Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: Human seizure disorders are a significant health concern due to the large number of affected individuals, the potentially devastating ramifications of untreated seizure episodes, and the limitations of anti-epileptic drug (AED) options. Studies of human seizure disorders have revealed that susceptibility to seizures is largely influenced by genetic factors. In addition to causing epilepsy, genetic factors can also suppress seizures and epileptogenesis. We propose that the fruit fly Drosophila provides a valuable genetic model for approaching basic biological insights and therapies for human epilepsy. During this project period, we investigated the seizure suppressor mutant gish that was identified from a single third chromosome deficiency (Ed10639) that reduced the bang-sensitivity in a heterozygous bss1 background. An additional deficiency (Exel7329) overlapping the original also strongly reduced bang-sensitivity. The region identified by the two overlapping deficiencies contained 6 genes. Individual mutations were collected for each available gene and the gish08495 allele was found that suppresses bss1 heterozygotes. The seizure threshold of bss1;; gish08945 and bss1;; Df Ed10639 trans-heterozygotes are significantly higher than balancer controls. Several different RNAi stocks have been obtained to reduce expression of gish. Stocks were constructed containing several GAL-4 drivers in a bss1 background. These drivers include repo-GAL-4, which allows selective expression of the RNAi construct in glia and hsp70-GAL-4, which allows ubiquitous expression following a heat-shock. The gish08495 allele is caused by a P-element insertion in the 5'UTR of 6 of the 9 transcripts. We are continuing investigation to determine if the gish suppression is via activation of the Wnt signaling pathway. Event: None. Services: None. Products: None. Dissemination: None. PARTICIPANTS: Mark A. Tanouye, Ph.D. (principal investigator) Arunesh Saras, Ph.D. (postdoctoral fellow) Iris Howlett (graduate student) Zeid Rusan (graduate student) TARGET AUDIENCES: Individuals afflicted with seizure disorders such as epilepsy. PROJECT MODIFICATIONS: No major changes to report.
Impacts
The Drosophila mutants that we work on have two dramatic phenotypes. They are seizure-susceptible which may ultimately provide basic information in developing new anti-convulsant drugs for human seizure disorders. They also suffer from paralysis that may provide a basis for designing new types of pesticides to fight insects.
Publications
- Parker L, Howlett IC, Rusan ZM, Tanouye MA (2011). Seizure and epilepsy: studies of seizure disorders in Drosophila. Intl Rev Neurobiol 99, 2-18.
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Progress 10/01/01 to 09/30/11
Outputs
OUTPUTS: The last report submitted was the final report. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The last report submitted was the final report including the Publications information.
Publications
- No publications reported this period
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: Outputs Activities Human seizure disorders are a significant health concern due to the large number of affected individuals, the potentially devastating ramifications of untreated seizure episodes, and the limitations of anti-epileptic drug (AED) options. Studies of human seizure disorders have revealed that susceptibility to seizures is largely influenced by genetic factors. In addition to causing epilepsy, genetic factors can also suppress seizures and epileptogenesis. We propose that the fruit fly Drosophila provides a valuable genetic model for approaching basic biological insights and therapies for human epilepsy. During this project period, we investigated bang senseless (bss), a Drosophila melanogaster mutant exhibiting seizure-like behaviors, and identified it as an allele of the paralytic (para) voltage-gated Na+ (NaV) channel gene. Mutants are more prone to seizure episodes than normal flies because of a lowered seizure threshold. The bss phenotypes are due to a missense mutation in a segment previously implicated in inactivation, termed the "paddle motif" of the NaV fourth homology domain. Heterologous expression of cDNAs containing the bss1 lesion, followed by electrophysiology show that mutant channels display altered voltage dependence of inactivation compared to wild type. The phenotypes of bss are the most severe of the bang sensitive (BS) mutants in Drosophila, and can be ameliorated, but not suppressed by treatment with anti-epileptic drugs. As such, bss-associated seizures resemble those of pharmacologically-resistant epilepsies caused by mutation of the human NaV SCN1A, such as severe myoclonic epilepsy in infants (SMEI) or intractable childhood epilepsy with generalized tonic-clonic seizures (ICEGTC). Events. None. Services. None. Products. None. Dissemination. None. PARTICIPANTS: Participants Mark A. Tanouye, Ph.D. (principal investigator) Louise Parker, Ph.D. (postdoctoral fellow) Iris Howlett (graduate student) Zeid Rusan (graduate student) TARGET AUDIENCES: Target audiences Individuals afflicted with seizure disorders such as epilepsy. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Outcomes Impacts The Drosophila mutants that we work on have two dramatic phenotypes. They are seizure-susceptible which may ultimately provide basic information in developing new anti-convulsant drugs for human seizure disorders. They also suffer from paralysis that may provide a basis for designing new types of pesticides to fight insects.
Publications
- Parker L, Padilla M, Du Y, Dong K, Mark A. Tanouye (2011). Drosophila as a model for epilepsy: bss is a gain-of-function mutation in the para sodium channel gene that leads to seizures. Genetics 187, 523-534.
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: Activities Human seizure disorders are a significant health concern due to the large number of affected individuals, the potentially devastating ramifications of untreated seizure episodes, and the limitations of anti-epileptic drug (AED) options. Studies of human seizure disorders have revealed that susceptibility to seizures is largely influenced by genetic factors. In addition to causing epilepsy, genetic factors can also suppress seizures and epileptogenesis. We propose that the fruit fly Drosophila provides a valuable genetic model for approaching basic biological insights and therapies for human epilepsy. During this project period, we investigated a new mutation that contributes to seizure-susceptibility in Drosophila. The mutation is named jitterbug and causes flies to be especially sensitive to seizure, about 3-fold more sensitive than normal flies. Interestingly, this seizure-sensitivity is itself also dependent on non-genetic factors. Mutants are seizure-sensitive when they are reared at colder temperatures (18degreesC), but show little seizure-sensitivity when reared at room temperature (25degreesC). Also, mutant jitterbug flies lose their seizure-sensitivity as they grow older; by a few days of age, their sensitivity to seizures is similar to normal flies. Several human epilepsies are childhood syndromes and we believe that jitterbug may be an important animal model for these. We also believe that jitterbug mutants may provide important clues about how genetic factors (such as mutations) and environmental factors (such as temperature in the case of jitterbug; alcohol, drugs, stress, etc in the case of human epilepsy) interact to build the overall seizure profile for individuals. The interaction between genetic and environmental factors in epilepsy is an important issue that has been extremely difficult to evaluate in human studies. We are especially hopeful that this issue modeled in Drosophila will provide new insights into these interactions. We are presently cloning the jitterbug gene to uncover the molecular identity of its gene product. Events. None. Services. None. Products. None. Dissemination. None. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The Drosophila mutants that we work on have two dramatic phenotypes. They are seizure-susceptible which may ultimately provide basic information in developing new anti-convulsant drugs for human seizure disorders. They also suffer from paralysis that may provide a basis for designing new types of pesticides to fight insects.
Publications
- Hekmat-Scafe DS, Mercado A, Fajilan AA, Lee AW, Hsu R, Mount DB, Tanouye MA (2010). Seizure sensitivity is ameliorated by targeted expression of K+-Cl- cotransporter function in the mushroom body of the Drosophila brain. Genetics 184,171-183. PMID: 19884312
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: Human seizure disorders are a significant health concern due to the large number of affected individuals, the potentially devastating ramifications of untreated seizure episodes, and the limitations of anti-epileptic drug (AED) options. Studies of human seizure disorders have revealed that susceptibility to seizures is largely influenced by genetic factors. In addition to causing epilepsy, genetic factors can also suppress seizures and epileptogenesis. We propose that the fruit fly Drosophila provides a valuable genetic model for approaching basic biological insights and therapies for human epilepsy. During this project period, we discovered that a Drosophila seizure mutant called bang senseless (bss) is due to a molecular lesion in a gene encoding a voltage-gated Na channel. This is an especially interesting finding because it provides an excellent animal model for several human epilepsies that are due to Na channel lesions. There are over 140 known mutations of the human Na channel that cause epilepsy. Mutations have been associated with GEFS+ (generalized epilepsy with febrile seizures plus), characterized by febrile seizures that persist beyond the age of 6y. They are also associated with the more severe SMEI (severe myoclonic epilepsy in infancy), an intractable epilepsy frequently resulting in convulsive status epilepticus. Na channel mutations are also associated with ICEGTC (intractable childhood epilepsy with generalized tonic-clonic), an atypical SMEI that does not show myoclonic seizures. The precise role of Na+ channel defects in epilepsy has not been completely clear, but now should become experimentally approachable in flies due to our discovery of the bss defect. PARTICIPANTS: Mark A. Tanouye, Ph.D. (principal investigator) Daria Hekmat-Scafe, Ph.D. (postdoctoral fellow) Louise Parker, Ph.D. (postdoctoral fellow) Iris Howlett (graduate student) Zeid Rusan (graduate student) TARGET AUDIENCES: Individuals afflicted with seizure disorders such as epilepsy. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The Drosophila mutants that we work on have two dramatic phenotypes. They are seizure-susceptible which may ultimately provide basic information in developing new anti-convulsant drugs for human seizure disorders. They also suffer from paralysis that may provide a basis for designing new types of pesticides to fight insects.
Publications
- Tanouye MA. (2008). Seymour Benzer 1921-2007 (obituary). Nat Genet. 40, 121. PMID: 18227864
- Song J, Parker L, Hormozi L & Tanouye MA (2008). DNA topoisomerase I inhibitors ameliorate seizure-like behaviors and paralysis in a Drosophila model of epilepsy. Neuroscience 156, 722-728. PMID: 18703119
- Song J & Tanouye MA (2008). From bench to drug: Human seizure modeling using Drosophila. Prog Neurobiol 84, 182-191. PMID: 18063465
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: Studies of human seizure disorders have revealed that susceptibility to seizures is largely influenced by genetic factors. In addition to causing epilepsy, genetic factors can also suppress seizures and epileptogenesis. Examination of seizure-suppressor genes is challenging in mammals, however, such genes are readily identified and analyzed in a Drosophila animal model of epilepsy. In this investigation, we examined the epilepsy phenotype of Drosophila neurological mutants with seizure disorders. A novel class of genes called seizure-suppressors is described, in detail. Mutations defining these suppressors revert the "epilepsy" phenotype of Drosophila neurological mutants. The major interest in seizure-suppressors is that they may lead to new and significant treatments for human epilepsy. Seizure-suppressor genes could help define targets for unexpected classes of anticonvulsant drugs that are effective treatments for epilepsy: treatments for intractable syndromes or
treatments with reduced side effects. Another possibility is to discover candidate genes that might be used for gene therapy. Among the several questions that arise are: what are seizure-suppressor genes and how might they lead to new therapeutics? What is the entire range of potential gene products that can act as seizure-suppressors? Is this range limited to nervous system-specific gene products, such as signaling molecules or does it include non-nervous system gene products as well? During this project period, we discovered that compounds that inhibit DNA topoisomerase I activity (top1 inhibitors) are potent anti-epileptic drugs (AEDs) when used to treat epilepsy in Drosophila mutants. They are comparable in efficacy to several currently utilized AEDs for humans, such as phenytoin and gabapentin. The top1 inhibitors examined were camptothecin and two related compounds, kaempferol and apigenin. The use of top1 inhibitors was inspired by the discovery in the previous project period
that top1 mutations are potent genetic suppressors of seizure. We were exploring the possibility here that other treatment that reduce top1 activity, such as the top1 inhibitor compounds, would also suppress seizures.
PARTICIPANTS: Mark A. Tanouye, Ph.D. (principal investigator) Daria Hekmat-Scafe, Ph.D. (postdoctoral fellow) Louise Parker, Ph.D. (postdoctoral fellow) Juan Song, Ph.D. (postdoctoral fellow)
Impacts
The Drosophila mutants that we work on have two dramatic phenotypes. They are seizure-susceptible which may ultimately provide basic information in developing new anti-convulsant drugs for human seizure disorders. They also suffer from paralysis that may provide a basis for designing new types of pesticides to fight insects.
Publications
- Song J & Tanouye MA (2007). Role for para sodium channel gene 3' UTR in the modification of Drosophila seizure susceptibility. Dev Neurobiol 67(14), 1944-1956. PMID: 17918245
- Song J, Hu J & Tanouye MA (2007). Seizure suppression by top1 mutations in Drosophila. J Neurosci 27, 2927-2937. PMID: 17360915
- Kim HJ, Kim SH, Shim SO, Park E, Kim C, Kim K, Tanouye MA & Yim J (2007). Drosophila homolog of APP-BP1 (dAPP-BP1) interacts antagonistically with APPL during Drosophila development. Cell Death Differ. 14, 103-115. PMID: 16628230
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Progress 01/01/06 to 12/31/06
Outputs
Studies of human seizure disorders have revealed that susceptibility to seizures is largely influenced by genetic factors. In addition to causing epilepsy, genetic factors can also suppress seizures and epileptogenesis. Examination of seizure-suppressor genes is challenging in mammals, however, such genes are readily identified and analyzed in a Drosophila animal model of epilepsy. In this investigation, we examined the epilepsy phenotype of Drosophila neurological mutants with seizure disorders. A novel class of genes called seizure-suppressors is described, in detail. Mutations defining these suppressors revert the "epilepsy" phenotype of Drosophila neurological mutants. The major interest in seizure-suppressors is that they may lead to new and significant treatments for human epilepsy. Seizure-suppressor genes could help define targets for unexpected classes of anticonvulsant drugs that are effective treatments for epilepsy: treatments for intractable syndromes or
treatments with reduced side effects. Another possibility is to discover candidate genes that might be used for gene therapy. Among the several questions that arise are: what are seizure-suppressor genes and how might they lead to new therapeutics? What is the entire range of potential gene products that can act as seizure-suppressors? Is this range limited to nervous system-specific gene products, such as signaling molecules or does it include non-nervous system gene products as well? During this project period, we discovered a seizure-suppressor gene encoding DNA topoisomerase I (top 1). Mutations of top 1 are especially effective at reverting the seizure phenotype of Drosophila epilepsy mutants. In addition, an unexpected class of anti-epileptic drugs has been identified. These are DNA topoisomerase 1 inhibitors such as camptothecin and its derivatives. We have conducted extensive investigations on this new class of drug and find that several candidates are as effective at reducing
seizures as traditional anti-epileptic drugs such as valproate. Further considerations of developmenting topo 1 inhibitors as anti-epileptic drugs for humans and other candidates arising from the Drosophila model are discussed.
Impacts
The Drosophila mutants that we work on have two dramatic phenotypes. They are seizure-susceptible which may ultimately provide basic information in developing new anti-convulsant drugs for human seizure disorders. They also suffer from paralysis that may provide a basis for designing new types of pesticides to fight insects.
Publications
- Hekmat-Scafe DS, Lundy MY, Ranga R & Tanouye MA (2006). Mutations in the K+/Cl- cotransporter gene kazachoc (kcc) increase seizure susceptibility in Drosophila. J Neurosci 26, 8943-8954. PMID: 16943550
- Allen MJ, Godenschwege TA, Tanouye MA & Phelan P (2006). Making an escape: development and function of the Drosophila giant fibre system. Semin Cell Dev Biol.17, 31-41. PMID: 16378740
- Song J & Tanouye MA (2006). Seizure suppression by shakB2, a gap junction mutation in Drosophila. J Neurophysiol 95, 627-635. PMID: 16192342
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Progress 01/01/05 to 12/31/05
Outputs
Gap junction proteins mediate electrical synaptic transmission. In Drosophila, flies carrying null mutations in the shakB locus, such as shakB2, have behavioral and electrophysiological defects in the giant fiber (GF) system neurocircuit consistent with a loss of transmission at electrical synapses. In this report, we describe investigations that show shakB2 mutations can also act as "seizure-suppressors." We have shown previously that seizure-suppressor mutations revert the "epilepsy" phenotype of Drosophila neurological mutants with seizure disorders. That is, suppressors are mutations that "cure" Drosophila epilepsy. The major interest in seizure-suppressors is that they may lead to new and significant treatments for human epilepsy. Seizure-suppressor genes could help define targets for unexpected classes of anticonvulsant drugs that are effective treatments for epilepsy: treatments for intractable syndromes or treatments with reduced side effects. Another
possibility is to discover candidate genes that might be used for gene therapy. Among the several questions that arise are: what are seizure-suppressor genes and how might they lead to new therapeutics? What is the entire range of potential gene products that can act as seizure-suppressors? Is this range limited to nervous system-specific gene products, such as signaling molecules or does it include non-nervous system gene products as well? The shakB2 mutation mutant flies are especially seizure-resistant and have a high threshold to evoked seizures. In addition, in some double mutant combinations with "epilepsy" mutations shakB2 appears to act as a seizure-suppressor mutation: shakB2 restores seizure-susceptibility to the wild-type range in the double mutant. In double mutant combinations, shakB2 completely suppresses seizures caused by slamdance (sda), knockdown (kdn), and jitterbug (jbug) mutations. Seizures caused by easily shocked (eas) and technical knockout (tko) mutations are
partially suppressed by shakB2. Seizures caused by bang-sensitive (bas2) and bang-senseless (bss1, bss2 alleles) mutations are not suppressed by shakB2. These results demonstrate the utility of Drosophila as a model system for studying the kinds of genetic interactions responsible for seizure susceptibility bringing us closer to unraveling the complexity of seizure disorders in humans.
Impacts
The Drosophila mutants that we work on have two dramatic phenotypes. They are seizure-susceptible which may ultimately provide basic information in developing new anti-convulsant drugs for human seizure disorders. They also suffer from paralysis that may provide a basis for designing new types of pesticides to fight insects.
Publications
- Glasscock, E., Singhania, A. & Tanouye, M.A. 2005. The mei-p26 gene encodes a RING finger B-box coiled-coil NHL protein that regulates seizure susceptibility in Drosophila. Genetics 170, 1677-1689.
- Hekmat-Scafe, D.S., Dang, K.N. & Tanouye, M.A. 2005. Seizure-suppression by gain-of-function escargot mutations. Genetics 169, 1477-1493.
- Song, J. & Tanouye, M.A. 2005. Seizure suppression by shakB2, a gap junction mutation in Drosophila. J Neurophysiol 95, 627-635. Allen MJ, Godenschwege TA, Tanouye MA & Phelan P (2006). Making an escape: development and function of the Drosophila giant fibre system. Sem Cell Dev Biol - in press.
- Kim, H.J., Kim, S.H., Shim, S.O., Park, E., Kim, C., Kim, K., Tanouye, M.A. & Yim, J. 2006. Drosophila homologue of APP-BP1(dAPP-BP1) interacts antagonistically with APPL during Drosophila development. Cell Differentiation & Dev - in press.
- Glasscock, E, & Tanouye, M.A. 2005. Drosophila couch potato mutants exhibit complex neurological abnormalities including epilepsy phenotypes. Genetics 169, 2137-2149.
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Progress 01/01/04 to 12/31/04
Outputs
We describe a novel class of genes called "seizure-suppressors." Mutations defining these suppressors revert the "epilepsy" phenotype of Drosophila neurological mutants with seizure disorders. That is, suppressors are mutations that "cure" Drosophila epilepsy. The major interest in seizure-suppressors is that they may lead to new and significant treatments for human epilepsy. Seizure-suppressor genes could help define targets for unexpected classes of anticonvulsant drugs that are effective treatments for epilepsy: treatments for intractable syndromes or treatments with reduced side effects. Another possibility is to discover candidate genes that might be used for gene therapy. Among the several questions that arise are: what are seizure-suppressor genes and how might they lead to new therapeutics? What is the entire range of potential gene products that can act as seizure-suppressors? Is this range limited to nervous system-specific gene products, such as signaling
molecules or does it include non-nervous system gene products as well? A screen utilizing the eas "epilepsy" mutant identified dominant gain-of-function mutations that suppressed seizures. We screened 1,800 mutations and found that neuronal escargot (esg) mutations reduced eas seizures substantially. For example, the suppressor mutation esgEP684 suppressed the epilepsy phenotype of eas by 96 percent. This appears to be due to an increase of the electrophysiological threshold of seizures from a low eas value of 7.5V to a suppressed value of 15.5V. This is a substantial improvement in eas seizure-susceptibility, although not quite to wild-type levels. The esg gene encodes a member of the snail family of transcription factors. Whereas esg is normally expressed in a limited number of neurons during a defined period of nervous system development, here normal esg was expressed in all neurons and throughout development via the ELAV neural promoter. This greatly ameliorated both the
electrophysiological and behavioral epilepsy phenotypes of eas.
Impacts
The Drosophila mutants that we work on have two dramatic phenotypes. They are seizure-susceptible which may ultimately provide basic information in developing new anti-convulsant drugs for human seizure disorders. They also suffer from paralysis, and this may provide a basis for designing new types of pesticides to fight insects.
Publications
- Tan, J.S., Lin, F. and Tanouye, M.A. 2004. Potassium bromide, an anticonvulsant, is effective at alleviating seizures in the Drosophila bang-sensitive mutant bang senseless. Brain Res 1020, 45-52.
- Glasscock, E. and Tanouye, M.A. 2005. Drosophila couch potato mutants exhibit complex neurological abnormalities including epilepsy phenotypes. Genetics (in press).
- Hekmat-Scafe, D.S., Dang, K.N. and Tanouye, M.A. 2005. Seizure-suppression by gain-of-function escargot mutations. Genetics (in press).
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Progress 01/01/03 to 12/31/03
Outputs
We have discovered a novel class of neurological genes in Drosophila called the "seizure-suppressor" gene class. To identify seizure-suppressor genes, we begin with mutant Drosophila that are afflicted with epilepsy. These are mutants such as slamdance (sda), bangsenseless (bss) and easily shocked (eas), and have been shown to be five to ten times more sensitive to seizure than normal wild type flies. We subject the epileptic flies to remutagenization and select for double mutants with revertant phenotypes. That is, we identify flies where a mutation in a seizure-suppressor gene restores seizure-susceptibility from the extremely sensitive epilepsy range to the normal wild type range. We have identified and characterized one seizure-suppressor gene called escargot. Mutants of escargot rescue epilepsy in about 95% of flies. This percentage is 100% effective when escargot is used in combination with human antiepileptic drugs. Additional experiments indicate that escargot
is most effective in "curing" the developing nervous system. The escargot protein is a DNA-binding protein that normally functions as a transcriptional repressor. We surmise that additional seizure suppressor genes that interact with escargot will provide a nice segue into the causes and cures of epilepsy, a disease that afflicts greater than 2 million Americans.
Impacts
The Drosophila mutants that we work on have two dramatic phenotypes. They are seizure-susceptible which may ultimately provide basic information in developing new anti-convulsant drugs for human seizure disorders. They also suffer from paralysis that may provide a basis for designing new types of pesticides to fight insects.
Publications
- No publications reported this period
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Progress 01/01/02 to 12/31/02
Outputs
Despite the frequency of seizure disorders in the human population, the genetic basis for these defects remains largely unclear. Currently, only a fraction of the epilepsies can be linked conclusively to a genetic determinant. In addition, a significant number of epileptics do not respond to the current anticonvulsant therapies. We have turned to Drosophila as a model to address these problems and have identified genetic mutants that are more sensitive to seizures, bang-sensitive (BS) mutants, such as slamdance (sda), bangsenseless (bss) and easily shocked (eas), as well as mutants that are resistant to seizures, such as paralytic, malelessnapts, shaking-B2 and Shaker. Here, we have developed a new method for evaluating compounds with anticonvulsant activity. The methodology uses Drosophila BS mutants to assay the ability of compounds to suppress the seizure susceptible phenotype normally seen in the BS mutants. To test the effectiveness of this method, two BS mutant
strains were administered the anticonvulsant valproate and in both cases the drug was able to suppress seizures. Valproate is a clinically-used antiepileptic drug that is effective for treating both generalized and partial seizures. Valproate has also been shown to be effective in suppressing seizures in a variety of animal models. We found valproate can suppress the seizure susceptible defect normally seen in the bss and sda mutant strains. We tested a range of increasing concentrations of valproate on both strains and assayed the susceptibility to an HFS wavetrain of fixed voltage (40 V, 0.5 ms pulses at 200 Hz for 400 ms). This voltage was chosen because it is about the seizure threshold for CS wild type flies. Thus, the ability of a given concentration to suppress seizures at 40 V indicates the ability to suppress seizures to wild type levels. We found that 5 mM valproate suppressed seizures in most sda flies and 10 mM completely suppressed seizures. In the case of bss, 10 mM
valproate suppressed seizures in most flies and 25 mM valproate completely suppressed seizures.
Impacts
The Drosophila mutants that we work on have two dramatic phenotypes. They are seizure-susceptible which may ultimately provide basic information in developing new anti-convulsant drugs for human seizure disorders. They also suffer from paralysis that may provide a basis for designing new types of pesticides to fight insects.
Publications
- Hekmat-Scafe DS, Scafe CR, McKinney AJ & Tanouye MA (2002). Genome-wide analysis of the odorant-binding protein gene family in Drosophila melanogaster. Genome Res 12, 1357-1369.
- Kuebler D & Tanouye MA (2002). The anticonvulsant valproate reduces seizure-susceptibility in mutant Drosophila. Brain Res 958, 36-42.
- Zhang HG, Tan J, Reynolds E, Kuebler D, Faulhaber S & Tanouye MA (2002). The Drosophila slamdance gene: a mutation in an aminopeptidase can cause seizure, paralysis, and neuronal failure. Genetics 162, 1283-1299.
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Progress 01/01/01 to 12/31/01
Outputs
Our continuing interest has been a class of behavioral mutants in Drosophila called bang-sensitive (BS) paralytics. Here, we report a behavioral, electrophysiological, genetic and molecular analysis of a BS paralytic called slamdance (sda). This mutant exhibits hyperactive behavior and paralysis following a mechanical 'bang' or electrical shock. Electrophysiological analyses have shown that this mutant is much more prone to seizure episodes than normal flies because they have a drastically lowered seizure threshold. In normal wild type flies, an electroconvulsive shock (ECS) of 44.5V (0.5 ms stimulus pulses delivered at 200 Hz for 300 ms) causes a 'seizure' consisting of aberrant high frequency firing (greater than 100 Hz) in all muscle fibers and motoneurons that have been examined in the fly that lasts for 2-3 sec. Mutants of sda are 5-7 times more seizure-sensitive than wild type and show seizures with ECS of 6.2V. Through genetic mapping, molecular cloning, and
RNA interference, we have demonstrated that the sda phenotype can be attributed to a mutation in the Drosophila homolog of the human aminopeptidase N (APN) gene. Genetic mapping showed that sda mutations map to location 97D1-5 on the third chromosome. Molecular access to the sda gene was via a P-element mutation. The sda gene was cloned by the method of plasmid rescue. Analysis of the sda sequence indicated that it is the Drosophila homolog to human APN. APN is a transmembrane ectoenzyme that catalyzes the removal of small neutral and basic amino acids from the N-termini of a number of small peptide substrates. The catalytic domain faces the exterior of the plasma membrane and is anchored by a transmembrane spanning domain. Human APN is identical to CD13, a cluster antigen expressed on the surface of myeloid leukemia cells. It is widely considered to enable tumor-cell invasion during metastasis of a primary tumor to vital organs by degrading collagen type IV of the basement membrane.
We are presently investigating if human APN may also play a role in seizure disorders such as epilepsy.
Impacts
The Drosophila mutants that we work on have two dramatic phenotypes. They are seizure-susceptible which may ultimately provide basic information in developing new anti-convulsant drugs for human seizure disorders. They also suffer from paralysis that may provide a basis for designing new types of pesticides to fight insects.
Publications
- Kuebler, D., Zhang, H., Ren, X. and Tanouye, M. 2001. Genetic suppression of seizure susceptibility in Drosophila. J Neurophysiol 86, 1211-1225.
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Progress 01/01/00 to 12/31/00
Outputs
Our continuing interest has been a class of behavioral mutants in Drosophila called bang-sensitive (BS) paralytics. Here, we report a behavioral, electrophysiological, genetic and molecular analysis of a new BS paralytic called jitterbug (jbug). The jbug mutation was identified in a screen that selected for genetic enhancers of the BS mutation slamdance (sda). Subsequent behavioral and genetic analysis determined that jbug is itself a BS mutation as well as an sda enhancer. The jbug mutation is a most interesting BS mutant: it appears to be poised on the boundary between normal behavior and BS abnormal behavior. About 80% of young (1-2d) adult jbug flies show BS paralysis following mechanical shock. This is different from any other Drosophila strain that we have examined which show either 100% bang-sensitive paralysis (all BS mutants and bss/+ heterozygotes); or 0% bang-sensitive paralysis (all wild type strains and non-BS neurological mutant strains). The jbug
mutants show developmental changes: as the flies get older, an increasingly smaller fraction of flies show bang-sensitive paralysis. The jbug mutants also show experiential effects most flies will paralyze only after the first mechanical stimulus. After the first bout of paralysis, they are completely resistant to the paralyzing effects of mechanical stimulation. That is, for most flies, they paralyze once and only once in their life. Electrophysiological analysis shows that seizures may be evoked in jbug mutants by low voltage electroconvulsive shock. The jbug mutants are about 3-fold more susceptible to seizure than normal flies. The location of the jbug mutation in the Drosophila genome was determined to be the 58F region of polytene chromosome 3R. Molecular access to the jbug gene was via a P-element mutation. The jbug gene was cloned by the method of plasmid rescue. Analysis of the jbug sequence indicated that it is the Drosophila homolog to human filamin. Filamin is a
cytoskeletal protein that is an actin-binding protein. The protein is characterized by an actin-binding domain and several "filamin repeat" domains. These domains are conserved in sequence and location in the Drosophila protein. Interestingly, the human protein is associated with a neurological syndrome called periventricular heterotopia. In this syndrome, some neurons are known to take up positions in abnormal locations. Epileptic seizures are observed in individuals afflicted with periventricular heterotopia.
Impacts
The Drosophila mutants that we work on have two dramatic phenotypes. They are seizure-susceptible which may ultimately provide basic information in developing new anti-convulsant drugs for human seizure disorders. They also suffer from paralysis that may provide a basis for designing new types of pesticides to fight insects.
Publications
- No publications reported this period
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Progress 01/01/99 to 12/31/99
Outputs
We have been studying a class of behavioral mutants in Drosophila called bang-sensitive (BS) paralytics. BS mutants are more susceptible to seizures than normal flies, and here we begin an examination of how genetic background acts to modulate this susceptibility. The approach is to examine seizures in animals carrying two neurological mutations (double mutant combinations). One mutation of the combination is a bang sensitive (BS) mutation which causes extreme seizure susceptibility. The other mutation of the combination is chosen with the expectation that it will provide a background that modifies seizure susceptibility. The BS mutations used are bangsenseless (bss), slamdance (sda), and easily shocked (eas). Seizures may be evoked in BS mutants by low voltage electric convulsive shock stimuli applied to the brain. BS mutants are 6- to 14-fold more susceptible to seizure than normal flies and each has a characteristic or signature threshold voltage at which it
seizes. To provide different genetic backgrounds for the BS mutants, Na+ channel mutants (para, nap) were chosen because they should provide a background with reduced nervous system excitability. K+ channel mutants (Sh, eag) were chosen because they should provide a background with increased nervous system excitability. Neuronal connectivity mutants (pas, netrin) were selected because they may disrupt neuronal circuits and interfere with feedback loops contributing to seizure generation and/or the spread of seizures through the nervous system. The results of this investigation show that seizure susceptibility can be affected by genetic background changes: double mutants showed substantially different seizure profiles and voltage thresholds than single BS mutants. The general findings were that nap mutations were the most effective at suppressing seizures. Sh mutations also were able to suppress seizures although they were the weakest suppressors. Pas mutations were somewhere in
between. Our findings also showed that bss mutations were the most difficult to suppress. Mutations of the eas gene were the easiest to suppress. Mutations of the sda gene were in between.
Impacts
The Drosophila mutants that we work on have two dramatic phenotypes. They are seizure-susceptible which may ultimately provide basic information in developing new anti-convulsant drugs for humans. They also suffer from paralysis that may provide a basis for designing new types of pesticides to fight insects.
Publications
- Kuebler, D. and Tanouye, M.A. 2000. Modifications of seizure susceptibility in Drosophila. J. Neurophysiol. IN PRESS.
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Progress 01/01/98 to 12/01/98
Outputs
We have been studying a class of mutants in Drosophila called bang-sensitive (BS) paralytics. Previously, we have shown that BS mutants are about 18-fold more susceptible to seizures than normal flies. Here, we show that within a single genotype, seizure susceptibility is also quite plastic and dependent on previous experience. Immediately following an initial seizure, susceptibility is modulated by some process which causes a large threshold jump. Seizures may still be elicited, however, a large stimulus voltage must be used, presumably to drive a much greater number of neurons. Following this threshold jump, there is a gradual change which eventually restores seizure susceptibility to that typical for the particular BS genotype. For example, sda flies were given an initial seizure at 6 V. At 60 sec intervals following the initial seizure, buzzes of various voltages were applied to determine susceptibility to a second seizure. One minute after the initial seizure,
the threshold for a second seizure is very high, about 90 V showing that at this time, sda is much less susceptible to seizure than wild type flies. The change in seizure susceptibility is transient: as the time following the first seizure increases, threshold continuously falls. At 2 min, seizure threshold is about 50 V; sda at this time has about the same susceptibility as wild type flies. At 5 to 6 min, seizure threshold falls to 6-7 V, near the value of the seizure activation voltage for an initial sda seizure. Similar results are seen in other BS mutants.
Impacts
(N/A)
Publications
- DOBSON, S. L. and TANOUYE, M. A. 1998. Interspecific movement of the paternal sex ratio chromosome. Heredity 81: 261-269.
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Progress 01/01/97 to 12/01/97
Outputs
We study DROSOPHILA MELANOGASTER mutants called bang-sensitive paralytics. In response to a mechanical stimulus, these mutants show abnormal behavioral hyperexcitability and paralysis. We have examined the hyperexcitability, in detail, and have shown that it is due to abnormal seizure activity in the central nervous system. Seizures may be elicited by delivering electrical stimuli to the brain or thoracic ganglion of the fly (200ms, 200Hz). Immediately after stimulation, many neurons in the fly show intense uncontrolled spiking activity that we have termed "seizures" because of similarity to mammalian seizures. Seizures last for about 2sec in flight motoneurons, for example, and reach firing frequencies greater than 150Hz. Normal animals will seize at stimulation voltages about 70V. In contrast, seizures in bang sensitive mutants may be elicited at much lower voltages (bss mutants at 4V, eas at 6V, sda at 15V). We interpret this to mean that the potential for seizure
is present in the brains of all flies, normal and mutant, but that the susceptibility may be modulated by genotype. We have cloned one of the genes, called bss, and discovered that it has strong sequence homology to a family of mammalian transcription factors called the HMG1 (high mobility group) family. Members of this family interact with DNA and other proteins to facilitate the formation of a transcriptional complex. We are determining how the bss transcription factor acts to disrupt normal nervous system excitability.
Impacts
(N/A)
Publications
- DOBSON, S.L. and TANOUYE, M.A. 1996. The paternal sex ratio chromosome induces chromosome loss independently of Wolbachia in the wasp NASONIA VITRIPENNIS. Dev. Genes Evol. 206:207-217.
- DOBSON, S.L. and TANOUYE, M.A. Interspecific transfer of a B chromosome. Heredity. IN PRESS.
- DOBSON, S.L. and TANOUYE, M.A. Evidence for a genomic imprinting sex determination mechanism in NASONIA VITRIPENNIS (Hymenoptera: Chalcidoidea). Genetics. IN PRESS.
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