【作者】 穆阿汉（Abuelnor Mohammed Mohammed Ahamed Ahamed）；

【导师】 李和；

【作者基本信息】 华中科技大学 ， 人体解剖学与组织胚胎学， 2012， 博士

【摘要】 亨廷顿病(Huntington’s disease, HD)是一种由于编码亨廷顿蛋白(huntingtin,Htt)的HD基因第一外显子中CAG重复序列异常扩展所致的遗传性神经退行性疾病。HD基因引起Htt在HD患者和HD动物模型脑内神经元胞质和核内错误折叠和聚集。突变Htt聚集物因募集多种细胞内重要成分而具有毒性,在HD发病中具有重要作用。错误折叠及异常聚集的突变Htt主要通过泛素-蛋白酶体系统(ubiquitin-proteasome system, UPS)、自噬系统和热休克蛋白等分子伴侣清除或纠正,UPS、自噬系统、分子伴侣功能损害会引起突变Htt聚集物的累积。突变Htt可导致氧化应激,氧化应激可通过损害UPS功能促进突变Htt聚集,导致细胞死亡,因此,抗氧化剂的应用是一种具有良好前景的减少突变Htt聚集物,抑制突变Htt毒性的策略。木犀草素(luteolin),也称四羟基黄酮漆黄素(tetrahydroxyflavone),是一种主要存在于多种植物包括药草中的黄酮类物质(flavonoids),能透过血脑屏障,具有抗炎、抗氧化、清除自由基和神经保护作用。本研究利用HD细胞模型和HD转基因小鼠观察了木犀草素对突变Htt毒性的影响及其机制,结果表明,木犀草素能抑制突变Htt毒性,其机制除了其抗氧化作用外,更重要的是促进突变Htt的蛋白酶体性和自噬性降解及通过激活热休克因子1(heat shock factor 1, HSF-1)而上调热休克蛋白分子伴侣的表达。1.木犀草素抑制突变Htt对N2a细胞的毒性和改善HD转基因小鼠症状为了分析木犀草素对突变Htt毒性的影响,用不同浓度的木犀草素处理表达正常Htt(20Q)或突变Htt(160Q)的N2a细胞,应用MTT方法检测细胞活力,结果显示,木犀草素处理能剂量依赖性抑制160Q细胞的活力下降。进一步的PI染色和激活型caspase 3水平的免疫印迹检测显示,木犀草素处理可明显降低160Q细胞中PI染色阳性细胞百分率,并剂量依赖性抑制突变Htt对caspase 3的激活。对表达Htt氨基末端1-171个氨基酸并含有82个谷氨酰胺重复序列的HD转基因小鼠(Tg小鼠)进行木犀草素处理(腹腔注射)后,与未进行木犀草素注射处理的Tg小鼠比较,木犀草素处理的Tg小鼠的体重下降、肢体握抱反应(limbclasping)等HD症状明显减轻,寿命延长,运动功能障碍(rotarod testing)明显改善。由此表明,木犀草素对突变Htt的毒性具有明显的抑制作用。2.木犀草素能降低表达突变Htt的N2a细胞和HD转基因小鼠脑内聚集型和可溶型突变Htt为了明确木犀草素能否减少突变Htt聚集物,对表达突变Htt的细胞内和HD转基因小鼠脑组织内突变Htt聚集物含量在木犀草素处理后的变化进行了检测。突变Htt聚集物在表达160Q的细胞内和Tg小鼠脑内呈时间或年龄依赖性增加,木犀草素处理后,不仅聚集型突变Htt显著减少,可溶型突变Htt含量也明显降低。对160Q细胞内报告基因Cherry mRNA的RT-PCR检测表明,木犀草素对160Q的表达无影响。因此,木犀草素引起的突变Htt含量减少可能由其促进突变Htt降解和/或抑制突变Htt聚集所致。3.木犀草素降低突变Htt聚集物的作用不依赖其抗氧化作用为了了解木犀草素降低突变Htt聚集物的作用是否与其抗氧化作用有关,对调节抗氧化反应元件(antioxidant responsive element, ARE)的转录因子核因子E2相关因子2(nuclear factor erythroid-2 related factor 2, Nrf2)在木犀草素减少突变Htt聚集物中的作用进行了检测。对Nrf2及其下游靶蛋白血红素氧合酶1(heme oxygenase 1,HO-1)的免疫印迹分析显示,突变Htt可升高Nrf2和HO-1水平；木犀草素处理后,Nrf2和HO-1水平在20Q细胞仅轻度升高,但在160Q细胞明显升高,并高于未用木犀草素处理的160Q细胞。然而,用Nrf2特异的SiRNA沉默Nrf2对木犀草素降低突变Htt聚集物的作用几乎没有阻断效应,因此,木犀草素对突变Htt聚集物累积的作用不依赖Nrf2通路的激活,即与木犀草素的抗氧化效应无关。4.木犀草素促进突变Htt的蛋白酶体性和自噬性降解为了明确蛋白酶体是否参与木犀草素对突变Htt的降解,首先检测了蛋白酶体抑制剂MG132是否阻断木犀草素对突变Htt的作用。免疫印迹检测显示,在木犀草素处理的160Q中加入MG132可阻断木犀草素减少可溶型突变Htt的作用,但不能阻断聚集型突变Htt的减少。接下来用稳定表达GFPu(反映UPS功能、羧基末端融合降解信号CL1的GFP)的N2a细胞(GFPu(?)田胞)分析了木犀草素对UPS功能的影响。与前人的报道一样,在GFPu细胞中转染表达160Q可明显减少GFPu的降解而使GFPu的荧光强度和蛋白水平明显升高,表明突变Htt可损害UPS的功能。木犀草素处理虽然仅轻微增加表达20Q的GFPu细胞中GFPu的降解,但可明显增加表达160Q的GFPu细胞中GFPu的降解。进而用小荧光底物分析法测定蛋白酶体活性(糜蛋白酶样活性)显示,虽然突变Htt对N2a细胞蛋白酶体活性无明显影响,但木犀草素处理可明显增加转染表达20Q或160Q的N2a细胞的蛋白酶体活性,其中对表达160Q的N2a细胞蛋白酶体活性的增加更为明显。这些结果表明,木犀草素可促进蛋白酶体对可溶型突变Htt的降解。木犀草素可减少160Q细胞中的突变Htt聚集物,而MG132不能阻断这一作用,因此自噬也可能参与木犀草素促进突变Htt聚集物降解效应。在木犀草素处理的160Q细胞中加入自噬抑制剂氯喹(chloroquine)可阻断木犀草素减少突变Htt聚集物的作用,对反映自噬激活的标志—微管相关蛋白1轻链3(microtubule-associated protein 1 light chain-3 (LC3)-Ⅰ向LC3-Ⅱ的转化检测显示,木犀草素处理可明显促进160Q细胞内LC3-Ⅰ向LC3-Ⅱ的转化。因此,木犀草素可通过诱导激活自噬而促进突变Htt聚集物的降解。5.木犀草素通过激活热休克因子1上调热休克蛋白的表达用木犀草素和MG132共同处理的160Q细胞中可溶型突变Htt水平不仅高于未用木犀草素处理的细胞,也高于仅用MG132处理的160Q细胞；而在木犀草素和氯喹共同处理的160Q细胞中,聚集型突变Htt水平低于仅用氯喹处理的160Q细胞,可溶型突变Htt水平高于仅用木犀草素处理的160Q细胞,由此提示,木犀草素可通过其他途径抑制可溶型突变Htt的聚集。热休克蛋白(HSP)40、HSP70、HSP105等分子伴侣可通过促进突变Htt的再折叠抑制其聚集。为了明确木犀草素处理引起的突变Htt聚集物的减少是否由其增加分子伴侣表达所致,应用免疫印迹和RT-PCR检测了木犀草素处理对HSP40、HSP70、HSP105表达的影响。转染表达160Q可明显下调这些热休克蛋白的表达,木犀草素处理可轻度增加20Q细胞内这些热休克蛋白的表达,并恢复(restore)这些热休克蛋白z在160Q细胞中的表达。活性HSF-1能转录激活热休克蛋白的表达,而HSF-1的活性受HSP90的调控,HSP90通过与HSF-1结合形成复合物将HSF-1滞留在细胞质内而抑制HSF-1的活性,而抑制HSP90活性可使HSF-1从复合物中释放,HSF-1因此发生磷酸化和向核内转位,启动热休克蛋白的表达,与HSF-1分离的HSP90变得不稳定而易被降解。木犀草素是HSP90活性的抑制剂,HSF-1活化后也能上调HSP90的表达。为了明确木犀草素对HSP40、HSP70、HSP105表达的上调由木犀草素抑制HSP90而激活HSF-1所致,继而检测了木犀草素处理对HSF-1磷酸化和核转位和HSP90降解的影响。在160Q细胞中,木犀草素处理明显促进HSF-1的磷酸化和核转位,上调HSP90 mRNA的表达,但HSP90的蛋白水平反而降低,表明木犀草素通过抑制HSP90活性激活了HSF-1。结论：木犀草素可通过多系统降低突变Htt：促进蛋白酶体降解可溶型突变Htt；诱导自噬活性,降解聚集型突变Htt；通过抑制HSP90激活HSF-1,上调热休克蛋白表达,进而抑制突变Htt聚集。木犀草素通过这些途径使突变Htt减少是其抑制突变Htt毒性的重要机制。更多还原

【Abstract】 Huntington’s disease (HD) is a hereditary progressive neurodegenerative disorder caused by expansion of CAG repeats in the first exon of HD gene that encodes huntingtin (Htt) protein. Mutation of HD gene leads to huntingtin protein misfolding and aggregation in the cytoplasm and the nucleus of the neurons in the brains of HD patients and animal models. It has been proposed that mutant huntingtin aggregates are toxic due to the sequestration of vital cellular components. The exact pathological mechanisms determining disease onset and progression remain unclear, however, impaired function of ubiquitin-proteasome system (UPS), defective autophagy function, oxidative stress, apoptosis, are also implicated for the disease pathogenesis mechanism. Accumulating evidences suggest that oxidative stress could promote mutant Htt aggregation, causing cell death by impairing UPS function, indicating that using of the antioxidant agents will be a promising strategy to reduce mutant Htt aggregates, preventing from mutant Htt toxicity. The intense search for small-molecular compounds that may modulate HD pathology has advanced the analysis of specific dietary substances from plants and herb.Luteolin, tetrahydroxyflavone, is a one kind of flavonoids found mainly in many types of plants including medicinal herbs, being considered as main anti-inflammatory, antioxidant and free radical scavenger. Luteolin consumption has been found to be able to improve spatial working memory and restore expression of inflammatory markers in aged mouse hippocampus, indicating its beneficial neuroprotection. In this study we examined the effect of luteolin on mutant Htt toxicity and its mechanism. Our results suggested that luteolin could reduce mutant huntingtin toxicity not only due to its antioxidative activity but, more importantly, also through promoting proteasomal and autophagy degradation of mutant huntingtin and heat shock factor 1 (HSF-1)-mediated upregulation of heat shock proteinsLuteolin protects against toxicity in N2a cells expressing mutant huntingting and improves symptoms of HD transgenic mice. To assess neuroprotection of luteolin against mutant Htt toxicity, the N2a cells transfected with normal (20Q) or mutant (160Q) Htt were treated with different concentrations of the luteolin, and the cell viability was evaluated with MTT assay. Treatment with luteolin resulted in dose-dependent inhibition of decrease in cell viability by mutant Htt. Further PI staining and detection of activated caspase 3 with Western blotting showed that luteolin could significantly reduce percentage of PI-stained cells and dose-dependently decrease level of activated caspase 3 in mutant Htt-transfected cells. Compared with those of untreated HD transgenic (Tg) mice that express the first 171 aa of Htt with 82 glutamines, the HD symptoms including body weight loss, limb clasping, lifespan abbreviation and motor dysfunction in Tg mice treated with intraperitoneal injection of luteolin were significantly improved. The results demonstrated that luteolin had protection against mutant Htt cytotoxicity in both mutant Htt-expressing cells and HD transgenic mice.Luteolin reduces both aggregated and soluble mutant Htt in N2a cells expressing mutant huntingtin and the brain of HD transgenic mice. To assess whether luteolin could reduce the mutant Htt aggregates, the change in amount of mutant Htt aggregates in the luteolin treated cells expressing mutant Htt and HD mice brains was detected. The cells expressing 160Q Htt and Tg mouse brains showed time-or age-dependent accumulation of the mutant Htt aggregates. After treatment with luteolin, the amount of both aggregated and soluble mutant Htt in both 160Q Htt transfected cells and Tg mouse brains was dramatically decreased. RT-PCR analysis did not detect any effect of luteolin on expression of mRNA for transfected mutant Htt in 160Q cells. Thus, the reduction of mutant Htt by luteolin indicates that degradation of mutant Htt was upregulated, or that aggregation of mutant Htt was inhibited, or both.Reduction of mutant huntingtin aggregates by luteolin is independent of activation of Nrf2 pathway. Oxidative stress has been found to be caused by mutant Htt, which in turn leads to aggregation of mutant Htt. To see whether reduction in Htt aggregates is resulted from antioxidation of luteolin, the role of nuclear factor erythroid-2 related factor 2 (Nrf2), the transcription factor that regulates antioxidant responsive elements (ARE), in reduction of mutant Htt aggregates by luteolin was examined. Western blotting analysis for expression levels of Nrf2 and its downstream target, heme oxygenase 1 (HO-1), showed that mutant Htt could elevate levels of Nrf2 and HO-1, and that luteolin treatment could slightly increase levels of Nrf2 and HO-1 in 20Q cells but greatly increase Nrf2 and HO-1 levels in 160Q cells, which was even higher than that in 160Q cells without luteolin treatment. Interestingly, in cells expressing 160Q Htt, the inhibition of mutant Htt aggregate accumulation and/or promotion of mutant Htt degradation by luteolin in 160Q cells was scarcely blocked by knocking down of Nrf2 with Nrf2 specific small interfering RNA (Nrf2 SiRNA). It appears that the effect of luteolin on mutant Htt aggregate accumulation is independent of the Nrf2 pathway activation.Luteolin enhances proteasomal and autophagy degradation of mutant huntingtin. To see the potential involvement of proteasome in the degradation of mutant Htt by luteolin, firstly, we checked if proteasome inhibitor MG132 could block the action of luteolin on mutant Htt. Western blot analysis showed that, addition of MG132 to 160Q transfected cells treated with luteolin blocked the decrease in soluble form of mutant Htt but did not block the decrease in the aggregated form. Next, the effect of luteolin on UPS function was assessed using N2a cells stably expressing a reporter of UPS function, which contains a short degron, CL1, fused to the COOH-terminus of GFP, called GFPu-cells. Consistent with previous study,160Q transfection in GFPu-cells significantly increased the fluorescent intensity and protein level of GFPu, indicating inhibition of UPS function by mutant Htt. Treatment with luteolin slightly decreased the fluorescent intensity and protein level of GFPu in 20Q GFPu-cells but dramatically inhibited increase in those in 160Q GFPu-cells. Furtherly, measurement of proteasome activity (chymotrypsin-like) using a fluorogenic peptide substrate assay showed that luteolin could activate proteasome in 20Q and 160Q cells with stronger activation in 160Q cells than in 20Q cells though 160Q Htt did not inhibit proteasome activity. It is thus suggested that luteolin can promote proteasome to degradate soluble mutan. Since MG132 did not block the decrease in the aggregated form in luteolin treated 160Q cells, autophagy is likely be involved in luteolin-promoted degradation of aggregated mutant Htt. As expected, addition of autophagy inhibitor chloroquine to 160Q cells treated with luteolin obviously blocked the decrease in the aggregated form, and the decrease in the soluble form was slightly inhibited. Moreover, in 160Q Htt-expressing N2a cells, conversion of microtubule-associated protein 1 light chain-3 (LC3)-Ⅰto LC3-Ⅱ, the indicator for autophagy activiation, was increased by treatment with luteolin. Thus, luteolin can promote degradation of mutant Htt aggregates through inducing activation of autophagy.Luteolin up-regulates expression of the heat shock proteins through activation of HSF-1. The level of soluble mutant Htt in 160Q Htt cells treated with luteolin and MG132 was higher not only than that in 160Q Htt cells without treatment but also than that in 160Q Htt cells treated with MG132 only. Furthermore, in 160Q Htt cells treated with luteolin and chloroquine, the level of aggregated mutant Htt was lower than that in 160Q Htt cells treated with chloroquine only, and the level of soluble form was higher than that in 160Q Htt cells treated with luteolin only. Therefore, it was suggested that luteolin might inhibit soluble mutant Htt to aggregate through other mechanism. Molecular chaperons, such as HSP40, HSP70, HSP105, can inhibit mutant Htt to aggregate through refolding them. To determine whether reduction of the mutant Htt aggregates after luteolin treatment might be caused by increased expression of molecular chaperons, we treated the cells transfected with 20Q or 160Q Htt and examined the expression level of HSP40, HSP70 and HSP105. Transfection of 160Q Htt caused decrease in expression level of these chaperons. Luteolin treatment slightly increased their expression in 20Q Htt transfected cells and restored expression of these chaperones in 160Q Htt transfected cells. The expression of HSP40, HSP70 and HSP105 are transcriptionally upregulated by active HSF-1 the function of which is regulated by chaperone HSP90. HSP90 inactivates HSF-1 through binding to and detaining HSF-1 in the cytoplasm. Inhibition of HSP90 leads to release of HSF-1 from HSP90 complex, which results in its phosphorylation, activation and translocation to the nucleus where it initiates heat shock protein expression. Luteolin has been found to be able to inhibit HSP90. To know if the upregulation of HSP40, HSP70 and HSP105 expression by luteolin is resulted from activation of HSF-1 through inhibition of HSP90 by luteolin, we checked if luteolin treatment could cause phosphorylation and nuclear translocation of HSF-1. The activation of HSF-1 was demonstrated by the results that luteolin treatment increased phosphorylation and nuclear localization of HSF-1 in 160Q Htt-expressing cells. Consistently, degradation of HSP90 was increased by luteolin since the protein level of HSP90 was decreased while its mRNA level was elevated after treating 20Q Htt-or 160Q Htt-expressing cells with luteolin, suggesting inhibition of HSP90 by luteolin (HSP90, when inhibited, is separated from HSF-1, becoming unstable).Taken together, luteolin can reduce mutant Htt through multiple system:promoting degradation of soluble mutant Htt by proteasome system, inducing activity of autophagy system to degrade aggregated form of mutant Htt, and activating HSF-1 via inhibition of HSP90 to upregulate expression of heat shock proteins, which, in turn, inhibits aggregation of mutant Htt. The role of luteolin in reducing mutant Htt could be the important contribution to the suppression of mutant Htt toxicity. These findings show neuroprotection of luteolin, providing a new perspective for using luteolin to treat HD and other polyglutamine neurodegenerative disorders.更多还原