Toll-like Receptor 3 Regulates Neural Stem Cell Proliferation by Modulating the Sonic Hedgehog Pathway Kavitha Yaddanapudi., Joari De Miranda . , Mady Hornig, W. Ian Lipkin* Center for Infection and Immunity, Mailman School of Public Health, Columbia University, New York, New York, United States of America Abstract Toll-like receptor 3 (TLR3) signaling has been implicated in neural stem/precursor cell (NPC) proliferation. However, the molecular mechanisms involved, and their relationship to classical TLR-mediated innate immune pathways, remain unknown. Here, we report investigation of the mechanics of TLR3 signaling in neurospheres comprised of epidermal growth factor (EGF)-responsive NPC isolated from murine embryonic cerebral cortex of C57BL/6 (WT) or TLR3 deficient (TLR32/2) mice. Our data indicate that the TLR3 ligand polyinosinic-polycytidylic acid (PIC) negatively regulates NPC proliferation by inhibiting Sonic Hedgehog (Shh) signaling, that PIC induces apoptosis in association with inhibition of Ras-ERK signaling and elevated expression of Fas, and that these effects are TLR3-dependent, suggesting convergent signaling between the Shh and TLR3 pathways. Citation: Yaddanapudi K, De Miranda J, Hornig M, Lipkin WI (2011) Toll-like Receptor 3 Regulates Neural Stem Cell Proliferation by Modulating the Sonic Hedgehog Pathway. PLoS ONE 6(10): e26766. doi:10.1371/journal.pone.0026766 Editor: Grainne M. McAlonan, The University of Hong Kong, Hong Kong Received May 20, 2011; Accepted October 4, 2011; Published October 25, 2011 Copyright: 2011 Yaddanapudi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by awards from the National Institutes of Health National Institute of Allergy and Infectious Diseases (NIH-NIAID), 5 U01 and AI070411 to WIL, and the Department of Defense. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: wil2001@columbia.edu . These authors contributed equally to this work. Introduction PIC is a synthetic analog of viral double-stranded (ds) RNA that activates immune responses through two dsRNA sensors, TLR3 and melanoma differentiation-associated protein 5 (MDA5). TLR3 senses PIC that has been internalized by endocytosis [1,2]. Upon binding PIC, TLR3 signals through the adaptor protein, Toll/IL-1 resistance domain-containing adaptor-inducing IFN-b (TRIF). Interaction with the adaptor activates an array of transcription factors, including IFN regulatory factor (IRF) 3, IRF7, IRF1, and NFkB. These factors induce the expression of genes encoding type I interferon (IFN, i.e., IFN-a and IFN-b) and proinflammatory cytokines [3,4]. Recent evidence suggests that TLR3 plays a role in neural development. TLR3 protein is present in mammalian brain cells in early embryonic stages of development and serves as a negative modulator of early embryonic NPC proliferation [5] and axonal growth [6]. In a mouse model of prenatal virus infection, we recently demonstrated that PIC-induced TLR3 activation inhibits embryonic NPC proliferation and decreases the number of neurons populating the upper layers of the cortex [7]. Single-cell suspensions of neural stem cells can be isolated from the embryonic telencephalon and propagated in vitro as suspended, spherical aggregates called neurospheres [8,9]. Epidermal growth factor (EGF)-responsive NPC in neurospheres express the neuroepithelial stem cell marker, nestin, and are derived from rapidly-cycling, radial glia (RG) in the embryonic telencephalon. Neural stem cells can give rise to all three major cell types of the central nervous system: neurons, oligodendrocytes, and astrocytes [10,11]. Accordingly, neurospheres contain a mixture of multip- otent stem cells, proliferating precursor cells, postmitotic neurons and glia [12,13,14]. Primary neurospheres can be clonally passaged in vitro and provide a useful tool for analysis of the proliferation and self-renewal capacity of neural stem and precursor cells. Regulation of the number of stem and precursor cells generated during neural development is important for control of brain size [15,16]. Shh signaling has been implicated in cell proliferation and growth of embryonic and postnatal dorsal brain [17,18,19,20,21,22,23]. Shh is a member of the hedgehog family of secreted glycoproteins that binds the cell surface receptor Patched (Ptch). Binding of Shh and related ligands to Ptch abrogates its inhibition of the G-protein-coupled receptor, Smoothened (Smo), resulting in increased expression of Gli1 zinc-finger transcription factors [24,25]. Three Gli proteins participate in the mediation of Shh signaling: Gli1 and Gli2 function as transcription activators, whereas the truncated form of Gli3, Gli3R, acts as a repressor [26,27,28,29]. Shh-Gli signaling induces formation of Gli activators (Gli1, Gli2) that are imported into the nucleus to transactivate target genes. Multiple effects of Shh signaling on cyclin-dependent kinases (Cdks), Cdk inhibitors, cyclins, N-myc or the transcription factor, E2F, acting at different points of the cell cycle, may account for the proliferative effects of Shh [30,31,32,33,34]. In mammals, Shh is the only hedgehog family member expressed in the normal central nervous system [35]. Shh PLoS ONE | www.plosone.org 1 October 2011 | Volume 6 | Issue 10 | e26766