【作者】 刘斌；

【导师】 王晓良； 程桂芳； 陈晓光；

【作者基本信息】 中国协和医科大学 ， 药理学， 2008， 博士

【摘要】 帕金森氏病(Parkinson’s Disease,PD)是一种老年人群中高发的神经退行性疾病,以运动能力障碍为其主要病理表现,部分患者还伴随痴呆症状。PD的神经病理学特点主要包括黑质多巴胺能神经元进行性丢失,纹状体中多巴胺含量减少以及神经元内涵体(路易小体,Lewy Body,LB)的形成。目前,PD的病理机制尚未明确,但许多研究显示为环境毒素与遗传因素共同作用的结果。本文利用MPTP小鼠模型和α-synuclein(A53T)转基因小鼠模型分别模拟了环境毒素和遗传因素诱导的PD模型,并结合PD患者血清样品,以差异蛋白质组学为主要手段对PD的病理机制进行了研究,试图发现与PD发病相关的新蛋白和药物治疗新靶标。第一部分MPTP模型小鼠纹状体的差异蛋白质组学及相关研究环境毒素是导致散发性PD的致病因子之一。目前已知可致PD的神经毒剂包括MPTP、杀虫剂(如Rotenon)、除草剂(如Paraquat)和金属锰等,它们的致病机制是特异性抑制多巴胺能神经元中线粒体呼吸链复合酶Ⅰ(ComplexⅠ)或Ⅲ(ComplexⅢ)。其中,MPTP可通过对ComplexⅠ的抑制,诱导C57BL/6小鼠表现出明显的PD样病理改变。因此该模型被广泛应用于PD的病理机制研究。在本研究中,我们对C57BL/6小鼠连续7天腹腔注射MPTP后,利用差异蛋白质组学的方法,对MPTP小鼠和生理盐水对照组小鼠纹状体线粒体总蛋白谱进行比较,发现MPTP小鼠蛋白谱中有5个蛋白点显著改变。其中,4个蛋白点表达显著降低,经质谱鉴定分别为:Zinc finger A20 domain containing 1、Lectin-related NK cell receptorLY49S、Protein similar to sodium channel associated protein 1 isoform 2和Proteasome19S ATPase Rpt6;而另外1个点表达显著升高,经质谱鉴定为α-synuclein(α-Syn)。在随后的研究中,我们集中探讨了差异点19S ATPase Rpt6(Rpt6)表达降低与差异点α-Syn升高间潜在的关系。已知,26S蛋白酶体复合物由1-2个19S调节单位和1个20S催化单位组成,而19S ATPase Rpt6是组成19S调节单位的亚基之一,因此提示26S蛋白酶体功能不足可能与α-Syn表达增加有关。Western Blot研究显示,MPTP小鼠纹状体Rpt6及其同源亚基Rpt4蛋白水平分别降低约85.0%和30.5%,而20S组成亚基α6无明显改变,同时α-Syn蛋白表达增加164.8%。但MPTP小鼠黑质中上述蛋白水平均无明显改变。此外,MPTP还可导致纹状体ATP浓度显著降低约36.4%,而对黑质中ATP浓度无影响。由于19S ATPase亚基的功能是ATP依赖的,因此,在MPTP小鼠纹状体中ATPase亚基表达降低和ATP浓度下降可直接导致19S功能障碍。为进一步研究Rpt6减少与α-Syn升高间是否具有直接的关系,我们利用RNA干扰技术,使用siRNA抑制原代培养的纹状体神经元中Rpt6基因的表达。结果显示,与control siRNA组相比,当Rpt6蛋白表达被Rpt6特异siRNA抑制后,神经元中α-Syn蛋白水平显著增加。为揭示Rpt6缺失与20S活性及底物α-Syn聚积的关系,我们进而检测了黑质及纹状体中20S的三种水解酶活性,即糜蛋白酶、胰蛋白酶和肽水解酶活性。结果显示,虽然黑质中20S的三种酶活性均被不同程度抑制,但纹状体中糜蛋白酶活性无明显改变,同时胰蛋白酶和肽水解酶活性均轻度升高。提示纹状体中Rpt6的缺失导致α-Syn水平升高,与20S的水解酶功能无直接关系。第二部分α-synuclein(A53T)转基因小鼠增龄过程中相关脑区的差异蛋白质组学研究α-Syn诱导的神经元毒性被认为是PD重要病理机制之一。在家族遗传性PD患者中已陆续发现α-Syn蛋白的三个突变位点,分别为A30P、A53T和E46K。而在散发性PD患者脑内广泛存在着以α-Syn蛋白为主要结构成分的神经元内涵体,即路易小体(LBs)和路易神经突(LNs)。为揭示α-Syn与PD的关系,近年来野生型和突变型α-Syn转基因小鼠模型被广泛应用于PD的研究中。在本部分研究中,我们首先评价了增龄过程中α-Syn(A53T)转基因小鼠与正常对照小鼠行为学以及特定脑区的免疫组织化学和神经递质水平的差异。结果显示,3月龄转基因小鼠的行为能力与同龄对照小鼠无差异;而增龄至9月龄后,转基因小鼠表现出一定程度的运动障碍。同时,3月龄转基因小鼠黑质多巴胺能神经元数量以及黑质、纹状体中凋亡细胞数量均与对照小鼠无差异;而增龄至9月龄后,转基因小鼠黑质多巴胺能神经元数量呈降低趋势,黑质、纹状体中凋亡细胞数量呈升高趋势。此外,3月龄转基因小鼠纹状体中HVA含量低于对照小鼠,其他递质如DA和DOPAC无差异;增龄至9月龄后,转基因小鼠纹状体中DA、DOPAC和HVA均呈降低的趋势。上述结果显示,9月龄转基因小鼠已处于由生理向病理状态过渡的阶段。为进一步研究α-Syn过表达及磷酸化程度与蛋白酶体功能间的关系,我们利用Western Blot方法检测了转基因小鼠及同龄对照小鼠黑质、纹状体中α-Syn、CaseinKinaseⅡ(CKⅡ)和19S ATPase Rpt6的蛋白表达,并同时检测了20S蛋白酶体的水解酶活性。结果显示,3月龄和9月龄转基因小鼠的黑质、纹状体中α-Syn均持续高表达。同时,3和9月龄转基因小鼠纹状体中CKⅡ表达水平均显著升高,而9月龄转基因小鼠黑质中CKⅡ表达水平降低。此结果提示,9月龄转基因小鼠黑质和纹状体中α-Syn的磷酸化水平可能发生不同的改变。此外,9月龄转基因小鼠黑质中19S ATPase Rpt6表达水平显著降低。然而,20S蛋白水解酶活性在3月龄和9月龄转基因小鼠黑质、纹状体中均无明显改变。提示α-Syn持续高表达可能首先影响蛋白酶体亚基的表达水平,但对20S活性无影响。基于上述结果,为进一步揭示α-Syn(A53T)过表达相关的生理病理学改变,我们采用差异蛋白质组学技术,比较了3月龄转基因及同龄对照小鼠,9月龄转基因和同龄对照小鼠黑质和纹状体的蛋白差异谱。黑质组织的差异蛋白质组学研究显示,3月龄转基因小鼠与同龄对照小鼠间存在9个差异蛋白点,其中2个点在转基因小鼠中表达显著增加,而另外7个点仅存在于转基因小鼠中。9月龄转基因小鼠与对照小鼠的蛋白差异谱研究共发现13个差异点,除与3月龄相同的9个差异点外,新出现4个差异点。纹状体组织的差异蛋白质组学研究显示,3月龄转基因小鼠与同龄对照小鼠间存在9个差异蛋白点,其中1个点在转基因小鼠中表达显著增加,1个点在转基因小鼠中显著减少,而另外7个点仅存在于转基因小鼠中。9月龄转基因小鼠与对照小鼠的蛋白差异谱研究共发现11个差异点,除与3月龄相同的9个差异点外,新出现2个差异点。对比黑质及纹状体的差异蛋白发现,8个差异点在黑质和纹状体中共同存在。它们在无病理异常的3月龄转基因小鼠中即已出现,且无组织特异性。此外,9月龄转基因动物黑质和纹状体中分别新出现4个和2个差异点,这些点不仅伴随动物病理进程而出现,且具有组织特异性,因此具有较高的研究价值。第三部分帕金森病患者与同龄正常人血清差异蛋白质组学研究虽然在老年人群中PD的患病率较高且严重影响患者的生活质量,但是目前尚无有效的方法可在PD患者表现出明显症状前对其进行预防和早期诊断。因此,分析生存状态下PD患者血液样本中蛋白质的改变情况,以寻找对临床诊断和机制研究有价值的生物标签蛋白显得尤为必要。由于血液中一些高丰度蛋白(如白蛋白、IgG等)的存在使许多具有重要意义的低丰度蛋白在传统的二维电泳中不易检出。因此,我们首先利用Agilent公司的亲和抗体柱结合高效液相系统,同时去除人血清中14种高丰度蛋白质,它们约占人血清总蛋白的94%。随后,我们利用二维电泳技术对10名男性PD患者及10名同龄男性健康志愿者血清中的低于10%的低丰度蛋白质进行分离和差异点分析,并利用MALDI-TOF-TOF技术对差异蛋白质进行鉴定。结果显示,在PD患者血清样品中共有16个蛋白点发生显著的改变,其中2个点升高,14个点降低。目前已有7个差异点被成功鉴定,质谱鉴定结果显示这7个差异点为5个已知蛋白质。其中,2个点共同鉴定为凝血酶(Thrombin,又名Coagulation factorⅡ);2个点共同鉴定为载脂蛋白A-Ⅳ(Apo A-Ⅳ);1个点为载脂蛋白A-Ⅰ(ApoA-Ⅰ);1个点为载脂蛋白L-Ⅰ(Apo L-Ⅰ);1个点为补体分子2(C2)。这5个蛋白在PD患者血清均中显著降低。生物信息学分析提示:由载脂蛋白参与的脂代谢过程,凝血酶参与的凝血系统和C2参与的经典补体激活通路可能与PD的病理改变密切相关,是病理机制研究和药物开发可供参考的靶点。更多还原

【Abstract】 Parkinson’s disease(PD) is the second most common neurodegenerative disorder, with a prevalence of about 2%among people over the age of 65 years.PD is typified clinically by motor symptoms and less frequent non-motor complications such as dementia.The neuropathological characters of PD include loss of dopaminergic neurons from the Substantia nigra(SN),depletion of striatal dopamine(DA) and presence of intraneuronal inclusions called Lewy body(LB).The pathogenesis of PD is still unknown, but it appears to be induced by genetic and environmental risk factors.In this work,the 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridine(MPTP)-treated mice model andα-synuclein(A53T) transgenic mice model were employed to represent the environmental toxin-induced and the genetic factor-induced PD-like models,respectively.The comparative proteomic researches were performed in these two mice models as well as the sera samples from PD patients,and the disease-related proteins were yielded and further investigated.PartⅠ.Comparative Proteomic Research of Striatal Proteins in MPTP-induced Mice Model and the Functional Investigation of Differential Proteins.It has been proved that exposure to environmental toxins(such as MPTP,Rotenone, Maneb and Paraquat) can produce Parkinson’s disease through specific inhibiting the mitochondrial respiratory chain complexⅠorⅢin dopaminergic neurons.MPTP is a potent and selective nigrostriatal dopaminergic neurotoxin that can induce parkinsonism when administered to primates and rodents,particularly in the C57BL/6 strain.In this part of work,MPTP was used to treat mouse for 7 d.The behaviors and neuropathological studies showed PD-like changes.The mitochondrial protein profiles in the striatum(STR) were compared between MPTP-treated mice and control by employing two-dimensional electrophoresis(2DE).After MPTP treatment,five proteins were predominantly changed abundance.Four of them were significantly decreased in MPTP-treated mice and identified as Zinc finger A20 domain containing 1,Lectin-related NK cell receptor LY49S,Protein similar to sodium channel associated protein 1 isoform 2 and Proteasome 19S ATPase Rpt6;and the other protein was highly increased in MPTP mice and identified asα-synuclein(α-Syn).The following investigations were focused on the potential relationships between 19S ATPase Rpt6(Rpt6) reduction andα-Syn elevation in MPTP mice STR.It is well-known that a full-functional 26S proteasome in mammaline cells is characterized by a catalytic 20S core particle and one or two 19S regulator particles.The 19S ATPase Rpt6 is one of the subunits involved in the 19S regulator particle,and whose deficit implied an insufficient role of 26S proteasome which might respond to the increased level ofα-Syn. Western blot analysis revealed that proteins expression of Rpt6 and Rpt4 were decreased by 85.0%and 30.5%,repectively;meanwhile,α-Syn level was increased by 164.8%in STR of MPTP-treated mice,however the protein level of 20Sα6 was not changed. Interestingly,none of the above protein subunits were altered in SN after MPTP treatment. In addition,the MPTP-induced reduction of ATP concentration(36.4%) was found in STR but not in SN.Because of the 19S ATPases function depends on the ATP level,the decline of ATP and deficit of subunits expression might lead to a dysfunction of 19S proteasome particle.To gain insight into the role of Rpt6 inα-Syn accumulation,RNA interference was performed in cultured striatal neurons.It showed that,compared with control,α-Syn was increased by 50%when Rpt6 was knocked down by siRNA.To further elucidate whetherα-Syn augment might partially be due to the Rpt6 deficit-induced suppression of 20S activities,the proteolytic activities of 20S were tested.However,the results combined with the Western blot analysis showed that Rpt6 deficit neither suppressed the striatal 20S activities nor effected on the striatal 20Sα6 subunit expression in MPTP-treated mice. Thus,the above data revealed that Rpt6-relatedα-Syn increase in STR might through a distinct mechanism which was not associated with 20S activities. Alpha-synuclein-induced neuronal toxicity was considered as one of the harmful risk factors contribute to PD pathogenesis.Three PD-linked missense mutations including A53T,A30P and E46K inα-Syn gene were discovered in familial PD.In sporadic PD, aggregatedα-Syn is the primary fibrillar component of LBs,which are hallmarks of PD and Lewy body dementia.To demonstrate the correlations betweenα-Syn and PD pathology,in the recent years,the wild-type and mutantα-Syn transgenic mice were wildly used in PD researches.In this part of work,the behavior,TH and TUNEL immunohistochemistry and the level of DA-related neurotransmitters were evaluated in a-Syn transgenic mice and control in different age.The behavioral tests revealed that no signigicant difference was found between 3-month-old transgenic mice and control,however,9-month-old transgenic mice presented a declined movement ability.Meanwhile,the TH and TUNEL immunostainning assays implied a decreased tendency of TH-positive dopaminergic neurons in SN and an increased trend of TUNEL-positive cells in both SN and STR of 9-month-old transgenic mice compared with control.In addition,the striatal level of HVA were decreased in 3-month-old transgenic mice,furthermore,DA and its metabolites,DOPAC and HVA, were consistently reduced in 9-month-old transgenic mice.These data implied that the 9-month-old transgenic mice gradually appeared the PD-like pathological changes.To further elucidate the correlation between the status ofα-Syn overexpression and phosphorylation and the proteasome function,we examed the proteins expression ofα-Syn, CKⅡand 19S ATPase Rpt6 and the enzymatic activity of 20S proteasome in SN and STR of transgenic mice and control.Data showed thatα-Syn was continously overexpressed in both SN and STR of 3-and 9-month-old transgenic mice compared with control. Meanwhile,the level of CKⅡwas significantly increased in STR of 3-and 9-month-old mice,whereas was attenuated in SN of 9-month-old mice.The distinct changes of CKII in SN and STR might lead to different status ofα-Syn phosphorylation,which would produce various physiopathological alterations.Moreover,the level of 19S ATPase Rpt6 was only decreased in SN of 9-month-old transgenic mice,while the 20S activity was not changed in both 3-and 9-month-old transgenic mice.The results indicated that high level ofα-Syn would prefer to affect the expression of 19S proteasome subunit rather than changed 20S activity.To gain insight into the protein targets involved inα-Syn(A53T) toxicity,the comparative proteomics investigation was carried out for analyzing the protein profiles in SN and STR between transgenic mice and age-matched control.The proteomics study of SN showed that 9 differential spots were viewed in 3-month-old transgenic mice,2 of which were dramatically elevated and the other 7 were newly appeared spots.Besides these 9 differential spots,in 9-month-old transgenic mice, another 4 fresh differential spots were yielded and presented declined abundances or almostly disappeared.The proteomics study of STR indicated that 9 differential spots were presented in 3-month-old transgenic mice,1 of which was significantly increased,1 of which was predominantly deficit and the other 7 were newly appeared spots.Besides these 9 differential spots,in 9-month-old transgenic mice,another 2 differential spots were discovered,and one spot was decreased while the other one was increased.In addition,we compared 13 differential spots in SN with 11 differential spots in STR of 9-month-old mice and found an overlap of 8 spots.The 8 spots were altered in 3-month-old transgenic mice without regional specificity.Furthermore,those differential spots which only presented in SN or STR of 9-month-old transgenic mice were thought to be more valuable for PD investigation.PartⅢ.Comparative Proteomic Study on Serum of Parkinson’s Disease Patients and Age-Matched Normal PeopleThe life quality of people suffered with PD is from bad to worse.However,no means exist to evaluate or predict the PD in the early-stage betbre the motor symptom onset. Moreover,the only current method for diagnosis of this disease is through physical examination and L-Dopa test,and no serum biochemical markers exist.Hence,to make efforts for PD prognosis and further advance a comprehensive understanding of PD pathology,we conducted a sera based comparative proteome study of healthy and diseased human in this part of work.The presence of high-abundant proteins(such as albumin,IgG,antitrypsin,etc.) in human serum always brings difficulty in separating and visualizing the meaningful low-abundant protein markers by 2DE.To address this problem,the Agilent Human 14 Multiple Affinity Removal System combinding with HPLC system were employed. Specfic removal of 14 high-abundant proteins depletes approximantly 94%of total protein mass of human serum.The low-abundant proteins in the flow-through fractions can be studied.Then,a comparative analysis of serum proteomes with low-abundant proteins was performed to discover proteins associated with sporadic PD.2DE patterns of human sera from 10 PD patients and 10 normal subjects were analyzed.The differentially expressed spots were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF-TOF-MS).In total,more than 1000 spots were separated in low-abundant protein fraction of human serum.The gross number of differentially regulated proteins in PD was 16,2 of which were significantly increased in PD samples and 14 of which were decreased in different levels.Among them,7 spots were successfully identified as five known proteins, which included thrombin,Apolipoprotein A-I,Apolipoprotein A-IV,Apolipoprotein L-! and Complement 2.These proteins were consistently decreased in PD patients.The results point towards a heterogeneous aetiopathogenesis of the disease,including alterations of Apolipoprotein-related lipid metabolism,alterations of the classical pathway involved in complement system as well as alterations of proteins involved in thrombin-induced coagulation system.更多还原