The role of nicotinamide mononucleotide dependent mitochondrial reactive oxygen species generation in acute brain injury

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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 contributes to neurodegeneration is unknown; and the mechanisms leading to mitochondrial failure are not well understood. Recently, it was suggested that an imbalance in mitochondrial fission/fusion dynamics can lead to neurodegeneration and brain damage. Furthermore, overactivation of nicotinamide adenine dinucleotide (NAD)+ degrading poly-ADP-ribose polymerase (PARP1) causes excessive cellular and mitochondrial NAD+ depletion resulting in impaired cell survival. We hypothesize that the nicotinamide mononucleotide (NMN) administration is inhibiting the post-ischemic neurodegeneration by (a) reversing excessive mitochondrial fission via stimulation of mitochondrial NAD+ synthesis that (b) stimulates deacetylation of mitochondrial proteins and leads to (c) reduction of mitochondrial superoxide production. Our preliminary data show that treatment of animals with NAD+ precursor NMN has dramatic neuroprotection effect, reverses the excessive mitochondrial fragmentation and increases the brain mitochondria NAD+ levels. As a downstream result NMN is decreasing mitochondrial proteins acetylation and inhibits mitochondrial reactive oxygen species (ROS) production. The primary goal of this study is to determine the mechanistic link(s) between NMN induced changes in mitochondrial NAD+ metabolism, protein acetylation, ROS generation and inhibition of fission. To address these questions, we propose to: 1. Determine the specific role of sirtuin 3 (SIRT3) in mitochondrial reactive oxygen species (ROS) production, nucleotide metabolism, mitochondrial bioenergetic functions, and dynamics. Cells will be prepared from our three transgenic animal models: (1) animals expressing mitochondria targeted enhanced yellow fluorescence protein (mito-eYFP) alone, (2) animals expressing mito-eYFP and overexpressing SIRT3 (mito-eYFP-SIRT3OE), or (3) mito-eYFP expressing SIRT3 knockout animals (mito-eYFP-SIRT3KO). The role of NMN-induced changes in mitochondrial protein acetylation on mitochondria ROS production, mitochondrial fragmentation and cell death will be determined. Cellular NAD+ metabolism, mitochondrial respiratory function, and mitochondrial fusion and fission will be analyzed and their role in NMN neuroprotection and oxygen glucose deprivation induced cell death will be determined. 2. To study the specific effect of NMN treatment on post-ischemic modulation of mitochondrial dynamics in brain, we will use our transgenic animals that will be subjected to transient forebrain ischemia and the post-ischemic alterations in neuronal mitochondrial morphometry will be examined. In addition, NMN- induced changes in NAD+ metabolism, mitochondrial protein acetylation and mitochondrial ROS generation will be determined. Additionally, NMN-induced changes in NAD+ metabolism, mitochondrial protein acetylation and mitochondrial ROS generation will be determined. Finally, we will assess the effect of NMN treatment on post-ischemic cellular and mitochondrial NAD+ metabolism and mitochondrial respiration. The significance of this work is that it proposes both mechanistic and translational approaches to unravel the mechanisms of NAD+ dependent mitochondrial ROS production, impairment in mitochondrial dynamics and determine its role in acute brain injury. Furthermore, the identification of a novel metabolic link between NAD+ catabolism, acetylation/deacetylation of mitochondrial proteins, mitochondrial ROS generation and inhibition of mitochondrial fission will identify new mechanisms for neuroprotection that could lead to the use of NMN as a therapeutic compound for acute brain injury such as global ischemia, stroke and TBI or chronic neurodegenerative disease, thus potentially have significant impact on the health of Veterans.