Originally Published: Declining NAD+ Induces a Pseudohypoxic State Disrupting Nuclear-Mitochondrial Communication during Aging Gomes, Ana P. et al. Cell , Volume 155 , Issue 7 , 1624 - 1638 DOI:
http://dx.doi.org/10.1016/j.cell.2013.11.037 Authors
Ana P. Gomes, Nathan L. Price, Alvin J.Y. Ling, Javid J. Moslehi, Magdalene K. Montgomery, Luis Rajman, James P. White, João S. Teodoro, Christiane D. Wrann, Basil P. Hubbard, Evi M. Mercken, Carlos M. Palmeira, Rafael de Cabo, Anabela P. Rolo, Nigel Turner, Eric L. Bell, David A. Sinclair
Highlights
•A specific decline in mitochondrially encoded genes occurs during aging in muscle
•Nuclear NAD+ levels regulate mitochondrial homeostasis independently of PGC-1α/β
•Declining NAD+ during aging causes pseudohypoxia, which disrupts OXPHOS function
•Raising nuclear NAD+ in old mice reverses pseudohypoxia and metabolic dysfunction
Summary
Ever since eukaryotes subsumed the bacterial ancestor of mitochondria, the nuclear and mitochondrial genomes have had to closely coordinate their activities, as each encode different subunits of the oxidative phosphorylation (OXPHOS) system. Mitochondrial dysfunction is a hallmark of aging, but its causes are debated. We show that, during aging, there is a specific loss of mitochondrial, but not nuclear, encoded OXPHOS subunits. We trace the cause to an alternate PGC-1α/β-independent pathway of nuclear-mitochondrial communication that is induced by a decline in nuclear NAD+ and the accumulation of HIF-1α under normoxic conditions, with parallels to Warburg reprogramming. Deleting SIRT1 accelerates this process, whereas raising NAD+ levels in old mice restores mitochondrial function to that of a young mouse in a SIRT1-dependent manner. Thus, a pseudohypoxic state that disrupts PGC-1α/β-independent nuclear-mitochondrial communication contributes to the decline in mitochondrial function with age, a process that is apparently reversible.
Introduction
One of the most conserved and robust phenomena in biology is a progressive decline in mitochondrial function with age, leading to a loss of cellular homeostasis and organismal health (Lanza and Nair, 2010, Wallace, 2010). There is considerable debate, however, about why mitochondrial homeostasis is disrupted in the first place. The original idea of Harman, that reactive oxygen species (ROS) from mitochondria are a primary cause of aging (Harman, 1972), has been challenged by recent studies of long-lived species and genetically altered animals (Lapointe and Hekimi, 2010).
Though most mitochondrial genes have been transferred to the nuclear genome, 13 subunits of the oxidative phosphorylation (OXPHOS) system remain, demanding functional communication between the nucleus and mitochondria to form stoichiometric OXPHOS complexes. This is mediated in large part by the peroxisome proliferator-activated receptor-γ coactivators α and β (PGC-1α and PGC1-1β), which along with NRF-1 and -2, induce nuclear-encoded proteins, such as TFAM (mitochondrial transcription factor A), that carry out the replication, transcription, and translation of mitochondrial DNA (mtDNA) (Larsson, 2010).
Mammalian sirtuins (SIRT1-7) are a conserved family of NAD+-dependent lysine-modifying acylases that control physiological responses to diet and exercise (Haigis and Sinclair, 2010). The expression of SIRT1, an NAD+-dependent deacetylase, is elevated in a number of tissues following calorie restriction (CR) (Cohen et al., 2004), an intervention that extends lifespan in diverse species. Overexpression or pharmacological activation of SIRT1 reproduces many of the health benefits of CR, including protection from metabolic decline, cardiovascular disease, cancer, and neurodegeneration (Haigis and Sinclair, 2010, Libert and Guarente, 2013). Some of the health benefits of SIRT1 have also been linked to improved mitochondrial function (Baur et al., 2006, Gerhart-Hines et al., 2007, Mouchiroud et al., 2013, Ptitsyn et al., 2006). Indeed, increased expression of neuronal SIRT1 extends mouse lifespan (Satoh et al., 2013), though its role in aging in lower organisms has been challenged (Burnett et al., 2011).
A hallmark of cancer is a shift away from OXPHOS toward anaerobic glycolysis that provides cells with sufficient substrates for biomass. This metabolic reprogramming, known as the Warburg effect (Warburg, 1956), is driven by several different pathways, including mTOR, c-Myc, and hypoxia-inducible factor 1 (HIF-1α) (Dang, 2012). Interestingly, SIRT1 increases HIF-1α transcriptional activity (Lim et al., 2010), SIRT3 destabilizes HIF-1α protein (Bell et al., 2011, Finley et al., 2011), and SIRT6 functions as a HIF-1α corepressor (Zhong et al., 2010), raising the possibility that HIF-1α may also be relevant to aging. Consistent with this, in C. elegans, Hif-1 regulates lifespan and the response to CR (Leiser and Kaeberlein, 2010). A role for HIF-1α in mammalian aging, however, has not been explored.
In this study, we provide evidence for a PGC-1α/β-independent pathway of mitochondrial regulation that plays a role in the aging process. Activity of this pathway declines during aging due to changes in nuclear NAD+ levels, causing a pseudohypoxia-driven imbalance between nuclear- and mitochondrially encoded OXPHOS subunits—a process that is prevented by CR and is reversed by raising NAD+, with implications for treating age-related diseases, including cancer.
Results
Aging Leads to a Specific Decline in Mitochondrially Encoded Genes
Aging is associated with disruption of mitochondrial homeostasis, but the underlying mechanisms are unclear. As in previous reports (Lanza and Nair, 2010), we observed a progressive, age-dependent decline in OXPHOS efficiency with age in skeletal muscle (Figures 1A and 1B ). By 22 months of age, ATP content and complex IV (COX) activity were decreased, even more so by 30 months of age. Although mtDNA content declined at both ages, the integrity of mtDNA was only lower in the 30 month olds (Figures 1C and 1D). Together with previous reports (Lapointe and Hekimi, 2010), this suggested an aging mechanism that disrupts OXPHOS prior to the accumulation of significant mtDNA damage.
A clue came from observations that the activity of OXPHOS complexes I, III, and IV decline with age, but complex II, the only complex composed exclusively of nuclear-encoded subunits, does not (Kwong and Sohal, 2000). Thus, we tested whether OXPHOS decline might be due to the specific loss of mitochondrially encoded transcripts. Mitochondrially encoded OXPHOS mRNAs (ND1, Cytb, COX1, ATP6) were all significantly lower at 22 months relative to 6 month olds, whereas those encoded by the nuclear genome (NDUFS8, SDHb, Uqcrc1, COX5, ATP5a) remained unchanged; but by 30 months of age, both the nuclear- and the mitochondrially encoded mRNAs were lower (Figures 1E). Protein levels of the mitochondrially encoded COX2 gene were decreased at 22 months, but COX4, a nuclear-encoded subunit, was only slightly lower. By 30 months, both proteins were reduced relative to young mice (Figure 1F). The mitochondrial unfolded protein response (mtUPR) has been recently linked to longevity (Durieux et al., 2011, Houtkooper et al., 2013, Minamishima et al., 2008); however, under these conditions there was no evidence of a mtUPR at 22 months of age (Figure S1A available online).
Knockout of SIRT1 Mimics Aging by Decreasing Mitochondrial, but Not Nuclear-Encoded, OXPHOS Components
We wondered whether the specific decline in mitochondrially encoded OXPHOS components in aged mice might be due, in part, to a loss of SIRT1 activity. To test this, we utilized an adult-inducible SIRT1 knockout mouse (SIRT1-iKO) (Price et al., 2012), which circumvents the developmental abnormalities of germline SIRT1 knockouts. SIRT1 was deleted at 2–4 months of age, and skeletal muscle was analyzed 2–6 months later. As expected, the mRNA levels of all 13 mitochondrially encoded OXPHOS genes and the two rRNAs were reduced in the SIRT1 iKO mice compared to wild-type controls (Figures 1G and S1B). Strikingly, there was no decrease in the expression of any of the nuclear-encoded components under fed conditions (Figure 1G). Again, protein levels of mitochondrially encoded COX2 were significantly decreased, whereas the nuclear-encoded COX4 was unaltered (Figure 1H), coincident with a decline in complex IV (COX), but not complex II (SDH), activity (Figures S1D and S1E). Similar to old mice, cellular ATP levels and mtDNA content were reduced (Figures 1I and 1J), with no apparent induction of mtUPR (Figure S1C).
Given that SIRT1 maintains mitochondrial mass by increasing PGC-1α activity, we were surprised to see that, under these basal conditions (i.e., the fed state), there was no effect of SIRT1 deletion on mitochondrial mass (Figure 1K). To understand why, we cultured SIRT1 iKO primary myoblasts and induced Cre-mediated deletion of the SIRT1 catalytic core ex vivo. After 12 hr, only the mitochondrially encoded OXPHOS mRNAs decreased (Figure 1L). Again, mtDNA content and mitochondrial membrane potential declined, with no change in mitochondrial mass (Figures 1M, S2A, and S2B). By 48 hr, mRNA from both the nuclear- and mitochondrially encoded genes had decreased, with a loss of mitochondrial mass and a further decrease in membrane potential (Figures 1L, 1M, S2A, and S2B). These data suggested that loss of SIRT1 results in a biphasic disruption of mitochondrial homeostasis.
Nuclear NAD+ Levels Regulate Mitochondrially Encoded Genes
Because there was no decline in SIRT1 protein with age (Figure S2E), we hypothesized that SIRT1 activity might be compromised in old mice due to a paucity of NAD+. Recent studies show that NAD+ levels are regulated independently in different cell compartments and that overall NAD+ levels decline during aging (Braidy et al., 2011, Massudi et al., 2012, Williamson et al., 1993). However, it is not clear in which cellular compartment(s) is NAD+ relevant to aging (Cantó and Auwerx, 2011)). Consistent with other reports (Braidy et al., 2011, Massudi et al., 2012), there was less total NAD+ in the skeletal muscle of elderly mice (Figure 2A). To determine which compartment might be responsible, we manipulated NAD+ levels in the different compartments by independently knocking down isoforms of nicotinamide mononucleotide adenylyltransferase, which regulate NAD+ levels in the nucleus (NMNAT1), golgi/cytoplasm (NMNAT2), and mitochondria (NMNAT3) (Berger et al., 2005). Knockdown of NMNAT2 or NMNAT3 had no effect on OXPHOS genes, whereas knockdown of NMNAT1 resulted in a specific reduction in the expression of mitochondrially encoded OXPHOS, mtDNA content, and ATP levels (Figures 2B–2F).
These results indicated that increasing the production of NAD+ within the nuclear pool might stimulate mitochondria. Overexpression of NMNAT1 in skeletal muscle of 10- to 12-month-old mice dramatically increased the expression of mitochondrially encoded OXPHOS genes (Figure 2G). Overexpression of NMNAT1 in primary myoblasts produced a similar effect that was SIRT1 dependent (Figure 2H). Together, these data indicated that mitochondria are regulated by nuclear NAD+ and that the impairment in OXPHOS function during aging may be precipitated by depletion of the nuclear NAD pool.