Effect Of3,5,3'-Triiodothyronine On Post-mitotic Neurons

Neurons in the CNS of mammals are typically considered as post-mitotic cells and not capable of dividing. However, the molecular mechanism that underlies the cell cycle exit of mature neurons is still unclear and it is always an attractive issue in Neuroscience research that whether post-mitotic neurons could re-enter cell cycle. In our previous study, N4was used to induce down-regulation of Necdin and neuronal dividing. And it was reported that3,5,3'-Triiodothyronin(T3), an ingredient of N4, could decrease the expression of Necdin in differentiated P19cells. Thus, the purpose of this study was to investigate the effect of down-regulated Necdin expression on post-mitotic neurons and the primary cultured rat cortical neurons would be our research model.Chapter one:Primary culture and evaluation of rat cortical neuronsPrimary culture of neurons is an important model for Neuroscience reaesrch. In this chapter, cortical neurons were obtained from E14-16d Wistar rats. Serum-free medium was used and1.5μg/ml Ara-C was added for48h to remove dividing cells so as to gain a satisfactory purity of neurons. Meanwhile, conditioned medium from glial cells was used to provide nutritional support. Subsequently, the purity was identified through immunocytochemistry and flow cytometry assay5days post-seeded.Immunocytochemistry staining was carried out and it was confirmed that most of the cells in the primary culture were neurons, and a small number of astrocytes were observed, while little precursor cell or nerve stem cell could be found. Meanwhile, result of flow cytometry assay showed that almost all the cells in the primary culture were post-mitotic neurons.Chapter Two:Effect of down-regulated Necdin expression by Lentivirus on neuronal cell cycleIn our previous study, a key molecular named Necdin was found to play an important role on cell cycle exit of post-mitotic neurons and maintaining the post-mitotic state. In this chapter, necdin-RNAi-LV was used to investigate the impact of down-regulated Necdin expression on neuronal cell cycle.Firstly, immunofluorescence labeling was used to detect the infection efficiency of the Lentivirus, with ShcC being the specific marker of neurons. The result showed that79.81%and81.85%of neurons in the primary culture were infected with control Lentivirus and necdin-RNAi-LV, respectively.Secondly, expression of Necdin interfered by Lentivirus was examined by Real-time PCR and Western Blot, which indicate that Necdin expression was decreased at both mRNA and protein level.Based on the above results, impact of necdin-RNAi-LV on neuronal cell cycle was investigated through immunofluorescence labeling of ShcC. Neurons in different mitotic phases were observed4days post-infection, suggesting that down-regulated Necdin expression could induce neuronal dividing. This was consistent with the previous results that N4could down-regulate the expression of Necdin and induce neuronal dividing, confirming the relation between down-regulated expression of Necdin and neuronal dividing.Chapter three:Effect of T3on primary cultured neuronsIn this chapter, the impact of T3on primary cultured neurons was evaluated, including expression of Necdin, subcellular location of E2F1, neuronal cell cycle, survival rate and cell apoptosis.In part one, expression of Necdin was examined with RT-PCR and Western Blot both showing a down-regulated tendency.In part two, expression and subcellular location of E2F1was assessed by Western Blot and immunofluorescence staining, respectively. No changes were found on expression of E2F1, but its subcellular location was tranferred from the whole cell body to the nucleus48h post-treatment with T3.In part three, neuronal cell cycle was evaluated through several methods, including immunofluorescence labeling, flow cytometry assay and Western Blot. Neurons in different mitotic phases were observed48h post-treatment with T3. While, results from flow cytometry assay showed no diffidence between control group and T3group. Similarly, no changes were observed on the expression of Cyclin D, and it failed to detect expression of Cyclin E and Cyclin A. These results probably suggest the amount of dividing neurons was too little to be detected by flow cytometry assay and Western Blot. Besides, the results could be affected by a small quantity of dividing cells in the primary culture.In part four, CCK-8method was utilized to detect survival rate of neurons after T3treatment. And a significant decrease on survival rate was found24and48hours post addition of T3.In part five, several methods, including TUNEL, Annexin V/PI and Western Blot, were used to investigate the effect of T3on neuronal apoptosis. It was indicated that T3played an important part in the activation of Caspase3and apoptosis of neurons.In part six, real-time PCR and Western Blot were used to assess the impact of T3on the expression of P53, Bax, Bcl-2, as well as the active form of P53. However, no significant changes were found on the expression of P53, Bax and Bcl-2, and it failed to detect the expression of phosphorylated P53. This suggests that the neuronal apoptosis induced by T3may through P53-independent pathways.In a word, primary cultured neurons were used as our research model in this study, and it was shown that both necdin-RNAi-LV and T3could down-regulate Necdin expression and induce neuronal dividing. Meanwhile, T3would decrease the survival rate of neurons and lead to neuronal apoptosis probably through P53-independent pathways.