Fellowship for Bolton: Role of Microglia in Acute Pathology of Repeated Concussion and CTE Development

2. Specific Aims Approximately 1.7-3.8million people in the United States sustain a traumatic brain injury (TBI) every year, and 70-90% of reported cases are considered “mild”. The likelihood of repeated mild TBI is increased for individuals involved in high risk activities including military combat and many athletics. However, much remains unknown about mechanisms of damage underlying repeated concussion and how these mechanisms determine the vulnerable interval for repeated TBI. Our goal is to bridge that knowledge gap using an innovative, mechanism-oriented approach with a rodent model. Animal models of repetitive mild TBI (rmTBI) have recapitulated persistent axonal injury and microglial activation as seen with rmTBI in humans. Increasing evidence points to inflammatory responses as a mediator of not only acute pathology but also chronic neurodegeneration initiated by rmTBI. My preliminary data in a mouse model show that concussions repeated at a 24h interval result in acute microglial activation accompanied by localized axonal injury and neurodegeneration. My overarching goal is to highlight the mechanisms underlying the pathology of repeated concussion, in order to elucidate potential therapeutic interventions. In Aim 1, I will use two experimental approaches to manipulate posttraumatic microglial activation to determine the causative role of microglia in the neuropathology of rmTBI. First, progranulin-deficient (GRN KO) mice, which exhibit a greatly exacerbated microglial response after penetrating brain injury, will be used to amplify microglial reactivity after rmTBI. Second, treatment with ibuprofen, a widely used anti-inflammatory drug, will be used to attenuate microglial reactivity after rmTBI. In Aim 2, I will examine long-term consequences of rmTBI, again utilizing GRN KO mice as a tool to facilitate early onset of neurodegenerative changes triggered by concussion. In the absence of any CNS insult, GRN KO mice show age-related increases in microglial activation, which precede late onset (12-18mo) accumulation of phosphorylated TAR DNA binding protein-43 (pTDP43) and deficits in learning and memory (18mo). pTDP43 accumulation is a signature pathology in Chronic Traumatic Encephalopathy (CTE), a neurodegenerative disease triggered by repeated concussions. CTE is associated with memory impairment, anxiety, motor deficits, and chronic activation of microglia but is only definitively diagnosed post-mortem by the accumulation of hyper-phosphorylated tau (pTau) and pTDP43 in the brain. Despite efforts of multiple laboratories no rodent model of rmTBI has yet recapitulated the full spectrum of CTE-related neuropathology. I postulate that GRN deficiency will serve as an catalyst to accelerate the onset of CTE-like changes in mice. My overall hypothesis is that repeated concussions induce persistent microgliosis, accelerating the onset of CTE-like behavioral and histopathological consequences. Aim 1: To test the hypothesis that repeated concussion results in persistent microglial activation and modulation of post-traumatic microgliosis affects the extent of concussion-related neuropathology. Mice will be randomized to receive either sham or three repeated midline CHI spaced at 24h intervals. Wildtype (WT) mice will be compared to GRN KO mice (enhanced microgliosis) and to WT mice treated with ibuprofen (attenuated microgliosis). 1.1) Memory, motor function, and anxiety-like behaviors will be evaluated prior to injury and at multiple time points over the course of 2wks following injury. 1.2) At 2d, 7d and 2wks postinjury, immunohistochemical analysis of microglial and astrocyte activation, blood brain barrier breakdown, axonal injury, and neuronal degeneration will be performed. 1.3) At 2d after the third CHI, ELISA analysis will be used to quantify cytokine expression to profile the M1 (cytotoxic) and M2 (repair) microglial responses after rmTBI. I anticipate that exacerbated microgliosis in GRN KO mice will lead to increased neuronal damage and behavioral deficits. Reduced microglial reactivity with ibuprofen is expected to result in less neuronal damage and milder behavioral deficits, implicating microglial activation as a causative factor in rmTBI pathology. Aim 2: To test the hypothesis that chronic microglial activation after repeated concussion accelerates the onset of CTE-like pathological and behavioral consequences in mice. GRN KO and WT mice will be randomized to receive either sham or three repeated CHI at 24h intervals. 2.1) Memory, motor function and anxiety-like behaviors will be evaluated prior to and 2 and 6 mo after rmTBI. 2.2) Immunohistochemical analysis of inflammation, cell death, pTDP43, and pTau accumulation will be performed at 2mo or 6mo post-injury. 2.3) Levels of pTDP43, pTau, and their respective kinases will be quantified by Western blot analysis. I anticipate that GRN KO mice will exhibit more prolonged microglial reactivity after rmTBI, more persistent behavioral deficits and earlier appearance of pTDP43 and pTau than will sham GRN KO or injured WT mice. These studies will provide me with an outstanding training in quantitative histological and protein assays, behavioral testing, in vivo TBI modeling and clinical and experimental neuropathology. Successful completion of these aims will provide the first evidence implicating microglia in the acute and chronic pathology of rmTBI and will establish the first model of CTE inclusive of pTDP43 pathology.