PP2ACα在小鼠大脑皮层神经元中的作用研究

发布时间：2018-06-18 08:45

  本文选题：蛋白磷酸酶2A(PP2A) + 新生死亡　； 参考：《北京协和医学院》2015年博士论文

【摘要】：蛋白磷酸酶2A (Protein Phosphatase 2A, PP2A)是真核生物蛋白磷酸酶家族中表达最为丰富的成员之一,它通过对蛋白质丝氨酸及苏氨酸残基的去磷酸化作用进而调控多种细胞进程,包括细胞增殖、分化、迁移及凋亡,细胞周期,DNA损伤应答甚至肿瘤的发生。PP2A全酶是由一个催化亚单位C,一个结构亚单位A和一个调节亚单位B所组成的异三聚体复合物。由于其调节亚单位B的底物特异性,PP2A能识别多种靶蛋白而发挥不同的生物学功能。其与相应的蛋白激酶共同调控生物体内重要蛋白质磷酸化水平与去磷酸化水平的动态平衡,这对于维持机体内正常信号转导是至关重要的。南京模式动物所首先建立了Ppp2cα基因全身敲除的小鼠模型,发现其约在胚胎第6.5天发生死亡。为了进一步研究其生物学功能,建立了Ppp2cα条件性敲除的小鼠模型,并发现在巨噬细胞、肝脏及心肌细胞中特异性敲除Ppp2cα后可分别导致机体免疫系统中Ⅰ型IFN信号通路应答增强；胚肝内红细胞生成障碍以及肝脏葡萄糖代谢异常；心肌肥大,甚至心功能不全。在此研究基础上,本课题组建立了PP2ACα中枢神经系统特异性敲除的小鼠模型(PP2ACαCNS-del)。首先,我们观察发现PP2ACαCNS-del小鼠在出生时即发生死亡,取其全脑称脑重发现较对照组相比有明显降低。随后对其全脑组织进行一系列形态学检测发现在胚胎发育期其大脑皮质中神经元存在凋亡增加以及增殖降低的现象。同时我们在体外对原代培养的大脑皮质神经元也进行了一系列增殖以及凋亡的检测,发现其结果与体内实验一致。由此我们推断,PP2ACα缺失确实可以引起皮质神经元的凋亡增加并伴有增殖降低。探究其原因,我们发现在PP2ACα缺失的皮质神经元中存在内源性DNA损伤应答ATR/Chk1信号通路的激活,并且在给予皮质神经元DNA单链损伤药物处理后,观察发现参与损伤应答的sensor-ATR与PP2AC有共定位现象。于是进一步利用免疫共沉淀的方法证实了PP2ACα缺失确实可以直接引起ATR磷酸化持续激活,进而激活下游Chk1等效应分子,从而导致内源性DNA损伤应答的激活。其过度激活进一步引起p53蛋白的表达上调,一方面通过p21靶蛋白导致细胞周期阻滞；另一方面可诱导促进凋亡的Bax表达上调,而抑制凋亡的Bcl-2表达下调。这种表达失衡最终导致了皮质神经元的异常凋亡增加。为了更全面地研究PP2ACα的生物学功能,我们又建立了在背侧皮质以及海马特异性敲除PP2ACα的小鼠模型(PP2ACαD6-del),我们发现其小鼠出生后能存活并伴有明显头小畸形(microcephaly)的表型,PP2ACα缺失的皮质神经元中也存在由于内源性DNA损伤应答激活而引起的凋亡增加现象。另外,由于海马及其对应皮质区域是调控学习和认知功能的高级中枢,我们采用以Morris水迷宫为主的行为学实验指标来评价其学习和记忆功能状态,发现PP2ACαD6-del小鼠与对照相比伴有明显的学习和记忆功能的降低,这与临床上microcephaly病人的临床体征相符合。综上所述,PP2ACα缺失可引起内源性DNA损伤应答缺陷,进而导致皮质神经元的凋亡增加并伴有增殖降低,最终表现为以皮质变薄及海马萎缩等为特征的头小畸形表型,这为探究microcephaly致病机制以及临床治疗提供了新的线索和理论依据。
[Abstract]:Protein phosphatase 2A (Protein Phosphatase 2A, PP2A) is one of the most abundant members of the eukaryotic protein phosphatase family. It regulates a variety of cell processes by dephosphorylation of protein serine and threonine residues, including cell proliferation, differentiation, migration and apoptosis, cell cycle, DNA damage response and even response. The.PP2A total enzyme is a heterogeneous tripolymer complex composed of a catalytic subunit, C, a structural subunit A and a regulatory subunit B. Because of its substrate specificity of modulating the subunit B, PP2A can recognize a variety of target proteins and play different biological functions. The dynamic balance of protein phosphorylation level and dephosphorylation level is essential to maintain normal signal transduction in the body. The Nanjing model animal first established a mouse model of Ppp2c alpha gene knockout, and found that it died on the 6.5 day of the embryo. In order to further study its biological function, Ppp2c alpha was established. The conditioned knockout mouse model found that the specific knockout of Ppp2c alpha in macrophages, liver and cardiac myocytes could lead to enhanced response of type I IFN signaling pathway in the immune system, dysfunction of erythrocyte in the liver and abnormal glucose metabolism in the liver, myocardial hypertrophy and even cardiac insufficiency. We have established a mouse model of PP2AC alpha central nervous system specific knockout (PP2AC alpha CNS-del). First of all, we observed that PP2AC alpha CNS-del mice died at birth, and the brain weight found in the whole brain was significantly lower than that of the control group. Then a series of morphological tests on the whole brain tissue of the mice were found to be found in the embryo. The apoptosis and proliferation of neurons in the cortex of the cerebral cortex were increased and the proliferation decreased. We also detected a series of proliferation and apoptosis in the primary cultured cortical neurons in vitro. We found that the results were consistent with the experiment in the body. Therefore, we infer that the deletion of PP2AC alpha does cause cortical neurons to wither. To explore the reason, we found that the activation of endogenous DNA damage response ATR/Chk1 signaling pathway was found in the cortical neurons of PP2AC - alpha deletion, and after the treatment of DNA single strand damage in cortical neurons, it was observed that the sensor-ATR and PP2AC involved in the injury response were Co located. The method of immunoprecipitation proved that PP2AC alpha deletion did directly cause the continuous activation of ATR phosphorylation, and then activated the downstream Chk1 and other effector molecules, which led to the activation of endogenous DNA damage response. Its overactivation further caused the up regulation of p53 protein expression, and one side through the p21 target protein resulted in cell cycle arrest. On the other hand, the expression of Bax, which promotes apoptosis, is up-regulated, and the inhibition of apoptosis is down down. This imbalance eventually leads to the abnormal apoptosis of cortical neurons. In order to study the biological function of PP2AC alpha more comprehensively, we also established a mouse model (PP2AC alpha D) in the dorsal cortex and the sea Matt heterosexual knockout of PP2AC a. 6-del), we found that the mice were able to survive and have a distinct head small deformity (microcephaly) phenotype after birth. There was also an increase in apoptosis caused by the activation of endogenous DNA damage responses in the cortical neurons of PP2AC alpha deletion. In addition, the hippocampus and its corresponding cortex area are the advanced Centers for regulating learning and cognitive function. We evaluated the learning and memory function of the Morris water maze, and found that the PP2AC alpha D6-del mice were associated with a significant decrease in learning and memory function compared with the control, which was consistent with the clinical signs of the clinical microcephaly patients. To sum up, the deletion of PP2AC alpha could cause endogenous DNA damage. The answer to the defect leads to the increase of cortical neuron apoptosis and the decrease of proliferation. Finally, the phenotype of the head small deformity characterized by the thinning of the cortex and the atrophy of the hippocampus, provides a new clue and theoretical basis for exploring the pathogenesis and clinical treatment of microcephaly.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R741
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本文编号：2034882