【作者】 曾梅；

【导师】 周江宁；

【作者基本信息】 中国科学技术大学 ， 神经生物学， 2008， 博士

【摘要】 细胞分化是一种重要的生理现象。例如在神经系统发育过程中,正是细胞分化使得神经干细胞衍生出了具有特定形态和功能的神经元或神经胶质细胞。而特定的蛋白合成对于进行分化和维持分化的状态可能是必不可少的。因此,细胞分化可能需要在合成和降解代谢之间达到一个新的平衡。本工作旨在研究一个重要的细胞内降解途径(自我吞噬)和控制细胞合成代谢的一个关键激酶(mTOR)在神经元样分化中的潜在作用。我们使用的模型是视黄酸诱导的小鼠成神经细胞瘤(N2a细胞)的神经元样分化。通过免疫荧光方法检测神经元特征性表达的蛋白——微管相关蛋白(MAP2)和神经元特异性核抗原(NeuN),观察到诱导分化的N2a细胞有较强的MAP2和NeuN免疫反应性,而未分化的细胞只有极弱的荧光强度。此外,诱导分化的细胞具有非常明显的突触样神经突起。所以这个模型在一定程度上可以用来模拟神经元的分化过程。1.自我吞噬对N2a细胞的神经元样分化必不可少为了了解自我吞噬这一重要的降解代谢途径在细胞分化中的潜在作用,我们分析了自我吞噬在细胞分化前后的水平。首先通过免疫印迹法,检测LC3(哺乳动物中广泛应用的自我吞噬标记物)在细胞内的蛋白表达水平。研究发现,诱导分化24小时后,LC3-Ⅱ的蛋白水平显著升高。我们还观察了GFP-LC3在转染细胞中的表达模式,发现分化细胞中的GFP-LC3呈点状分布,而未分化细胞则是均一分布。这些结果表明自我吞噬在细胞分化过程中被显著激活。自我吞噬在细胞分化中的上调提示其可能对细胞分化有重要作用。为了检验这一假设,我们首先用小分子药物来抑制自我吞噬,发现两种抑制剂(3-MA和LY294002)强烈抑制了细胞分化。我们还采用了RNA干扰技术(RNAi)来降低Beclin 1(一种对自我吞噬必不可少的蛋白)的表达水平,从而下调自我吞噬。研究发现对beclin 1基因的RNAi降低了其蛋白表达水平,明显延缓了细胞分化的进程。这些结果提示自我吞噬在N2a细胞的分化过程中是必需的。2.mTOR在细胞分化中的重要作用mTOR是细胞生长的重要调控蛋白,同时也负调节自我吞噬。为了揭示自我吞噬在分化过程中被激活的分子机制,我们检测了mTOR信号通路中一系列蛋白的磷酸化水平,包括mTOR的底物S6K1和4E-BP1,以及上游的Akt和TSC2。研究发现细胞分化过程中mTOR信号通路显著下调,并且在时间上对应于自我吞噬的上调。这提示mTOR活性的降低可能导致了自我吞噬的激活。考虑到mTOR对蛋白合成的重要调节作用,我们试图通过雷帕霉素(rapamycin)充分抑制mTOR活性来观察其对细胞分化的影响。结果发现雷帕霉素显著抑制了细胞分化。在雷帕霉素的处理下,分化细胞的突起明显变细变短;细胞胞体变小;神经元特异性标记蛋白(MAP-2和NeuN)的免疫反应性也均有下降。这些结果提示,mTOR活性对细胞分化过程中大分子合成与降解之间的平衡可能至关重要。一方面,下调的mTOR信号促进了自我吞噬,而自我吞噬对细胞分化是必需的;另一方面,适当的mTOR水平对于分化细胞内的蛋白合成可能必不可少。自我吞噬的分子机制和生理功能,以及mTOR的众多调控机制在最近几年取得了大量的研究进展。我们工作的创新之处在于:运用N2a细胞的神经元样分化为模型,首次揭示了自我吞噬和mTOR在神经分化过程中的重要作用。细胞分化伴随着自我吞噬的上调和mTOR活性的下调,并且自我吞噬和适当的mTOR活性对于正常的细胞分化都是必需的。我们的研究提示细胞分化需要合成和降解代谢之间的准确调节。更多还原

【Abstract】 Cell differentiation is a critical physiological process, through which neural stem cells give rise to neurons, which display the unique morphology and functions. Due to the lack of cell proliferation, terminally differentiated cells generally show decreased cell growth. On the other hand, synthesis of specific proteins could be essential for cell differentiation and the maintenance of the differentiated status. Therefore, cell differentiation may need to acquire a new shifted balance between macromolecule synthesis and degradation. Our study aims to investigate the potential roles of autophagy (an important intracellular degradation pathway) and mTOR (a key regulator of cell growth) in neuronal differentiation.In our study, we employed the model of retinoic acid-induced neuronal differentiation in mouse neuroblastoma N2a cells. Differentiated cells showed strong immunostaining for two neuronal markers, microtubule-associated protein 2 (MAP2) and neuronal nuclear specific protein (NeuN). While uninduced cells only had weak staining. In addition, differentiated cells diplayed the typical axon-like processes.1. Autophagy is required for neuronal differentiation in N2a cells.To evaluate the potential role of autophagy in neuronal differentiation, the level of autophagy was examined during the course of differentiation. First, we analyzed the expression of LC3, a widely used marker of mammalian autophagy. After the induction of differentiation, the level of LC3-II was markedly increased, revealed by immunoblotting using anti-LC3. The expression pattern of LC3 in cells transfected with GFP-LC3 was also measured. Differentiated cells displayed the punctate pattern of GFP-LC3, while Undifferentiated cells had the diffusive distribution. These results indicate that autophagy is activated during cell differentiation.The upregulation of autophagy indicates a possible role in cell differentiation. To test this hypothesis, we inhibited autophagy by chemical inhibitors, 3-methyladenine and LY294002. Both of them strongly inhibited the differentiation of N2a cells. We further employed small interference RNA (siRNA) to knock down the expression of beclin 1,an Atg gene essential for autophagy. The beclin 1 knockdown cells exhibited a 50% reduction in Beclin 1 expression and a remarkable delay in neuronal differentiation. Therefore autophagy might be required for cell differentiation. 2. The important role of mTOR in neuronal differentiation.mTOR is a central controller of cell growth and a negative regulator of autophagy. To understand how autophagy is activated during cell differentiation, we examined the components of mTOR signaling pathway, including two well-characterized substrates of mTOR, S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E binding protein 1 (4E-BP1), as well as Akt and TSC2 (which are upstream of mTOR). Consistent with the increased level of autophagy, mTOR signaling pathway is downregulated during cell differentiation. This result indicates that the reduction of mTOR signaling may be responsible for the activation of autophagy. Regarding to the critical role of mTOR in protein synthesis, we proceeded to test whether complete inhibition of mTOR affects cell differentiation. Rapamycin, a specific inhibitor of mTOR, further inhibited mTOR activity in differentiated cells and dramatically impaired cell differentiation. Rapamycin-treated cells showed a reduction in neurite outgrowth, cell size, and immunoreactivities for MAP2 and NeuN.Our study indicate that an appropriate level of mTOR activity is important in cell differentiation, possibly via contributing to establish a new balance between macromolecule synthesis and degradation. The moderate reduction in mTOR activity may promote autophagy and at the same time enable mTOR-regulated protein synthesis involved in differentiation and cellular functions.Recent studies have gained enormous insights into the moleculear mechanisms and physiological functions of autophagy and mTOR. The novelty of this study lies in that we revealed, for the first time, the significant roles of autophagy and mTOR signaling in neuronal differentiation of N2a cells. Neuronal differentiation is accompanied by activation of autophagy and reduction of mTOR signaling. Both autphagy and mTOR signaling are vital for this physiological process. Out study indicate that the precise regulation between synthesis and degradation is critical for neuronal differentiation.更多还原