Fig 1.
Expression of neural precursor cell markers and GABAB receptor subunits in secondary neurospheres derived from the subventricular zone.
Microphotographs showing the immunoreactivity to precursor cell markers and GABAB subunits of cultured neurospheres. The secondary neurospheres exhibited a mean ratio of 70 ± 5 um. The nuclei of the proliferative cells within the neurosphere were labeled with DAPI staining. Immunoreactivity signals were detected for A) nestin (red) and B) KI67 (green), indicating the presence of neural progenitor cells and active proliferation, respectively. Likewise, the neurospheres exhibited immunoreactivity to the GABAB receptor subunits, C) GABABR1 (red), and D) GABABR2 (red). All the microphotographs were taken with a 20x magnification lens. The scale bar applies for all the panels.
Fig 2.
The interaction of the GABAA receptor and taurine promotes neurite outgrowth of NPC SVZ.
A) Representative bright-field and immunofluorescence assays microphotographs against DCX for immature neurons in the control condition or in taurine (10 mM). The nuclei were stained with DAPI (blue) and DCX (red). Arrowheads indicate cells with neuronal-type morphology used for the analyses included in this study. B) Scatter plot showing the percentage of DCX+ cells in the control and in taurine (n = 8 cell cultures for each experimental condition). C) Scatter plot with the total number of neurites in the control condition (n = 36) and in taurine (n = 40) DCX+ cells). D) Sholl-like analysis contrasting the number of neurite intersections in the control condition and the taurine-treated cells. Upper inset: area under the curve (AUC) chart showing the total area obtained from the Sholl-like analysis performed in the control and taurine-treated cells (n = 8 DCX+ cells). E) Cumulative probability chart summarizing the number of neurite intersections in the control cells versus the taurine-treated cells. The right shift of the taurine-treated cells mirrors the increased number of neurite intersections (n = 8 for each experimental condition). F) Upper panels: representative digital reconstructions and concentric rings (distance between rings = 10 μM) used for the Sholl-like morphometric analysis. Bottom panel: examples of the increased number of primary, secondary, and tertiary neurites of the taurine-treated cells. G) Left panel: a patch-clamp pipette attached to an NPC SVZ incubated with taurine. Notice the cell body and the neurite outgrowths. Middle panel: representative current traces obtained in a control and a taurine-treated cell. The blue arrowheads indicate the inward current, the black arrowheads indicate the fast-inactivating and the persistent, steady-state outward current. Right panel: magnification of the inward currents, black trace control, red trace, inward current of a taurine-treated cell. Bottom left panel: plot of the voltage dependence and maximal amplitude of the inward current. Middle panel: plot showing the maximal peak amplitude of the fast-inactivating outward current. Right panel: plot depicting the amplitude of the non-inactivating steady-state outward current. **p < 0.01; ***p < 0.001 or higher statistical significance. Error bars indicate SEM.
Fig 3.
Pharmacological blockade of GABAA receptor inhibits neurite outgrowth of NPC SVZ.
A) Bright-field and immunofluorescence assays. Nuclei were stained with DAPI (blue) and DCX (red). Arrowheads show cells with a neuronal-type morphology obtained in the indicated experimental conditions. B) Scatter plot summarizing the percentage of DCX+ cells in the control and in the presence of taurine and taurine + PTX (n = 8 cell cultures for each experimental condition). C) Scatter plot showing the total number of neurites in the indicated experimental conditions. PTX prevented the neurite outgrowth observed stimulated with taurine (control n = 34 cells; taurine n = 38 cells; taurine + PTX n = 27 DCX+ cells). D) Sholl-like analysis with the number of neurite intersections in the indicated experimental conditions. Upper inset: AUC chart summarizing the findings of the Sholl-like analysis (n = 8 DCX+ cells). E) Cumulative probability chart. The left shift of the taurine + PTX–treated cells (blue line) mirrors the decreased number of neurite intersections when GABAA receptors were blocked (control n = 34 cells; taurine n = 38 cells; taurine + PTX n = 27 DCX+ cells). F) Representative digital reconstructions and concentric rings used for the Sholl-like analysis. Notice the decreased number of primary, secondary, and tertiary neurites of the taurine + PTX–treated cells (right panel). G) Upper panel: representative current traces obtained from cells treated with taurine or taurine + PTX. Blue arrowheads indicate the inward currents, black arrowheads the fast-inactivating and the persistent, steady-state outward current. Upper right panel: magnification of the inward current from a taurine-treated cell (red trace) and a taurine + PTX–treated cell (blue trace). Bottom left panel: voltage dependence and maximal amplitude of the inward current. Middle panel: the maximal amplitude of the fast-inactivating outward current. Right panel: the steady-state amplitude of the non-inactivating outward current. ns = non-significant statistical difference; **p < 0.01; ***p < 0.001 or higher statistical significance. Error bars indicate SEM.
Fig 4.
Activation of the GABAB receptor with baclofen does not stimulate the differentiation process of NPC SVZ.
A) Bright-field and immunofluorescence assays microphotographs for DCX in the control cells, taurine (10mM), and taurine + baclofen (100 μM)–treated cells. Nuclei were stained with DAPI (blue) and DCX (red). Arrowheads show cells with a neuronal-type morphology obtained in the indicated conditions. B) Scatter plot showing the percentage of DCX+ cells in the indicated experimental conditions. Stimulation with baclofen did not alter the number of DCX+ cells (n = 8 cell cultures for each experimental condition). C) Comparison of the total number of neurites in the indicated experimental conditions. The combination of taurine + baclofen did not stimulate neurite outgrowths compared to the taurine-treated cells (control n = 33 cells; taurine n = 43 cells; taurine + baclofen n = 37 DCX+ cells). D) Sholl-like analysis obtained from the indicated experimental conditions. Upper inset: AUC chart summarizing the findings of the Sholl-like analysis (n = 8 DCX+ cells). E) Cumulative probability chart of the number of neurite intersections in the three experimental conditions (control n = 33 cells; taurine n = 43 cells; taurine + baclofen n = 37 DCX+ cells). F) Digital reconstructions and concentric rings used for morphometric analyses. Notice the subtle changes in the number of primary, secondary, and tertiary neurites of the taurine + baclofen–treated cells (right panel). G) Upper panel: representative current traces obtained from cells treated with taurine or taurine + baclofen. Blue arrowheads indicate the inward currents, black arrowheads the fast-inactivating and the persistent, steady-state outward current. Upper right panel: magnification of the inward current from a taurine-treated cell (red trace) and taurine + baclofen–treated cell (blue trace). Bottom left panel: voltage dependence and maximal amplitude of the inward current. Middle panel: the maximal amplitude of the fast-inactivating outward current. Right panel: the steady-state amplitude of the non-inactivating outward current. ns = non-statistical significance; **p < 0.01; ***p < 0.001 or higher statistical significance. Error bars indicate SEM.
Fig 5.
The blockade of GABAB receptors with CGP 55845 favors the differentiation process and neurite outgrowth of NPC SVZ.
A) Bright-field and immunofluorescence assays microphotographs for DCX in the control condition, taurine, and taurine + CGP 55845 (5 μM). Arrowheads indicate cells with a neuronal-type morphology. B) Scatter plot summarizing the percentage of DCX+ cells in the indicated experimental conditions (n = 8 cell cultures for each experimental condition). C) Scatter plot summarizing the percentage of total neurites. Taurine + CGP 55845 increased the total number of neurites (control n = 38 cells; taurine n = 47 cells; taurine + CGP 55845 n = 43 DCX+ cells). D) Sholl-like plot contrasting the number of intersections in the indicated experimental conditions. Upper inset: AUC chart (n = 8 cells for each experimental condition). E) Cumulative probability chart summarizing the number of neurite intersections. The right shift of the taurine + CGP 55845–treated cells mirrors the increased number of neurite intersections (n = 6 DCX+ cells). F) Representative digital reconstructions and concentric rings. Notice the increased number of primary, secondary, and tertiary neurites of the taurine + CGP 55845–treated cells. G) Upper panel: representative current traces obtained from NPC SVZ treated with taurine or taurine + CGP 55845. Blue arrowheads indicate the inward currents, black arrowheads the fast-inactivating and the persistent, steady-state outward current. Upper right panel: magnification of the inward current from a taurine-treated cell (red trace) and a taurine + CGP 55845–treated cell (blue trace). Bottom left panel: voltage dependence and maximal amplitude of the inward current. Middle panel: the maximal amplitude of the fast-inactivating outward current. Right panel: the steady-state amplitude of the non-inactivating outward current. ns = non-statistical significance; *p < 0.05; **p < 0.01; ***p < 0.001 or higher statistical significance. Error bars indicate SEM.
Fig 6.
Taurine involves intracellular calcium mobilization for the differentiation process and neurite outgrowth of NPC SVZ.
A) Scatter plot showing the number of neurites of NPC SVZ exposed to taurine or taurine + BAPTA-AM. Intracellular calcium chelation prevented neurite outgrowth. B) Scatter plot showing neurite length in the indicated experimental conditions. BAPTA incubation inhibited neurite outgrowth stimulated with taurine (control n = 31 cells; taurine n = 36 cells; taurine + BAPTA-AM n = 12 MAP2+ cells). C) Sholl-like plot contrasting the number of intersections in the indicated experimental conditions. D) AUC chart summarizing the findings of the Sholl-like analysis (n = 6 MAP2+ cells). E) Immunofluorescence assay microphotographs against MAP2 in taurine- and taurine + BAPTA–treated cells. BAPTA treatment reduced immunoreactivity to MAP2 (n = 6 cells for each experimental condition). ns = non-statistical significance; **p < 0.01; ***p < 0.001 or higher statistical significance. Error bars indicate SEM.
Fig 7.
Involvement of signaling cascades in the differentiation process and neurite outgrowth of NPC SVZ mediated by taurine.
A, B) Scatter plots showing the total number (A) and total length of neurites (B) of NPC SVZ treated with taurine or taurine + the CaMKII inhibitor KN93, taurine + the ERK1/2 kinase inhibitor FR1805, and taurine + the Src kinase inhibitor Srcl. The blockade of these signaling cascades prevented the neurite outgrowth promoted with taurine (control n = 25 cells; taurine n = 42 cells; taurine + KN93 n = 15 cells; taurine + FR1805 n = 16 cells; taurine + SrcI n = 19 MAP2+ cells). C) Sholl-like plot contrasting the number of neurite intersections in the control condition and the blockade of the indicated signaling cascades (n = 6 MAP2+ cells). D) AUC chart showing the total area obtained from the Sholl-like analysis performed in the control cells, in cells treated with taurine, and following the blockade of the indicated signaling cascades (n = 6 MAP2+ cells for each experimental condition). E) Immunofluorescence assay microphotographs against MAP2 in taurine and taurine + the signaling cascade blockers. The nuclei were stained with DAPI (blue). The blockade of CaMKII, ERK½, or Src kinase reduced the immunoreactivity to MAP2 (n = 6 cell cultures for each experimental condition). **p < 0.01; ***p < 0.001 or higher statistical significance. Error bars indicate SEM.
Fig 8.
Schematic representation of the possible mechanisms necessary for the differentiation of neural precursor cells of the SVZ mediated by taurine.
Taurine binds to the GABAA receptor and promotes chloride extrusion and the blockade of potassium channels, favoring cellular depolarization, the activation of calcium channels, and activation of the calcium–calmodulin (CaM)–dependent protein kinase II (CaMKII) enzyme that promotes phosphorylation of the cellular transcription factor cAMP response element-binding protein (p-CREB). Cellular depolarization also activates the non-receptor tyrosine kinase Src and phosphorylates protein kinase B or Akt, which is involved in the PI3K/AKT/mTOR signaling cascade. The blockade of the Gi/Go-linked GABABR with CGP55485 may increase the concentration of cAMP, possibly leading to increased CREB phosphorylation via PKA activation or MAPK cascades. Furthermore, its coupling to calcium channels through the Gβγ subunits of the protein G coupled to GABABR can allow high intracellular Ca2+ levels, activating the CAMKII signaling pathway, thus continuing the pathway mentioned with the GABAA receptors that could ultimately induce CREB activation.