Elsevier

Brain Research

Volume 1424, 18 November 2011, Pages 53-59
Brain Research

Research Report
Neurotrophin-3 mRNA a putative target of miR21 following status epilepticus

https://doi.org/10.1016/j.brainres.2011.09.039Get rights and content

Abstract

Status epilepticus induces a cascade of protein expression changes contributing to the subsequent development of epilepsy. By identifying the cascade of molecular changes that contribute to the development of epilepsy we hope to be able to design therapeutics for preventing epilepsy. MicroRNAs influence gene expression by altering mRNA stability and/or translation and have been implicated in the pathology of multiple diseases. MiR21 and its co-transcript miR21*, microRNAs produced from either the 5′ or 3′ ends of the same precursor RNA strand, are increased in the hippocampus following status epilepticus.

We have identified a miR21 binding site, in the 3′ UTR of neurotrophin-3 that inhibits translation. Neurotrophin-3 mRNA levels decrease in the hippocampus following SE concurrent with the increase in miR21. MiR21 levels in cultured hippocampal neurons inversely correlate with neurotrophin-3 mRNA levels. Treatment of hippocampal neuronal cultures with excess K+Cl, a depolarizing agent mimicking the episode of status epilepticus, also results in an increase in miR21 and a decrease in neurotrophin-3 mRNA. MiR21 is a candidate for regulating neurotrophin-3 signaling in the hippocampus following status epilepticus.

Highlights

►Following status epilepticus miR21 increases in the hippocampus. ►Following status epilepticus neurotrophin-3 mRNA decreases in the hippocampus. ►The 3′ UTR of neurotrophin-3 mRNA has a miR21 binding site. ►In neuronal cultures miR21 levels are inversely related to neurotrophin-3 mRNA levels.

Introduction

Epilepsy, a disorder of recurrent unprovoked seizures, has a lifetime prevalence of ~ 0.5%. Currently treatments are aimed at suppressing seizures but are not effective in 30% of patients and no medications to prevent or reverse the development of epilepsy have been developed. An improved understanding of which molecules contribute to the development of epilepsy will allow identification of therapeutic targets and anti-epileptogenic medications.

Pilocarpine treatment of rodents provokes a prolonged seizure, status epilepticus (SE), and leads to changes that eventually cause spontaneous seizures or epilepsy. This model has been used to identify suspected molecular and cellular changes contributing to the development of epilepsy. Molecular changes shown to influence the development of epilepsy include altered expression of neurotransmitters, neurotransmitter receptors, transcription factors, neurotrophins and neurotrophin receptors (Lin et al., 2003, He et al., 2004, Raol et al., 2006, Noe et al., 2008). In several models of epileptogenesis, neurotrophin-3 mRNA (NT-3) levels decrease following an insult that leads to the development of epilepsy (Bengzon et al., 1993, Schmidt-Kastner and Olson, 1995, Mudo et al., 1996, Kim et al., 1998). NT-3 deprivation decreases neurite outgrowth, neuronal cell survival and suppresses the development of epilepsy (Lowenstein and Arsenault, 1996, Elmer et al., 1997). To date, no one has identified the mechanism that suppresses NT-3 levels following SE.

MicroRNAs bind to the 3′ tails of mRNAs, decreasing protein levels either by blocking translation and/or by destabilizing mRNAs leading to increased degradation. MicroRNA genes are interspersed throughout the genome and following transcription of a primary transcript, pri-miRNA, the Drosha complex cleaves it to a pre-miRNA and finally a dicer containing complex cleaves it to mature ~ 21 base pair length miRNAs. Additional members of the microRNA-processing complex have been identified and add an additional layer of regulation of mature microRNA products (for review see Nilsen (2007)). MicroRNAs are controlled at a transcription, translation or stabilization level (Thomson, et al., 2006). Increased levels of miR21 can occur through specific activation of a post-translational complex of Drosha and SMAD, increasing miR21 production or stability (Davis et al., 2008, Davis et al., 2010). MiR21 is an extensively studied microRNA, as it is widely increased in multiple cancers including those of the nervous system and following hypoxia (Kulshreshtha et al., 2007, Gabriely et al., 2008, van Rooij et al., 2006). MiR21 has been shown to be increased in several brain injury models, stroke, trauma and as demonstrated here following SE (Buller et al., 2010, Hu et al., 2011, Redell et al., 2011, Ziu et al., 2011). MiR21 targets to date have included proteins involved in neurotrophin signaling, sprouty family members SPY-1 and SPY-2 but not NT-3 (Sayed et al., 2008, Thum et al., 2008). Using a rodent epilepsy model, we show that miR21 is a candidate for regulating NT-3 mRNA following an episode of SE.

Section snippets

Results

A microRNA array of rat hippocampus was carried out using whole RNA isolated from animals treated with high dose pilocarpine to induce SE or low dose pilocapine (control) and sacrificed at 4, 48 h and 3 weeks following SE. MiR21 and 21* increased at several time points following SE (Table 1). We then carried out real-time PCR analysis of hippocampal RNA from a new set of animals to confirm the microRNA array results. MiR21 increased at 48 h and 3 weeks following SE but not at 4 h (Fig. 1A, 4 h SE

Discussion

Here we have shown that miR21 and miR21* increase in the hippocampus following pilocarpine induced SE. Prior studies have shown an increase in miR21 following hypoxia in heart tissue or with increased inflammation in the lungs (van Rooij et al., 2006, Lu et al., 2009) and following hypoxia or trauma in the brain (Redell et al., 2011, Ziu et al., 2011). Together with our findings of an increase in miR21 following SE, miR21 appears to be upregulated following biologic stresses in a variety of

Experimental procedures

Induction of SE. The Institutional Animal Care and Use Committee of the Children's Hospital of Philadelphia approved the experimental protocol. Adult male CD (a Sprague–Dawley) rats from Charles River between 60 and 90 days of age underwent pilocarpine induced SE. All animals received methyl-scopalamine, 1 mg/kg intraperitoneal (IP) 30 min prior to pilocarpine to block peripheral cholinergic effects. SE was induced with pilocarpine (385 mg/kg, IP), with a half dose given 1 h later if a stage V

Acknowledgments

The authors thank Margie Maronsky and Mark Dichter for providing hippocampal neurons.

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