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.concussion | insect | chronic traumatic encephalopathy T raumatic brain injury (TBI) is a leading cause of neurological deficits and mortality worldwide (1, 2). TBI outcomes result from primary and secondary injuries that cause cell damage and death in the brain. Primary injuries occur during the initial impact and are triggered by external mechanical forces that deform the brain, whereas secondary injuries are triggered by cellular and molecular responses that occur over time in reaction to the primary injuries. TBI outcomes are heterogeneous in the human population owing to variation in the location and strength of primary injuries as well as genetic and environmental factors that affect the severity of primary and secondary injuries. This heterogeneity is one of the most significant barriers to the development of therapeutic interventions (3-5). Therefore, research aimed at determining the genetic and environmental factors that affect the severity of primary and secondary injuries is essential for developing treatments for TBI.To investigate the underlying cellular and molecular basis of TBI, we developed a Drosophila melanogaster model. Key advantages of flies are that (i) large numbers of animals can be rapidly and inexpensively analyzed to establish causality between injuries and outcomes, (ii) many molecular and genetic tools are available to investigate the molecules and pathways that underlie injuries, and (iii) outcomes can readily be evaluated over the entire animal lifespan. Thus, flies provide unique opportunities to understand TBI.It is reasonable to expect that TBI can be modeled in flies because Drosophila has already proved to be an extremely useful model for studying other human neurodegenerative disorders (6). In fact, research in flies has already provided novel insights into neurodegeneration, memory, and sleep, all of which are affected in human TBI (6-8)...

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

Neurodegeneration is a hallmark of the human disease ataxia-telangiectasia (A-T) that is caused by mutation of the A-T mutated (ATM) gene. We have analyzed Drosophila melanogaster ATM mutants to determine the molecular mechanisms underlying neurodegeneration in A-T. Previously, we found that ATM mutants upregulate the expression of innate immune response (IIR) genes and undergo neurodegeneration in the central nervous system. Here, we present evidence that activation of the IIR is a cause of neurodegeneration in ATM mutants. Three lines of evidence indicate that ATM mutations cause neurodegeneration by activating the Nuclear Factor-kB (NF-kB) transcription factor Relish, a key regulator of the Immune deficiency (Imd) IIR signaling pathway. First, the level of upregulation of IIR genes, including Relish target genes, was directly correlated with the level of neurodegeneration in ATM mutants. Second, Relish mutations inhibited upregulation of IIR genes and neurodegeneration in ATM mutants. Third, overexpression of constitutively active Relish in glial cells activated the IIR and caused neurodegeneration. In contrast, we found that Imd and Dif mutations did not affect neurodegeneration in ATM mutants. Imd encodes an activator of Relish in the response to gram-negative bacteria, and Dif encodes an immune responsive NF-kB transcription factor in the Toll signaling pathway. These data indicate that the signal that causes neurodegeneration in ATM mutants activates a specific NF-kB protein and does so through an unknown activator. In summary, these findings suggest that neurodegeneration in human A-T is caused by activation of a specific NF-kB protein in glial cells.

Deacetylation of histones by the SIN3 complex is a major mechanism utilized in eukaryotic organisms to repress transcription. Presumably, developmental and cellular phenotypes resulting from mutations in SIN3 are a consequence of altered transcription of SIN3 target genes. Therefore, to understand the molecular mechanisms underlying SIN3 mutant phenotypes in Drosophila, we used full-genome oligonucleotide microarrays to compare gene expression levels in wild type Drosophila tissue culture cells versus SIN3-deficient cells generated by RNA interference. Of the 13,137 genes tested, 364 were induced and 35 were repressed by loss of SIN3. The ϳ10-fold difference between the number of induced and repressed genes suggests that SIN3 plays a direct role in regulating these genes. The identified genes are distributed throughout euchromatic regions but are preferentially excluded from heterochromatic regions of Drosophila chromosomes suggesting that the SIN3 complex can only access particular chromatin structures. A number of cell cycle regulators were repressed by loss of SIN3, and functional studies indicate that repression of string, encoding the Drosophila homologue of the yeast CDC25 phosphatase, contributes to the G 2 cell cycle delay of SIN3-deficient cells. Unexpectedly, a substantial fraction of genes induced by loss of SIN3 is involved in cytosolic and mitochondrial energy-generating pathways and other genes encode components of the mitochondrial translation machinery. Increased expression of mitochondrial proteins in SIN3-deficient cells is manifested in an increase in mitochondrial mass. Thus, SIN3 may play an important role in regulating mitochondrial respiratory activity.

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.

Alternative pre-mRNA splicing is a major mechanism utilized by eukaryotic organisms to expand their protein-coding capacity. To examine the role of cell signaling in regulating alternative splicing, we analyzed the splicing of the Drosophila melanogaster TAF1 pre-mRNA. TAF1 encodes a subunit of TFIID, which is broadly required for RNA polymerase II transcription. We demonstrate that TAF1 alternative splicing generates four mRNAs, TAF1-1, TAF1-2, TAF1-3, and TAF1-4, of which TAF1-2 and TAF1-4 encode proteins that directly bind DNA through AT hooks. TAF1 alternative splicing was regulated in a tissue-specific manner and in response to DNA damage induced by ionizing radiation or camptothecin. Pharmacological inhibitors and RNA interference were used to demonstrate that ionizing-radiation-induced upregulation of TAF1-3 and TAF1-4 splicing in S2 cells was mediated by the ATM (ataxia-telangiectasia mutated) DNA damage response kinase and checkpoint kinase 2 (CHK2), a known ATM substrate. Similarly, camptothecin-induced upregulation of TAF1-3 and TAF1-4 splicing was mediated by ATR (ATM-RAD3 related) and CHK1. These findings suggest that inducible TAF1 alternative splicing is a mechanism to regulate transcription in response to developmental or DNA damage signals and provide the first evidence that the ATM/CHK2 and ATR/CHK1 signaling pathways control gene expression by regulating alternative splicing.Alternative splicing is a major mechanism utilized by higher eukaryotic organisms to regulate gene expression during development and in response to stress (8,44,48,50). In fact, 35 to 74% of human genes may encode pre-mRNAs that are alternatively spliced (10,22,23,29,34). Alternative splicing can regulate whether or not a protein is produced, or it can generate pre-mRNAs that encode proteins with distinct functions (7,17). By analogy to other gene expression-regulatory mechanisms, such as transcription, it is probable that signal transduction pathways play a widespread role in controlling alternative splicing. However, documented examples of this phenomenon are limited, and a complete pathway has not been described.One of the most thoroughly understood examples of signaldependent alternative splicing is Ras signal-induced splicing of the CD44 pre-mRNA in humans (28,32,57). The Ras GTPase and the downstream mitogen-activated protein kinase (MAPK) signaling cascade specify inclusion of exon 5 (v5) in the mature CD44 mRNA. Stimuli that activate Ras lead to activation of MAPK, which in turn phosphorylates SAM68, an RNA-binding protein that interacts with an exonic splicing silencer element within v5. Phosphorylated SAM68 is then thought to interfere with the repressive activity of hnRNP A1 and allow factors bound to a v5 exonic splicing enhancer element to enhance v5 inclusion. Signal-dependent alternative splicing has also been implicated in the regulation of cellular processes, including apoptosis and the cell cycle (44, 47, 49). For instance, many genes encoding apoptotic regulators are alternatively spliced; however, littl...

Dynamic changes in chromatin structure, induced by posttranslational modification of histones, play a fundamental role in regulating eukaryotic transcription. Here we report that histone H2B is phosphorylated at evolutionarily conserved Ser33 (H2B-S33) by the carboxyl-terminal kinase domain (CTK) of the Drosophila TFIID subunit TAF1. Phosphorylation of H2B-S33 at the promoter of the cell cycle regulatory gene string and the segmentation gene giant coincides with transcriptional activation. Elimination of TAF1 CTK activity in Drosophila cells and embryos reduces transcriptional activation and phosphorylation of H2B-S33. These data reveal that H2B-S33 is a physiological substrate for the TAF1 CTK and that H2B-S33 phosphorylation is essential for transcriptional activation events that promote cell cycle progression and development.

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. Video LinkThe video component of this article can be found at