Glial immune-related pathways as mediators of closed head TBI effects on behavior in Drosophila

Abstract

20In traumatic brain injury (TBI) the initial injury phase is followed by a secondary phase that 21 contributes to neurodegeneration. Yet the mechanisms leading to neuropathology in vivo remain 22 to be elucidated. To address this question, we developed a Drosophila head-specific model for 23 TBI, which we term Drosophila Closed Head Injury (dCHI), where well-controlled, non-24 penetrating strikes are directly delivered to the head of unanesthetized flies. This assay 25 recapitulates many TBI phenotypes, including increased mortality, impaired motor control, 26 fragmented sleep, and increased neuronal cell death. To discover novel mediators of TBI, we 27 used glial targeted translating ribosome affinity purification in combination with RNA 28 sequencing. We detected significant changes in the transcriptome at various times after TBI 29 including in genes involved in innate immunity within 24 hours after TBI. To test the in vivo 30 functional role of these changes, we examined TBI-dependent behavior and lethality in mutants 31 of the master immune regulator NF-κB and found that while lethality effects were still evident, 32 changes in sleep and motor function were substantially reduced. These studies validate a new 33 head-specific model for TBI in Drosophila and identify glial immune pathways as candidate in 34 vivo mediators of TBI effects. 35 36 37 Traumatic brain injury (TBI) is one of the leading causes of death and disability in the developed 38 world [1][2][3]. Yet the underlying mechanisms that lead to long term physical, emotional, and 39 cognitive impairment remain unclear. 40 41Unlike in most forms of trauma, a large percentage of people killed by traumatic brain injuries do 42 not die immediately but rather days or weeks after the insult [4]. TBI consists of a primary and a 43 secondary phase. The primary brain injury is the result of an external mechanical force, 44 resulting in damaged blood vessels, axonal shearing [5], cell death, disruption of the blood-45 brain barrier, edema, and the release of damage associated molecular patterns (DAMPs) and 46 excitotoxic agents [6]. In response, local glia and infiltrating immune cells upregulate cytokines 47 (tumor necrosis factor α) and interleukins (IL-6 and IL-1β) that drive post-traumatic 48 neuroinflammation [7][8][9][10]. This secondary injury develops over a much longer time course, 49 ranging from hours to months after the initial injury and is the result of a complex cascade of 50 metabolic, cellular and molecular processes [11][12][13]. Neuroinflammation is beneficial when it is 51 promoting clearance of debris and regeneration [14] but can become harmful, mediating 52 neuronal death, progressive neurodegeneration, and neurodegenerative disorders [15][16][17][18]. The 53 mechanisms underlying these opposing outcomes are largely unknown, but are thought to 54 depend of the location and timing of the neuroinflammatory response [19, 20]. It remains to be 55 determined what the relative roles of TBI-induced neuroinflammation and other TBI-induced 56 c...

Drosophila Exhibit Divergent Sex-Based Responses in Transcription and Motor Function After Traumatic Brain Injury

“…Previous studies have explored activation of JAK-STAT (60) in response to injury but we did not see any change in gene expression associated with this pathway. We did, however, observe changes in transcript levels for genes involved in Toll, Imd pathway and JNK pathway as seen in other Drosophila TBI models (30,57,61). Although the genetic components for activation of Toll and Imd pathways are independent, induction of antimicrobial peptide genes like, Drosomycin (Drs), Defensin increased upregulation of expression is observed in AttA, AttC, and CecA2 at 2 h. Unlike females, males have no significant transcript level changes in the antibacterial and antifungal genes assessed ( Figure 3B).…”

“…In males, however, the transcriptional response is more subtle at these early time-points. (30,47,48). TBI-related damage from secondary injuries due to activated immune response was shown previously in a similar fly model of head injury (57).…”

“…Consistent with mammalian and human TBI studies, flies subjected to rapid acceleration and impact exhibit TBI related secondary phase symptoms including innate immune response, neurodegeneration, disrupted sleep cycles and a decreased lifespan (27,28). The few studies that looked at responses to TBI in Drosophila have reported data either from one sex only (29,30), both sexes combined (31) or only studied epigenetic changes in offspring after injury in both sexes (32). Therefore, we sought to compare response to traumatic injury in both male and female fly brains.…”

“…Sex as a confounding biological variable to TBI outcome has not been widely considered in previous transcriptomic studies (81)(82)(83)(84)(85). In flies, genome-wide mRNA expression profiles were studied in whole male flies to compare age and diet related mechanisms that contribute to injuries after TBI (27,29) and transcriptional changes at 1-, 3-, and 7-days post-injury in whole heads using Translating Ribosome Affinity Purification and Sequencing (TRAP-seq) (30). We have also previously shown selective intron retention in genes associated with tricarboxylic acid (TCA) cycle 24 hr post-TBI in whole heads from male and female flies pooled together after 1-or 2-strikes (27,33).…”

“…To identify genes involved in these pathways, we used 3 ′ mRNA-Seq to study genome wide gene expression changes between control and TBI flies in both sexes. The majority of TBI data identifying transcriptional changes has focused on investigating gene profiles several hours or days post-injury (29,30,33). In our study, we collected individual brains from control and 1-, 2-, and 4-hr post-injury (single strike) time points to capture gene expression changes within the immediate timeframe of TBI which may include primary and secondary effects.…”