| Neurogenesis is a multi-stage process in which neural stem cells (NSCs) self-renew, convert into progenitors by asymmetrical cell division, differentiate, migrate into the destinations, mature and finally integrate into the exiting neural circuits for physiology function, which is also referred to the brain plasticity. In the adult mammalian central nervous system, active neurogenesis under normal conditions is spatially restricted to two specific "neurogenic" brain regions, the subventricular zone (SVZ) of the lateral ventricles and the subgranular zone (SGZ) in the dentate gyrus of the hippocampus. In the SVZ, the neural precursors derived from NSCs migrate along the rostral migratory stream (RMS) into the olfactory bulb to become interneurons. In the SGZ, newly generated neurons become granule neurons at the inner granule cell layer after a short-distance migration, extend their long axonal projects through the mossy fiber pathway, and reach their target and form a functional synapse in the CA3 pyramidal cell layer within 4 to 10 days after division. Due to the importance in learning, memory, and possibly mood (depression), neurogenesis in the hippocampus is gaining more and more attention in recent years.Phosphofructokinase-1 (PFK-1) is a rate limiting enzyme in glycolysis by phosphorylating fructose-6-phosphate to form fructose-1,6-bisphosphate, and plays an essential role in energy metabolism. Evidence showed that PFK-1 is crucial for the growth and development of the tumor and become a potential therapeutic target. In addition, PFK-1 is also involved in mediating the different functions of neural cells, such as neurons and astrocytes in the central nervous system. However, little is known about the effect of PFK-1 on the NSCs, another important population in the central nervous system.Here, we investigated the following questions:(1) whether and how PFK-1from NSCs is involved in mediating neurogenesis from NSCs in physiology? (2) Dose PFK-1 play a similar role to the one under physiological conditions in neuronal differentiation of NSCs after hypoxia? (3) What are the potential mechanisms responsible for the neurogenesis mediated by PFK-1?Chapter 1. Role of PFK-1 in neurogenesis from NSCs in physiologyTo investigate the role of PFK-1 in the function of NSCs in physiology, we analyzed the effect of PFK-1 knockdown and overexpression on neurogenesis in vitro by generating a lentiviral vector containing shRNA of PFK-1 (LV-PFK-1-shRNA) and another one expressing full-length PFK-1 (LV-PFK-1-GFP), respectively. After 4 days of differentiation, expression of β-Ⅲ-Tubulin, a cell-type-specific marker for neurons, was assessed by immunofluorescence. The results showed that LV-PFK-1-shRNA-infected NSCs displayed a significantly increased neuronal differentiation and enhanced percentage of neurons with multi-neurites, whereas LV-PFK-1-GFP-infected NSCs exhibited a substantially decreased neuronal differentiation, as well as a lowered number of neurons with multi-neurites, suggesting a negative role of PFK-1 in neurogenesis from NSCs. Meanwhile, compared with LV-Control-shRNA-infected NSCs, the percentage of Nestin+cells in LV-PFK-1-shRNA-infected NSCs decreased significantly and spread out uniformly when cultivated in proliferation medium, while Nestin+NSCs with LV-Control-shRNA infected showed a tendency to form neurospheres, a biological character of NSCs, indicating a weakened capacity for NSCs to maintain self-renewal, or an enhanced tendency to differentiate. All these observations here, consistent with the results above, confirm that PFK-1 negatively regulates neuronal differentiation and development in physiology.We next examined whether PFK-1 affects the neurogenesis in the DG of hippocampus in physiology. To address this notion, we treated mice with BrdU, a marker of dividing cells, to label proliferating cells at day 4 and day 5 after microinjecting LV-PFK-1-shRNA or LV-Control-shRNA into the DG of hippocampus. Mice were sacrificed at day 14 to estimate the number of GFP+/BrdU+ (a marker for proliferating and dividing cells infected by the lentivirus) cells and GFP+/BrdU+/DCX+(a marker for immature neurons differentiated from the infected NSCs) cells. In line with the results in vitro, compared with LV-Control-shRNA treatment, LV-PFK-1-shRNA injection both significantly raised the numbers of GFP+/BrdU+/DCX+newborn neurons and GFP+/BrdU+dividing cells in granular cell layer (GCL) of the DG, suggesting an increased neurogenesis from the LV-PFK-1-shRNA-infected NSCs in the DG of hippocampus.However, lentiviral delivery of shRNA would result in an overall knockdown of PFK-1 in the entire population of DG cells (e.g., NSCs, neurons and glia cells), which might interfere with the effect of PFK-1 knockdown on neurogenesis from NSCs. To solve this problem, we assessed neurogenesis, the numbers of newborn neurons (GFP-/BrdU+/DCX+) and dividing cells (GFP-/BrdU+/DCX+), from the NSCs which were not infected by the lentivirus. The results proved no significant difference for both between LV-Control-shRNA treatment and LV-PFK-1-shRNA treatment, indicating that non-cell-type-specific knockdown of PFK-1 dose not play a role in neurogenesis from the uninfected NSCs. Thus, it can be deduced that non-cell-type-specific knockdown of PFK-1 dose not affect neurogenesis from the lentiviral infected NSCs, either. On the whole, these results, in combined with the observations in vitro, indicate that knockdown of PFK-1 enhances neurogenesis from NSCs in the DG.The results above has proved that PFK-1 knockdown promotes neuronal differentiation, which might be attributed to better neuronal survival, enhanced proliferation of progenitors, and/or elevated neuronal cell fate commitment. Firstly, the roles of the former two factors in PFK-1 knockdown-mediated neurogenesis were analyzed by incubating NSCs with Hoechst and BrdU, respectively. The results showed that, compared with LV-Control-shRNA, LV-PFK-1-shRNA markedly promoted proliferation of neural progenitors, but had no significant impact on survival of neural progenitors. Then, to illuminate whether the neurogenesis is ascribed to increased neuronal fate commitment of NSCs, we established a mathematical description of neurogenesis, in which in which βj was defined as the conversion rate of progenitors (β-ⅢI-Tubulin-) to neurons (β-Ⅲ-Tubulin+) at the jth day. The maximum and minimum of βj were estimated by the assumption that no neurons were dead or all dead cells were neurons, respectively. The results showed that both the maximum (βmax) and minimum (βmin) conversion rate of progenitors increased markedly in LV-PFK-1-shRNA infected NSCs as compared with NSCs infected with nontargeting shRNA-expressing control virus. Taken together, PFK-1 knockdown upregulates neurogenesis by enhancing proliferation rate of progenitors and potentiating neuronal fate commitment of NSCs, but has no effect on cell survival.Chapter 2. Effect of PFK-1 on neruogenesis from NSCs after hypoxiaThe role of PFK-1 on neuronal differentiation after hypoxia is investigated in this chapter. We subjected cultured NSCs with PFK-1 knockdown or overexpression to hypoxia for different time periods, followed by western blot analysis immediately or immunofluorescence after 4 days of normoxic culture. The expression level of PFK-1 was significantly upregulated by hypoxia in a time-dependent manner after hypoxia. Meanwhile, slightly increase of neuronal differentiation of NSCs was also observed, which, however, was inconsistent with the observation in physiology that overexpressing PFK-1 in NSCs markedly inhibited neuronal differentiation. This inconsistency might be ascribed to some factors that were involved in the hypoxia-induced neurogenesis were activate, and counteract the effect of PFK-1 knockdown induced neurogenesis. In addition, the LV-PFK-1-shRNA treatment, as expected, significantly elevated the percentage of neurons and neurons with multi-neurites, as compared with LV-Control-shRNA treatment after hypoxia. In contrast, overexpression of PFK-1 in NSCs markedly reversed the phenotype above. All together, the results above illustrate that PFK-1 inhibits neurogenesis from NSCs after hypoxia.Chapter 3. Potential mechanisms responsible for the role of PFK-1 in neurogenesis.In this chapter, mechanisms underlying PFK-1-mediated neurogenesis were investigated in this chapter. Accordingly, we investigated whether knockdown or overexpression of PFK-1 alters the expression of Mash 1, NeuroD and Sox2 in vitro. Firstly, in physiology, we allowed NSCs with PFK-1 knockdown or overexpression to differentiate for 6 h followed by western blot analysis, and found that PFK-1 knockdown significantly increased the expression levels of Mash 1, NeuroD and Sox2, contrastively, overexpression of PFK-1 decreased the expression levels, indicating that PFK-1 might attenuate neurogenesis via regulation of neural transcription factors. Additionally, expression levels of Mash 1, NeuroD and Sox2 were also analyzed after 5 h of hypoxia. Similarly, knockdown of PFK-1 increased and overexpression of PFK-1 decreased the expression of the transcriptional factors. Collectively, these data suggest that PFK-1 may negatively regulate neurogenesis from NSCs via regulating Mash 1, NeuroD and Sox2. Moreover, we examined whether PFK-1 negatively mediated neurogenesis via Wnt/β-catenin signaling, which was proved to be unaffected by Wnt/β-catenin signaling.Taken togther, our results therefore indicate that:(1) under physiological conditions, PFK-1 negatively modulates the the neuronal differentiation and the development of dendrites by targeting the proliferation and neuronal fate commitment of progenitors. (2) After hypoxia, PFK-1 also exerts a negative control on neuronal differentiation and development. (3) The effect of PFK-1 neurogenesis is probably achieved by targeting the neural transcriptional factors. |
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