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Mek inhibition, NOS inhibition, and nNOS knockout

ABSTRACT: embryonic cortical neuron treated with CSS, NMDA and NMDA with Mek inhibitor: replicate 1: GSM49556, GSM49557, GSM49558 replicate 2: GSM49559, GSM49560, GSM49561 nNOS KO brain cortex and wild-type littermate brain cortex: GSM49562, GSM49563 embryonic cortical neuron treated with CSS, NMDA and NMDA with NOS inhibitor: replicate 1: GSM49618, GSM49619, GSM49620 replicate 2: GSM49621, GSM49622, GSM49623 Keywords: other

ORGANISM(S): Mus musculus Rattus norvegicus

PROVIDER: GSE2572 | GEO | 2006/04/26

SECONDARY ACCESSION(S): PRJNA91979

REPOSITORIES: GEO

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Project description:Primary cortical neurons were isolated from E15 mice and after 5 days in vitro were untreated or treated for 24 h with mesenchymal stem cell conditioned medium and then untreated or treated for a further 24 h with NMDA. Neuron gene expression was profiled and compared between the four different conditions (neurons, neurons+MSC cm, neurons+NMDA, neurons+MSC cm+NMDA) to investigate the molecular mechanisms of MSC neuroprotection. Mesenchymal stem cells (MSC) promote functional recovery in experimental models of central nervous system (CNS) pathology and are currently being tested in clinical trials for stroke, multiple sclerosis and CNS injury. Their beneficial effects are attributed to activation of endogenous CNS repair processes and immune regulation but their mechanisms of action are poorly understood. Here we investigated the neuroprotective effects of MSC in simplified MSC-neuron co-culture systems and in mice using models of glutamate excitotoxicity. MSC protected primary cortical neurons against glutamate (NMDA) receptor-induced death and conditioned medium from MSC (MSC cm), but not control NIH3T3 cells, was sufficient for this effect. MSC cm neuroprotection in mouse cortical neurons was reduced by neutralizing antibodies to bFGF and associated with altered gene expression in neurons towards an immature phenotype as well as reduced neuronal Grin1, Grin2a and Grin2b mRNA levels in response to NMDA stimulation. Further, MSC cm neuroprotection in rat retinal ganglion cells was associated with absence of glutamate-induced calcium influx. Adoptive transfer of EGFP+MSC in a mouse kainic acid seizure model reduced CA3 neuron damage and hippocampal astrocytosis and resulted in the increased expression of neuronal genes that are upregulated by MSC cm, Bmi1, Ddx4, Ezh1, in the hippocampus. These results show that MSC mediate direct neuroprotection against glutamate excitotoxicity by secreting bFGF, reducing glutamate receptor expression and function and altering neuron gene expression towards an immature pattern, and provide evidence for a link between the therapeutic effects of MSC and the activation of endogenous repair processes following CNS injury. In vitro cultures primary cortical neurons from mice were protected from glutamate excitotoxicity when pre-treated with MSC cm. Global gene expression changes induced in neurons before and after treatment with MSC cm and/or NMDA were investigated using a cDNA spotted macroarray filter. Four samples were analysed in duplicate: neurons alone (untreated), neurons+MSC cm, neurons+NMDA, neurons+MSC cm+NMDA.

Project description:Nicotinic receptors (nAChR) are widely distributed in the adult brain where they mediate excitatory postsynaptic transmission and presynaptic release of other neurotransmitters. Functional nicotinic receptors are also present in the embryonic brain. Several studies have suggested a role for nAChRs in cognitive functions (learning and memory) and cellular events such as degeneration and survival. In vitro, activation of nAChR can differentially modulate neuritic outgrowth, and enhance early gene expression prior to neuronal differentiation. Furthermore, several reports have shown that nicotine can prevent cell death induced by activation of glutamatergic receptors, NMDA-subtype. Little is known of these events in the cerebral cortex. Knowledge of the genes activated by exposure of cortical neurons to nicotine or to an excitotoxic agent such as the glutamatergic agonist NMDA could potentially lead to new strategies for adult brain protection and repair. The effect of nicotine and the cembranoid 4R, a novel nicotinic receptor non competitive antagonist, will be analyzed in primary cultures of cortical neurons, in the presence or the absence of NMDA, to identify which genes affected by NMDA receptor activation are modulated nicotine or 4R. We hypothesize that the neuroprotective effect of nAChRs is mediated by modulation of the changes in gene expressions elicited by NMDA. In order to detect early changes in gene expression, primary culture of cortical neurons prepared from 16 day old Sprague Dawley rat embryos at 11 days in culture will be treated for different times (10, 30, 60, 180, 360, min) with 1 mM NMDA and then harvested. In a second set of experiments, neuronal cultures will be treated with NMDA for 10, 30 and 60 min then the drug will be washed out and the cells will be harvested 24 h later. It was reported that 8-24 h pretreatment of neurons with 10 micromolar nicotine is neuroprotective, therefore we will treat separate cultures with either 10 micromolar nicotine or 2 micromolar 4R for 18 h and then with NMDA for 30 min or 1 h. The cells will be either harvested soon after NMDA treatment or the drugs will be washed out and the cells harvested 24 h later. We will also examine sample treated only with either 4R or nicotine for 15 min or 18 h in order to determine which genes are differentially regulated by these drugs. These experiments will allow us to detect any change in gene expression elicited soon by each treatment or with some delay. The samples in Trizol will be kept at -70 until all the 3 or 5 replicates are harvested and then the RNA will be purified. RNA from 3-5 independent experiments for each different protocol or treatment together with controls will be sent to the facility.

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Mek inhibition, NOS inhibition, and nNOS knockout

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