For example Riluzole, an FDA approved therapeutic for the treatment of amyotrophic lateral sclerosis (ALS), has been proposed to act as an antagonist of both glutamate receptors and glutamate transporters (Villmann and Becker, 2007), in addition to a tetrodotoxin-sensitive sodium channel blocker (Song et al., 1997), and a two-pore potassium channel agonist (Mathie and Veale, 2007). of targeting NDMA receptors may be due to poor relevance of animal models or suboptimal design of clinical trials (Hoyte et al., 2004). The disconnect may also arise from an oversimplified standard model of excitotoxicity, which links cell death to a linear cascade of signaling events following receptor overstimulation (Besancon et al., 2008). For example, NMDA receptors (NMDA-R) may stimulate cell survival or cell death signals, depending on their subcellular localization. Whereas extra-synaptic NMDA-R activation may preferentially trigger cell death cascades, synaptic NMDA-R activation may promote neuroprotection, (Hardingham and Bading, 2010). The release of axonal glutamate can be preceded by large Na+ influxes which have been suggested to be more detrimental than the ultimate Ca2+ imbalance of the standard model (Besancon et al., 2008). Moreover, an expanded repertoire of glutamate and Ca2+ sensing receptors and transporters in the CNS continues to unfold (Villmann and Becker, 2007, Besancon et al., 2008, Trapp and Stys, 2009). Neuroprotective agents may have multiple mechanistic roles in neuroprotection. For example Riluzole, an FDA approved therapeutic for the treatment of amyotrophic lateral sclerosis (ALS), has been proposed to act as an antagonist of both glutamate receptors and glutamate transporters (Villmann and Becker, 2007), in addition to a tetrodotoxin-sensitive sodium channel blocker (Song et al., 1997), and a two-pore potassium channel agonist (Mathie and Veale, 2007). Also, the standard model (3β,20E)-24-Norchola-5,20(22)-diene-3,23-diol has been limited by a neuronal centric view. However, astrocytes and oligodendrocytes are critical players in glutamate regulation and express a similar complement of ionotropic and metabotropic (3β,20E)-24-Norchola-5,20(22)-diene-3,23-diol glutamate receptors that render them vulnerable to excitotoxic injury (Bolton and Paul, 2006). Finally, while many pathogenic mechanisms of glutamate excitotoxicity and cell death pathways have been well established, we still do not fully understand the complexities and multiplicity of networks, pathways, and intracellular signaling cascades that promote neuroprotection and cell survival (Lau and Tymianski, 2010). To increase our understanding of the intracellular mechanisms of neuroprotection, the current study used genome-wide expression analysis followed by a multi-step analytical approach that included text and database mining, as well as biological systems analysis. By (3β,20E)-24-Norchola-5,20(22)-diene-3,23-diol employing primary mouse cortical neurons exposed to an excitotoxic insult of NMDA in the presence or absence of neuroprotective molecules, we were able to identify expression profiles that may represent shared signatures of neuroprotection. Interestingly, while diverging chemically and acting through different putative mechanisms of action, we found that these molecules converged at the level of whole-genome transcription. Namely, these signatures include MAPK signaling, calcium ion transport, and cellular adhesion, as well as pathways related to ischemic tolerance, such as the hypoxic inducible factor (HIF) and Toll-like receptor (TLR) pathways. Activation of these pathways may underlie a fundamental mechanism driving neuronal survival. Experimental Procedures Primary Cortical Neuron Generation Generation of cortical neurons from postnatal day-0 CD-1 mice brains (Charles River Laboratories) was achieved by papain (Worthing Biochemical Corporation, “type”:”entrez-nucleotide”,”attrs”:”text”:”LS003126″,”term_id”:”1321651598″,”term_text”:”LS003126″LS003126) dissociation and manual trituration (Chen et al., 2005). Dissociated cells (6 105 cells/ml) were cultured on poly-ornithine/poly-lysine (Sigma P3655, P5282) coated 10-cm plates in neurobasal A medium (NBA) (Invitrogen, 10888-022) supplemented with B-27 (Invitrogen, 17504-044) and penicillin/streptomycin (Invitrogen, 15140-122). Neurons were cultured for seven days at which time the NBAM was replaced and all molecule testing and treatment was performed. Neuroprotection Assays Neuroprotection was assessed using the Cell-Titer Glo? Luminescent Cell Viability Assay (Promega, G7571) according to the manufacturers protocol. Initially, each molecule was titrated over a 2-fold dilution curve (eight technical replicates were concentration) to determine neuroprotective efficacy (3β,20E)-24-Norchola-5,20(22)-diene-3,23-diol Rabbit Polyclonal to USP32 following a NDMA induced excitotoxic shock. Molecule concentrations that resulted in the highest level of cell viability (Table 1) (3β,20E)-24-Norchola-5,20(22)-diene-3,23-diol were used for subsequent for RNA extraction and microarray analysis. Of the 20 molecules used, 14 were classified as protective and 6 non-protective. The experimental design included single replicates for treatments with the 20 molecules and five biological replicates for non-treatment/vehicle controls. For RNA isolation, culture neuorons were pre-treated for 1 hr in NBAM+ media (NBAM with either media alone, vehicle, or molecule), followed by a 1 hr incubation in excitotoxic media (EXM+, 120 mM NaCl, 5.3 mM KCL, 1.8 mM CaCl2, 15 mM D-glucose, 25 mM Tris, pH 7.4 supplemented with 10 M glycine and 100 M NMDA) containing the respective molecule additives as in the NBAM+. Following incubation, neurons were washed with NBAM, and incubated for an additional 16.