TLR3 Regulates Neural Stem Cell Proliferation Figure 5. Shh pathway activation reverses PIC-mediated inhibition of GLI1 expression in WT NPCs. Primary neurospheres were obtained from cerebral cortex cells isolated from WT embryos at GD14. Neurospheres were cultured for 7 days with EGF (5 ng/ml) either in the presence or in the absence of cyclopamine (5 mM) and treated with PIC for the last 24 hours. The control group includes cells cultured with EGF (5 ng/ml) and ethanol and treated with PBS for the last 24 hours (vehicle control group) (a) Dot plots showing Gli1+nestin+ cells in PIC-treated WT neurospheres cultured either in the presence or absence of cyclopamine and vehicle control group. Numbers in quadrants represent the percentages of each subpopulation. Three independent cell culture assays were performed with cells isolated from embryos (n = 12); data from one representative assay is shown. (b) Dot plots showing Gli1+nestin+ cells expression in PBS- and PIC-treated neurospheres incubated in EGF-containing medium (5 ng/ml) with or without added recombinant Shh protein (5 nM). Numbers in quadrants represent the percentages of each subpopulation. Three independent cell culture assays were performed with cells isolated from embryos (n = 12); data from one representative assay is shown. doi:10.1371/journal.pone.0026766.g005 embryonic neocortex at GD18 [7]. Results reported here confirm that TLR3 receptors are expressed on NPC and regulate PIC signaling. PIC treatment inhibited the proliferation of NPC and increased the ratio of GFAP+- and bIII-tubulin+-differentiated subpopulations in primary WT neurospheres. Although we observed comparable NPC multipotency in control and PIC- treated WT neurospheres (data not shown), we cannot ascertain from current data whether PIC induces NPC differentiation. The Ras-MEK-ERK signaling pathway is involved in cell proliferation, apoptosis and differentiation. EGF stimulates cell proliferation via the Ras-MEK-ERK signaling cascade [60,61,62]. Furthermore, Ras-ERK activation is implicated both in the maintenance of tumor stem cells [63], and in regulation of Fas transcription [56,64]. Our results in EGF-responsive NPC indicate that Shh signaling machinery is present in nestin+ NPC in primary cortical neurospheres. PIC treatment inhibited Shh signaling in WT primary neurospheres, resulting in reduced proliferation of NPC. PIC also induced apoptosis in the WT neurospheres. In Figure 9, we present a model of PIC-mediated effects on NPC. PIC inhibits the Shh-mediated Ras-MEK-ERK signaling path- way, resulting in reduced MEK (data not shown) and ERK phosphorylation and increased Fas expression. Thus, PIC- mediated inhibition of Ras-MEK-ERK activation may contribute to both suppression of proliferation and induction of apoptosis. PIC signals via a TLR3-dependent NFkB pathway to induce IFN-a, IFN-b and proinflammatory cytokines [3,65]. Li and colleagues have shown that IFN-a inhibits the Shh-activated Ras- MEK-ERK pathway and induces apoptosis in human cancer cells [46]. Our experiments with neurospheres from TLR3-deficient mice indicate that PIC interferes with Shh signaling and NPC proliferation in a TLR3-dependent manner: PIC treatment of TLR32/2 neurospheres neither induced apoptosis nor decreased proliferation. The factor(s) that mediate cross-talk between TLR3 and Shh signaling cascades are unknown. However, a recent study describing Shh as a direct transcriptional target of NFkB [66] suggests that the NFkB pathway may be pivotal. Brain structures require Shh for normal proliferation and dorsal- ventral patterning [67]. In humans, loss of Shh signaling causes holoprosencephaly, a birth defect characterized by failure of forebrain development [68,69]. Conversely, enhanced Shh signaling has been implicated in cancers of prostate, pancreas, muscle and skin as well as the brain [70,71]. In mice, development of the telencephalon commences at GD11 and peaks between GD14 and 16; this time period is characterized by rapid expansion of NPC PLoS ONE | www.plosone.org 7 October 2011 | Volume 6 | Issue 10 | e26766