青光眼神经节细胞退行性病变相关研究
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摘要
青光眼是一种复杂的致盲性神经退行性疾病。青光眼的神经退行性病变发生在视网膜神经节细胞及视神经轴突,涉及先天遗传倾向,后天危险因素以及与年龄增长相关的环境刺激。眼内压升高被认为是青光眼疾病发生发展的重要危险因素之一,也是目前可以干预的因素。至今青光眼的治疗还是基于降低眼压。近年来研究发现,除了眼压升高,在青光眼中多种刺激因素影响了细胞内环境稳定,而且神经元对刺激因素的抵抗力也决定了其内在易感性。有证据显示当视网膜神经节细胞暴露在青光眼性压力刺激下,导致其出现一系列相关联的细胞下游通路活动,包括线粒体功能异常,蛋白水解级联反应,内质网压力和氧化应激等。引起神经起始损伤和青光眼性神经退行性病变的具体分子机制一直在被广泛研究并存在很大争议。
青光眼神经节细胞损伤的因素有很多,目前有很多学说试图解释青光眼的发病机制,如机械压力学说、氧化应激学说等,而最近研究的热点是青光眼的自身免疫和异常蛋白沉淀这两大方面：研究发现青光眼神经退行性病变涉及免疫系统,包括天然免疫和获得免疫在内的不同成分,还有越来越多的证据将青光眼的发病机制指向补体系统的激活,补体激活的产物,如Clq, C3和膜攻击复合物(MAC)在青光眼模型动物和青光眼病人的视网膜中被发现。本研究的前两个部分试图探讨补体系统的激活在青光眼发病机制中所扮演的角色。第一部分为体内研究,连续观察补体系统相关蛋白在大鼠青光眼模型中的变化。第二部分为体外研究,探讨补体系统激活终产物膜攻击复合物C5b-9对视网膜神经节细胞凋亡和程序化坏死的影响。另一方面,神经退行性病变是指神经元细胞在结构及功能上的逐渐损失,最终死亡的病变,其发病机理涉及蛋白的异常加工,错误折叠以及在蛋白在细胞内外的异常聚集。视网膜也是中枢神经系统的一部分,因此在神经退行性病变的进程中,视网膜和视神经会受到相似的影响。异常蛋白在神经元的沉积,包括Ap和异常Tau蛋白的沉积,是神经节细胞另一损伤因素。本研究第三部分是观察Tau蛋白在青光眼视网膜中的变化并探讨氯化锂在慢性青光眼神经退行性病变的作用及治疗机制。
第一部分：大鼠青光眼模型中补体系统相关蛋白的变化
目的：研究补体系统的激活对慢性青光眼高眼压模型中视网膜神经节细胞的影响。
方法：用DA大鼠构建慢性高眼压模型,将动物分成4组,每组5只,左眼为手术眼,通过往巩膜静脉注射高渗盐水的方法提高眼内压,右眼不做处理作为正常对照眼。动物自手术后1周,3周,8周和16周分别处死,免疫荧光法测试高眼压眼和对照眼视网膜内的膜攻击复合物(MAC, C5b-9), C3和衰变加速因子(DAF)的表达水平。
结果：C5b-9自青光眼大鼠眼压升高8周起表达明显上调,C3的表达在高眼压中也有所提高。而衰变加速因子DAF的表达发现在8周和16周高眼压组明显降低。
结论：在慢性青光眼动物模型视网膜节细胞层补体活动产物上调和补体调节蛋白衰变加速因子DAF的下降提示补体激活和补体调节的失衡,此现象是否在青光眼的发病机制中扮演潜在作用还需进一步研究。
第二部分：膜攻击复合物C5b-9对视网膜神经节细胞的凋亡和程序化坏死的影响
目的：视网膜神经节细胞的损失是青光眼的特征,但是神经节细胞死亡的调节机制仍未研究清楚。本部分的研究目的是确定膜攻击复合物C5b-9是否诱导神经节细胞的死亡和／或者调节了视网膜神经节细胞对其他细胞压力作用的敏感性。
方法：本研究使用RGC-5细胞系为研究对象,用抗CD59抗体阻断CD59后, RGC-5细胞作用于正常人血清,用免疫荧光染色法观察细胞形态学,流式细胞技术以及用PARP剪切和活化的caspase-3作为探针的western blot技术检测细胞凋亡的程度。同时用程序化坏死抑制剂AGK2作用于细胞,用流式细胞技术检测细胞程序性坏死的程度。在有或没C5b-9生成的条件下,ionomycin, staurosporine,双氧化氢和chelerythrine对RGC-5细胞的敏感性的变化也被观察。
结果：在暴露于C5b-9的条件下,RGC-5细胞出现凋亡。RGC-5细胞还在staurosporine作用后出现凋亡,但对ionomycin和过氧化氢存在抵抗作用。C5b-9能够明显增加staurosporine引发RGC-5细胞凋亡和程序化坏死的敏感度。
结论：低水平的C5b-9可以引起RGC-5细胞的凋亡,应用不同的激动剂证实C5b-9能特异地敏感化一些凋亡和程序化坏死的通路。研究结果揭示了补体系统在视网膜神经节细胞损失中的潜在作用。
第三部分：Tau蛋白在鼠慢性青光眼模型中的变化和机制研究
目的：研究在慢性青光眼性损害时视网膜神经节细胞层中Tau蛋白磷酸化的变化,并通过利用氯化锂抑制Tau上游激酶GSK-3β活性的实验,为在临床上用氯化锂治疗慢性青光眼神经退行性病变提供实验依据。
方法：建立慢性高眼压SD大鼠模型,分为两组各10只。一组为高眼压未治疗组,右眼为高眼压模型眼,左眼为正常对照眼；另一组为高眼压氯化锂治疗组,治疗组于建模当天起每日腹腔注射0.6M氯化锂(GSK-3β抑制剂)。在第2周和第4周从两组中各取5只大鼠处死并摘取眼球,用免疫荧光和Western blot观察视网膜总Tau和磷酸化Tau蛋白的变化。
结果：1)高眼压模型视网膜Tau蛋白及其磷酸化水平发生了改变：总Tau含量在第2周时下降为对照眼的77.3%,在第4周时下降为对照组的60.4%; p-Tau与总Tau的比值,在第2周时几乎没有变化,在第4周时增加到对照眼的135.4%,磷酸化明显增加。2)氯化锂对高眼压模型鼠的影响：在第4周时,氯化锂治疗组视网膜总Tau含量较未治疗组明显升高,为对照眼的99%;p-Tau与总Tau的比值较未治疗组明显下降,几乎降到对照眼水平。结论：持续高眼压刺激导致大鼠视网膜总Tau蛋白表达量降低,Tau蛋白磷酸化水平增加；氯化锂能减轻由持续高眼压引起的Tau蛋白的过度磷酸化,其机制还需进一步研究。
研究总结
一、主要研究结果
1.在慢性高眼压模型鼠中,膜攻击复合物C5b-9在视网膜神经节细胞层的表达在眼压升高第8周明显上升,补体激活途径枢纽蛋白C3在慢性高眼压模型中的表达也上调。补体调节蛋白衰变加速因子(DAF)的表达在高眼压第8周明显下降,与C5b-9的表达变化相对应。
2.体外研究发现膜攻击复合物C5b-9可在RGC-5细胞上被诱导生成。C5b-9的生成可诱导RGC-5细胞的发生凋亡。C5b-9能够明显增加staurosporine引发细胞凋亡和程序化坏死的敏感度。
3.在慢性高眼压模型鼠中,总Tau含量在高眼压第2周时已下降,在第4周进一步降低,在第4周Tau蛋白的磷酸化明显增加；氯化锂治疗组在第4周时视网膜总Tau含量较未治疗组明显升高,磷酸化程度较未治疗组明显下降。
二、研究结论
1.在慢性青光眼动物模型中,视网膜节细胞层各补体活动产物上调和补体条件蛋白DAF的下降提示补体激活和补体调节的失衡可能在青光眼的发病机制中扮演潜在作用。
2.低水平的C5b-9可以引起RGC-5细胞的凋亡,并通过多种不同的激动剂的运用,进一步证实了C5b-9能特异地使细胞的一些凋亡和程序化坏死的通路敏感化。
3.在慢性青光眼动物模型中,持续高眼压刺激导致大鼠视网膜总Tau蛋白表达量降低,Tau蛋白磷酸化水平增加；氯化锂能减轻由持续高眼压引起的Tau蛋白的过度磷酸化。
Glaucoma is a complex neurodegenerative disease leading to blindness. Glaucomatous neurodegeneration involves specific combinations of genetic predispositions, epigenetic risk factors, and environmental stressors that increase the aging-related stress in retinal ganglion cells (RGCs) and optic nerve axons. Intraocular pressure, however, has been one of the risk factors for the development and progression of the disease that has been identified to date and the only one that is modifiable. IOP reduction forms the basis for our current therapy of glaucoma. Present evidence indicates highly interconnected downstream pathways of cellular processes in RGCs exposed to glaucomatous stress, which include mitochondrial dysfunction, proteolytic caspase cascade, endoplasmic reticulum stress, and oxidative stress. The precise molecular mechanism of initiating the neuronal injury and the glaucomatous neurodegeneration are under intensive investigation and great debate.
There are many factors of glaucomatous ganglion cell damage, and some theories attempt to explain the pathogenesis of glaucoma, such as the mechanical theory, oxidative stress theory, recently scentists pay more attention on two aspects:autoimmunity and abnormal protein precipitation:Studies highlight the invovement of the immune system in glaucomatous neurodegeneration. Different components, including both innate and adaptive immunity, exhibit prominent activity in glaucoma. Recently, growing evidence points to the involvement of complement activation in the pathogenesis of glaucoma. Complement activation products, such as Clq, C3and membrane attack complex (MAC) have been found both in animal glaucoma models and in the retinas of glaucoma patients. Part Ⅰ and part Ⅱ of the research attempted to investigate the role of complement system activation in the pathogenesis of glaucoma. Part Ⅰ was in vivo work, continuous observing the alteration of complement associated proteins in the chronic hypertention rat model of glaucoma, while part Ⅱ was in vitro work, discussing the effect of exposure to the complement C5b-9complex sensitizes retinal ganglion cells to both apoptosis and necroptosis. Neurodegeneration is a collective term for the progressive loss of the structure, function, or even death of neurons. The pathogenesis of neurodegeneration involves the aberrant processing, misfolding, and subsequent aggregation of normal proteins that are then deposited both intracellularly and extracellularly. The retina is part of the central nervous system (CNS), therefore, the retina and optic nerve could be affected by similar degenerative processes during neurodegeneration Abnormal protein deposition in neurons, including A(3and abnormal Tau protein deposition is the other factor of the injure of ganglion cells. Part Ⅲ of the research is observation of the variation of Tau protein in glaucomatous retina and discussion the effect and mechanism of treatment of LiCl on chronic glaucomatous neurodegeneration.
Part I Alteration of complement associated proteins in the chronic hypertention rat model of glaucoma.
PURPOSE:This study tempted to investigate the role of complement system in retinal ganglion cells (RGCs) in chronic ocular hypertension model of glaucoma.
METHODS:Construct of a chronic ocular hypertension model on DA rats which were divided into4groups,5of each. Intraocular pressure (IOP) was elevated in the left eye of DA rats by injection of hypertonic saline into episcleral veins, while right eyes did not receive treatment and served as control. Animals were sacrificed at lweek,3weeks,8weeks and16weeks post-injection. Levels of membrane attack complex (MAC, C5b-9), C3and Decay accelerating facor (DAF) were elevated by immunefluorescence in the retina of eyes with increased IOP and the control.
RESULTS:C5b-9was found to be upregulated in the retina of glaucomatowus rat from8weeks after IOP elevated. The express of C3on RGC layer of IOP elevated eyes was also upregulated. And complement activation inhibitor DAF was found decreased in both8weeks and16weeks high IOP groups.
CONCLUSIONS:Complement cascade produts were upregulated and the complement regulatory protein DAF was deceased on the GCL of in chronic glaucoma models suggests that complement activation and the imbalance of complement regulation plays a potential role in the pathogenesis of glaucoma.
Part II Exposure to the complement C5b-9complex sensitizes retinal ganglion cells to both apoptosis and necroptosis
PURPOSE:Retinal ganglion cell (RGC) loss is the defining characteristic of glaucoma, but the mechanisms that regulate RGC death are not fully understood. The aim of this work was to determine whether exposure to the terminal complement complex C5b-9induces RGC death and/or modulates the sensitivity of RGC cells to other cellular stressors.
METHODS:RGC-5cells were exposed to normal human serum following antibody blockade of CD59. Apoptosis induction was assessed morphologically, by flow cytometry, and on western blotting by probing for cleaved PARP and activated caspase-3. Necroptosis was assessed by flow cytometry and inhibition using AGK2. The sensitivity of RGC-5cells to ionomycin, staurosporine, peroxide and chelerythrine was also investigated, with or without prior formation of C5b-9.
RESULTS:RGC-5cells underwent apoptotic cell death following exposure to C5b-9, as judged by PARP cleavage and activation of caspase-3. We also observed apoptotic cell death in response to staurosporine, but RGC-5cells were resistant to both ionomycin and peroxide. Interestingly, C5b-9significantly increased RGC-5sensitivity to staurosporine-but not chelerythrine-induced apoptosis and necroptosis.
CONCLUSIONS:We have shown that low levels of C5b-9on RGC-5cells can induce apoptosis, and by using a range of different agonists we have further demonstrated that C5b-9specifically sensitizes RGC-5cells to certain apoptotic and necroptotic pathways. Our observations provide new insight into the potential role of the complement system in RGC loss, with implications for the cellular aetiology of glaucoma.
Part Ⅲ Alteration of Tau protein in the chronic hypertention rat model of glaucoma.
Objective This study attempted to investigate the variation of tau phosphorylation in chronic glaucomatous retina, with the treatment of lithium chloride (LiCl), inhibited the activity of the up stream kinase Glycogen synthase kinase-3beta (GSK-3β), and provided experimental evidence for the clinical treatment of LiCl on chronic glaucomatous neurodegeneration.
Methods Construct of a chronic ocular hypertension model on SD rats which were divided into2groups,10of each. Group1were high intra ocular pressure (IOP) ones with no treatment. The right eyes were high IOP ones, and the left eyes were normal control. Group2were high IOP ones treated with lithium chloride (LiCl). On the first day of constructing the model, the SD rat of group2were intraperitoneal injected with0.6M LiCl, which was the inhibitor of GSK3beta.5SD rats of each group were executed at the week2and week4, and their retina was taken for immunofluorescence stain and Western Blot, and the variation of tau phosphorylation in retina was investigated.
Results1) Variations of Tau and phosphorylation of Tau on chronic hyperocular model::the amount of total tau was decreased to77.3%and60.4%of the control at week2and4respectively. The ratio of p-tau/total tau was unchanged at week2and increased to135.4%of the control at week4, and the phosphorylation of tau was increased significantly.2) The effect of LiCl on high IOP models:The total tau in retina was increased compared to the no treated group, reached to99%of the normal control. The ratio of p-tau/total tau declined as well, almost to the level of normal control.
Conclusion In ocular hypertension model, total-Tau was decreased in retina compared with controls. Meanwhile expression of phospho-Tau was increased. The Tau phosphorylation could be inhibited by blocking the activation of GSK3beta with LiCl.
SUMMARY and CONCLUSIONS
Main findings:
1. C5b-9was found to be increased in the ganglion cell layer of retina in chronic ocular hypertention rat model from8weeks after IOP elevated. The express of C3which is a important protein in three pathway of complement activation was also upgrade in the chronic ocular hypertention model. And complement regulating protein Decay Accelerating Facor (DAF) was found decreased obviously from8weeks and. the decrease was relatively with the increase of C5b-9.
2. In vitro, the membrane attack complex C5b-9could be formed on RGC-5. C5b-9formation promotes apoptosis of RGC-5cells. C5b-9significantly increased RGC-5sensitivity to staurosporine-induced apoptosis and necroptosis.
3. In retina of chronic ocular hypertension rat model, the amount of total tau began decreased from2weeks time point and was decreased dramatically at4weeks time point. The phosphorylation of tau was increased significantly at week4.2) The total tau in retina was increased with the treatment of LiCl compared to the no treated group. The phosphorylation of tau declined as well.
Conclusions:
1. Complement cascade produts were upregulated and the complement regulatory protein DAF was deceased on the GCL of in chronic glaucoma models suggests that complement activation and the imbalance of complement regulation plays a potential role in the pathogenesis of glaucoma.
2. Low levels of C5b-9on RGC-5cells can induce apoptosis, and by using a range of different agonists we have further demonstrated that C5b-9specifically sensitizes RGC-5cells to certain apoptotic and necroptotic pathways.
3. In ocular hypertension model, total-Tau was decreased in retina compared with control. Meanwhile expression of phospho-Tau was increased. The Tau phosphorylation caused by sustained ocular hypertension could be inhibited by LiCl.
青光眼神经节细胞损伤的因素有很多,目前有很多学说试图解释青光眼的发病机制,如机械压力学说、氧化应激学说等,而最近研究的热点是青光眼的自身免疫和异常蛋白沉淀这两大方面：研究发现青光眼神经退行性病变涉及免疫系统,包括天然免疫和获得免疫在内的不同成分,还有越来越多的证据将青光眼的发病机制指向补体系统的激活,补体激活的产物,如Clq, C3和膜攻击复合物(MAC)在青光眼模型动物和青光眼病人的视网膜中被发现。本研究的前两个部分试图探讨补体系统的激活在青光眼发病机制中所扮演的角色。第一部分为体内研究,连续观察补体系统相关蛋白在大鼠青光眼模型中的变化。第二部分为体外研究,探讨补体系统激活终产物膜攻击复合物C5b-9对视网膜神经节细胞凋亡和程序化坏死的影响。另一方面,神经退行性病变是指神经元细胞在结构及功能上的逐渐损失,最终死亡的病变,其发病机理涉及蛋白的异常加工,错误折叠以及在蛋白在细胞内外的异常聚集。视网膜也是中枢神经系统的一部分,因此在神经退行性病变的进程中,视网膜和视神经会受到相似的影响。异常蛋白在神经元的沉积,包括Ap和异常Tau蛋白的沉积,是神经节细胞另一损伤因素。本研究第三部分是观察Tau蛋白在青光眼视网膜中的变化并探讨氯化锂在慢性青光眼神经退行性病变的作用及治疗机制。
第一部分：大鼠青光眼模型中补体系统相关蛋白的变化
目的：研究补体系统的激活对慢性青光眼高眼压模型中视网膜神经节细胞的影响。
方法：用DA大鼠构建慢性高眼压模型,将动物分成4组,每组5只,左眼为手术眼,通过往巩膜静脉注射高渗盐水的方法提高眼内压,右眼不做处理作为正常对照眼。动物自手术后1周,3周,8周和16周分别处死,免疫荧光法测试高眼压眼和对照眼视网膜内的膜攻击复合物(MAC, C5b-9), C3和衰变加速因子(DAF)的表达水平。
结果：C5b-9自青光眼大鼠眼压升高8周起表达明显上调,C3的表达在高眼压中也有所提高。而衰变加速因子DAF的表达发现在8周和16周高眼压组明显降低。
结论：在慢性青光眼动物模型视网膜节细胞层补体活动产物上调和补体调节蛋白衰变加速因子DAF的下降提示补体激活和补体调节的失衡,此现象是否在青光眼的发病机制中扮演潜在作用还需进一步研究。
第二部分：膜攻击复合物C5b-9对视网膜神经节细胞的凋亡和程序化坏死的影响
目的：视网膜神经节细胞的损失是青光眼的特征,但是神经节细胞死亡的调节机制仍未研究清楚。本部分的研究目的是确定膜攻击复合物C5b-9是否诱导神经节细胞的死亡和／或者调节了视网膜神经节细胞对其他细胞压力作用的敏感性。
方法：本研究使用RGC-5细胞系为研究对象,用抗CD59抗体阻断CD59后, RGC-5细胞作用于正常人血清,用免疫荧光染色法观察细胞形态学,流式细胞技术以及用PARP剪切和活化的caspase-3作为探针的western blot技术检测细胞凋亡的程度。同时用程序化坏死抑制剂AGK2作用于细胞,用流式细胞技术检测细胞程序性坏死的程度。在有或没C5b-9生成的条件下,ionomycin, staurosporine,双氧化氢和chelerythrine对RGC-5细胞的敏感性的变化也被观察。
结果：在暴露于C5b-9的条件下,RGC-5细胞出现凋亡。RGC-5细胞还在staurosporine作用后出现凋亡,但对ionomycin和过氧化氢存在抵抗作用。C5b-9能够明显增加staurosporine引发RGC-5细胞凋亡和程序化坏死的敏感度。
结论：低水平的C5b-9可以引起RGC-5细胞的凋亡,应用不同的激动剂证实C5b-9能特异地敏感化一些凋亡和程序化坏死的通路。研究结果揭示了补体系统在视网膜神经节细胞损失中的潜在作用。
第三部分：Tau蛋白在鼠慢性青光眼模型中的变化和机制研究
目的：研究在慢性青光眼性损害时视网膜神经节细胞层中Tau蛋白磷酸化的变化,并通过利用氯化锂抑制Tau上游激酶GSK-3β活性的实验,为在临床上用氯化锂治疗慢性青光眼神经退行性病变提供实验依据。
方法：建立慢性高眼压SD大鼠模型,分为两组各10只。一组为高眼压未治疗组,右眼为高眼压模型眼,左眼为正常对照眼；另一组为高眼压氯化锂治疗组,治疗组于建模当天起每日腹腔注射0.6M氯化锂(GSK-3β抑制剂)。在第2周和第4周从两组中各取5只大鼠处死并摘取眼球,用免疫荧光和Western blot观察视网膜总Tau和磷酸化Tau蛋白的变化。
结果：1)高眼压模型视网膜Tau蛋白及其磷酸化水平发生了改变：总Tau含量在第2周时下降为对照眼的77.3%,在第4周时下降为对照组的60.4%; p-Tau与总Tau的比值,在第2周时几乎没有变化,在第4周时增加到对照眼的135.4%,磷酸化明显增加。2)氯化锂对高眼压模型鼠的影响：在第4周时,氯化锂治疗组视网膜总Tau含量较未治疗组明显升高,为对照眼的99%;p-Tau与总Tau的比值较未治疗组明显下降,几乎降到对照眼水平。结论：持续高眼压刺激导致大鼠视网膜总Tau蛋白表达量降低,Tau蛋白磷酸化水平增加；氯化锂能减轻由持续高眼压引起的Tau蛋白的过度磷酸化,其机制还需进一步研究。
研究总结
一、主要研究结果
1.在慢性高眼压模型鼠中,膜攻击复合物C5b-9在视网膜神经节细胞层的表达在眼压升高第8周明显上升,补体激活途径枢纽蛋白C3在慢性高眼压模型中的表达也上调。补体调节蛋白衰变加速因子(DAF)的表达在高眼压第8周明显下降,与C5b-9的表达变化相对应。
2.体外研究发现膜攻击复合物C5b-9可在RGC-5细胞上被诱导生成。C5b-9的生成可诱导RGC-5细胞的发生凋亡。C5b-9能够明显增加staurosporine引发细胞凋亡和程序化坏死的敏感度。
3.在慢性高眼压模型鼠中,总Tau含量在高眼压第2周时已下降,在第4周进一步降低,在第4周Tau蛋白的磷酸化明显增加；氯化锂治疗组在第4周时视网膜总Tau含量较未治疗组明显升高,磷酸化程度较未治疗组明显下降。
二、研究结论
1.在慢性青光眼动物模型中,视网膜节细胞层各补体活动产物上调和补体条件蛋白DAF的下降提示补体激活和补体调节的失衡可能在青光眼的发病机制中扮演潜在作用。
2.低水平的C5b-9可以引起RGC-5细胞的凋亡,并通过多种不同的激动剂的运用,进一步证实了C5b-9能特异地使细胞的一些凋亡和程序化坏死的通路敏感化。
3.在慢性青光眼动物模型中,持续高眼压刺激导致大鼠视网膜总Tau蛋白表达量降低,Tau蛋白磷酸化水平增加；氯化锂能减轻由持续高眼压引起的Tau蛋白的过度磷酸化。
Glaucoma is a complex neurodegenerative disease leading to blindness. Glaucomatous neurodegeneration involves specific combinations of genetic predispositions, epigenetic risk factors, and environmental stressors that increase the aging-related stress in retinal ganglion cells (RGCs) and optic nerve axons. Intraocular pressure, however, has been one of the risk factors for the development and progression of the disease that has been identified to date and the only one that is modifiable. IOP reduction forms the basis for our current therapy of glaucoma. Present evidence indicates highly interconnected downstream pathways of cellular processes in RGCs exposed to glaucomatous stress, which include mitochondrial dysfunction, proteolytic caspase cascade, endoplasmic reticulum stress, and oxidative stress. The precise molecular mechanism of initiating the neuronal injury and the glaucomatous neurodegeneration are under intensive investigation and great debate.
There are many factors of glaucomatous ganglion cell damage, and some theories attempt to explain the pathogenesis of glaucoma, such as the mechanical theory, oxidative stress theory, recently scentists pay more attention on two aspects:autoimmunity and abnormal protein precipitation:Studies highlight the invovement of the immune system in glaucomatous neurodegeneration. Different components, including both innate and adaptive immunity, exhibit prominent activity in glaucoma. Recently, growing evidence points to the involvement of complement activation in the pathogenesis of glaucoma. Complement activation products, such as Clq, C3and membrane attack complex (MAC) have been found both in animal glaucoma models and in the retinas of glaucoma patients. Part Ⅰ and part Ⅱ of the research attempted to investigate the role of complement system activation in the pathogenesis of glaucoma. Part Ⅰ was in vivo work, continuous observing the alteration of complement associated proteins in the chronic hypertention rat model of glaucoma, while part Ⅱ was in vitro work, discussing the effect of exposure to the complement C5b-9complex sensitizes retinal ganglion cells to both apoptosis and necroptosis. Neurodegeneration is a collective term for the progressive loss of the structure, function, or even death of neurons. The pathogenesis of neurodegeneration involves the aberrant processing, misfolding, and subsequent aggregation of normal proteins that are then deposited both intracellularly and extracellularly. The retina is part of the central nervous system (CNS), therefore, the retina and optic nerve could be affected by similar degenerative processes during neurodegeneration Abnormal protein deposition in neurons, including A(3and abnormal Tau protein deposition is the other factor of the injure of ganglion cells. Part Ⅲ of the research is observation of the variation of Tau protein in glaucomatous retina and discussion the effect and mechanism of treatment of LiCl on chronic glaucomatous neurodegeneration.
Part I Alteration of complement associated proteins in the chronic hypertention rat model of glaucoma.
PURPOSE:This study tempted to investigate the role of complement system in retinal ganglion cells (RGCs) in chronic ocular hypertension model of glaucoma.
METHODS:Construct of a chronic ocular hypertension model on DA rats which were divided into4groups,5of each. Intraocular pressure (IOP) was elevated in the left eye of DA rats by injection of hypertonic saline into episcleral veins, while right eyes did not receive treatment and served as control. Animals were sacrificed at lweek,3weeks,8weeks and16weeks post-injection. Levels of membrane attack complex (MAC, C5b-9), C3and Decay accelerating facor (DAF) were elevated by immunefluorescence in the retina of eyes with increased IOP and the control.
RESULTS:C5b-9was found to be upregulated in the retina of glaucomatowus rat from8weeks after IOP elevated. The express of C3on RGC layer of IOP elevated eyes was also upregulated. And complement activation inhibitor DAF was found decreased in both8weeks and16weeks high IOP groups.
CONCLUSIONS:Complement cascade produts were upregulated and the complement regulatory protein DAF was deceased on the GCL of in chronic glaucoma models suggests that complement activation and the imbalance of complement regulation plays a potential role in the pathogenesis of glaucoma.
Part II Exposure to the complement C5b-9complex sensitizes retinal ganglion cells to both apoptosis and necroptosis
PURPOSE:Retinal ganglion cell (RGC) loss is the defining characteristic of glaucoma, but the mechanisms that regulate RGC death are not fully understood. The aim of this work was to determine whether exposure to the terminal complement complex C5b-9induces RGC death and/or modulates the sensitivity of RGC cells to other cellular stressors.
METHODS:RGC-5cells were exposed to normal human serum following antibody blockade of CD59. Apoptosis induction was assessed morphologically, by flow cytometry, and on western blotting by probing for cleaved PARP and activated caspase-3. Necroptosis was assessed by flow cytometry and inhibition using AGK2. The sensitivity of RGC-5cells to ionomycin, staurosporine, peroxide and chelerythrine was also investigated, with or without prior formation of C5b-9.
RESULTS:RGC-5cells underwent apoptotic cell death following exposure to C5b-9, as judged by PARP cleavage and activation of caspase-3. We also observed apoptotic cell death in response to staurosporine, but RGC-5cells were resistant to both ionomycin and peroxide. Interestingly, C5b-9significantly increased RGC-5sensitivity to staurosporine-but not chelerythrine-induced apoptosis and necroptosis.
CONCLUSIONS:We have shown that low levels of C5b-9on RGC-5cells can induce apoptosis, and by using a range of different agonists we have further demonstrated that C5b-9specifically sensitizes RGC-5cells to certain apoptotic and necroptotic pathways. Our observations provide new insight into the potential role of the complement system in RGC loss, with implications for the cellular aetiology of glaucoma.
Part Ⅲ Alteration of Tau protein in the chronic hypertention rat model of glaucoma.
Objective This study attempted to investigate the variation of tau phosphorylation in chronic glaucomatous retina, with the treatment of lithium chloride (LiCl), inhibited the activity of the up stream kinase Glycogen synthase kinase-3beta (GSK-3β), and provided experimental evidence for the clinical treatment of LiCl on chronic glaucomatous neurodegeneration.
Methods Construct of a chronic ocular hypertension model on SD rats which were divided into2groups,10of each. Group1were high intra ocular pressure (IOP) ones with no treatment. The right eyes were high IOP ones, and the left eyes were normal control. Group2were high IOP ones treated with lithium chloride (LiCl). On the first day of constructing the model, the SD rat of group2were intraperitoneal injected with0.6M LiCl, which was the inhibitor of GSK3beta.5SD rats of each group were executed at the week2and week4, and their retina was taken for immunofluorescence stain and Western Blot, and the variation of tau phosphorylation in retina was investigated.
Results1) Variations of Tau and phosphorylation of Tau on chronic hyperocular model::the amount of total tau was decreased to77.3%and60.4%of the control at week2and4respectively. The ratio of p-tau/total tau was unchanged at week2and increased to135.4%of the control at week4, and the phosphorylation of tau was increased significantly.2) The effect of LiCl on high IOP models:The total tau in retina was increased compared to the no treated group, reached to99%of the normal control. The ratio of p-tau/total tau declined as well, almost to the level of normal control.
Conclusion In ocular hypertension model, total-Tau was decreased in retina compared with controls. Meanwhile expression of phospho-Tau was increased. The Tau phosphorylation could be inhibited by blocking the activation of GSK3beta with LiCl.
SUMMARY and CONCLUSIONS
Main findings:
1. C5b-9was found to be increased in the ganglion cell layer of retina in chronic ocular hypertention rat model from8weeks after IOP elevated. The express of C3which is a important protein in three pathway of complement activation was also upgrade in the chronic ocular hypertention model. And complement regulating protein Decay Accelerating Facor (DAF) was found decreased obviously from8weeks and. the decrease was relatively with the increase of C5b-9.
2. In vitro, the membrane attack complex C5b-9could be formed on RGC-5. C5b-9formation promotes apoptosis of RGC-5cells. C5b-9significantly increased RGC-5sensitivity to staurosporine-induced apoptosis and necroptosis.
3. In retina of chronic ocular hypertension rat model, the amount of total tau began decreased from2weeks time point and was decreased dramatically at4weeks time point. The phosphorylation of tau was increased significantly at week4.2) The total tau in retina was increased with the treatment of LiCl compared to the no treated group. The phosphorylation of tau declined as well.
Conclusions:
1. Complement cascade produts were upregulated and the complement regulatory protein DAF was deceased on the GCL of in chronic glaucoma models suggests that complement activation and the imbalance of complement regulation plays a potential role in the pathogenesis of glaucoma.
2. Low levels of C5b-9on RGC-5cells can induce apoptosis, and by using a range of different agonists we have further demonstrated that C5b-9specifically sensitizes RGC-5cells to certain apoptotic and necroptotic pathways.
3. In ocular hypertension model, total-Tau was decreased in retina compared with control. Meanwhile expression of phospho-Tau was increased. The Tau phosphorylation caused by sustained ocular hypertension could be inhibited by LiCl.
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4. Liang, Y. B., et al., Prevalence of primary open angle glaucoma in a rural adult Chinese population:the Handan eye study. Invest Ophthalmol Vis Sci,2011.52(11):p.8250-7.
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7. Wostyn, P., K. Audenaert, and P. P. De Deyn, Alzheimer's disease and glaucoma:is there a causal relationship? Br J Ophthalmol,2009. 93(12):p.1557-9.
8. Bayer, A. U. and F. Ferrari, Severe progression of glaucomatous optic neuropathy in patients with Alzheimer's disease. Eye (Lond),2002. 16(2):p.209-12.
9. McKinnon, S. J., et al., Caspase activation and amyloid precursor protein cleavage in rat ocular hypertension. Invest Ophthalmol Vis Sci, 2002.43(4):p.1077-87.
10. Guo, L., et al., Targeting amyloid-beta in glaucoma treatment. Proc Natl Acad Sci U S A,2007.104(33):p.13444-9.
11. Gupta, N., et al., Retinal tau pathology in human glaucomas. Can J Ophthalmol,2008.43(1):p.53-60.
12. Noble, W., et al., Inhibition of glycogen synthase kinase-3 by lithium correlates with reduced tauopathy and degeneration in vivo. Proc Nat1 Acad Sci U S A,2005.102(19):p.6990-5.
13. McKinnon, S. J., Glaucoma:ocular Alzheimer's disease? Front Biosci, 2003.8:p. s1140-56.
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17. Pei, J. J., et al., Distribution of active glycogen synthase kinase 3beta (GSK-3beta) in brains staged for Alzheimer disease neurofibrillary changes. J Neuropathol Exp Neurol,1999.58(9):p. 1010-9.
18. Leroy, K., Z. Yilmaz, and J. P. Brion, Increased level of active GSK-3beta in Alzheimer's disease and accumulation in argyrophilic grains and in neurones at different stages of neurofibrillary degeneration. Neuropathol Appl Neurobiol,2007.33(1):p.43-55.
19. LaPointe, N. E., et al., The amino terminus of tau inhibits kinesin-dependent axonal transport:implications for filament toxicity. J Neurosci Res,2009.87(2):p.440-51.
20. Hong, M., et al., Lithium reduces tau phosphorylation by inhibition of glycogen synthase kinase-3. J Biol Chem,1997.272(40):p. 25326-32.
21. Stambolic, V., L. Ruel, and J. R. Woodgett, Lithium inhibits glycogen synthase kinase-3 activity and mimics wingless signalling in intact cells. Curr Biol,1996.6(12):p.1664-8.
22. Munoz-Montano, J.R., et al., Lithium inhibits Alzheimer's disease-like tau protein phosphorylation in neurons. FEBS Lett,1997. 411(2-3):p.183-8.
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24. Fuentealba, L. C., et al., Integrating patterning signals:Wnt/GSK3 regulates the duration of the BMP/Smadl signal. Cell,2007.131(5): p.980-93.
25. Ozaita, A., E. Puighermanal, and R. Maldonado, Regulation of PI3K/Akt/GSK-3 pathway by cannabinoids in the brain. J Neurochem,2007. 102(4):p.1105-14.
26. Doble, B. W. and J. R. Woodgett, GSK-3:tricks of the trade for a multi-tasking kinase. J Cell Sci,2003.116(Pt 7):p.1175-86.
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10. Guo, L., et al., Targeting amyloid-beta in glaucoma treatment. Proc Natl Acad Sci U S A,2007.104(33):p.13444-9.
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19. LaPointe, N. E., et al., The amino terminus of tau inhibits kinesin-dependent axonal transport:implications for filament toxicity. J Neurosci Res,2009.87(2):p.440-51.
20. Hong, M., et al., Lithium reduces tau phosphorylation by inhibition of glycogen synthase kinase-3. J Biol Chem,1997.272(40):p. 25326-32.
21. Stambolic, V., L. Ruel, and J. R. Woodgett, Lithium inhibits glycogen synthase kinase-3 activity and mimics wingless signalling in intact cells. Curr Biol,1996.6(12):p.1664-8.
22. Munoz-Montano, J.R., et al., Lithium inhibits Alzheimer's disease-like tau protein phosphorylation in neurons. FEBS Lett,1997. 411(2-3):p.183-8.
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26. Doble, B. W. and J. R. Woodgett, GSK-3:tricks of the trade for a multi-tasking kinase. J Cell Sci,2003.116(Pt 7):p.1175-86.
1. Huber-Lang, M., et al., Generation of C5a in the absence of C3:a new complement activation pathway. Nat Med,2006.12(6):p.682-7.
2. Rahpeymai, Y., et al., Complement:a novel factor in basal and ischemia-induced neurogenesis. EMBO J,2006.25(6):p.1364-74.
3. Strey, C. W., et al., The proinflammatory mediators C3a and C5a are essential for liver regeneration. J Exp Med,2003.198(6):p.913-23.
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