For decades, a number of labs have been involved in isolation Drosophila mutants that have abnormal behavior (Homyk et al., 1980; Suzuki, 1970). A large subset of these mutants were isolated based on reversible paralysis induced by trauma, usually either mechanical shock or a rapid change in temperature. Again, a subset of these mutants have been described as undergoing epileptic-type seizures prior to paralysis. In parallel screens, mutants were selected by visual observation as being uncoordinated or impaired in locomotion. A number of these mutants were described as undergoing localized seizures or muscle spasms, particularly in the movement of the legs and wings. The combination of these screens have generated a pool of over 20 mutants with epilepsy-like symptoms which can be have divided into three groups; mechanical shock-sensitive, temperature-sensitive and movement defective mutants.

The progress on characterizing these intriguing mutants has been somewhat slow and uneven. By far the most exciting results have been on the mechanical shock-sensitive class. For example, mutations in the bang-senseless (bss) gene respond to a mechanical shock with a brief seizure followed by reversible paralysis, the length of which increases with age (Pavlidis and Tanouye, 1995). These epilepsy symptoms are associated with abnormal, prolonged release of neurotransmitter at the NMJ, apparently caused by multiple, synchronous firing of action potentials in the nerve (Pavlidis and Tanouye, 1995). It is known that this defect is suppressed by mutations in no action potential (nap) which decrease the number of functional Na+ channels in the neuronal membrane (Ganetzky and Wu, 1986). Likewise, mutations in the bang-sensitive (bas) show a similar trauma-induced generalized seizure followed by paralysis (Ganetzky and Wu, 1982). In this case a neuronal defect is indicated since the mutant displays an abnormal ERG and prolonged PDA in the retina (Homyk and Pye, 1989). However, unlike bss, no defect has yet been uncovered at the NMJ. These mutant analyses indicate that neuronal mechanisms can be associated with epilepsy-like behavioral defects. However, much more detailed analyses are necessary and, in the majority of cases, even assays at this level have yet to be attempted.

Molecular characterization of beavioral/epilepsy mutants has been even slower. The notable exception is the easily shocked (eas) gene. Like bss and bas, the eas mutant responds to mechanical shock with a brief seizure followed by reversible paralysis (i.e. unconsciousness). The gene has been characterized and found to encode ethanolamine kinase, an enzyme involved in membrane manufacture (Pavlidis et al., 1994). This characterization has led to the hypothesis that eas mutants have a neuronal excitability defect caused by altered membrane lipid composition. Another example is seizure (sei) a mutant which responds to temperature-shock with an epileptic phenotype (Jackson et al., 1984). The sei mutants are known to increase the spontaneous activity in recording from muscle. Two series of experiments suggest the molecular nature of the sei gene; Na+ current is dramatically reduced in the neuronal membrane and there is a high Kd for saxitoxin (STX) binding activity to neuronal membranes in sei mutants (Jackson et al., 1984; O'Dowd and Aldrich, 1988). This evidence suggests that sei might code for a voltage-gated sodium channel expressed in neuronal membranes but, despite efforts, this identity has not been confirmed. Interestingly, a related gene, enhancer of seizure, both enhances sei mutant defect and, by itself, causes trauma-induced epilepsy-like symptoms (Kasbecker et al., 1987). However, e(sei) does not affect STX binding to membrane extracts. The final example is a mutation in the Na+ pump a subunit which was isolated from a p-element insertion resulting in a temperature-sensitive epilepsy-like phenotype (Schubiger et al., 1994). These molecular analyses indicate that molecular identities can be logically linked to the neuronal mechanisms underlying epilepsy symptoms. However, as yet very few of the available mutants have been characterized to a molecular level.

In summary, previous work has isolated a large pool of Drosophila mutants which show convincing epilepsy symptoms. In a small number of cases, the neuronal defects underlying the mutant behavior have been assayed at the NMJ and in the retina. These studies have revealed neuronal mechanisms consistent with the behavioral phenotype and consistent with the causes believed to underlie epilepsy in man. In an even smaller number of cases, the molecular identity of the mutant gene has been identified. Again, these molecular identities have been consistent with the neuronal defects known to underlie epilepsy. However, in both mutant assays and molecular characterizations, the take-home message is that there is a great deal left to accomplish. Most of the known epilepsy-inducing mutations in Drosophila have not been characterized at a cellular level and even fewer at a molecular level. This pool of mutants represents a treasure-trove which should be exploited as a priority in epilepsy research.

The long-term objective of this research is to generate a forward genetic system for the characterization of the causes of behavioral defects, especially epilepsy. From such a system we demand that 1) epilepsy can be accurately modeled, 2) that the system permits systematic molecular genetic analysis and 3) that the neurological mechanisms underlying epilepsy can be assayed using detailed electrophysiological and anatomical measurements. The only system that fulfills these three criteria is the fruit fly, Drosophila melanogaster. First, genetic mutations have been generated in Drosophila which accurately model the symptoms of both generalized epilepsy and partial (or focal) epilepsy. To date, more than 20 mutations have the characteristics of trauma-induced general seizure followed by unconsciousness (generalized epilepsy) or local spasms/muscle contractions leading to uncoordination and movement difficulties (partial epilepsy) . In the few cases where these mutations have been characterized in more detail, it has been shown that these "epileptic seizures" result from hyperactive discharge in neurons. Thus, Drosophila mutants have both the symptoms and causes common to epilepsy. Second, Drosophila is the classic genetic system and has perhaps the most advanced molecular genetic techniques available. The primary strength of the Drosophila system is forward genetic screens designed to systematically investigate biological problems. Finally, Drosophila is the only advanced genetic system which permits detailed electrophysiological investigation. For over twenty years, Drosophila has been used to genetically investigate the mechanisms of neural and synaptic function. These basic neural functions have been linked to higher brain functions such as behavior, learning and memory. Therefore, Drosophila is the only system which links convincing models of epilepsy, forward genetic analysis and the ability to uncover the functional mechanisms underlying the causes of epilepsy.

Three general classes of epileptic mutants have been uncovered in Drosophila. First, mutants which respond to mechanical shock with generalized seizures followed by paralysis/unconsciousness. These mutants belong to the bang-sensitive class (e.g. bang-senseless, easily shocked, technical knockout, etc.) and the stress-sensitive class (stoned, stress sensitive A-H). Second, mutants which respond to temperature shock with generalized seizures followed by paralysis. These temperature-sensitive mutants includes seizure and enhancer of seizure. Third, mutants which show localized seizures resulting in uncoordinated behavior. These movement-defective mutants include spastic, palsied and shudderer.

Despite the riches of this genetic resource, few of these epileptic mutants have been analyzed in detail. We therefore propose to begin a systematic investigation of the neurological basis of the epileptic mutant defects in Drosophila. This work will concentrate on examining neuronal membrane excitability and synaptic function in epileptic mutant backgrounds. Analyses will be accomplished with a combination of intracellular and patch-clamp physiology and anatomical measurements at both the light microscopic and ultrastructural levels. We will focus in two particular directions. First, on the analyses of these defined conditional, epileptic mutants. The priority will be on the collection of stress-sensitive mutants (ses A-H) and the stoned mutant locus. Second, we will focus on characterizing the null mutant phenotypes of these conditional mutants. Many of these genes encode essential neural function products which result in early lethality if the gene is deleted. These phenotype analyses should allow us to pinpoint the location of genetic defects leading to epileptic seizures. The second stage of our investigation will be to clone out such genes and molecularly characterize them and their products. This approach should allow us to gain a systematic understanding of the genetic and molecular mechanisms underlying epilepsy. This knowledge will be invaluable in understanding the inherited causes of epilepsy as well as devising cures and treatments to circumvent the defect and effectively eliminate epilepsy symptoms.

Fragile X syndrome

Fragile X Syndrome (FraX) is a broad-spectrum neurological disorder with symptoms ranging from hyperexcitability to mental retardation and autism. Loss of the fragile X mental retardation 1 (fmr1) gene product, the mRNA-binding translational regulator FMRP, causes structural over-elaboration of dendritic and axonal processes as well as functional alterations in synaptic plasticity at maturity. It is unclear, however, whether FraX is primarily a disease of development, a disease of plasticity or both; a distinction vital for engineering intervention strategies. We are using the power of Drosophila genetics to investigate this critical question. There are three distinct advantages to using the Drosophila FraX model versus its mammalian counterpart. First, the Drosophila genome encodes only a single fragile X gene, compared to the 3 member fragile X gene family in mammals (fmr, fxr1, fxr2) (9). Perhaps for this reason, dfmr1 null phenotypes in central, peripheral sensory, and motor neurons appear stronger in flies than fmr defects in mammals. These more robust phenotypes significantly aid our investigations. Second, the well characterized and easily visualized glutamatergic neuromuscular junction (NMJ) is a highly accessible model for studying synaptic development and plasticity in the Drosophila system. We have pioneered complementary techniques that allow us to analyze dfmr1 function in central neurons, providing a powerful combination of approaches. Third, the genetic tools available in Drosophila allow rapid identification of specific disease related genetic interactions. For example, our lab identified the critical relationship between the microtubule-associated protein futsch/MAP1b and dFMRP. We determined that dFMRP negatively regulates futsch translation, and by genetic approaches showed that returning futsch protein levels to normal in a dfmr1 null background rescues FraX phenotypes. This misregulation has since been confirmed in mammals. Thus, Drosophila has already been used to identify a potential point of therapeutic intervention for FXS. We are continuing these and other experiments in the hopes of understanding not only the molecular basis of FMRP mediated neuronal dysfunction but also the basic mechanisms of neuronal maturation and synaptogenesis.

Niemann-Pick type C

Synaptic transmission requires specific lipid composition in vesicle and plasma membranes, and is tightly regulated by lipid-based signaling mechanisms. Of particular interest is the involvement of sterol/sphingolipid rich lipid microdomains (lipid rafts). Many aspects of these domains remain highly controversial, including how to define them, their size, lipid and protein composition, and biological relevance. Nevertheless, lipid microdomains in neuronal membranes have documented roles in regulating ion channels, localizing the synaptic vesicle exocytic machinery and modulating neurotransmitter receptors. Altered lipid domain organization and lipid mistrafficking/aggregation are also associated with several diseases including Niemann-Pick, Alzheimer's and lysosomal storage diseases. The central role sterol/sphingolipids may play in synaptic function and neurological disease dictates the need to understand the mechanisms of sterol/sphingolipid trafficking/signaling in neurons. The power of Drosophila genetics has proven to be an invaluable resource for modeling neurological diseases. Furthermore, the Drosophila neuromuscular junction (NMJ) is commonly employed for studying synaptic mechanisms, making this an ideal genetic system to better understand the requirement of sterol/sphingolipid microdomain function in regulating neurotransmission. The goals of our research are: (i), to determine the effects of disrupting sterol/sphingolipid metabolism on neuronal lipid trafficking/composition and lipid domain formation, (ii), to determine the requirement of sterol/sphingolipid rich domains in neurotransmission and protein trafficking in neurons, and (iii), to test if altered synaptic activity caused by changes in membrane lipid composition leads to age-progressive neurodegeneration. Our hypothesis is that sterol/sphingolipid microdomains are necessary for protein/membrane trafficking in the synapse and that disruption of these domains may be causative in neurodegeneration.

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