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 µm.
Figure 2.
Effects of taurine treatment on proliferation of P5 hippocampal stem/progenitor cells.
Cells were treated with five different concentrations (10 µM, 100 µM, 500 µM, 2.5 mM and 5 mM) of taurine for 48 h and labeled with EdU (10 µM) in the last 3 h of incubation. Data are presented as percentage of EdU positive cells normalized to control (A). Data are expressed as mean ± SEM. * P<0.05 and ** P<0.01. Representative microscopic images showing EdU-labeled P5 hippocampal progenitor cells in control (left panel) and taurine treated groups (right panel)) (B). Scale bar = 20 µm.
Figure 3.
Effects of taurine treatment on cell proliferation in the dentate gyrus of cultured hippocampal slices.
EdU labeling was used to assess cell proliferation. The culture medium was changed to serum free condition on day 7 and taurine was added to the medium on day 9. EdU (10 µM) was added to the culture medium at day 11 and the slices were fixed and processed for EdU staining on day 17. Numbers of EdU positive cells in dentate gyrus were analyzed and the data are presented as mean ± SEM. (* P<0.05) (A). Images showing EdU-labeled cells in the dentate gyrus of control (upper panel) and taurine treated groups (lower panel) (B). SGZ and GCL denote subgranular zone and granule cell layer respectively. Scale bar = 20 µm.
Figure 4.
Effects of taurine treatment on cell proliferation in the dentate gyrus of embryonic hippocampus.
The embryonic brains were fixed at E-17. EdU intensity was measured for 6–7 identical sections per brain and at least 5–6 fetuses were used. The data of the percentage changes in EdU intensity are presented as mean ± SEM. (A). Representative images showing EdU-labeled cells (red) in the dentate gyrus of control (left panel) and taurine treated groups (right panel) (B). P>0.05, Scale bar = 20 µm.
Figure 5.
Effects of taurine treatment on cell differentiation.
EdU labeling and differentiated cell markers (DCX labels neurons and GFAP labels astrocytes) were used to assess the differentiation and fate choice of newly generated cells in NPCs and in DG of cultured hippocampal slices. Representative images of cultured P5 hippocampal stem/progenitor cells treated with taurine (100 µM) and allowed to differentiate for 5 days before fixation for immunohistochemical processing (A). Quantitative analysis of cells positive for EdU and DCX or GFAP indicating that the percentage of DCX or GFAP was not altered by taurine treatment (B). Representative images showing cells positive for EdU and DCX or GFAP in DG of hippocampal slices (C and D). Quantitative analysis of cells positive for EdU and DCX or GFAP in the dentate gyrus indicating that the percentage of DCX or GFAP was not altered by taurine treatment (E). All quantitative data are expressed as mean ± SEM. Scale bar = 20 µm.
Figure 6.
ERK1/2 is involved in taurine induced NPC proliferation.
The cells were incubated with or without 100 µM taurine for 48 hrs. ERK1/2 inhibitor, PD98059 was added to the media 30 min before taurine treatment. Representative immunoblots showing protein levels of phosphorylated ERK1/2 (A) and total ERK1/2 (C). Graphs showing the ratio of densitometric measurements of phosphorylated ERK1/2 and total ERK1/2, normalized with total protein levels of α-tubulin. (B and D). Graph showing fold change in phosphorylated ERK1/2 upon treatments as indicated, normalized with total ERK1/2 (E) * P<0.0 5 and ** P<0.01 as compared with the control. The western blots are representative blots from at least three individual experiments.
Figure 7.
Taurine-induced proliferation of P5 hippocampal stem/progenitor cells is dependent on the activation of ERK1/2.
Cultured hippocampal stem/progenitor cells were incubated with ERK1/2 inhibitor PD98059 for 30 min followed by treatment with taurine for 48 h and labeling with EdU (10 µM) in the last 3 h of incubation. Quantitative data presented as percentage of EdU positive cells normalized to control, indicates pretreatment with ERK1/2 inhibitor attenuates the taurine induced increase in the number of EdU-labeled cells (A). Representative images showing EdU -labeled P5 hippocampal progenitor cells in control (left panel), taurine treated groups (centre), and ERK1/2 inhibitor + taurine (right panel) (B). Scale bar = 20 µm. All data presented here are expressed as mean ± SEM. * P<0.05.
Figure 8.
The effects of taurine on neurite outgrowth of NPCs and primary neurons.
The average total neurite length (A and D) and neurite numbers (B and E) were measured. Images in C and F show representative primary neurons and NPCs treated with vehicle (left panel) or taurine (right panel) respectively. Taurine was added to the media on day 2. Primary neurons and NPCs were then fixed on day 5 and 7 respectively. Differentiated neurons were then analyzed and data presented as mean ± SEM. Taurine shows a small non-significant effect on neurite development of primary neurons as well as newborn neurons. Scale bar = 20 µm.
Figure 9.
The relative percentage of the effect of taurine on synaptic puncta and protein levels.
Taurine was added to the primary neurons on day 2 and the neurons were then fixed and processed at day 9 and 15. The quantification of synapsin 1 and PSD 95 positive puncta was done by measuring the number of puncta per µm. Data are presented as percentages normalized to the control (100%) ± SEM and puncta from at least 20 different neurons of each batch from at least three repeated experiments were quantified. Taurine treatment significantly increased the number of Synapsin 1 (A) and PSD 95 (B) puncta. Representative images show synapsin 1 (top panel) and PSD 95 puncta (bottom panel) respectively (C). The protein levels of synapsin 1 (D) and PSD95 (E) in protein lysates in primary neurons were shown using western blotting analysis (D-F). Data were normalized with total protein levels of α-tubulin. * P<0.05 and ** P<0.01. Scale bar = 1 µm.