| Traditionally, it is believed that neurons can not divide. However, in the previous work in our lab, neurons from multiple sources were observed still having the ability to divide, and dividing neurons at various mitotic phases were also visualized through serial microphotography. In addition, we found the dividing neurons possessed the typical markers and electrophysiological characters of mature neurons through modern biological and medical methods.Why do neurons withdraw from the mitotic cycle? And why do neurons divide under certain conditions? This investigation provides us an opportunity about the regeneration of the central nervous system and renews the hope in developing strategies for treatment and cure of traumatic nervous system injuries, nervous system hereditary defects and degenerative diseases, such as Parkinson's disease and Alzheimer's disease.In this paper, we mainly study two molecules, Mage-H1 and Mage-D1, which have highly similar sequence structure yet possess distinct biological functions, and investigate their roles in cell cycle progression, cell proliferation and differentiation, which may provide clues in better understanding the molecular mechanisms of the withdrawal of neurons from the mitotic cycle.At the beginning, the major B cell epitopes of Mage-H1 and Mage-D1were analyzed. Then a fusion protein between His-tag and the N-terminal region (amino acids 53-295) of Mage-D1 protein was expressed in bacterium and specific antibodies were prepared.Semi-quantitative RT-PCR and Western-blot using specific PCR primers and antibodies towards Mage-H1 and Mage-D1 on native and differentiated PC12 cells revealed that Mage-H1 was up-regulated and Mage-D1 was down-regulated. So, Mage-H1 may play some functional roles in the induction and maintenance of the terminally differentiated state of cells while to some extent Mage-D1 may promote proliferation and maintain undifferentiated state of cells. When PC12 cells were transfected with pEGFP-Mage-H1 plasmid, which could express Mage-H1 protein with GFP-tag in eukaryotic cells, some PC12 cells with green fluorescence showed morphological differentiation. FACS analysis showed that PC12 cells which transiently over-expressed Mage-H1 were significantly arrested at G0-G1 phase while the percentage of cells at S phase was reduced. Then, we analyzed the amounts of cell cycle-related proteins, such as cyclin D1, cyclin E, and Cdk2, which are believed to be involved in the cell cycle control in Mage-H1 cDNA transfected PC12 cells by Western-blot. At 24 hrs, expression of Cdk2 was strikingly reduced in Mage-H1 cDNA transfectants and this reduction could also be detected at 72 hrs while the expression levels of these proteins in undifferentiated cells and cell transfected with the empty vector remained high. Moreover, stably Mage-H1-overexpressed PC12 cell lines (PC12 [myc-Mage-H1]) were established through selection with G418 antibiotic. Versus native PC12 cells, stably Mage-H1-overexpressed PC12 cells also showed arrest at G0-G1 phase and could differentiate into neurons more easily under the stimulation of NGF.In the yeast two-hybrid screening, we identified a Mage-D1 binding partner termed Rit2 from rat brain cDNA library. Ectopic expression of active Rit in PC12 cells promotes neuronal differentiation through sustained activation of the extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinase signaling pathways. We presume that the high expression levels of Mage-D1 in native PC12 cell may inhibit the activation of Rit2 to maintain the undifferentiated state of PC12 cells. |
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