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.

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.

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.

Developmental arrest of Drosophila larvae elicits presynaptic depression and enables prolonged studies of neurodegeneration.

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.

A Novel Mutation in Brain Tumor Causes Both Neural Over-Proliferation and Neurodegeneration in Adult Drosophila.

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.

Neurodegeneration and locomotor dysfunction in Drosophila scarlet mutants.

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.

Mito-Nuclear Interactions Affecting Lifespan and Neurodegeneration in a Drosophila Model of Leigh Syndrome.

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.

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.

NF-κB Immunity in the Brain Determines Fly Lifespan in Healthy Aging and Age-Related Neurodegeneration.

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.

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.

Non-cell autonomous cell death caused by transmission of Huntingtin aggregates in Drosophila.

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.

Persistent Activation of the Innate Immune Response in Adult Drosophila Following Radiation Exposure During Larval Development.

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.

Transcellular spreading of huntingtin aggregates in the Drosophila brain.

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.

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.