Mitochondrial translation deficiency impairs NAD+ -mediated lysosomal acidification.

Mitochondrial translation dysfunction is associated with neurodegenerative and cardiovascular diseases. Cells eliminate defective mitochondria by the lysosomal machinery via autophagy. The relationship between mitochondrial translation and lysosomal function is unknown. In this study, mitochondrial translation-deficient hearts from p32-knockout mice were found to exhibit enlarged lysosomes containing lipofuscin, suggesting impaired lysosome and autolysosome function. These mice also displayed autophagic abnormalities, such as p62 accumulation and LC3 localization around broken mitochondria. The expression of genes encoding for nicotinamide adenine dinucleotide (NAD+ ) biosynthetic enzymes-Nmnat3 and Nampt-and NAD+ levels were decreased, suggesting that NAD+ is essential for maintaining lysosomal acidification. Conversely, nicotinamide mononucleotide (NMN) administration or Nmnat3 overexpression rescued lysosomal acidification. Nmnat3 gene expression is suppressed by HIF1α, a transcription factor that is stabilized by mitochondrial translation dysfunction, suggesting that HIF1α-Nmnat3-mediated NAD+ production is important for lysosomal function. The glycolytic enzymes GAPDH and PGK1 were found associated with lysosomal vesicles, and NAD+ was required for ATP production around lysosomal vesicles. Thus, we conclude that NAD+ content affected by mitochondrial dysfunction is essential for lysosomal maintenance.

Nmnat3 deficiency in hemolytic anemia exacerbates malaria infection.

Malaria is an infectious disease caused by Plasmodium parasites and has high mortality rates, especially among children in African and Southeast Asian countries. Patients with hemolytic anemia are suggested to adapt protective measures against malarial infection. Nicotinamide adenine dinucleotide (NAD+) is a crucial cofactor associated with numerous biological processes that maintain homeostasis in all living organisms. In a previous study, we had demonstrated that the deficiency of nicotinamide mononucleotide adenylyltransferase 3 (Nmnat3), an enzyme catalyzing NAD+ synthesis, causes hemolytic anemia accompanied by a drastic decline in the NAD+ levels in the erythrocytes. It is well known that hemolytic anemia is linked to a reduced risk of malarial infections. In the present study, we investigated whether hemolytic anemia caused by Nmnat3 deficiency is beneficial against malarial infections. We found that Nmnat3 deficiency exacerbated malarial infection and subsequently caused death. Moreover, we demonstrated that the NAD+ levels in malaria-infected Nmnat3 red blood cells significantly increased and the glycolytic flow was largely enhanced to support the rapid growth of malarial parasites. Our results revealed that hemolytic anemia induced by the deletion of Nmnat3 was harmful rather than protective against malaria.

NMNAT3 is involved in the protective effect of SIRT3 in Ang II-induced cardiac hypertrophy.

Pathological cardiac hypertrophy is a maladaptive response in a variety of organic heart disease (OHD), which is characterized by mitochondrial dysfunction that results from disturbed energy metabolism. SIRT3, a mitochondria-localized sirtuin, regulates global mitochondrial lysine acetylation and preserves mitochondrial function. However, the mechanisms by which SIRT3 regulates cardiac hypertrophy remains to be further elucidated. In this study, we firstly demonstrated that expression of SIRT3 was decreased in Angiotension II (Ang II)-treated cardiomyocytes and in hearts of Ang II-induced cardiac hypertrophic mice. In addition, SIRT3 overexpression protected myocytes from hypertrophy, whereas SIRT3 silencing exacerbated Ang II-induced cardiomyocyte hypertrophy. In particular, SIRT3-KO mice exhibited significant cardiac hypertrophy. Mechanistically, we identified NMNAT3 (nicotinamide mononucleotide adenylyltransferase 3), the rate-limiting enzyme for mitochondrial NAD biosynthesis, as a new target and binding partner of SIRT3. Specifically, SIRT3 physically interacts with and deacetylates NMNAT3, thereby enhancing the enzyme activity of NMNAT3 and contributing to SIRT3-mediated anti-hypertrophic effects. Moreover, NMNAT3 regulates the activity of SIRT3 via synthesis of mitochondria NAD. Taken together, these findings provide mechanistic insights into the negative regulatory role of SIRT3 in cardiac hypertrophy.

Subcellular Distribution of NAD+ between Cytosol and Mitochondria Determines the Metabolic Profile of Human Cells.

The mitochondrial NAD pool is particularly important for the maintenance of vital cellular functions. Although at least in some fungi and plants, mitochondrial NAD is imported from the cytosol by carrier proteins, in mammals, the mechanism of how this organellar pool is generated has remained obscure. A transporter mediating NAD import into mammalian mitochondria has not been identified. In contrast, human recombinant NMNAT3 localizes to the mitochondrial matrix and is able to catalyze NAD(+) biosynthesis in vitro. However, whether the endogenous NMNAT3 protein is functionally effective at generating NAD(+) in mitochondria of intact human cells still remains to be demonstrated. To modulate mitochondrial NAD(+) content, we have expressed plant and yeast mitochondrial NAD(+) carriers in human cells and observed a profound increase in mitochondrial NAD(+). None of the closest human homologs of these carriers had any detectable effect on mitochondrial NAD(+) content. Surprisingly, constitutive redistribution of NAD(+) from the cytosol to the mitochondria by stable expression of the Arabidopsis thaliana mitochondrial NAD(+) transporter NDT2 in HEK293 cells resulted in dramatic growth retardation and a metabolic shift from oxidative phosphorylation to glycolysis, despite the elevated mitochondrial NAD(+) levels. These results suggest that a mitochondrial NAD(+) transporter, similar to the known one from A. thaliana, is likely absent and could even be harmful in human cells. We provide further support for the alternative possibility, namely intramitochondrial NAD(+) synthesis, by demonstrating the presence of endogenous NMNAT3 in the mitochondria of human cells.

Overexpression of Nmnat3 efficiently increases NAD and NGD levels and ameliorates age-associated insulin resistance.

Nicotinamide adenine dinucleotide (NAD) is an important cofactor that regulates various biological processes, including metabolism and gene expression. As a coenzyme, NAD controls mitochondrial respiration through enzymes of the tricarboxylic acid (TCA) cycle, ß-oxidation, and oxidative phosphorylation and also serves as a substrate for posttranslational protein modifications, such as deacetylation and ADP-ribosylation by sirtuins and poly(ADP-ribose) polymerase (PARP), respectively. Many studies have demonstrated that NAD levels decrease with aging and that these declines cause various aging-associated diseases. In contrast, activation of NAD metabolism prevents declines in NAD levels during aging. In particular, dietary supplementation with NAD precursors has been associated with protection against age-associated insulin resistance. However, it remains unclear which NAD synthesis pathway is important and/or efficient at increasing NAD levels in vivo. In this study, Nmnat3 overexpression in mice efficiently increased NAD levels in various tissues and prevented aging-related declines in NAD levels. We also demonstrated that Nmnat3-overexpressing (Nmnat3 Tg) mice were protected against diet-induced and aging-associated insulin resistance. Moreover, in skeletal muscles of Nmnat3 Tg mice, TCA cycle activity was significantly enhanced, and the energy source for oxidative phosphorylation was shifted toward fatty acid oxidation. Furthermore, reactive oxygen species (ROS) generation was significantly suppressed in aged Nmnat3 Tg mice. Interestingly, we also found that concentrations of the NAD analog nicotinamide guanine dinucleotide (NGD) were dramatically increased in Nmnat3 Tg mice. These results suggest that Nmnat3 overexpression improves metabolic health and that Nmnat3 is an attractive therapeutic target for metabolic disorders that are caused by aging.