Perturbed Brain Glucose Metabolism Caused by Absent SIRT3 Activity.

Acetylation is a post-translational modification that regulates the activity of enzymes fundamentally involved in cellular and mitochondrial bioenergetic metabolism. NAD+ dependent deacetylase sirtuin 3 (SIRT3) is localized to mitochondria where it plays a key role in regulating acetylation of TCA cycle enzymes and the mitochondrial respiratory complexes. Although the SIRT3 target proteins in mitochondria have been identified, the effect of SIRT3 activity on mitochondrial glucose metabolism in the brain remains elusive. The impact of abolished SIRT3 activity on glucose metabolism was determined in SIRT3 knockout (KO) and wild type (WT) mice injected with [1,6-13C]glucose using ex vivo 13C-NMR spectroscopy. The 1H-NMR spectra and amino acid analysis showed no differences in the concentration of lactate, glutamate, alanine, succinate, or aspartate between SIRT3 KO and WT mice. However, glutamine, total creatine (Cr), and GABA were lower in SIRT3 KO brain. Incorporation of label from [1,6-13C]glucose metabolism into lactate or alanine was not affected in SIRT3 KO brain. However, the incorporation of the label into all isotopomers of glutamate, glutamine, GABA and aspartate was lower in SIRT3 KO brain, reflecting decreased activity of mitochondrial and TCA cycle metabolism in both neurons and astrocytes. This is most likely due to hyperacetylation of mitochondrial enzymes due to suppressed SIRT3 activity in the brain of SIRT3 KO mice. Thus, the absence of Sirt3 results in impaired mitochondrial oxidative energy metabolism and neurotransmitter synthesis in the brain. Since the SIRT3 activity is NAD+ dependent, these results might parallel changes in glucose metabolism under pathologic reduction in mitochondrial NAD+ pools.

Engineering CAR-NK cells to secrete IL-15 sustains their anti-AML functionality but is associated with systemic toxicities.

BACKGROUND: The prognosis of patients with recurrent/refractory acute myelogenous leukemia (AML) remains poor and cell-based immunotherapies hold promise to improve outcomes. Natural Killer (NK) cells can elicit an antileukemic response via a repertoire of activating receptors that bind AML surface ligands. NK-cell adoptive transfer is safe but thus far has shown limited anti-AML efficacy. Here, we aimed to overcome this limitation by engineering NK cells to express chimeric antigen receptors (CARs) to boost their anti-AML activity and interleukin (IL)-15 to enhance their persistence. METHODS: We characterized in detail NK-cell populations expressing a panel of AML (CD123)-specific CARs and/or IL-15 in vitro and in AML xenograft models. RESULTS: CARs with 2B4.ζ or 4-1BB.ζ signaling domains demonstrated greater cell surface expression and endowed NK cells with improved anti-AML activity in vitro. Initial in vivo testing revealed that only 2B4.ζ Chimeric Antigen Receptor (CAR)-NK cells had improved anti-AML activity in comparison to untransduced (UTD) and 4-1BB.ζ CAR-NK cells. However, the benefit was transient due to limited CAR-NK-cell persistence. Transgenic expression of secretory interleukin (sIL)-15 in 2B4.ζ CAR and UTD NK cells improved their effector function in the setting of chronic antigen simulation in vitro. Multiparameter flow analysis after chronic antigen exposure identified the expansion of unique NK-cell subsets. 2B4.ζ/sIL-15 CAR and sIL-15 NK cells maintained an overall activated NK-cell phenotype. This was confirmed by transcriptomic analysis, which revealed a highly proliferative and activated signature in these NK-cell groups. In vivo, 2B4.ζ/sIL-15 CAR-NK cells had potent anti-AML activity in one model, while 2B4.ζ/sIL-15 CAR and sIL-15 NK cells induced lethal toxicity in a second model. CONCLUSION: Transgenic expression of CD123-CARs and sIL-15 enabled NK cells to function in the setting of chronic antigen exposure but was associated with systemic toxicities. Thus, our study provides the impetus to explore inducible and controllable expression systems to provide cytokine signals to AML-specific CAR-NK cells before embarking on early-phase clinical testing.

Role of NAD+-Modulated Mitochondrial Free Radical Generation in Mechanisms of Acute Brain Injury.

It is commonly accepted that mitochondria represent a major source of free radicals following acute brain injury or during the progression of neurodegenerative diseases. The levels of reactive oxygen species (ROS) in cells are determined by two opposing mechanisms-the one that produces free radicals and the cellular antioxidant system that eliminates ROS. Thus, the balance between the rate of ROS production and the efficiency of the cellular detoxification process determines the levels of harmful reactive oxygen species. Consequently, increase in free radical levels can be a result of higher rates of ROS production or due to the inhibition of the enzymes that participate in the antioxidant mechanisms. The enzymes' activity can be modulated by post-translational modifications that are commonly altered under pathologic conditions. In this review we will discuss the mechanisms of mitochondrial free radical production following ischemic insult, mechanisms that protect mitochondria against free radical damage, and the impact of post-ischemic nicotinamide adenine mononucleotide (NAD+) catabolism on mitochondrial protein acetylation that affects ROS generation and mitochondrial dynamics. We propose a mechanism of mitochondrial free radical generation due to a compromised mitochondrial antioxidant system caused by intra-mitochondrial NAD+ depletion. Finally, the interplay between different mechanisms of mitochondrial ROS generation and potential therapeutic approaches are reviewed.

NAD+ precursor modulates post-ischemic mitochondrial fragmentation and reactive oxygen species generation via SIRT3 dependent mechanisms.

Global cerebral ischemia depletes brain tissue NAD+, an essential cofactor for mitochondrial and cellular metabolism, leading to bioenergetics failure and cell death. The post-ischemic NAD+ levels can be replenished by the administration of nicotinamide mononucleotide (NMN), which serves as a precursor for NAD+ synthesis. We have shown that NMN administration shows dramatic protection against ischemic brain damage and inhibits post-ischemic hippocampal mitochondrial fragmentation. To understand the mechanism of NMN-induced modulation of mitochondrial dynamics and neuroprotection we used our transgenic mouse models that express mitochondria targeted yellow fluorescent protein in neurons (mito-eYFP) and mice that carry knockout of mitochondrial NAD+-dependent deacetylase sirt3 gene (SIRT3KO). Following ischemic insult, the mitochondrial NAD+ levels were depleted leading to an increase in mitochondrial protein acetylation, high reactive oxygen species (ROS) production, and excessive mitochondrial fragmentation. Administration of a single dose of NMN normalized hippocampal mitochondria NAD+ pools, protein acetylation, and ROS levels. These changes were dependent on SIRT3 activity, which was confirmed using SIRT3KO mice. Ischemia induced increase in acetylation of the key mitochondrial antioxidant enzyme, superoxide dismutase 2 (SOD2) that resulted in inhibition of its activity. This was reversed after NMN treatment followed by reduction of ROS generation and suppression of mitochondrial fragmentation. Specifically, we found that the interaction of mitochondrial fission protein, pDrp1(S616), with neuronal mitochondria was inhibited in NMN treated ischemic mice. Our data thus provide a novel link between mitochondrial NAD+ metabolism, ROS production, and mitochondrial fragmentation. Using NMN to target these mechanisms could represent a new therapeutic approach for treatment of acute brain injury and neurodegenerative diseases.