NAD Abstracts 8

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Increased nuclear NAD biosynthesis and SIRT1 activation prevent axonal degeneration.
(Araki et al., 2004) Download
Axonal degeneration is an active program of self-destruction that is observed in many physiological and pathological settings. In Wallerian degeneration slow (wlds) mice, Wallerian degeneration in response to axonal injury is delayed because of a mutation that results in overexpression of a chimeric protein (Wlds) composed of the ubiquitin assembly protein Ufd2a and the nicotinamide adenine dinucleotide (NAD) biosynthetic enzyme Nmnat1. We demonstrate that increased Nmnat activity is responsible for the axon-sparing activity of the Wlds protein. Furthermore, we demonstrate that SIRT1, a mammalian ortholog of Sir2, is the downstream effector of increased Nmnat activity that leads to axonal protection. These findings suggest that novel therapeutic strategies directed at increasing the supply of NAD and/or Sir2 activation may be effective for treatment of diseases characterized by axonopathy and neurodegeneration.

Loss of NAD Homeostasis Leads to Progressive and Reversible Degeneration of Skeletal Muscle.
            (Frederick et al., 2016) Download
NAD is an obligate co-factor for the catabolism of metabolic fuels in all cell types. However, the availability of NAD in several tissues can become limited during genotoxic stress and the course of natural aging. The point at which NAD restriction imposes functional limitations on tissue physiology remains unknown. We examined this question in murine skeletal muscle by specifically depleting Nampt, an essential enzyme in the NAD salvage pathway. Knockout mice exhibited a dramatic 85% decline in intramuscular NAD content, accompanied by fiber degeneration and progressive loss of both muscle strength and treadmill endurance. Administration of the NAD precursor nicotinamide riboside rapidly ameliorated functional deficits and restored muscle mass despite having only a modest effect on the intramuscular NAD pool. Additionally, lifelong overexpression of Nampt preserved muscle NAD levels and exercise capacity in aged mice, supporting a critical role for tissue-autonomous NAD homeostasis in maintaining muscle mass and function.


 

NAD+ modulates p53 DNA binding specificity and function.
            (McLure et al., 2004) Download
DNA damage induces p53 DNA binding activity, which affects tumorigenesis, tumor responses to therapies, and the toxicities of cancer therapies (B. Vogelstein, D. Lane, and A. J. Levine, Nature 408:307-310, 2000; K. H. Vousden and X. Lu, Nat. Rev. Cancer 2:594-604, 2002). Both transcriptional and transcription-independent activities of p53 contribute to DNA damage-induced cell cycle arrest, apoptosis, and aneuploidy prevention (M. B. Kastan et al., Cell 71:587-597, 1992; K. H. Vousden and X. Lu, Nat. Rev. Cancer 2:594-604, 2002). Small-molecule manipulation of p53 DNA binding activity has been an elusive goal, but here we show that NAD(+) binds to p53 tetramers, induces a conformational change, and modulates p53 DNA binding specificity in vitro. Niacinamide (vitamin B(3)) increases the rate of intracellular NAD(+) synthesis, alters radiation-induced p53 DNA binding specificity, and modulates activation of a subset of p53 transcriptional targets. These effects are likely due to a direct effect of NAD(+) on p53, as a molecule structurally related to part of NAD(+), TDP, also inhibits p53 DNA binding, and the TDP precursor, thiamine (vitamin B(1)), inhibits intracellular p53 activity. Niacinamide and thiamine affect two p53-regulated cellular responses to ionizing radiation: rereplication and apoptosis. Thus, niacinamide and thiamine form a novel basis for the development of small molecules that affect p53 function in vivo, and these results suggest that changes in cellular energy metabolism may regulate p53.

NAD⁺ metabolism: a therapeutic target for age-related metabolic disease.
            (Mouchiroud et al., 2013) Download
Nicotinamide adenine dinucleotide (NAD) is a central metabolic cofactor by virtue of its redox capacity, and as such regulates a wealth of metabolic transformations. However, the identification of the longevity protein silent regulator 2 (Sir2), the founding member of the sirtuin protein family, as being NAD⁺-dependent reignited interest in this metabolite. The sirtuins (SIRT1-7 in mammals) utilize NAD⁺ to deacetylate proteins in different subcellular compartments with a variety of functions, but with a strong convergence on optimizing mitochondrial function. Since cellular NAD⁺ levels are limiting for sirtuin activity, boosting its levels is a powerful means to activate sirtuins as a potential therapy for mitochondrial, often age-related, diseases. Indeed, supplying excess precursors, or blocking its utilization by poly(ADP-ribose) polymerase (PARP) enzymes or CD38/CD157, boosts NAD⁺ levels, activates sirtuins and promotes healthy aging. Here, we discuss the current state of knowledge of NAD⁺ metabolism, primarily in relation to sirtuin function. We highlight how NAD⁺ levels change in diverse physiological conditions, and how this can be employed as a pharmacological strategy.


Role of sirtuins in lifespan regulation is linked to methylation of nicotinamide.
            (Schmeisser et al., 2013) Download
Sirtuins, a family of histone deacetylases, have a fiercely debated role in regulating lifespan. In contrast with recent observations, here we find that overexpression of sir-2.1, the ortholog of mammalian SirT1, does extend Caenorhabditis elegans lifespan. Sirtuins mandatorily convert NAD(+) into nicotinamide (NAM). We here find that NAM and its metabolite, 1-methylnicotinamide (MNA), extend C. elegans lifespan, even in the absence of sir-2.1. We identify a previously unknown C. elegans nicotinamide-N-methyltransferase, encoded by a gene now named anmt-1, to generate MNA from NAM. Disruption and overexpression of anmt-1 have opposing effects on lifespan independent of sirtuins, with loss of anmt-1 fully inhibiting sir-2.1-mediated lifespan extension. MNA serves as a substrate for a newly identified aldehyde oxidase, GAD-3, to generate hydrogen peroxide, which acts as a mitohormetic reactive oxygen species signal to promote C. elegans longevity. Taken together, sirtuin-mediated lifespan extension depends on methylation of NAM, providing an unexpected mechanistic role for sirtuins beyond histone deacetylation.

NAD Deficiency, Congenital Malformations, and Niacin Supplementation.
            (Shi et al., 2017) Download
BACKGROUND:  Congenital malformations can be manifested as combinations of phenotypes that co-occur more often than expected by chance. In many such cases, it has proved difficult to identify a genetic cause. We sought the genetic cause of cardiac, vertebral, and renal defects, among others, in unrelated patients. METHODS:  We used genomic sequencing to identify potentially pathogenic gene variants in families in which a person had multiple congenital malformations. We tested the function of the variant by using assays of in vitro enzyme activity and by quantifying metabolites in patient plasma. We engineered mouse models with similar variants using the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 system. RESULTS:  Variants were identified in two genes that encode enzymes of the kynurenine pathway, 3-hydroxyanthranilic acid 3,4-dioxygenase (HAAO) and kynureninase (KYNU). Three patients carried homozygous variants predicting loss-of-function changes in the HAAO or KYNU proteins (HAAO p.D162*, HAAO p.W186*, or KYNU p.V57Efs*21). Another patient carried heterozygous KYNU variants (p.Y156* and p.F349Kfs*4). The mutant enzymes had greatly reduced activity in vitro. Nicotinamide adenine dinucleotide (NAD) is synthesized de novo from tryptophan through the kynurenine pathway. The patients had reduced levels of circulating NAD. Defects similar to those in the patients developed in the embryos of Haao-null or Kynu-null mice owing to NAD deficiency. In null mice, the prevention of NAD deficiency during gestation averted defects. CONCLUSIONS:  Disruption of NAD synthesis caused a deficiency of NAD and congenital malformations in humans and mice. Niacin supplementation during gestation prevented the malformations in mice. (Funded by the National Health and Medical Research Council of Australia and others.).

 


References

Araki, T, Y Sasaki, and J Milbrandt (2004), ‘Increased nuclear NAD biosynthesis and SIRT1 activation prevent axonal degeneration.’, Science, 305 (5686), 1010-13. PubMed: 15310905
Frederick, DW, et al. (2016), ‘Loss of NAD Homeostasis Leads to Progressive and Reversible Degeneration of Skeletal Muscle.’, Cell Metab, 24 (2), 269-82. PubMed: 27508874
McLure, KG, M Takagi, and MB Kastan (2004), ‘NAD+ modulates p53 DNA binding specificity and function.’, Mol Cell Biol, 24 (22), 9958-67. PubMed: 15509798
Mouchiroud, L, RH Houtkooper, and J Auwerx (2013), ‘NAD⁺ metabolism: a therapeutic target for age-related metabolic disease.’, Crit Rev Biochem Mol Biol, 48 (4), 397-408. PubMed: 23742622
Schmeisser, K, et al. (2013), ‘Role of sirtuins in lifespan regulation is linked to methylation of nicotinamide.’, Nat Chem Biol, 9 (11), 693-700. PubMed: 24077178
Shi, H, et al. (2017), ‘NAD Deficiency, Congenital Malformations, and Niacin Supplementation.’, N Engl J Med, 377 (6), 544-52. PubMed: 28792876