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-Screen for eas suppressors reveals multiple escargot alleles.The three strongest gain-of-function eas suppressors
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Suppressor mutations provide potentially powerful tools for examining mechanisms underlying neurological disorders and identifying novel targets for pharmacological intervention. Here we describe mutations that suppress seizures in a Drosophila model of human epilepsy. A screen utilizing the Drosophila easily shocked (eas) "epilepsy" mutant identif...
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... In Drosophila, mutants such as easily shocked (eas), julius seizure (jus), and para (paralytic) are used as models for human epilepsy. Ectopic pan-neural expression of esg throughout larval development suppresses the behavioral and electrophysiological epileptic phenotypes observed in eas and sda mutants (Hekmat-Scafe et al., 2005). The potential neuronal Esg gene targets that mediate seizure suppression are yet to be determined. ...
The Snail superfamily of transcription factors plays a crucial role in metazoan development; one of the most important vertebrate members of this family is Snai1 which is orthologous to the Drosophila melanogaster esg gene. This review offers a comprehensive examination of the roles of the esg gene in Drosophila development, covering its expression pattern and downstream targets, and draws parallels between the vertebrate Snai1 family proteins on controlling the epithelial‐to‐mesenchymal transition and esg . This gene regulates stemness, ploidy, and pluripontency. esg is expressed in various tissues during development, including the gut, imaginal discs, and neuroblasts. The functions of the esg include the suppression of differentiation in intestinal stem cells and the preservation of diploidy in imaginal cells. In the nervous system development, esg expression also inhibits neuroblast differentiation, thus regulating the number of neurons and the moment in development of neuronal differentiation. Loss of esg function results in diverse developmental defects, including defects in intestinal stem cell maintenance and differentiation, and alters imaginal disc and nervous system development. Expression levels of esg also play a role in regulating longevity and metabolism in adult stages. This review provides an overview of the current understanding of esg's developmental role, emphasizing cellular and tissue effects that arise from its loss of function. The insights gained may contribute to a better understanding of evolutionary conserved developmental mechanisms and certain metabolic diseases.
... The zinc-finger transcription factor esg is another novel seizure-suppressor mutation found by mutant screening. Seizure-like activity in the seizure-prone eas mutants was suppressed when the esg gene mutation product was over-expressed ectopically in all nerve cells, suggesting gain-of-function seizure suppressor; the seizure suppression was associated with elevated seizure threshold without change in neuronal excitability [39]. The esg mutations also increase the seizure threshold in other seizureprone Drosophila models, including sda and para bss1/+ [39]. ...
... Seizure-like activity in the seizure-prone eas mutants was suppressed when the esg gene mutation product was over-expressed ectopically in all nerve cells, suggesting gain-of-function seizure suppressor; the seizure suppression was associated with elevated seizure threshold without change in neuronal excitability [39]. The esg mutations also increase the seizure threshold in other seizureprone Drosophila models, including sda and para bss1/+ [39]. ...
Introduction:
Epilepsies are disorders of neuronal excitability characterized by spontaneously recurrent focal and generalized seizures, some of which result from genetic mutations. Despite the availability of antiseizure medications, pharmaco-resistant epilepsy is seen in about 23% of epileptic patients worldwide. Therefore, there is an urgent need to develop novel therapeutic strategies for epilepsies. Several epilepsy-associated genes have been found in humans. Seizure susceptibility can also be induced in Drosophila mutants, some showing features resembling human epilepsies. Interestingly, several second-site mutation gene products have been found to suppress seizure susceptibility in the seizure genetic model Drosophila. Thus, these so-called 'seizure-suppressor' gene variants may lead to the development of a novel class of antiseizure medications.
Area covered:
This review evaluates the potential therapeutic of seizure-suppressor gene variants.
Expert opinion:
Studies on epilepsy-associated genes have allowed analyses of mutations linked to human epilepsy by reproducing these mutations in Drosophila using reverse genetics to generate potential antiseizure therapeutics. As a result, about fifteen seizure-suppressor gene mutants have been identified. Furthermore, some of these epilepsy gene mutations affect ligand-and voltage-gated ion channels. Therefore, a better understanding of the antiseizure activity of seizure-suppressor genes is essential in advancing gene therapy and precision medicine for epilepsy.
... The daughterless(da)-GAL4 was obtained from Bloomington Drosophila Stock Center. Elav3A-Gal4 was a gift of M. Tanouye (72). Glass multimer reporter gmr-GAL4(III) was a gift from Y. Hiromi. ...
Spinocerebellar ataxia type 2 is a polyglutamine (polyQ) disease associated with an expanded polyQ domain within the protein product of the ATXN2 gene. Interestingly, polyQ repeat expansions in ATXN2 are also associated with amyotrophic lateral sclerosis (ALS) and parkinsonism depending upon the length of the polyQ repeat expansion. The sequence encoding the polyQ repeat also varies with disease presentation: a pure CAG repeat is associated with SCA2, whereas the CAG repeat in ALS and parkinsonism is typically interrupted with the glutamine encoding CAA codon. Here we asked if the purity of the CAG sequence encoding the polyQ repeat in ATXN2 could impact the toxicity of the ataxin-2 protein in vivo in Drosophila. We found that ataxin-2 encoded by a pure CAG repeat conferred toxicity in the retina and nervous system, whereas ataxin-2 encoded by a CAA-interrupted repeat or CAA-only repeat failed to confer toxicity, despite expression of the protein at similar levels. Furthermore, the CAG-encoded ataxin-2 protein aggregated in the fly eye, while ataxin-2 encoded by either a CAA/G or CAA repeat remained diffuse. The toxicity of the CAG-encoded ataxin-2 protein was also sensitive to the translation factor eIF4H, a known modifier of the toxic GGGGCC repeat in flies. These data indicate that ataxin-2 encoded by a pure CAG vs interrupted CAA/G polyQ repeat domain is associated with differential toxicity, indicating that mechanisms associated with the purity of the sequence of the polyQ domain contribute to disease.
... Our results demonstrated that a decrease in esg transcription in the nervous system prolonged life span, thus confirming that the neuronal function of esg is indeed relevant to life span control. Microarray analysis revealed approximately 100 esg primary targets whose transcription was induced or repressed by neuronal esg overexpression in Drosophila larvae (Hekmat-Scafe et al., 2005). Targets of esg encoded enzymes involved in the biosynthesis of neurotransmitters, neuropeptides, cationic transporters and other proteins. ...
... Our preliminary data showed that insertion mutations in genes that are also involved in the control of asymmetric neuroblast division, such as inscuteable, shaggy, and aPKC, are also able to increase life span; however, these results only represent the first steps toward investigating the molecular mechanisms underlying esg effects on life span. Interestingly, ectopic neuronal expression of esg is a general seizure suppressor and esg must be ectopically expressed during nervous system development to produce adults that are less seizure prone (Hekmat-Scafe et al., 2005). Our suggestions agree well with these data. ...
In recent years, several genes involved in complex neuron specification networks have been shown to control life span. However, information on these genes is scattered, and studies to discover new neuronal genes and gene cascades contributing to life span control are needed, especially because of the recognized role of the nervous system in governing homeostasis, aging, and longevity. Previously, we demonstrated that several genes that encode RNA polymerase II transcription factors and that are involved in the development of the nervous system affect life span in Drosophila melanogaster. Among other genes, escargot (esg) was demonstrated to be causally associated with an increase in the life span of male flies. Here, we present new data on the role of esg in life span control. We show that esg affects the life spans of both mated and unmated males and females to varying degrees. By analyzing the survival and locomotion of the esg mutants, we demonstrate that esg is involved in the control of aging. We show that increased longevity is caused by decreased esg transcription. In particular, we demonstrate that esg knockdown in the nervous system increased life span, directly establishing the involvement of the neuronal esg function in life span control. Our data invite attention to the mechanisms regulating the esg transcription rate, which is changed by insertions of DNA fragments of different sizes downstream of the structural part of the gene, indicating the direction of further research. Our data agree with the previously made suggestion that alterations in gene expression during development might affect adult lifespan, due to epigenetic patterns inherited in cell lineages or predetermined during the development of the structural and functional properties of the nervous system.
... The transcription factor Escargot (Esg), a homologue of mammalian Slug, encodes a zinc finger motif present in genes of the Snail family of transcription factors 8 . Previous studies in Drosophila showed that Esg maintains the diploidy of imaginal cells 9 , regulates cell adhesion and motility in trachea 10 , and acts as a Seizure repressor in a Drosophila epilepsy model 11 . Esg can directly interact with Daughterless (Da), thereby preventing Da protein degradation and thus promoting neuronal differentiation 12 . ...
The balanced maintenance and differentiation of local stem cells is required for Homeostatic renewal of tissues. In the Drosophila midgut, the transcription factor Escargot (Esg) maintains undifferentiated states in intestinal stem cells, whereas the transcription factors Scute (Sc) and Prospero (Pros) promote enteroendocrine cell specification. However, the mechanism through which Esg and Sc/Pros coordinately regulate stem cell differentiation is unknown. Here, by combining chromatin immunoprecipitation analysis with genetic studies, we show that both Esg and Sc bind to a common promoter region of pros. Moreover, antagonistic activity between Esg and Sc controls the expression status of Pros in stem cells, thereby, specifying whether stem cells remain undifferentiated or commit to enteroendocrine cell differentiation. Our study therefore reveals transcription factor antagonism between Esg and Sc as a novel mechanism that underlies fate specification from intestinal stem cells in Drosophila.
... We found that the distribution of Cac-GFP remains unchanged in wildtype and eas KO mutants ( Figures S3G-S3L), suggesting that eas does not regulate the localization of Cacophony. Although overexpressing escargot (esg, a member of the snail family of transcription factors) (Hekmat-Scafe et al., 2005) or kazachoc (kcc, K + -Cl − cotransporter) (Hekmat-Scafe et al., 2010) was able to suppress seizure-like phenotype in eas mutants, their overexpression in class IV da neurons did not suppress the dendrite phenotype in eas KO mutants ( Figures S3C-S3F). Therefore, the increased Ca 2+ influx, rather than hyperexcitability per se, likely contribute to dendrite morphogenesis defects in eas KO mutants. ...
Disruptions in lipid homeostasis have been observed in many neurodevelopmental disorders that are associated with dendrite morphogenesis defects. However, the molecular mechanisms of how lipid homeostasis affects dendrite morphogenesis are unclear. We find that easily shocked (eas), which encodes a kinase with a critical role in phospholipid phosphatidylethanolamine (PE) synthesis, and two other enzymes in this synthesis pathway are required cell autonomously in sensory neurons for dendrite growth and stability. Furthermore, we show that the level of Sterol Regulatory Element-Binding Protein (SREBP) activity is important for dendrite development. SREBP activity increases in eas mutants, and decreasing the level of SREBP and its transcriptional targets in eas mutants largely suppresses the dendrite growth defects. Furthermore, reducing Ca2+ influx in neurons of eas mutants ameliorates the dendrite morphogenesis defects. Our study uncovers a role for EAS kinase and reveals the in vivo function of phospholipid homeostasis in dendrite morphogenesis.
... The comparably simple nervous system of both adult fly and larvae, coupled to genetic tractability, makes this insect attractive for studying the mechanisms that underlie seizure events. Molecular screens have also been used to identify seizure-suppressor genes, the identity of which can greatly facilitate not only better understanding of the seizure process but also identify possible targets for AED design [6][7][8]. However, molecular screens often generate lengthy lists of genes which require validation. ...
Although there are now a number of antiepileptic drugs (AEDs) available, approximately one-third of epilepsy patients respond poorly to drug intervention. The reasons for this are complex, but are probably reflective of the increasing number of identified mutations that predispose individuals to this disease. Thus, there is a clear requirement for the development of novel treatments to address this unmet clinical need. The existence of gene mutations that mimic a seizure-like behaviour in the fruit fly, Drosophila melanogaster, offers the possibility to exploit the powerful genetics of this insect to identify novel cellular targets to facilitate design of more effective AEDs. In this study we use neuronal expression of GCaMP, a potent calcium reporter, to image neuronal activity using a non-invasive and rapid method. Expression in motoneurons in the isolated CNS of third instar larvae shows waves of calcium-activity that pass between segments of the ventral nerve cord. Time between calcium peaks, in the same neurons, between adjacent segments usually show a temporal separation of greater than 200 ms. Exposure to proconvulsants (picrotoxin or 4-aminopyridine) reduces separation to below 200 ms showing increased synchrony of activity across adjacent segments. Increased synchrony, characteristic of epilepsy, is similarly observed in genetic seizure mutants: bangsenseless1 (bss1) and paralyticK1270T (paraK1270T). Exposure of bss1 to clinically-used antiepileptic drugs (phenytoin or gabapentin) significantly reduces synchrony. In this study we use the measure of synchronicity to evaluate the effectiveness of known and novel anticonvulsive compounds (antipain, isethionate, etopiside rapamycin and dipyramidole) to reduce seizure-like CNS activity. We further show that such compounds also reduce the Drosophila voltage-gated persistent Na+ current (INaP) in an identified motoneuron (aCC). Our combined assays provide a rapid and reliable method to screen unknown compounds for potential to function as anticonvulsants.
... Identifying seizure suppressor genes in Drosophila has proven effective for identifying mechanisms underlying seizure and identifying novel targets for AED design (Kuebler et al., 2001;Hekmat-Scafe et al., 2005;Parker et al., 2011a). For example, topoisomerase 1 (top1 JS ) and gilgamesh mutant flies, as well as the topoisomerase 1 inhibitor, camptothecin, reduce the severity of bss 1 seizure behaviour (Song et al., 2007;Howlett et al., 2013). ...
Seizure can result from increased voltage-gated persistent sodium current expression. Although many clinically-approved antiepileptic drugs target voltage-gated persistent sodium current, none exclusively repress this current without also adversely affecting the transient voltage-gated sodium current. Achieving a more selective block has significant potential for the treatment of epilepsy. Recent studies show that voltage-gated persistent sodium current amplitude is regulated by alternative splicing offering the possibility of a novel route for seizure control. In this study we identify 291 splicing regulators that, on knockdown, alter splicing of the Drosophila voltage-gated sodium channel to favour inclusion of exon K, rather than the mutually exclusive exon L. This change is associated with both a significant reduction in voltage-gated persistent sodium current, without change to transient voltage-gated sodium current, and to rescue of seizure in this model insect. RNA interference mediated knock-down, in two different seizure mutants, shows that 95 of these regulators are sufficient to significantly reduce seizure duration. Moreover, most suppress seizure activity in both mutants, indicative that they are part of well conserved pathways and likely, therefore, to be optimal candidates to take forward to mammalian studies. We provide proof-of-principle for such studies by showing that inhibition of a selection of regulators, using small molecule inhibitors, is similarly effective to reduce seizure. Splicing of the Drosophila sodium channel shows many similarities to its mammalian counterparts, including altering the amplitude of voltage-gated persistent sodium current. Our study provides the impetus to investigate whether manipulation of splicing of mammalian voltage-gated sodium channels may be exploitable to provide effective seizure control.
© The Author (2015). Published by Oxford University Press on behalf of the Guarantors of Brain.
... and Kyoto (http:// kyotofly.kit.jp/cgi-bin/stocks/index.cgi). Screens using these libraries or novel lines have successfully identified novel genetic enhancers or suppressors related to human diseases, such as in cell outgrowth control [33], lifespan determination [34], epilepsy [35] and neuronal degeneration [36][37][38][39]. ...
... The combined use of gene manipulation with transcriptome analysis has generated data for a number of disease model systems, including renal disease [61], epilepsy [35], Parkinson's diseases [62], polyglutamine [63], Alzheimer's disease [36] and repeated RNA toxicity [64]). Notably a comparative genomic approach was integrated to identify taupathy regulators by parallel screens using tau mutant mouse brain RNA microarray and the fly rough eye modifier [65] (see below sections for similar strategy for interactomics). ...
It is occasionally observed that common sporadic diseases have rare familial counterparts in which mutations at a single locus result in a similar disorder exhibiting simple Mendelian inheritance. Such an observation is often sufficient justification for the creation of a disease model in the fly. Whether the system is based on the over-expression of a toxic variant of a human protein or requires the loss of function of an orthologous fly gene, the consequent phenotypes can be used to understand pathogenesis through the discovery of genetic modifiers. Such genetic screening can be completed rapidly in the fly and in this review we outline how libraries of mutants are generated and how consequent changes in disease-related phenotypes are assessed. The bioinformatic approaches to processing the copious amounts of data so generated are also presented. The next phase of fly modelling will tackle the challenges of complex diseases in which many genes are associated with risk in the human. There is growing interest in the use of interactomics and epigenetics to provide proteome- and genome-scale descriptions of the regulatory dysfunction that results in disease.
... For example, utilized mutagenesis by P-element transposons in an eas genetic background. Primary screening is behavioral, selecting for exceptional bang-resistant eas flies that indicate a reversion of the eas BS paralytic phenotype Hekmat-Scafe et al., 2005;. Suppressor mutations (second-site suppressors) are separated from eas genetically, then mapped, cloned, and characterized (Table II). ...
... A total of nine seizure-suppressor mutations have been identified, to date by forward genetics screens, including two that validated screening procedures: a new allele of the Na + channel gene (para JS1 ) and a new allele of the gap junction channel gene (shakB JS ). Several novel seizure-suppressor mutants were also identified including DNA topoisomerase I (top1 JS ), a Zn 2+ -finger transcription factor (esg), and a ring finger B-box coiled-coil-NHL protein (mei-P26 EG16 ) Hekmat-Scafe et al., 2005;. ...
... Gap junction channel escargot (esg EP684 + 4 alleles) Zn 2+ -finger transcription factor (Hekmat-Scafe et al., 2005) meiosis-P26 (mei-P26 EG16 , mei-P26 1 ) Ring finger B-box coiled-coil-NHL protein topoisomerase I (top1 JS + 3 alleles) DNA topoisomerase type I kazal-domain protein-1 (kdp1) Kazal-type serine protease inhibitor (Hekmat-Scafe et al., 2005) kazal-domain protein-2 (kdp2) Kazal-type serine protease inhibitor (Hekmat-Scafe et al., 2005) suppressor of eas7 (su(eas7)) Unknown suppressor of eas13 (su(eas13)) Unknown This Seizure susceptibility for some BS mutants are completely reverted to wild-type levels (Class III). Some mutations (Class II) show partial suppression. ...
Despite the frequency of seizure disorders in the human population, the genetic and physiological basis for these defects has been difficult to resolve. Although many genetic contributions to seizure susceptibility have been identified, these involve disparate biological processes, many of which are not neural specific. The large number and heterogeneous nature of the genes involved makes it difficult to understand the complex factors underlying the etiology of seizure disorders. Examining the effect known genetic mutations have on seizure susceptibility is one approach that may prove fruitful. This approach may be helpful in both understanding how different physiological processes affect seizure susceptibility and identifying novel therapeutic treatments. We review here factors contributing to seizure susceptibility in Drosophila, a genetically tractable system that provides a model for human seizure disorders. Seizure-like neuronal activities and behaviors in the fruit fly are described, as well as a set of mutations that exhibit features resembling some human epilepsies and render the fly sensitive to seizures. Especially interesting are descriptions of a novel class of mutations that are second-site mutations that act as seizure suppressors. These mutations revert epilepsy phenotypes back to the wild-type range of seizure susceptibility. The genes responsible for seizure suppression are cloned with the goal of identifying targets for lead compounds that may be developed into new antiepileptic drugs.
