本文選題：ALS + 自噬　； 參考：《蘇州大學(xué)》2016年博士論文

【摘要】：肌萎縮側(cè)索硬化癥(amyotrophic lateral sclerosis,ALS)是一類神經(jīng)退行性疾病,主要累及脊髓以及大腦皮質(zhì)和腦干運動神經(jīng)元,其主要臨床表現(xiàn)為肌肉逐漸萎縮無力,患者最后會因呼吸衰竭而死亡。在ALS病人中,主要病理表現(xiàn)為異常蛋白的積聚和神經(jīng)炎癥。TDP-43是近年發(fā)現(xiàn)的一個重要的ALS致病相關(guān)蛋白,但其致病機制尚不清楚。本論文主要研究TDP-43對ALS病理形成的作用,以期闡述其在疾病發(fā)生中的分子機制。結(jié)果一肌萎縮側(cè)索硬化癥(ALS)相關(guān)蛋白TDP-43對自噬溶酶體通路的調(diào)控及其致病機理目的:觀察TDP-43對自噬溶酶體通路的調(diào)控;探索TDP-43對自噬溶酶體通路調(diào)控的機制與ALS病理的關(guān)系。方法:體外培養(yǎng)HEK 293、HeLa、SH-SY5Y、ATG5-WT MEF和ATG5-KO MEF等細胞系,用RNAiMax轉(zhuǎn)染siRNA敲減目的基因,構(gòu)建TDP-43缺陷細胞模型。利用TDP-43缺陷細胞模型進行以下一系列實驗:過表達TFEB-EGFP,在熒光顯微鏡下觀察TFEB入核情況,同時進行核質(zhì)分離實驗,采用免疫印跡檢測TFEB的定位;進行mTOR的免疫熒光實驗,在熒光顯微鏡下觀察mTOR的定位,并通過免疫印跡法檢測mTORC1下游的靶基因p70S6K的磷酸化水平;利用免疫印跡檢測溶酶體上Ragulator-Rag-mTORC1復(fù)合物的各個組分的蛋白量,并通過qRT-PCR檢測各個組分的mRNA水平。采用RNA-蛋白質(zhì)結(jié)合實驗(RIP),檢測TDP-43和raptor的mRNA是否直接結(jié)合;在TDP-43缺陷的細胞模型中敲減raptor或者回補raptor,在熒光顯微鏡下觀察mTOR和TFEB的定位;利用流式細胞術(shù)檢測溶酶體的熒光強度,通過免疫熒光檢測自噬相關(guān)標記物的定位及表達強度,并利用免疫印跡和qRT-PCR檢測自噬和溶酶體相關(guān)基因的蛋白含量和mRNA水平;在TFEB和TDP-43都缺陷的細胞中,利用免疫印跡和qRT-PCR手段分別去檢測自噬和溶酶體相關(guān)基因的蛋白量和mrna水平,并通過電鏡實驗觀察自噬小體和溶酶體的數(shù)量;tdp-43缺陷的細胞給予bafilomycina1或者饑餓處理,利用免疫印跡檢測自噬相關(guān)基因的蛋白量;在tdp-43缺陷的細胞中,過表達mcherry-egfp-lc3,熒光顯微鏡下觀察并統(tǒng)計自噬小體和自噬溶酶體的數(shù)量,檢測自噬流的變化;在tdp-43缺陷的atg5-wtmef和atg5-komef細胞模型中過表達egfp-nhtt-150q(亨廷頓蛋白片段,含多聚谷氨酰胺),采用mtt法檢測細胞的存活率;在tdp-43缺陷的細胞中,利用免疫印跡檢測自噬小體和溶酶體融合相關(guān)蛋白的量,并且通過免疫熒光實驗檢測lamp1-rfp和lc3的共定位。在tbph(tdp-43果蠅同源基因)缺陷的果蠅模型中:利用免疫印跡檢測mtorc1下游靶基因p70s6k和4e-bp1的磷酸化水平;利用qrt-pcr檢測raptor,自噬和溶酶體相關(guān)基因的mrna水平;對tbph缺陷的果蠅給予pa或者rapamycin處理,觀察果蠅幼蟲運動及發(fā)育過程的變化。結(jié)果:在tdp-43缺陷的細胞中過表達tfeb-egfp,發(fā)現(xiàn)tfeb明顯進核;tdp-43敲減后mtor不能在溶酶體上定位,mtorc1下游的靶基因p70s6k磷酸化水平下調(diào);tdp-43敲減后只有raptor的蛋白量和mrna水平下調(diào),而ragulator-rag-mtorc1復(fù)合物的其它組分mtor、ragb、p18的蛋白量和mrna都未見改變;rna-蛋白質(zhì)結(jié)合實驗(rip)表明tdp-43和raptor的mrna直接結(jié)合;在tdp-43缺陷的細胞中敲減raptor,mtor不能在溶酶體上定位,tfeb明顯進核,而在tdp-43缺陷的細胞中回補raptor,mtor可恢復(fù)到溶酶體的定位,tfeb則恢復(fù)到胞質(zhì)的定位,說明tdp-43是通過mtorc1調(diào)控tfeb的核定位。由于tfeb是一個轉(zhuǎn)錄因子,調(diào)控許多溶酶體和自噬基因的表達,我們進一步研究了tdp-43對自噬的影響。tdp-43缺陷細胞自噬小體標記物lc3的點狀分布明顯增加,自噬和溶酶體相關(guān)蛋白含量和mrna水平都上調(diào),而在tfeb缺陷的細胞中再敲減tdp-43,這些基因的蛋白量和mrna水平都不再上調(diào),提示tdp-43對自噬調(diào)控依賴于tfeb。在tdp-43缺陷的細胞中,電鏡觀察發(fā)現(xiàn)自噬小體和溶酶體數(shù)量明顯增多,而在tfeb缺陷的細胞中即使再敲減tdp-43自噬小體和溶酶體的數(shù)量也不再增加。在過表達了egfp-nhtt-150q的atg5-wtmef細胞中,tdp-43敲減后細胞存活率明顯降低,而在過表達了egfp-nhtt-150q的atg5-komef細胞中,tdp-43敲減后細胞存活率未見改變;在tdp-43缺陷的細胞中,dynactin1的含量顯著下調(diào),自噬小體和溶酶體的共定位顯著降低。上述結(jié)果提示tdp-43敲減后自噬小體形成增加,但由于dynactin1的減少,與溶酶體融合減少,從而在自噬缺陷細胞(ATG5-KO MEF)中反而增加了細胞存活。進一步的果蠅實驗顯示,在TBPH缺陷的果蠅中,免疫印跡發(fā)現(xiàn)p70S6K和4E-BP1的磷酸化水平下調(diào),qRT-PCR檢測發(fā)現(xiàn)raptor的mRNA水平下調(diào),而自噬和溶酶體相關(guān)基因的mRNA水平上調(diào);TBPH缺陷的果蠅給予Rapamycin處理,加重了果蠅的幼蟲運動障礙和發(fā)育過程的缺陷,而給予PA處理,可以明顯改善果蠅的幼蟲運動和發(fā)育過程。結(jié)論:TDP-43特異性的調(diào)控mTORC1的關(guān)鍵成分raptor,降低mTORC1的活性,從而引起TFEB明顯進核,導(dǎo)致自噬小體和溶酶體生成增加;同時TDP-43缺陷降低dynactin 1的表達水平,導(dǎo)致自噬小體和溶酶體融合障礙。這兩者共同作用的結(jié)果是導(dǎo)致細胞內(nèi)自噬小體和溶酶體的過度堆積,且自噬小體由于與溶酶體融合障礙不能降解,造成細胞的壓力,引起神經(jīng)毒性。結(jié)果二肌萎縮側(cè)索硬化癥相關(guān)蛋白TDP-43對神經(jīng)炎癥的調(diào)控及其致病機理目的:在TDP-43功能缺失的模型中探索小膠質(zhì)細胞和星型膠質(zhì)細胞介導(dǎo)的神經(jīng)毒性作用。方法:體外培養(yǎng)原代小膠質(zhì)細胞、原代星型膠質(zhì)細胞和BV2細胞,用RNAiMax轉(zhuǎn)染si RNA敲減目的基因,構(gòu)建TDP-43缺陷細胞模型。TDP-43缺陷細胞給予LPS處理,采用免疫印跡檢測炎癥因子COX-2和i NOS的蛋白量,并通過q RT-PCR檢測COX-2和i NOS的m RNA水平;利用免疫印跡檢測TDP-43缺陷的小膠質(zhì)細胞中的信號通路;TDP-43缺陷的小膠質(zhì)細胞給予一系列抑制劑處理后進行免疫印跡和q RT-PCR實驗檢測COX-2的表達;TDP-43缺陷的小膠質(zhì)細胞用U0126處理后采用熒光素酶報告基因?qū)嶒灆z測AP1的活性;TDP-43缺陷的小膠質(zhì)細胞給予U0126或celecoxib處理,利用ELISA試劑盒檢測釋放的PGE2的量;用TDP-43缺陷的小膠質(zhì)細胞和星型膠質(zhì)細胞的培養(yǎng)基對皮層神經(jīng)元和運動神經(jīng)元進行條件性培養(yǎng)基處理并觀察細胞存活;TDP-43缺陷的小膠質(zhì)細胞給予celecoxib處理后進行皮層神經(jīng)元和運動神經(jīng)元的條件性培養(yǎng)基實驗檢測細胞存活。結(jié)果:免疫印跡實驗表明TDP-43缺陷的小膠質(zhì)細胞中COX-2的蛋白量上調(diào),但i NOS的蛋白量未見上調(diào),而在TDP-43缺陷的星型膠質(zhì)細胞中COX-2和i NOS的蛋白量都未見改變;q RT-PCR檢測發(fā)現(xiàn)TDP-43缺陷的小膠質(zhì)細胞中COX-2的m RNA水平上調(diào),而i NOS的m RNA水平未見上調(diào);免疫印跡實驗表明TDP-43缺陷的小膠質(zhì)細胞中磷酸化的MEK和ERK上調(diào),而磷酸化的JNK和p38未見上調(diào),此外,總的JNK、p38、MEK和ERK的蛋白水平都未見改變;TDP-43缺陷的小膠質(zhì)細胞給予U0126(一個MEK1抑制劑)處理,免疫印跡和q RT-PCR檢測發(fā)現(xiàn)COX-2的蛋白和m RNA水平都不再上調(diào),而用其它激酶抑制劑,包括SP600125(JNK抑制劑)、SB216763(GSK3β抑制劑)、BAY(NF-κB抑制劑)和SB203580(p38抑制劑)處理對TDP-43介導(dǎo)的COX-2的表達都沒有影響,上述結(jié)果提示TDP-43特異性地影響小膠質(zhì)細胞的ERK信號通路從而調(diào)節(jié)COX-2的表達。進一步熒光素酶報告基因?qū)嶒灡砻鱐DP-43缺陷的小膠質(zhì)細胞中AP1的轉(zhuǎn)錄活性上調(diào),而給予U0126處理后AP1的轉(zhuǎn)錄活性不再上調(diào);ELISA試劑盒檢測發(fā)現(xiàn)TDP-43缺陷的小膠質(zhì)細胞中PGE2的產(chǎn)生呈時間依賴的上調(diào),而給予U0126或celecoxib(COX-2特異性的抑制劑)處理后PGE2的產(chǎn)生不再上調(diào);條件性培養(yǎng)基實驗表明TDP-43缺陷的小膠質(zhì)細胞,而不是TDP-43缺陷的星型膠質(zhì)細胞的條件培養(yǎng)基可引起皮層神經(jīng)元和運動神經(jīng)元能夠顯著的引起細胞死亡,而TDP-43缺陷的小膠質(zhì)細胞用celecoxib處理后再進行條件性培養(yǎng)基實驗發(fā)現(xiàn)在一定程度上可以減少皮層神經(jīng)元和運動神經(jīng)元損傷。結(jié)論:TDP-43缺陷特異性激活小膠質(zhì)細胞,而不影響星型膠質(zhì)細胞。小膠質(zhì)細胞的激活由MAPK/ERK信號通路介導(dǎo),誘導(dǎo)COX-2和PGE2的表達,引起神經(jīng)炎癥和毒性。
[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.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R744.8
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本文編號：1964079