Adult hippocampal neurogenesis is the procedure whereby adult neural precursor cells

Adult hippocampal neurogenesis is the procedure whereby adult neural precursor cells (aNPCs) in the subgranular area (SGZ) of the dentate gyrus (DG) generate adult-born functional neurons in the hippocampus. apoptosis and inhibits proliferation in SGZ-aNPCs. In addition lead significantly impairs spontaneous neuronal differentiation and maturation. Furthermore we found that activation of the c-Jun NH2-terminal kinase (JNK) and p38 mitogen triggered protein (MAP) Zosuquidar kinase signaling pathways are important for lead cytotoxicity. Our data suggest that lead can directly take action on adult neural stem cells and impair essential processes in adult hippocampal neurogenesis which may contribute to its neurotoxicity and adverse effects on cognition in Zosuquidar adults. 2012 b; Pan found no effect of lead on neuronal differentiation (Verina et al. 2007 Although these studies are very interesting the inconsistent results warrant further investigation. Moreover the early life exposure paradigms used in these studies may have launched potential confounding factors due to the adverse effects of lead during development (Gilbert 2005; Jaako-Movits model system to test the hypothesis that lead exposure impairs adult hippocampal neurogenesis and to elucidate the underlying signaling mechanisms. Materials and Methods Reagents The preparation use and disposal of hazardous providers were carried out according to the Environmental Health and Security Office in the University or college of Washington. Lead (II) acetate trihydrate (Cat. 316512 Sigma-Aldrich St. Louis MO) was dissolved in deionized distilled water (H2O) to make a 5 mM stock solution and stored at ?20°C. Z-VAD-FMK (Cat. FMK001 R&D Systems Minneapolis MN) was dissolved in dimethyl sulfoxide (DMSO) to make a 20 mM stock and used relating the manufacturer’s Zosuquidar specifications. The p38 (Cat. SB2021990 EMD Millipore Zosuquidar Calbiochem Billerica MA) and JNK (Cat. SP600125 EMD Millipore Calbiochem) inhibitors were dissolved in DMSO to yield 3 mM stock solutions and stored at ?20°C. 5-bromo-2′-deoxyuridine (BrdU) was from Sigma (Cat. B9285) and stored as a 65 mM stock solution. The primary antibodies and dilutions used in immunocytochemistry were rat anti-BrdU (1:500 Bio-Rad Laboratories AbD Serotec Raleigh NC) mouse anti-βIII-tubulin (1:500 Promega Madison WI) and mouse Rabbit polyclonal to TDGF1. anti-SOX2 (1:500 R&D Systems). Goat anti-rat and goat anti-mouse Alexa Fluor-conjugated secondary antibodies as well as Hoechst 33342 were from Invitrogen (Carlsbad CA). For Western Blot analysis the following rabbit primary antibodies from Cell Signaling (Beverly MA) were used at Zosuquidar a 1:1000 dilution unless otherwise specified: monoclonal anti-phospho-Akt (Cat. 4060 1 polyclonal anti-phospho-p38 (Cat. 9211) monoclonal anti-phospho JNK (Cat. 4668) monoclonal anti-JNK (Cat. 9258) polyclonal anti-phospho-c-Jun (Cat. Zosuquidar 9164) and monoclonal anti-GAPDH (Cat. 2118). Horseradish peroxidase-conjugated secondary antibodies were from EMD Millipore (Billerica MA). All of the primary and secondary antibodies were diluted into the appropriate blocking buffer. Cell culture The University of Washington Institutional Animal Care and Use Committee approved all experimental procedures. The primary aNPCs were prepared as previously described (Guo et al. 2012 Pan et al. 2013 from the SGZ of the DG from 6-7 week-old male C57BL/6J mice (Taconic Hudson NY). The solutions and media used during the aNPC isolation were filter sterilized. Briefly the whole brain from four adult male mice was harvested and placed in HBSS (Invitrogen). Each brain was then sliced into 1 mm sections using an adult mouse brain matrix (Kent Scientific Torrington CT) and then the SGZ was isolated from these sections via microdissection under a dissection microscope. The SGZ tissue was placed in Solution A (30 mM Glucose 26 mM NaCO3 2 HEPES pH 7.4 (Invitrogen) in HBSS (Invitrogen)) and spun down for 10 min at 1 0 rpm. The pelleted tissue was then resuspended and a combination of mechanical and enzymatic digestion (MACS Neural Tissue Dissociation Kit Miltenyi Biotec San Diego CA) was used to dissociate the tissue. To stop the digestion DMEM/F-12 medium (Invitrogen) with 10% Fetal Bovine Serum (FBS Invitrogen) was added and the SGZ tissue was then filtered through a cell strainer (70 μm cell strainer Fisher Scientific Waltham MA) and spun down for 3 min at 1 0 rpm. The pellet was washed once with DMEM/F-12 medium with 10% FBS and once with DMEM/F-12 medium with 10% FBS plus Percoll (GE Healthcare Life Sciences Pittsburgh PA) solution (1:10 Percoll in PBS) accompanied by spins at 1 0 rpm for 3 and 15 min respectively. The pellet was cleaned once with Remedy A as soon as with.