【作者】 台运春；

【导师】 王慧君；

【作者基本信息】 南方医科大学 ， 法医学， 2013， 博士

【摘要】 研究背景：甲基苯丙胺(Methamphetamine, METH)是一种新型毒品,属于苯丙胺类神经兴奋剂(Amphetamine-Typed Stimulant, ATS),其盐酸盐为透明的结晶体,外观状如冰,俗称“冰毒”。因具有见效快、兴奋作用维持时间长、价格低廉、化学合成技术简单、多途径摄取等特点,METH的滥用及蔓延速度极快,成为当今我国危害最为严重和滥用的两大毒品之一。METH的滥用不仅给个人生理及心理带来极大痛苦,更给家庭和社会带来沉重负担。因此,对METH神经毒性损伤的机制及其防治手段的研究已成为世界面临的重大课题和研究热点。METH为拟交感胺类兴奋剂,具有中枢神经兴奋性、致幻、食欲抑制和拟交感神经能效应等药理、毒理学特性。大量临床资料表明,METH可引起体内重要实质器官的损害,包括心、脑、肺、肝、肾等,但更主要的是对中枢神经系统的毒性。METH滥用能引起明显的行为和精神改变,导致大脑黑质、纹状体、海马皮质多部位损伤,可致动物大脑多巴胺能及五羟色胺能末梢损伤,包括纹状体内多巴胺(DA)含量下降,多巴胺转运体(DAT)降低,酪氨酸羟化酶(TH)活性减少,5-HT及代谢产物的耗竭,单胺囊泡转运体2(VMAT-2)及多巴胺摄取位点缺失,导致神经元、轴索损伤,神经细胞凋亡。目前,关于METH的神经毒性机制尚未完全明确。现阶段的研究结果显示多种机制和途径参与了METH的神经毒性。这些机制主要包括：①过量多巴胺的氧化作用及氧化应激损伤；②谷氨酸的兴奋性作用所致的Ca2+超载和细胞稳态破坏；③线粒体损伤和功能障碍；④激活多条凋亡相关通路、诱发神经元凋亡等。其中氧化应激是METH所致神经毒性损伤的重要作用机制。在我们前一阶段的研究中,对METH注射后大鼠纹状体、皮质、海马等部位的差异表达蛋白质进行了鉴定,认为氧化应激、能量代谢障碍、蛋白酶体功能失调及凋亡等是METH神经毒性的主要机制。同时也发现METH大鼠模型纹状体区活性氧(ROS)、一氧化氮合酶(NOS)和过氧亚硝酸盐(ONOO)、NO和脂质过氧化产物丙二醛(MDA)明显增加,超氧化物歧化酶(SOD)含量下降,伴有神经元凋亡,发现二甲基精氨酸二甲基氨基水解酶(DDAH)表达增高,提出DDAH/ADMA/NOS系统可能是NO过表达引起的中枢神经系统损伤病理过程的重要调控机制。在上述研究中,我们首次发现METH大鼠模型组纹状体、皮质、海马三个脑区中a-突触核蛋白(a-synuclein, a-syn)表达明显升高。α-突触核蛋白由140个氨基酸构成的酸性可溶性蛋白,主要分布于神经元的核膜及突触前末梢。a-syn参与正常突触功能的维持,在调节突触可塑性和递质释放等方面有重要的作用；可以与许多信号蛋白、骨架蛋白、酶和离子等结合,有伴侣分子样活性的作用。a-syn基因敲除小鼠却未发现有明显功能损害和形态改变,表明其生理功能具有代偿机制。a-syn高浓度或过表达时又对神经元有细胞毒的效应。在高浓度时,a-syn形成细胞毒性更大的p-折叠寡聚物,这预示着该蛋白质的异常聚集和纤维化与帕金森病(PD)、阿尔茨海默病(AD)等神经退行性疾病密切相关。METH可导致大脑出现与上述神经退行性病变相类似的改变,并且METH使用者患PD的比率明显升高。最近的研究显示METH可以导致小鼠黑质纹状体区产生类似Lewys小体的包涵体。在PD等许多神经退行性疾病中,多巴胺递质释放减少、氧化应激、Ca2+超载、线粒体功能损伤和细胞凋亡等几种损伤机制密切相关、协同作用,而a-syn在其中扮演着重要的角色。当其浓度升高时既参与了神经损伤的启动,同时也变成损伤过程的效应蛋白,促进神经元变性直至死亡。a-syn的硝基化促进了其寡聚体的稳定性导致其毒性持续增强,因此,它有可能是METH神经毒性损伤中的靶蛋白。正如前面所述,METH所致神经损伤也包含上述几种损伤机制,而a-syn在皮质、海马和纹状体表达升高,这预示着a-syn与上述损伤机制之间也存在着必然的联系,作为重要的靶蛋白参与了神经损伤的过程。本课题组前一阶段通过RNAi技术沉默人多巴胺能神经细胞系SH-SY5Y细胞中a-syn基因,发现干扰a-syn表达可抑制METH引起的细胞活力下降、TH、 DAT、VMAT-2等基因水平下降、多巴胺耗竭和ROS、NOS、NO水平升高,能抵抗METH引起的线粒体△Ψm下降、MPTP开放、细胞外钙离子内流,抑制线粒体的细胞色素C释放进入胞浆,从而在一定程度上保护线粒体的正常功能、抵抗METH引起的细胞死亡。但这一结果尚未在体内得到验证,且a-syn究竟是通过何种路径参与METH的神经毒性损伤、其调控的靶蛋白究竟有哪些及其可能的机制尚未见报道,也正是本研究的目的。目前,RNA干扰(RNAi)技术已成为研究基因功能的主要工具,与传统的基因抑制相比,RNAi技术具有更强大、更持久地抑制基因表达的能力,其效能是反义寡核苷酸的100-10000倍,并且具有高度的序列特异性,无免疫原性,能克服蛋白抑制剂应用中所存在的不足。iTRAQ (isobaric tags for relative and absolute quantitation)技术是一种多肽体外标记技术。该技术采用4种或8种同位素的标签,通过特异性标记多肽的氨基基团,进行串联质谱分析,可同时比较4种或8种不同样品中蛋白质的相对含量或绝对含量。iTRAQ技术灵敏度高,样品需求量少,相对于传统技术能够筛选出更多的差异蛋白。因此,本研究拟利用RNAi技术在体内抑制a-syn的表达,为研究a-syn在METH介导的神经毒性损伤中的机制提供载体；并应用iTRAQ技术筛选差异蛋白并进行定性和定量分析,探讨这些蛋白和METH神经毒性之间的联系。目的：建立a-syn沉默大鼠模型,运用行为学、酶化学等方法,全面了解a-syn抑制后对METH导致的多巴胺系统和氧化应激系统损伤的影响；在此基础上,利用iTRAQ技术,筛选和鉴定出给予METH刺激及RNA干扰a-syn后,大鼠纹状体的差异表达蛋白质,结合已知蛋白质的功能,探讨a-syn在METH神经毒性机制中的作用。方法：1.RNA干扰a-syn大鼠模型的建立成年雄性Wistar大鼠,随机分成4组,①对照组(control, CON);②模型组(model, model);③阴性病毒组(empty vector);④阳性病毒组(RNAi):使用本课题组前期合成并鉴定有效的a-syn-ShRNA重组病毒。利用脑立体定向注射技术向大鼠纹状体注射相应的物质,CON组和model组均注射生理盐水,empty vector组注射阴性病毒,RNAi组注射a-syn-ShRNA重组病毒。注射后二周,取各组大鼠纹状体检测a-syn的表达,实时荧光定量PCR、Western Blot分别检测a-syn在基因和蛋白水平的表达变化。2.RNA干扰a-syn对METH毒性损伤的影响利用成功建立的a-syn沉默大鼠模型,给予METH刺激,CON组腹腔注射生理盐水,model组、empty vector组和RNAi组动物腹腔注射METH,剂量15mg/kg,每天早8：00点、晚6：00点各注射一次,连续注射4天,共8次。观察动物的行为变化,体重、进食量、饮水量的变化；酶联免疫吸附试验方法(ELISA)检测各组大鼠纹状体多巴胺(DA)和络氨酸羟化酶(TH)的含量；TUNEL法检测细胞凋亡；酶化学方法检测ROS、NOS、NO的活性变化,分别用ROS荧光检测试剂盒、NOS活性检测试剂盒、NOS检测试剂盒检测。3.RNA干扰a-syn与METH神经毒性相关差异蛋白质的筛选和鉴定每组取6个纹状体组织,取适量纹状体组织,加入SDT裂解液,匀浆均匀后超声裂解：离心取上清,利用BCA法进行蛋白质浓度测定。各取20μg蛋白质样品进行SDS-PAGE电泳、考马斯亮蓝染色检测蛋白质量。各组取400μg样品进行酶解和肽段定量。分别取约64gg酶解后的肽段,用iTRAQ Reagent-4plexMultiplex Kit进行肽段标记后,采用纳升流速HPLC液相系统Easy nLC进行分离,用Q-Exactive质谱仪进行肽段的定性和定量分析。结果：1.RNA干扰a-syn大鼠模型成功建立。α-syn-shRNA重组慢病毒成功转染至大鼠纹状体细胞,可抑制a-syn基因mRNA表达达到80%,Western Blot实验也证实RNAi组大鼠纹状体内α-syn蛋白表达显著下降。2.METH注射后动物进食量明显减少(P<0.001),具有明显的食欲抑制作用,并可导致动物体重下降(P<0.001)；METH导致动物出现明显的行为学改变,主要表现为好动、易激惹等,动物刻板行为评分明显高于对照组(P<0.001)。METH促进动物活动量增加,从而导致动物口渴口干,饮水量上升；RNAi组动物与model组比较,上述异常表现均有所减少(P<0.001)。3. METH注射后动物纹状体内DA含量和TH活性均明显下降,RNAi组动物与model组比较,DA含量和TH活性均显著上升(P<0.001)。4. METH注射后动物纹状体内ROS上升,RNAi组动物与model组比较,ROS含量显著下降(P<0.001)。5. METH注射后动物纹状体内NOS活性和NO含量均明显下降,RNAi组动物与model组比较,NOS活性和NO含量均显著上升(P<0.001)。6.利用iTRAQ技术鉴定到的蛋白质组2327个,其中65个蛋白同时在RNAi组和empty vector组以及model组CON组存在差异。这65个蛋白质中,包括9个细胞骨架蛋白、5个突触功能相关蛋白、5个细胞增殖和凋亡相关蛋白、3个泛素相关蛋白、2个核糖体蛋白和涉及神经递质、线粒体功能、能量代谢等功能的蛋白。结论：1.成功建立RNA干扰a-syn大鼠模型,在基因和蛋白水平都证实了a-syn表达量的下降。2.干扰a-syn表达可抑制METH中毒大鼠动物行为学上异常表现；通过提高TH的活性从而提高DA的含量,缓解METH导致的多巴胺系统的紊乱；直接抑制ROS的含量,通过降低NOS活性从而降低NO的含量,缓解METH导致的氧化应激和硝化应激的损伤。3.蛋白质精氨酸甲基转移酶5可能是a-突触核蛋白参与METH神经毒性的效应蛋白,通过上调蛋白质精氨酸甲基转移酶5活性而增加NO生成,增强氧化应激损伤；a-突触核蛋白通过调节泛素羧基端水解酶24和VCIP135的表达参与了泛素-蛋白酶体系统的调控。4.在甲基苯丙胺的神经毒性作用中,α-突触核蛋白扮演了重要角色,参与了氧化应激、凋亡、细胞骨架损伤和神经递质障碍等神经损伤过程。更多还原

【Abstract】 Backgroud:Methamphetamine (METH) is an emerging addictive drug, belonging to Amphetamine-Typed Stimulants (ATS). METH can elicit a wide spectrum of pharmacological and toxicological responses, such as central excitability, hallucination, appetite repression and sympathomimetic effects. Due to its fast-acting and sustained effects, low cost, ease of production through chemical synthesis, METH has become one of the two heavily abused drugs, which poses a tremendous burden to the succumbed individuals, their family and the whole society as well. Therefore, it is imperative to elucidate the mechanisms underlying METH’s neurotoxicity and addiction.It has been shown by many clinical studies that METH intoxication causes serious damage of a number of vital organs such as the heart, brain, lung, live and kidney. However, the brain is the most affected organ. METH abuse causes overt psychobehaviral changes, affording lesions in multiple areas such as substantia nigra, striatum and hippocampus. It is demonstrated that dopaminergic and5-HT-ergic termini in the brain were damaged by METH abuse, resulting in decreased level of dopamine (DA), dopamine transporter (DAT) and tyrosine hydroxylase (TH) activity, exhaustion of5-HT and its metabolites, depletion of dopamine binding sites on vesicular monoamine transporter2(VMAT-2), resulting in axonal damage and neuronal demise.The mechanisms underlying the neurotoxicity of METH are still not well understood, however, it has been shown that the following cascades are involved:1) oxidative stress damage by excessive dopamine oxidation,2) calcium overload and disrupted cellular homeostasis resulted from glutamate-induced excitotoxcity,3) mitochondrial damage and malfunction4) activation of apoptotic pathways, oxidative stress is considered one of the most important mechanisms underpinning METH-induced neurotoxicity.In our previous study, we have investigated the differentially expressed genes in the rat striatum, cortex and hippocampus after METH injection. The results showed that oxidative stress, perturbed energy metabolism, proteasome dysfunction and apoptosis are responsible for the neurotoxicity of METH. In addition, in striatum of METH-injected rat, we found that ROS, NOS, ONOO-、NO, MDA and DDAH were significantly increased, whereas SOD level was decreased. These findings suggest that NO may afford damage to the CNS through DDAH/ADMA/NOS pathway. Of notice, we found that the expression of a-synuclein (a-sys) was remarkably increased in the striatum, cortex and hippocampus of METH-injected rat.Physiologically, a-sys is an acidic soluble protein localized in neuronal nuclear membrane and presynaptic terminus, consisting of140amino acid residues.α-sys plays pivotal role in neural transmission and plasticity, capable of binding to a number of signaling molecules, scaffold proteins, enzymes, irons and chaperones. a-sys knock-out mouse shows no overt phenotype, suggesting potential redundancy may exist in vivo. High concentration or overexpression of a-sys is toxic to neurons. At high concentration, a-sys forms oligomers of P-sheet, which are more neurotoxic, hinting that abnormal aggregation and fibrillogenesis may underline neurodegenerative disorders such as Parkinson’s disease (PD), and Alzheimer’s disease. METH-injected brains show neuopathological changes similar to what was observed in the aforementioned neurodegenerative diseases, moreover, the incidence of PD is increased in METH abusers. Recently, striatal Lewys body-like inclusions have been observed in METH-injected mouse. Reduced dopamine release, oxidative stress, calcium overload, mitochondrial damage and apoptosis are contributing together to the pathogenesis of PD, for which a-sys plays an important role. When a-sys is of high concentration, it engages in the initiating the process of neuronal damage, in return, a-sys itself becomes an effector protein to drive neurodegeneration and neuronal loss. Nitration of a-sys makes its oligomer more stable and neurotoxic. Therefore, a-sys is a potential target of METH-induced neurotoxicity.We found that in dopaminergic cell line SH-SY5Y, silencing α-sys via RNAi inhibited METH-induced cell viability reduction, downregulation of TH,DAT, VMAT-2, dopamine exhaustion, elevation of ROS,NOS and NO, reduction of mitochondrial ΔΨm, calcium influx and cytochrome C release to cytosol, preventing METH-induced cell death. However, it is unclear as to how a-sys is involved in METH-induced neurotoxicity and what the target proteins are. Therefore, we aim to sort out these questions.RNAi has become an instrumental tool to delineate gene function. Compared with antisense-mediated gene suppression, RNAi is more potent, sustained in terms of silencing off target genes (100to10,000times more potent), providing high sequence-specificity and avoidance of immunogenicity. iTRAQ (isobaric tags for relative and absolute quantitation) is an in vitro polypeptide labeling technique that is based on the covalent labeling of the N-terminus and side chain amines of peptides from protein digestions with tags of varying mass of4to8different istotopes, enabling simultaneous quantification and comparison of proteins from4or8different samples when coupled with tandem mass spectrometer. Due to its higher sensitivity, iTRAQ can identify more differentially regulated proteins.Therefore we aim to knock down a-sys expression using RNAi, which may serve as a good platform to study the role played by a-sys in METH-induced neurotoxicity. Furthermore, iTRAQ technique will be utilized to identify differentially expressed proteins, whose roles in METH-induced neurotoxicity will be investigated. Aims:To establish rat model of a-sys-RNAi, in which the effect of knockdown of a-sys gene expression on METH-induced dopaminergic toxicity and oxidative stress damage will be investigated using behavioral and enzymology methods. In addition, proteins differentially regulated in the striatum in response to a-sys RNAi in METH-induced neurotoxicity rat model will be identified using iTRAQ technique and their roles with respect to a-sys in the pathogenesis of METH-induced neurotoxicity will be characterized.Methods:1. The establishment of rat model of a-sys-RNAiAdult male Wistar rats were randomized into four groups,①Control (CON);②Model;③Empty vector;④RNAi:a-sys-ShRNA lentiviruses were produced and characterized in our previous work. The intended substances were delivered into rat striatum via stereotaxic injection as following:saline for CON and Model groups,) for Empty vector group, a-sys-ShRNA lentivirus for RNAi group。Striatum was collected2weeks postinjection and a-sys expression was examined using real-time quantitative PCR and Western blot.2. The effect of a-sys-RNAi on METH-induced neurotoxicity in vivoSaline was intraperitoneally administered to rats in CON group, while METH (15mg/kg, one injection at8am and6pm respectively,4consecutive days,8injection in total) was intraperitoneally injected to rats in Model, Empty vector and RNAi groups. Animal behavior, weight, food intake and water consumption were closely monitored. Striatal DA and TH levels were assayed with ELISA; activity of ROS, NOS was examined with fluorescence enzymology assay kits.3. The identification and characterization of differentially expressed proteins in METH-induced neurotoxicity in response to α-sys-RNAiStriatal tissues from various groups (n=6/group) were homogenized using SDT lysis buffer, sonicated and spun, protein concentration of the supernatants was measured using BCA method. Subsequently, the quality of the protein was checked by subjecting20ug protein sample to SDS-PAGE and Coomassie blue staining.400microgram protein was subjected to trypsin digestion and peptide quantification.64microgram of tryptic peptides were labeled using iTRAQ Reagent-4plex Multiplex Kit and separated through nanoflow liquid chromatography system (Easy nLC), followed by peptide identification and validation through Q-Exactive mass spectrometer.Results:1. Establishment of a-sys-RNAi rat model:a-sys mRNA level was reduced80%in a-sys-shRNA lentivirus transduced rat striatum, striatal a-sys protein level was also significantly decreased as evidenced by Western blot.2. Food intake was significantly (p<0.001) after METH injection, confirming METH’s suppression of appetite; animal weight loss was significant (p<0.001). Behavioral changes observed in METH-injected rat manifested as hyperactivity and irritability, with stereotypical behavior score was higher than control group (p<0.001). Water consumption was also increased in METH-injected rats, presumably due to increased activity. These abnormalities were all lessened in RNAi group.3. Striatal DA level and TH activity were significantly decreased in METH-injected rats, while DA level and TH activity in RNAi-treated rats were significantly higher than METH-injcted rats (p<0.001)4. Striatal ROS level was significantly increased ater METH injection, while ROS in RNAi treated rats were significantly decreased compared with METH-injected rats (p<0.001)5. Striatal NOS activity and NO level were significantly lowered in METH-injected rat, while NOS activity and NO level were increased in RNAi rats compared with METH-injected rats(p<0.001).6Using iTRAQ techniques, a total of2327proteins in striatum were identified,65of which were associated with a-syn. Among these65proteins,9proteins are cytoskeleton proteins,5proteins are involved in synaptic transmission,5proteins are involved in cell proliferation and apoptosis,3proteins are concerned with ubiquitin,2 proteins are ribosomal proteins.Conclusions1. Rat model a-sys-RNAi was established and a-sys knockdown were confirmed at transcriptional and translational level.2. METH-induced behavioral phenotypes were suppressed by RNAi-mediated silencing of a-sys. This was accompanied with increased TH activity and DA level, decreased ROS, reduced NOS activity and NO level.3. a-syn knockdown led to downregulation of protein arginine methyltransferase5(PRMT5), resulting in NO production; a-syn regulates ubiquitin-proteasome system through regulating ubiquitin carboxyl-terminal hydrolase24and VCIP1354. a-syn plays a pivotal role in METH-induced neurotoxicity, contributing to oxidative stress, apoptosis, cytoskeletal damage and synaptic transmission dysfunction.更多还原