Traumatic brain injury (TBI) is one of the leading causes of death and disability in adult humans including service personnel and veterans. TBI promotes significant motor, cognitive and neuropsychiatric dysfunction. The effects of TBI in surviving veterans can often be seen for decades after the initial injury. However, there is no efficacious therapy to prevent post-TBI secondary brain damage and neurologic deficits. Vitamin C (ascorbate) is highly concentrated in brain and known to promote neuroprotection after acute and chronic CNS insults. However, its efficacy in TBI was not yet tested comprehensively. In preliminary studies, we observed that ascorbate treatment reduces secondary brain damage and promotes better motor and cognitive functional outcome in adult mice subjected to controlled cortical impact (CCI)- induced TBI. Based on this, we will test the hypothesis “ascorbate treatment is neuroprotective after TBI.” Epigenetic changes are known to significantly influence the gene expression and outcome after many diseases. Of particular interest, cytosine in DNA undergoes methylation to form 5-methylcytosine (5mC) which is a transcriptional silencer. 5mC will be oxidized by TET hydroxylases to form 5- hydroxymethylcytosine (5hmC) which is a transcriptional derepression mark that increases cell survival under adverse conditions. In particular, brain contains ~10 fold higher 5hmC levels than other organs of the body. Preliminary studies showed that that ascorbate treatment enhances cerebral TET activity and 5hmC levels after TBI. Hence, we further hypothesize that “the mechanism of ascorbate-induced neuroprotection after TBI is by the epigenetic modulation of 5hmC.” Aim 1: To test the neuroprotective potential of ascorbate after TBI. For any drug to be translated clinically after TBI, robust preclinical testing that includes, but not limited to, a window of therapeutic opportunity, long-term outcomes, efficacy in both sexes using a mouse CCI injury model that is rigorously established in our lab. Aim 2: To evaluate if DNA hydroxymethylation is mechanistically responsible for ascorbate neuroprotection after TBI. Understanding the mechanisms of a drug actions help fine tune the therapy as needed. Based on established work and preliminary data, we will test the functional significance of 5hmC in post-TBI pathophysiology by knockdown of TET/5hmC together with ascorbate treatment. Genomic sites where 5hmC is increased by ascorbate treatment after TBI will be mapped by hMeDIP- seq following TET knockdown. This profiling will show the ascorbate-induced (TET-dependent) gene expression that helps to understand the downstream effects of ascorbate protection. We will then analyze the effect of ascorbate therapy on post-TBI gene expression (RNA-seq) in both male and female mice. Overall, the present project will help us to identify if ascorbate minimizes secondary brain damage and neurologic dysfunction after TBI by modulating the neuroprotective epigenet...