Dietary Supplementation with Milk Lipids Leads to Suppression of Developmental and Behavioral Phenotypes of Hyperexcitable Drosophila Mutants.

Dietary modifications often have a profound impact on the penetrance and expressivity of neurological phenotypes that are caused by genetic defects. Our previous studies in Drosophila melanogaster revealed that seizure-like phenotypes of gain-of-function voltage-gated sodium (Nav) channel mutants (paraShu, parabss1, and paraGEFS+), as well as other seizure-prone "bang-sensitive" mutants (eas and sda), were drastically suppressed by supplementation of a standard diet with milk whey. In the current study we sought to determine which components of milk whey are responsible for the diet-dependent suppression of their hyperexcitable phenotypes. Our systematic analysis reveals that supplementing the diet with a modest amount of milk lipids (0.26% w/v) mimics the effects of milk whey. We further found that a minor milk lipid component, α-linolenic acid, contributed to the diet-dependent suppression of adult paraShu phenotypes. Given that lipid supplementation during the larval stages effectively suppressed adult paraShu phenotypes, dietary lipids likely modify neural development to compensate for the defects caused by the mutations. Consistent with this notion, lipid feeding fully rescued abnormal dendrite development of class IV sensory neurons in paraShu larvae. Overall, our findings demonstrate that milk lipids are sufficient to ameliorate hyperexcitable phenotypes in Drosophila mutants, providing a foundation for future investigation of the molecular and cellular mechanisms by which dietary lipids modify genetically induced abnormalities in neural development, physiology, and behavior.

Reduced Function of the Glutathione S-Transferase S1 Suppresses Behavioral Hyperexcitability in Drosophila Expressing Mutant Voltage-Gated Sodium Channels.

Voltage-gated sodium (Nav) channels play a central role in the generation and propagation of action potentials in excitable cells such as neurons and muscles. To determine how the phenotypes of Nav-channel mutants are affected by other genes, we performed a forward genetic screen for dominant modifiers of the seizure-prone, gain-of-function Drosophila melanogaster Nav-channel mutant, paraShu Our analyses using chromosome deficiencies, gene-specific RNA interference, and single-gene mutants revealed that a null allele of glutathione S-transferase S1 (GstS1) dominantly suppresses paraShu phenotypes. Reduced GstS1 function also suppressed phenotypes of other seizure-prone Nav-channel mutants, paraGEFS+ and parabss Notably, paraShu mutants expressed 50% less GstS1 than wild-type flies, further supporting the notion that paraShu and GstS1 interact functionally. Introduction of a loss-of-function GstS1 mutation into a paraShu background led to up- and down-regulation of various genes, with those encoding cytochrome P450 (CYP) enzymes most significantly over-represented in this group. Because GstS1 is a fly ortholog of mammalian hematopoietic prostaglandin D synthase, and in mammals CYPs are involved in the oxygenation of polyunsaturated fatty acids including prostaglandins, our results raise the intriguing possibility that bioactive lipids play a role in GstS1-mediated suppression of paraShu phenotypes.

Milk-whey diet substantially suppresses seizure-like phenotypes of paraShu, a Drosophila voltage-gated sodium channel mutant.

The Drosophila mutant paraShu harbors a dominant, gain-of-function allele of the voltage-gated sodium channel gene, paralytic (para). The mutant flies display severe seizure-like phenotypes, including neuronal hyperexcitability, spontaneous spasms, ether-induced leg shaking, and heat-induced convulsions. We unexpectedly found that two distinct food recipes used routinely in the Drosophila research community result in a striking difference in severity of the paraShu phenotypes. Namely, when paraShu mutants were raised on the diet originally formulated by Edward Lewis in 1960, they showed severe neurological defects as previously reported. In contrast, when they were raised on the diet developed by Frankel and Brousseau in 1968, these phenotypes were substantially suppressed. Comparison of the effects of these two well-established food recipes revealed that the diet-dependent phenotypic suppression is accounted for by milk whey, which is present only in the latter. Inclusion of milk whey in the diet during larval stages was critical for suppression of the adult paraShu phenotypes, suggesting that this dietary modification affects development of the nervous system. We also found that milk whey has selective effects on other neurological mutants. Among the behavioral phenotypes of different para mutant alleles, those of paraGEFS+ and parabss were suppressed by milk whey, while those of paraDS and parats1 were not significantly affected. Overall, our study demonstrates that different diets routinely used in Drosophila labs could have considerably different effects on neurological phenotypes of Drosophila mutants. This finding provides a solid foundation for further investigation into how dietary modifications affect development and function of the nervous system and, ultimately, how they influence behavior.

Lithium-Responsive Seizure-Like Hyperexcitability Is Caused by a Mutation in the Drosophila Voltage-Gated Sodium Channel Gene paralytic.

Shudderer (Shu) is an X-linked dominant mutation in Drosophila melanogaster identified more than 40 years ago. A previous study showed that Shu caused spontaneous tremors and defects in reactive climbing behavior, and that these phenotypes were significantly suppressed when mutants were fed food containing lithium, a mood stabilizer used in the treatment of bipolar disorder (Williamson, 1982). This unique observation suggested that the Shu mutation affects genes involved in lithium-responsive neurobiological processes. In the present study, we identified Shu as a novel mutant allele of the voltage-gated sodium (Nav) channel gene paralytic (para). Given that hypomorphic para alleles and RNA interference-mediated para knockdown reduced the severity of Shu phenotypes, Shu was classified as a para hypermorphic allele. We also demonstrated that lithium could improve the behavioral abnormalities displayed by other Nav mutants, including a fly model of the human generalized epilepsy with febrile seizures plus. Our electrophysiological analysis of Shu showed that lithium treatment did not acutely suppress Nav channel activity, indicating that the rescue effect of lithium resulted from chronic physiological adjustments to this drug. Microarray analysis revealed that lithium significantly alters the expression of various genes in Shu, including those involved in innate immune responses, amino acid metabolism, and oxidation-reduction processes, raising the interesting possibility that lithium-induced modulation of these biological pathways may contribute to such adjustments. Overall, our findings demonstrate that Nav channel mutants in Drosophila are valuable genetic tools for elucidating the effects of lithium on the nervous system in the context of neurophysiology and behavior.

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

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

The amnesiac gene is involved in the regulation of thermal nociception in Drosophila melanogaster.

Nociception is a mechanism fundamental to the ability of animals to avoid noxious stimuli capable of causing serious tissue damage. It has been established that in the fruit fly Drosophila melanogaster, the transient receptor potential (TRP) channel encoded by the painless gene (pain) is required for detecting thermal and mechanical noxious stimuli. Little is known, however, about other genetic components that control nociceptive behaviors in Drosophila. The amnesiac gene (amn), which encodes a putative neuropeptide precursor, is important for stabilizing olfactory memory, and is involved in various aspects of other associative and nonassociative learning. Previous studies have indicated that amn also regulates ethanol sensitivity and sleep. Here the authors show that amn plays an additional critical role in nociception. Their data show that amn mutant larvae and adults are significantly less responsive to noxious heat stimuli (greater than approximately 40 degrees C) than their wild-type counterparts. The phenotype of amn mutants in thermal nociception, which closely resembles that of pain mutants, was phenocopied in flies expressing amn RNAi, and this phenotype was rescued by the expression of a wild-type amn transgene. These results provide compelling evidence that amn is a novel genetic component of the mechanism that regulates thermal nociception in Drosophila.

Neuronal mechanisms of learning and memory revealed by spatial and temporal suppression of neurotransmission using shibire, a temperature-sensitive dynamin mutant gene in Drosophila melanogaster.

The fruit fly Drosophila melanogaster is an excellent model organism to identify genes and genetic pathways important for learning and memory. However, its small size makes surgical treatment and electrophysiological manipulation technically difficult, hampering the functional analysis of neuronal circuits that play critical roles in memory processing. To circumvent this problem, we developed a unique experimental strategy that uses the temperature-sensitive allele of the Drosophila dynamin gene, shibire(ts1) (shi(ts1)), in combination with the GAL4/UAS expression system. This strategy allows for rapid and reversible perturbation of synaptic neurotransmission in identifiable neurons, and analysis of the behavioral consequences of such manipulation in free-moving animals. Since its introduction in 2001, this GAL4/UAS-shi(ts1) strategy has been widely used to study the neuronal basis of learning and memory. This review focuses on how this strategy has revitalized Drosophila memory research, and contributed to our understanding of dynamic neuronal processes that control various aspects of memory.

Effects of lithium chloride on the gene expression profiles in Drosophila heads.

To gain insight into the basic neurobiological processes regulated by lithium--an effective drug for bipolar disorder--we used Affymetrix Genome Arrays to examine lithium-induced changes in genome-wide gene expression profiles of head mRNA from the genetic model organism Drosophila melanogaster. First, to identify the individual genes whose transcript levels are most significantly altered by lithium, we analyzed the microarray data with stringent criteria (fold change>2; p<0.001) and evaluated the results by RT-PCR. This analysis identified 12 genes that encode proteins with various biological functions, including an enzyme responsible for amino acid metabolism and a putative amino acid transporter. Second, to uncover the biological pathways involved in lithium's action in the nervous system, we used less stringent criteria (fold change>1.2; FDR<0.05) and assigned the identified 66 lithium-responsive genes to biological pathways using DAVID (Database for Annotation, Visualization and Integrated Discovery). The gene ontology categories most significantly affected by lithium were amino acid metabolic processes. Taken together, these data suggest that amino acid metabolism is important for lithium's actions in the nervous system, and lay a foundation for future functional studies of lithium-responsive neurobiological processes using the versatile molecular and genetic tools that are available in Drosophila.