5.无镁组PAR蛋白表达量显著高于对照组(p<0.05),无镁+DPQ组PAR表达量显著低于在无镁组(p<0.05),而无镁天+NAD组PAR表达量与无镁组相比在明显差异,各组PARP-1蛋白水平无显著差异,
结论
无镁诱导大鼠海马神经元癫痫模型中,PARP-1被过度激活,胞内NAD急剧消耗,SIRT1去乙酰化活性下降,AIF由线粒体向细胞枋核内转位,神经元大量死亡。补充外源性NAD+能抑制AIF转位,减少神经元死亡；而SIRT1作为PARP-1和NAD+的下游分子,其活性(而不是蛋白表达量)与神经元死亡率呈负相关。因此神经元内NAD+含量与SIRT1去乙酰化活性的下降为PARP-1介导的无镁致痫神经元的死亡机制提供了另一条通中。
Epilepsy is a common and diverse set of chronic clinic syndrome characterized by paroxysmal, transient, repetitive and inflexible neurological disorders caused by hyper-synchronous neuronal discharges in the brain. Epilepsy is one of the most common neurological diseases which affects about50million patients around the world and9million in China. And about25%of patients suffer from intractable epilepsy with poor effects of medication and surgery. The pathophysiologic mechanisms are complicated. It has been suggested that the behavior, electroencephalogram (EEG) and the hippocampal neuronal injury of seizures induced by lithium-pilocarpine and kainic acid in rats is similar to that in patients with temporal lobe epilepsy. So the two methods have been the most frequently used animal models to research human epilepsy. Sombati established the hippocampal neuronal culture (HNC) model of acquired epilepsy (AE) which displayed spontaneous recurrent epileptiform discharges (SREDs) induced by Mg2+-free treatment in1995. It is convenient to observe the electrophysiological changes and drug effect in this model. Moreover, the pharmacological interference of epileptogenic drugs can be properly excluded to reveal the underlying mechanisms of epilepsy.
Poly (ADP-ribose) polymerase-1(PAR.P-1) is a kind of non-histone chromosomal proteins which exists in the nucleus of eucaryotes, which is proved to be involved in DNA repair, gene transcription. DNA duplication and cell death. In physiological conditions, PARP-1activated by DNA breaks cleaves nicotinamide adenine dinucleotide (NAD) into nicotinamide and ADP-ribose. The latter can be synthesized into long-branching poly(ADP-ribose)(PAR) polymers which covalently attach to acceptor proteins including histones, DNA repair enzymes, transcription factors and PARP-1itself, facilitating DNA repair and cell survival. However, PARP-1can be hyper-activated by excessive DNA damage, triggering apoptosis or necrotic cell death which depletes a large amount of NAD+and ATP.
Cumulative evidence suggested that the inhibition of PARP-1has cytoprotective effects against neurotoxicity, central nervous system injury, ncurodcgcnerative disorders, inflammation and epilepsy. The mechanisms of neuronal damages mediated by hyper-activation of PARP-1are complicated, including classical caspase-dependent apoptosis pathway, caspase-independent cell death pathway, accumulation of inflammatory mediators, hyper-activation of Ca2+/Mg2+-dependent endonuclease and energy failure. It is noteworthy that PARP-1-dependent cell death is unique in terms of its biochemical and morphological features, which is called "parthanatos". The biochemical features of parthanatos are distinct from classically defined pathways of cell death and involve rapid PARP-1activation, early PAR accumulation, mitochondrial depolarization, early nuclear apoptosis-inducing factor (AIF) translocation, cellular NAD+and ATP consumption and late caspase activation. And silent mating type information regulation2homolog1(SIRT1) has recently confirmed to be related to cell survival conditions. Therefore, it will be prospective to invent new therapies of epilepsy by regulating the target proteins involved in the pathway.
In this study, the I INC model of AE was established to mimic epileptic conditions caused by hippocampus damages. Based on this, we examined the response of related cell signaling pathway and critical proteins to reveal the underlying mechanisms of caspase-independent cell death mediated by PARP-1hyper-activation.
PART I A study of morphological and electrophysiological characteristics of neurons in the HNC model of AE in rats
Objective
To establish the HNC model of AE induced by Mg2+-free treatment in rats, and investigate the morphological and electrophysiological characteristics of neurons in this model.
Methods
Hippocampal cells were separated from24h postnatal Sprague-Dawley rats, plated and maintained in proper conditions. The purity of neurons in primary cultures was detected by immunocytochemical technique using primary antibodies to NSE. The growth and morphological features were carefully observed through the process. Two weeks later, the neuron cultures were exposed to Mg2+-free solution for3h and then returned to normal maintenance medium. Whole-cell patch clamp (WCC) recordings were performed at different time points to investigate the electrophysiological characteristics of neurons and verify establishment of the HNC model of AE.
Results
1. Morphological characteristics of neuron culture:The isolated round hippocampal neurons adhered to the plate surface with growing neuritis after12-24h. On the7th day. the neurons became more stereoscopic with classic axons and dendrites. Synaptic connections were observed on the10th day, which became intensive network during the10～14th day. Part of the neurons were cracked on the25～28th day.
2. Purity of neuron culture:The cytoplasm and neuritis of neurons were stained by NSE antibody on the14th day. The purity of neuron culture was92.4%.
3. Electrophysiological characteristics of neurons:WCC recordings of control neurons revealed "normal" baseline recordings, displaying spontaneously occurring action potentials, excitatory and inhibitory postsynaptic potentials. During the3h Mg+-free exposure, neuronal firing behavior changed to continuous tonic high-frequency burst disclarges with "wedge-shaped depolarization". And WCC recordings demonstrated recurrent seizure activity with paroxysmal depolarizing shifts (PDSs) and sustained spike discharges alter the restoration of the normal Mg" concentration. The SREDs could be detected at96h after Mg2+-free treatment.
Conclusions
1. The hippocampal neurons of rats in this model were suitable for patch clamp studies because of the smooth surfaces, stereoscopic shapes and high purity.
2. The hippocampal neurons in this model could form intensive synaptic connections in vitro, which provided the base of studies of information transformation.
3. The electrophysiological characteristics of this model suggested that the SREDs induced by Mg2+-free treatment were similar to electrical activities of epileptic seizure. This neuronal culture model of AE provided a useful tool to epilepsy study.
PART Ⅱ The effects of PARP-1on AIF translocation and PI3K/Akt signal pathway in the HNC model of AE in rats
Objective
To investigate the neuronal survival conditions and the effects of PARP-1on regulating mitochondria-nucleus translocation of AIF and PI3K/Akt signal pathway in the HNC model of AE in rats.
Methods
1. The hippocampal neurons of rats were randomly divided into control, Mg-free, Mg-free+Z-VAD-FMK (Caspase inhibitor), Mg-free+DPQ (PARP-1inhibitor) groups. The conditions of neuronal death were detected by TUNEL and LDH detection at1and24h after Mg2+-free treatment.
2. The hippocampal neurons were randomly divided into control, Mg-free, Mg-free+DMSO, Mg-free+DPQ, Mg-free+WTN (PI3K inhibitor) groups. Proteins were extracted from whole cells, nuclei or mitochondrial fractions, which were used to detect the expressions of PARP-1, AIF,(p-)Akt and (p-)Gsk3p by Western blot analysis.
3. The distributions of AIF were detected by cell immunofluorescence and confocal laser scanning microscopy.
Results
1. The TUNEL-positive nuclei ratio was71.2%at24h after Mg2+-free treatment, which decreased to45.25%after pretreatment of Z-VAD-FMK and26.88%by the addition of DPQ.
2. PAR expression was elevated intensively after Mg2+-free treatment with decreased expression of AIF in mitochondria and increased amount in nuclei (P<0.05). And DPQ inhibited the expression of PAR, made more amount of AIF in mitochondria and less in nuclei than Mg-free+DMSO group (P<0.05).
3. Phosphorylation of Akt and Gsk3β temporally decreased at1h after Mg2+-free treatment, then returned to the control level at12h. The phosphorylation was increased by DPQ addition and decreased by WTN (P<0.05). WTN also made less amount of AIF in mitochondria and more in nuclei than Mg-free+DPQ group (P<0.05).
Conclusions
PARP-1was hyper-activated by Mg2+-free treatment, which resulted in mitochondria-nucleus translocation of AIF and neuronal death. And the PARP-1inhibitor, DPQ, could block the AIF translocation and protect neurons from cell death by activating the PI3K/Akt pathway.
PART Ⅲ The effects of PARP-1on cellular NAD+and SIRT1levels in the HNC model of AE in rats
Objective
To investigate the linkage among PARP-1-mediated neuronal death, cellular NAD+level, expression and deacetylase activity of SIRT1, and the effect of NAD+on mitochondria-nucleus translocation of AIF in the HNC model of AE in rats.
Methods
The hippocampal neurons were randomly divided into control, Mg-free. Mg-free+DPQ, Mg-free+NAD (5,10mM). Mg-free+resveratrol (activator of SIRT1) and Mg-free+sirtinol (inhibitor of SIRT1) groups. Cellular NAD+content. expression and deacetylase activity of SIRT1were detected at1h,3h,6h,12h and24h after Mg2+-free treatment. The effects of DPQ or NAD on cellular NAD+content. TUNEL positive ratio. PARP-1conditions. AIF translocation. expression and activity of SIRT1were studied at24h after Mg2+-free treatment.
Results
1. Cellular NAD' content was decreased with time alter Mg2+-free treatment. Compared with Mg-free group, DPQ pretreatment increased cellular NAD content and attenuated TUNHL positive ratio significantly (p<0.05).
2. The data of cell immunofluorescence and Western blot demonstrated that All' expression was decreased in nuclei and increased in mitochondria significantly (p<0.05), compared with Mg-free group, at24h after Mg2+-free treatment.
3. The mRNA levels of SIRT1were gradually elevated with time after Mg2+-free treatment (p<0.05), but protein levels remained stable. The mRNA levels were significantly decreased by DPQ or NAD addition without changes of protein levels, compared with Mg-free group (p<0.05).
4. The deacetylase activity of SIRT1was elevated by more than3times by addition of DPQ and NAD, accompanied by lower TUNEL positive ratio, compared with Mg-free group. And Mg-free+sirtinol group was quite the opposite.
5. The PAR protein level was markedly decreased in Mg-free+DPQ group (p<0.05), and had no distinct changes in Mg-free+NAD group, compared with Mg-free group. There were no significant changes of PARP-1protein levels in each group.
Conclusions
Mg2+-free treatment hyper-activated PARP-1, depleted cellular NAD+, attenuated deacetylase activity of SIRT1, induced AIF translocation and led to severe neuronal death. And NAD+repletion and SIRT1activation could both protect neurons from cell death mediated by PARP-1activation, providing another pathway to reveal the mechanisms of neuronal death in the HNC model of AE.
聚腺苷二磷酸核糖多聚酶[poly(ADP-ribose) polymerase, PARP]主要存在于真核细胞核中,是一类非组蛋白染色体蛋白质,其主要生物学功能包括：(1)修复DNA损伤并维持基因稳定性；(2)调节相关蛋白的转录水平,影响其表达；(3)影响复制和分化,参与维持端粒长度；(4)清除机体内部损伤细胞,调控细胞死亡。其中PARP-1是PARP家族成员中含量最高(90%以上)、研究最清楚的蛋白。在生理状态下,PARP-1被DNA断裂损伤激活,形成同型二聚体并将烟酰胺腺嘌呤二核甘酸(nicotinamide adenine dinucleotide, NAD)分解为烟酰胺和腺苷二磷酸核糖大分子聚合物,聚腺苷二磷酸核糖[poly(ADP-ribose), PAR]可与组蛋白、转录因子及PARP-1结合,调节其活性和功能,利JDNA修复的进行。而DNA受损严重时,PARP-1被过度激活,大量消耗细胞内NAD+和ATP,激活细胞死亡相关蛋白,引起炎症反应,可见PARP-1的生物学效应是一把双刃剑。
研究发现,PARP-1的药物抑制或基因敲除在神经系统退行性病变、外伤、炎症、缺血再灌注和癫痫等疾病中发挥了明显的神经保护作用。PARP-1过度激活介导细胞损伤的机制很复杂,主要包括：Caspase依赖性细胞凋亡,非Caspase依赖性细胞死亡,炎性介质过度表达,能量代谢障碍和Ca2+/Mg2+依赖性核酸内切酶活性增高等。近期观点认为,PARP介导细胞死亡的形态学与分子生物学特征同经典的凋亡与坏死存在明显的区别,为非Caspase依赖性细胞死亡的一种,并被命名为Parthanatos。其生化特征包括：PARP迅带激活,早期PAR的积累,线粒体膜电位去极化并通透性改变,凋亡诱导因子(apoptosis-inducing factor, AIF)向细胞核转位,细胞内NAD+(?)(?)ATP大量消耗,晚期Caspase激活,细胞萎缩等。近期研究发现沉默信息调节因子2同源蛋白1(silencing information regulator2homolog1,SIRT1)能够通过调节细胞能量代谢、生长分化发挥保护作用,但其生物学效应受NAD+水平制约。因此对PARP-1, NAD+, AIF, SIRT1等潜在靶点的调控可为PARP(?)(?)关性疾病的治疗开辟新的途径。然而,Parthanatos的具体机制尚未完全明确,在癫痫病理生理过程中的作用研究较少。
为此,本课题建立无镁诱导大鼠海马神经元癫痫放电模型,排除体内众多因素的影响,模拟边缘系统受损性癫痫,探讨癫痫体外模型中海马神经元的电生理特性,并应用多种药物干预手段,检测PARP-1介导的细胞死亡信号通路和相关蛋白变化,以此深入研究癫痫的分子生物学机制并为癫痫的治疗提供新的靶点。本研究为三个部分：
第一部分无镁诱导大鼠海马神经元癫痫放电模型的建立及形态学、电生理学观察
目的
建立无镁诱导大鼠海马神经元癫痫放电模型,观察自发性癫痫放电对体外培养大鼠海马神经元细胞形态学及电生理学特征的影响。
方法
采用新生24h内的Sprague-Dawley大鼠断头取脑,显微镜下分离海马神经元进行的原代培养。培养过程中观察神经元的形态学特征,并用培养至14d的细胞用神经元特异性烯醇化酶(neuron-specific enolase, NSE)抗体,采用免疫细胞化学技术鉴定神经元纯度。培养至14d的海马神经元经过无镁细胞外液处理3h后恢复至正常培养基,在不同时间点采用全细胞膜片钳技术记录神经元放电情况,验证无镁诱导大鼠海马神经元癫痫放电模型建立成功。
结果
1.细胞形态学特征：刚接种至六孔板培养基内的海马神经元为小圆形,散在分布。大部分神经元在培养12-24h后贴壁,突起明显增多,长短不一。培养至7d,已具有典型的轴突与树突,细胞结构立体,折光感强,细胞核呈空泡状,神经元间尚未形成突触联系,为单个生长的细胞。10d左右神经元间形成突触联系,呈网状结构。培养10-14d时,细胞体积最大,突起最为清晰,网络最为密集。至25-28d,培养液中有悬浮的细胞碎片。
2.神经元纯度检验：培养至14d的神经元细胞爬片采用免疫细胞化学技术染色后,神经元的胞浆和突起被染成棕色,镜下计数后神经元纯度可达94.7%。
3.电生理学特征：在正常生理外液中,大部分神经元可记录到正常突触后电活动,包括兴奋性突触后电位(excitatory postsynaptic potential, EPSP)、抑制性突触后电位(inhibitory postsynaptic potential, IPSP)和偶发动作电位(action potential, AP)。其静息电位范围为-60--40mV,动作电位幅度为15～30mV。神经元在无镁刺激过程中,放电形式发生改变,表现为持续强直性高频爆发和“楔形”去极化。经无镁处理3h并恢复正常培养基后,表现为阵发性持续棘波样爆发,另外存在有“楔形”去极化及阵发性去极化偏移(paroxysmal depolarizing shifts,PDS)。无镁刺激恢复正常后持续至96h仍可检测到周期性自发放电表现。
结论
1.本研究所用方法培养的大鼠海马神经元在数量、纯度、存活时间方而较好,表面光滑,轮廓清楚,有较强的立体感,具有适于膜片钳研究的细胞结构基础。
2.本模型中海马神经元虽为体外培养,但神经元之间能形成广泛的突触网状联系,具有信息传递的形态学基础。
3.无镁刺激能诱发神经元产生持久的自发性反复性放电,与癫痫发作时电生理活动类似,具有体外癫痫细胞模型研究的电生理基础。
4.无镁诱导海马神经元癫痫放电模型稳定可靠、可控性强,是良好的癫痫细胞模型,为研究癫痫病理生理机制及新型抗癫痫药物的研发提供了良好平台。
第二部分PARP-1调控无镁致痫大鼠海马神经元中AIF表达、分布及PI3K/Akt信号通路的机制研究
目的
评估无镁诱导大鼠海马神经元癫痫放电模型中的细胞生存状态：探讨神经元内AIF的表达、分布及PI3K/Akt信号通路随时间的变化规律；探讨PARP-1及其抑制剂DPQ(3,4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)一isoquinolinone)对上述指标的调节作用及相关机制。
方法
1.将体外培养至14d的大鼠海马神经元随机分为对照组、尤镁组、在镁+Z-VAD-FMK(Caspase抑制剂)组、无镁饮+DPQ组,均选用致痫后1h、24h两个时间点,采用FUNEI及LDH检测法,评估无镁诱导后神经元死亡情况,并探讨DPQ对其影响。
2.将大鼠海马神经元随机分为对照组、无镁组、无镁+DMSO祖、无镁(?)DPQ组、无镁+dpq+WTN(wortmannin,PI3K抑制剂组,提取总蛋白、细胞核及线粒体蛋白,采用Western blot法检测PAR、PARP-1\AIF、(p-)Akt、(p-)GSK3β的表达
3.采用细胞免疫荧光共聚集技术,检测AIF在神经元内的分布。
结果
1.无镁诱导后24h,TUNEL细胞核阳性率可达71.2%：而在镁+FMK组及无镁+DPQ组的该比例分别降至45.25%和26.88%,LDH漏出率分别为在镁24h组的57.35%和36.94%。
2.PAR的蛋白量在无镁诱导3h后显著升高,24h达最高水平。线粒体中AIF表达量随时间降低,诱导后24h达最低水平(p<0.05)；与之对应,细胞核中AIF表达逐渐增高。无镁+DPQ组中PAR表达量下降,线粒体内AIF的含量明显高于无镁+DMSO组(p<0.05)；细胞核内AIF含量则显著降低(p<0.05)。细胞免疫荧光检测结果与上述趋势相一致。
3.无镁诱导后,Akt、GSK3β磷酸化水平在1h显著降低(p<0.05),在12h基本恢复至对照组水平。无镁+DPQ组Akt及GSK3β磷酸化水平显著高于无镁+DMSO组(p<0.05)。而无镁+DPQ+WTN组,Akt、GSK3P磷酸化水平及线粒体内AIF表达显著低于无镁+DPQ组(p<0.05),细胞核内AIF表达则与之相反。
结论
无镁诱导大鼠海马神经元癫痫模型中,PARP-1被过度激活,导致AIF由线粒体向细胞核内转位,神经元大量死亡。而PARP-1抑制剂DPQ通过激活PI3K/Akt/GSK3β信号通路,抑制AIF向细胞核的转位,减少神经元死亡,发挥了细胞保护作用。
第三部分PARP-1调节无镁致痫大鼠海马神经元中NAD+及SIRT1水平的机制研究
目的
探讨无镁诱导大鼠海马神经元癫痫放电模型中,PARP-1介导的细胞死亡与神经元内NAD+含量、SIRT1的表达及去乙酰化活性的关系；探讨细胞内NAD+含量对AIF由线粒体向细胞核转位的作用。
方法
将体外培养至14d的大鼠海马神经元随机分为对照组、无镁组、无镁+DPQ组、无镁+NAD (5,10mM)组、无镁+白藜芦醇(resveratrol, SIRT1激动剂)组及无镁+sirtinol (SIRT1抑制剂)组。检测无镁诱导后NAD+的含量、SIRT1的mRNA和蛋白水平随时间的变化规律：探讨应用DPQ或NAD干预后,TUNEL阳性细胞率与NAD+水平的变化关系、PARP-1的表达与活性状态、AIF的胞内分布以及SIRT1(?)的表达和活性对神经元存活状况的影响。
结果
1.无镁组大鼠海马神经元内NAD+含量随时间逐渐下降。与无镁组相比,无镁+DPQ组的NAD+含量显著增高(p<0.05),TUNEL阳性细胞率显著降低(p<0.05)。
2.细胞免疫荧光和Western blot(?)支术均证实,无镁24h+NAD组神经元细胞核AIF的蛋白表达量显著低于无镁24h组(p<0.05),而线粒体AIF表达量高于无镁组(p<0.05)。
3.SIRT1的mRNA水平随无镁诱导后时间延长而增高,但蛋白水平与对照{组无显著差异；应用DPQ或NAD干预后SIRT1的mRNA水平较在镁明显著降低(p<0.05),但蛋白水平与无镁组相比无显著差异。
4.在镁+DPQ组SIRT1去乙酰化活性是无镁组的3倍,TUNEL阳性率为27.62%。无镁+resveratrol组将SIRT1(?)活性提高至无镁组的3.72倍,TUNEL阳性率为35.73%。无镁+sirtinol组SIRT1活性显著降低,TUNEL(?)(?)性率与无镁组无显著差异(p>0.05)。
5.无镁组PAR蛋白表达量显著高于对照组(p<0.05),无镁+DPQ组PAR表达量显著低于在无镁组(p<0.05),而无镁天+NAD组PAR表达量与无镁组相比在明显差异,各组PARP-1蛋白水平无显著差异,
结论
无镁诱导大鼠海马神经元癫痫模型中,PARP-1被过度激活,胞内NAD急剧消耗,SIRT1去乙酰化活性下降,AIF由线粒体向细胞枋核内转位,神经元大量死亡。补充外源性NAD+能抑制AIF转位,减少神经元死亡；而SIRT1作为PARP-1和NAD+的下游分子,其活性(而不是蛋白表达量)与神经元死亡率呈负相关。因此神经元内NAD+含量与SIRT1去乙酰化活性的下降为PARP-1介导的无镁致痫神经元的死亡机制提供了另一条通中。
Epilepsy is a common and diverse set of chronic clinic syndrome characterized by paroxysmal, transient, repetitive and inflexible neurological disorders caused by hyper-synchronous neuronal discharges in the brain. Epilepsy is one of the most common neurological diseases which affects about50million patients around the world and9million in China. And about25%of patients suffer from intractable epilepsy with poor effects of medication and surgery. The pathophysiologic mechanisms are complicated. It has been suggested that the behavior, electroencephalogram (EEG) and the hippocampal neuronal injury of seizures induced by lithium-pilocarpine and kainic acid in rats is similar to that in patients with temporal lobe epilepsy. So the two methods have been the most frequently used animal models to research human epilepsy. Sombati established the hippocampal neuronal culture (HNC) model of acquired epilepsy (AE) which displayed spontaneous recurrent epileptiform discharges (SREDs) induced by Mg2+-free treatment in1995. It is convenient to observe the electrophysiological changes and drug effect in this model. Moreover, the pharmacological interference of epileptogenic drugs can be properly excluded to reveal the underlying mechanisms of epilepsy.
Poly (ADP-ribose) polymerase-1(PAR.P-1) is a kind of non-histone chromosomal proteins which exists in the nucleus of eucaryotes, which is proved to be involved in DNA repair, gene transcription. DNA duplication and cell death. In physiological conditions, PARP-1activated by DNA breaks cleaves nicotinamide adenine dinucleotide (NAD) into nicotinamide and ADP-ribose. The latter can be synthesized into long-branching poly(ADP-ribose)(PAR) polymers which covalently attach to acceptor proteins including histones, DNA repair enzymes, transcription factors and PARP-1itself, facilitating DNA repair and cell survival. However, PARP-1can be hyper-activated by excessive DNA damage, triggering apoptosis or necrotic cell death which depletes a large amount of NAD+and ATP.
Cumulative evidence suggested that the inhibition of PARP-1has cytoprotective effects against neurotoxicity, central nervous system injury, ncurodcgcnerative disorders, inflammation and epilepsy. The mechanisms of neuronal damages mediated by hyper-activation of PARP-1are complicated, including classical caspase-dependent apoptosis pathway, caspase-independent cell death pathway, accumulation of inflammatory mediators, hyper-activation of Ca2+/Mg2+-dependent endonuclease and energy failure. It is noteworthy that PARP-1-dependent cell death is unique in terms of its biochemical and morphological features, which is called "parthanatos". The biochemical features of parthanatos are distinct from classically defined pathways of cell death and involve rapid PARP-1activation, early PAR accumulation, mitochondrial depolarization, early nuclear apoptosis-inducing factor (AIF) translocation, cellular NAD+and ATP consumption and late caspase activation. And silent mating type information regulation2homolog1(SIRT1) has recently confirmed to be related to cell survival conditions. Therefore, it will be prospective to invent new therapies of epilepsy by regulating the target proteins involved in the pathway.
In this study, the I INC model of AE was established to mimic epileptic conditions caused by hippocampus damages. Based on this, we examined the response of related cell signaling pathway and critical proteins to reveal the underlying mechanisms of caspase-independent cell death mediated by PARP-1hyper-activation.
PART I A study of morphological and electrophysiological characteristics of neurons in the HNC model of AE in rats
Objective
To establish the HNC model of AE induced by Mg2+-free treatment in rats, and investigate the morphological and electrophysiological characteristics of neurons in this model.
Methods
Hippocampal cells were separated from24h postnatal Sprague-Dawley rats, plated and maintained in proper conditions. The purity of neurons in primary cultures was detected by immunocytochemical technique using primary antibodies to NSE. The growth and morphological features were carefully observed through the process. Two weeks later, the neuron cultures were exposed to Mg2+-free solution for3h and then returned to normal maintenance medium. Whole-cell patch clamp (WCC) recordings were performed at different time points to investigate the electrophysiological characteristics of neurons and verify establishment of the HNC model of AE.
Results
1. Morphological characteristics of neuron culture:The isolated round hippocampal neurons adhered to the plate surface with growing neuritis after12-24h. On the7th day. the neurons became more stereoscopic with classic axons and dendrites. Synaptic connections were observed on the10th day, which became intensive network during the10～14th day. Part of the neurons were cracked on the25～28th day.
2. Purity of neuron culture:The cytoplasm and neuritis of neurons were stained by NSE antibody on the14th day. The purity of neuron culture was92.4%.
3. Electrophysiological characteristics of neurons:WCC recordings of control neurons revealed "normal" baseline recordings, displaying spontaneously occurring action potentials, excitatory and inhibitory postsynaptic potentials. During the3h Mg+-free exposure, neuronal firing behavior changed to continuous tonic high-frequency burst disclarges with "wedge-shaped depolarization". And WCC recordings demonstrated recurrent seizure activity with paroxysmal depolarizing shifts (PDSs) and sustained spike discharges alter the restoration of the normal Mg" concentration. The SREDs could be detected at96h after Mg2+-free treatment.
Conclusions
1. The hippocampal neurons of rats in this model were suitable for patch clamp studies because of the smooth surfaces, stereoscopic shapes and high purity.
2. The hippocampal neurons in this model could form intensive synaptic connections in vitro, which provided the base of studies of information transformation.
3. The electrophysiological characteristics of this model suggested that the SREDs induced by Mg2+-free treatment were similar to electrical activities of epileptic seizure. This neuronal culture model of AE provided a useful tool to epilepsy study.
PART Ⅱ The effects of PARP-1on AIF translocation and PI3K/Akt signal pathway in the HNC model of AE in rats
Objective
To investigate the neuronal survival conditions and the effects of PARP-1on regulating mitochondria-nucleus translocation of AIF and PI3K/Akt signal pathway in the HNC model of AE in rats.
Methods
1. The hippocampal neurons of rats were randomly divided into control, Mg-free, Mg-free+Z-VAD-FMK (Caspase inhibitor), Mg-free+DPQ (PARP-1inhibitor) groups. The conditions of neuronal death were detected by TUNEL and LDH detection at1and24h after Mg2+-free treatment.
2. The hippocampal neurons were randomly divided into control, Mg-free, Mg-free+DMSO, Mg-free+DPQ, Mg-free+WTN (PI3K inhibitor) groups. Proteins were extracted from whole cells, nuclei or mitochondrial fractions, which were used to detect the expressions of PARP-1, AIF,(p-)Akt and (p-)Gsk3p by Western blot analysis.
3. The distributions of AIF were detected by cell immunofluorescence and confocal laser scanning microscopy.
Results
1. The TUNEL-positive nuclei ratio was71.2%at24h after Mg2+-free treatment, which decreased to45.25%after pretreatment of Z-VAD-FMK and26.88%by the addition of DPQ.
2. PAR expression was elevated intensively after Mg2+-free treatment with decreased expression of AIF in mitochondria and increased amount in nuclei (P<0.05). And DPQ inhibited the expression of PAR, made more amount of AIF in mitochondria and less in nuclei than Mg-free+DMSO group (P<0.05).
3. Phosphorylation of Akt and Gsk3β temporally decreased at1h after Mg2+-free treatment, then returned to the control level at12h. The phosphorylation was increased by DPQ addition and decreased by WTN (P<0.05). WTN also made less amount of AIF in mitochondria and more in nuclei than Mg-free+DPQ group (P<0.05).
Conclusions
PARP-1was hyper-activated by Mg2+-free treatment, which resulted in mitochondria-nucleus translocation of AIF and neuronal death. And the PARP-1inhibitor, DPQ, could block the AIF translocation and protect neurons from cell death by activating the PI3K/Akt pathway.
PART Ⅲ The effects of PARP-1on cellular NAD+and SIRT1levels in the HNC model of AE in rats
Objective
To investigate the linkage among PARP-1-mediated neuronal death, cellular NAD+level, expression and deacetylase activity of SIRT1, and the effect of NAD+on mitochondria-nucleus translocation of AIF in the HNC model of AE in rats.
Methods
The hippocampal neurons were randomly divided into control, Mg-free. Mg-free+DPQ, Mg-free+NAD (5,10mM). Mg-free+resveratrol (activator of SIRT1) and Mg-free+sirtinol (inhibitor of SIRT1) groups. Cellular NAD+content. expression and deacetylase activity of SIRT1were detected at1h,3h,6h,12h and24h after Mg2+-free treatment. The effects of DPQ or NAD on cellular NAD+content. TUNEL positive ratio. PARP-1conditions. AIF translocation. expression and activity of SIRT1were studied at24h after Mg2+-free treatment.
Results
1. Cellular NAD' content was decreased with time alter Mg2+-free treatment. Compared with Mg-free group, DPQ pretreatment increased cellular NAD content and attenuated TUNHL positive ratio significantly (p<0.05).
2. The data of cell immunofluorescence and Western blot demonstrated that All' expression was decreased in nuclei and increased in mitochondria significantly (p<0.05), compared with Mg-free group, at24h after Mg2+-free treatment.
3. The mRNA levels of SIRT1were gradually elevated with time after Mg2+-free treatment (p<0.05), but protein levels remained stable. The mRNA levels were significantly decreased by DPQ or NAD addition without changes of protein levels, compared with Mg-free group (p<0.05).
4. The deacetylase activity of SIRT1was elevated by more than3times by addition of DPQ and NAD, accompanied by lower TUNEL positive ratio, compared with Mg-free group. And Mg-free+sirtinol group was quite the opposite.
5. The PAR protein level was markedly decreased in Mg-free+DPQ group (p<0.05), and had no distinct changes in Mg-free+NAD group, compared with Mg-free group. There were no significant changes of PARP-1protein levels in each group.
Conclusions
Mg2+-free treatment hyper-activated PARP-1, depleted cellular NAD+, attenuated deacetylase activity of SIRT1, induced AIF translocation and led to severe neuronal death. And NAD+repletion and SIRT1activation could both protect neurons from cell death mediated by PARP-1activation, providing another pathway to reveal the mechanisms of neuronal death in the HNC model of AE.
引文
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3 R.J. DeLorenzo, S. Pal, S. Sombati, Prolonged activation of the N-methyl-D-aspartate reeeptor-Ca2+ transduction pathway causes spontaneous recurrent epileptiform discharges in hippocampal neurons in culture, Proceedings of the National Academy of Sciences of the United States of America 95 (1998) 14482-14487.
4 R.E. Blair, S. Sombati, S.B. Churn, R.J. Delorenzo, Epileptogenesis causes an N-methyl-d-aspartate receptor/Ca2+-dependent decrease in Ca2+/calmodulin-dependent protein kinase Ⅱ activity in a hippocampal neuronal culture model of spontaneous recurrent epileptiform discharges, European journal of pharmacology 588 (2008) 64-71.
5 I. Guillemain, P. Kahanc, A. Depaulis, Animal models to study actiopathology of epilepsy: what are the features to model?, Epileptic disorders : international epilepsy journal with videotape 14 (2012) 217-225.
6 PH. Stypulkowski, J.E. Giftakis, T.M. Billstrom, Development of a large animal model for investigation of deep brain stimulation for epilepsy, Stereotactic and functional neurosurgery 89 (2011) 111 -122.
7 K. Biziere, J.P. Chambon, [Animal models of epilepsy and experimental seizures]. Revue neurologique 143 (1987) 329-340.
8 A. Pitkanen, T.P. Sutula, Is epilepsy a progressive disorder? Prospects for new therapeutic approaches in temporal-lobe epilepsy, Lancet neurology 1 (2002) 173-181.
9 G. Popescu, Principles of N-methyl-D-aspartate receptor allosteric modulation, Molecular pharmacology 68 (2005) 1148-1155.
10 S. Pal, S. Sombati, D.D. Limbrick, Jr., R.J. DeLorenzo, In vitro status epilepticus causes sustained elevation of intracellular calcium levels in hippocampal neurons, Brain research 851 (1999)20-31.
11 N.P. Poolos, D. Johnston, Dendritic ion channelopathy in acquired epilepsy, Epilepsia 53 Suppl 9 (2012) 32-40.
12 M. Oliva, S.F. Berkovic, S. Petrou, Sodium channels and the neurobiology of epilepsy, Epilepsia 53 (2012) 1849-1859.
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20 S.D. Skaper, Poly(ADP-ribosyl)ation enzyme-1 as a target for neuroprotection in acute central nervous system injury. Current drug targets. CNS and neurological disorders 2 (2003) 279-291.
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