【作者】 胡军；

【导师】 范卫民；

【作者基本信息】 南京医科大学 ， 外科学， 2012， 博士

【摘要】 帕金森病（Parkinson’s disease, PD）是老年人群中发病率很高的一种神经系统退行性疾病，随着我国逐渐步入老龄化社会，预期发病率呈逐年增高的趋势。目前认为PD是由于黑质（Substantia nigra pars compacta, SNpc）多巴胺（Dopamine,DA）神经元进行性变性、死亡导致纹状体内DA绝对含量减少所致，但对其确切的病因机制目前仍不清楚。流行病学统计显示，吸烟者患PD和老年痴呆症（Alzheimer’s disease,AD）的概率远低于不吸烟者，大量研究表明烟碱型乙酰胆碱受体（nicotinic acetylcholine receptors, nAChRs）参与了其中的神经保护机制，发展以nAChRs为靶标、调节其功能的药物是目前PD等神经退行性疾病治疗学研究的热点之一。nAChRs是介导突触间快速信号传递的配体门控的离子通道蛋白。nAChRs大体可分为中枢型和外周型两类。中枢型nAChRs包括脑nAChRs和神经元nAChRs；外周型nAChRs包括成熟型ε-nAChRs和胚胎型γ-nAChRs。nAChRs由不同的亚单位构成，至今，已确认的16种nAChRs亚单位分别为α1-7，α9-10，β1-4，δ，ε和γ，不同亚单位的不同的组合表现出不同的药理学和生理学特征。中枢神经系统中分布最多的是α4β2和α7两种受体亚型。其中α7-nAChRs是较为特殊的一种亚型，它由5个相同的亚基构成．对钙离子有相当高的通透性，能调节钙的活化及递质乙酰胆碱的释放，近年来，大量研究发现α7-nAChRs与PD等神经退行性疾病中神经炎性机制密切相关，α7-nAChRs在“胆碱能抗炎通路”中发挥关键作用，有望成为包括PD在内的神经退行性疾病的新型治疗药物靶点。星形胶质细胞是脑内数量众多的一大类细胞群体，其在维持神经电活动正常环境、调节神经递质代谢、保护神经元及促神经再生和修复过程中发挥重要的作用。大量研究表明，星形胶质细胞功能障碍能够影响神经元的存活，与PD等神经退行性疾病的发生发展密切相关。在PD患者和MPTP造模动物脑内黑质区均存在星形胶质细胞活化，活化的星形胶质细胞产生大量的活性氧和炎性因子，这些毒性物质促发神经元变性，促进了PD的发生发展。因此，靶向于星形胶质细胞功能的调节已经成为PD等神经退行性疾病神经保护的重要策略。本文工作第一部分首先应用MPTP建立PD小鼠模型，系统研究脑内nAChRs主要亚基α7及α4和β2在小鼠黑质、海马、前皮质和纹状体脑区的表达改变，阐明nAChRs与PD发生、发展的相关性。第二部分继续以MPTP制备的PD模型小鼠为实验对象，整体水平研究α7-nAChRs的神经保护作用及其机制，同时，本部分工作以原代培养的小鼠星形胶质细胞为研究对象，探讨α7-nAChRs对MPP⁺或LPS诱导的星形胶质细胞活化及其生成TNF-α致炎因子的调节作用；及调节星形胶质细胞活化的分子信号传导机制，从而探索调控星形胶质细胞活化的新靶标，进一步阐明α7-nAChRs神经保护作用的抗炎机制。此外，脑内多巴胺神经元主要分布于中脑黑质致密部和腹侧被盖区（Ventraltegmental area, VTA）。前者通过黑质-纹状体通路调节机体的运动功能，后者通过中脑-边缘系统，中脑-皮层通路广泛投射到前额叶、颞叶、扣带回、海马、杏仁体等脑区，与认知、记忆等神经活动有关，但二者的投射部位有部分重叠，提示PD的病理学变化可能有中脑腹侧被盖区多巴胺神经元的改变。我们前期的研究也证实PD模型小鼠中脑VTA区多巴胺神经元较对照组有不同程度的变性、坏死。对中脑SNpc和VTA多巴胺神经元上nAChRs亚型（如α7-nAChRs）的功能研究将深化α7-nAChRs在PD中枢神经系统功能的认识，丰富α7-nAChRs研究的神经生物学内容，因此，第三部分我们通过膜片钳技术就VTA多巴胺神经元表达的α7-nAChRs等烟碱受体亚型进行生理药理学功能分类，为探索α7烟碱乙酰胆碱受体的神经保护作用及其机制积累必要的实验与学术基础。第一部分烟碱受体亚基在MPTP急性PD小鼠相关脑区的表达变化目的：研究脑内nAChRs主要亚基α7及α4和β2在小鼠黑质、海马、前皮质和纹状体等脑区的表达改变，探索nAChRs与PD发生的相关性。方法：MPTP模型组鼠腹腔注射MPTP-HCl，一天四次，每次20mg·kg^-1，间隔2h，总量为80mg·kg^-1。最后一次给药后3h、1d、2d和7d时处死小鼠进行实验。处死小鼠前进行行为学检测；免疫组织化学法检测脑黑质区DA能神经元的存活（TH染色）；同时检测黑质部位星形胶质细胞的活化（GFAP染色）；RT-PCR法检测脑内nAChRs主要亚基α7及α4和β2在小鼠黑质、海马、前皮质和纹状体等脑区的mRNA表达改变。结果：α7亚基在MPTP急性小鼠模型黑质、纹状体脑区3h明显增高，1天后α7、α4和β2亚基与对照组相比均显著性增高，但2d和7d时，α7、α4和β2亚基恢复至对照组水平，而海马、皮质区，并没有发现这种短暂的升高现象，但模型组海马区α7、α4和β2亚基mRNA在注射MPTP7d后明显降低。急性给予MPTP后小鼠SNpc区TH阳性细胞计数在一天后即显著降低，7d时这种降低最为明显。而星形胶质细胞则在黑质区建模一天后明显增多活化，7d时这种活化最为明显。小鼠行为学的改变似乎与黑质致密部多巴胺神经元的损伤无明显联系，而似与nAChRs α7、α4和β2亚基在小鼠黑质和纹状体脑区的mRNA表达时程规律有关，急性MPTP注射后3h，小鼠的行为学改变最为明显，建模后第7d，小鼠行为学部分恢复。结论：脑内nAChRs主要亚基α7及α4和β2在小鼠黑质、海马、前皮质和纹状体等脑区的表达改变，为研究胆碱能信号通路与PD病理机制的相关性打下了实验基础。第二部分α7-nAChRs对MPTP致PD模型小鼠的神经保护作用及其机制研究目的：研究α7-nAChRs对MPTP制备的PD模型小鼠的神经保护作用及其机制。在鉴证原代培养的小鼠星形胶质细胞表达α7-nAChRs的基础上，研究α7-nAChRs调制MPP+和LPS诱导的星形胶质细胞活化的作用及其机制。方法：小鼠分别给予腹腔注射烟碱(0.25或0.5mg·kg^-1)持续一周，每天五次，于第八天，B、C、D、E组小鼠腹腔注射MPTP-HCl，一天四次，每次20mg·kg^-1，间隔2h，总量为80mg·kg^-1。造模当天，继续给予烟碱，剂量、次数不变，前四次在给予MPTP前30分钟给药，第九天继续腹腔注射烟碱(0.25或0.5mg·kg^-1)持续一周，每天五次，对照组小鼠给予相同剂量的生理盐水。α7-nAChRs特异性的阻断剂MLA在给予烟碱前20-30分钟给药，一天两次。免疫组织化学法检测脑SNpc区DA能神经元的存活（TH染色），同时检测黑质部位星形胶质细胞的活化（GFAP染色）。应用RT-PCR和Western blotting检测原代培养的小鼠星形胶质细胞上α7-nAChRs的表达；ELISA法检测细胞培养液中TNF-α的含量；Western blotting检测星形胶质细胞内Erk1/2和p38磷酸化水平。结果：我们发现Nicotine可明显改善PD模型小鼠的行为学变化，减少SNpc区多巴胺神经元的损伤及黑质区星形胶质细胞的活化，其机制与Nicotine激活α7-nAChRs有关，应用原代培养的星形胶质细胞进一步深入探讨激活α7-nAChRs的神经保护机制，发现MPP+和LPS可显著诱导星形胶质细胞活化，致炎因子TNF-α生成增加，Nicotine预处理能显著抑制星形胶质细胞的活化，抑制TNF-α的释放，机制与Erk1/2和p38MAPK信号通路有关，选择性α7-nAChRs阻断剂MLA可逆转Nicotine的上述效应。结论：Nicotine能够显著缓解MPTP诱导的小鼠帕金森样症状，促进黑质多巴胺能神经元存活，其神经保护作用机制与通过α7-nAChRs抑制星形胶质细胞活化介导的神经炎性反应相关。第三部分α7-nAChRs在中脑腹侧被盖区多巴胺神经元上的电生理学机制研究目的：研究VTA多巴胺神经元表达的α7-nAChRs等烟碱受体亚型的功能特点。方法：应用多巴胺神经元急性分离技术、穿孔膜片钳技术和U管快速给药系统，观察VTA多巴胺神经元表达的α7-nAChRs等烟碱受体亚型的功能特点。结果：根据全细胞电流动力学及药理学特点，我们将VTA区nAChRs分为ID、IID、IIID型受体。动力学分析揭示ID和IID型受体可被快速激活及衰减，而IIID型受体则相对较慢。药理学分析揭示ID型受体介导的电流对α4β2-nAChRs特异的激动剂RJR-2403和拮抗剂DHβE敏感，IID型受体介导的电流对α7-nAChRs特异的激动剂choline和拮抗剂MLA敏感，而IIID型受体介导的电流对含β4的nAChRs特异的激动剂cytisine和拮抗剂MEC敏感。IID型受体激动剂EC50明显高于ID和IIID型受体，表明其对激动剂的亲合力较低。结论：VTA多巴胺神经元表达多种不同的功能性nAChRs，有趣的是，每个多巴胺神经元似乎只有一种nAChRs亚型，该研究深化了α7-nAChRs在PD中枢神经系统功能的认识，丰富了α7-nAChRs研究的神经生物学内容。综上所述，本文工作的主要创新之处在于：1.揭示了Nicotine对MPTP制备的PD模型鼠具有显著的神经保护作用，其机制与抑制星形胶质细胞活化及神经炎性反应相关，α7-nAChRs在其中发挥关键作用，为PD的治疗提供了新的思路，为研发靶向于α7-nAChRs的抗神经炎性保护剂提供了新的理论依据。2.通过膜片钳技术就VTA多巴胺神经元表达的α7-nAChRs等烟碱受体亚型进行功能学分类，为探索α7-nAChRs的神经保护作用及其机制积累必要的实验与学术基础。更多还原

【Abstract】 Parkinson’s disease （PD）, a common neurodegenerative disorder, is characterizedby both motor symptoms （including resting tremor, cogwheel rigidity, bradykinesiaand loss of postural reflex） and disturbed cognitive function. These abnormalities areattributed to the depletion of dopamine （DA） in the brain that results from thedramatic loss of dopaminergic neurons in the substantia nigra pars compacta （SNpc）and their projecting nerve fibers in the striatum. According to the pathomechanismgoverning dopaminergic loss, various hypotheses, including excitotoxicity,mitochondrial dysfunction, oxidative stress and inflammation have been proposed tobe involved in the pathogenesis of PD. Therapeutic strategies derived from these,such as L-DOPA administration, are essentially symptomatical or accompanied withserious side effects, and cannot stop the progressive loss of dopaminergic neurons.Hence, many studies are focused on searching for new potential therapeutic targetsand agents for the treatment of PD.Epidemiologic studies show that the prevalence of Parkinson’s disease （PD） islower in smokers than in nonsmokers. Nicotine, a potent agonist of nicotinicacetylcholine receptors （nAChRs）, excites midbrain dopaminergic neurons and thismay contribute to the anti-parkinsonian effects. An understanding of the molecularbasis for the roles of nAChRs on the reduced occurrence of PD in tobacco users iscrucial for the development of intervention strategies to reduce or halt diseaseprogression.Neuronal nAChRs are transmitter-gated ion channels with nine different αsubunits （α2-α10） and three β subunits （β2-β4） been cloned to date. Different combinations of α and β subunits, or of α subunits alone, produce various receptorsubtypes exhibiting different physiological, pharmacological and anatomicalproperties. The α4β2and α7nAChR subtypes are the most abundant nicotinicreceptor subtypes in the mammalian brain. Recent studies suggest the implications ofthe α7-nAChRs as a critical link between inflammation and neurodegeneration in PD.α7-nAChRs are not only expressed in neurons but also in glia cells where they canmodulate the immunological responses contributing to PD. Successful therapeuticstrategies against PD should consider the connections between inflammation andneurodegeneration. Among them, α7-nAChRs may represent a pharmacologicaltarget to control these two mechanisms during the pathogenesis of neurodegenerativeand behavioral disorders.Astrocyte, the most abundant glial cell type in the brain, provides metabolic andtrophic support to neurons and modulates synaptic activity. Accordingly, astrocytedysfunction and even dysregulation of astrocyte-specific functions can criticallyinfluence neuronal survival. Emerging evidences, including from our laboratory, hasrevealed that modulation of astrocyte function may provide a novel therapeuticstrategy for neurodegenerative disorders. Increasing evidence demonstrates thatastrocyte activation involves in the pathogenesis of PD. Indeed, activated astrocytesproduce broad array of neurotoxic molecules, including proinflammatory factors,reactive oxygen species and reactive nitrogen species. These factors-mediatedinflammatory processes contribute to PD. Thus, inhibition of astrocyte dysfunctionmay provide therapeutic benefits for neurodegenerative disorders such as PD.In the present study, we first investigated the alterations of gene expression ofnicotinic acetylcholine receptor α7, α4and β2subunits in an acute MPTP-lesionedmouse model. The alterations in the level of α7, α4and β2nAChR mRNA mayprovide some insight into changes in cholinergic signaling associated with PDpathogenesis and may provide valuable information for new drug therapies. Then, we determine whether nicotine, acting at α7-nAChRs, could inhibit astrocyte-mediatedneuroinflammation triggered by MPTP/MPP+utilizing in vivo and in vitro models.The potential anti-inflammatory mechanisms of and regulation of astrocyte activationby nicotine were further investigated by utilizing lipopolysaccharide （LPS）, which isthe most extensively utilized glial activator due to its induction of inflammatorychanges and consequent dopaminergic neurodegeneration. Furthermore, weinvestigated whether α7-nAChR function is associated with modulation ofphosphorylation of Erk1/2and p38mitogen-activated protein kinases （MAPKs）.Finally, we studied distinctive nAChR functional phenotypes of rat ventral tegmentalarea dopaminergic neurons.Part Ⅰ Dynamic alterations of gene expression of nicotinic acetylcholine receptorα7, α4and β2subunits in an acute MPTP-lesioned mouse modelAIM: To investigate the alterations of gene expression of nicotinic acetylcholinereceptor α7, α4and β2subunits in an acute MPTP-lesioned mouse modelMETHODS: For acute MPTP treatment, the mice received four intraperitonealinjections of MPTP-HCl （20mg/kg） in saline at2hr intervals on the same day. TheMPTP-treated mice were classified into four groups and recaptured at different timecourses since their last injection of MPTP. The time periods for the four groups were3hr,1,2and7days, after the time of the last injection. The pole test was used for theassessment of MPTP-induced parkinsonian symptoms. Immunostaining was taken forTH and GFAP expression in mouse substantia nigra. A comparative RT-PCRapproach was used for the assessment of the time course of nAChR α7, α4and β2subunit expression levelsRESULTS: The results fall into four categories.（1） MPTP treatment transientlyincreased nAChR α7（after last injection of MPTP3and24hr）, α4and β2（24hr） mRNA expression in the substantia nigra （SN） and striatum.（2） Compared to corticaland hippocampal tissues, this transient increase of nAChR subunit expressionspecifically occurred in the SN and striatum.（3） In the acute MPTP model,time-courses of altered expression for nAChR α7, α4and β2subunits closelymirrored the deficits observed in animal motor activity.（4） Stereological data showedthat after administration of MPTP for24hr, there was a robust astrogliosis in the SNassociated with significant dopaminergic neurodegeneration. These changes followedor paralleled MPTP-induced elevation in the levels of α7, α4and β2mRNAs.CONCLUSION: Collectively, our results demonstrate that nAChRs are importanttargets in the MPTP neurotoxic process. These data suggest that therapeutic strategiestargeted toward nAChR α7, α4and β2subunits may have potential for developingnew treatments for PD.Part Ⅱ α7nicotinic acetylcholine receptor-mediated neuroprotection againstdopaminergic neuron loss in an MPTP mouse model via inhibition of astrocyteactivationAIM: The objective of the present study was to determine whether nicotine-inducedneuroprotection in an1-methyl-4-phenyl^-1,2,3,6-tetrahydropyridine （MPTP） mousemodel occurs via α7-nAChR-mediated inhibition of astrocytes.METHODS: Both in vivo （MPTP） and in vitro （1-methyl-4-phenylpyridinium ion,MPP⁺; lipopolysaccharide, LPS） models of PD were used to investigate the roles andthe possible mechanisms whereby α7-nAChRs protect against dopaminergic neuronloss. Multiple experimental approaches, including behavioral tests, immunochemistryand stereology experiments, astrocyte cell cultures, reverse transcription-polymerasechain reaction, TNF-α assays and Western blotting were employed to elucidate themechanisms of α7-nAChR-mediated neuroprotection.RESULTS: Systemic administration of nicotine alleviated MPTP-induced behavioral symptoms, improved motor coordination, protected against dopaminergic neuron lossand activated astrocytes in the substantia nigra. The protective effects of nicotinewere abolished by administration of the α7-nAChR-selective antagonistmethyllycaconitine （MLA）. In mouse primary cultured astrocytes, pretreatment withnicotine suppressed MPP+-or LPS-induced astrocyte activation evidenced by bothdecreased production of TNF-α and inhibition of Erk1/2and p38activation inastrocytes, and these effects also were reversed by MLA.CONCLUSION: Taken together, our results suggest that α7-nAChR-mediatedinhibition of astrocyte activation is an important mechanism underlying the protectiveeffects of nicotine.Part Ⅲ Distinctive nicotinic acetylcholine receptor functional phenotypes of ratventral tegmental area dopaminergic neuronsAIM: To investigate distinctive nicotinic acetylcholine receptor functionalphenotypes of rat ventral tegmental area dopaminergic neurons.METHODS: A patch-clamp recordings were used in single DAergic neurons freshlydissociated from rat VTA.RESULTS: We clarified three functional subtypes of nAChRs （termed ID, IID andIIID receptors） based on whole-cell current kinetics and pharmacology. Kineticanalysis demonstrated that comparing to ID, IID receptor-mediated current had fasteractivation and decay constant and IIID receptor-mediated current had larger currentdensity. Pharmacologically, ID receptor-mediated current was sensitive to theα4β2-nAChR agonist RJR-2403and antagonist dihydro-β-erythroidine （DHβE）; IIDreceptor-mediated current was sensitive to the selective α7-nAChR agonist cholineand antagonist methyllycaconitine （MLA）; while IIID receptor-mediated current wassensitive to the β4-containing nAChR agonist cytisine and antagonist mecamylamine （MEC）. The agonist concentration–response relationships demonstrated that IIDreceptor-mediated current exhibited the highest EC50value compared to ID and IIIDreceptors, suggesting a relatively low agonist affinity of type IID receptors. Theseresults suggest that the type ID, IID and IIID nAChR-mediated currents arepredominately mediated by activation of α4β2-nAChR, α7-nAChR and a novelnAChR subtypes, respectively.CONCLUSION: Collectively, these findings indicate that the VTA DAergic neuronsexpress diversity and multiplicity of functional nAChR subtypes. Interestingly, eachDAergic neuron predominantly expresses only one particularly functional nAChRsubtype, which may have distinct but important roles in regulation of VTA DAneuronal function, DA transmission and nicotine dependence.The major contributions of the present study lie in:1. The underlying mechanisms of α7-nAChR-mediated neuroprotection againstdopaminergic neuron loss in an MPTP mouse model are related to the inhibitionof astrocyte activation. α7-nAChRs in astrocytes may be a novel target foranti-neuroinflammtion agents.2. Studies of distinctive nAChR functional phenotypes of rat VTA dopaminergicneurons could improve our understanding of the roles of VTA nAChRs innicotine reinforcement and dependence in VTA function, and in the control ofDAergic signalling.更多还原