Dr. Singhal's long-term goal is to be a VA physician investigator, elucidating mechanisms of neuronal bioenergetics in order to improve outcomes for veterans with acute brain injuries and other neurological diseases. The mechanisms linking cellular bioenergetics and cell death in ischemic brain diseases such as stroke are incompletely understood, and a greater understanding of these details will lead to the development of urgently needed neuroprotective agents for Veterans. During the Career Development Award-2 period, Dr. Singhal's goal is to acquire the training and implement the studies needed to understand and develop novel treatment strategies for conditions he treats in the Neurointensive care unit such as stroke. As such, he proposes a training program focused on identifying the mechanisms underlying the dramatic ischemia tolerance observed in one of nature's most resilient animals, the Arctic ground squirrel. Through comparative genomics and cell resilience-based cDNA expression screens, he discovered unique amino acid substitutions in cytoprotective proteins conferring increased resilience to metabolic stressors. The research proposed will build on his preliminary data and bring two candidate genes forward for rigorous study in neural cells using metabolic and cell death assays, visualization techniques, and biochemical measurements. He will use CRISPR-Cas9 knock-in technology to precisely edit the genes of interest, which are not fully characterized to date, and study the effect of their editing on neuronal cell resilience (Aim 1) and elucidate their cell physiologic mechanisms of action (Aim 2). Finally, he will test the candidate genes in the transient middle cerebral artery occlusion stroke model in vivo (Aim 3). The in-depth characterization using in vivo and in vitro models will establish a critical connection between the Arctic ground squirrel cytoprotective proteins and neuroprotective mechanisms, and importantly, provide valuable insights into novel drug targets. Dr. Singhal has assembled a diverse mentorship team comprised of experts in the fields of stroke, genetics, statistics, and mitochondrial physiology. Dr. Singhal's career development plan includes a clear timeline for individual tutorials with mentors and scientific advisors, hand-on experience, formal seminars, dissemination of findings, and plans for independent projects and funding. The mentored research and training in gene editing, neurovascular biology, mitochondrial physiology, and statistics described in this proposal will complement Dr. Singhal's training and facilitate his goal of launching an independent research career at the VA.