Roles Of Autophagy And MTOR Signaling In Neuronal Differentiation Of Mouse Neuroblastoma Cells

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.