TDP-43在肌萎缩侧索硬化症病理形成中的作用

发布时间：2018-06-01 11:49

  本文选题：ALS + 自噬　； 参考：《苏州大学》2016年博士论文

【摘要】：肌萎缩侧索硬化症(amyotrophic lateral sclerosis,ALS)是一类神经退行性疾病,主要累及脊髓以及大脑皮质和脑干运动神经元,其主要临床表现为肌肉逐渐萎缩无力,患者最后会因呼吸衰竭而死亡。在ALS病人中,主要病理表现为异常蛋白的积聚和神经炎症。TDP-43是近年发现的一个重要的ALS致病相关蛋白,但其致病机制尚不清楚。本论文主要研究TDP-43对ALS病理形成的作用,以期阐述其在疾病发生中的分子机制。结果一肌萎缩侧索硬化症(ALS)相关蛋白TDP-43对自噬溶酶体通路的调控及其致病机理目的:观察TDP-43对自噬溶酶体通路的调控;探索TDP-43对自噬溶酶体通路调控的机制与ALS病理的关系。方法:体外培养HEK 293、HeLa、SH-SY5Y、ATG5-WT MEF和ATG5-KO MEF等细胞系,用RNAiMax转染siRNA敲减目的基因,构建TDP-43缺陷细胞模型。利用TDP-43缺陷细胞模型进行以下一系列实验:过表达TFEB-EGFP,在荧光显微镜下观察TFEB入核情况,同时进行核质分离实验,采用免疫印迹检测TFEB的定位;进行mTOR的免疫荧光实验,在荧光显微镜下观察mTOR的定位,并通过免疫印迹法检测mTORC1下游的靶基因p70S6K的磷酸化水平;利用免疫印迹检测溶酶体上Ragulator-Rag-mTORC1复合物的各个组分的蛋白量,并通过qRT-PCR检测各个组分的mRNA水平。采用RNA-蛋白质结合实验(RIP),检测TDP-43和raptor的mRNA是否直接结合;在TDP-43缺陷的细胞模型中敲减raptor或者回补raptor,在荧光显微镜下观察mTOR和TFEB的定位;利用流式细胞术检测溶酶体的荧光强度,通过免疫荧光检测自噬相关标记物的定位及表达强度,并利用免疫印迹和qRT-PCR检测自噬和溶酶体相关基因的蛋白含量和mRNA水平;在TFEB和TDP-43都缺陷的细胞中,利用免疫印迹和qRT-PCR手段分别去检测自噬和溶酶体相关基因的蛋白量和mrna水平,并通过电镜实验观察自噬小体和溶酶体的数量;tdp-43缺陷的细胞给予bafilomycina1或者饥饿处理,利用免疫印迹检测自噬相关基因的蛋白量;在tdp-43缺陷的细胞中,过表达mcherry-egfp-lc3,荧光显微镜下观察并统计自噬小体和自噬溶酶体的数量,检测自噬流的变化;在tdp-43缺陷的atg5-wtmef和atg5-komef细胞模型中过表达egfp-nhtt-150q(亨廷顿蛋白片段,含多聚谷氨酰胺),采用mtt法检测细胞的存活率;在tdp-43缺陷的细胞中,利用免疫印迹检测自噬小体和溶酶体融合相关蛋白的量,并且通过免疫荧光实验检测lamp1-rfp和lc3的共定位。在tbph(tdp-43果蝇同源基因)缺陷的果蝇模型中:利用免疫印迹检测mtorc1下游靶基因p70s6k和4e-bp1的磷酸化水平;利用qrt-pcr检测raptor,自噬和溶酶体相关基因的mrna水平;对tbph缺陷的果蝇给予pa或者rapamycin处理,观察果蝇幼虫运动及发育过程的变化。结果:在tdp-43缺陷的细胞中过表达tfeb-egfp,发现tfeb明显进核;tdp-43敲减后mtor不能在溶酶体上定位,mtorc1下游的靶基因p70s6k磷酸化水平下调;tdp-43敲减后只有raptor的蛋白量和mrna水平下调,而ragulator-rag-mtorc1复合物的其它组分mtor、ragb、p18的蛋白量和mrna都未见改变;rna-蛋白质结合实验(rip)表明tdp-43和raptor的mrna直接结合;在tdp-43缺陷的细胞中敲减raptor,mtor不能在溶酶体上定位,tfeb明显进核,而在tdp-43缺陷的细胞中回补raptor,mtor可恢复到溶酶体的定位,tfeb则恢复到胞质的定位,说明tdp-43是通过mtorc1调控tfeb的核定位。由于tfeb是一个转录因子,调控许多溶酶体和自噬基因的表达,我们进一步研究了tdp-43对自噬的影响。tdp-43缺陷细胞自噬小体标记物lc3的点状分布明显增加,自噬和溶酶体相关蛋白含量和mrna水平都上调,而在tfeb缺陷的细胞中再敲减tdp-43,这些基因的蛋白量和mrna水平都不再上调,提示tdp-43对自噬调控依赖于tfeb。在tdp-43缺陷的细胞中,电镜观察发现自噬小体和溶酶体数量明显增多,而在tfeb缺陷的细胞中即使再敲减tdp-43自噬小体和溶酶体的数量也不再增加。在过表达了egfp-nhtt-150q的atg5-wtmef细胞中,tdp-43敲减后细胞存活率明显降低,而在过表达了egfp-nhtt-150q的atg5-komef细胞中,tdp-43敲减后细胞存活率未见改变;在tdp-43缺陷的细胞中,dynactin1的含量显著下调,自噬小体和溶酶体的共定位显著降低。上述结果提示tdp-43敲减后自噬小体形成增加,但由于dynactin1的减少,与溶酶体融合减少,从而在自噬缺陷细胞(ATG5-KO MEF)中反而增加了细胞存活。进一步的果蝇实验显示,在TBPH缺陷的果蝇中,免疫印迹发现p70S6K和4E-BP1的磷酸化水平下调,qRT-PCR检测发现raptor的mRNA水平下调,而自噬和溶酶体相关基因的mRNA水平上调;TBPH缺陷的果蝇给予Rapamycin处理,加重了果蝇的幼虫运动障碍和发育过程的缺陷,而给予PA处理,可以明显改善果蝇的幼虫运动和发育过程。结论:TDP-43特异性的调控mTORC1的关键成分raptor,降低mTORC1的活性,从而引起TFEB明显进核,导致自噬小体和溶酶体生成增加;同时TDP-43缺陷降低dynactin 1的表达水平,导致自噬小体和溶酶体融合障碍。这两者共同作用的结果是导致细胞内自噬小体和溶酶体的过度堆积,且自噬小体由于与溶酶体融合障碍不能降解,造成细胞的压力,引起神经毒性。结果二肌萎缩侧索硬化症相关蛋白TDP-43对神经炎症的调控及其致病机理目的:在TDP-43功能缺失的模型中探索小胶质细胞和星型胶质细胞介导的神经毒性作用。方法:体外培养原代小胶质细胞、原代星型胶质细胞和BV2细胞,用RNAiMax转染si RNA敲减目的基因,构建TDP-43缺陷细胞模型。TDP-43缺陷细胞给予LPS处理,采用免疫印迹检测炎症因子COX-2和i NOS的蛋白量,并通过q RT-PCR检测COX-2和i NOS的m RNA水平;利用免疫印迹检测TDP-43缺陷的小胶质细胞中的信号通路;TDP-43缺陷的小胶质细胞给予一系列抑制剂处理后进行免疫印迹和q RT-PCR实验检测COX-2的表达;TDP-43缺陷的小胶质细胞用U0126处理后采用荧光素酶报告基因实验检测AP1的活性;TDP-43缺陷的小胶质细胞给予U0126或celecoxib处理,利用ELISA试剂盒检测释放的PGE2的量;用TDP-43缺陷的小胶质细胞和星型胶质细胞的培养基对皮层神经元和运动神经元进行条件性培养基处理并观察细胞存活;TDP-43缺陷的小胶质细胞给予celecoxib处理后进行皮层神经元和运动神经元的条件性培养基实验检测细胞存活。结果:免疫印迹实验表明TDP-43缺陷的小胶质细胞中COX-2的蛋白量上调,但i NOS的蛋白量未见上调,而在TDP-43缺陷的星型胶质细胞中COX-2和i NOS的蛋白量都未见改变;q RT-PCR检测发现TDP-43缺陷的小胶质细胞中COX-2的m RNA水平上调,而i NOS的m RNA水平未见上调;免疫印迹实验表明TDP-43缺陷的小胶质细胞中磷酸化的MEK和ERK上调,而磷酸化的JNK和p38未见上调,此外,总的JNK、p38、MEK和ERK的蛋白水平都未见改变;TDP-43缺陷的小胶质细胞给予U0126(一个MEK1抑制剂)处理,免疫印迹和q RT-PCR检测发现COX-2的蛋白和m RNA水平都不再上调,而用其它激酶抑制剂,包括SP600125(JNK抑制剂)、SB216763(GSK3β抑制剂)、BAY(NF-κB抑制剂)和SB203580(p38抑制剂)处理对TDP-43介导的COX-2的表达都没有影响,上述结果提示TDP-43特异性地影响小胶质细胞的ERK信号通路从而调节COX-2的表达。进一步荧光素酶报告基因实验表明TDP-43缺陷的小胶质细胞中AP1的转录活性上调,而给予U0126处理后AP1的转录活性不再上调;ELISA试剂盒检测发现TDP-43缺陷的小胶质细胞中PGE2的产生呈时间依赖的上调,而给予U0126或celecoxib(COX-2特异性的抑制剂)处理后PGE2的产生不再上调;条件性培养基实验表明TDP-43缺陷的小胶质细胞,而不是TDP-43缺陷的星型胶质细胞的条件培养基可引起皮层神经元和运动神经元能够显著的引起细胞死亡,而TDP-43缺陷的小胶质细胞用celecoxib处理后再进行条件性培养基实验发现在一定程度上可以减少皮层神经元和运动神经元损伤。结论:TDP-43缺陷特异性激活小胶质细胞,而不影响星型胶质细胞。小胶质细胞的激活由MAPK/ERK信号通路介导,诱导COX-2和PGE2的表达,引起神经炎症和毒性。
[Abstract]:Amyotrophic lateral sclerosis (amyotrophic lateral sclerosis, ALS) is a class of neurodegenerative diseases that mainly involve the spinal cord, the cerebral cortex and the motor neurons of the brain stem. The main clinical manifestation is the gradual atrophy of the muscles and the death of the patients due to respiratory failure. In ALS patients, the main pathological manifestation is the accumulation of abnormal protein. .TDP-43 is an important ALS pathogenic protein found in recent years, but its pathogenesis is not clear. This paper mainly studies the role of TDP-43 on the pathogenesis of ALS, in order to explain its molecular mechanism in the pathogenesis of the disease. Results the modulation of autophagosome pathway in amyotrophic lateral sclerosis (ALS) related protein TDP-43 Control and pathogenesis: To observe the regulation of autophagosome pathway by TDP-43; explore the relationship between the mechanism of TDP-43's regulation of autophagic lysosome pathway and the pathology of ALS. Methods: in vitro culture of HEK 293, HeLa, SH-SY5Y, ATG5-WT MEF and ATG5-KO MEF, using RNAiMax transfection to knock down the target gene and construct a defective cell model. The following series of experiments were carried out with the TDP-43 defect cell model: overexpressing TFEB-EGFP, observing the nucleation of TFEB under the fluorescence microscope, carrying out the nuclear separation experiment, using immunoblotting to detect the location of TFEB, the immunofluorescence test of mTOR, observing the location of mTOR under the fluorescence microscope, and detecting the mTORC1 under the immunoblotting method. The phosphorylation level of the target gene, p70S6K, was used to detect the protein amount of each component of the Ragulator-Rag-mTORC1 complex on the lysosome by Western blot, and the mRNA level of each component was detected by qRT-PCR. RNA- protein binding assay (RIP) was used to detect the direct combination of mRNA in TDP-43 and Raptor, and in the cell model of TDP-43 defects. The localization of mTOR and TFEB, the fluorescence intensity of the lysosomes were detected under the fluorescence microscope, the fluorescence intensity of the lysosomes were detected by flow cytometry, and the localization and expression intensity of autophagy related markers were detected by immunofluorescence, and the protein content and mRNA levels of autophagy and lysosome related genes were detected by immunoblotting and qRT-PCR by flow cytometry. In TFEB and TDP-43 deficient cells, immunoblotting and qRT-PCR were used to detect the protein and mRNA levels of autophagy and lysosome related genes, and the number of autophagosomes and lysosomes were observed by electron microscopy. The cells with TDP-43 deficiency were treated with bafilomycina1 or starvation, and the correlation of autophagy was detected by immunoblotting. The protein amount of the gene; over expression of mcherry-egfp-lc3 in the TDP-43 deficient cells. The number of autophagosomes and autophagosomes were observed under the fluorescence microscope and the autophagic lysosomes were measured. The changes in autophagic flow were detected; egfp-nhtt-150q (Huntington protein fragment containing polyglutamine) was overexpressed in the atg5-wtmef and atg5-komef cell model of the TDP-43 defect. MTT method was used to detect the cell survival rate; in TDP-43 deficient cells, the amount of autophagosome and lysosome fusion related proteins were detected by immunoblotting, and the co localization of lamp1-rfp and LC3 was detected by immunofluorescence test. In the fruit fly model of TBPH (TDP-43 fruit fly homologous gene), the target base of mTORC1 downstream was detected by immunoblotting. The level of phosphorylation of P70S6K and 4E-BP1; mRNA levels of raptor, autophagy and lysosome related genes by qRT-PCR; PA or rapamycin treatment for Drosophila melanogaster deficient in TBPH; the changes in the movement and development of Drosophila larvae were observed. Results: tfeb-egfp was overexpressed in the TDP-43 deficient cells, and TFEB obviously entered the nucleus; TDP-43 knocked down. MTOR could not be located on the lysosome, and the phosphorylation level of the target gene of the downstream mTORC1 was downregulated, and the protein amount and mRNA level of the Raptor decreased after the TDP-43 knockout, while the other components of the ragulator-rag-mtorc1 complex, mTOR, ragb, P18, were not changed. Binding; knocking down raptor in the cells with TDP-43 defects, mTOR can not be located on the lysosome, TFEB is obviously nucleating, and raptor in the TDP-43 deficient cells, mTOR can be restored to the localization of the lysosome, TFEB is restored to the cytoplasm location, indicating that TDP-43 is controlled by mTORC1 TFEB nuclear localization. TFEB is a transcription factor, regulation The expression of many lysosomes and autophagy genes, we further studied the effect of TDP-43 on autophagy, the dot like distribution of autophagic marker LC3 was significantly increased, the content of autophagy and lysosome related proteins and mRNA levels were up-regulated, and TDP-43 in TFEB deficient cells, and the protein and mRNA levels of these genes. The regulation of autophagy depended on tfeb. in TDP-43 deficient cells. The number of autophagosomes and lysosomes increased significantly by electron microscopy, while the number of TDP-43 autophagic corpuscles and lysosomes in TFEB deficient cells was no longer increased. TDP in the atg5-wtmef cells over the expression of egfp-nhtt-150q in atg5-wtmef cells. The survival rate of cells decreased significantly after -43 knockout, but the survival rate of TDP-43 after TDP-43 knockout was not changed in atg5-komef cells overexpressing egfp-nhtt-150q; in TDP-43 deficient cells, the content of dynactin1 decreased significantly, and the co localization of autophagosomes and lysosomes decreased significantly. These results suggest that the autophagic corpuscle is formed after TDP-43 knockout. Increase, but because of the decrease of dynactin1, the fusion with lysosome decreased, and thus increased cell survival in the autophagic defect cell (ATG5-KO MEF). Further Drosophila experiments showed that in the TBPH deficient Drosophila, the immunoblotting showed that the phosphorylation level of p70S6K and 4E-BP1 was down, and the mRNA level of Raptor was down regulated by qRT-PCR detection. The mRNA level of the genes related to the lysosome related genes was up-regulated, and the Drosophila melanogaster deficient in the TBPH was treated with Rapamycin, which aggravated the dyskinesia and developmental defects of the Drosophila, while PA treatment could obviously improve the larval movement and development of the Drosophila. Conclusion: TDP-43 specifically regulates the key component of mTORC1, raptor, and reduces the survival of mTORC1. Sex, resulting in obvious TFEB nucleation, resulting in an increase in the formation of autophagosomes and lysosomes, while TDP-43 deficiency reduces the expression level of dynactin 1, leading to a barrier to fusion of autophagosomes and lysosomes. The two results are the result of the accumulation of autophagosomes and lysosomes in the cells, and the autophagic body is fused with the lysosome. No degradation, resulting in cell stress and neurotoxicity. Results two the regulation of amyotrophic lateral sclerosis related protein TDP-43 on neuroinflammation and its pathogenic mechanism: To explore the neurotoxicity mediated by microglia and astrocytes in the model of TDP-43 dysfunction. Methods: in vitro culture of microglia microglia Cells, primary astrocytes and BV2 cells were transfected by RNAiMax to Si RNA to knock down the target gene, and the.TDP-43 deficient cells of the TDP-43 deficient cell model were constructed by LPS treatment. The protein amount of the inflammatory factors COX-2 and I NOS was detected by immunoblotting and the level of the Q RT-PCR was detected by Q RT-PCR, and the defect was detected by Western blot. Signal pathways in microglia; TDP-43 deficient microglia were treated with a series of inhibitors to perform immunoblotting and Q RT-PCR tests to detect the expression of COX-2; the microglia of TDP-43 deficient microglia used luciferase reporter gene assay to detect AP1 viability after U0126 treatment; TDP-43 deficient microglia was given U0126 or Celecoxib was used to detect the amount of PGE2 released by ELISA kit; the conditioned medium of cortical neurons and motoneurons was treated with the medium of TDP-43 deficient microglia and astrocytes, and the cell survival was observed. The microglia of the TDP-43 deficient microglia was given to the cortical neurons and transport after the celecoxib treatment. Results: the immunoblot test showed that the protein amount of COX-2 in TDP-43 deficient microglia was up up, but the protein amount of I NOS was not up, but the protein amount of COX-2 and I NOS in the TDP-43 deficient astrocytes had not changed; Q RT-PCR detection found TDP-43 defect. The level of M RNA in microglia was up-regulated, while the m RNA level of I NOS was not up. The immunoblotting experiment showed that the phosphorylation of MEK and ERK in the microglia of TDP-43 deficient microglia was up, while the phosphorylation JNK and p38 was not up. 0126 (a MEK1 inhibitor) treatment, immunoblotting and Q RT-PCR detection found that both COX-2 protein and m RNA levels were no longer up-regulated, while other kinase inhibitors, including SP600125 (JNK inhibitor), SB216763 (GSK3 beta inhibitor), BAY (NF- kappa inhibitor) and COX-2 inhibitors did not affect the expression. The results suggest that TDP-43 specifically affects the ERK signaling pathway of microglia and thus regulates the expression of COX-2. Further luciferase reporter gene experiment indicates that the transcriptional activity of AP1 is up regulated in the microglia of TDP-43 defect, and the transcriptional activity of AP1 is no longer up after U0126 treatment; ELISA kit detection found the small defect of TDP-43 The production of PGE2 in glial cells was time dependent, and the production of PGE2 was no longer up-regulated after the treatment of U0126 or celecoxib (COX-2 specific inhibitor). The conditioned medium experiment showed that the TDP-43 deficient microglia, rather than the conditional culture of the TDP-43 deficient star glial cells, could cause cortical neurons and motor deity. The cell death can be significantly caused by the trans element, and the conditioned medium of TDP-43 deficient microglia after celecoxib treatment can reduce the damage of cortical neurons and motor neurons to some extent. Conclusion: TDP-43 deficiency specifically activates microglia cells without affecting astrocytes. Microglia cells. Activation is mediated by the MAPK/ERK signaling pathway, inducing the expression of COX-2 and PGE2, resulting in neuroinflammation and toxicity.
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R744.8
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本文编号：1964079