【作者】 王琳辉；

【导师】 秦正红；

【作者基本信息】 苏州大学 ， 药理学， 2010， 博士

【摘要】 目的:构建亨廷顿舞蹈病(Huntington’s disease, HD)的体外原代星形胶质细胞模型;研究亨廷顿蛋白(Hungtingtin, htt)氨基端片段htt552在星形胶质细胞中的代谢途径及其对自噬水平的影响;研究htt552对星形胶质细胞脑源性神经营养因子(brain-derived neurotrophic factor , BDNF)转录与分泌的影响。方法:采用腺病毒介导野生型与突变型htt552片段转染的方法构建HD的原代星形胶质细胞模型,利用Western Blot法和免疫荧光双标记法检测htt552在细胞内表达的水平及时程,采用MTT法测定腺病毒载体对细胞生存率的影响。运用Western Blot法及免疫荧光双标记法检测自噬抑制剂3-甲基腺嘌呤(3-MA)、自噬激活剂雷帕霉素(Rapamycin)和蛋白酶体抑制剂乳胞素(Lactacystin)对htt552在细胞内蓄积的影响。利用Western Blot法和Real-time PCR法检测细胞模型中自噬相关蛋白LC3II和Beclin1的表达情况。采用MDC法检测细胞自噬体形成的情况。采用ELISA和Western Blot法检测表达htt552后细胞内以及分泌的BDNF含量,采用Real-time PCR法测定htt552对细胞内BDNF mRNA及其不同转录本水平的影响。采用免疫荧光双标记法测定htt552与CREB结合蛋白(CBP)或高尔基复合体的共定位,从而研究htt552影响星形胶质细胞内BDNF转录和分泌的机制。结果:腺病毒成功介导野生型与突变型htt552在原代星形胶质细胞中表达,该蛋白在细胞内主要分布于胞浆,而核内表达较少,部分细胞内可见突变型片段形成明显的聚集体。为了提高感染率和降低腺病毒对细胞的影响,以m.o.i为40的剂量加入病毒最为合适,感染率可达80%以上,感染后12hr直到7d都有蛋白表达,并且感染24hr到4d蛋白保持高表达。加入自噬抑制剂3-MA或者加入蛋白酶体抑制剂Lactacystin后,htt552在细胞内的蓄积量明显增加,其中突变型htt552蓄积更为显著且聚集体明显增多;加入自噬激活剂Rapamycin后,细胞内突变型htt552及其聚集体的含量显著降低,而对野生型片段无显著影响;在表达突变型和野生型htt552的两种细胞模型中,LC3II蛋白表达量显著增加,Beclin1的mRNA和蛋白表达水平均显著增高,MDC染色阳性的自噬囊泡数目显著增加;与野生型片段相比,表达突变型片段的细胞内LC3II与MDC染色阳性自噬囊泡的水平更高。免疫荧光实验显示,表达htt552-100Q的星形胶质细胞条件培养基(ACM)使原代培养的神经细胞突起分支数目减少、发育不良;ELISA和Western Blot法检测ACM中BDNF含量,结果显示htt552-100Q抑制BDNF的产生,而野生型片段无显著影响;Real-time PCR实验显示htt552-100Q抑制了细胞内BDNF mRNA水平,其原因和该突变片段显著抑制BDNF促进子II、III、IV有关,而野生型片段对BDNF的转录无显著影响;免疫荧光双标记法实验结果显示,htt552-100Q的可溶性片段及其聚集体和核内的CBP存在共定位,可能影响BDNF的转录过程;ELISA和Western Blot法检测细胞内BDNF含量,结果显示与表达野生型片段相比,表达htt552-100Q的细胞内BDNF含量显著升高,其中前体含量显著升高,而成熟的BDNF含量显著降低,提示htt552-100Q抑制BDNF的转运和成熟过程;免疫荧光双标记法实验显示,htt552-100Q的可溶性片段及其聚集体与BDNF囊泡和高尔基体共定位,并且使高尔基体的形态和分布发生变化,从而影响了BDNF的转运和分泌。结论:成功构建高效表达htt552的体外HD星形胶质细胞模型,为研究HD的发病机制提供了一个新的平台;泛素-蛋白酶体途径和自噬/溶酶体途径均参与了htt552在细胞内的降解过程,而自噬-溶酶体途径是突变型htt552及其聚集体代谢的主要途径;htt552特别是突变型片段能够提高细胞自噬水平,从而加速自身代谢;突变型htt552能够抑制星形胶质细胞内BDNF的转录和分泌,从而对其周围神经元的生长发育造成影响;突变型htt552的可溶性片段及其聚集体与CBP、分泌囊泡及高尔基体结合,干扰了这些结构的正常功能,从而抑制BDNF转录和分泌。更多还原

【Abstract】 Aim: To establish a glia Huntington’s disease (HD) model expressing wild-type and mutant N-terminal huntingtin fragment 1–552 amino acids (htt552), to study the contribution of autophagy/lysosomal pathway on the metabolism of the wild-type and mutant htt552 and to study the effects of wild-type and mutant htt552 on the production and secretion of brain-derived neurotrophic factor (BDNF) in astrocytes.Method: In this study, we developed an in vitro model of HD by infecting primary cortical astrocytes of new-born rat with adenoviral vectors encoding the first 552 amino acids of wild-type (18Q) and mutant (100Q) htt. Western Blot analysis and double immunofluorescence (IF) were used to measure the levels and duration of htt552 expression in astrocytes. MTT assay was used to detect the viability of astrocytes after viral infection. The effects of autophagy specific inhibitor 3-methyadenine (3-MA), autophagy activator rapamycin, and the proteasome inhibitor lactacystin on htt552 accumulation were assessed in astrocytes expressing htt552 with Western Blot analysis and double immunofluorescence (IF). Proteins related to autophagy -- LC3 and Beclin1 were determined with Western Blot analysis and Real-time PCR. Autophagosomes were detected by monodansylcardeverine (MDC) immuofluorescence. Protein levels of BDNF in cell lysates and medium were detected by enzyme-linked immunosorbent assay (ELISA) and Western Blot analysis. The levels of BDNF mRNA and its four transcripts were detected with Real-time PCR to estimate the influence of htt552 on the transcription of BDNF. The co-location of htt552 with cAMP-response element binding protein (CBP) or Golgi complex was detected by double IF to estimate the mechanisms by which htt552 affects the transcription and secretion of BDNF.Results: Wild-type and mutant htt552 transfected by adenovirus successfully expressed in primary astrocytes. They distributed predominantly in the cytoplasm with relatively low levels in the nucleus. Meanwhile, htt552-100Q produced the characteristic HD pathology by the appearance of cytoplasmic aggregates and intranuclear inclusions. To obtain high infection rate and low toxicity, the viral dose with an m.o.i. of 40 was optimal to our cell model, the duration of expression of htt552 lasted for about 7 days with a relatively high levels from 1 to 4 days post-infection. Treatments with the autophagy inhibitor (3-MA) or proteasome inhibitor (lactacystin) resulted in the augments of htt552 levels in cells, especially in those expressing mutant htt552. The activator of autophagy, rapamycin, significantly decreased the levels of mutant htt552 and the number of aggregates, however, had little effect on htt552-18Q accumulation. Western Blot analysis and Real time PCR revealed that the expression of LC3 and Beclin1 were up-regulated, and autophagosomes detected by MDC increased in cells expressing wild-type htt552 but it was more robust in the cells expressing mutant htt552. The result of IF assay shown that neurons cultured with astrocyte conditioned medium (ACM) from the cell expressing htt552-100Q exhibited shorter processes and fewer branchings than those with either ACM from the cell cultured normally or ACM from the cell expressing htt552-18Q. ELISA analysis revealed that total BDNF in the cell expressing htt552-100Q was increased compared with the cells expressing htt552-18Q. Western blot and ELISA analysis revealed that BDNF in ACM was down-regulated by htt552-100Q, and was not significantly changed by wild-type htt552. Western Blot assay further showed that proBDNF in astrocytes expressing with htt552-100Q was increased, but mature BDNF was decreased compared with the astrocytes expressing with htt552-18Q. Real time PCR revealed that BDNF mRNA levels were decreased in the astrocytes expressing htt552-100Q, as a result of the inhibition of promoter II、III、IV in BDNF gene. The result also showed that the levels of BDNF and its four transcripts were not significant change in the astrocytes expressing htt552-18Q. The result of double IF showed that htt552-100Q co-localized with CBP in the nucleus, which may have influence on the function of CBP. The result of double IF showed that htt552-100Q and its aggregates colocalized with BDNF puncta or Golgi complex, moreover, the morphology of Golgi complex and size of Golgi complex were changed by mutant htt552. Conclusion: In vitro astrocytes HD model expressing wild-type and mutant N-terminal huntingtin fragment htt552 had been successfully established, which provided an advantageous system for research of HD pathogenesis in primary cortical astrocyte cultures. The ubiquitin-proteasome pathway (UPP) and autophagy/lysosomal pathway participated in the catabolism of htt552, and autophagy/lysosomal pathway was the major pathway in the catabolism of mutant htt552 and its aggregates. Wild-type and mutant htt552 fragments can both activate autophagy in cells and mutant fragments activated autophagy more robustly. Htt552-100Q can inhibit the transcription and secretion of BDNF in astrocytes, and then have influence on the development and function of neurons. Htt552-100Q and its aggregates may sequester CBP in the nucleus and BDNF vesicles in the cytoplasm. In addition, Htt552-100Q may have an aberrant effect on Golgi complex, thus disrupt the processing and secretion of BDNF.更多还原