Figure 4. Shh pathway activation reverses PIC-mediated inhibition of neurosphere-derived NPC proliferation. Primary neurospheres were obtained from cerebral cortex cells isolated from WT embryos at GD14. Neurospheres were cultured for 7 days in the presence of EGF (5 ng/ml) and treated with PBS or PIC for 24 hours. BrdU was added for the last 12 hours of the culture. (a) Proliferative response of neurospheres in EGF-containing medium with or without added recombinant Shh protein (5 nM). Representative histograms showing the mean fluorescence intensity of BrdU-labeled cells represented as a percentage of maximum BrdU incorporation in WT neurospheres: green line: EGF, red line: EGF + Shh, and gray line: isotype control. (b) Representative histograms showing the mean fluorescence intensity of BrdU labeling in PBS- or PIC-treated neurospheres cultured in EGF. (c) Representative histograms showing the mean fluorescence intensity of BrdU labeling in PBS- or PIC-treated neurospheres cultured in the presence of EGF (5 ng/ml) and Shh (5 nM): red line: PBS-treated cells, blue line: PIC-treated cells, and gray line: isotype control. (d) Box plot showing the mean fluorescent intensity of BrdU-labeled cells in PBS- and PIC-treated WT neurospheres cultured in the presence of EGF TLR3 Regulates Neural Stem Cell Proliferation and EGF + Shh. Results are expressed as percentages of maximum expression (*, p,0.05 relative to PBS control group; Mann-Whitney U test). Three independent cell culture assays were performed with cells isolated from embryos (n = 12). Height of box plot shows inter-quartile range; horizontal line, median. Due to the low numbers involved in the flow cytometry experiments, the box plot upper and lower interquartile ranges also represent the maximum and minimum scores. White bars: PBS-treated mice, hatched bars: PIC-treated mice. doi:10.1371/journal.pone.0026766.g004 comparison to cells from the PBS-treated WT control group (Fig. 5b), confirming our earlier observation that in neurosphere cultures, under conditions of sub-maximal EGF stimulation, recombinant Shh treatment abrogates the PIC-mediated effects on Shh signaling. PIC induces apoptosis in primary cortical neurospheres To test whether PIC altered viability of NPC, we measured apoptosis in GD14 WT and TLR32/2 neurospheres after 7 days of culture in proliferation medium supplemented with EGF (20 ng/ml) that included 24 hours of treatment with PIC or PBS. PIC resulted in a close to two-fold increase in the percentage of annexin V+ cells in WT neurospheres (n = 12; Mann-Whitney U, p = 0.04; relative to PBS-treated WT control group; Fig. 6a, c). In contrast, no significant differences were observed between PIC- and PBS-treated TLR32/2 neurospheres (n = 12; Mann- Whitney U, p = 0.27; relative to PBS-treated TLR32/2 control group; Fig. 6b, c). These results indicate that PIC augments apoptosis in NPC and that the effect is TLR3-dependent. PIC prevents EGF/Shh-mediated ERK activation and induces Fas expression in primary cortical neurospheres To elucidate the molecular basis by which PIC mediates inhibition of Shh pathway activation, we examined the effects of PIC on the Ras-ERK pathway, a downstream effector of Shh and EGF signaling in GD14 WT primary cortical neurospheres cultured in the presence of EGF. PIC treatment decreased the percentage of neurosphere-derived cells expressing phosphorylated-ERK, the active form of the protein (n = 12; Mann-Whitney U, p = 0.02; relative to PBS-treated WT control group; Fig. 7a, e). The mean fluorescent intensity of phosphorylated-ERK was also reduced in PIC-treated cells (n = 12; Mann-Whitney U, p = 0.04; relative to PBS-treated WT control group; Fig. 7c). Consistent with studies reporting regulation of Fas (CD95) expression via the Ras-ERK pathway [56,57,58] Fas expression was elevated in WT cortical NPC treated with PIC (n = 12; Mann-Whitney U, p = 0.02; relative to PBS-treated WT control group; Fig. 7b, d, f). Collectively, these results suggest that inhibition of Ras-ERK signaling by PIC may lead to elevated levels of Fas and Fas-mediated apoptosis in NPC. To elucidate that Poly IC-mediated effects are dependent on Shh, neurosphere cultures were grown for 7 days in media supplemented with recombinant Shh (5 nM) in the presence of sub-maximal concentration of EGF (5 ng/ml) and treated with PIC or PBS as described above. Recombinant Shh pre-treatment in the presence of EGF abrogated PIC-mediated inhibition of phospho-ERK expression in WT neurosphere cells as compared with PIC-treated cells cultured in the presence of EGF alone (Fig. 8a, b). Discussion The majority of EGF-responsive nestin+ cells in the cortical neurosphere model correspond to the rapidly-cycling, PAX6+ RG stem cells and support the hypothesis that stem cells are contained within the neuroepithelial-radial glia-astrocyte lineage [59]. We recently reported that PAX6+ RG cells express TLR3 in PLoS ONE | www.plosone.org 6 October 2011 | Volume 6 | Issue 10 | e26766