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

Bart van Alphen*, Samuel Stewart, Marta Iwanaszko, Fangke Xu, Eugenie Bang, Sydney Rozenfeld, Anujaianthi Ramakrishnan, Taichi Q. Itoh, Rosemary I. Braun, Ravi Allada

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract

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, which we term Drosophila Closed Head Injury (dCHI), where well-controlled, non-penetrating strikes are directly 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. To discover novel mediators of TBI, we used glial targeted translating ribosome affinity purification in combination with RNA sequencing. We detected significant changes in the transcriptome at various times after TBI including in genes involved in innate immunity within 24 hours after TBI. 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 and found that while lethality effects were still evident, changes in sleep and motor function were substantially reduced. These studies validate a new head-specific model for TBI in Drosophila and identify glial immune pathways as candidate in vivo mediators of TBI effects.Traumatic brain injury (TBI) is one of the leading causes of death and disability in the developed world [1-3]. Yet the underlying mechanisms that lead to long term physical, emotional, and cognitive impairment remain unclear.Unlike in most forms of trauma, a large percentage of people killed by traumatic brain injuries do not die immediately but rather days or weeks after the insult [4]. TBI consists of a primary and a secondary phase. The primary brain injury is the result of an external mechanical force, resulting in damaged blood vessels, axonal shearing [5], cell death, disruption of the blood– brain barrier, edema, and the release of damage associated molecular patterns (DAMPs) and excitotoxic agents [6]. In response, local glia and infiltrating immune cells upregulate cytokines (tumor necrosis factor α) and interleukins (IL-6 and IL-1β) that drive post-traumatic neuroinflammation [7-10]. This secondary injury develops over a much longer time course, ranging from hours to months after the initial injury and is the result of a complex cascade of metabolic, cellular and molecular processes [11-13]. Neuroinflammation is beneficial when it is promoting clearance of debris and regeneration [14] but can become harmful, mediating neuronal death, progressive neurodegeneration, and neurodegenerative disorders [15-18]. The mechanisms underlying these opposing outcomes are largely unknown, but are thought to depend of the location and timing of the neuroinflammatory response [19, 20]. It remains to be determined what the relative roles of TBI-induced neuroinflammation and other TBI-induced changes are in mediating short and long-term impairments in brain function in vivo.To study the mechanisms that mediate TBI pathology in vivo over time, we employ the fruit fly Drosophila melanogaster, a model organism well suited to understanding the in vivo genetics of brain injury. Despite considerable morphological differences between flies and mammals, the fly brain operates on similar principles through a highly conserved repertoire of neuronal signaling proteins, including a large number of neuronal cell adhesion receptors, synapse-organizing proteins, ion channels and neurotransmitter receptors, and synaptic vesicle-trafficking proteins [21]. This homology makes Drosophila a fruitful model to study neurodegenerative disorders [22], including ALS [23], Alzheimer’s disease [24], Huntington’s disease [25] and Parkinson’s disease [26].Trauma-induced changes in glial gene expression are a highly conserved feature of both mammalian [27, 28] and Drosophila glia [29-32] (reviewed in [33]). In Drosophila, glia are able to perform immune-related functions [32, 34]. Ensheathing glia can act as phagocytes and contribute to the clearance of degenerating axons from the fly brain [29, 31, 35]. The Drosophila innate immune system is highly conserved with that of mammals and consists primarily of the Toll, Immunodeficiency (Imd) and Janus Kinase protein and the Signal Transducer and Activator of Transcription (JAK-STAT) pathways, which together combat fungal and bacterial infections [36, 37]. Dysregulation of cerebral innate immune signaling in Drosophila glial cells can lead to neuronal dysfunction and degeneration [38, 39], suggesting that changes in glia cells could underlie secondary injury mechanisms in our Drosophila model of TBI.Existing Drosophila TBI models [40, 41] deliver impacts to the entire body, not just the head, and thus, one cannot definitively attribute ensuing phenotypes to TBI. To remove the confound of bodily injury, we have developed a novel, head-specific Drosophila model for TBI, Drosophila Closed Head Injury (dCHI). Here we show that by delivering precisely controlled, non-penetrating strikes to an unanesthetized fly’s head, we can induce cell death and increased mortality in a dose-dependent manner. In addition, TBI results in impaired motor control and decreased, fragmented sleep. Impaired motor control persists for many days after TBI while the sleep phenotype disappears after three days. These TBI-induced behavioral phenotypes do not occur in mutants lacking the master immune regulator NF-κB Relish (Rel), even though TBI-induced mortality is greatly induced in these mutants. In wild type flies, TBI results in changes in glial gene expression, where many immune related genes are upregulated 24 hours after injury. Together, these results establish a platform where powerful Drosophila genetics can be utilized to study the complex cascade of secondary injury mechanisms that occur after TBI in order to genetically disentangle its beneficial and detrimental effects.

Original languageEnglish (US)
JournalUnknown Journal
DOIs
StatePublished - Sep 20 2018

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)
  • Immunology and Microbiology(all)
  • Neuroscience(all)
  • Pharmacology, Toxicology and Pharmaceutics(all)

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