Taurine Effect on Stem Cells and Synaptogenesis Figure 1. Neural progenitor cells (NPCs) isolated from 5 days old mouse hippocampus are multipotent. These NPCs can proliferate and self-renew in culture, as demonstrated by the expression of proliferation marker, Ki67 and the standard NPCs markers, Nestin, Sox2, BLBP and Vimentin (A). All DAPI positive cells are positive for Nestin and the dividing cells are labeled with EdU (B). Scale bar = 20 mm. doi:10.1371/journal.pone.0042935.g001 ERK1/2 is involved in the stimulatory effect of taurine on NPC proliferation. The Effect of Taurine on Neuritogenesis by NPC Differentiated Neurons and Rat Embryonic Primary Neurons To further investigate the effects of taurine on neuronal development, we studied the effect of taurine on neurite growth in neurons differentiated from P5 neural progenitor/stem cells and cultured E18 rat embryonic primary neurons. We found that taurine has a moderate influence on neurite growth. It modestly increased the neurite length in NPCs (16%) and in primary neurons (18%). However, no significant difference in neurite number was observed between control and treated cells (Fig. 8A– F). Higher concentration of taurine (for example, at 2.7 mM) significantly decreased the neurite length and number of neurite in neurons differentiated from NPCs, therefore, we used only lower concentration (100 mM) to check on its effect on primary neurons. Taurine Increased Synaptic Proteins Expression in Hippocampal Primary Neurons and Cultured Hippocampal Slices To determine if taurine induces synaptogenesis in the neurons of the brain, we added taurine (100 mM) to cultured hippocampal primary neurons from 2 to 15 days in vitro (DIV). At the end of the incubation period, the neurons were immunostained using an antibody against PSD-95, an excitatory postsynaptic marker. Taurine treatment led to 60% increase (P,0.01) in the density of PSD-95 positive puncta (Fig. 9B, C). We also immunostained for synapsin 1, the marker of the presynaptic terminal, at the end of 9 DIV and also observed a significant increase (P,0.01) in the density of synapsin 1 positive puncta after taurine treatment (Fig. 9A, C). We also determined the protein expression levels of synapsin 1 and PSD 95 using western blot analysis and observed an increase in expression of synapsin 1 (Fig 9D, F, P,0.05) and PSD 95 (Fig. 9E, F, P,0.05). The increased expression levels of PSD-95 and synapsin 1 indicate that taurine also has important role in synaptogenesis. Discussion Our results documented the importance of taurine in the developing brain cells by enhancing proliferation and synapto- genesis. We demonstrated in this study that taurine supplemen- tation induces proliferation of cultured P5 neural stem cell/ progenitor cells, and stem cells in dentate gyrus of cultured organotypic hippocampus slices derived from P5 mice, signifying the effect of taurine in early postnatal development. It has been reported that monkeys fed with formulas without taurine showed a prominent defect in organization of cortical layers in the visual cortex [26]. Cats born from taurine-deficient mothers have smaller brain weight and an abnormal morphology in the cerebellum and the visual cortex [19,20]. Delayed migration of neuroblasts and PLOS ONE | www.plosone.org 3 August 2012 | Volume 7 | Issue 8 | e42935