DNA topoisomerase I inhibitors ameliorate seizure-like behaviors and paralysis in a Drosophila model of epilepsy.

The Drosophila DNA topoisomerase type I mutant allele, top1JS is an effective general seizure-suppressor mutation, reverting seizure-sensitive phenotypes of several mutant strains in a genetic model of epilepsy. Seizure-suppression is caused by reduced transcription of the top1 (topoisomerase I gene) gene [Song J, Hu J, Tanouye MA. (2007) Seizure suppression by top1 mutations in Drosophila. J Neurosci 27(11):2927-2937]. Here, we examine the possibility that pharmaceutical inhibition of Top1 (topoisomerase I protein) enzymatic activity may also be effective at reducing seizure phenotypes. We investigate the effect of vertebrate Top1 inhibitor camptothecin (CPT) along with two related compounds, apigenin and kaempferol, when fed to seizure-sensitive mutant Drosophila. All three Top1 inhibitors were found to suppress phenotypes in these mutants. In particular, for drug treatments, the recovery time from seizure and paralysis is greatly reduced compared with untreated animals. Intriguingly we find that chronic drug treatments result in a small reduction in seizure sensitivity. Taken together, the results suggest that Top1 inhibitors may have the potential to be developed into effective anti-epileptic drugs, especially for brain tumor patients presenting with epilepsy.

Drosophila homolog of APP-BP1 (dAPP-BP1) interacts antagonistically with APPL during Drosophila development.

beta-Amyloid precursor protein binding protein 1 (APP-BP1) was previously identified based on its binding to the carboxyl terminal of beta-amyloid precursor protein. In this report, we have discovered that a mutation of dAPP-BP1 (Drosophila ortholog of APP-BP1) hinders tissue development, causes apoptosis in imaginal disc cells, and blocks the NEDD8 conjugation pathway. We show that dAPP-BP1 specifically binds the intracellular domain of APP-like protein (APPL). The dAPP-BP1 mutation partially suppresses the abnormal macrochaete phenotype of Appl(d), while overexpression of dAPP-BP1 causes abnormal macrochaetes. When APPL is overexpressed, the normal bristle pattern in the fly thorax is disturbed and apoptosis is induced in wing imaginal discs. APPL overexpression phenotypes are enhanced by reducing the level of dAPP-BP1. APPL overexpression is shown to inhibit the NEDD8 conjugation pathway. APPL-induced apoptosis is rescued by overexpression of dAPP-BP1. Our data suggest that APPL and dAPP-BP1 interact antagonistically during Drosophila development.

Genetic suppression of seizure susceptibility in Drosophila.

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 defects that cause seizure susceptibility have been identified, the defects 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 both in understanding how different physiological processes affect seizure susceptibility and in identifying novel therapeutic treatments. In this study, we have taken advantage of Drosophila, a genetically tractable system, to identify factors that suppress seizure susceptibility. Of particular interest has been a group of Drosophila mutants, the bang-sensitive (BS) mutants, which are much more susceptible to seizures than wild type. The BS phenotypic class includes at least eight genes, including three examined in this study, bss, eas, and sda. Through the generation of double-mutant combinations with other well-characterized Drosophila mutants, the BS mutants are particularly useful for identifying genetic factors that suppress susceptibility to seizures. We have found that mutants affecting Na+ channels, mle(napts) and para, K+ channels, Sh, and electrical synapses, shak-B(2), can suppress seizures in the BS mutants. This is the first demonstration that these types of mutations can suppress the development of seizures in any organism. Reduced neuronal excitability may contribute to seizure suppression. The best suppressor, mle(napts), causes an increased stimulation threshold for the giant fiber (GF) consistent with a reduction in single neuron excitability that could underlie suppression of seizures. For some other double mutants with para and Sh(KS133), there are no GF threshold changes, but reduced excitability may also be indicated by a reduction in GF following frequency. These results demonstrate the utility of Drosophila as a model system for studying seizure susceptibility and identify physiological processes that modify seizure susceptibility.

Modifications of seizure susceptibility in Drosophila.

In a given population, certain individuals are much more likely to have seizures than others. This increase in seizure susceptibility can lead to spontaneous seizures, such as seen in idiopathic epilepsy, or to symptomatic seizures that occur after insults to the nervous system. Despite the frequency of these seizure disorders in the human population, the genetic and physiological basis for these defects remains unclear. The present study makes use of Drosophila as a potentially powerful model for understanding seizure susceptibility in humans. In addition to the genetic and molecular advantages of using Drosophila, it has been found that seizures in Drosophila share much in common with seizures seen in humans. However, the most powerful aspect of this model lies in the ability to accurately measure seizure susceptibility across genotypes and over time. In the current study seizure susceptibility was quantified in a variety of mutant and wild-type strains, and it was found that genetic mutations can modulate susceptibility over an extremely wide range. This genetic modulation of seizure susceptibility apparently occurs without affecting the threshold of individual neurons. Seizure susceptibility also varied depending on the experience of the fly, decreasing immediately after a seizure and then gradually increasing over time. A novel phenomenon was also identified in which seizures are suppressed after certain high-intensity stimuli. These results demonstrate the utility of Drosophila as a model system for studying human seizure disorders and provide insights into the possible mechanisms by which seizure susceptibility is modified.

Interspecific movement of the paternal sex ratio chromosome.

Here we examine the potential for interspecific movement of a supernumerary or B chromosome and its subsequent stability. B chromosomes differ from autosomes in that they are nonvital, nonpairing chromosomes which vary in number between conspecific individuals. According to one generally accepted hypothesis, B chromosomes originate from the autosomes of their host. However, previous comparisons of B chromosome and host autosome sequences have suggested an additional evolutionary route: that B chromosomes originating in one species may be subsequently transferred into another species via interspecific hybridization. To examine B chromosome movement, hybrid crosses were utilized to introduce the paternal sex ratio chromosome (PSR) of Nasonia vitripennis into N. giraulti and N. longicornis. The results demonstrate that these transfers occur at high rates and that the meiotic drive system of PSR continues to function in both species, resulting in the maintenance of PSR at high frequencies. The relevance of these results to origin hypotheses of PSR is discussed here, as are the potential ecological effects of naturally occurring PSR movement and the potential applied uses of the mechanisms of PSR.

Evidence for a genomic imprinting sex determination mechanism in Nasonia vitripennis (Hymenoptera; Chalcidoidea).

Five different models have been proposed for the sex determination mechanism of Chalcidoidea (Hymenoptera). Except for the most recently proposed model (genomic imprinting sex determination; GISD), each of these models has required complicating additions to explain observed phenomena. This report provides the first experimental test of the GISD model while simultaneously examining the four previously proposed models of sex determination. This test utilizes the parasitic wasp Nasonia vitripennis, crossing polyploid females with males harboring the paternal sex ratio chromosome (PSR). The results of this study support the GISD model as the mechanism of sex determination in Chalcidoidea. Specifically, crosses demonstrate that sex determination is independent of embryonic heterozygosity, ploidy, and gametic syngamy but is directly correlated with the embryonic presence of correctly imprinted chromosomes of paternal origin. These crossing experiments also provide information about the poorly characterized mechanisms of PSR, a supernumerary chromosome that induces paternal autosome loss in early embryos. The results demonstrate that the poor transmission of PSR through females is not a result of the ploidy of the host but of an alternative sex-dependent process. Crossing data reveal that PSR consistently induces the loss of the entire paternal complement that it accompanies, regardless of whether this complement is haploid or diploid.

Identification of a Drosophila muscle development gene with structural homology to mammalian early growth response transcription factors.

In Drosophila, stripe (sr) gene function is required for normal muscle development. Some mutations disrupt embryonic muscle development and are lethal. Other mutations cause total loss of only a single muscle in the adult. Molecular analysis shows that sr encodes a predicted protein containing a zinc finger motif. This motif is homologous to the DNA binding domains encoded by members of the early growth response (egr) gene family. In mammals, expression of egr genes is induced by intercellular signals, and there is evidence for their role in many developmental events. The identification of sr as an egr gene and its pattern of expression suggest that it functions in muscle development via intercellular communication.

Seizures and failures in the giant fiber pathway of Drosophila bang-sensitive paralytic mutants.

Drosophila bang-sensitive paralytic mutants suffer from hyperactivity and paralysis following a mechanical shock; after recovery from paralysis, they cannot be paralyzed for a refractory period lasting up to 1 hr. Previously, we have shown that in easily shocked (eas), a typical bang-sensitive mutant, electrical shocks delivered to the brain cause seizure-like activity in the dorsal longitudinal flight motor neurons (DLMmns), and failure of giant fiber (GF) stimulation to evoke DLM potentials via the escape response pathway (Pavlidis et al., 1994). Here, we show that seizure and failure in the GF pathway with a refractory period is common to all six members of the bang-sensitive class. This syndrome was not found in any of eight other excitability mutants, including those affecting voltage-gated sodium or potassium-channel function. We show that failure occurs at the synapse between a peripherally synapsing interneuron (PSI) and the DLMmns, while the DLMmn-DLM neuromuscular junctions remain functional. Additionally, failure occurs in all other GF pathway-activated muscles. Failures occurred without seizures in the tergotrochanteral jump muscle (TTM), as was also found in approximately 10% of DLM tests, suggesting that seizures and failures may be independent events. This hypothesis is supported by the finding that, in double mutant combination with mlenapts, which suppresses behavioral bang sensitivity, DLM failures, but not seizures, were reduced.

The Drosophila easily shocked gene: a mutation in a phospholipid synthetic pathway causes seizure, neuronal failure, and paralysis.

We have characterized easily shocked (eas), a Drosophila "band-sensitive" paralytic mutant. Electrophysiological recordings from flight muscles in the giant fiber pathway of adult eas flies reveal that induction of paralysis with electrical stimulation results in a brief seizure, followed by a failure of the muscles to respond to giant fiber stimulation. Molecular cloning, germline transformation, and biochemical experiments show that eas mutants are defective in the gene for ethanolamine kinase, which is required for a pathway of phosphatidylethanolamine synthesis. Assays of phospholipid composition reveal that total phosphatidylethanolamine is decreased in eas mutants. The data suggest that eas bang sensitivity is due to an excitability defect caused by altered membrane phospholipid composition.

bendless, a Drosophila gene affecting neuronal connectivity, encodes a ubiquitin-conjugating enzyme homolog.

The Drosophila bendless (ben) gene was originally isolated as a mutation affecting the escape jump response. This behavioral defect was ascribed to a single lesion affecting the connectivity between the giant fiber and the tergotrochanter motor neuron. A closer examination of the ben phenotype suggests that ben activity is broader and affects a variety of other neurons including photoreceptor cells and their axons. Mosaic analysis indicates that the focus of ben activity is presynaptic. We have cloned the ben gene through a chromosomal walk and show that it is homologous to a class of ubiquitin-conjugating enzymes. The major role of ubiquitination in the protein degradative pathway suggests that ben regulates neural developmental processes such as growth cone guidance by targeting specific proteins for degradation.

Tandem linkage of Shaker K+ channel subunits does not ensure the stoichiometry of expressed channels.

Shaker K+ channels are multimeric, probably tetrameric proteins. Substitution of a conserved leucine residue to valine (V2) at position 370 in the Drosophila Shaker 29-4 sequence results in large alterations in the voltage dependence of gating in the expressed channels. In order to determine the effects of this mutation in hybrid channels with a fixed stoichiometry of V2 and wild-type (WT) subunits we generated cDNA constructs of two linked-monomeric subunits similar to the tandem constructs previously reported by Isacoff, E. Y., Y. N. Jan, and L. Y. Jan. (1990. Nature (Lond.). 345:530-534). In addition, we constructed a tandem cDNA containing a wild-type subunit and a truncated nonfunctional subunit (Sh102) that suppresses channel expression. We report that the voltage-dependence of the channels produced with WT and V2 subunits varied significantly with the order of the subunits in the construct (WT-V2 or V2-WT), while the WT-Sh102 construct yielded currents that were much larger than expected. These results suggest that the tandem linkage of Shaker subunits does not guarantee the stoichiometry of the expressed channel proteins.

The size of gating charge in wild-type and mutant Shaker potassium channels.

The high sensitivity of voltage-gated ion channels to changes in membrane potential implies that the process of channel opening is accompanied by large charge movements. Previous estimates of the total charge displacement, q, have been deduced from the voltage dependence of channel activation and have ranged from 4 to 8 elementary charges (e0). A more direct measurement of q in Drosophila melanogaster Shaker 29-4 potassium channels yields a q value of 12.3 e0. A similar q value is obtained from mutated Shaker channels having reduced voltage sensitivity. These results can be explained by a model for channel activation in which the equilibria of voltage-dependent steps are altered in the mutant channels.

A role for hydrophobic residues in the voltage-dependent gating of Shaker K+ channels.

A leucine heptad repeat is well conserved in voltage-dependent ion channels. Herein we examine the role of the repeat region in Shaker K+ channels through substitution of the leucines in the repeat and through coexpression of normal and truncated products. In contrast to leucine-zipper DNA-binding proteins, we find that the subunit assembly of Shaker does not depend on the leucine heptad repeat. Instead, we report that substitutions of the leucines in the repeat produce large effects on the observed voltage dependence of conductance voltage and prepulse inactivation curves. Our results suggest that the leucines mediate interactions that play an important role in the transduction of charge movement into channel opening and closing.

Expression of ion channel genes in Drosophila.

Ion channel transcripts from three genes were localized by the method of tissue in situ hybridization. The genes examined were the Drosophila Na+ channel genes, paralytic (para) and Drosophila Sodium Channel (DSC), and the K+ channel gene, Shaker( Sh). All three of the genes were expressed in cell bodies of the fly central nervous system, including optic lobes, central brain, suboesophageal ganglion, and thoracico-abdominal ganglion. Sh was additionally expressed in the photoreceptor cells of the retina and pupal flight muscle, while para and DSC were not. The temporal expression pattern of Sh in muscle was different from that in the central nervous system: muscle expression was transient and limited to mid-pupal stage while nervous system expression was observed throughout pupation, apparently peaking at the late-pupal stage. Only one class of 5' end was found in pupal muscle, possibly indicating regulation of splicing pathways.

Alteration of potassium channel gating: molecular analysis of the Drosophila Sh5 mutation.

The Drosophila Shaker (Sh) gene encodes a family of voltage-gated K+ channels. Mutant alleles of Sh alter the currents expressed from these channels in a variety of ways. To identify the molecular basis of these alterations, Sh transcript sequences were amplified using the polymerase chain reaction after reverse transcription of mutant RNA. Amplified products from each mutant were cloned and sequenced. Two alleles, Sh102 and Sh5, had single base substitutions in the central conserved region shared by all Sh channels. RNA synthesized in vitro from a cDNA construct carrying the Sh5 mutation was injected into Xenopus oocytes. Currents expressed by the mutant RNA were altered in their voltage dependence of activation and inactivation, similar to the alterations in Sh currents recorded from different preparations of Sh5 fly tissue. The changes in current properties and the location of the mutation are consistent with the participation of a novel region of the channel in voltage gating.

Molecular cloning and functional expression of a potassium channel cDNA isolated from a rat cardiac library.

A full-length K+ channel cDNA (RHK1) was isolated from a rat cardiac library using the polymerase chain reaction (PCR) method and degenerate oligonucleotide primers derived from K+ channel sequences conserved between Drosophila Shaker H4 and mouse brain MBK1. Although RHK1 was isolated from heart, its expression was found in both heart and brain. The RHK1-encoded protein, when expressed in Xenopus oocytes, gated a 4-aminopyridine (4-AP)-sensitive transient outward current. This current is similar to the transient outward current measured in rat ventricular myocytes with respect to voltage-dependence of activation and inactivation, time course of activation and inactivation, and pharmacology.

Genetic analysis of the Shaker gene complex of Drosophila melanogaster.

The Shaker complex (ShC) spans over 350 kb in the 16F region of the X chromosome. It can be dissected by means of aneuploids into three main sections: the maternal effect (ME), the viable (V) and the haplolethal (HL) regions. The mutational analysis of ShC shows a high density of antimorphic mutations among 12 lethal complementation groups in addition to 14 viable alleles. The complex is the structural locus of a family of potassium channels as well as a number of functions relevant to the biology of the nervous system. The constituents of ShC seem to be linked by functional relationships in view of the similarity of the phenotypes, antimorphic nature of their mutations and the behavior in transheterozygotes. We discuss the relationship between the genetic organization of ShC and the functional coupling of potassium currents with the other functions encoded in the complex.

Human potassium channel genes: Molecular cloning and functional expression.

Complementary DNAs representing three voltage-gated K(+) channels from humans (HuKI, HuKII, and HuKIV) were isolated, their nucleotide sequences determined, and their functional products examined electrophysiologically. The three human K(+) channels are closely related to the Shaker gene of Drosophila and possess several canonical structural features including multiple hydrophobic segments which are potentially membrane spanning, a positively charged S4 segment which may be the voltage sensor, and a leucine heptad repeat which may be involved in channel gating. Members of the human gene family have specific, highly conserved homologs in rodents, suggesting that the individual members arose prior to the mammalian radiation. The degree of homology indicates that these are among the most highly conserved proteins known. The three human channels expressed in Xenopus oocytes vary in voltage dependence, kinetics, and sensitivity to pharmacological blockers of K(+) channels. HuKII is a rapidly inactivating channel; HuKI and HuKIV are noninactivating. Also, although all three channels are sensitive to the K(+) channel blocker, 4-aminopyridine, only HuKI has tetraethylammonium sensitivity; only HuKIV has charybdotoxin sensitivity. Differences are observed between the pharmacological sensitivities of human channels and the reported sensitivities of their rat homology.

Expression of Drosophila Shaker potassium channels in mammalian cells infected with recombinant vaccinia virus.

A recombinant vaccinia virus containing a Drosophila potassium channel (Shaker H4) cDNA was constructed by homologous recombination between wild-type vaccinia virus DNA and a transfer plasmid. The new virus was used to infect four types of mammalian cells in culture. Electrophysiological recording 24-72 hr after infection revealed the expression of voltage-gated transient potassium channels in all four cell types. The properties of the induced currents were identical to those previously observed following injection of the Shaker H4 transcript into oocytes. Vaccinia promises to be an effective vehicle for the heterologous expression of transmembrane ion channels in a variety of cell types.

Two sodium-channel genes in Drosophila: implications for channel diversity.

We describe two Drosophila melanogaster transcription units that are highly homologous to a rat Na+-channel cDNA. They appear to encode the major subunits of two distinct Na+-channel proteins. One of these maps to the second chromosome and is identical to a Na+-channel gene whose partial sequence has been previously reported [Salkoff, L., Butler, A., Wei, A., Scavarda, N., Giffen, K., Ifune, K., Goodman, R. & Mandel, G. (1987) Science 237, 744-749]. The other transcription unit maps to position 14C/D, on the X chromosome, close to the paralyzed (para) gene. Mutations in para affect membrane excitability in Drosophila neurons [Ganetzky, B. & Wu, C.F. (1986) Annu. Rev. Genet. 20, 13-44]. Sequence comparisons suggest that two Na+-channel genes arose early in evolution, before the divergence of vertebrate and invertebrate lines.