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Global analysis of excitotoxicity-induced alterations in RNA structure and RNA-protein binding in neurons identifies subsets of transcripts and RNA-binding proteins involved in synaptic plasticity and neurodegenerative disorders [RNA-seq]

ABSTRACT: Excitotoxicity and altered RNA regulation by RNA-binding proteins (RBPs) are two prevalent hallmarks in multiple neurodegenerative disorders. However, global analysis of RNA-protein interactions in primary neurons in the context of excitotoxicity has not been assessed. To address this, in this work we have applied the protein interaction profile sequencing (PIP-seq) method to primary neurons treated with NMDA to induce excitotoxicity. Our results reveal that NMDA dramatically changes RNA secondary structure at the UTR regions, and that specific RNA structure reduction at the 3’UTR correlates with increased mRNA abundance as assessed by mRNA-Seq. Protein binding sites increase in response to excitotoxicity and, interestingly, different RNA-protein binding in this context defines subsets of transcripts functionally associated with synaptic functions and neurodegenerative disorders. We also identify two RNA motifs enriched in protein binding in the context of excitotoxicity and a list of proteins binding to them in vitro. Accordingly, we observe that two of these RBPs, CELF6 and YBX3 alter their protein levels in response to NMDA treatment, suggesting their involvement in RNA regulation in the context of excitotoxicity. Overall, we describe for the first time the global RNA-protein interactions of primary cortical neurons in baseline conditions and in an in vitro model of excitotoxicity, providing extensive datasets that can be leveraged to bridge the mechanistic gap between two major hallmarks common in multiple neurodegenerative disorders: excitotoxicity and RNA regulation by RBPs

ORGANISM(S): Rattus norvegicus

PROVIDER: GSE268844 | GEO | 2025/04/28

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.

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Global analysis of excitotoxicity-induced alterations in RNA structure and RNA-protein binding in neurons identifies subsets of transcripts and RNA-binding proteins involved in synaptic plasticity and neurodegenerative disorders [RNA-seq]

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