br Materials and methods br Results br Discussion To evaluat

Materials and methods
Results
Discussion To evaluate the effects of proNGF on NSC proliferation, we performed cell viability assay, BrdU incorporation assay and neurosphere assay. Our results showed that proNGF significantly reduced the total number of viable cells and the proportion of BrdU-positive cells, as well as the neurosphere size, suggesting that proNGF exerted an inhibitory role on NSC proliferation. Proliferation is intimately controlled by a progression in the cell cycle. Mitogenic control of proliferation occurs early in G1, allowing cells to bypass the first decision point (R1) in this cycle when a cell may either continue to proliferate or exit the mi2 (Lange et al., 2009). The results of flow cytometry demonstrate that proNGF increases the distribution of cells in the G1/G0 phase at the expense of the S phase and downregulates the expression of cyclin E, an important positive regulator driving cells to pass the G1 phase. These findings are in accordance with the inhibitory effect of proNGF on NSC proliferation. Our data showed that proNGF reduced the proportion of oligodendrocyte lineage after NSCs differentiated, accompanied by a slight increase in the proportion of astrocytes. However, the ratio of neurons was not substantially changed. Previous reports have shown that transcription factors regulate cell-type-specific differentiation during CNS development. Olig1 and Olig2, two basic helix–loop–helix (bHLH) transcription factors, are believed to facilitate oligodendrogenesis during CNS development (Rowitch et al., 2002). Moreover, downregulation of Olig2 has been shown to cause a switch from an oligodendrocyte to an astrocyte fate in neural stem cells (Zhu et al., 2012). In this study, Olig2 and Olig1 were found to be downregulated under the differentiation conditions after 24-hour and 4-day treatments of proNGF respectively, indicating that proNGF might inhibit oligodendrogenesis partially through regulating the expression of oligodendrocyte lineage-specific transcription factors. p75NTR, initially recognized as a low-affinity pan-receptor for the neurotrophin family, has been demonstrated to preferentially bind proNGF with the highest specificity and affinity (Lee et al., 2001). Recently, several studies have provided evidence for the involvement of p75NTR in NSC proliferation and differentiation. It was proposed that p75NTR defined a discrete population of highly proliferative SVZ precursor cells and that the absence of p75NTR disrupted neurotrophin-regulated neurogenesis in vivo (Young et al., 2007). p75NTR deficiency also significantly decreased the number of BrdU-positive cells in the hippocampus of adult mice (Bernabeu and Longo, 2010). In addition, p75NTR is essential for the action of BDNF and NT3 on oligodendrocyte differentiation (Du et al., 2006). In this study, we confirmed the expression of p75NTR on hippocampal NSCs in culture. The addition of p75NTR/Fc fusion protein into the culture medium as a scavenger of proNGF significantly reversed its effects on NSC proliferation and oligodendrocyte differentiation. Moreover, deletion of the ligand-binding extracellular domain of p75NTR abolished the responsiveness of NSCs to proNGF. These results demonstrated an important role of proNGF/p75NTR interaction in the functions of proNGF. It is interesting to note that p75NTR deficiency resulted in a remarkable decrease in NSC proliferation and oligodendrogenesis, which is consistent with previous studies (Bernabeu and Longo, 2010; Du et al., 2006). It is likely that p75NTR deficiency depletes the beneficial effects of certain ligands such as NGF and NT3 secreted by NSCs themselves, which has been shown to promote NSC proliferation and oligodendrogenesis. Another possibility is that p75NTR functions in a ligand-independent manner which might be inhibited by proNGF binding as proposed by Bredesen and Rabizadeh (1997). However, neither p75NTR deficiency (data not shown) nor proNGF treatment exerted an obvious effect on neurogenesis, which seems to be contradictory to previous observations on SVZ NSCs (Young et al., 2007). It might be partially explained by a relatively low level of neuron differentiation in our experimental condition. Alternatively, NSCs from different regions of the brain are not all equivalent and have specific responses to stimulators (Thiriet et al., 2011). Furthermore, the downstream signaling pathways of p75NTR are so complicated that proNGF may induce quite different responses from those induced by mature neurotrophins.