【作者】 陈锶；

【导师】 肖波； 龙小艳；

【作者基本信息】 中南大学 ， 神经病学， 2009， 博士

【摘要】 目的拟探讨亨廷顿蛋白相关蛋白1（HAP1）与脑源性神经营养因子（BDNF）及其受体p75NTR的胞吞和逆向囊泡运输关系,深入阐述亨廷顿舞蹈病的分子生物学机制。方法1、首先构建荧光融合蛋白质粒,利用基因转染、细胞免疫荧光染色等技术,在激光共聚焦显微镜下观察HAP1与BDNF、p75NTR在细胞中的表达和共定位现象:1)构建荧光融合蛋白质粒HAPlA-CFP和BDNF-DsRed、pro-BDNF-DsRed、pre-BDNF-DsRed;制备含BDNF-DsRed、pro-BDNF-DsRed和pre-BDNF-DsRed荧光融合蛋白的培养液,以及生物素标记BDNF、pro-BDNF、pre-BDNF蛋白。2)分为BDNF组、pro-BDNF组和pre-BDNF组三部分。利用荧光融合蛋白质粒HAP1A-CFP和（或）BDNF-DsRed转染PC12细胞,分别在含不同成分的培养液孵育后,应用激光共聚焦显微镜研究HAP1与BDNF在细胞中的表达和定位。3)荧光融合蛋白质粒HAP1A-CFP和p75NTR-YFP转染PC12细胞,分别在含有或无BDNF-DsRed的培养液孵育后,应用激光共聚焦显微镜研究HAP1与p75NTR在细胞中的表达和定位。4)采用免疫荧光染色的方法,将PC12细胞分二组,在含有或无生物素标记的BDNF培养基中孵育后进行免疫荧光标记（双标或三标）,在激光共聚焦显微镜下观察HAP1、p75NTR和BDNF的表达与定位。2、通过转基因鼠皮层神经元培养、基因转染和蛋白免疫印迹（Western-Blot）技术对HAP1与BNDF的胞吞和逆向囊泡运输的相关性进行更加深入的探讨和寻求相关性证据:1)根据PCR扩增基因产物鉴定鼠尾DNA结果,将离体培养的新生鼠皮层神经元为三组:正常型(HAP1^+/+)、HAP1基因敲除型(HAP1^-/-)和HAP1杂合型(HAP1^+/-)。细胞在含有生物素标记的BDNF培养基中孵育,采用免疫荧光标记（双标或三标）技术,利用激光共聚焦显微镜观察和对照三种神经元的BDNF胞吞情况。同样的方法应用于pro-BDNF和pre-BDNF。2)荧光融合蛋白质粒HAP1A—CFP转染入HAP1^-/-神经元,在同样含有lnM生物素标记BDNF蛋白（BDNF组）的Neurobasal培养基中孵育后,在激光共聚焦显微镜下监测HAP1^-/-神经元的胞吞情况。同样的方法应用于pro-BDNF组和pre-BDNF组。3)培养在25cm²培养瓶中的新生鼠皮层神经元分为二组,正常组和HAP1基因敲除组。细胞在含有生物素标记的BDNF培养基中孵育,提取细胞总蛋白,采用Western-Blot技术,对照不同基因型的神经元胞吞BDNF的情况。3、通过生物素标记胞膜受体蛋白、受体漂白荧光能量转移和免疫共沉淀技术进一步探讨在BDNF的胞吞过程中,HAP1和BDNF的受体p75NTR之间的相互关系:1)离体培养新生鼠皮层神经元,按照基因分型分为二组HAP1^+/+组和HAP1^-/-组,采用sulfo-NHS-S-S-biotin（可逆性的不可透膜的生物素交联剂）标记活细胞膜表面蛋白,二组细胞分别在含有或无BDNF的培养基中孵育,然后利用免疫沉淀（Precipitaion）和Western-Blot技术对胞吞的p75NTR受体进行检测,探讨在BDNF胞吞过程中,HAP1是否对p75NTR的信号传递也起着重要调控作用。2)以CFP和YFP作为FRET荧光对,将载有这二种荧光蛋白探针的重组质粒—HAP1A-CFP和p75NTR-YFP共转染PC12细胞,分别在含有或无BDNF的培养基中孵育后,应用激光共聚焦显微镜和受体漂白FRET技术研究HAP1A-CFP和p75NTR-YFP的胞内能量传递和相互作用。3)将重组质粒HAP1A-CFP和p75NTR-YFP共转染HEK293细胞,将细胞在含有或无BDNF的培养基中孵育1hr,提取细胞总蛋白,采用免疫共沉淀和Western-Blot技术,并进行蛋白浓度的内参校正,检测HAP1蛋白与p75NTR是否存在生理性的相互作用。结果1、HAP1蛋白与经胞吞途径进入细胞的BDNF有高度的共定位现象,但是,与细胞内表达的BDNF几乎没有共定位,BDNF抗体或者p75NTR抗体可以完全阻断后者的共定位。2、HAP1蛋白与细胞内表达的和经胞吞途径进入细胞的pro-BDNF均有高度共定位现象;BDNF抗体可以完全阻断后者的共定位,p75NTR抗体或者sortilin抗体可以部分阻断共定位。3、HAP1蛋白与细胞内表达的和经胞吞途径进入细胞的pre-BDNF蛋白均有高度共定位现象;pre-BDNF抗体或者sortilin抗体可以完全阻断后者的共定位。4、PC12细胞转染荧光融合蛋白质粒HAP1A-CFP和p75NTR-YFP后,激光共聚焦显微镜显示BDNF、p75NTR和HAP1三种蛋白分别存在共定位,并且,BDNF可增强胞浆内HAP1蛋白与p75NTR的共定位,与对照组相比P＜0.05。5、采用细胞免疫荧光染色技术,激光共聚焦显微镜显示分化的PC12细胞中的内源性HAP1、p75NTR蛋白仅存在部分共定位,BDNF可增强胞浆内HAP1与p75NTR的共定位,与对照组相比P＜0.01。6、离体培养新生鼠皮层神经元,在含生物素标记的BDNF的培养基孵育后,经细胞免疫荧光染色技术,激光共聚焦显微镜显示HAP1^+/+和HAP1^+/-神经元的胞膜、树突和胞体中都有BDNF的荧光,但是HAP1^-/-神经元中没有,提示BDNF的胞吞和逆向囊泡运输需要HAP1的参与,p＜0.01。7、和HAP1^+/+神经元相比,HAP1^+/-神经元内摄的BDNF荧光强度减弱,p＜0.05,提示皮层神经元对BDNF的胞吞可能受HAP1基因表达水平的影响。8、HAP1^-/-神经元成功转染质粒HAP1A-CFP后,可以恢复对BDNF的胞吞,进一步证实HAP1是BDNF胞吞的关键蛋白。9、Western-Blot实验证明,HAP1^+/+神经元中可以检测到胞吞的BDNF,分子量14kD左右;但是HAP1^-/-神经元中没有。10、离体培养新生鼠皮层神经元,采用sulfo-NHS-S-S-biotin标记活细胞膜表面蛋白,然后利用免疫沉淀和Western-Blot技术对胞吞的受体进行检测。结果显示HAP1^-/-神经元中检测不到胞吞的p75NTR,提示在皮层神经元胞吞BNDF的过程中,HAP1蛋白对p75NTR的信号传递起着重要的调控作用。11、利用受体漂白荧光共振能量转移（FRET）技术研究HAP1A-CFP和p75NTR-YFP之间的能量传递,结果显示,BDNF可以使HAP1A-CFP和p75NTR-YFP之间出现能量传递,即HAP1与p75NTR蛋白之间的距离在1-10nm范围,表明在活细胞生理条件下,二种蛋白之间很可能存在相互作用,与对照组相比p＜0.05。12、采用免疫共沉淀技术检测生理条件下HAP1与p75NTR之间是否相互结合。结果显示,p75NTR抗体和蛋白质A Sepharose可以从细胞蛋白质提取液中拉下HAP1,证实HAP1蛋白可以与p75NTR存在生理性的结合和相互作用。结论本研究在国际上首次发现HAP1蛋白是BDNF及其受体p75NTR的胞吞和逆向囊泡运输的关键蛋白,并且可以与p75NTR存在生理性的结合,从而介导BDNF的胞吞和逆向囊泡运输。更多还原

【Abstract】 Objective This project aims to elucidate whether Huntingtin associated protein 1（HAP1） plays a role in the endocytosis and retrograde transport of BDNF and its receptors,which may offer a way to investigate the molecular biology mechanism of Huntington disease.Methods 1.Plasmids were constructed firstly.PC12 cells were transfected with plasmids,or followed by triple labeling with an immunofluorescence method.Co-localization was analyzed and quantified with confocal imaging software:1) Plasmids of HAP1A-CPF,BDNF-DsRed,pro-BDNF-DsRed and pre-BDNF-DsRed were constructed.The biotin labeled BDNF, pro-BDNF,pre-BDNF,and medium containing DsRed labeled mature BDNF,were prepared.2) We have co-transfected PC12 cells with HAP1A-CPF and BDNF-DsRed,and incubated cells with the conditioning medium containing exogenous DsRed labeled mature BDNF,in the absence,or presence,of the antibodies to mature BDNF or to p75NTR extracellular domain.Confocal images were taken and merged.The same methods were used with pro-BDNF and pre-BDNF.3) PC12 cells were used to co-transfected with the plasmid HAP1-A-CFP and p75NTR-YFP.After co-transfection,differentiated PC12 cells were incubated in the presence or absence of DsRed-labeled BDNF conditioning medium to allow internalization,followed by immunofluorescence confocal imaging.4) We compared the intracellular distribution of p75NTR and HAP1 in differentiated PC12 cells which were incubated with biotin labeled BDNF,followed by triple labeling with an immunofluorescence method. Co-localization was analyzed and quantified with confocal imaging software.2.Cortical neurons from transgenic mice were incubated with biotin-labeled BDNF,followed by triple labeling with an immunofluorescence confocal imaging,or analyzed by Western blot,in order to elucidate whether HAP1 plays a role in the endocytosis and retrograde transport of BDNF:1) Cortical neurons from HAP1^+/+,HAP1^+/-,and HAP1^-/- from mice at postnatal day 1 were cultured.Then applied biotin labeled BDNF to trigger endocytosis,followed by immunostaining experiments and confocal imaging.2) We transfected the HAP1^-/- neurons with HAP1-CFP plasmids and incubated with incubated with biotin-labeled BDNF,followed by immunostaining experiments and confocal imaging.3) Cortical neurons were incubated with biotin labeled BDNF for 1 hour,lysed and analyzed by Western blot. 3.The p75NTRs on the surface of cultured cortical neurons from transgenic mice were biotin labeled,followed by incubation,precipitation and Western-Blot;the apFRET technology was used to see the fluorescence resonance energy transfer between HAP1 A-CFP and p75NTR-YFP;finally,a Co-immunoprecipitation assay was done to elucidate whether and how HAP1 plays a role in the endocytosis and retrograde transport of BDNF receptors-p75NTR:1) The p75NTRs on the surface of cultured cortical neurons from HAP1^+/+ and HAP1^-/- neonatal mice were biotin labeled using a reversible, membrane-impermeable cross-linker（sulfo-NHS-S-S-biotin）.Then we incubated neurons with BDNF（25nM） or not for 1 hour.The cell surface biotin was then cleaved from the remaining surface- exposed receptors as described,and the neurons were lysed in RIPA buffer.Equal amounts of protein lysates containing internalized biotinylated receptors were subjected to precipitation with streptavidin- agarose.Immunoblotting was performed with p75 antibodies.2) The apFRET technology was used to see the fluorescence resonance energy transfer between HAP1 A-CFP and p75NTR-YFP, which were expressed by co- transfected PC12 cells with plasmids of HAP1A-CFP and p75NTR-YFP,by BDNF stimulation.3) A Co-immunoprecipitation assay was done using HEK293 cell lysate which co-expressed HAP1A-CFP and p75NTR-YFP by co-transfection,followed by precipitation with p75NTR antibody and protein A Sepharose and immunoblotting.Results 1.Internalized BDNF in PC12 cells is highly co-localized with HAP1 but the co-localization was completely abolished by the antibodies to BDNF or to p75NTR.2.Internalized pro-BDNF in PC12 cells is highly co-localized with HAP1 but the co-localization was completely abolished by the antibodies to BDNF,and partly to p75NTR or sortilin.3.Internalized pre-BDNF in PC12 cells is highly co-localized with HAP1 but the co-localization was completely abolished by the antibodies to BDNF or to sortilin,not to p75NTR.4.The co-localization of the HAP1A-CFP with p75NTR-YFP,which were expressed by co-transfected PC12 cells with plasmids of HAP1A-CFP and p75NTR- YFP,was found and the co-localization was increased by BDNF stimulation.5.Substantial co-localization of the intracellular distribution of HAP1 with p75NTR was found in differentiated PC12 cells,and the co-localization was increased by BDNF stimulation.6.Upon incubation of cortical neurons with exogenous labeled BDNF,the labeled factor was detected on the cell surface,and within neurites and cell bodies,in most HAP1^+/+ and HAP1^+/-,but not in HAP1^-/- neurons,suggesting HAP1 is required for BDNF endocytosis and intracellular trafficking.7.Moreover,the intensity of internalized BDNF was attenuated in HAP1^+/- neurons compared with WT neurons.8.The defect in BDNF endocytosis in HAP1^-/- neurons could be rescued by HAP1 plasmid transfection.9.A Western blot assay showed endocytosed BDNF at 14 kDa was detected in HAP1^+/+,but not in HAP1^-/- neurons.10.Incubation of cortical neurons with sulfo-NHS-S-S-biotin,a reversible membrane-impermeable cross-linker of biotin,followed by precipitation and immunoblotting,showed that the endocytosed p75NTR were abolished in HAP1^-/- mice,suggesting HAP1 is significant for the signaling of p75NTR in the endocytosis of BDNF in cortical neurons.11.The FRET technology suggested that there were fluorescence resonance energy transfer between HAP1 A-CFP and p75NTR-YFP, which were expressed by co-transfected PC12 cells with plasmids of HAP1A-CFP and p75NTR-YFP,by BDNF stimulation,suggesting that HAP1 may interact with p75NTR.12.A Co-immunoprecipitation assay showed that HAP1 A-CFP at about 100Kd was detected by precipitation with p75NTR antibody and protein A Sepharose using HEK293 cell lysate which co-expressed HAP1A-CFP and p75NTR-YFP by co-transfection,and immunoblotting. Results verify that HAP1 could binds with p75NTR in the endocytosis and retrograde transport of BDNF.Conclusions We found that HAP1 is an essential molecule required for the endocytosis and trafficking of BDNF and its associated receptor p75NTR.The mechanism underlying the effect is that HAP1 could binds with p75NTR in the endocytosis and retrograde transport of BDNF.更多还原