Role of Astrocyte-Derived ATP Signaling in Traumatic Brain Injury

Abstract: It has become clear that astrocytes, the most abundant glial cell types in the brain play a plethora of critical roles in both normal and disease states. Under physiological conditions, astrocytes exert various homeostatic and metabolic functions. They are actively involved in the modulation of neural circuits and synaptic plasticity by releasing signaling metabolites, small peptides, and neurotransmitters. In conjunction with other glial cell types, astrocytes serve as a key immunomodulatory player in various neuropathology and brain injuries. Our previous research and recent exciting preliminary studies strongly suggest that a dynamic regulation of astrocytic exocytosis of ATP is key to the modulatory roles of astrocytes in healthy and disease conditions. On one hand, under normal conditions, exocytosis of ATP in astrocytes is important to maintain normal activity of neuronal pathways. For instance, our lab has recently shown that astrocytic exocytosis of ATP is critical in supporting dopamine release and motivation for reward. On the other hand, and maybe more importantly, excess ATP exocytosis from astrocytes may exacerbate neuroinflammation and cellular dysfunction in response to chronic neuropathology or secondary to acute brain injuries. In agreement with this, our exploratory studies show a beneficial effect on cognitive performance following controlled cortical impact (CCI) in mice with genetic ablation of astrocytic ATP exocytosis machinery. These findings strongly indicate an astrocyte-dependent protective mechanism critical for the functional preservation after CCI in the mice lacking astrocytic exocytosis of ATP. Here, we hypothesize that excess ATP release via astrocytic exocytosis is detrimental for functional recovery following traumatic brain injury, and the loss of astrocytic exocytosis of ATP provides beneficial effects by tampering neuro-inflammation, clearing cellular debris, and promoting functional recovery after traumatic brain injury. To release ATP via exocytosis, cytosolic ATP is loaded to secretory vesicles by the vesicular nucleotide transporter (Vnut). We have developed a unique conditional knock-out mouse, in which the Vnut gene is floxed to allow astrocyte-specific Cre- dependent deletion. With the strong support from our excellent research team, specific genetic models, and a collection of preliminary data, we will test our central hypothesis in 3 specific aims. Aim 1. Assess the impact of astrocytic exocytosis of ATP on cortical lesion and cognitive function following controlled cortical impact. Based on our preliminary findings that loss of Vnut in astrocytes significantly improves cognitive function after CCI, both male and female astrocyte-specific Vnut KO (iA- VnutKO) mice and their Vnutf/f littermates will be subjected to controlled cortical impact or receive sham control procedure at 12 weeks of age. We will assess both locomotor and cognitive behavior followed by measurement of the lesion volume and cell death in these mice at 3 days, 2 weeks and 4 weeks after injury. We carefully selected these time points for analysis to understand the dynamics of the responses to injury. Aim 2. Examine the role of astrocytic exocytosis of ATP on glial reactivity and neuroinflammation following controlled cortical impact. Glial cell reactivity and the persistent neuroinflammation play critical roles in leading to secondary injuries after TBI. Excess extracellular ATP triggers astrocytic exocytosis of ATP, which creates a vicious cycle for pro-inflammatory microenvironment. In this aim, we will assess whether loss of Vnut in astrocytes effectively blocks this vicious cycle and tampers neuroinflammation after CCI. Thus, we will perform microdialysis to determine the extracellular ATP levels in the hippocampus of iA-VnutKO mice and their Vnutf/f littermates subjected to CCI or sham procedure at different time points as in Aim 1. Further immunohistological and biomedical analyses will be performed to assess the reactivity of astrocytes and microglia, and the expression of inflammatory cytokines. Aim 3. Determine the phagocytosis of degenerated neurons by Vnut-/- astrocytes following controlled cortical impact. Clearance of degenerated neurons and cell debris by glial cells has been shown to play beneficial effects at the recovery phase after CCI. Our preliminary studies show that loss of Vnut dramatically upregulates the phagocytic activity of primary cultured astrocytes by ~2 folds. Build upon these findings, we will examine the impact of Vnut deletion in astrocytes on phagocytic activity in animals following CCI. Briefly, we will use RiboTag system to specifically determine the transcriptional regulations, particularly the regulation on the phagocytic pathway in astrocytes in response to Vnut deletion and CCI. Phagocytosis of degenerated neurons by astrocytes after CCI will be determined and quantified by Fluoro-Jade and GFAP co-staining.