亨廷顿蛋白相关蛋白1与脑源性神经营养因子的胞吞关系的机制研究

Huntingtin associated protein 1 is involved with the endocytosis of mature brain derived neurotrophic factor

摘要:

目的 探讨亨廷顿蛋白相关蛋白1(HAP1)与脑源性神经营养因子(mBDNF)胞吞的相关性和可能的机制。 方法 神经营养因子(NGF)诱导分化PC12细胞,将荧光质粒HAP1A-CFP和(或)mBDNF-ds-red转染进入细胞,培养48 h后在含有BDNF或p75NTR抗体的培养基中继续培养,激光共聚焦显微镜观察荧光的表达情况及其在细胞中的定位;利用小鼠皮层神经元(正常型和HAP1基因敲除型)在生物素标记mBDNF的培养基中孵育60 min,激光共聚焦显微镜观察皮层神经元免疫荧光的效果。结果 共转染HAP1A-CFP和mBDNF-ds-red质粒的细胞,2种荧光蛋白存在部分共定位34%。共转染的细胞在抗BDNF培养基孵育下,或者单转染HAP1A-CFP质粒的细胞在mBDNF-ds-red荧光蛋白+抗BDNF/抗p75NTR培养基孵育下,2种荧光蛋白几乎没有共定位现象。单转染HAP1A-CFP质粒的细胞在mBDNF-ds-red荧光蛋白培养基孵育下,mBDNF与HAP1蛋白的共定位比例高达93%。正常新生小鼠皮层神经元内可见内吞的mBDNF免疫荧光,HAP1基因敲除小鼠的皮层神经元内未见。结论 mBDNF的胞吞必需HAP1的表达和参与。

Abstract:

Objective To investigate the roles of Huntingtin associated protein 1 (HAP1) in the endocytosis of mature brain derived neurotrophic factor (mBDNF) and the underlying mechanism.Methods PC12 cells were differentiated by NGF and co-transfected with plasmid of HAP1A-CFP and (or) mBDNF-ds-red.The cells were incubated with either recombinant ds-red-labeled mBDNF, or in combination of sheep anti-BDNF antibodies or rabbit anti-p75NTR.The expression of fluorescence and its intercellular location were determined by laser scanning confocal microscope.Cortical neurons from HAP1^+/+ and HAP1^-/- mice at postnatal day 1 were cultured and treated with biotin labeled mBDNF to trigger endocytosis.After 60 minutes, the cells were washed and fixed, followed by immunostaining and confocal imaging.Results Co-transfected PC12 cells expressed partly co-localization of HAP1 with mBDNF.Cells transfected with HAP1A-CFP which were incubated with recombinant ds-red-labeled mBDNF showed almost complete co-localization of HAP1 with mBDNF.The antibodies to BDNF and p75NTR abolished co-internalization of HAP1 with mBDNF.In addition, the labeled mBDNF was detected in almost all HAP1^+/+, but not in HAP1^-/- cortical neurons.Conclusions HAP1 plays an important role in the endocytosis of mBDNF.