线粒体自噬在肌萎缩侧索硬化症中的相关研究以及大蒜素的神经保护作用的机制探讨
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摘要
背景：肌萎缩侧索硬化[Amyotrophic lateral sclerosis,(ALS)]，是一个致死性的神经变性疾病，临床表现为肌萎缩，肌强直，无力等。通常在诊断后的3-5年就可以死亡。最近，在ALS和伴有泛素阳性包涵体的额颞叶痴呆病人[frontotemporal lobar dementia with ubiquitin-positive inclusions,(FTLD-U)]的脑中发现了泛素化的包涵体，其中TDP-43是主要的组成成分。所以，TDP-43在神经变性疾病中受到越来越多的人的关注。TDP-43,它是普遍存在的核蛋白，由414个氨基酸组成。它是由1号染色体上的TARDBP基因编码的。它的作用包括了特异性前RNA的剪切和转录，信使RNA的稳定性以及小RNA的生物发生等。至今，已在散发和遗传性的ALS病人的TDP-43基因中发现40多种的显性突变，就为TDP-43功能异常和变性疾病中提供了直接的证据。TDP-43的发现为我们研究ALS和FTLD-U的发病机制开拓了新的视野。在ALS的发病机制中，线粒体是多个发病机制的汇合点。大量的研究表明在突变的SOD1相关的ALS的病人，细胞以及动物模型中，线粒体损伤参与了发病机制过程。其中突变和野生型的SOD1在线粒体上聚集是一个非常关键的环节。相对应的是在转染TDP-43的细胞中，同样存在线粒体膜电位的降低，ROS的生成增多以及呼吸链的受损等线粒体损伤的证据。线粒体选择性的自噬在线粒体的质量控制机制中发挥着重要的作用，它参与了帕金森病、阿尔茨海默病的发病机制。但是在ALS的模型中，研究的却非常少。
大蒜素，它是大蒜油中有机硫化合物的主要成分，由于其强大的抗癌效应受到越来越多的关注。在动物模型中，它不但可以通过多种机制保护化学物质诱导的癌症，抗癌机制包括调控致癌物的代谢，抑制肿瘤细胞的增殖来阻止肿瘤细胞的增长，诱导凋亡，组氨酸的修饰，抑制血管的再生。大蒜素其他的健康益处包括抗血栓效应，改善免疫功能，降低血糖，防止辐射和微生物的侵入。另外，其神经保护作用在ALS的脊髓培养模型和SOD1的转基因鼠中均得到验证，但是在ALS的细胞模型中的疗效却没有证实。
第一部分过表达TDP-43及其片段对线粒体的功能和形态的影响
目的：本研究的目的是探讨TDP-43及其片段对线粒体的功能和形态的影响。
方法：在NSC34细胞中瞬时表达含有空质粒对照、野生型、Q331K、M337V、TDP-25和TDP-35的质粒。在细胞转染48h后，收集细胞并用western印迹验证细胞的表达效率；用免疫荧光观察外源性蛋白的分布特点；用流式细胞计数观察TDP-43及其片段对线粒体膜电位和活性氧簇的影响；用免疫荧光观察外源性蛋白对线粒体形态的影响。
结果：（1）在NSC34细胞中可以顺利表达外源性的TDP-43蛋白。并且表达全长的TDP-43蛋白水平和内源性TDP-43蛋白水平差不多，表达片段的TDP-43水平大约是内源性TDP-43蛋白水平的一半。
（2）在对照细胞中，内源性的TDP-43主要在胞核中。在转染的细胞中，全长的TDP-43蛋白主要分布在胞核中，一部分在胞浆中存在。而片段化的TDP-43更容易形成在胞核和胞浆致密的包涵体。
（3）通过流式细胞术检测结果表明，表达全长及片段的TDP-43细胞和空质粒对照的细胞相比，线粒体的膜电位降低了，活性氧簇的水平明显上升。
（4）通过激光共聚焦显微镜观察，转染全长及片段的TDP-43的细胞中，线粒体的分布不均匀和聚集更多见。
结论：表达全长及片段TDP-43的NSC34细胞和对照细胞相比，明显降低了线粒体膜电位的水平，提高了活性氧簇的水平，并且干扰了线粒体的正常分布。
第二部分过表达TDP-43及其片段对线粒体自噬的影响
目的：本研究的目的是探讨TDP-43及其片段对线粒体自噬的影响。
方法：在NSC34细胞中瞬时表达含有空质粒对照、野生型、Q331K、M337V、TDP-25和TDP-35的质粒。在细胞转染48h后，用匀浆器离心方法提取胞浆和线粒体组分，然后用透射电镜和Western印迹检测提取线粒体的纯度和形态；并用Western印迹技术检测各组分外源性和内源性TDP-43的表达，用免疫荧光观察外源性TDP-43和线粒体的分布关系，用western印迹技术检测线粒体和胞浆中LC3B、P62的蛋白水平。
结果：（1）用透射电镜技术检测提取的线粒体形态保存尚好，纯度约在90%以上。用western印迹检测线粒体部分的标记物VDAC1和胞浆部分的标记物actin，彼此并不掺杂，说明提取线粒体的纯度高。
（2）用western印迹检测转染细胞中胞浆和线粒体部分的外源性TDP-43，在线粒体上可有少量的外源性TDP-43的存在，而且野生型和突变型的水平差不多。用免疫荧光检测结果表明，外源性TDP-43可以和线粒体的标记物共定位。
（3）用western印迹分别检测转染细胞中胞浆和线粒体部分的外源性TDP-43、内源性TDP-43、LC3B、P62的蛋白表达，转染全长TDP-43细胞比空质粒对照细胞中，线粒体部分的内源性TDP-43的表达明显升高，LC3B蛋白水平明显升高，P62的蛋白水平显著降低。在转染片段的TDP-43的细胞中，线粒体外膜蛋白VDAC1的水平降低，而线粒体部分的LC3B蛋白水平明显升高，P62的蛋白水平显著降低。
结论：匀浆离心法提取的线粒体形态完整，纯度高；外源性TDP-43在线粒体部分少量存在；转染外源性TDP-43的细胞，LC3B蛋白水平明显升高，P62的蛋白水平显著降低，激活了线粒体自噬。
第三部分大蒜素在肌萎缩侧索硬化模型中神经保护作用机制的探讨
目的：本研究的目的是探讨大蒜素在肌萎缩侧索硬化模型中的神经保护作用和机制。
方法：用CCK-8法检测不同浓度和时间的大蒜素处理的NSC34细胞后，对细胞活力影响；采用合适的浓度及时间的大蒜素干预NSC34细胞后，用western印迹技术检测LC3B蛋白水平；在转染携带GFP-LC3质粒的细胞中，在不同浓度的大蒜素处理后，用免疫荧光技术观察GFP-LC3点状聚集的情况；在转染TDP-43的细胞中，选择合适浓度的大蒜素干预后，用western印迹技术检测外源性TDP-43变化；分别选用自噬通路阻断剂（Bafilomycin A1）和蛋白酶体通路阻断剂（MG132）处理细胞，观察大蒜素是通过什么途径影响蛋白水平；在转染TDP-43的细胞中，选择合适浓度的大蒜素干预后，用Western印迹技术检测胞核部分Nrf2的表达情况与细胞中抗氧化酶的表达水平。
结果：（1）分别用不同浓度（0-100μM）的大蒜素处理NSC34细胞24h后,在0-10μΜ的大蒜素对于细胞活力没有明显区别，在20-100μM之间的大蒜素依据药物浓度逐渐增大，细胞毒性逐渐增加。在10μM的大蒜素处理下，处理48h后，细胞活力明显下降。
（2）分别用不同浓度（0-10μM）的大蒜素处理NSC34细胞24h后,用western印迹检测结果表明，LC3-II的水平是逐渐升高；分别用10μM的大蒜素处理NSC34细胞不同时间（0-36h）后, Western印迹检测结果表明，处理24h后，LC3-II的水平是最高的；用免疫荧光技术观察GFP-LC3点状聚集，结果表明在处理24h后，携带点状聚集的细胞数目是依浓度增加而逐渐最多的。
（3）在NSC34细胞中瞬时表达含有空质粒对照、野生型、Q331K、M337V、TDP-25和TDP35的质粒，给予10μM的大蒜素处理24h后，Western印迹检测结果表明，外源性TDP-43水平都有显著的下降；给予自噬通路阻断剂（Bafilomycin A1）和蛋白酶体通路阻断剂（MG132）阻断处理后，western印迹检测结果表明，大蒜素在Bafilomycin A1存在的情况下，不能降解TDP-25蛋白水平，而在MG132存在的情况下，仍然可以降解。结果表明大蒜素是通过自噬途径来促进蛋白的降解。
（4）在NSC34细胞中瞬时表达含有空质粒对照、野生型、Q331K、M337V、TDP-25和TDP-35的质粒，给予10μM的大蒜素处理24h后，Western印迹检测结果表明，可以上调胞核中Nrf2蛋白的表达，并且上调了抗氧化酶HO-1和NQO-1的蛋白水平。
结论：在TDP-43相关的ALS细胞模型中，低剂量的大蒜素可以上调自噬水平，通过自噬途径促进外源性TDP-43的降解，并激活了Nrf2/ARE通路，上调了二相酶HO-1和NQO-1的蛋白表达，从而发挥它的神经保护作用。
Background: Amyotrophic lateral sclerosis (ALS), a fatalneurodegenerative disease, is characterized by muscle atrophy, spasticity andweakness, and leads to death within three to five years of diagnosis. Recently,TAR DNA binding protein of43kDa (TDP-43) was identified as the majorprotein constituent of ubiquitinated inclusions in brains of ALS andfrontotemporal lobar degeneration. TDP-43is encoded by the gene TARDBPon human chromosome1. It may be involved in specific pre-mRNA splicingand transcription, mRNA stability and the biosynthesis of microRNAs. Up tonow, over40dominant mutations in the TDP-43gene have been linked tosporadic and familial ALS, which provided strong evidence for a direct linkbetween TDP-43dysfunction and neurodegeneration. TDP-43C-terminalfragments are recovered in the sarkosyl-insoluble fraction of affected brainregions of ALS patients, it has been hypothesized that they serve to seedTDP-43aggregation and play an important role in disease pathogenesis. It iswidely accepted that mitochondria is a converging point of multiplepathological pathways in ALS. A large number of researches indicated thatmitochondrial dysfunction participated in the degeneration of ALS patients,animal models for mutant SOD1-linked ALS, in which the accumulation ofmutants or wild type (WT) SOD1in mitochondria represents a critical point.Correspondingly, TDP-43-expressing models also showed mitochondrialdysfunction, indicated by accumulation of reactive oxygen species (ROS), lossof mitochondrial membrane potential (Ψm), and impairment of therespiratory chain. Mitophagy plays an important role in the quality control ofmitochondria, which has been implicated in Parkinson’s disease andAlzheimer’s disease, but very little is known about their role in ALS.
Diallyl trisulfide (DATS), one of the major organosulfur compounds in garlic oil，has received considerable attention because of its anticancer effect.It can not only ofer protection against chemically induced cancer in animalmodels by modulation of carcinogen metabolism, but also suppress growth ofcancer cells in vitro and in vivo by inhibition of cancer cell proliferation andcell cycle arrest, induction of apoptosis, histone modification, and suppressionof angiogenesis. The other known health benefits of DATS includeantithrombotic effects, improvement of the immune function, lowering ofblood glucose level, radioprotection, and protection against microbialinfections. In addition, its neuroprotective effects were demonstrated bystudies from our laboratory using organotypic spinal cord culture andtransgenic mice model of ALS. However, that was not confirmed in cellularmodel.
Part Ⅰ: The effects of full-length TDP-43and its C-terminal fragmentson mitochondrial function and morphology
Objective: To investigate whether full-length TDP-43and its C-terminalfragments impair mitochondrial function and morphology.
Methods: NSC34cells were transiently transfected with different humanTDP-43plasmids, including WT, Q331K, M337V TDP-43and twoC-terminal fragments termed as TDP-25and TDP-35, and the cells wereharvested after48h of transfection. We first determined the expression levelsby western blots. We next observed the subcellular localization of TDP-43byconfocal microscopy, and further investigated the effects of human TDP-43onmitochondrial function, indicators of mitochondrial activity such asmitochondrial membrane potential and levels of cellular ROS were determined.Finally we examined morphological changes of mitochondria in transfectedcells by confocal microscopy.
Results:(1) Human TDP-43plasmids were sucessfully expressed inNSC34cells. Western blot data showed the expression levels of full-lengthTDP-43were very similar to endogenous TDP-43, but the expression levels ofTDP-25and TDP-35were approximately50%of that endogenous TDP-43.
(2) In untransfected cells, TDP-43was localized to the nucleus. In cells transfected with full-length TDP-43, WT and mutant TDP-43predominantlyexpressed in the nucleus, but TDP-43signals were also detected in thecytoplasm. Compared with full-length TDP-43, C-terminal fragments weremore likely to form compact cytoplasmic and nuclear inclusions.
(3) Quantification by flow cytometry showed that both WT and mutantTDP-43could lead to decrease in mitochondrial membrane potential.Compared with control cells, full-length TDP-43and its C-terminal fragmentssignificantly increased ROS production.
(4) We also investigated morphological changes of mitochondria byconfocal microscopy, and clustered and uneven distributed mitochondria weremore common in full-length and truncated TDP-43transfection cells.
Conclusion: Compared with control cells, full-length TDP-43and itsC-terminal fragments significantly decreased mitochondrial membranepotential levels, increased ROS production, and disturbed mitochondrianormal distribution.
Part Ⅱ: The effects of full-length TDP-43and its C-terminal fragmentson mitophagy in NSC34cells
Objective: To investigate whether full-length TDP-43and its C-terminalfragments activate mitophagy.
Methods: NSC34cells were transiently transfected with different humanTDP-43plasmids, including WT, Q331K, M337V TDP-43and twoC-terminal fragments termed as TDP-25and TDP-35, and the cells wereharvested after48h of transfection. We first confirmed the purity ofmitochondrial isolation, obtained from the homogenization method, bytransmission electron microscope. Then we examined the protein level ofhuman and endogenous TDP-43in the mitochondria and cytoplasm bywestern blot, observed distributive characters of TDP-43and mitochondria byconfocal microscopy, and determined LC3B and P62expression levels inmitochondrial and cytosol fractions.
Results:(1) We first confirmed the purity of mitochondrial isolation,obtained from the homogenization-centrifugation method, by transmission electron microscope; the micrograph showed that mitochondria isolated bythis technique preserved morphological integrity very well，and the purity ofmitochondria maintained in90%above. Western blot data showed that actin(a cytosol maker) was undetectable in mitochondrial fraction, and VDAC1(amitochondrial maker) was also undetectable in cytosol fraction.
(2) We examined TDP-43in the mitochondrial fractions by western blotsand confocal microscopy. Our data showed that WT and mutant TDP-43could be present and localized in the mitochondria, and the expression level ofWT TDP-43in mitochondrial fraction was very similar to mutant TDP-43.
(3) We further evaluated mitophagy in purified mitochondria bymeasuring the levels of LC3-II and p62. expression of WT and mutantTDP-43led to sequestration of endogenous nuclear TDP-43after48htransfection. Furthermore, in cells transfected with WT or mutant TDP-43, thelevel of LC3-II markedly increased and p62decreased in the mitochondrialfractions. In addition, VDAC1, decreased in cells expressed TDP-25andTDP-35, compared with control. Moreover, in the mitochondrial fractions,TDP-25and TDP-35clearly up-regulated the levels of LC3-II but reduced theexpression of p62.
Conclusion: Mitochondria isolated by homogenization-centrifugationmethod preserved morphological integrity very well and high purity, and aportion of exogenous TDP-43were localized in the mitochondria. WT, mutantTDP-43and its C-terminal fragments all induced LC3-II but reduced p62inthe mitochondrial fractions, indicating that mitophagy was activated at thesubcellular level.
Part Ⅲ: The protective effect of Diallyl trisulfide on cellular model ofamyotrophic lateral sclerosis
Objective: To investigate the protective effect and mechanism Diallyltrisulfide on cellular model of amyotrophic lateral sclerosis
Methods: NSC34cells were exposed to different concentrations andtimes of DATS, and cell viability was measured by the CCK-8assay. NSC34cells were treated with appropriate DATS for indicated time, and we examined LC3B level by western blot. In addition, cells expressing pEGFP-LC3weretreated with DATS for indicated concentration and time, and the accumulationof pEGFP-LC3in response to treatment were analyzed by fluorescencemicroscopy. We also investiged the effect of DATS on human TDP-43intransfected cells by western blots，and determined which means DATS causedthe clearance of TDP-43using the autophagy inhibitor Baf A1and theproteasome inhibitor MG132. We finally examined Nrf2in the nucleus andantioxidase levels after the treatment of DATS in transfected cells.
Results:(1) There was no marked difference in the degree of toxicityfrom0to10μM for24h. In contrast, cell viability measured by the CCK-8assay was significantly decreased from20to100μM in NSC34cells in adose-dependent manner. Next the cells were treated with10μM DATS atincreasing times and cell viability was analyzed. The CCK-8leveldramatically decreased at48h after the addition of DATS.
(2) NSC34cells were exposed to DATS from0to10μM for24h, andwestern blotting analysis revealed a steadily increasing quantity of theLC3-II form. In addition, time-course analysis showed that24h of exposure toDATS stimulated the greatest amount LC3-II protein. Furthermore, we alsotreated the pEGFP-LC3transfected cells with DATS and found that there wereincreased numbers of punctate pEGFP-LC3dots in the treated cells indose-dependent manner.
(3) NSC34cells were transiently transfected with different humanTDP-43plasmids, including WT, Q331K, M337V TDP-43and twoC-terminal fragments termed as TDP-25and TDP-35. Treatment with10μMDATS for24h resulted in decrease in human TDP-43protein levels, both inWT and mutants transfected cells by western blots. Furthermore, we treatedTDP-25transfected cells with the autophagy inhibitor Baf A1and theproteasome inhibitor MG132. These inhibitor data revealed that TDP-25levels were not decreased by24h in cells treated with DATS and Baf A1compared to DATS alone. In virtually, they were decreased after exposure ofDATS and MG132, indicating that DATS promotes clearance of misfolded proteins by induction of autophagy.
(4) NSC34cells were transiently transfected with different humanTDP-43plasmids, including WT, Q331K, M337V TDP-43and twoC-terminal fragments termed as TDP-25and TDP-35. After treatment with10μM DATS for24h, western blot data showed the treatment of DATSsignificantly induced nuclear translocation of Nrf2, increased antioxidaseHO-1and NQO1erxpression.
Conclusion: In TDP-43-associated ALS cellular models, low-doseDATS could up-regulate autophagy flux, cause the clearance of exogenousTDP-43by autophagy, activate Nrf2/ARE pathway, enhance the expression ofHO-1and NQO-1, and confer its robust neuroprotection.
大蒜素，它是大蒜油中有机硫化合物的主要成分，由于其强大的抗癌效应受到越来越多的关注。在动物模型中，它不但可以通过多种机制保护化学物质诱导的癌症，抗癌机制包括调控致癌物的代谢，抑制肿瘤细胞的增殖来阻止肿瘤细胞的增长，诱导凋亡，组氨酸的修饰，抑制血管的再生。大蒜素其他的健康益处包括抗血栓效应，改善免疫功能，降低血糖，防止辐射和微生物的侵入。另外，其神经保护作用在ALS的脊髓培养模型和SOD1的转基因鼠中均得到验证，但是在ALS的细胞模型中的疗效却没有证实。
第一部分过表达TDP-43及其片段对线粒体的功能和形态的影响
目的：本研究的目的是探讨TDP-43及其片段对线粒体的功能和形态的影响。
方法：在NSC34细胞中瞬时表达含有空质粒对照、野生型、Q331K、M337V、TDP-25和TDP-35的质粒。在细胞转染48h后，收集细胞并用western印迹验证细胞的表达效率；用免疫荧光观察外源性蛋白的分布特点；用流式细胞计数观察TDP-43及其片段对线粒体膜电位和活性氧簇的影响；用免疫荧光观察外源性蛋白对线粒体形态的影响。
结果：（1）在NSC34细胞中可以顺利表达外源性的TDP-43蛋白。并且表达全长的TDP-43蛋白水平和内源性TDP-43蛋白水平差不多，表达片段的TDP-43水平大约是内源性TDP-43蛋白水平的一半。
（2）在对照细胞中，内源性的TDP-43主要在胞核中。在转染的细胞中，全长的TDP-43蛋白主要分布在胞核中，一部分在胞浆中存在。而片段化的TDP-43更容易形成在胞核和胞浆致密的包涵体。
（3）通过流式细胞术检测结果表明，表达全长及片段的TDP-43细胞和空质粒对照的细胞相比，线粒体的膜电位降低了，活性氧簇的水平明显上升。
（4）通过激光共聚焦显微镜观察，转染全长及片段的TDP-43的细胞中，线粒体的分布不均匀和聚集更多见。
结论：表达全长及片段TDP-43的NSC34细胞和对照细胞相比，明显降低了线粒体膜电位的水平，提高了活性氧簇的水平，并且干扰了线粒体的正常分布。
第二部分过表达TDP-43及其片段对线粒体自噬的影响
目的：本研究的目的是探讨TDP-43及其片段对线粒体自噬的影响。
方法：在NSC34细胞中瞬时表达含有空质粒对照、野生型、Q331K、M337V、TDP-25和TDP-35的质粒。在细胞转染48h后，用匀浆器离心方法提取胞浆和线粒体组分，然后用透射电镜和Western印迹检测提取线粒体的纯度和形态；并用Western印迹技术检测各组分外源性和内源性TDP-43的表达，用免疫荧光观察外源性TDP-43和线粒体的分布关系，用western印迹技术检测线粒体和胞浆中LC3B、P62的蛋白水平。
结果：（1）用透射电镜技术检测提取的线粒体形态保存尚好，纯度约在90%以上。用western印迹检测线粒体部分的标记物VDAC1和胞浆部分的标记物actin，彼此并不掺杂，说明提取线粒体的纯度高。
（2）用western印迹检测转染细胞中胞浆和线粒体部分的外源性TDP-43，在线粒体上可有少量的外源性TDP-43的存在，而且野生型和突变型的水平差不多。用免疫荧光检测结果表明，外源性TDP-43可以和线粒体的标记物共定位。
（3）用western印迹分别检测转染细胞中胞浆和线粒体部分的外源性TDP-43、内源性TDP-43、LC3B、P62的蛋白表达，转染全长TDP-43细胞比空质粒对照细胞中，线粒体部分的内源性TDP-43的表达明显升高，LC3B蛋白水平明显升高，P62的蛋白水平显著降低。在转染片段的TDP-43的细胞中，线粒体外膜蛋白VDAC1的水平降低，而线粒体部分的LC3B蛋白水平明显升高，P62的蛋白水平显著降低。
结论：匀浆离心法提取的线粒体形态完整，纯度高；外源性TDP-43在线粒体部分少量存在；转染外源性TDP-43的细胞，LC3B蛋白水平明显升高，P62的蛋白水平显著降低，激活了线粒体自噬。
第三部分大蒜素在肌萎缩侧索硬化模型中神经保护作用机制的探讨
目的：本研究的目的是探讨大蒜素在肌萎缩侧索硬化模型中的神经保护作用和机制。
方法：用CCK-8法检测不同浓度和时间的大蒜素处理的NSC34细胞后，对细胞活力影响；采用合适的浓度及时间的大蒜素干预NSC34细胞后，用western印迹技术检测LC3B蛋白水平；在转染携带GFP-LC3质粒的细胞中，在不同浓度的大蒜素处理后，用免疫荧光技术观察GFP-LC3点状聚集的情况；在转染TDP-43的细胞中，选择合适浓度的大蒜素干预后，用western印迹技术检测外源性TDP-43变化；分别选用自噬通路阻断剂（Bafilomycin A1）和蛋白酶体通路阻断剂（MG132）处理细胞，观察大蒜素是通过什么途径影响蛋白水平；在转染TDP-43的细胞中，选择合适浓度的大蒜素干预后，用Western印迹技术检测胞核部分Nrf2的表达情况与细胞中抗氧化酶的表达水平。
结果：（1）分别用不同浓度（0-100μM）的大蒜素处理NSC34细胞24h后,在0-10μΜ的大蒜素对于细胞活力没有明显区别，在20-100μM之间的大蒜素依据药物浓度逐渐增大，细胞毒性逐渐增加。在10μM的大蒜素处理下，处理48h后，细胞活力明显下降。
（2）分别用不同浓度（0-10μM）的大蒜素处理NSC34细胞24h后,用western印迹检测结果表明，LC3-II的水平是逐渐升高；分别用10μM的大蒜素处理NSC34细胞不同时间（0-36h）后, Western印迹检测结果表明，处理24h后，LC3-II的水平是最高的；用免疫荧光技术观察GFP-LC3点状聚集，结果表明在处理24h后，携带点状聚集的细胞数目是依浓度增加而逐渐最多的。
（3）在NSC34细胞中瞬时表达含有空质粒对照、野生型、Q331K、M337V、TDP-25和TDP35的质粒，给予10μM的大蒜素处理24h后，Western印迹检测结果表明，外源性TDP-43水平都有显著的下降；给予自噬通路阻断剂（Bafilomycin A1）和蛋白酶体通路阻断剂（MG132）阻断处理后，western印迹检测结果表明，大蒜素在Bafilomycin A1存在的情况下，不能降解TDP-25蛋白水平，而在MG132存在的情况下，仍然可以降解。结果表明大蒜素是通过自噬途径来促进蛋白的降解。
（4）在NSC34细胞中瞬时表达含有空质粒对照、野生型、Q331K、M337V、TDP-25和TDP-35的质粒，给予10μM的大蒜素处理24h后，Western印迹检测结果表明，可以上调胞核中Nrf2蛋白的表达，并且上调了抗氧化酶HO-1和NQO-1的蛋白水平。
结论：在TDP-43相关的ALS细胞模型中，低剂量的大蒜素可以上调自噬水平，通过自噬途径促进外源性TDP-43的降解，并激活了Nrf2/ARE通路，上调了二相酶HO-1和NQO-1的蛋白表达，从而发挥它的神经保护作用。
Background: Amyotrophic lateral sclerosis (ALS), a fatalneurodegenerative disease, is characterized by muscle atrophy, spasticity andweakness, and leads to death within three to five years of diagnosis. Recently,TAR DNA binding protein of43kDa (TDP-43) was identified as the majorprotein constituent of ubiquitinated inclusions in brains of ALS andfrontotemporal lobar degeneration. TDP-43is encoded by the gene TARDBPon human chromosome1. It may be involved in specific pre-mRNA splicingand transcription, mRNA stability and the biosynthesis of microRNAs. Up tonow, over40dominant mutations in the TDP-43gene have been linked tosporadic and familial ALS, which provided strong evidence for a direct linkbetween TDP-43dysfunction and neurodegeneration. TDP-43C-terminalfragments are recovered in the sarkosyl-insoluble fraction of affected brainregions of ALS patients, it has been hypothesized that they serve to seedTDP-43aggregation and play an important role in disease pathogenesis. It iswidely accepted that mitochondria is a converging point of multiplepathological pathways in ALS. A large number of researches indicated thatmitochondrial dysfunction participated in the degeneration of ALS patients,animal models for mutant SOD1-linked ALS, in which the accumulation ofmutants or wild type (WT) SOD1in mitochondria represents a critical point.Correspondingly, TDP-43-expressing models also showed mitochondrialdysfunction, indicated by accumulation of reactive oxygen species (ROS), lossof mitochondrial membrane potential (Ψm), and impairment of therespiratory chain. Mitophagy plays an important role in the quality control ofmitochondria, which has been implicated in Parkinson’s disease andAlzheimer’s disease, but very little is known about their role in ALS.
Diallyl trisulfide (DATS), one of the major organosulfur compounds in garlic oil，has received considerable attention because of its anticancer effect.It can not only ofer protection against chemically induced cancer in animalmodels by modulation of carcinogen metabolism, but also suppress growth ofcancer cells in vitro and in vivo by inhibition of cancer cell proliferation andcell cycle arrest, induction of apoptosis, histone modification, and suppressionof angiogenesis. The other known health benefits of DATS includeantithrombotic effects, improvement of the immune function, lowering ofblood glucose level, radioprotection, and protection against microbialinfections. In addition, its neuroprotective effects were demonstrated bystudies from our laboratory using organotypic spinal cord culture andtransgenic mice model of ALS. However, that was not confirmed in cellularmodel.
Part Ⅰ: The effects of full-length TDP-43and its C-terminal fragmentson mitochondrial function and morphology
Objective: To investigate whether full-length TDP-43and its C-terminalfragments impair mitochondrial function and morphology.
Methods: NSC34cells were transiently transfected with different humanTDP-43plasmids, including WT, Q331K, M337V TDP-43and twoC-terminal fragments termed as TDP-25and TDP-35, and the cells wereharvested after48h of transfection. We first determined the expression levelsby western blots. We next observed the subcellular localization of TDP-43byconfocal microscopy, and further investigated the effects of human TDP-43onmitochondrial function, indicators of mitochondrial activity such asmitochondrial membrane potential and levels of cellular ROS were determined.Finally we examined morphological changes of mitochondria in transfectedcells by confocal microscopy.
Results:(1) Human TDP-43plasmids were sucessfully expressed inNSC34cells. Western blot data showed the expression levels of full-lengthTDP-43were very similar to endogenous TDP-43, but the expression levels ofTDP-25and TDP-35were approximately50%of that endogenous TDP-43.
(2) In untransfected cells, TDP-43was localized to the nucleus. In cells transfected with full-length TDP-43, WT and mutant TDP-43predominantlyexpressed in the nucleus, but TDP-43signals were also detected in thecytoplasm. Compared with full-length TDP-43, C-terminal fragments weremore likely to form compact cytoplasmic and nuclear inclusions.
(3) Quantification by flow cytometry showed that both WT and mutantTDP-43could lead to decrease in mitochondrial membrane potential.Compared with control cells, full-length TDP-43and its C-terminal fragmentssignificantly increased ROS production.
(4) We also investigated morphological changes of mitochondria byconfocal microscopy, and clustered and uneven distributed mitochondria weremore common in full-length and truncated TDP-43transfection cells.
Conclusion: Compared with control cells, full-length TDP-43and itsC-terminal fragments significantly decreased mitochondrial membranepotential levels, increased ROS production, and disturbed mitochondrianormal distribution.
Part Ⅱ: The effects of full-length TDP-43and its C-terminal fragmentson mitophagy in NSC34cells
Objective: To investigate whether full-length TDP-43and its C-terminalfragments activate mitophagy.
Methods: NSC34cells were transiently transfected with different humanTDP-43plasmids, including WT, Q331K, M337V TDP-43and twoC-terminal fragments termed as TDP-25and TDP-35, and the cells wereharvested after48h of transfection. We first confirmed the purity ofmitochondrial isolation, obtained from the homogenization method, bytransmission electron microscope. Then we examined the protein level ofhuman and endogenous TDP-43in the mitochondria and cytoplasm bywestern blot, observed distributive characters of TDP-43and mitochondria byconfocal microscopy, and determined LC3B and P62expression levels inmitochondrial and cytosol fractions.
Results:(1) We first confirmed the purity of mitochondrial isolation,obtained from the homogenization-centrifugation method, by transmission electron microscope; the micrograph showed that mitochondria isolated bythis technique preserved morphological integrity very well，and the purity ofmitochondria maintained in90%above. Western blot data showed that actin(a cytosol maker) was undetectable in mitochondrial fraction, and VDAC1(amitochondrial maker) was also undetectable in cytosol fraction.
(2) We examined TDP-43in the mitochondrial fractions by western blotsand confocal microscopy. Our data showed that WT and mutant TDP-43could be present and localized in the mitochondria, and the expression level ofWT TDP-43in mitochondrial fraction was very similar to mutant TDP-43.
(3) We further evaluated mitophagy in purified mitochondria bymeasuring the levels of LC3-II and p62. expression of WT and mutantTDP-43led to sequestration of endogenous nuclear TDP-43after48htransfection. Furthermore, in cells transfected with WT or mutant TDP-43, thelevel of LC3-II markedly increased and p62decreased in the mitochondrialfractions. In addition, VDAC1, decreased in cells expressed TDP-25andTDP-35, compared with control. Moreover, in the mitochondrial fractions,TDP-25and TDP-35clearly up-regulated the levels of LC3-II but reduced theexpression of p62.
Conclusion: Mitochondria isolated by homogenization-centrifugationmethod preserved morphological integrity very well and high purity, and aportion of exogenous TDP-43were localized in the mitochondria. WT, mutantTDP-43and its C-terminal fragments all induced LC3-II but reduced p62inthe mitochondrial fractions, indicating that mitophagy was activated at thesubcellular level.
Part Ⅲ: The protective effect of Diallyl trisulfide on cellular model ofamyotrophic lateral sclerosis
Objective: To investigate the protective effect and mechanism Diallyltrisulfide on cellular model of amyotrophic lateral sclerosis
Methods: NSC34cells were exposed to different concentrations andtimes of DATS, and cell viability was measured by the CCK-8assay. NSC34cells were treated with appropriate DATS for indicated time, and we examined LC3B level by western blot. In addition, cells expressing pEGFP-LC3weretreated with DATS for indicated concentration and time, and the accumulationof pEGFP-LC3in response to treatment were analyzed by fluorescencemicroscopy. We also investiged the effect of DATS on human TDP-43intransfected cells by western blots，and determined which means DATS causedthe clearance of TDP-43using the autophagy inhibitor Baf A1and theproteasome inhibitor MG132. We finally examined Nrf2in the nucleus andantioxidase levels after the treatment of DATS in transfected cells.
Results:(1) There was no marked difference in the degree of toxicityfrom0to10μM for24h. In contrast, cell viability measured by the CCK-8assay was significantly decreased from20to100μM in NSC34cells in adose-dependent manner. Next the cells were treated with10μM DATS atincreasing times and cell viability was analyzed. The CCK-8leveldramatically decreased at48h after the addition of DATS.
(2) NSC34cells were exposed to DATS from0to10μM for24h, andwestern blotting analysis revealed a steadily increasing quantity of theLC3-II form. In addition, time-course analysis showed that24h of exposure toDATS stimulated the greatest amount LC3-II protein. Furthermore, we alsotreated the pEGFP-LC3transfected cells with DATS and found that there wereincreased numbers of punctate pEGFP-LC3dots in the treated cells indose-dependent manner.
(3) NSC34cells were transiently transfected with different humanTDP-43plasmids, including WT, Q331K, M337V TDP-43and twoC-terminal fragments termed as TDP-25and TDP-35. Treatment with10μMDATS for24h resulted in decrease in human TDP-43protein levels, both inWT and mutants transfected cells by western blots. Furthermore, we treatedTDP-25transfected cells with the autophagy inhibitor Baf A1and theproteasome inhibitor MG132. These inhibitor data revealed that TDP-25levels were not decreased by24h in cells treated with DATS and Baf A1compared to DATS alone. In virtually, they were decreased after exposure ofDATS and MG132, indicating that DATS promotes clearance of misfolded proteins by induction of autophagy.
(4) NSC34cells were transiently transfected with different humanTDP-43plasmids, including WT, Q331K, M337V TDP-43and twoC-terminal fragments termed as TDP-25and TDP-35. After treatment with10μM DATS for24h, western blot data showed the treatment of DATSsignificantly induced nuclear translocation of Nrf2, increased antioxidaseHO-1and NQO1erxpression.
Conclusion: In TDP-43-associated ALS cellular models, low-doseDATS could up-regulate autophagy flux, cause the clearance of exogenousTDP-43by autophagy, activate Nrf2/ARE pathway, enhance the expression ofHO-1and NQO-1, and confer its robust neuroprotection.
引文
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