【作者】 张欣；

【导师】 宋学琴；

【作者基本信息】 河北医科大学 ， 神经病学， 2012， 硕士

【摘要】 目的：肌萎缩侧索硬化（Amyotrophic lateral sclerosis, ALS）是一种进行性加重的神经系统变性疾病，累及上下运动神经元，主要临床表现为进行性加重的肌肉萎缩、肌无力，其感觉系统一般不受累，缺乏有效的治疗方法，病人常在发病后3-5年因呼吸肌麻痹而死亡。90%～95%的ALS为散发性，5%～10%的ALS为家族性，其中约20%的家族性肌萎缩侧索硬化被证实由Cu/Zn超氧化物歧化酶（SOD1）基因突变所致。目前ALS的发病机制尚不清楚，可能是多元的，包括氧化应激、谷氨酸兴奋毒性、线粒体功能障碍、异常的蛋白质聚集、轴索运输障碍和凋亡等。近年来线粒体功能障碍在ALS的发生发展中所起的作用越来越受到人们的重视。线粒体是细胞内最重要细胞器之一，提供细胞生存所需能量，传递细胞信息，介导细胞凋亡，调节钙离子浓度等。运动神经元的线粒体功能是否正常至关重要。在生理条件下，机体可以通过自噬途径清除异常的蛋白质和受损的细胞器，如线粒体、内质网、过氧化物酶体等，以实现细胞器的更新，维持细胞稳态，保持旺盛的生理状态，自噬障碍可导致很多神经变性病，过多的或过少的自噬都是有害的。线粒体经自噬途径被选择性的清除，即线粒体自噬。线粒体不断的进行增殖和自噬，来维持线粒体的正常形态、数目和功能等。LC3II（Microtubule-associated protein1light chain3-II, LC3II）特异地参与自噬泡形成，被认为是自噬泡的“标志物”。P62是一种LC3/Atg8连接蛋白，在自噬过程中绝大多数被清除，常被人们作为自噬途径通畅与否的标志物。有研究报道自噬相关基因7(Autophagy-related gene7, Atg7)缺陷的小鼠骨骼肌中出现线粒体肿胀、嵴断裂、呼吸链活性下降等。在ALS中，人们也已发现：线粒体嵴断裂、消失、空泡化、呼吸链活性下降等改变。那么，ALS中线粒体自噬是否存在障碍，这和SOD1蓄积是否存在相关性，仍需进一步研究。SOD1-G93A转基因小鼠是目前研究ALS最为理想的动物模型之一。本实验旨在研究线粒体自噬在SOD1-G93A转基因小鼠中的情况，探讨其在ALS的发生和发展中所起的作用。方法：选取SOD1-G93A转基因雌性小鼠为实验组，根据病程分为60天组、症状早期组、终末期组，选取90天的雌性阴性小鼠作为对照组，各组12只。以10%水合氯醛(350mg/Kg体重)腹腔注射麻醉，处死小鼠，取腰髓迅速投入液氮冷冻，之后保存于-80℃冰箱；采用Percoll密度梯度离心法分离得到小鼠脊髓线粒体组分和去线粒体胞浆组分；4%多聚甲醛经心脏灌注，之后剥离小鼠腰髓，用4%多聚甲醛浸泡或用2.5%戊二醛固定组织。利用电镜技术、Western blot技术和激光共聚焦技术观察SOD1-G93A转基因小鼠腰髓运动神经元的形态并检测LC3II和P62在小鼠腰髓各组分中的含量。结果：1．SOD1-G93A转基因小鼠腰髓总的LC3II的含量：利用Western blot技术检测SOD1-G93A转基因小鼠腰髓总的LC3II表达量，示症状早期组和终末期组较对照组增加，60天组较对照组无明显变化。利用激光共聚焦技术检测LC3II与SMI32共定位情况，发现在症状早期和终末期出现荧光斑点状聚集，60天组和阴性对照组均无斑点状聚集。SOD1-G93A转基因小鼠腰髓前角细胞形态学变化：利用电镜我们观察到，症状早期组及终末期组SOD1-G93A转基因小鼠腰髓前角细胞中出现大量空泡聚集，60天组和阴性对照组无明显空泡。2．线粒体组分和去线粒体胞浆组分中LC3II的含量：利用Percoll密度梯度离心法分离得到线粒体组分和去线粒体胞浆组分，利用Westernblot技术分别检测两组分中LC3II的蛋白含量，发现症状早期组和终末期组线粒体组分中LC3II的含量较对照组明显增多，去线粒体胞浆组分中LC3II的含量较对照组明显减少。利用激光共聚焦技术检测LC3II与线粒体标记物VDAC共定位情况，示在症状早期组和终末期组VDAC与LC3II共定位增加。3．线粒体组分中P62的含量：利用Percoll密度梯度离心法提取线粒体，利用Western blot技术检测线粒体组分中P62含量，发现随着疾病的进展，症状早期组和终末期组P62的含量较阴性对照组明显增加，60天组较对照组无明显变化。结论：通过对SOD1-G93A转基因小鼠腰髓前角细胞的形态学观察及对LC3II和P62的测定，本实验结果表明，随着疾病的进展，SOD1-G93A转基因小鼠腰髓总LC3II的蛋白水平是增加的，线粒体组分中LC3II和P62的含量明显增加，去线粒体胞浆组分中LC3II的含量明显减少。这证实了线粒体自噬在SOD1-G93A转基因小鼠中存在障碍，过多的线粒体滞留在线粒体自噬的早期阶段，成为ALS的始动环节或恶化因素。更多还原

【Abstract】 Objective: Amyotrophic lateral sclerosis (ALS) is a progressiveneurodegenerative disease, affecting upper and lower motor neurons (MNs),characterized by progressive limb weakness and muscle atrophy, whilesensory systems are generally spared. Lack of effective treatment and most ofthe patients die of respiratory failure3-5years later. ALS can be divided intofamilial amyotrophic lateral sclerosis (fALS) accounting for about5%-10%and sporadic amyotrophic lateral sclerosis (sALS) accounting for about90%-95%according to its speciality of episode and heredity. Approximately20%of fALS patients are caused by mutations of copper-zinc superoxidedismutase (SOD1) gene. Until now, the aetiology and pathogenesis of ALSremain largely unknown. Oxidative stress, glutamate excitotoxicity,mitochondrial dysfunction, abnormal protein aggregation, axonal transportbarriers, and apoptosis have been proved to participate in its pathogenesis.Recent years the dysfunction of mitochondria in ALS attracts more and moreattentions. Mitochondria are one of the most important organelles, providingenergy for cell survival, participating in information transmission, mediatingapoptosis, regulating calcium concentration and so on. Normal mitochondrialfunction is crucial for motor neurons.In physiological conditions, the organism can remove aberrant proteinsand damaged organelles by autophagy pathway, such as mitochondria,endoplasmic reticulum, peroxisomes, to update cell, maintain cellularhomeostasis and keep its physiological state. Impaired autophagy can lead tomany neurodegenerative diseases, and too much or too little autophagy isharmful. Mitochondria can be selectively cleared by autophagy, termedmitophagy. Mitophagy plays an important role to maintain mitochondrialnormal morphology, number and function. LC3II (Microtubule-associatedprotein1light chain3-II, LC3II) is specifically bound to autophagosome membranes and serves as an autophagic marker protein. P62servers as abridge between LC3II and the ubiquitin-conjugated cargo, which is degradedin the process of autophagy, so people often take p62as a marker ofautophagic flux. Research reported that skeletal muscle in Atg7(autophagyrelated gene7, Atg7) deficient mice showed mitochondria swollen, cristaebroken, respiratory chain activity declined and so on. In ALS, people have alsofound mitochondria cristae fragmentation, vacuolization and the respiratorychain activity decreased. Whether mitophagy in ALS is impaired? Are thereany relation between mitophagy and the accumulation of mutant SOD1? Itsrelevant reports are still poor, and need further study.At present, the SOD1-G93A transgenic mice are one of the most idealALS models. Here we focus on mitophagy in SOD1-G93A transgenic mice, tostudy its role in the pathogenesis of ALS.Methods: Female SOD1-G93A transgenic mice were used as theexperimental animals. The90days’ female wild type controls served ascontrol group. There were four groups: control group,60-day-old group, onsetstage group and ending stage group. Each group included twelve mice. After10%hydration aldehydes (350mg/kg body weight) intraperitoneal injectionanesthesia, decapitated, extracted the lumbar spinal cord of mice immediately,and they were frozen in liquid nitrogen and stored at-80℃; isolated spinalcord mitochondria and cytoplasm without mitochondria using percoll densitygradient centrifugation; fixated tissues via heart perfusion by4%paraformaldehyde, dissected lumbar spinal cord of mice and fixated them in4%paraformaldehyde or2.5%glutaraldehyde. Using electron microscopy,western blot and confocal microscopy to detect the morphology; the proteinexpression of LC3II and p62in mitochondria and cytoplasm withoutmitochondria.Results:1. The protein levels of LC3II in the lumbar spinal cord of ALS mice:using western blot, we find the protein levels of LC3II increase at the onsetstage and ending stage compared with WT mice. There is no obvious difference between the60-day-old group and control group. Confocalmicroscopy shows that LC3II immunofluorescence turns into a population ofpuncta in SMI32labeled MNs at the onset and ending stages of ALS, whileLC3II immunofluorescence lightly distributed homogeneously in thecytoplasm of MNs in WT mice and60d ALS mice. The morphologicalchanges in the lumbar spinal cord MNs of SOD1-G93A transgenic mice: usingelectron microscopy, we find there are a lot of autophagosomes in the MNs atthe onset and ending stage of ALS, while no accumulation of autophagosomesat the control group and60-day-old group.2. The protein levels of LC3II in isolated mitochondria and cytoplasmwithout mitochondria by western blot: the protein levels of LC3II inmitochondria increase at the onset and ending stages of ALS mice, while itdecreased in cytoplasm without mitochondria. By double stainings of LC3IIand mitochondria marker VDAC, we find VDAC immunopositive clustersco-localized with LC3II-positive puncta in MNs of ALS at the onset andending stages of ALS.3. The protein levels of p62in isolated mitochondria by western blot: theprotein levels of p62combined with mitochondria increase at the onset andending stages of ALS mice; there is no significant difference of the proteinlevels of p62combined with mitochondria between WT mice and60d groupof ALS mice.Conclusions: By morphological observation, protein quantitation andco-localization observation, we find: with the progress of ALS, the proteinlevels of LC3II in lumbar spinal cord increase; the protein levels of LC3II andp62in mitochondria component increase; the protein levels of LC3II incytoplasm without mitochondria decrease. This indicates that mitophagy inSOD1transgenic mice is impaired: excessive mitochondria stay at the earlystage of mitophagy, which may be the initiating or deteriorative factor of ALS.更多还原