Role of calcineurin/NFAT signaling in traumatic brain injury

The overarching hypothesis of this grant is that excessive astrocyte activation in the hippocampus negatively modulates neurologic recovery following traumatic brain injury (TBI). Historically, it has been very difficult to assess the functional impact of activated astrocytes in intact brain, due mainly to the lack of experimental tools and reagents for directly and selectively modulating astrocyte signaling cascades. The proposed work will overcome this difficulty in a rat model of TBI using adeno-associated virus (AAV) vectors bearing the astrocyte-specific promoter, Gfa2. AAV-Gfa2 vectors carry a payload (i.e. the peptide VIVIT) that interferes with the calcineurin (CN)-dependent transcription factor NFAT (Nuclear Factor of Activated T cells), shown by our lab and others to orchestrate biochemical cascades leading to astrocyte activation. In our previous cell culture work, viral-mediated delivery of VIVIT to astrocytes was shown to prevent most morphological and biochemical features of astrocyte activation, while simultaneously protecting neurons from inflammatory and excitotoxic insults. This grant will extend these potentially important findings to an intact animal model of TBI. In Aim 1 studies, rats will receive intrahippocampal infusions of AAV-Gfa2-VIVIT eight weeks prior to the delivery of a unilateral cortical contusion injury (CCI). Rats will then be evaluated at 2, 7, and 14 days post-CCI for signs of neuroinflammation, oxidative stress, and synaptic dysfunction using biochemical and electrophysiologic techniques. Hippocampal dependent cognition will also be investigated at 14 days post injury in a subgroup of injured/infected rats using the water T maze. In Aim 2, we will test the feasibility of NFAT inhibition as a possible treatment strategy for TBI. Rats will receive a lateral CCI as described above followed immediately by intraventricular infusion of VIVIT peptides (i.e. non-viral). The same outcome measures used in Aim 1 will be assessed at 2, 7, and 14 days post-CCI. Based on our earlier work, we predict that AAV-Gfa2-VIVIT vectors (Aim 1) and post-injury administration of VIVIT peptides (Aim 2) will ameliorate neuroinflammation and oxidative stress and improve synaptic and cognitive function in injured rats. To summarize, the proposed studies use cutting-edge approaches to determine the role of astrocyte activation in acute CNS injury. The focus of this work on a novel molecular target (i.e. NFATs) may also stimulate promising new treatment strategies for neurotrauma and other neurodegenerative conditions.