Transient P2X7 Receptor Antagonism Produces Lasting Reductions in Spontaneous Seizures and Gliosis in Experimental Temporal Lobe Epilepsy

Neuroinflammation is thought to contribute to the pathogenesis and maintenance of temporal lobe epilepsy, but the underlying cell and molecular mechanisms are not fully understood. The P2X7 receptor is an ionotropic receptor predominantly expressed on the surface of microglia, although neuronal expression has also been reported. The receptor is activated by the release of ATP from intracellular sources that occurs during neurodegeneration, leading to microglial activation and inflammasome-mediated interleukin 1β release that contributes to neuroinflammation. Using a reporter mouse in which green fluorescent protein is induced in response to the transcription of P2rx7, we show that expression of the receptor is selectively increased in CA1 pyramidal and dentate granule neurons, as well as in microglia in mice that developed epilepsy after intra-amygdala kainic acid-induced status epilepticus. P2X7 receptor levels were increased in hippocampal subfields in the mice and in resected hippocampus from patients with pharmacoresistant temporal lobe epilepsy. Cells transcribing P2rx7 in hippocampal slices from epileptic mice displayed enhanced agonist-evoked P2X7 receptor currents, and synaptosomes from these animals showed increased P2X7 receptor levels and altered calcium responses. A 5 d treatment of epileptic mice with systemic injections of the centrally available, potent, and specific P2X7 receptor antagonist JNJ-47965567 (30 mg/kg) significantly reduced spontaneous seizures during continuous video-EEG monitoring that persisted beyond the time of drug presence in the brain. Hippocampal sections from JNJ-47965567-treated animals obtained >5 d after treatment ceased displayed strongly reduced microgliosis and astrogliosis. The present study suggests that targeting the P2X7 receptor has anticonvulsant and possibly disease-modifying effects in experimental epilepsy. SIGNIFICANCE STATEMENT: Temporal lobe epilepsy is the most common and drug-resistant form of epilepsy in adults. Neuroinflammation is implicated as a pathomechanism, but the upstream mechanisms driving gliosis and how important this is for seizures remain unclear. In our study, we show that the ATP-gated P2X7 receptor is upregulated in experimental epilepsy and resected hippocampus from epilepsy patients. Targeting the receptor with a new centrally available antagonist, JNJ-47965567, suppressed epileptic seizures well beyond the time of treatment and reduced underlying gliosis in the hippocampus. The findings suggest a potential disease-modifying treatment for epilepsy based on targeting the P2X7 receptor.</p

Systematic Analysis of microRNA Targeting Impacted by Small Insertions and Deletions in Human Genome

<div><p>MicroRNAs (miRNAs) are small noncoding RNA that play an important role in posttranscriptional regulation of mRNA. Genetic variations in miRNAs or their target sites have been shown to alter miRNA function and have been associated with risk for several diseases. Previous studies have focused on the most abundant type of genetic variations, single nucleotide polymorphisms (SNPs) that affect miRNA-mRNA interactions. Here, we systematically identified small insertions and deletions (indels) in miRNAs and their target sites, and investigated the effects of indels on miRNA targeting. We studied the distribution of indels in miRNAs and their target sites and found that indels in mature miRNAs, experimentally supported miRNA target sites and PAR-CLIP footprints have significantly lower density compared to flanking regions. We identified over 20 indels in the seed regions of miRNAs, which may disrupt the interactions between these miRNAs and their target genes. We also identified hundreds of indels that alter experimentally supported miRNA target sites. We mapped these genes to human disease pathways to identify indels that affect miRNA targeting in these pathways. We also used the results of genome-wide association studies (GWAS) to identify potential links between miRNA-related indels and diseases.</p> </div

Somatic mutations that alter miRNA targeting in linkage disequilibrium blocks of association study markers.

a<p>: The number in parentheses below the name of the polymorphism is the distance, in bp, from the germline mutation to the somatic mutation.</p

Frequency of single base substitutions.

<p>The percentage of each class of substitution among somatic mutations in 3′UTRs (black bar) or across the entire genome (white bar) is shown for (A) lung cancer, (B) SCLC, (C) melanoma, and (D) prostate cancer.</p

Selected human disease pathways containing genes with indels and SNPs in miRNA target sites.

<p>Selected human disease pathways containing genes with indels and SNPs in miRNA target sites.</p

Genes in the pancreatic cancer pathway containing SNPs and indels that altered experimentally supported target sites.

<p>Genes containing only indels (pink), only SNPs (yellow), and both SNPs and indels (green) in target sites are within colored rectangles. The miRNAs that have been shown to target these genes are shown with red text for disrupted sites and blue text for created sites.</p

Additional file 1 of OCDD: an obesity and co-morbid disease database

Supplementary material. (PDF 66.9 kb

Selected somatic mutations that alter miRNA target sites in cancer-related genes.

a<p>:Mutations in bold type indicate somatic mutations in genes over-expressed in cancers that create or enhance miRNA target sites or somatic mutations in genes under-expressed in cancer that disrupt or hinder miRNA target sites.</p

Indels in miRNAs and miRNA target sites in linkage disequilibrium block for high-scoring markers from association studies.

<p>Indels in miRNAs and miRNA target sites in linkage disequilibrium block for high-scoring markers from association studies.</p

Density of all genetic variants (a) and indels (b) in dbSNP 135 as well as indels (c) from the GATK resource bundle in PAR-CLIP footprints and flanking regions, entire 3′ UTR and experimentally validated target sites.

<p>Flanking regions 1 and 2 represent successive sequences adjacent to PAR-CLIP footprints that were equal to the length of the footprints (∼41 bp). Error bars indicate the standard error. The density of all genetic variants (a) in PAR-CLIP footprints was significantly different from the density in flanking regions (**10⁻⁵−3, ***p = 0.04). The density of indels in PAR-CLIP footprints (b) and (c) was significantly different from the density in flanking regions (*p<10⁻¹²). The density of all genetic variants (a) in experimentally validated targets was significantly different from the density in entire 3′ UTR regions (**p = 5.7×10⁻⁷). The density of indels in experimentally validated targets (b) and (c) was significantly different from the density in 3′ UTR regions (*p<10⁻¹²).</p