【作者】 张幸鼎；

【导师】 秦正红；

【作者基本信息】 苏州大学 ， 药理学， 2011， 博士

【摘要】 目的:硕士期间的研究工作表明,大鼠纹状体内给予线粒体毒素3-硝基丙酸(3-NP)引起的纹状体神经元死亡伴有自噬和凋亡激活,p53诱导的DRAM1表达在自噬激活和细胞死亡中有重要作用。本论文研究DRAM1调节细胞线粒体失能引起的自噬激活和细胞死亡的分子机制。方法:(1)建立3-NP所致A549细胞线粒体失能毒性模型。3-NP处理细胞24, 48和72h后,Western blot法测定DRAM1,BAX,p62和LC3等凋亡和自噬蛋白的表达和线粒体细胞色素C的释放,免疫荧光法检测LC3的表达,观察自噬和凋亡通路的激活。(2)观察自噬和凋亡过程在3-NP所致的细胞线粒体毒性引起的细胞死亡中的作用。Western blot法检测Atg5的siRNA干扰效果。WST-1法检测Atg5 siRNA干扰和Z-VAD-FMK对3-NP诱导的细胞死亡的影响。(3)检测DRAM1在3-NP诱导的细胞自噬流量变化中的作用。Western Blot法检测DRAM1的siRNA后观察p62,LC3等自噬蛋白的表达。免疫组化法检测DRAM1蛋白下调后3-NP所致的LC3表达的变化。(4)研究DRAM1调节3-NP诱导的细胞自噬流量的作用机制。免疫荧光双标法观察DRAM1和溶酶体标志物LysoTracker在细胞内的共定位情况。为了观察DRAM1在细胞清除自噬-溶酶体中的作用,野生型细胞和DRAM1 siRNA干扰细胞分别用雷帕霉素处理24h以及处理6h后撤去雷帕霉素,采用Western blot法检测LC3的蓄积情况,并对LC3点状聚集进行量化,以及用免疫组化法观察LC3和Lamp2检测在胞质内的降解情况。为了观察DRAM1影响自噬体的降解机制,用Western blot法检测Cathepsin D的激活以及使用分子探针观察氢质子泵和溶酶体的酸化情况。(5)验证DRAM1是否是通过溶酶体来调节自噬流量。使用溶酶体抑制剂E64d和氯喹抑制溶酶体后观察是否会产生LC3的蓄积,采用Western blot法观察了抑制溶酶体后Cathepsin D的变化,以及用WST-1法观察是否影响细胞活力。(6)检测DRAM1在3-NP诱导的细胞凋亡过程中的作用。Western Blot法观察DRAM1表达降低后BAX的表达和线粒体细胞色素C的释放以及caspase-3的激活。(7)检测DRAM1调节3-NP所致细胞凋亡的机制。Western Blot法验证BAX siRNA干扰和过表达的效率。为了研究BAX在DRAM1调节凋亡中的作用机制,采用Western Blot法观察BAX的表达降低后对于Cathepsin D和casepase 3的激活以及细胞色素C的释放的影响。WST-1法检测BAX表达降低后对3-NP引起的细胞死亡的影响。结果: (1)在不同时间点3-NP所致A549细胞线粒体失能毒性模型中,Western blot和免疫荧光结果显示DRAM1,LC3自噬相关蛋白在24-48h达到高峰(p<0.01),自噬底物蛋白p62蓄积减少(p<0.01)。同时,凋亡相关蛋白BAX和细胞色素C的释放在48-72h达到高峰(p<0.01)。表明3-NP导致了自噬和凋亡过程的激活。(2) Western blot结果显示Atg5 siRNA干扰后,Atg5蛋白表达明显降低(p<0.01)。WST-1法结果显示Atg5表达降低和Z-VAD-FMK处理对3-NP所致细胞死亡有显著抑制作用。表明自噬和凋亡过程都参与了3-NP诱导的细胞死亡。(3) Western blot结果显示DRAM1的siRNA干扰效果明显(p<0.01),并且DRAM1蛋白表达降低后对3-NP所致自噬相关蛋白LC3的升高有明显抑制作用(p<0.01),自噬底物蛋白p62表达升高(p<0.01)。说明siRNA干扰DRAM1后细胞自噬流量降低。(4)免疫荧光双标结果显示DRAM1和溶酶体标志物LysoTracker在胞质内的共定位现象明显。雷帕霉素处理24h组和处理6h后撤去雷帕霉素组的结果显示,siRNA干扰DRAM1细胞组在撤去雷帕霉素后LC3和Lamp2的清除情况明显比野生型细胞要缓慢。LC3的量化结果表明了siRNA干扰DRAM1组LC3-II在撤除雷帕霉素后下降减慢。Western blot结果显示siRNA干扰DRAM1对3-NP所致Cathepsin D的激活有明显抑制作用(p<0.01)。说明siRNA干扰DRAM1细胞对比野生型细胞对胞内自噬溶酶体的清除能力降低。溶酶体酸化指示剂和量化结果显示siRNA干扰DRAM1对3-NP所致的溶酶体酸化程度降低(p<0.01)。氢质子泵结果显示siRNA干扰DRAM1对3-NP所致的氢质子泵的激活有抑制作用。说明siRNA干扰DRAM1的细胞溶酶体酸化作用降低,清除自噬溶酶体能力减弱。(5)免疫荧光双染结果显示,当线粒体毒性药物3-NP处理后,LC3和Lamp2表达明显增加,并且共定位现象明显。E64d和氯喹处理后,LC3和Lamp2蓄积明显增加。Western blot结果表明3-NP诱导的LC3Ⅱ在E64d处理后显著上升(p<0.05), 3-NP诱导的LC3Ⅱ在氯喹处理后也有显著上升(p<0.01)。Western blot结果表明在E64d和氯喹处理后3-NP诱导的细胞色素C从线粒体内的释放显著减少(p<0.01)。WST-1结果显示WT细胞在溶酶体抑制剂处理后,细胞活力没有很明显的变化(p>0.05),但溶酶体抑制剂显著抑制了3-NP导致的细胞死亡(p<0.05)。说明抑制了溶酶体就抑制了自噬的流量和3-NP诱导的细胞死亡,而DRAM1就是通过溶酶体来达到调节自噬流量的作用。(6) Western blot法结果显示DRAM1蛋白siRNA干扰后对3-NP所致BAX蛋白升高有明显抑制作用(p<0.01),线粒体细胞色素C的释放减少(p<0.01), caspase-3的激活减少(p<0.01)。(7) Western blot结果显示BAX siRNA干扰后,BAX蛋白表达明显下降(p<0.01)。Western blot结果显示,细胞内BAX表达降低会抑制3-NP诱导的Cathepsin D和casepase-3的激活(p<0.01)和3-NP诱导的细胞色素C的释放(p<0.01)。WST-1结果显示BAX的siRNA对3-NP所致的细胞死亡有明显抑制作用(p<0.01)。说明DRAM1有可能通过BAX来调节3-NP诱导的凋亡过程。结论:本文研究结果表明DRAM1通过促进溶酶体酸化和激活溶酶体酶来调节自噬流量,同时DRAM1通过上调BAX来调节线粒体介导的凋亡通路。正在进行中的工作:自噬体和溶酶体的融合对于自噬体的降解是一个很重要的过程,因此对于深刻的了解DRAM1在自噬体和溶酶体融合中的作用正在研究中。DRAM1通过BAX来调节线粒体信号凋亡通路是一个重要的发现,DRAM1如何调节BAX的表达正在进一步研究中。更多还原

【Abstract】 Aim: Previous studies demonstrated that intrastiatal administration of 3-nitropropionic acid induced death of striatal neurons acopmnied by activation of autophagy and apoptosis. The p53-dependent induction of damage-regulated autophagy modulator 1 (DRAM1) played an important role of in autophagy activation and cell death. This study was designed to investgate the molecular mechanisms by which DRAM1 regulates autophagy and apoptosis.Methods: (1) To evaluate activation of autophagy and apoptotic signaling in a cellular model of mitochondrial dysfunction-induced cellular toxicity, A549 cells were treated with 3-nitropropionic Acid (3-NP) for various length of time. The protein levels of DRAM1, BAX, p62, LC3 and release of cytochrome c were determined with Western blot analysis. Expression of GFP-LC3 was determined with fluorescence. (2) To evaluate if an autophagic and apoptotic mechanism contributes to 3-NP-induced cell death, the effects of knockdown of autophagy gene Atg5 and pan-caspase inhibitor Z-VAD-FMK on cell viability determined with WST - 1 method. (3) To understand the role of DRAM1 in the regulation of autophagy, the induction of protein levels of p62 and LC3 induced by 3-NP were determined with Western blot analysis after sirencing DRAM1. Expression of GFP-LC3 induced by 3-NP after DRAM1 knockdown was determined with fluorescence. (4) To study the mechanisms of DRAM1 in regulating autophagy, the lysosomal localization of DRAM1 was examined with LysoTracker. Clearance of autophagosomes is a measure of autophagy flux, and the present study employed double immunofluorescence of cathepsin D and LysoTracker to explore the role of DRAM1 in lysosomal function. To assess lysosomal acidification, we used LysoSensor DND-167 and measured lysosomal pH in quantization.Then we examined the ATP-dependent lysosomal acidification using the pH sensitive dye FITC-dextran. (5) The lysosomal inhibitors E64d (10 ?M) and chloroquine (20 ?M) were used to evaluate if inhibition of lysosomal functions produces effects similar to knockdown of DRAM1. (6) To understand the role of DRAM1 in the regulation of apoptosis, the induction of protein levels of BAX and activation of lysosomal enzyme cathepsin D and release of cyt-c following 3-NP were determined with Western blot analysis after sirencing DRAM1.(7)Next, we analyzed if DRAM1 affects apoptosis through up-regulation of BAX. The connections between BAX and activation of lysosomal enzyme cathepsin D and release of cyt-c following 3-NP treatment were examined with sirecing BAX. The effects of knockdown of BAX on cell viability were determined with WST - 1 method.Results: (1) The results showed that 3-NP-induced a significant increase in the protein levels of DRAM1 and LC3-II from 24-72 h, and 3-NP induced a time-dependent increase in GFP-LC3 in A549 cells, while p62 decreased 24-72 h after 3-NP treatment. 3-NP induced expression of BAX 24 h after treatment. Western blot analysis showed that cyt-c levels decreased in the mitochondrial fractions but increased in the cytosolic fractions starting 24 h after treatment with 3-NP and persisted to 72 h. These results indicate that autophagic and apoptotic proteins were induced by 3-NP. (2) Western blot analysis showed that ATG5 siRNAs caused a down-regulation of ATG5 proteins and a reduction in 3-NP-induced cell death. Z-Vad-FMK (20-40 ?M) had no effect on cell survival under normal conditions, whereas inhibition of caspase activation effectively inhibited the cell death induced by 3-NP. These results suggest that both autophagy contribute to 3-NP-induced cell death. (3) Following treatment of cells with DRAM1 siRNA, the induction of LC3-II, GFP-LC3 puncta by 3-NP was markedly reduced. p62 levels increased in DRAM1 siRNA-treated cells. These lines of evidence support an important role of DRAM1 in autophagy activation. (4) Marked co-localization of DRAM1 and LysoTracker was seen, suggesting that DRAM1 predominantly localizes to lysosomes. In control cells, acute autophagy induction with rapamycin elevated LC3-II levels as revealed by immunoblotting. After removing rapamycin from the medium for 6 h, LC3-II returned towards baseline levels.While in DRAM1 siRNA-treated cells, LC3-II remained elevated 6 h after removing rapamycin. These suggest that the clearance of autophagic vacuoles is impaired in DRAM1 siRNA-treated A549 cells. 3-NP treatment increased the expression of Cathepsin D and the number of LysoTraker labeled lysosomes. The results of immunoblotting showed that knockdown of DRAM1 significantly inhibited 3-NP-induced production of the active form of Cathepsin D. In control cells, the fluorescence of LysoSensor was enhanced from 24 to 72 h after 3-NP exposure. By contrast, in DRAM1 siRNA-treated cells, the fluorescence was lower than that in the control cells. WT cells exhibited an intralysosomal pH of 4.75, and lysosomal pH decreased following 3-NP treatment. In contrast, the lysosomal pH values in DRAM1 siRNA-treated cells were 5.23 and decreased to a lesser extent following 3-NP treatment. These results suggest that there is a defective lysosomal acidification in DRAM1 siRNA-treated cells. In DRAM1 siRNA-treated cells, ATP addition-induced drop in fluorescence emission was reduced, reflecting a reduction in internal lysosomal acidification. Thus, the impairment of acidification in DRAM1 siRNA-treated cells might be due to decreased V-ATPase activity. (5) Compared with cells treated with 3-NP alone, LC3 in E64d and chloroquine-treated cells accumulated more Lamp2-positive vacuoles. LC3-II accumulated after E64d or chloroquine treatment. Western blot analysis showed that cyt-c levels decreased in the cytosolic fractions and increased in the mitochondrial fractions 24 h after treatment of E64d or chloroquine in the presence 3-NP (48 h). Although E64d and chloroquine had no effect on cell survival under normal conditions, they significantly reduced 3-NP-induced cell death 48 h after 3-NP treatment. These results suggest a defective clearance of autophagic vacuoles and a reduction of 3-NP-induced cell death in E64d- and chloroquine-treated cells. (6) Following treatment of cells with DRAM1 siRNA, the induction of BAX、release of cyt-c and activation of caspase-3 by 3-NP was markedly reduced. (7) The efficiency of over-expression and knock-down of BAX was validated by the Western blot analysis. Knock-down of BAX significantly reduced the activation of Cathepsin D. Western blot analysis showed that 3-NP-induced release of cyt-c release from mitochondria and activation of caspase-3 was significantly attenuated after knock-down of BAX with siRNA. The results showed that over-expression of BAX increased 3-NP-induced cell death, while silencing of BAX had no significant effect on cell survival under normal conditions, but significantly reduced 3-NP–induced cell death. It is indicated that DRAM1 affects apoptosis, at least, partially through up-regulation of BAX. Conclusions: The present study demonstrates that DRAM1 regulates autophagy flux through promoting lysosomal acidification and activation of lysosomal enzymes. DRAM1 also has a regulatory role in mitochondrial apoptotic signaling pathway through up-regulating BAX.Under going works: The fusion of autophagosomes with lysosomes is an important step for autophagic degradation. For a full understand of a role of DRAM1 in autophagy flux, the effects of DRAM1 on the fusion process between autophagosomes and lysosomes are currently under investigation. The cross-talk to mitochondrial apoptotic pathway through BAX is interesting. How does DRAM1 regulate the BAX expression is also under investigation.更多还原