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Traumatic brain injury (TBI) pathologies are caused by primary and secondary injuries. Primary injuries result from physical damage to the brain, and secondary injuries arise from cellular responses to primary injuries. A characteristic cellular response is sustained activation of inflammatory pathways commonly mediated by nuclear factor-κB (NF-κB) transcription factors. Using a Drosophila melanogaster TBI model, we previously found that the main proximal transcriptional response to primary injuries is triggered by activation of Toll and Imd innate immune response pathways that engage NF-κB factors Dif and Relish (Rel), respectively. Here, we found by mass spectrometry that Rel protein level increased in fly heads at 4–8 hr after TBI. To investigate the necessity of Rel for secondary injuries, we generated a null allele, Rel(del), by CRISPR/Cas9 editing. When heterozygous but not homozygous, the Rel(del) mutation reduced mortality at 24 hr after TBI and increased the lifespan of injured flies. Additionally, the effect of heterozygosity for Rel(del) on mortality was modulated by genetic background and diet. To identify genes that facilitate effects of Rel(del) on TBI outcomes, we compared genome-wide mRNA expression profiles of uninjured and injured +/+, +/Rel(del), and Rel(del)/Rel(del) flies at 4 hr following TBI. Only a few genes changed expression more than twofold in +/Rel(del) flies relative to +/+ and Rel(del)/Rel(del) flies, and they were not canonical innate immune response genes. Therefore, Rel is necessary for TBI-induced secondary injuries but in complex ways involving Rel gene dose, genetic background, diet, and possibly small changes in expression of innate immune response genes.

Traumatic brain injury (TBI) is a common neurological disorder whose outcomes vary widely depending on a variety of environmental factors, including diet. Using a Drosophila melanogaster TBI model that reproduces key aspects of TBI in humans, we previously found that the diet consumed immediately following a primary brain injury has a substantial effect on the incidence of mortality within 24 h (early mortality). Flies that receive equivalent primary injuries have a higher incidence of early mortality when fed high-carbohydrate diets versus water. Here, we report that flies fed high-fat ketogenic diet (KD) following TBI exhibited early mortality that was equivalent to that of flies fed water and that flies protected from early mortality by KD continued to show survival benefits weeks later. KD also has beneficial effects in mammalian TBI models, indicating that the mechanism of action of KD is evolutionarily conserved. To probe the mechanism, we examined the effect of KD in flies mutant for Eip75B, an ortholog of the transcription factor PPARγ (peroxisome proliferator-activated receptor gamma) that contributes to the mechanism of action of KD and has neuroprotective effects in mammalian TBI models. We found that the incidence of early mortality of Eip75B mutant flies was higher when they were fed KD than when they were fed water following TBI. These data indicate that Eip75B/PPARγ is necessary for the beneficial effects of KD following TBI. In summary, this work provides the first evidence that KD activates PPARγ to reduce deleterious outcomes of TBI and it demonstrates the utility of the fly TBI model for dissecting molecular pathways that contribute to heterogeneity in TBI outcomes.

Background General anesthetics influence mitochondrial homeostasis, placing individuals with mitochondrial disorders and possibly carriers of recessive mitochondrial mutations at increased risk of perioperative complications. In Drosophila, mutations in the ND23 subunit of complex I of the mitochondrial electron transport chain–analogous to mammalian NDUFS8–replicate key characteristics of Leigh syndrome, an inherited mitochondrial disorder. The authors used the ND23 mutant for testing the hypothesis that anesthetics have toxic potential in carriers of mitochondrial mutations. Methods The authors exposed wild-type flies and ND23 mutant flies to behaviorally equivalent doses of isoflurane or sevoflurane in 5%, 21%, or 75% oxygen. The authors used percent mortality (mean ± SD, n ≥ 3) at 24 h after exposure as a readout of toxicity and changes in gene expression to investigate toxicity mechanisms. Results Exposure of 10- to 13-day-old male ND23 flies to isoflurane in 5%, 21%, or 75% oxygen resulted in 16.0 ± 14.9% (n = 10), 48.2 ± 16.1% (n = 9), and 99.2 ± 2.0% (n = 10) mortality, respectively. Comparable mortality was observed in females. In contrast, under the same conditions, mortality was less than 5% for all male and female groups exposed to sevoflurane, except 10- to 13-day-old male ND23 flies with 9.6 ± 8.9% (n = 16) mortality. The mortality of 10- to 13-day-old ND23 flies exposed to isoflurane was rescued by neuron- or glia-specific expression of wild-type ND23. Isoflurane and sevoflurane differentially affected expression of antioxidant genes in 10- to 13-day-old ND23 flies. ND23 flies had elevated mortality from paraquat-induced oxidative stress compared with wild-type flies. The mortality of heterozygous ND23 flies exposed to isoflurane in 75% oxygen increased with age, resulting in 54.0 ± 19.6% (n = 4) mortality at 33 to 39 days old, and the percent mortality varied in different genetic backgrounds. Conclusions Mutations in the mitochondrial complex I subunit ND23 increase susceptibility to isoflurane-induced toxicity and to oxidative stress in Drosophila. Asymptomatic flies that carry ND23 mutations are sensitized to hyperoxic isoflurane toxicity by age and genetic background. Editor’s Perspective What We Already Know about This Topic What This Article Tells Us That Is New

A screen for neuroprotective genes in Drosophila melanogaster led to the identification of a mutation that causes extreme, progressive loss of adult brain neuropil in conjunction with massive brain overgrowth. We mapped the mutation to the brain tumor (brat) locus, which encodes a tripartite motif-NCL-1, HT2A, and LIN-41 (TRIM-NHL) RNA-binding protein with established roles limiting stem cell proliferation in developing brain and ovary. However, a neuroprotective role for brat in the adult Drosophila brain has not been described previously. The new allele, bratcheesehead (bratchs), carries a mutation in the coiled-coil domain of the TRIM motif, and is temperature-sensitive. We demonstrate that mRNA and protein levels of neural stem cell genes are increased in heads of adult bratchs mutants and that the over-proliferation phenotype initiates prior to adult eclosion. We also report that disruption of an uncharacterized gene coding for a presumptive prolyl-4-hydroxylase strongly enhances the over-proliferation and neurodegeneration phenotypes. Together, our results reveal an unexpected role for brat that could be relevant to human cancer and neurodegenerative diseases.

Synapses exhibit an astonishing degree of adaptive plasticity in healthy and disease states. We have investigated whether synapses also adjust to life stages imposed by novel developmental programs for which they were never molded by evolution. Under conditions in which Drosophila larvae are terminally arrested, we have characterized synaptic growth, structure and function at the neuromuscular junction (NMJ). Although wild-type larvae transition to pupae after 5 days, arrested third instar (ATI) larvae persist for 35 days, during which time NMJs exhibit extensive overgrowth in muscle size, presynaptic release sites and postsynaptic glutamate receptors. Remarkably, despite this exuberant growth, stable neurotransmission is maintained throughout the ATI lifespan through a potent homeostatic reduction in presynaptic neurotransmitter release. Arrest of the larval stage in stathmin mutants also reveals a degree of progressive instability and neurodegeneration that was not apparent during the typical larval period. Hence, an adaptive form of presynaptic depression stabilizes neurotransmission during an extended developmental period of unconstrained synaptic growth. More generally, the ATI manipulation provides a powerful system for studying neurodegeneration and plasticity across prolonged developmental timescales.

Outcomes of traumatic brain injury (TBI) vary because of differences in primary and secondary injuries. Primary injuries occur at the time of a traumatic event, whereas secondary injuries occur later as a result of cellular and molecular events activated in the brain and other tissues by primary injuries. We used a Drosophila melanogaster TBI model to investigate secondary injuries that cause acute mortality. By analyzing mortality percentage within 24 hr of primary injuries, we previously found that age at the time of primary injuries and diet afterward affect the severity of secondary injuries. Here, we show that secondary injuries peaked in activity 1–8 hr after primary injuries. Additionally, we demonstrate that age and diet activated distinct secondary injuries in a genotype-specific manner, and that concurrent activation of age- and diet-regulated secondary injuries synergistically increased mortality. To identify genes involved in secondary injuries that cause mortality, we compared genome-wide mRNA expression profiles of uninjured and injured flies under age and diet conditions that had different mortalities. During the peak period of secondary injuries, innate immune response genes were the predominant class of genes that changed expression. Furthermore, age and diet affected the magnitude of the change in expression of some innate immune response genes, suggesting roles for these genes in inhibiting secondary injuries that cause mortality. Our results indicate that the complexity of TBI outcomes is due in part to distinct, genetically controlled, age- and diet-regulated mechanisms that promote secondary injuries and that involve a subset of innate immune response genes.

Parkinson's disease (PD) is characterized by the loss of dopaminergic neurons, resulting in progressive locomotor dysfunction. Identification of genes required for the maintenance of these neurons should help to identify potential therapeutic targets. However, little is known regarding the factors that render dopaminergic neurons selectively vulnerable to PD. Here, we show that Drosophila melanogaster scarlet mutants exhibit an age-dependent progressive loss of dopaminergic neurons, along with subsequent locomotor defects and a shortened lifespan. Knockdown of Scarlet specifically within dopaminergic neurons is sufficient to produce this neurodegeneration, demonstrating a unique role for Scarlet beyond its well-characterized role in eye pigmentation. Both genetic and pharmacological manipulation of the kynurenine pathway rescued loss of dopaminergic neurons by promoting synthesis of the free radical scavenger kynurenic acid (KYNA) and limiting the production of the free radical generator 3-hydroxykynurenine (3-HK). Finally, we show that expression of wild-type Scarlet is neuroprotective in a model of PD, suggesting that manipulating kynurenine metabolism may be a potential therapeutic option in treating PD. This article has an associated First Person interview with the first author of the paper.

Genetic variability affects the response to numerous xenobiotics but its role in the clinically-observed irregular responses to general anesthetics remains uncertain. To investigate the pharmacogenetics of volatile general anesthetics (VGAs), we developed a Serial Anesthesia Array apparatus to expose multiple Drosophila melanogaster samples to VGAs and behavioral assays to determine pharmacokinetic and pharmacodynamic properties of VGAs. We studied the VGAs isoflurane and sevoflurane in four wild type strains from the Drosophila Genetic Reference Panel, two commonly used laboratory strains (Canton S and w 1118 ), and a mutant in Complex I of the mitochondrial electron transport chain (ND23 60114 ). In all seven strains, isoflurane was more potent than sevoflurane, as predicted by their relative lipid solubilities, and emergence from isoflurane was slower than from sevoflurane, reproducing cardinal pharmacokinetic and pharmacodynamic properties in mammals. In addition, ND23 60114 flies were more sensitive to both agents, as observed in worms, mice, and humans carrying Complex I mutations. Moreover, we found substantial variability among the fly strains both in absolute and in relative pharmacokinetic and pharmacodynamic profiles of isoflurane and sevoflurane. These data indicate that naturally occurring genetic variations measurably influence cardinal pharmacologic properties of VGAs and that flies can be used to identify relevant genetic variations.

Children undergoing cranial radiation therapy (CRT) for pediatric central nervous system malignancies are at increased risk for neurological deficits later in life. We have developed a model of neurotoxic damage in adult Drosophila following irradiation during the juvenile stages with the goal of elucidating underlying neuropathological mechanisms and of ultimately identifying potential therapeutic targets. Wild-type third-instar larvae were irradiated with single doses of γ-radiation, and the percentage that survived to adulthood was determined. Motor function of surviving adults was examined with a climbing assay, and longevity was assessed by measuring lifespan. Neuronal cell death was assayed by using immunohistochemistry in adult brains. We also tested the sensitivity at different developmental stages by irradiating larvae at various time points. Irradiating late third-instar larvae at a dose of 20 Gy or higher impaired the motor activity of surviving adults. A dose of 40 Gy or higher resulted in a precipitous reduction in the percentage of larvae that survive to adulthood. A dose-dependent decrease in adult longevity was paralleled by a dose-dependent increase in activated Death caspase-1 (Dcp1) in adult brains. Survival to adulthood and adult lifespan were more severely impaired with decreasing larval age at the time of irradiation. Our initial survey of the Drosophila Genetic Reference Panel demonstrated that differences in genotype can confer phenotypic differences in radio-sensitivity for developmental survival and motor function. This work demonstrates the usefulness of Drosophila to model the toxic effects of radiation during development, and has the potential to unravel underlying mechanisms and to facilitate the discovery of novel therapeutic interventions., Highlighted Article: To model delayed neurological deficits resulting from pediatric cranial radiation therapy, neurotoxic damage in adult Drosophila is assessed following larval irradiation with the goal of elucidating underlying pathological mechanisms.

Traumatic brain injury (TBI) is a complex disorder that affects millions of people worldwide. The complexity of TBI partly stems from the fact that injuries to the brain instigate non-neurological injuries to other organs such as the intestine. Additionally, genetic variation is thought to play a large role in determining the nature and severity of non-neurological injuries. We recently reported that TBI in flies, as in humans, increases permeability of the intestinal epithelial barrier resulting in hyperglycemia and a higher risk of death. Furthermore, we demonstrated that genetic variation in flies is also pertinent to the complexity of non-neurological injuries following TBI. The goals of this review are to place our findings in the context of what is known about TBI-induced intestinal permeability from studies of TBI patients and rodent TBI models and to draw attention to how studies of the fly TBI model can provide unique insights that may facilitate diagnosis and treatment of TBI.

A common feature of many neurodegenerative diseases is the accumulation of toxic proteins that disrupt vital cellular functions. Degradative pathways such as autophagy play an important protective role in breaking down misfolded and long-lived proteins. Neurons are particularly vulnerable to defects in these pathways, but many of the details regarding the link between autophagy and neurodegeneration remain unclear. We previously found that temperature-sensitive paralytic mutants in Drosophila are enriched for those exhibiting age-dependent neurodegeneration. Here we show that one of these mutants, comatose (comt), in addition to locomotor defects, displays shortened lifespan and progressive neurodegeneration, including loss of dopaminerigic (DA) neurons. comt encodes N-ethyl-maleimide sensitive fusion protein (NSF1), which has a well-documented role in synaptic transmission. However, the neurodegenerative phenotypes we observe in comt mutants do not appear to depend on defects in synaptic transmission, but rather from their inability to sustain autophagy under stress, due at least in part to a defect in trafficking of lysosomal proteases such as cathepsin-L. Conversely, overexpression of NSF1 rescues α-synuclein-induced toxicity of DA neurons in a model of Parkinson’s disease. Our results demonstrate a neuroprotective role for NSF1 that involves mediation of fusion events crucial for degradative pathways such as autophagy, providing greater understanding of cellular dysfunctions common to several neurodegenerative diseases.

Proper mitochondrial activity depends upon proteins encoded by genes in the nuclear and mitochondrial genomes that must interact functionally and physically in a precisely coordinated manner. Consequently, mito-nuclear allelic interactions are thought to be of crucial importance on an evolutionary scale, as well as for manifestation of essential biological phenotypes, including those directly relevant to human disease. Nonetheless, detailed molecular understanding of mito-nuclear interactions is still lacking, and definitive examples of such interactions in vivo are sparse. Here we describe the characterization of a mutation in Drosophila ND23, a nuclear gene encoding a highly conserved subunit of mitochondrial complex 1. This characterization led to the discovery of a mito-nuclear interaction that affects the ND23 mutant phenotype. ND23 mutants exhibit reduced lifespan, neurodegeneration, abnormal mitochondrial morphology, and decreased ATP levels. These phenotypes are similar to those observed in patients with Leigh syndrome, which is caused by mutations in a number of nuclear genes that encode mitochondrial proteins, including the human ortholog of ND23. A key feature of Leigh syndrome, and other mitochondrial disorders, is unexpected and unexplained phenotypic variability. We discovered that the phenotypic severity of ND23 mutations varies depending on the maternally inherited mitochondrial background. Sequence analysis of the relevant mitochondrial genomes identified several variants that are likely candidates for the phenotypic interaction with mutant ND23, including a variant affecting a mitochondrially encoded component of complex I. Thus, our work provides an in vivo demonstration of the phenotypic importance of mito-nuclear interactions in the context of mitochondrial disease.

Summary During aging, innate immunity progresses to a chronically active state. However, what distinguishes those that “age well” from those developing age-related neurological conditions is unclear. We used Drosophila to explore the cost of immunity in the aging brain. We show that mutations in intracellular negative regulators of the IMD/NF-κB pathway predisposed flies to toxic levels of antimicrobial peptides, resulting in early locomotor defects, extensive neurodegeneration, and reduced lifespan. These phenotypes were rescued when immunity was suppressed in glia. In healthy flies, suppressing immunity in glial cells resulted in increased adipokinetic hormonal signaling with high nutrient levels in later life and an extension of active lifespan. Thus, when levels of IMD/NF-κB deviate from normal, two mechanisms are at play: lower levels derepress an immune-endocrine axis, which mobilizes nutrients, leading to lifespan extension, whereas higher levels increase antimicrobial peptides, causing neurodegeneration. Immunity in the fly brain is therefore a key lifespan determinant., Graphical Abstract, Highlights • Constitutive immunity predisposes to short lifespan with severe neurodegeneration • Blocking constitutive immunity in glia rescues predisposed flies • Suppression of immunity in glia of healthy flies triggers adipokinetic signaling • This immune-endocrine axis mobilizes nutrients and extends active lifespan, In humans, both healthy aging and age-dependent neurodegeneration are accompanied by an upregulation of innate immunity. What is the cause and what is the consequence remain unclear. Kounatidis et al. show that, in flies, NF-κB immune signaling controls lifespan in healthy flies, as well as in those predisposed to early neurodegeneration.

Although evolutionary changes must take place in neural connectivity and synaptic architecture as nervous systems become more complex, we lack understanding of the general principles and specific mechanisms by which these changes occur. Previously, we found that morphology of the larval neuromuscular junction (NMJ) varies extensively among different species of Drosophila but is relatively conserved within a species. To identify specific genes as candidates that might underlie phenotypic differences in NMJ morphology among Drosophila species, we performed a genetic analysis on one of two phenotypic variants we found among 20 natural isolates of Drosophila melanogaster. We discovered genetic polymorphisms for both positive and negative regulators of NMJ growth segregating within the variant line. Focusing on one subline, that displayed NMJ overgrowth, we mapped the phenotype to Mob2 [Monopolar spindle (Mps) one binding protein 2)], a gene encoding a Nuclear Dbf2 (Dumbbell formation 2)-Related (NDR) kinase activator. We confirmed this identification by transformation rescue experiments and showed that presynaptic expression of Mob2 is necessary and sufficient to regulate NMJ growth. Mob2 interacts in a dominant, dose-dependent manner with tricornered but not with warts, to cause NMJ overgrowth, suggesting that Mob2 specifically functions in combination with the former NDR kinase to regulate NMJ development. These results demonstrate the feasibility and utility of identifying genetic variants affecting NMJ morphology in natural populations of Drosophila. These variants can lead to discovery of new genes and molecular mechanisms that regulate NMJ development while also providing new information that can advance our understanding of mechanisms that underlie nervous system evolution.

Recent evidence indicates that protein aggregates can spread between neurons in several neurodegenerative diseases but much remains unknown regarding the underlying mechanisms responsible for this spreading and its role in disease progression. We recently demonstrated that mutant Huntingtin aggregates spread between cells within the Drosophila brain resulting in non-cell autonomous loss of a pair of large neurons in the posterior protocerebrum. However, the full extent of neuronal loss throughout the brain was not determined. Here we examine the effects of driving expression of mutant Huntingtin in Olfactory Receptor Neurons (ORNs) by using a marker for cleaved caspase activity to monitor neuronal apoptosis as a function of age. We find widespread caspase activity in various brain regions over time, demonstrating that non-cell autonomous damage is widespread. Improved understanding of which neurons are most vulnerable and why should be useful in developing treatment strategies for neurodegenerative diseases that involve transcellular spreading of aggregates.

[Figure][1] Great articles often begin with an intriguing paradox, describe an elegant experimental approach, provide lasting and important data, and change the course of their discipline. [Sturtevant and Beadle (1936)][2] meets all of those criteria and stands as a paradigm for the genetic

Neuropeptides are ubiquitous in both mammals and invertebrates and play essential roles in regulation and modulation of many developmental and physiological processes through activation of G-protein-coupled-receptors (GPCRs). However, the mechanisms by which many of the neuropeptides regulate specific neural function and behaviors remain undefined. Here we investigate the functions of Drosulfakinin (DSK), the Drosophila homolog of vertebrate neuropeptide cholecystokinin (CCK), which is the most abundant neuropeptide in the central nervous system. We provide biochemical evidence that sulfated DSK-1 and DSK-2 activate the CCKLR-17D1 receptor in a cell culture assay. We further examine the role of DSK and CCKLR-17D1 in the regulation of larval locomotion, both in a semi-intact larval preparation and in intact larvae under intense light exposure. Our results suggest that DSK/CCKLR-17D1 signaling promote larval body wall muscle contraction and is necessary for mediating locomotor behavior in stress-induced escape response.

TheDrosophilalarval neuromuscular junction (NMJ) is a powerful system for the genetic and molecular analysis of neuronal excitability, synaptic transmission, and synaptic development. However, its use for studying age-dependent processes, such as maintenance of neuronal viability and synaptic stability, are temporally limited by the onset of pupariation and metamorphosis. Here we characterize larval NMJ growth, growth regulation, structure, and function in a developmental variant with an extended third instar (ETI). RNAi-knockdown of the prothoracicotropic hormone receptor,torso, in the ring gland of developing larvae leaves the timing of first and second instar molts largely unchanged, but triples duration of the third instar from 3 to 9.5 d (McBrayer et al., 2007; Rewitz et al., 2009). During this ETI period, NMJs undergo additional growth (adding >50 boutons/NMJ), and this growth remains under the control of the canonical regulators Highwire and the TGFβ/BMP pathway. NMJ growth during the ETI period occurs via addition of new branches, satellite boutons, and interstitial boutons, and continues even after muscle growth levels off. Throughout the ETI, organization of synapses and active zones remains normal, and synaptic transmission is unchanged. These results establish the ETI larval system as a viable model for studying motor neuron diseases and for investigating time-dependent effects of perturbations that impair mechanisms of neuroprotection, synaptic maintenance, and response to neural injury.

Neuropeptide signaling, which is known to affect synaptic function in neural communication, also promotes neuromuscular junction growth in Drosophila., Neuropeptide signaling is integral to many aspects of neural communication, particularly modulation of membrane excitability and synaptic transmission. However, neuropeptides have not been clearly implicated in synaptic growth and development. Here, we demonstrate that cholecystokinin-like receptor (CCKLR) and drosulfakinin (DSK), its predicted ligand, are strong positive growth regulators of the Drosophila melanogaster larval neuromuscular junction (NMJ). Mutations of CCKLR or dsk produced severe NMJ undergrowth, whereas overexpression of CCKLR caused overgrowth. Presynaptic expression of CCKLR was necessary and sufficient for regulating NMJ growth. CCKLR and dsk mutants also reduced synaptic function in parallel with decreased NMJ size. Analysis of double mutants revealed that DSK/CCKLR regulation of NMJ growth occurs through the cyclic adenosine monophosphate (cAMP)–protein kinase A (PKA)–cAMP response element binding protein (CREB) pathway. Our results demonstrate a novel role for neuropeptide signaling in synaptic development. Moreover, because the cAMP–PKA–CREB pathway is required for structural synaptic plasticity in learning and memory, DSK/CCKLR signaling may also contribute to these mechanisms.

To signal properly, excitable cells must establish and maintain the correct balance of various types of ion channels that increase or decrease membrane excitability. The mechanisms by which this balance is regulated remain largely unknown. Here, we describe a regulatory mechanism uncovered by a Drosophila behavioral mutant, down and out ( dao ). At elevated temperatures, dao loss-of-function mutants exhibit seizures associated with spontaneous bursts of neural activity. This phenotype closely resembles that of seizure mutations, which impair activity of ether-a-go-go-related gene (Erg)-type potassium channels. Conversely, neural over-expression of wild-type Dao confers dominant temperature-sensitive paralysis with kinetics reminiscent of paralytic sodium-channel mutants. The over-expression phenotype of dao is suppressed in a seizure mutant background, suggesting that Dao acts by an effect on Erg channels. In support of this hypothesis, functional expression of Erg channels in a heterologous system is dependent on the presence of Dao. These results indicate that Dao has an important role in establishing the proper level of neuronal membrane excitability by regulating functional expression of Erg channels.

The ubiquitin–proteosome and autophagy pathways work together to regulate synaptic growth and plasticity in response to environmental conditions., Autophagy, a lysosome-dependent degradation mechanism, mediates many biological processes, including cellular stress responses and neuroprotection. In this study, we demonstrate that autophagy positively regulates development of the Drosophila melanogaster larval neuromuscular junction (NMJ). Autophagy induces an NMJ overgrowth phenotype closely resembling that of highwire (hiw), an E3 ubiquitin ligase mutant. Moreover, like hiw, autophagy-induced NMJ overgrowth is suppressed by wallenda (wnd) and by a dominant-negative c-Jun NH2-terminal kinase (bskDN). We show that autophagy promotes NMJ growth by reducing Hiw levels. Thus, autophagy and the ubiquitin–proteasome system converge in regulating synaptic development. Because autophagy is triggered in response to many environmental cues, our findings suggest that it is perfectly positioned to link environmental conditions with synaptic growth and plasticity.

A key feature of many neurodegenerative diseases is the accumulation and subsequent aggregation of misfolded proteins. Recent studies have highlighted the transcellular propagation of protein aggregates in several major neurodegenerative diseases, although the precise mechanisms underlying this spreading and how it relates to disease pathology remain unclear. Here we use a polyglutamine-expanded form of human huntingtin (Htt) with a fluorescent tag to monitor the spreading of aggregates in the Drosophila brain in a model of Huntington's disease. Upon expression of this construct in a defined subset of neurons, we demonstrate that protein aggregates accumulate at synaptic terminals and progressively spread throughout the brain. These aggregates are internalized and accumulate within other neurons. We show that Htt aggregates cause non-cell-autonomous pathology, including loss of vulnerable neurons that can be prevented by inhibiting endocytosis in these neurons. Finally we show that the release of aggregates requires N-ethylmalemide-sensitive fusion protein 1, demonstrating that active release and uptake of Htt aggregates are important elements of spreading and disease progression.

Cranial radiation therapy (CRT) is an effective treatment for pediatric central nervous system malignancies, but survivors often suffer from neurological and neurocognitive side effects that occur many years after radiation exposure. Although the biological mechanisms underlying these deleterious side effects are incompletely understood, radiation exposure triggers an acute inflammatory response that may evolve into chronic inflammation, offering one avenue of investigation. Recently, we developed a Drosophila model of the neurotoxic side effects of radiation exposure. Here we use this model to investigate the role of the innate immune system in response to radiation exposure. We show that the innate immune response and NF-ĸB target gene expression is activated in the adult Drosophila brain following radiation exposure during larval development, and that this response is sustained in adult flies weeks after radiation exposure. We also present preliminary data suggesting that innate immunity is radioprotective during Drosophila development. Together our data suggest that activation of the innate immune response may be beneficial initially for survival following radiation exposure but result in long-term deleterious consequences, with chronic inflammation leading to impaired neuronal function and viability at later stages. This work lays the foundation for future studies of how the innate immune response is triggered by radiation exposure and its role in mediating the biological responses to radiation. These studies may facilitate the development of strategies to reduce the deleterious side effects of CRT.

Traumatic brain injury (TBI) affects millions of people each year, causing impairment of physical, cognitive, and behavioral functions and death. Studies using Drosophila have contributed important breakthroughs in understanding neurological processes. Thus, with the goal of understanding the cellular and molecular basis of TBI pathologies in humans, we developed the High Impact Trauma (HIT) device to inflict closed head TBI in flies. Flies subjected to the HIT device display phenotypes consistent with human TBI such as temporary incapacitation and progressive neurodegeneration. The HIT device uses a spring-based mechanism to propel flies against the wall of a vial, causing mechanical damage to the fly brain. The device is inexpensive and easy to construct, its operation is simple and rapid, and it produces reproducible results. Consequently, the HIT device can be combined with existing experimental tools and techniques for flies to address fundamental questions about TBI that can lead to the development of diagnostics and treatments for TBI. In particular, the HIT device can be used to perform large-scale genetic screens to understand the genetic basis of TBI pathologies.

Neuronal Na+ and K+ channels elicit currents in opposing directions and thus have opposing effects on neuronal excitability. Mutations in genes encoding Na+ or K+ channels often interact genetically, leading to either phenotypic suppression or enhancement for genes with opposing or similar effects on excitability, respectively. For example, the effects of mutations in Shaker (Sh), which encodes a K+ channel subunit, are suppressed by loss-of-function mutations in the Na+ channel structural gene para, but enhanced by loss-of-function mutations in a second K+ channel encoded by eag. Here we identify two novel mutations that suppress the effects of a Sh mutation on behavior and neuronal excitability. We used recombination mapping to localize both mutations to the eag locus, and we used sequence analysis to determine that both mutations are caused by a single amino acid substitution (G297E) in the S2–S3 linker of Eag. Because these novel eag mutations confer opposite phenotypes to eag loss-of-function mutations, we suggest that eagG297E causes an eag gain-of-function phenotype. We hypothesize that the G297E substitution may cause premature, prolonged, or constitutive opening of the Eag channels by favoring the “unlocked” state of the channel.

Most of us sleep 7-8 h per night, and if we are deprived of sleep our performance suffers greatly; however, a few do well with just 3-4 h of sleep-a trait that seems to run in families. Determining which genes underlie this phenotype could shed light on the mechanisms and functions of sleep. To do so, we performed mutagenesis in Drosophila melanogaster, because flies also sleep for many hours and, when sleep deprived, show sleep rebound and performance impairments. By screening 9,000 mutant lines, we found minisleep (mns), a line that sleeps for one-third of the wild-type amount. We show that mns flies perform normally in a number of tasks, have preserved sleep homeostasis, but are not impaired by sleep deprivation. We then show that mns flies carry a point mutation in a conserved domain of the Shaker gene. Moreover, after crossing out genetic modifiers accumulated over many generations, other Shaker alleles also become short sleepers and fail to complement the mns phenotype. Finally, we show that short-sleeping Shaker flies have a reduced lifespan. Shaker, which encodes a voltage-dependent potassium channel controlling membrane repolarization and transmitter release, may thus regulate sleep need or efficiency.

To investigate the cellular and molecular etiology of early onset torsion dystonia, we have established a Drosophila model of this disorder. Expression of mutant human torsinA deleted for a single glutamic acid residue (DeltaE HtorA), but not normal HtorA, elicits locomotor defects in Drosophila. As in mammalian systems, DeltaE HtorA in flies forms protein accumulations that localize to synaptic membranes, nuclei and endosomes. Various morphological defects at the neuromuscular junction in larvae expressing DeltaE HtorA were observed at the EM level, some of which resemble those recently reported for mutants with defects in TGF-beta signaling. These results together with the distribution patterns and localizations of DeltaE HtorA accumulations suggested that DeltaE HtorA could interfere with some aspect of TGF-beta signaling from synapses to endosomes or nuclei. Consistent with this possibility, neuronal overexpression of Drosophila or human Smad2, a downstream effector of the TGF-beta pathway, suppressed the behavioral and ultrastructural defects of DeltaE HtorA flies. These results raise the possibility that a defect in TGF-beta signaling might also underlie early onset torsion dystonia in humans.

The Na(+)/K(+) ATPase asymmetrically distributes sodium and potassium ions across the plasma membrane to generate and maintain the membrane potential in many cell types. Although these pumps have been hypothesized to be involved in various human neurological disorders, including seizures and neurodegeneration, direct genetic evidence has been lacking. Here, we describe novel mutations in the Drosophila gene encoding the α (catalytic) subunit of the Na(+)/K(+) ATPase that lead to behavioral abnormalities, reduced life span, and severe neuronal hyperexcitability. These phenotypes parallel the occurrence of extensive, age-dependent neurodegeneration. We have also discovered that the ATPalpha transcripts undergo alternative splicing that substantially increases the diversity of potential proteins. Our data show that maintenance of neuronal viability is dependent on normal sodium pump activity and establishDrosophila as a useful model for investigating the role of the pump in human neurodegenerative and seizure disorders.

Segregation Distorter ( SD ) is a meiotic drive system in Drosophila that causes preferential transmission of the SD chromosome from SD / SD + males owing to the induced dysfunction of SD + spermatids. The key distorter locus, Sd , is a dominant neomorphic allele encoding a truncated, but enzymatically active, RanGAP (RanGTPase-activating protein) whose nuclear mislocalization underlies distortion by disrupting the Ran signaling pathway. Here, we show that even wild-type RanGAP can cause segregation distortion when it is overexpressed in the male germ line or when the gene dosage of a particular modifier locus is increased. Both manipulations result in substantial nuclear accumulation of RanGAP. Distortion can be suppressed by overexpression of Ran or Ran guanine nucleotide exchange factor (RanGEF) in the male germ line, indicating that the primary consequence of nuclear mislocalization of RanGAP is reduction of intranuclear RanGTP levels. These results prove that segregation distortion does not depend on any unique properties of the mutant RanGAP encoded by Sd and provide a unifying explanation for the occurrence of distortion in a variety of experimental situations.

Kvbeta2 binds to K(+) channel alpha subunits from at least two different families (Kv1 and Kv4) and is a member of the aldo-ketoreductase (AKR) superfamily. Proposed functions for this protein in vivo include a chaperone-like role in Kv1 alpha subunit biogenesis and catalytic activity as an AKR oxidoreductase. To investigate the in vivo function of Kvbeta2, Kvbeta2-null and point mutant (Y90F) mice were generated through gene targeting in embryonic stem cells. In Kvbeta2-null mice, Kv1.1 and Kv1.2 localize normally in cerebellar basket cell terminals and the juxtaparanodal region of myelinated nerves. Moreover, normal glycosylation patterns are observed for Kv1.1 and Kv1.2 in whole brain lysates. Thus, loss of the chaperone-like activity does not appear to account for the phenotype of Kvbeta2-null mice, which include reduced life spans, occasional seizures, and cold swim-induced tremors similar to that observed in Kv1.1-null mice. Mice expressing Kvbeta2, mutated at a site (Y90F) that abolishes AKR-like catalytic activity in other family members, have no overt phenotype. We conclude that Kvbeta2 contributes to regulation of excitability in vivo, although not directly through either chaperone-like or typical AKR catalytic activity. Rather, Kvbeta2 relies upon as yet unidentified mechanisms in the regulation of K(+) channel and/or oxidoreductive functions.

Traumatic brain injury (TBI) is a major cause of death and disability worldwide. Unfavorable TBI outcomes result from primary mechanical injuries to the brain and ensuing secondary non-mechanical injuries that are not limited to the brain. Our genome-wide association study of Drosophila melanogaster revealed that the probability of death following TBI is associated with single nucleotide polymorphisms in genes involved in tissue barrier function and glucose homeostasis. We found that TBI causes intestinal and blood–brain barrier dysfunction and that intestinal barrier dysfunction is highly correlated with the probability of death. Furthermore, we found that ingestion of glucose after a primary injury increases the probability of death through a secondary injury mechanism that exacerbates intestinal barrier dysfunction. Our results indicate that natural variation in the probability of death following TBI is due in part to genetic differences that affect intestinal barrier dysfunction. DOI: http://dx.doi.org/10.7554/eLife.04790.001, eLife digest Traumatic brain injury (TBI) caused by a violent blow to the head or body and the resultant collision of the brain against the skull is a major cause of disability and death in humans. Primary injury to the brain triggers secondary injuries that further damage the brain and other organs, generating many of the detrimental consequences of TBI. However, despite decades of study, the exact nature of these secondary injuries and their origin are poorly understood. A better understanding of secondary injuries should help to develop novel therapies to improve TBI outcomes in affected individuals. To obtain this information, in 2013 researchers devised a method to inflict TBI in the common fruit fly, Drosophila melanogaster, an organism that is readily amenable to detailed genetic and molecular studies. This investigation demonstrated that flies subjected to TBI display many of the same symptoms observed in humans after a brain injury, including temporary loss of mobility and damage to the brain that becomes worse over time. In addition, many of the flies die within 24 hr after brain injury. Now Katzenberger et al. use this experimental system to investigate the secondary injuries responsible for these deaths. First, genetic variants were identified that confer increased or decreased susceptibility to death after brain injury. Several of the identified genes affect the structural integrity of the intestinal barrier that isolates the contents of the gut—including nutrients and bacteria—from the circulatory system. Katzenberger et al. subsequently found that the breakdown of this barrier after brain injury permits bacteria and glucose to leak out of the intestine. Treating flies with antibiotics did not increase survival, whereas reducing glucose levels in the circulatory system after brain injury did. Thus, Katzenberger et al. conclude that high levels of glucose in the circulatory system, a condition known as hyperglycemia, is a key culprit in death following TBI. Notably, these results parallel findings in humans, where hyperglycemia is highly predictive of death following TBI. Similarly, individuals with diabetes have a significantly increased risk of death after TBI. These results suggest that the secondary injuries leading to death are the same in flies and humans and that further studies in flies are likely to provide additional new information that will help us understand the complex consequences of TBI. Important challenges remain, including understanding precisely how the brain and intestine communicate, how injury to the brain leads to disruption of the intestinal barrier, and why elevated glucose levels increase mortality after brain injury. Answers to these questions could help pave the way to new therapies for TBI. DOI: http://dx.doi.org/10.7554/eLife.04790.002

Although Obaid Siddiqi’s major research focus in neurogenetics was on chemosensation and olfaction in Drosophila, he made seminal contributions to the study of temperature-sensitive paralytic mutants that paved the way for research that we and many other investigators have continued to pursue. Here we recount Siddiqi’s investigation and the impact it had on our own studies especially at a formative stage of our careers. We acknowledge our debt to Obaid Siddiqi and remember him fondly as an inspired and inspiring scientist, mentor, role model and human being.

The Toll-like receptor Tollo positively regulates growth of the Drosophila larval neuromuscular junction through the JNK pathway after activation by the neurotrophin Spätzle3., Toll-like receptors (TLRs) are best characterized for their roles in mediating dorsoventral patterning and the innate immune response. However, recent studies indicate that TLRs are also involved in regulating neuronal growth and development. Here, we demonstrate that the TLR Tollo positively regulates growth of the Drosophila melanogaster larval neuromuscular junction (NMJ). Tollo mutants exhibited NMJ undergrowth, whereas increased expression of Tollo led to NMJ overgrowth. Tollo expression in the motoneuron was both necessary and sufficient for regulating NMJ growth. Dominant genetic interactions together with altered levels of phosphorylated c-Jun N-terminal kinase (JNK) and puc-lacZ expression revealed that Tollo signals through the JNK pathway at the NMJ. Genetic interactions also revealed that the neurotrophin Spätzle3 (Spz3) is a likely Tollo ligand. Spz3 expression in muscle and proteolytic activation via the Easter protease was necessary and sufficient to promote NMJ growth. These results demonstrate the existence of a novel neurotrophin signaling pathway that is required for synaptic development in Drosophila.

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an oil-coated graduated cylinder; landing height provided a measure of flight performance by assessing how far flies will fall before producing enough thrust to make contact with the wall of the cylinder. Here we describe an updated version of the flight tester with four major improvements. First, we added a "drop tube" to ensure that all flies enter the flight cylinder at a similar velocity between trials, eliminating variability between users. Second, we replaced the oil coating with removable plastic sheets coated in Tangle-Trap, an adhesive designed to capture live insects. Third, we use a longer cylinder to enable more accurate discrimination of flight ability. Fourth we use a digital camera and imaging software to automate the scoring of flight performance. These improvements allow for the rapid, quantitative assessment of flight behavior, useful for large datasets and large-scale genetic screens.

Expression of multiple reporter or effector transgenes in the same cell from a single construct is increasingly necessary in various experimental paradigms. The discovery of short, virus-derived peptide sequences that mediate a ribosome-skipping event enables generation of multiple separate peptide products from one mRNA. Here we describe methods and vectors to facilitate easy production of polycistronic-like sequences utilizing these 2A peptides tailored for expression in Drosophila both in vitro and in vivo. We tested the separation efficiency of different viral 2A peptides in cultured Drosophila cells and in vivo and found that the 2A peptides from porcine teschovirus-1 (P2A) and Thosea asigna virus (T2A) worked best. To demonstrate the utility of this approach, we used the P2A peptide to co-express the red fluorescent protein tdTomato and the genetically-encoded calcium indicator GCaMP5G in larval motorneurons. This technique enabled ratiometric calcium imaging with motion correction allowing us to record synaptic activity at the neuromuscular junction in an intact larval preparation through the cuticle. The tools presented here should greatly facilitate the generation of 2A peptide-mediated expression of multiple transgenes in Drosophila.

Soluble N- ethylmaleimide-sensitive fusion attachment protein receptor (SNARE)-mediated fusion of synaptic vesicles with the presynaptic-plasma membrane is essential for communication between neurons. Disassembly of the SNARE complex requires the ATPase N -ethylmaleimide-sensitive fusion protein (NSF). To determine where in the synaptic-vesicle cycle NSF functions, we have undertaken a genetic analysis of comatose (dNSF-1) in Drosophila . Characterization of 16 comatose mutations demonstrates that NSF mediates disassembly of SNARE complexes after synaptic-vesicle fusion. Hypomorphic mutations in NSF cause temperature-sensitive paralysis, whereas null mutations result in lethality. Genetic-interaction studies with para demonstrate that blocking evoked fusion delays the accumulation of assembled SNARE complexes and behavioral paralysis that normally occurs in comatose mutants, indicating NSF activity is not required in the absence of vesicle fusion. In addition, the entire vesicle pool can be depleted in shibire comatose double mutants, demonstrating that NSF activity is not required for the fusion step itself. Multiple rounds of vesicle fusion in the absence of NSF activity poisons neurotransmission by trapping SNAREs into cis-complexes. These data indicate that NSF normally dissociates and recycles SNARE proteins during the interval between exocytosis and endocytosis. In the absence of NSF activity, there are sufficient fusion-competent SNAREs to exocytose both the readily released and the reserve pool of synaptic vesicles.

Calcium-activated protein for secretion (CAPS) is proposed to play an essential role in Ca2+-regulated dense-core vesicle exocytosis in vertebrate neuroendocrine cells. Here we report the cloning, mutation, and characterization of the Drosophila ortholog (dCAPS). Null dCAPS mutants display locomotory deficits and complete embryonic lethality. The mutant NMJ reveals a 50% loss in evoked glutamatergic transmission, and an accumulation of synaptic vesicles at active zones. Importantly, dCAPS mutants display a highly specific 3-fold accumulation of dense-core vesicles in synaptic terminals, which was not observed in mutants that completely arrest synaptic vesicle exocytosis. Targeted transgenic CAPS expression in identified motoneurons fails to rescue dCAPS neurotransmission defects, demonstrating a cell nonautonomous role in synaptic vesicle fusion. We conclude that dCAPS is required for dense-core vesicle release and that a dCAPS-dependent mechanism modulates synaptic vesicle release at glutamatergic synapses.

Synaptotagmin has been proposed to function as a Ca(2+) sensor that regulates synaptic vesicle exocytosis, whereas the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex is thought to form the core of a conserved membrane fusion machine. Little is known concerning the functional relationships between synaptotagmin and SNAREs. Here we report that synaptotagmin can facilitate SNARE complex formation in vitro and that synaptotagmin mutations disrupt SNARE complex formation in vivo. Synaptotagmin oligomers efficiently bind SNARE complexes, whereas Ca(2+) acting via synaptotagmin triggers cross-linking of SNARE complexes into dimers. Mutations in Drosophila that delete the C2B domain of synaptotagmin disrupt clathrin AP-2 binding and endocytosis. In contrast, a mutation that blocks Ca(2+)-triggered conformational changes in C2B and diminishes Ca(2+)-triggered synaptotagmin oligomerization results in a postdocking defect in neurotransmitter release and a decrease in SNARE assembly in vivo. These data suggest that Ca(2+)-driven oligomerization via the C2B domain of synaptotagmin may trigger synaptic vesicle fusion via the assembly and clustering of SNARE complexes.

Genetic analysis of ion channel dysfunction in Drosophila. To dissect the molecular mechanisms of electrical activity in the nervous system, an extensive collection of mutations affecting various types of voltage-gated ion channels was identified and characterized in Drosophila. Most of these mutations were generated by chemical mutagenesis and were recognized on the basis of defects in motor behavior. These were the first genetically determined ion channelopathies to be characterized in any multicellular organism. Drosophila is a particularly attractive model system for such studies because of the availability of powerful genetic, electrophysiological, and molecular techniques for generating new mutations, characterizing their phenotypes, and cloning the genes thus defined. Consequently, a number of ion channels, including various types of K+ channels that had not yielded previously to biochemical approaches, were first identified via a genetic strategy in Drosophila. Evolutionary conservation of these genes enabled subsequent isolation of the corresponding genes from various mammals, including humans. Several of these human homologues have been found to be associated with heritable neuromuscular disorders. Studies of ion channel mutations in Drosophila have thus provided important biological information concerning the molecular and functional diversity of ion channels, their evolutionary relationships, and their in vivo functions in the nervous system. Similar studies of additional new mutations should now facilitate the analysis of ion channel regulatory mechanisms.

Communication within the nervous system is mediated by Ca2+-triggered fusion of synaptic vesicles with the presynaptic plasma membrane. Genetic and biochemical evidence indicates that synaptotagmin I may function as a Ca2+ sensor in neuronal exocytosis because it can bind Ca2+ and penetrate into lipid bilayers1,2,3,4. Chronic depolarization or seizure activity results in the upregulation of a distinct and unusual isoform of the synaptotagmin family, synaptotagmin IV (ref. 5). We have identified a Drosophila homologue of synaptotagmin IV that is enriched on synaptic vesicles and contains an evolutionarily conserved substitution of aspartate to serine that abolishes its ability to bind membranes in response to Ca2+ influx. Synaptotagmin IV forms hetero-oligomers with synaptotagmin I, resulting in synaptotagmin clusters that cannot effectively penetrate lipid bilayers and are less efficient at coupling Ca2+ to secretion in vivo : upregulation of synaptotagmin IV, but not synaptotagmin I, decreases evoked neurotransmission. These findings indicate that modulating theexpression of synaptotagmins with different Ca2+-binding affinities can lead to heteromultimers that can regulate the efficiency of excitation–secretion coupling in vivo and represent a new molecular mechanism for synaptic plasticity.

ONE cannot help but admire a gene as fiendishly clever as Segregation distorter ( Sd ). Not only has it figured out a way to cheat the system, but it is extraordinarily good at what it does. It violates the fundamental principle of Mendelian inheritance regularly and with remarkable potency by

Members of the Ether à go-go (Eag) K+channel subfamilies Eag, Erg, and Elk are widely expressed in the nervous system, but their neural functionsin vivoremain largely unknown. The biophysical properties of channels from the Eag and Erg subfamilies have been described, and based on their characteristic features and expression patterns, Erg channels have been associated with native currents in the heart. Little is known about the properties of channels from the Elk subfamily. We have identified a mouse gene,Melk2, that encodes a predicted polypeptide with 48% amino acid identity toDrosophilaElk but only 40 and 36% identity with mouse Erg (Merg) and Eag (Meag), respectively.Melk2RNA appears to be expressed at high levels only in brain tissue. Functional expression ofMelk2inXenopusoocytes reveals large, transient peaks of current at the onset of depolarization. Like Meag currents, Melk2 currents activate relatively quickly, but they lack the nonsuperimposable Cole–Moore shift characteristic of the Eag subfamily. Melk2 currents are insensitive to E-4031, a class III antiarrhythmic compound that blocks the Human Ether-à-go-go-Related Gene (HERG) channel and its counterpart in native tissues, IKr. Melk2 channels exhibit inward rectification because of a fast C-type inactivation mechanism, but the slower rate of inactivation and the faster rate of activation results in less inward rectification than that observed in HERG channels. This characterization of Melk currents should aid in identification of native counterparts to the Elk subfamily of channels in the nervous system.

Mutations of eag, first identified in Drosophila on the basis of their leg-shaking phenotype, cause repetitive firing and enhanced transmitter release in motor neurons. The encoded EAG polypeptide is related both to voltage-gated K+ channels and to cyclic nucleotide-gated cation channels. Homology screens identified a family of eag-related channel polypeptides, highly conserved from nematodes to humans, comprising three subfamilies: EAG, ELK, and ERG. When expressed in frog oocytes, EAG channels behave as voltage-dependent, outwardly rectifying K+-selective channels. Mutations of the human eag-related gene (HERG) result in a form of cardiac arrhythmia that can lead to ventricular fibrillation and sudden death. Electrophysiological and pharmacological studies have provided evidence that HERG channels specify one component of the delayed rectifier, IKr, that contributes to the repolarization phase of cardiac action potentials. An important role or HERG channels in neuronal excitability is also suggested by the expression of these channels in brain tissue. Moreover, mutations of ERG-type channels in the Drosophila sei mutant cause temperature-induced convulsive seizures associated with aberrant bursting activity in the flight motor pathway. The in vivo function of ELK channels has not yet been established, but when these channels are expressed in frog oocytes, they display properties intermediate between those of EAG- and ERG-type channels. Coexpression of the K+-channel b subunit encoded by Hk with EAG in oocytes dramatically increases current amplitude and also affects the gating and modulation of these currents. Biochemical evidence indicates a direct physical interaction between EAG and HK proteins. Overall, these studies highlight the diverse properties of the eag family of K+ channels, which are likely to subserve diverse functions in vivo.

Clathrin-mediated endocytosis is thought to involve the activity of the clathrin adaptor protein AP180. However, the role of this protein in endocytosis in vivo remains unknown. Here, we show that a mutation that eliminates an AP180 homolog (LAP) in Drosophila severely impairs the efficiency of synaptic vesicle endocytosis and alters the normal localization of clathrin in nerve terminals. Most importantly, the size of both synaptic vesicles and quanta is significantly increased in lap mutants. These results provide novel insights into the molecular mechanism of endocytosis and reveal a role for AP180 in regulating vesicle size through a clathrin-dependent reassembly process.

The neuronal SNARE complex is formed via the interaction of synaptobrevin with syntaxin and SNAP-25. Purified SNARE proteins assemble spontaneously, while disassembly requires the ATPase NSF. Cycles of assembly and disassembly have been proposed to drive lipid bilayer fusion. However, this hypothesis remains to be tested in vivo. We have isolated a Drosophila temperature-sensitive paralytic mutation in syntaxin that rapidly blocks synaptic transmission at nonpermissive temperatures. This paralytic mutation specifically and selectively decreases binding to synaptobrevin and abolishes assembly of the 7S SNARE complex. Temperature-sensitive paralytic mutations in NSF (comatose) also block synaptic transmission, but over a much slower time course and with the accumulation of syntaxin and SNARE complexes on synaptic vesicles. These results provide in vivo evidence that cycles of assembly and disassembly of SNARE complexes drive membrane trafficking at synapses.

Assembly of K channel alpha subunits of the Shaker (Sh) family occurs in a subfamily specific manner. It has been suggested that subfamily specificity also applies in the association of beta subunits with Sh channels (Rhodes, K. J., Keilbaugh, S. A., Barrezueta, N. X., Lopez, K. L., and Trimmer, J. S. (1995) J. Neurosci. 15, 5360-5371; Sewing, S., Roeper, J. and Pongs, O. (1996) Neuron 16, 455-463; Yu, W., Xu, J., and Li, M. (1996) Neuron 16, 441-453). Here we show that the Drosophila beta subunit homologue Hyperkinetic (Hk) associates with members of the ether go-go (eag), as well as Sh, families. Anti-EAG antibody coprecipitates EAG and HK indicating a physical association between proteins. Heterologously expressed Hk dramatically increases the amplitudes of eag currents and also affects gating and modulation by progesterone. Through their ability to interact with a range of alpha subunits, the beta subunits of voltage-gated K channels are likely to have a much broader impact on the signaling properties of neurons and muscle fibers than previously suggested.

The steroid hormone ecdysone directs Drosophila metamorphosis via three heterodimeric receptors that differ according to which of three ecdysone receptor isoforms encoded by the EcR gene (EcR-A, EcR-B1, or EcR-B2) is activated by the orphan nuclear receptor USP. We have identified and molecularly mapped two classes of EcR mutations: those specific to EcR-B1 that uncouple metamorphosis, and embryonic-lethal mutations that map to common sequences encoding the DNA- and ligand-binding domains. In the larval salivary gland, loss of EcR-B1 results in loss of activation of ecdysone-induced genes. Comparable transgenic expression of EcR-B1, EcR-B2, and EcR-A in these mutant glands results, respectively, in full, partial, and no repair of that loss.

The Drosophila para sodium channel α subunit was expressed in Xenopus oocytes alone and in combination with tipE, a putative Drosophila sodium channel accessory subunit. Coexpression of tipE with para results in elevated levels of sodium currents and accelerated current decay. Para/TipE sodium channels have biophysical and pharmacological properties similar to those of native channels. However, the pharmacology of these channels differs from that of vertebrate sodium channels: (a) toxin II from Anemonia sulcata, which slows inactivation, binds to Para and some mammalian sodium channels with similar affinity (Kd ≅ 10 nM), but this toxin causes a 100-fold greater decrease in the rate of inactivation of Para/TipE than of mammalian channels; (b) Para sodium channels are >10-fold more sensitive to block by tetrodotoxin; and (c) modification by the pyrethroid insecticide permethrin is >100-fold more potent for Para than for rat brain type IIA sodium channels. Our results suggest that the selective toxicity of pyrethroid insecticides is due at least in part to the greater affinity of pyrethroids for insect sodium channels than for mammalian sodium channels.

Traumatic brain injury (TBI) is a substantial health issue worldwide, yet the mechanisms responsible for its complex spectrum of pathologies remains largely unknown. To investigate the mechanisms underlying TBI pathologies, we developed a model of TBI in Drosophila melanogaster. The model allows us to take advantage of the wealth of experimental tools available in flies. Closed head TBI was inflicted with a mechanical device that subjects flies to rapid acceleration and deceleration. Similar to humans with TBI, flies with TBI exhibited temporary incapacitation, ataxia, activation of the innate immune response, neurodegeneration, and death. Our data indicate that TBI results in death shortly after a primary injury only if the injury exceeds a certain threshold and that age and genetic background, but not sex, substantially affect this threshold. Furthermore, this threshold also appears to be dependent on the same cellular and molecular mechanisms that control normal longevity. This study demonstrates the potential of flies for providing key insights into human TBI that may ultimately provide unique opportunities for therapeutic intervention.

A growing body of evidence in humans implicates chronic activation of the innate immune response in the brain as a major cause of neuropathology in various neurodegenerative conditions, although the mechanisms remain unclear. In an unbiased genetic screen for mutants exhibiting neurodegeneration in Drosophila, we have recovered a mutation of dnr1 (defense repressor 1), a negative regulator of the Imd (immune deficiency) innate immune-response pathway. dnr1 mutants exhibit shortened lifespan and progressive, age-dependent neuropathology associated with activation of the Imd pathway and elevated expression of AMP (antimicrobial peptide) genes. To test the hypothesis that overactivation of innate immune-response pathways in the brain is responsible for neurodegeneration, we demonstrated that direct bacterial infection in the brain of wild-type flies also triggers neurodegeneration. In both cases, neurodegeneration is dependent on the NF-κB transcription factor, Relish. Moreover, we found that neural overexpression of individual AMP genes is sufficient to cause neurodegeneration. These results provide a mechanistic link between innate immune responses and neurodegeneration and may have important implications for the role of neuroinflammation in human neurodegenerative diseases as well.