Abstracts

Rationale: Neuron restrictive Silencing factor (NRSF/REST) is a transcriptional repressor of neuronal genes in non-neuronal tissue, which is expressed at very low levels in mature hippocampus. NRSF levels are upregulated in hippocampus by insults promoting epilepsy, such as epileptogenesis that follows kainic-acid (KA)-induced status epilepticus (SE) (MCClelland, 2011). This enhancement promotes repression of a number of key neuronal genes, including HCN1. Inhibition of NRSF function using an oligodeoxynucleotide decoy strategy resulted in rescue of the repressed HCN1 (and likely other genes). Importantly, blocking NRSF function attenuated epileptogenesis (McClelland et al, 2011). These data provided strong support for a role of NRSF upregulation in epileptogenesis. Therefore, we examined how epilepsy-provoking insults upregulate NRSF expression?Methods: SE was induced in P10 rats by systemic KA administration. Because NRSF might be regulated by microRNAs (Packer et al., 2008), and because reduced miR124 was found in epileptic brain (Kan et al., 2012), we examined levels of NRSF, REST4, pri-mir-124 and miR-124 levels at specific time points using qRT-PCR. If reduction of miR-124 directly augments NRSF levels, then increasing miR124 levels following KA should prevent the upregulation of NRSF. Therefore, organotypic hippocampal slice cultures were treated with KA to produce long (3h) seizure-like events. Hippocampi were then exposed to either miR-124 mimics or scrambled controls for 48h and expression levels of NRSF, REST4 and a target gene of NRSF; HCN1, were determined using qRT-PCR. Results: NRSF mRNA levels increased 4h following SE and this upreglation lasted for a week. Levels of miR-124 decreased as early as 90 min following KA administration, prior to reduction of NRSF levels,and corelated inversely with those of NRSF. In the organotypic slice culture, a molecular agonist (mimic) of miR-124, given at the end of KA-induced seizure-like events, attenuated increases in NRSF levels. HCN1, a target gene of NRSF, was repressed by seizure-like events, and this represion was prevented by the application of the miR124 mimic. Conclusions: Together, these data support the scenario that seizures suppress miR-124 expression, resulting in enhanced NRSF levels and function. Because augmented NRSF repression of key neuronal genes promotes epilepsy, miR-124 based therapeutics may provide a selective strategy option in the prevention of epileptogenesis.