In traumatic brain injury (TBI), the initial injury phase is followed by a secondary phase that contributes to neurodegeneration, yet the mechanisms leading to neuropathology in vivo remain to be elucidated. To address this question, we developed a Drosophila head-specific model for TBI termed Drosophila Closed Head Injury (dCHI), where well-controlled, nonpenetrating strikes are delivered to the head of unanesthetized flies. This assay recapitulates many TBI phenotypes, including increased mortality, impaired motor control, fragmented sleep, and increased neuronal cell death. TBI results in significant changes in the transcriptome, including up-regulation of genes encoding antimicrobial peptides (AMPs). To test the in vivo functional role of these changes, we examined TBI-dependent behavior and lethality in mutants of the master immune regulator NF-κB, important for AMP induction, and found that while sleep and motor function effects were reduced, lethality effects were enhanced. Similarly, loss of most AMP classes also renders flies susceptible to lethal TBI effects. These studies validate a new Drosophila TBI model and identify immune pathways as in vivo mediators of TBI effects.

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...