Stress Pathways Induced by Volatile Anesthetics and Failure of Preconditioning in a Mitochondrial Complex I Mutant.

BACKGROUND: Carriers of mutations in the mitochondrial electron transport chain are at increased risk of anesthetic-induced neurotoxicity. To investigate the neurotoxicity mechanism and to test preconditioning as a protective strategy, this study used a Drosophila melanogaster model of Leigh syndrome. Model flies carried a mutation in ND23 (ND2360114) that encodes a mitochondrial electron transport chain complex I subunit. This study investigated why ND2360114 mutants become susceptible to lethal, oxygen-modulated neurotoxicity within 24 h of exposure to isoflurane but not sevoflurane. METHODS: This study used transcriptomics and quantitative real-time reverse transcription polymerase chain reaction to identify genes that are differentially expressed in ND2360114 but not wild-type fly heads at 30 min after exposure to high- versus low-toxicity conditions. This study also subjected ND2360114 flies to diverse stressors before isoflurane exposure to test whether isoflurane toxicity could be diminished by preconditioning. RESULTS: The ND2360114 mutation had a greater effect on isoflurane- than sevoflurane-mediated changes in gene expression. Isoflurane and sevoflurane did not affect expression of heat shock protein (Hsp) genes (Hsp22, Hsp27, and Hsp68) in wild-type flies, but isoflurane substantially increased expression of these genes in ND2360114 mutant flies. Furthermore, isoflurane and sevoflurane induced expression of oxidative (GstD1 and GstD2) and xenobiotic (Cyp6a8 and Cyp6a14) stress genes to a similar extent in wild-type flies, but the effect of isoflurane was largely reduced in ND2360114 flies. In addition, activating stress response pathways by pre-exposure to anesthetics, heat shock, hyperoxia, hypoxia, or oxidative stress did not suppress isoflurane-induced toxicity in ND2360114 mutant flies. CONCLUSIONS: Mutation of a mitochondrial electron transport chain complex I subunit generates differential effects of isoflurane and sevoflurane on gene expression that may underlie their differential effects on neurotoxicity. Additionally, the mutation produces resistance to preconditioning by stresses that protect the brain in other contexts. Therefore, complex I activity modifies molecular and physiologic effects of anesthetics in an anesthetic-specific manner.

The Serial Anesthesia Array for the High-Throughput Investigation of Volatile Agents Using Drosophila melanogaster.

Volatile general anesthetics (VGAs) are used worldwide on millions of people of all ages and medical conditions. High concentrations of VGAs (hundreds of micromolar to low millimolar) are necessary to achieve a profound and unphysiological suppression of brain function presenting as "anesthesia" to the observer. The full spectrum of the collateral effects triggered by such high concentrations of lipophilic agents is not known, but interactions with the immune-inflammatory system have been noted, although their biological significance is not understood. To investigate the biological effects of VGAs in animals, we developed a system termed the serial anesthesia array (SAA) to exploit the experimental advantages offered by the fruit fly (Drosophila melanogaster). The SAA consists of eight chambers arranged in series and connected to a common inflow. Some parts are available in the lab, and others can be easily fabricated or purchased. A vaporizer, which is necessary for the calibrated administration of VGAs, is the only commercially manufactured component. VGAs constitute only a small percentage of the atmosphere flowing through the SAA during operation, as the bulk (typically over 95%) is carrier gas; the default carrier is air. However, oxygen and any other gases can be investigated. The SAA's principal advantage over prior systems is that it allows the simultaneous exposure of multiple cohorts of flies to exactly titrable doses of VGAs. Identical concentrations of VGAs are achieved within minutes in all the chambers, thus providing indistinguishable experimental conditions. Each chamber can contain from a single fly to hundreds of flies. For example, the SAA can simultaneously examine eight different genotypes or four genotypes with different biological variables (e.g., male vs. female, old vs. young). We have used the SAA to investigate the pharmacodynamics of VGAs and their pharmacogenetic interactions in two experimental fly models associated with neuroinflammation-mitochondrial mutants and traumatic brain injury (TBI).

Mutations in Complex I of the Mitochondrial Electron-Transport Chain Sensitize the Fruit Fly (Drosophila melanogaster) to Ether and Non-Ether Volatile Anesthetics.

The mitochondrial electron transport chain (mETC) contains molecular targets of volatile general anesthetics (VGAs), which places carriers of mutations at risk for anesthetic complications. The ND-2360114 and mt:ND2del1 lines of fruit flies (Drosophila melanogaster) that carry mutations in core subunits of Complex I of the mETC replicate numerous characteristics of Leigh syndrome (LS) caused by orthologous mutations in mammals and serve as models of LS. ND-2360114 flies are behaviorally hypersensitive to volatile anesthetic ethers and develop an age- and oxygen-dependent anesthetic-induced neurotoxicity (AiN) phenotype after exposure to isoflurane but not to the related anesthetic sevoflurane. The goal of this paper was to investigate whether the alkane volatile anesthetic halothane and other mutations in Complex I and in Complexes II-V of the mETC cause AiN. We found that (i) ND-2360114 and mt:ND2del1 were susceptible to toxicity from halothane; (ii) in wild-type flies, halothane was toxic under anoxic conditions; (iii) alleles of accessory subunits of Complex I predisposed to AiN; and (iv) mutations in Complexes II-V did not result in an AiN phenotype. We conclude that AiN is neither limited to ether anesthetics nor exclusive to mutations in core subunits of Complex I.

Lissencephaly-1 mutations enhance traumatic brain injury outcomes in Drosophila.

Traumatic brain injury (TBI) outcomes vary greatly among individuals, but most of the variation remains unexplained. Using a Drosophila melanogaster TBI model and 178 genetically diverse lines from the Drosophila Genetic Reference Panel (DGRP), we investigated the role that genetic variation plays in determining TBI outcomes. Following injury at 20-27 days old, DGRP lines varied considerably in mortality within 24 h ("early mortality"). Additionally, the disparity in early mortality resulting from injury at 20-27 vs 0-7 days old differed among DGRP lines. These data support a polygenic basis for differences in TBI outcomes, where some gene variants elicit their effects by acting on aging-related processes. Our genome-wide association study of DGRP lines identified associations between single nucleotide polymorphisms in Lissencephaly-1 (Lis-1) and Patronin and early mortality following injury at 20-27 days old. Lis-1 regulates dynein, a microtubule motor required for retrograde transport of many cargoes, and Patronin protects microtubule minus ends against depolymerization. While Patronin mutants did not affect early mortality, Lis-1 compound heterozygotes (Lis-1x/Lis-1y) had increased early mortality following injury at 20-27 or 0-7 days old compared with Lis-1 heterozygotes (Lis-1x/+), and flies that survived 24 h after injury had increased neurodegeneration but an unaltered lifespan, indicating that Lis-1 affects TBI outcomes independently of effects on aging. These data suggest that Lis-1 activity is required in the brain to ameliorate TBI outcomes through effects on axonal transport, microtubule stability, and other microtubule proteins, such as tau, implicated in chronic traumatic encephalopathy, a TBI-associated neurodegenerative disease in humans.

Anesthetic Preconditioning of Traumatic Brain Injury Is Ineffective in a Drosophila Model of Obesity.

We tested the hypothesis that obesity influences the pharmacodynamics of volatile general anesthetics (VGAs) by comparing effects of anesthetic exposure on mortality from traumatic brain injury (TBI) in lean and obese Drosophila melanogaster We induced TBI with a high-impact trauma device. Starvation-selection over multiple generations resulted in an obese phenotype (SS flies). Fed flies served as lean controls (FC flies). Adult (1-8-day-old) SS and FC flies were exposed to equianesthetic doses of isoflurane or sevoflurane either before or after TBI. The principal outcome was percent mortality 24 hours after injury, expressed as the Mortality Index at 24 hours (MI24). TBI resulted in a lower MI24 in FC than in SS flies [21 (2.35) and 57.8 (2.14), respectively n = 12, P = 0.0001]. Pre-exposure to isoflurane or sevoflurane preconditioned FC flies to TBI, reducing the risk of death to 0.53 (0.25 to 1.13) and 0.82 (0.43 to 1.58), respectively, but had no preconditioning effect in SS flies. Postexposure to isoflurane or sevoflurane increased the risk of death in SS flies, but only postexposure to isoflurane increased the risk in FC flies [1.39 (0.81 to 2.38)]. Thus, obesity affects the pharmacodynamics of VGAs, thwarting the preconditioning effect of isoflurane and sevoflurane in TBI. SIGNIFICANCE STATEMENT: Inadvertent preconditioning in models of traumatic brain injury (TBI) is a recognized confounder. The findings in a fruit fly (Drosophila melanogaster) model of closed-head TBI indicate that anesthetic pharmacodynamics are profoundly affected by obesity. Specifically, obesity thwarts the brain-protective effect of anesthetic preconditioning. This finding is important for experimental studies of TBI and supports the versatility of the fruit fly as a model for the exploration of anesthetic pharmacodynamics in a wide parameter space.

Beta-blockers reduce intestinal permeability and early mortality following traumatic brain injury in Drosophila.

Traumatic brain injury (TBI) frequently leads to non-neurological consequences such as intestinal permeability. The beta-blocker drug labetalol, which inhibits binding of catecholamine neurotransmitters to adrenergic receptors, reduces intestinal permeability in a rat TBI model. Using a Drosophila melanogaster TBI model, we previously found a strong positive correlation between intestinal permeability and mortality within 24 hours of TBI in a standard laboratory line (w1118 ) and across genetically diverse inbred lines from the Drosophila Genetic Reference Panel (DGRP). Here, we report that feeding injured w1118 flies the beta-blockers labetalol and metoprolol reduced intestinal permeability and mortality. Additionally, metoprolol reduced intestinal permeability when 18 DGRP fly lines were analyzed in aggregate, but neither beta-blocker affected mortality. These data indicate that the mechanism underlying disruption of the intestinal barrier by adrenergic signaling following TBI is conserved between humans and flies and that mortality following TBI in flies is not strictly dependent on disruption of the intestinal barrier. Thus, the fly TBI model is useful for shedding light on the mechanism and consequences of adrenergic signaling between the brain and intestine following TBI in humans.

Ketogenic diet reduces early mortality following traumatic brain injury in Drosophila via the PPARγ ortholog Eip75B.

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.

Effects of blunt force injuries in third-instar Drosophila larvae persist through metamorphosis and reduce adult lifespan.

Blunt force injuries are a significant cause of disability and death worldwide. Here, we describe a Drosophila melanogaster model of blunt force injury that can be used to investigate cellular and molecular mechanisms that underlie the short-term and long-term effects of injuries sustained at a juvenile stage of development. Injuries inflicted in late third-instar larvae using the spring-based High-Impact Trauma (HIT) device robustly activated the humoral defense response process of melanization and caused larval and pupal lethality. Additionally, adults that developed from injured larvae had reduced lifespans, indicating that cellular and molecular mechanisms activated by blunt force injuries in larvae persist through metamorphosis and adult development. Previously, the HIT device has been used to investigate genetic and environmental factors underlying mechanisms that contribute to consequences of blunt force injuries incurred in adult flies. This work expands use of the HIT device to a juvenile stage of development, offering the opportunity to investigate whether the consequences of blunt force injuries involve different factors and mechanisms at different stages of development.

Survival Following Traumatic Brain Injury in Drosophila Is Increased by Heterozygosity for a Mutation of the NF-κB Innate Immune Response Transcription Factor Relish.

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, Reldel , by CRISPR/Cas9 editing. When heterozygous but not homozygous, the Reldel mutation reduced mortality at 24 hr after TBI and increased the lifespan of injured flies. Additionally, the effect of heterozygosity for Reldel on mortality was modulated by genetic background and diet. To identify genes that facilitate effects of Reldel on TBI outcomes, we compared genome-wide mRNA expression profiles of uninjured and injured +/+, +/Reldel , and Reldel /Reldel flies at 4 hr following TBI. Only a few genes changed expression more than twofold in +/Reldel flies relative to +/+ and Reldel /Reldel 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.

Interactions among Genetic Background, Anesthetic Agent, and Oxygen Concentration Shape Blunt Traumatic Brain Injury Outcomes in Drosophila melanogaster.

Following traumatic brain injury (TBI), the time window during which secondary injuries develop provides a window for therapeutic interventions. During this time, many TBI victims undergo exposure to hyperoxia and anesthetics. We investigated the effects of genetic background on the interaction of oxygen and volatile general anesthetics with brain pathophysiology after closed-head TBI in the fruit fly Drosophila melanogaster. To test whether sevoflurane shares genetic risk factors for mortality with isoflurane and whether locomotion is affected similarly to mortality, we used a device that generates acceleration-deceleration forces to induce TBI in ten inbred fly lines. After TBI, we exposed flies to hyperoxia alone or in combination with isoflurane or sevoflurane and quantified mortality and locomotion 24 and 48 h after TBI. Modulation of TBI-induced mortality and locomotor impairment by hyperoxia with or without anesthetics varied among fly strains and among combinations of agents. Resistance to increased mortality from hyperoxic isoflurane predicted resistance to increased mortality from hyperoxic sevoflurane but did not predict the degree of locomotion impairment under any condition. These findings are important because they demonstrate that, in the context of TBI, genetic background determines the latent toxic potentials of oxygen and anesthetics.

Mitochondrial Complex I Mutations Predispose Drosophila to Isoflurane Neurotoxicity.

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.

Loss of the Antimicrobial Peptide Metchnikowin Protects Against Traumatic Brain Injury Outcomes in Drosophila melanogaster.

Neuroinflammation is a major pathophysiological feature of traumatic brain injury (TBI). Early and persistent activation of innate immune response signaling pathways by primary injuries is associated with secondary cellular injuries that cause TBI outcomes to change over time. We used a Drosophila melanogaster model to investigate the role of antimicrobial peptides (AMPs) in acute and chronic outcomes of closed-head TBI. AMPs are effectors of pathogen and stress defense mechanisms mediated by the evolutionarily conserved Toll and Immune-deficiency (Imd) innate immune response pathways that activate Nuclear Factor kappa B (NF-κB) transcription factors. Here, we analyzed the effect of null mutations in 10 of the 14 known Drosophila AMP genes on TBI outcomes. We found that mutation of Metchnikowin (Mtk) was unique in protecting flies from mortality within the 24 h following TBI under two diet conditions that produce different levels of mortality. In addition, Mtk mutants had reduced behavioral deficits at 24 h following TBI and increased lifespan either in the absence or presence of TBI. Using a transcriptional reporter of gene expression, we found that TBI increased Mtk expression in the brain. Quantitative analysis of mRNA in whole flies revealed that expression of other AMPs in the Toll and Imd pathways as well as NF-κB transcription factors were not altered in Mtk mutants. Overall, these results demonstrate that Mtk plays an infection-independent role in the fly nervous system, and TBI-induced expression of Mtk in the brain activates acute and chronic secondary injury pathways that are also activated during normal aging.

Ageing and genetic background influence anaesthetic effects in a D. melanogaster model of blunt trauma with brain injury†.

BACKGROUND: General anaesthetics interact with the pathophysiological mechanisms of traumatic brain injury (TBI). We used a Drosophila melanogaster (fruit fly) model to test the hypothesis that ageing and genetic background modulate the effect of anaesthetics and hyperoxia on TBI-induced mortality in the context of blunt trauma. METHODS: We exposed flies to isoflurane or sevoflurane under normoxic or hyperoxic conditions and TBI, and subsequently quantified the effect on mortality 24 h after injury. To determine the effect of age on anaesthetic-induced mortality, we analysed flies at 1-8 and 43-50 days old. To determine the effect of genetic background, we performed a genome-wide association study (GWAS) analysis on a collection of young inbred, fully sequenced lines. RESULTS: Exposure to anaesthetics and hyperoxia differentially affected mortality in young and old flies. Pre-exposure of young but not old flies to anaesthetics reduced mortality. Post-exposure selectively increased mortality. For old but not young flies, hyperoxia enhanced the effect on mortality of post-exposure to isoflurane but not to sevoflurane. Post-exposure to isoflurane in hyperoxia increased the mortality of young fly lines in the Drosophila Genetic Reference Panel collection to different extents. GWAS analysis of these data identified single nucleotide polymorphisms in genes involved in cell water regulation and oxygen sensing as being associated with the post-exposure effect on mortality. CONCLUSIONS: Ageing and genetic background influence the effects of volatile general anaesthetics and hyperoxia on mortality in the context of traumatic brain injury. Polymorphisms in specific genes are identified as potential causes of ageing and genetic effects.

Anesthetics Influence Mortality in a Drosophila Model of Blunt Trauma With Traumatic Brain Injury.

BACKGROUND: Exposure to anesthetics is common in the majority of early survivors of life-threatening injuries. Whether and to what degree general anesthetics influence outcomes from major trauma is unknown. Potential confounding effects of general anesthetics on outcome measures are usually disregarded. We hypothesized that exposure to isoflurane or sevoflurane modulates the outcome from blunt trauma with traumatic brain injury (bTBI). METHODS: We tested the hypothesis in a novel model of bTBI implemented in Drosophila melanogaster. Fruit flies of the standard laboratory strain w were cultured under standard conditions. We titrated the severity of bTBI to a mortality index at 24 hours (MI24) of approximately 20% under control conditions. We administered standard doses of isoflurane and sevoflurane before, before and during, or after bTBI and measured the resulting MI24. We report the MI24 as mean ± standard deviation. RESULTS: Isoflurane or sevoflurane administered for 2 hours before bTBI reduced the MI24 from 22.3 ± 2.6 to 10.4 ± 1.8 (P < 10, n = 12) and from 19.3 ± 0.9 to 8.9 ± 1.1 (P < .0001, n = 8), respectively. In contrast, administration of isoflurane after bTBI increased the MI24 from 18.5% ± 4.3% to 25.3% ± 9.1% (P = .0026, n = 22), while sevoflurane had no effect (22.4 ± 7.1 and 21.5 ± 5.8, n = 22). CONCLUSIONS: In a whole animal model of bTBI, general anesthetics were not indifferent with respect to early mortality. Therefore, collateral effects of general anesthetics should be considered in the interpretation of results obtained in vertebrate trauma models. Invertebrate model organisms can serve as a productive platform to interrogate anesthetic targets that mediate collateral effects and to inform trauma research in higher organisms about the potential impact of anesthetics on outcomes.

Genetic variability affects absolute and relative potencies and kinetics of the anesthetics isoflurane and sevoflurane in Drosophila melanogaster.

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.

A pharmacological screen for compounds that rescue the developmental lethality of a Drosophila ATM mutant.

Ataxia-telangiectasia (A-T) is a neurodegenerative disease caused by mutation of the A-T mutated (ATM) gene. ATM encodes a protein kinase that is activated by DNA damage and phosphorylates many proteins, including those involved in DNA repair, cell cycle control, and apoptosis. Characteristic biological and molecular functions of ATM observed in mammals are conserved in Drosophila melanogaster. As an example, conditional loss-of-function ATM alleles in flies cause progressive neurodegeneration through activation of the innate immune response. However, unlike in mammals, null alleles of ATM in flies cause lethality during development. With the goals of understanding biological and molecular roles of ATM in a whole animal and identifying candidate therapeutics for A-T, we performed a screen of 2400 compounds, including FDA-approved drugs, natural products, and bioactive compounds, for modifiers of the developmental lethality caused by a temperature-sensitive ATM allele (ATM8) that has reduced kinase activity at non-permissive temperatures. Ten compounds reproducibly suppressed the developmental lethality of ATM8 flies, including Ronnel, which is an organophosphate. Ronnel and other suppressor compounds are known to cause mitochondrial dysfunction or to inhibit the enzyme acetylcholinesterase, which controls the levels of the neurotransmitter acetylcholine, suggesting that detrimental consequences of reduced ATM kinase activity can be rescued by inhibiting the function of mitochondria or increasing acetylcholine levels. We carried out further studies of Ronnel because, unlike the other compounds that suppressed the developmental lethality of homozygous ATM8 flies, Ronnel was toxic to the development of heterozygous ATM8 flies. Ronnel did not affect the innate immune response of ATM8 flies, and it further increased the already high levels of DNA damage in brains of ATM8 flies, but its effects were not harmful to the lifespan of rescued ATM8 flies. These results provide new leads for understanding the biological and molecular roles of ATM and for the treatment of A-T.

Age and Diet Affect Genetically Separable Secondary Injuries that Cause Acute Mortality Following Traumatic Brain Injury in Drosophila.

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.

The gut reaction to traumatic brain injury.

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 Method to Inflict Closed Head Traumatic Brain Injury in Drosophila.

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.