The effect of taurine increasing NPC number may result

The effect of taurine increasing NPC number may result from a protective action improving cell viability or/and from an increase in proliferation. This was examined by measuring viability by the Trypan blue exclusion and MTT assays, and proliferation by BrdU incorporation. Fig. 2A shows the time course of BrdU incorporation, in cells cultured with or without taurine. BrdU incorporation was monitored at short times in culture, 1.5, 3, 6 and 12h, and thereafter every 24h up to 96h (Fig. 2A). In control cultures, the percentage of BrdU+ cells found immediately after neurosphere disaggregation in the previous passage was 14% (initial point of the curve) and this number remained essentially unchanged during the first 12h in the new culture. During the next hours (24–48h) the number of BrdU+ cells increased up to 24% and thereafter a decline was observed (Fig. 2A). The BrdU incorporation pattern in the presence of taurine was markedly different than in controls. The percentage of BrdU in cells from dissociated neurospheres was about 12%, but once seeded for a new culture, a striking and fast increase in BrdU incorporation occurred, so that as early as 1.5h of culture the number of BrdU+ cells has doubled attaining 26% of NPCs. At this time a BrdU incorporation peak was observed (Fig. 2A). BrdU incorporation rate slightly increased in the following days up to 48h, and declined thereafter. Upon neurosphere disaggregation to start a new cycle of expansion, the effect of taurine increasing BrdU incorporation occurs again, with the same window time, and this sequence of events reproduces at every passage. Comparing the BrdU incorporation curve in control and taurine cultures, it is evident that taurine was markedly increasing the number of NPCs in DNA synthesis phase during the first hours after seeding (Fig. 2A). In contrast, taurine did not affect cell viability, examined by Trypan blue exclusion and MTT assays. No difference was found at any time between 1.5 and 96h between taurine and control cultures (Figs. 2B, C). Apoptotic death estimated by TUNEL was found to be lower than 3%, in cultures either in the presence or absence of taurine (results not shown). Taurine effect increasing NPC number was examined at taurine concentrations ranging 0.1 to 30mM. Maximal effect was found at 10mM, but some increase was observed already at 0.5mM although it was significant only from 1mM (Fig. 3A). Other AS-1404 such as GABA, glycine or alanine, at the same concentration, did not modify NPC number. Only the amino acids structurally similar to taurine, hypotaurine and β-alanine, exhibited some effect increasing NPC number although it is clearly lower than that of taurine (Fig. 3B). In embryonic NPCs, GABA shows a proliferative effect at low doses (Nakamichi et al., 2009). In adult NPCs, GABA (1–10mM) induced a slight decrease in cell number (Fig. 3C). NPCs is used as a generic term including stem cell and progenitor cells. To investigate whether the effect of taurine above described occurs on the neurosphere stem cells as well as on progenitor cells, NPCs were seeded at low-density on a 96 wells plate (25 cells per well; 0.25cells/μL) and the total number of neurospheres generated after 7days was counted. Under these conditions, control cultures generated 31 (±4) neurospheres per plate while in the presence of taurine 86 (±14) neurospheres per plate were formed, an increase of 177% (Fig. 4). The clonal efficiency in cultures with or without taurine was calculated and results showed a markedly higher efficiency in the presence of taurine than in controls (Fig. 4). Taurine concentration in the SVZ of the lateral ventricles from which NPCs were obtained was 14.5μmol/g protein, but the taurine content in cells forming neurospheres in the absence of taurine is of only 2μmol/g protein. Upon taurine addition to the culture medium, this concentration strikingly increased up to 240μmol/g protein (Fig. 5B), a level higher than that of other adult brain regions (Fig. 5A), and comparable to that found in fetal brain. This ability to concentrate taurine indicates the presence of an efficient mechanism for taurine uptake into the cell. In most cell types, taurine accumulates by the operation of a well characterized energy-dependent transporter TauT. The molecular expression of TauT in NPCs was detected by immunocytochemistry. As shown in Fig. 6A TauT is expressed in essentially all (92.8±2.6%) NPCs. The functional expression of TauT was also examined. Fig. 6B shows taurine uptake by NPCs incubated with increasing concentrations of unlabelled taurine in a range of 1 to 200μM and 0.8μCi/mL of 3H-taurine. Total taurine uptake include a nonsaturable and a saturable component. The non-saturable component was identified by measuring 3H-taurine uptake when cells were incubated in a Na+-free medium (NaCl replaced by KNO3). Subtraction of this non-saturable component from total uptake revealed the saturable component. The kinetic constant values of the saturable taurine uptake calculated from the Lineweaver–Burk equation were a Vmax of 493pmol/mg protein/min and a Km of 41.7μM (Fig. 6B).