【作者】 李超；

【导师】 李新钢； 乐卫东；

【作者基本信息】 山东大学 ， 外科学， 2010， 博士

【摘要】 【研究背景与目的】一、帕金森病的简介及发病机制1817年,英国学者James Parkinson首先发现一种在老年人中常见的中枢神经系统变性疾病,临床表现以运动徐缓,静止性震颤,僵直等运动系统障碍为主,因此命名为帕金森病(Parkinson’s disease, PD)。此后,人们通过大量研究逐渐对帕金森病的流行病学特征、临床特点、发病机制、疾病转归及治疗有了更深入系统的认识。帕金森病的主要病理学特征是：中脑黑质致密部(substantia nigra pars compacta, SNpc)为主的多巴胺(dopamine, DA)神经元的进行性丢失以及胞浆内包涵体物质的出现,称为路易小体(Lewy bodies, LBs)。虽然在帕金森疾病中发生的神经变性机制尚未明了,但是广大学者认为帕金森病是一种由多个因素所导致的综合性疾病,这些因素包括遗传因素中a-synuclein、parkin、UCH-L1、PINK1、DJ-1、LRRK2等基因的影响,环境因素如除草剂、鱼藤酮、MPTP等化学毒物的作用,人口老龄化,现代生活习惯及其它因素。这些影响因素可能会引起线粒体功能障碍,氧化应激,炎症反应,兴奋性中毒(图1),从而导致帕金森病的发生。近年来,经过许多研究证实了异常聚集的错构蛋白在帕金森发病机制中的作用,这个领域的研究越来越受到重视。泛素蛋白酶体系统(ubiquitin-proteasome system)是清除这些错构蛋白的主要途径,泛素蛋白酶体系统的功能障碍与帕金森的关系成为帕金森病的研究热点。与此同时,在帕金森发病机制研究中,越来越多的研究者认识到自噬溶酶体通路(autophagy-lysosome pathway)对于修复和清除异常聚集蛋白所起到的重要作用二、多巴胺受体激动剂在帕金森病治疗中的作用帕金森病的治疗以对症治疗和恢复纹状体内多巴胺神经元的功能为主。由于具有良好的耐受性,易于吸收以及经济实惠的特点,左旋多巴一直以来是对症治疗帕金森疾病症状的首选药物。但是它的副作用也很明显,尤其是长期使用会造成严重并发症,例如症状波动,开关现象和运动障碍。除此之外,左旋多巴还对多巴胺神经元有毒性作用,并会导致具有细胞毒性的自由基的生成。这样会破坏残存的多巴胺神经元并加剧帕金森疾病的恶化程度。现在作为左旋多巴的辅助药品,多巴胺受体激动剂在治疗帕金森症状中起到了更为重要的作用,它们通过模拟内源性的神经递质,直接作用于多巴胺受体,使其激活,起到类似多巴胺的作用(图2)。与左旋多巴合用时,可以减少左旋多巴的用量,预防和缓解长期服用左旋多巴所引起的运动障碍。多巴胺受体激动剂有两种亚型：麦角类和非麦角类激动剂。后者包括prarmipexole和ropinirole,比前者更具效用。这种新型的多巴胺受体激动剂具有高度选择性和副作用较少的优点。三、帕金森疾病动物模型的构建和应用为了直观比较和量化检测帕金森病的表现症状和实验药物的治疗效果,我们需要成功构建帕金森病动物模型进行体内科学实验研究(图3)。神经毒性药物,例如1-甲基-4-苯基-1,2,3,6-四氢吡啶(MPTP),导致的帕金森病动物模型对于研究该病具有重要作用,这些模型可以帮助阐明针对黑质区的多巴胺神经元损伤和退行性病变的进展过程。MPTP会产生类似于特发性帕金森病的改变,例如临床的,生化的,神经病理学的改变,并牵涉到诸多细胞死亡的机制,例如对线粒体电子传递链中线粒体复合物Ⅰ的抑制,导致其缺失；毒性分子活性氧的产生：炎症反应；凋亡；自噬等。接触过MPTP的人或灵长类动物会产生类似于帕金森病的症状,并造成多巴胺神经元的变性,对于我们研究帕金森病提供了较为可靠的动物模型。猴的MPTP模型被认为是评估帕金森疾病新的研究策略和治疗方法的金标准,但是缺乏2种帕金森病的特有属性：一是与单胺能神经核内的神经元丢失不一致；二是尚未大量证实神经细胞内路易小体的出现。基于实际考虑,猴MPTP模型不是一种普遍的应用于多巴胺神经变性机制研究的动物模型,而使用MPTP诱导的小鼠模型对于研究以下3种可能导致帕金森疾病的功能障碍：氧化应激,线粒体缺陷,蛋白异常聚集,具有较好的适用性。小鼠模型不但易于构建,而且成本较低,故一直以来是被最广泛应用的帕金森动物模型。但是小鼠模型仍然存在一定的局限性：首先,小鼠对于MPTP的敏感性较差,故需要大剂量的MPTP才能产生足够明显的症状,这就带来了增大环境污染的问题。其次,小鼠的帕金森体征不如猴的典型。最后,黑质纹状体通路受影响的程度取决于MPTP的剂量和注射程序。泛素蛋白酶体系统的主要作用是清除细胞内正常或异常聚集的蛋白。lactacystin是一种高选择性的泛素蛋白酶体抑制剂,通过抑制或破坏泛素蛋白酶体系统的作用,能导致聚集的蛋白不能降解并最终导致神经元的功能障碍和死亡。在前期实验中,我们已经证实直接向内侧前脑束进行单侧或双侧立体定向注射lactacystin会产生类似于帕金森病的神经病理特性：严重的多巴胺神经元丧失以及包涵体样蛋白的聚集。此外,我们还发现纹状体内的多巴胺及其代谢产物高香草酸(HVA)及二羟苯乙酸(DOPAC)的含量显著降低,而5-羟色胺(5-HT)、5-羟吲哚乙酸(5-HIAA)的含量去变化不明显。这说明lactacystin是高度选择性的作用于黑质纹状体多巴胺通路。四、小结本论文的实验是在山东大学与美国Baylor College of Medicine神经科学研究所进行博士生联合培养期间完成的。本文将充分、严谨的研究结果与帕金森病的研究现状相结合。通过在不同类型的帕金森小鼠模型上研究多巴胺受体激动剂对于神经退行性疾病的保护作用,旨在通过不同的角度来论证多巴胺受体激动剂神经保护作用的机制,并从一些新的角度分析,提出新的观点,以期能为将来神经外科与其他相关专业合作研究和治疗中枢神经系统的疾病提供理论依据和实验基础。第一部分新型多巴胺受体D3激动剂-D264对两种类型帕金森病动物模型的神经保护作用【研究目的】建立两种不同类型的帕金森疾病(Parkinson’s disease, PD)小鼠(C57BL／6)动物模型：MPTP(1-甲基-4-苯基-1,2,3,6-四氢吡啶)模型及lactacystin模型,观察不同剂量新型多巴胺受体D3激动剂-D264对帕金森疾病小鼠模型的作用,以及特异性D3受体拮抗剂U99194干预D264对D3受体的激动作用,联合研究行为学改变(疲劳转棒测试,自主活动测试,爬杆测试),病理学特征(中脑黑质致密区多巴胺神经元),中枢神经系统内生化改变(多巴胺、5-羟色胺及其代谢产物),蛋白酶体活动的改变,脑源性神经营养因子(Brain derived neurotrophic factor, BDNF)和胶质源性神经营养因子(Glial derived neurotrophic factor, GDNF)的改变。分析上述指标,明确这两种帕金森疾病小鼠模型成功构建,以及D264的神经保护作用及机制。【研究方法】12周龄的雄性C57BL／6小鼠随机分成12个组：对照组1(MPTP),对照组2(lactacystin), MPTP组,lactacystin组,低剂量D264(1 mg／kg)预处理MPTP组,高剂量D264(5 mg／kg)预处理MPTP组,低剂量D264(1 mg／kg)预处理lactacystin组,高剂量D264(5 mg／kg)预处理lactacystin组,独立D264组1(MPTP),独立D264组2(lactacystin),联合使用U99194与D264预处理MPTP组,联合使用U99194与D264预处理lactacystin组。在对小鼠进行MPTP和lactacytin处理的前一天以及处理过后的每周进行行为学测试,并分别于第14天(MPTP组)和第21天(lactacystin组)处死小鼠,取出小鼠的脑组织分别进行低温保存和免疫组化处理,测定中脑黑质酪氨酸羟化酶(TH)阳性细胞数,多巴胺(dopamine, DA)、5-羟色胺(5-HT)及其代谢产物的含量,蛋白酶体活动以及BDNF和GDNF的水平。【结果】(1)与对照组相比,MPTP和lactacystin处理的小鼠活动能力均大幅降低(P<0.01),TH阳性细胞数降低51.9％和47.9％(P<0.01),DA和5-HT减少87.3％和70.9%(P<0.01), lactacystin抑制蛋白酶体活动48.5％(P<0.01),并降低了BDNF和GDNF的表达水平(P<0.01)。(2)与MPTP和lactacystin处理组相比,D264预处理组分别提高了小鼠的活动能力,TH阳性细胞数,DA和5-HT的含量,蛋白酶体活动以及BDNF和GDNF的表达水平,具有明显的神经保护作用。(3)与D264预处理组相比,U99194降低了D264的保护作用。【结论】(1)MPTP和lactacystin的小鼠模型可以表现出大部分帕金森疾病的特点,可以作为本研究中的帕金森疾病动物模型。(2)新型多巴胺受体D3激动剂-D264能够减轻MPTP和lactacystin对于小鼠模型的损害,具有明显的保护作用。(3)特异性的D3受体拮抗剂U99194可以部分减弱D264的保护作用,说明D264是通过调节D3受体起到保护作用的。第二部分Pramipexole在蛋白酶体抑制剂致帕金森动物模型神经损伤中的保护机制研究【研究目的】帕金森疾病是以中脑黑质区多巴胺神经元的进展性减少和蛋白质的异常聚集形成路易小体为主要特征。导致帕金森疾病中神经退行性病变的原因目前尚未明确。有学者认为泛素-蛋白酶体系统的障碍在帕金森的发病中起重要作用。我们的研究旨在探讨pramipexole在蛋白酶体抑制导致的帕金森小鼠模型中的神经保护作用,以及自噬对其损伤多巴胺神经元的可能保护机制。【研究方法】10-12周龄的雄性C57BL／6小鼠随机分成6个组：对照组,lactacystin组,独立pramipexole组,低剂量pramipexole (0.1 mg/kg)预处理lactacystin组,高剂量pramipexole (0.5 mg/kg)预处理lacatcystin组,联合使用U99194与pramipexole预处理lactacystin组。在立体定向(小鼠的中前脑束)注射lactacystin的7天前使用pramipexole预处理小鼠,并持续4周。第28天处死小鼠,取出小鼠的脑组织分别进行低温保存和免疫组化处理,测定中脑黑质酪氨酸羟化酶(TH)阳性细胞数,多巴胺(dopamine,DA)、5-羟色胺(5-HT)及其代谢产物的含量,蛋白酶体活动,脑源性神经营养因子(Brain derived neurotrophic factor, BDNF)和胶质源性神经营养因子(Glial derived neurotrophic factor, GDNF)的水平以及在电镜下观察自噬活动。【结果】我们发现：pramipexole能显著改善小鼠的行为障碍,减少多巴胺神经元的丧失,提高蛋白酶体的活动。我们还指出pramipexole能增加BDNF和GDNF的水平。此外我们还观察到自噬活动在pramipexole处理小鼠中的增强。与pramipexole预处理组相比,U99194降低了D264的保护作用。【结论】这些结果表明,(1)立体定向注射lactacystin的小鼠模型可以表现出大部分帕金森疾病的特点,可以作为本研究中的帕金森疾病动物模型。(2)pramipexole在蛋白酶体抑制剂致帕金森动物模型神经损伤的神经保护机制可能通过多种分子通路来完成。(3)自噬-溶酶体通路的障碍也可能导致错构蛋白的异常聚集并成为导致神经退行性疾病的原因,包括帕金森病。本研究的结果为大家提供了一个崭新的思路来探讨治疗帕金森疾病的机制与方法。更多还原

【Abstract】 1. The Pathogenesis of Parkinson’s diseaseParkinson’s disease (PD) is named after James Parkinson, who made a first and detailed description of the disease in the year 1817. PD is a neurodegenerative disorder, usually affects old people at the age of 60, clinically characterized by bradykinesia, rigidity, resting tremor, and a variety of other motor.The pathological hallmark of PD is progressive loss of dopamine (DA) neurons in the substantia nigra pars compacta (SNpc), and the presence of cytoplasmic inclusions, termed Lewy bodies. There is growing consensus among parkinsonologists, that PD is probably not a homogenous disease, but a syndrome of different disorders, caused by genetic, environmental, aging, and other etiologies. Although the mechanism of neurodegeneration in PD is not clear, the pathogenesis of PD has been postulated to result from a complex interaction between environmental and genetic factors leading to mitochondrial dysfunction, oxidative stress, inflammation, and excitotoxicity, eventually leading to nigral DAergic neuron degeneration.Impaired degradation of misfolded and aggregated proteins is being increasingly recognized to play an important role in the pathogenesis of PD. Dysfunction of the ubiquitin-proteasome system (UPS) has been already strongly implicated in the pathogenesis of this disease and growing interest has been shown in identifying the role of autophagy-lysosome pathway (ALP) in repair and removal of misfolded proteins.2. The role of dopamine agonist in the treatment of Parkinson’s diseaseCurrent drug therapy in Parkinson’s disease is symptomatic and primarily aimed at restoring dopaminergic function in the striatum. Levodopa is still the most effective symptomatic treatment. Levodopa is well tolerated, easy to administer, and inexpensive. However, long term use is associated with disabling complications such as fluctuating motor responses and dyskinesias. Besides, it is reported that levodopa is toxic in vitro to dopaminergic neurons and in vivo its use could lead to formation of cytotoxic free radicals when exogenous dopamine is decarboxylated, these would cause damage to surviving dopaminergic neurons and potentially exacerbate the disease . Dopamine agonists were introduced as an adjunct to levodopa treatment and play an important role in antiparkinsonian by acting directly on dopamine receptors and imitating the endogenous neurotransmitter. There are two subclasses of dopamine agonists:ergoline and non-ergoline agonists. The non-ergoline agonists such as pramipexole and ropinirole are more effective than the ergoline agonist, the reason may be that pramipexole is a selective D3 dopamine receptor agonist and with less side effects.3. The animal models of Parkinson’s disease:construction and applicationThe successful application of animal models to the study of Parkinson’s disease has advanced our knowledge of Parkinson’s disease. Neurotoxin-based models have been important in clarifying aspects of the disease, such as selective vulnerability of substantia nigra dopaminergic neurons to the degenerative process. It produces clinical, biochemical, and neuropathological changes of those occurring in idiopathic PD. Several cell death mechanisms have been implicated in MPTP toxicity, including an inhibition of complex I in the mitochondrial electron transport chain, the generation of reactive oxygen species (ROS), inflammation, apoptosis, and autophagia.Exposure of humans to MPTP causes a syndrome that mimics the core neurological symptoms and relatively selective dopaminergic neurodegeneration of PD. Although the monkey MPTP model is the gold standard for the assessment of novel strategies and agents for the treatment of PD symptoms, the monkey MPTP model does not include two characteristic features of PD. First, neurons are not consistently lost from other monaminergic nuclei, a typical feature of PD . Second, although intraneuronal inclusions resembling LBs have been described classical LBs have not been demonstrated convincingly in the brains of MPTP-intoxicated patients or monkeys. Because of practical considerations, MPTP monkeys have not generally been used to explore the molecular mechanisms of dopaminergic neurodegeneration; the MPTP mouse model is typically used for such studies. MPTP toxicity in mice has become the most commonly used animal model of PD for both technical and financial reasons. These studies have focused on three types of dysfunction that may be important in the pathogenesis of PD:oxidative stress, mitochondrial defect, and abnormal protein aggregation.However, several problems need to be emphasized. First, mice are much less sensitive to MPTP than monkeys; thus, much higher doses are required to produce significant SNpc damage in this animal species, presenting a far greater hazardous situation. Second, in contrast to the situation in monkeys, mice treated with MPTP do not develop parkinsonism. Third, the magnitude of nigrostriatal damage depends on the dose and dosing schedule.Lactacystin is a selective inhibitor of ubiquitin proteasome system (UPS), which is responsible for the degradation of normal processed cellular proteins as well as misfolded proteins, it is believed that inhibition or impairment of UPS could lead to the accumulation of toxic proteins and ultimately result in neuronal dysfunction and death. In our lab’s previous study, we have showed that direct stereotactic injection with proteasome inhibitor-lactacystin, into the unilateral or bilateral mouse medial forebrain bundle (MFB) can cause severe nigral cell degeneration and inclusion body-like protein aggregate formation, which resemble some neuropathological features of PD. In addition, we have found that, in the lactacystin-injected mice, the striatal levels of dopamine and its metabolites, DOPAC and HVA, were significantly reduced, whereas the levels of 5-HT and 5-HIAA were unchanged, indicating that the toxicity of lactacystin might be relatively selective to nigro-striatal dopaminergic pathway.4. ConclusionThe thesis is based on my study during the Joint training of Shandong University and Baylor College of Medicine.Our study on the neuroprotective property of dopamine receptor in two kinds of Parkinson’s disease animal models has not only improved the knowledge on neurodegeneration disease but also focus on novel therapeutic strategies. It may provide us further communication between neurology and neurosurgery in different fields of central nervous system diseases. PART ONE The neuroprotective property of novel D3 dopamine receptor preferring agonist D-264 in two kinds of Parkinson’s disease animal modelsObjectivesThe animal models of Parkinson’s disease, induced by MPTP and lactacystin, were constructed. We used a combination study, including a blocking experiment with D3 receptor antagonist U99194, to better understand the mechanism of neuroprotection of selective D3 receptor agonist-D264. We observed the changes of behavioral performances (rotarod, locomotion, pole), the number of dopamine neurons in substantia nigra area, the concentration of dopamine and its metabolites, proteasomal activity, the levels of BDNF and GDNF. After analyzing these changes, we get the conclusions that the mouse models of Parkinson’s disease were successfully constructed and the neuroprotective property of D264 was well evaluated.MethodsMale C57BL/6 mice, aged 12 weeks, were randomly assigned into twelve groups:control for MPTP, control for lactacystin, MPTP, lactacystin, D-264 Low Dose (1 mg/kg)+MPTP, D-264 High Dose (5 mg/kg)+MPTP, D-264 Low Dose+lactacystin, D-264 High Dose+lactacystin, D-264 non-lesioned control for MPTP, D-264 non-lesioned control for lactacystin, U99194+D-264 high dose+MPTP, and U99194+D-264 high dose+lactacystin, respectively. The mice were examined 1 day prior to MPTP and lactacystin treatment as a base level and then every week. The mice were sacrificed on day 14 (MPTP) and day 21 (lactacystin), the brains were immediately removed and stored at-80℃until analysis.Results(1) Compared to the control, behavioral performances, the number of TH positive cells in substantia nigra area, the concentration of dopamine and its metabolites, proteasomal activity, the levels of BDNF and GDNF were significantly reduced in MPTP and lactacystin treated mice.(2) Compare to the mice treated with MPTP and lactacystin, pretreatment with D264 at low and high dose significantly attenuated behavioral impairment, showed neuroprotection against both MPTP and lactacystin induced DA neuron loss and depletion of DA and its metabolites in the SN, alleviated lactacystin induced proteasomal inhibition, and increased the BDNF and GDNF levels in MPTP and lactacystin lesioned mice.(3) Pretreatment with U99194 partially but significantly altered the neuroprotective effect of selective D3 agonist-D-264.Conclusion(1) The mouse model induced by MPTP and lactacystin replicated many of the features of PD and were expected to be suitable for our study.(2) D-264 can prevent neurodegeneration induced by the selective neurotoxin MPTP and UPS inhibitor lactacystin, be potentially served as a both symptomatic and neuroprotective treatment agent for PD.(3) Pretreatment with D3 receptor antagonist U99194 significantly altered the effect of neuroprotection conferred by D-264, which showed that the effect of D-264 was mediated partly or completely by D3 receptor. PART TWO Pramipexole in proteasome inhibition induced animal model of Parkinson’s disease:Underlying neuroprotective mechanismsObjectivesParkinson’s disease (PD) is characterized by the progressive loss of nigral dopamine (DA) neurons in substantia nigra (SN) area and the accumulation of inclusion bodies, known as Lewy bodies (LBs). The cause of the neurodegenerative process in PD remains unclear. Ubiquitin-proteasome system (UPS) impairment has been proposed to play an important role in the pathogenesis of PD. In the present study, we attempt to better evaluate the mechanism of neuroprotection of pramipexole (PPX) in a mouse model of DA neuron degeneration induced by UPS impairment, in addition to test the possible mechanisms of autophagy in prevention of the proteasome inhibition-induced DA neuron degeneration.MethodsMale C57BL/6 mice at the age of 10-12 weeks were were randomly assigned into six groups:vehicle control, lactacystin, PPX alone, PPX Low Dose (01 mg/kg)+lactacystin, PPX High Dose (0.5 mg/kg)+lactacystin, and U99194+PPX High Dose+lactacystin, respectively. Mice treated with PPX (low dose 0.1 mg/kg or high dose 0.5 mg/kg, i.p, twice a day) started 7 days before, and continued after microinjection of proteasome inhibitor lactacystin in the medial forebrain bundle (MFB) for a total 4 weeks. Animal behaviors, pathological and biochemical assays were performed to determine the neuroprotective effect of PPX.ResultsWe found that administration with PPX significantly improved behavioral performance, attenuated DA neuron loss and striatal DA reduction, and alleviated proteasomal inhibition and microglial activation in the SN of lactacystin-lesioned mice. We also documented that PPX can increase the levels of brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) in the lactacystin-lesioned mice. In addition, we demonstrated an activation of autophagy in PPX treated mice. Furthermore, pretreatment with D3 receptor antagonist U99194 significantly altered the neuroprotection conferred by PPX.ConclusionOur study indicates that, (1) The mouse model microinjected by lactacystin replicated many of the features of PD and were expected to be suitable for our study. (2) Multiple molecular pathways may be attributed to the neuroprotective effects of PPX in the UPS impairment induced animal model of PD. (3) Dysfunction in the autophagy-lysosome pathway (ALP) may cause aggregation of misfolding proteins and attribute to the neurodegenerative diseases, including Parkinson’s disease. The results of the study may provide us new insight into the potential novel mechanisms for the treatment of PD.更多还原