DESCRIPTION (provided by applicant): Impairments in mitochondrial functions have been frequently implicated in ischemic brain injury associated with cardiac arrest or stroke. However, the extent to which mitochondrial dysfunction in neurons and astrocytes contributes to neurodegeneration is unknown, and the mechanisms leading to mitochondrial failure are elusive. Recently, it was suggested that an imbalance in mitochondrial dynamics could lead to neurodegeneration and brain damage. Furthermore, over-activation of NAD+ degrading poly-ADP-ribose polymerase (PARP1) causes excessive cellular and mitochondrial NAD+ depletion resulting in detrimental effects for cell survival. We hypothesize that the activity of ADP-ribose catabolizing enzyme, NUDIX hydrolase NUDT9, leads to downstream mitochondrial GTP consumption and consequently to inhibition of GTPases that control the organelle's fusion. Since ischemic insult triggers an extensive fission of mitochondria, the ability to refuse these organelles is essential for cell viability. This notio is strongly supported by our data showing irreversible fragmentation of mitochondria in ischemia vulnerable regions, re-fusion of mitochondria in ischemia resistant brain cells, and genotoxic stress induced depletion of mitochondrial NAD+ and GTP pools. Our preliminary data also show that over-expression of mitochondrial NUDT9 isoform is aggravating cell death; the mitochondria are more sensitive to ischemic insult and cell death maturation is accelerated in transgenic animals with increased levels of NUDT9. The primary goal of this study is to determine whether neuronal or astrocytic activity of NUDT9 is a major contributor to cell death mechanisms following ischemia. To address these questions we propose to: 1. Determine the specific role of NUDT9 in mitochondrial nucleotide metabolism, mitochondrial bioenergetics functions, dynamics and cell death. The role of NUDT9 in cell death of astrocytes and neurons will be examined by utilizing pure neuronal and astrocytic cell cultures prepared from brain tissue of our new transgenic animals that conditionally express mito-eYFP and NUDT9 either in neurons or in astrocytes. These cell cultures will be exposed to oxygen-glucose deprivation (OGD) and the effect of NUDT9 on bioenergetics metabolism, mitochondrial respiratory functions, mitochondrial dynamics and cell death will be determined. In addition, cellular and mitochondrial metabolism, mitochondrial respiratory functions, and mitochondrial fusion and fission will be analyzed. Small interference RNA (siRNA)-induced knockdown of the NUDT9 gene will be used to confirm the NUDT9 effect on impairment of mitochondrial dynamics and cell death mechanisms. 2. To study the specific role of NUDT9 in post-insult impairment of mitochondrial dynamics, we will use our transgenic animals that will be subjected to transient forebrain ischemia and at designated recovery periods the alterations in mitochondrial morphometry specifically in neurons or astrocytes in brain will be examined. Finally, we will assesse the effect of NUDT9 over-expression in neurons or astrocytes on the histological and neurological outcome after ischemic insult. Similarly, we will determine the role of NUDT9 enzyme activity in the mechanism of ischemic brain damage by assessing post-ischemic histological and neurological outcome of animals pre-treated with NUDT9 targeted siRNA. This work proposes both mechanistic and translational approaches to unravel the mechanisms of impairment in neuronal and astrocytic mitochondrial dynamics and determine its role in acute brain injury. Furthermore, the identification of a novel metabolic link between NAD+ catabolism and inhibition of mitochondrial fusion will offer new protective mechanisms that could significantly impact the clinical application of NUDT9 inhibitors as therapeutic compounds for acute brain injury such as global ischemia, stroke, TBI or chronic neurodegenerative disease.

PUBLIC HEALTH RELEVANCE With the high prevalence of heart attack and stroke risk factors among Veterans, including age, it is not surprising that these diseases causing acute ischemic brain damage are extremely common in this population, with approximately 50,000 strokes per year. Stroke with heart attack are the 3rd leading cause of death and the leading cause of disability in the US, placing a great demand on VA to provide disability and long-term care. By focusing on understanding injury mechanisms that leads to mitochondrial and ultimately cellular bioenergetic failure, the research in this grant will promote the development of treatments with the ability to improve the long-term clinical outcome for stroke victims. Since cell death mechanisms triggered by ischemia are very similar to those causing brain damage due to traumatic brain injury, the new therapeutic approaches studied in this project will also have significant implication for treatment of TBI victims.