Downregulation of oxidative stress-mediated glial innate immune response suppresses seizures in a fly epilepsy model.

Previous work in our laboratory has shown that mutations in prickle (pk) cause myoclonic-like seizures and ataxia in Drosophila, similar to what is observed in humans carrying mutations in orthologous PRICKLE genes. Here, we show that pk mutant brains show elevated, sustained neuronal cell death that correlates with increasing seizure penetrance, as well as an upregulation of mitochondrial oxidative stress and innate immune response (IIR) genes. Moreover, flies exhibiting more robust seizures show increased levels of IIR-associated target gene expression suggesting they may be linked. Genetic knockdown in glia of either arm of the IIR (Immune Deficiency [Imd] or Toll) leads to a reduction in neuronal death, which in turn suppresses seizure activity, with oxidative stress acting upstream of IIR. These data provide direct genetic evidence that oxidative stress in combination with glial-mediated IIR leads to progression of an epilepsy disorder.

Characterization of prickle isoform-specific pk pk1 and pk sple1 mutations in Drosophila melanogaster.

We used paired-end next generation sequencing (NGS) to characterize the classic isoform-specific pk pk1 and pk sple1 mutations of the prickle gene in Drosophila melanogaster . Here we provide evidence that these previously reported null mutations are caused by either a tirant transposon insertion into the 5' UTR of pk pk1 or a premature stop codon in the second exon of pk sple1 . Additional likely benign missense mutations were identified in both mutant isoforms.

The Dm-Myb Oncoprotein Contributes to Insulator Function and Stabilizes Repressive H3K27me3 PcG Domains.

Drosophila Myb (Dm-Myb) encodes a protein that plays a key role in regulation of mitotic phase genes. Here, we further refine its role in the context of a developing tissue as a potentiator of gene expression required for proper RNA polymerase II (RNA Pol II) function and efficient H3K4 methylation at promoters. In contrast to its role in gene activation, Myb is also required for repression of many genes, although no specific mechanism for this role has been proposed. We now reveal a critical role for Myb in contributing to insulator function, in part by promoting binding of insulator proteins BEAF-32 and CP190 and stabilizing H3K27me3 Polycomb-group (PcG) domains. In the absence of Myb, H3K27me3 is markedly reduced throughout the genome, leading to H3K4me3 spreading and gene derepression. Finally, Myb is enriched at boundaries that demarcate chromatin environments, including chromatin loop anchors. These results reveal functions of Myb that extend beyond transcriptional regulation.

Mutation of orthologous prickle genes causes a similar epilepsy syndrome in flies and humans.

OBJECTIVE: Genetically tractable fruit flies have been used for decades to study seizure disorders. However, there is a paucity of data specifically correlating fly and human seizure phenotypes. We have previously shown that mutation of orthologous PRICKLE genes from flies to humans produce seizures. This study aimed to determine whether the prickle-mediated seizure phenotypes in flies closely parallel the epilepsy syndrome found in PRICKLE patients. METHODS: Virtually all fly seizure studies have relied upon characterizing seizures that are evoked. We have developed two novel approaches to more precisely characterize seizure-related phenotypes in their native state in prickle mutant flies. First, we used high-resolution videography to document spontaneous, unprovoked seizure events. Second, we developed a locomotion coordination assay to assess whether the prickle mutant flies were ataxic. Third, we treated the mutant flies with levetiracetam to determine whether the behavioral phenotypes could be suppressed by a common antiepileptic drug. RESULTS: We find that the prickle mutant flies exhibit myoclonic-like spontaneous seizure events and are severely ataxic. Both these phenotypes are found in human patients with PRICKLE mutations, and can be suppressed by levetiracetam, providing evidence that the phenotypes are due to neurological dysfunction. These results document for the first time spontaneous, unprovoked seizure events at high resolution in a fly human seizure disorder model, capturing seizures in their native state. INTERPRETATION: Collectively, these data underscore the striking similarities between the fly and human PRICKLE-mediated epilepsy syndromes, and provide a genetically tractable model for dissecting the underlying causes of the human syndromic phenotypes.