Parkin和Interleukin-1β在帕金森病中的作用研究

【作者】 卞敏娟；

【导师】 黄芳； 孙凤艳；

【作者基本信息】 复旦大学 ， 神经生物学， 2009， 博士

【摘要】 第一部分Parkin基因的突变与常染色体青少年帕金森症密切相关,而且早发性PD中超过50%的病例发现有Parkin基因的突变。Parkin蛋白是一种E3泛素连接酶,它紧密联系着参与PD进程的蛋白泛素化修饰和泛素蛋白酶体系统。目前发现的Parkin底物有:CDCrel-1,CDCrel-2a,synphilin-1,糖基化的α-synuclein,Pael-R,错误折叠的DJ-1和Hsp70等。大量研究显示Parkin对于由底物蛋白过表达(如突变形式的α-synuclein、Pael-R、CDCrel-1和错误折叠的DJ-1等)造成的细胞毒性有明显的保护作用。其神经保护作用同样见于MPTP或6-OHDA诱导的PD动物模型中。另外,在内质网应激、蛋白酶体抑制、海人藻酸引起的兴奋性毒和神经酰胺诱导的线粒体凋亡等情况下,Parkin的高表达同样能挽救受损的神经元。因此Parkin在多巴胺能神经元存活方面发挥重要作用。但是Parkin剔除小鼠脑内并未发现多巴胺能神经元的改变,却显示出线粒体功能的异常。为研究Parkin在体内的PD进程中的保护作用和机制,我们构建了神经元特异性表达Parkin的转基因小鼠,并分析了这种小鼠对于MPTP的神经毒性是否具有抵抗作用及其相关机制。研究结果如下:1.Parkin转基因小鼠在mRNA和蛋白水平都有转基因Parkin的表达。我们分别从转录水平和蛋白水平检测各Founder子代小鼠脑内转基因的表达情况,最终得到两株转基因小鼠品系。转录水平上,转基因Parkin在小鼠皮层、海马、纹状体和黑质部都有表达,且Parkin蛋白在转基因小鼠各脑区表达增加。焦油紫(CV)染色显示转基因与野生型小鼠之间没有明显的形态改变。2.青年和老年Parkin转基因小鼠对于MPTP诱导多巴胺神经毒性都具有保护作用。青年和老年转基因及野生型小鼠在MPTP注射后1天和3天后,我们观察了脑内纹状体TH蛋白的表达和黑质区TH阳性细胞的数量。结果显示青年Parkin转基因小鼠与野生型小鼠相比,在MPTP注射3天后纹状体TH蛋白呈现显著升高的表达,而在MPTP注射1天后黑质区TH阳性细胞数量的降低明显减小。MPTP在老年Parkin转基因小鼠脑内引起的TH蛋白和TH阳性细胞数量的下降幅度明显低于野生型小鼠。该结果提示Parkin在各年龄小鼠脑内均具有神经保护作用。3.青年Parkin转基因与野生型小鼠之间纹状体内DA及其代谢产物的含量没有明显差异。HPLC用于检测青年小鼠脑内纹状体DA及其代谢产物的含量,结果显示MPTP引起纹状体DA及其代谢产物含量的减少,Parkin转基因和野生型小鼠之间未见明显差异。说明Parkin可能不影响DA的代谢过程。4.MPTP所引起的黑质区线粒体损伤在Parkin转基因小鼠脑内明显降低。MPTP的神经毒性作用首先损伤线粒体功能,Parkin是否可以改善由MPTP引发的线粒体损伤,为此,我们利用电镜观察了黑质致密部神经元内线粒体的形态变化。结果显示MPTP在野生型小鼠脑内引起的线粒体肿胀、空泡和嵴减少等损伤现象在Parkin转基因小鼠脑内有明显的改善。说明Parkin可对毒性物质造成的线粒体损伤产生保护作用。5.Parkin转基因小鼠黑质区呈现bcl-2,bax,iNOS和DJ-1等基因在转录水平上的改变。我们进一步检测了线粒体功能相关基因bcl-2、bax、DJ-1和PINK1,炎症相关因子iNOS和TNFα,及其他相关基因CHIP和UCH-L1的转录水平变化。结果显示bcl-2和DJ-1的表达在青年Parkin转基因小鼠脑内显著上调,而老年转基因小鼠在MPTP注射3天后出现bcl-2的表达上调、bax和CHIP的表达下调。提示Parkin的神经保护作用可能与bcl-2和DJ-1的上调或bax等基因的下调有关。6.α-synuclein蛋白的表达在老年转基因小鼠的纹状体显著降低;而Hsp70的表达在各组之间没有明显改变。Hsp70是与Parkin相互作用的蛋白,还可以通过上调bcl-2或下调bax等机制抑制线粒体相关的凋亡通路。我们检测了各组小鼠纹状体Hsp70的表达,结果并没有发现Hsp70蛋白表达的显著改变。而与PD进程中Lewy Body的形成密切相关的蛋白α-synuclein却在老年Parkin转基因小鼠脑内出现显著的下调。以上结果提示Parkin在老年小鼠脑内可能部分通过下调α-synuclein的表达从而减少脑内的蛋白聚集以达到保护DA神经元的作用。结论:1.Parkin通过减少线粒体损伤改善MPTP引起的多巴胺能神经退行性改变。2.Parkin并不影响MPTP导致的纹状体DA及其代谢物的含量下降。3.Parkin在老年小鼠脑内下调α-synuclein的表达,可部分解释Parkin的神经保护作用。第二部分神经免疫是一种重要的PD发病机制。在PD病人和MPTP诱导的动物模型脑内发现有大量小胶质细胞和星形胶质细胞的激活。胶质细胞释放的细胞因子IL-1β在中枢神经系统损伤情况下可以调节细胞的反应。PD病人脑内黑质和纹状体区都检测到IL-1β的增加,提示IL-1β可能参与PD进程。但目前对于IL-1β在大脑损伤过程中的确切作用仍无定论。PD的流行病学研究发现男性的发病率明显高于女性,雌激素被认为是发挥神经保护作用的物质,在人和PD小鼠模型中都证明了这一点,说明雌激素是PD发病率中雌雄差异的基础之一。为探究PD发病中IL-1β的变化规律及IL-1β和老年雌性与雄性小鼠PD发病差异之间的相关性,我们利用一种人IL-1β基因的启动子驱动萤火虫酶表达的转基因报告小鼠建立了MPTP诱导的亚急性PD模型,研究老年雌性与雄性小鼠中IL-1β的表达模式,研究结果如下:1.MPTP引起老年雄鼠脑内急剧升高的萤火虫酶表达,并呈现明显波动变化,而雌鼠脑内的升高变化趋于平稳。在MPTP或生理盐水注射过程中不同的时间点进行萤火虫酶活性的检测。MPTP均引起雌性与雄性小鼠脑内酶活性的增加,49h雌鼠脑内的信号已回复至基础水平,而雄鼠仍然维持在较高水平。量化后的信号显示在雌性和雄性小鼠脑内MPTP引起的萤火虫酶升高呈现不同的变化趋势,雄鼠变化急剧且波动明显,雌鼠的变化则较缓与平稳。2.MPTP在老年雌鼠脑内引起的多巴胺神经毒性较雄鼠减小。MPTP末次注射后65h,老年雌性和雄性小鼠纹状体TH蛋白的表达都明显下降,且雄鼠的下降幅度比雌鼠更大。MPTP引起的雄鼠脑内黑质部TH阳性神经细胞数量的显著减少,而雌鼠脑内TH阳性神经细胞数量的变化却不明显。3.黑质部几种炎症因子和凋亡相关分子的转录水平表达在各组之间差异不大。我们用实时定量PCR的方法检测了MPTP末次注射65h后,黑质部多种基因在转录水平上的表达变化,包括细胞因子TNF-α和IL-6,促炎分子iNOS,及凋亡相关分子bcl-2和bax。结果显示在这个时间点除IL-6在雌鼠MPTP注射和生理盐水对照组之间的p值等于0.07外,其余基因在各组之间均无显著变化。4.MPTP引起雌性和雄性小鼠纹状体GFAP蛋白表达显著升高,BDNF的表达在各组之间无明显变化。我们通过Western blot检测MPTP末次注射65h后纹状体GFAP和BDNF蛋白的表达变化。结果显示MPTP引起老年雌性和雄性鼠GFAP蛋白的显著上升,雌雄之间无明显差异;BDNF在各组之间无显著变化。结论:1.老年雄鼠脑内MPTP诱导的升高且变化剧烈波动的IL-1β可能会导致DA神经元对MPTP易感性增加。2.MPTP在老年雌鼠脑内引起的神经毒性明显低于雄鼠。更多还原

【Abstract】 PartⅠMutations in the Parkin gene are associated with autosomal recessive juvenile parkinsonism(AR-JP),accounting for more than 50%of early onset PD cases.Parkin, as an ubiquitin E3 ligase,provides an important link between protein ubiquitination and the ubiquitin-proteasome system(UPS) in the pathogenesis of PD.A number of Parkin substrates have been identified,including CDCrel-1,CDCrel-2a,synphilin-1, glycosylatedα-synuclein,Pael-R,misfolded DJ-1,Hsp70,etc.Several recent papers have shown that expression of parkin is beneficial to dopaminergic neurons as it protects them from a number of damaging events such as overexpression of mutant forms ofα-synuclein,Pael-R,CDCrel-1,misfolded DJ-1 or neurotoxin MPTP and 6-OHDA.Parkin is also reported to be protective against ER stress,proteasomal inhibition,kainic acid ’excitotoxicity’ and ceramide-induced mitochondrial apoptosis. However,Parkin deficient mice display little or no alteration in dopamine neuron survival,but show reduced mitochondrial function.To determine the protective effect of parkin in PD process in vivo,we generated Parkin transgenic mice in which the expression of parkin is confined to neurons.We examined whether MPTP induced less neurotoxicity in Parkin transgenic mice,the results are summarized as follows:1.Transgenic mice showed transgene expression at mRNA and protein levels.Offsprings of different founder mice were screened for transgene expression at both mRNA and protein levels,we finally got two transgenic mouse lines.The transgenic mice showed exogenous Parkin transcription and elevated Parkin protein expression in the cortex,hippocampus,striatum and substantia nigra.Creysl violet staining of different brain regions revealed no change between transgenic mice and wild type littermates.2.Young and old Parkin transgenic mice displayed different protection patterns of dopamine neurons after MPTP administration.We examined TH protein expression in the striatum and TH positive neurons in the substantia nigra after MPTP administration.Compared to wild type litermates,young Parkin transgenic mice showed less reduction of TH protein expression in the striatum 3 days after MPTP toxication,while TH positive cells in the SN were less decreased 1day after MPTP treatment.MPTP induced less neurotoxicity both at 1 day and 3 days after MPTP administration in old transgenic mice.These data indicate that Parkin protects DA neurons from MPTP toxicity.3.DA and its metabolites didn’t show significant change in young Parkin transgenic mice and wild type littermates.Measured by HPLC,striatal DA and its metabolites showed decreased concentrations after MPTP treatment.No significant change was observed between Parkin transgenic mice and wild type littermates.It suggests Parkin may be not involved in DA metabolism.4.MPTP elicited less mitochondrial impairment in Parkin transgenic mice.MPTP is reported to impair mitochondrial function.In order to examine whether Parkin could protect mitochondria,we observed morphology of mitochondria in the substantia nigra pars compacta after MPTP administration.Electron microscopy showed increased abnormal mitochondria with numerous vacuoles and fragmented cristae in wild type mice after MPTP treatment,while less impaired mitochondria were found in Parkin transgenic mice.This indicates that Parkin can protect mitochondria from MPTP toxicity.5.Transcriptional expression of bcl-2,bax,iNOS and DJ-1 altered within groups.We then examined the transcriptional expression of mitochondrial related genes, such as bcl-2,bax,DJ-1 and PINK1,pro-inflammation molecules iNOS and TNFα, and other related genes such as CHIP and UCH-L1.We found that bcl-2 and DJ-1 remained up-regulated in the young Parkin transgenic mice,while the up-regulation of bcl-2 and down-regulation of bax and CHIP were detected in the old Parkin transgenic mice 3 days after MPTP administration.These data may in part explain the beneficial role of Parkin.6.α-synuclein protein expression in the striatum was reduced in old Parkin transgenic mice,while Hsp70 expression remained unchanged.As one of the substrate proteins of Parkin,Hsp70 can inhibit mitochondrial apoptosis.Thus,we detected the protein expression of Hsp70 in the striatum within groups and revealed no significant change,α-synuclein,which is involved in the formation of Lewy Body in PD,showed decreased protein expression in old Parkin transgenic mice.This may in part explain that Parkin protects DA neurons through down regulatingα-synuclein to reduce protein aggregation during PD process. Conclusion:1.Parkin attenuates dopaminergic neurodegeneration induced by MPTP through protection of mitochondria.2.MPTP-induced depletion of the striatal DA and its metabolites is not protected by Parkin.3.Downregulation ofα-synuclein protein expression in the old Parkin transgenic mice may in part explain the protection of Parkin. PartⅡThere is increasing evidence that recognition of the possible role of neuroinflammation is a causative factor in the pathogenesis of PD.Glial reaction, which is featured by activation of microglia and astrocytes,is observed in brain tissues of postmortem PD patients and MPTP-induced animal models.IL-1β,an inflammatory cytokine released mainly from glial cells,plays important roles in mediating cellular responses to injuries in the central nerve system.The expression of IL-1βhas been reported to be elevated in the striatum and substantia nigra of PD patients,suggesting its association with PD.However,findings about the role of IL-1βduring brain damage were contradictoryreported.Moreover,greater prevalence of PD in men compared to women is reported.Estrogen is conceived reported to exert dopaminergic neuroprotection both in human and PD mice model,this may be one of the explanations of the gender difference in PD.To determine the changes of IL-1βexpression and the association between the IL-1βexpression and the prevalence of PD in aged mice.By using a sensitive transgenic mice model in which the expression of luciferase reporter gene is under the control of human IL-1β.gene promoter,we examined the IL-1βgene expression pattern in vivo after subacute MPTP toxication in old male and female mice.The results are summarized as follows:1.Old male mice showed dramatically elevated luciferase signals in a flexuous manner at early period of time,meanwhile,the changes of luciferase signals in female were more moderate.Old male and female mice were screened for luciferase expression at different time points during and after MPTP or saline administration.The luciferase activity increased in both MPTP groups.By 49h,the enzyme activity in female mice treated with MPTP had returned back to baseline,while the activity in male mice still sustained at a higher level.Data quantification showed different patterns of MPTP-induced luciferase signals between male and female.Though both sexes revealed significant higher levels of luciferase expression,the changes in male mice were especially vigorous,but quite moderate in female.2.MPTP elicited less dopaminergic toxicity in old female mice than male ones.65h after MPTP administration,we observed a significant depletion of striatal TH protein expression in both sexes,however,TH reduction was significantly greater in old male mice when compared to female.The TH positive neurons in the substantia nigra of old female mice didn’t show prominent difference between the saline and MPTP group,while significant decrease of TH positive cells was seen in male MPTP intoxicated mice3.The transcriptions of some inflammatory and apoptotic molecules in the substantia nigra showed no significant change between groups.65h after the last MPTP injection,transcriptional levels of cytokine TNF-αand IL-6,pro-inflammatory molecule iNOS,together with apoptosis related molecules bcl-2 and bax in the substantia nigra were detected by real-time PCR.The expression of these genes showed no significant alteration between groups at this time point, although IL-6 gene trancription was increased after MPTP administration,the p-value of IL-6 between the control and MPTP group in female mice was 0.07.4.MPTP intoxication increased GFAP protein expression,but didn’t change BDNF expression in the striatum of both old male and female mice.GFAP and BDNF protein expression in the striatum was were measured through western blot analysis.MPTP intoxication induced robust increase of GFAP expression in both sexes 65h after the last MPTP injection.Only slightly higher expression of BDNF was observed in old female mice,however,no significant difference was observed obtained between groups.Conclusion:1.Elevated and dramatic changes of IL-1βgene expression in old male mice at early period of time may be responsible for the DA neuron susceptibility to MPTP in old male mice.2.MPTP elicited less dopaminergic toxicity in old female than male mice.更多还原