EFTA00611136.pdf

Cel Declining NAD÷ Induces a Pseudohypoxic State Disrupting Nuclear-Mitochondrial Communication during Aging Ana P. Comes,12.3 Nathan L. Price. Alvin J.Y. Ling, Javid J. Moslehi. • Magdalene K. Montgomery," Luis Rajman,' James P. White/ Joao S. Teodoro.' Christiane D. Wrann. Basil P. Hubbard. . Evi M. Mercken," Carlos M. Palmeira,2•3 Rafael de Cabo,BAnabela P. Rolo, Nigel Turner,' Eric L. Bell,' and David A. Sinclair'-6'* 'Glenn Labs for the Biological Mechanisms of Aging, Department of Genetics. Harvard Medical School. Boston. MA 02115. USA 'Center for Neurosciences and Cell Biology, 3004.517 Coimbra, Portugal *Department of Life Sciences. Faculty of Science and Technology. University of Coimbra, 3004.517 Coimbra, Portugal 'Department of Medical Oncology. Brigham and Women's Hospital and Dana-Farber Cancer Institute. Harvard Medical School, Boston, MA 02115. USA *Division of Cardiovascular Medicine. Department of Medicine, Brigham and Women's Hospital. Harvard Medical School. Boston, MA 02115. USA *Department of Pharmacology. School of Medical Sciences. The University of New South Wales, Sydney NSW 2052, Australia 'Dana-Farber Cancer Institute, Department of Cell Biology. Harvard Medical School. Boston. MA 02115. USA *Laboratory of Experimental Gerontology, National Institute on Aging. National Institutes of Health, Baltimore. MD 21224. USA *Department of Biology. University of Aveiro. 3810.193 Aveiro, Portugal '*Department of Biology. Massachusetts Institute of Technology. Paul F. Glenn Laboratory for the Science of Aging. Cambridge, MA 02139, USA 'Correspondence: david sinclair@hms.harvard.edu httpfidx.doi.org/10.10164.cell.2013.11.0.37 SUMMARY Ever since eukaryotes subsumed the bacterial ancestor of mitochondria, the nuclear and mitochon- drial genomes have had to closely coordinate their activities, as each encode different subunits of the oxidative phosphorylation (OXPHOS) system. Mito- chondria] 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-1a/frindependent path- way of nuclear-mitochondrial communication that is induced by a decline in nuclear HAD' and the accu- mulation of HIF-1a under normoxic conditions, with parallels to Warburg reprogramming. Deleting SIRT1 accelerates this process, whereas raising HAD' levels in old mice restores mitochondrial function to that of a young mouse in a SIRT1-depen- dent manner. Thus, a pseudohypoxic state that disrupts PGC-1O3-independent nuclear-mitochon- drial 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 et al., 2010). There is considerable debate, however, about why mitochondria' homeostasis is disrupted in the first place. The original idea of Hannan, 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 (La- pointe and Hekimi, 2010). Though most mitochondrial genes have been transferred to the nuclear genome, 13 subunits of the oxidative phos- phorylation (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-y coacti- vators ct and p (PGC-1 x and PGC1-1p), which along with NRF-1 and -2, induce nuclear-encoded proteins, such as TFAM (mito- chondria] 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, can- cer. and neurodegeneration (Haig is and Sinclair, 2010: Li bert and Guarente. 2013). Some of the health benefits of SIRT1 have also been linkedto improved mitochondria' function (Baur et al., 2006: Gerhart-Hines et al.. 2007: Price et al.. 2012: Rodgers et al., 2005). Indeed, increased expression of neuronal SIRT1 extends 1624 Col 155. 1624-1638, December 19, 2013 *2013 Elsevier Inc. CrossMatk EFTA00611136 Cell 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-1a) (Deng, 2012). Interestingly, SIRT1 increases HIF-1 a transcriptional activity (Lim et al.. 2010), SIRT3 destabilizes HIF-la protein (Bell et al.. 2011; Finley et al., 2011), and SIRT6 functions as a HIF-la corepressor (Thong et al.. 2010), raising the possibility that HIF-1a may also be relevant to aging. Consis- tent with this, in C. slogans, Hif-1 regulates lifespan and the response to CR (Leiser and Kaeberlein. 2010). A role for HIF-la in mammalian aging, however, has not been explored. In this study, we provide evidence for a PGC-1a/11-indepen- dent pathway of mitochondria' 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 pseudo- hypoxia-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 homeosta- sis. 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 skel- etal muscle (Figures 1A and 1B). By 22 months of age. ATP content and complex IV (CO4 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, Ill, and IV decline with age, but complex II, the only complex composed exclusively of nuclear-encoded sub- units, 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, Cylb, COX1 , A W6) were all significantly lower at 22 months relative to 6 month olds, whereas those encoded by the nuclear genome (NDUFS8, SDHb, Uqcrcl, 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 en- coded 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: Mouchiroud et al.. 2013); however, under these conditions there was no evidence of a mtUPR at 22 months of age (Figure SIA available online). Knockout of SIRT1 Mimics Aging by Decreasing Mitochondria!, 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 (SIRTHKO) (Price et al.. 2012), which circumvents the developmental abnormal- ities of germline SIRT1 knockouts. VAT, 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 mito- chondrially encoded OXPHOS genes and the two rRNAs were reduced in the SIRT1 iKO mice compared to wild-type controls (Figures 1G and SIB). Strikingly. there was no decrease in the expression of any of the nuclear-encoded components under fed conditions (Figure 1G). Again, protein levels of mitochondri- ally encoded COX2 were significantly decreased, whereas the nuclear-encoded COX4 was unaltered (Figure 1I-1), coincident with a decline in complex IV (CO)Q, but not complex II (SDH). activity (Figures S1I3 and S1E). Similar to old mice, cellular ATP levels and mtDNA content were reduced (Figures 11 and 1J). with no apparent induction of mtUPR (Figure S1C). Given that SIRT1 maintains mitochondrial mass by increasing PGC-1 a activity, we were surprised to see that, under these basal conditions 0.e., the fed state), there was no effect of SIRT1 deletion on mitochondria] mass (Figure 1K). To under- stand 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 11..). Again, mtDNA content and mito- chondria! membrane potential declined, with no change in mito- chondria! mass (Figures 1M, S2A, and S2B). By 48 hr, mRNA from both the nuclear- and mitochondrially encoded genes had decreased, with a loss of mitochondria' 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 mitochondria' homeostasis. Nuclear NAD' Levels Regulate Mitochondrially Encoded Genes Because there was no decline in SIRT1 protein with age (Fig- ure S2E), we hypothesized that SIRT1 activity might be com- promised 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; Yang et al.. 2007). However, it is not clear in which cellular compartment(s) is NAD` relevant to aging (Canto and Auwenc. 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 com- partments by independently knocking down isoforms of nico- tinamide mononucleotide adenylyltransferase, which regulate NAD' levels in the nucleus (NMNAT1). golgi/cytoplasm Cell 155.1624-1638. December 19. 2013 *2013 Elsevier Inc. 1625 EFTA00611137 Cell A ATP (pmolimg protein) 10 8 - 6 - 4 2 0 6 30 Age (months) 1.2 E 1.0 0.8 Z.' 0.6 0.4 O • 0.2 0.0 E F 1.2 1.0 0.8 0.6 0.4 0.2 0.0 6.0 .5 so ar 4.0 E a 2.0 a. l— 1.0 3.0 0.0 enc-encoded emt-encoded • COX 2 COX4 Tubulin 6 22 30 Age (months) C 1.2 1.0 - E g0.8- Z 0.6 o ci is 0.4 0.2 0.0 6 mo 22 mo 30 mo Er'd 1.0 E 0.8 6 22 30 Age (months) 0.2 0.0 6 Age (months) G H O WT • SIRT1 iKO win 2.0 1.5 0.5 0.0 10 20 30 40 nc-encoded mt-encoded Age (months) J 1.2 9 is 0.6 E .? a 0.4 0.2 0.0 WT SIRT1 iKO WT SIRT1 iKO L K WT SIRT1 iKO M 1.2 enc-encoded 60 i 1.0 emt-encoded ca 1 50 3 Fl 1 L6) 0.8 •a rn 40 cc I. 0.6 I i 30 E co i 0.4 10 20 rp- 0.2 E 1 10 0.0 0 0 20 40 60 48 Time of SIRT1 excision (hours) Time of SIRT1 excision (hours) 12 22 COX 2 (mt) COX4 (nc) Tubulin 1.4 1.2 1.0 - To — 0.8 .g 0.6 0.4 0.2 0.0 24 30 WT SIRT I iKO M.M110111,1= WT SIRT1 iKO Figure 1. Aging and Loss of SIRT1 Leads to a Specific Decline in Mitochondrial-Encoded Genes and Impairment in Mitochondria' Homeostasis in Skeletal Muscle (A) ATP content of 6-. 22-. and 30-month-old mice (n = 5. <0.05 versus 6-month-old mice). (8) Cytochrome c oxidase (COX) activity (n = 5. 'p < 0.05 versus 6-month-old animals). (C and 0) Mitochondria' DNA content (C) and DNA integrity (D) (n = 5. < 0.05 versus 6-incoth-old animals). (legend continued on next page) 1626 Cell 155, 1624-1638. December 19, 2013 O2013 Elsevier Inc. EFTA00611138 Cell (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-29. These results indicated that increasing the production of NAD' within the nuclear pool might stimulate mitochondria. Overex- pression 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 indi- cated 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. SIRTI Can Regulate Mitochondria through a PGC-1a/ (3-Independent Pathway A central dogma in the sirtuin field is that SIRTI promotes mito- chondria' function in response to fasting and CR by deacetylat- ing PGC-la (Gerhart-Hines et al., 2007: Rodgers et al.. 2005). Consistent with this, SIRTI iKO animals failed to upregulate both nuclear- and mitochondrially encoded OXPHOS genes in response to fasting (Figure S2C). However, our findings in fed animals (see Figure 1) indicated that SIRTI can regulate mito- chondria' genes independently of PGC-12. To test this. we examined primary myotubes from PGC-1a/II knockout (KO) mice (Zechner et al., 2010) and from PGC-1 a muscle-specific null mice (Handschin et al.. 2007), and we saw no defect in the ability of SIRT1 and NMNAT1 to induce mitochondrially encoded OXPHOS genes (Figures 21 and 24 Thus. SIRT1 can induce OXPHOS genes in the absence of PGC-12/p (Figure S2D). SIRTI Regulates Mitothondrially Encoded Genes through HIF-1 Next. we sought to understand how SIRT1 regulates mitochon- dria independently of PGC-17.43. Analysis of SIRTI iKO animals indicated that genes involved in glycolysis were upregulated, with increased lactate levels (Figures 3A and 3B) and a switch from slow-twitch oxidative fibers (MyHCIla) to fast-twitch glyco- lytic fibers (MyHCllb) (Figure S1 F). These metabolic changes were reminiscent of Warburg remodeling of metabolism in cancer cells, which is known to be mediated, in part, by the stabilization of the transcription factor HIF-12 (Majmundar et al.. 2010). The levels of HIF-12 and the expression of HIF-1a target genes were considerably higher in the SIRT1 iKO (Figures 3C and S3A). Despite being cultured under normoxic conditions, primary myoblasts deleted for SIRT1 also had increased HIF-12 protein levels and activity of a HIF-1a reporter (Figures 3C and S3B). Reducing NAD' levels. either by knocking down NMNAT1 or by treating cells with lactate (which decreases the NAD'/ NADH ratio), also caused HIF-12 protein stabilization (Figures 3D, 3E, and S3C). HIF-12 has been studied extensively in cancer and during hypoxia; however, its role in normal physiology remains largely unknown. To better understand this, HIF-la was stabilized ectopically in vivo by deleting the EgIN1 gene encoding HIF prolyl hydroxylase 2 (PHD2) (Minamishima et al.. 2008). Upon EgIN1 deletion and HIF-la stabilization in muscle. there was a specific decline in mtDNA content and decreased levels of mitochondrially encoded, but not nuclear-encoded, OXPHOS mRNA, paralleling the effects of SIRTI deletion and normal aging (Figures 3F-3H). Pharmacological stabilization of HIF-la in PGC- 1 a/I3 knockout myotubes reduced expression of mitochondrially encoded genes (Figures 31 and S3D), whereas treating PGC- /gip KO cells with pyruvate (to increase NAD' levels) up- regulated mitochondrially encoded genes, an effect that was prevented by stabilization of HIF-1a (Figure S3E). Stabilization of HIF-la in primary cells and transgenic mice blocked the ability of SIRT1 to upregulate mitochondrially encoded genes and increase ATP levels, with a specific loss of mitochondrially encoded mRNAs (Figures 31-3L and S3F-SFI). Overexpression of a stabilized mutant version of the related factor HIF-2a did not have the same effect (Figures 3J-3L and S3I), demonstrating that the inhibition of OXPHOS and mitochondrially encoded genes is HIF-1a specific. In primary myoblasts lacking HIF-la, deletion of WATT had no effect on mtDNA content, mitochond- rially encoded gene expression, or ATP levels (Figures 3M-3P). Together, our results show that HI F-12, but not HIF-2a, regulates mitochondria in response to SIRT1 activity, which is under the control of nuclear NAD' levels. SIRT1 Stabilizes HIF-1a via VHL HIF-la can be stabilized by ROS originating from complex ill of the ETC as part of retrograde response (Bell et al., 2007). Six hours after inducing SIRTI deletion in primary myoblasts, HIF-la levels increased (Figure 59, and by 12 hr, mitochondria] homeostasis was impaired (Figures 1L, S2A, and S2B). Yet, ROS levels did not increase until the 24 hr time point (Figure 54A). Myoblasts depleted of mitochondria' DNA (rho0), which are (0 Expression of nuclear- and mitochcothialy encoded genes (n = 5. p <0.05 versus 6-month-old animals). (F) Imrnunobbt foe COX2 and COX4 in 6-. 22-. and 30-month-old mite. (0) Expression of nuclear- WOUFS8.NOUFAS.SDHb.SDHd.Uqcrcl. Uqcrc2. COX5b. Cox641. ATPS41. and ATPcfland mitocnondzialty encoded genes WO I. ND2. ND3. N04. NO4 NOS. ND6. Cytb. COX, COX2. COX3. A7P6. and ATP8) in WT and SIRTI il<0 mice (n = 5. *p < 0.05 versus WT). (H and I) (H) Immunoblot for COX2 and COX4 and (I) ATP content in WT and SIRT1 iK0 mice (n = S. < 0.05 versus WT). (J) MilochcadrialDNA content of WT and SAT, iK0 mice (1 = 5.'p <0.05 versus WT). (K) Electron microscopy of gastrocnerrius from WT and SIR'", il<0 mice and mitochondrial area in = 4). (L) Expression of nuclear- and ntoctondrially encoded genes in SlAT? flog/Sox Cre-ERT2 primary myoblasts treated with vehicle (0 h0 or tamoxifen (Offl) to induce SIRT1 excision for 6. 12.24. and 48 hr (n = 4.'p < 0.05 versus vehicle). (M) Mitochondria! mass by NAO fluorescence ri SIRT7 floxfflox Cre-ERT2 primary myoblasts treated with veNcle (0 h0or OHT to induce SIRT1 excision for & 12. 24. and 48 hr (n = 4. p< 0.05 versus vehicle). Nuclear- and mitochondrially encoded genes were ND). Cytb. COX1. ATP6 and NDUFSS. SOHb. Uqcrcl. COX5b. ATPSal. respectively. Tissue samples are gastrocnemius unless otherwise stated. Values are expressed as mean x SEM. See also Ftgise Si. Cell 155.1624-1838. December 19. 2013 02013 Elsevier Inc. 1627 EFTA00611139 Cell A 250 S 200 •••••• 3.0 2 2.5 .2 g 2.0 ge 150 • < E g 1.5 E 100 • Ea 1.0 a 50 - as 0 0.0 G 20 < z 2 15 re E o r, v a x 10 v o c., c. .17.5 5 0 6 22 Age (months) ■ shNT • shNMNAT1 #1 O shNMNAT1 #2 C 1.4 2 1.2 .2 S 1 0 0.8 re ro E e 0.6 11! 0.4.2 0.0 NANAT2 Tubtin yi nc-encoded mt-encoded nc-e coded mt-en oded D E 1.6 1.2 1.4 1.2 1.0 _ 1.0 ... a 0.8 E 0.8 0.6 a shNT a shNMNAT3 #1 O shNMNAT3 #2 Z (12 0.6 0 m t a OA 2 0.4 i 0.2 0.2 0.0 0.0 nc-encoded mt-encoded EmOy MfflaTIOE MINATI Taal, 2.0 LI H Vehicle NMNAT1 OE <2 E .,.° 1.5 • N.to • 05 • E E O Empty • NMNATI OE 0.0 r. Vehide SIRT1 KO F O shNT 9 shNMNAT1 #1 shNMNAT1 N2 0 0.10 10.05 a. I 0.00 O shNT K shNMNAT2 x1 O shNMNAT2 P*2 • WT Empty • PGC-1a/6 KO Empty ▪ WT SIRT1 OE • PGC-le) KO SIRTI OE 2.5 2.0 1 1105 e • 0.5 0.0 nc-encoded mt-e coded PGC-1aIS KO myotubes 2.5 2.0 - 1.5 - 1.0 - 0.5 • 0.0 Empty NMNAT1 OE Figure 2. Nuclear NAIY Levels Regulate Mitochondrial-Encoded Genes and Mitochondrial Homeostasis through SIRT1, Independently of PGC-1,./(1 (A) MAD' levels in gastrocnemius of 6-. 22-. and 30-month-old mice (n = 5.13 < 0.05 versus 6-month-old mice). (3—D) Expression of nuclear- and mitochondrially encoded genes in primary myoblaststransduced with NMIVAT1(3).NMNAT2 (C).NMNATS (D). or nontargeting shRNA (n = 4.13 < 0.05 versus shNT). (E and F) Mitochondrial DNA content and (H)ATP content (I) in primary myoblaststransduced with NMNATI or nontargeting shRNA(n = 4.13 <0.05 versus shNT). (G) Expression of rritochondrially encoded genes in tibialis of 10- to 12-month-old mice overexpressing NMNATI compared to the contraleteral tibialis muscle treated with vehicle (n = 4.13 < 0.05 versus vehicle). (H) Expression of mitochondrially encoded genes in Sail flox/flox Cie-ERT2 primary myoblasts treated with vehicle or OHT to induce SIRTI excision infected with adenovirus overexpressing NMNATI a empty vector (n = 4.13 < 0.05 versus vehicle empty vector). (I and J) Expression of nuclear-and mitochondrially encoded genes in WT and PGC-7w6 knockout myotubes treated with adenovirus overexpressing &ATI (I) or NMNATI (J) (n = 4. 'p < 0.05 versus WT empty: tip < 0.05 versus P3C-1710 KO empty). Nuclear- and mitochandrially encoded genes were NO1. Cyrb. COX1. A7P6 and NDUFSS. SOHb. Uqorl. COXSb. A7P581. respecWely. Tissue samples are gastrocnemius muscle unless otherwise stated. Values are expressed as mean * SEM. See also Fgure S2. 1628 Cell 155, 1624-1638, December 19, 2013 ic)2013 Elsevier Inc. EFTA00611140 Cell A WT • SIRT1 KO D shNT sh•MNAT1 HIF-la Tubulin E Vehicle Pyruvate Lactate -IIF-1a Tubulin PGC-1a/p KO myotubes 3.5 g yé 3.0- É 2.5 •2 9. 2.0 - §i ° 1 s - 41 m 1.0 - É$ 0.0 • D DMSO • DMOG Iì i Empty SIRT1 OE M HIF-1 a Tubulin shNT shHIF-1a J N F 5.0 E" 4.0 2 3.0 1 2.0 E -E- 1.0 0.0 WT EgIN1 KO o o QP sN 4, O WT • SIRTI iKO G a 2 HIF-lo E E 0 Tubulin C Skeletal muscle WT HIF-la Tubulin Primary myoblasts SIRT1 iKO HIF-la Tubulin wr • EgIN1 KO t4 1.2 - 1.0 • 0.8 • 0.6 - 0.4 • 0.2 1 0.0 no -encoded mt-e coded K L • Empty • HIF-la DPA O I-IIF-2o DPA 1.4 7 .2r703 11.20 4 11- < 2 6 i I 5 g I. 0.8 -1 4 rc 0 HA E >CD 0.6 .§ — : 3 :§" OA (7) f 5 2 Tubulin ED, -7- 0.2 E .?_. 1 0.0 • 0 nc-encoded mt-encoded 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 • shNT + Vehide • shNT + SIRTI iKO P H 1.2 - 1.0 02 g 0.6 E a i 0.4 ; - 0.2 0.0 O shHIF-lo + SIRTI iKO 1.2 12 rg . 1.0 eta 1.0 o ••••••. E e 0.8 a 0.8 ,p 0.6 0.6 o 0.4 ci 0.4 0.2 u- 0.2 0.0 00 VVT ■ EgIN1 KO • Empty • SIRT1 OE O HIF-la DPA + SIRTI OE O HIF-2a DPA + SIRT1 OE (legend on next page) Cell /55. 1624-1638. December 19. 2013 E2O13 Elsevier Inc. 1629 EFTA00611141 Cell unable to produce ROS and signal to the nucleus (Chandel and Schumacker. 1999), were similar to the parental control cells (Figure S4B). indicating that ROS and retrograde signaling are not the cause of HIF-1 a stabilization. HIF-1 a stability has been previously reported to be regulated by acetylation of lysine 709 (Geng et al.. 2011). To test whether SIRT1-mediated deacetytation was the mechanism, we mutated K709 to glutamine (an acetylation mimetic) or to arginine (nonacetylated mimetic), with K674 serving as a negative control (Um et al.. 2010). Neither of the K709 substitutions stabilized HIF-la, nor were they affected by SIRT1 deletion (Figure S4C), indicating that SIRT1 does not regulate HIF-la protein stability by deacetylating K709. HIFa proteins are regulated by a proteasomal degradation mechanism mediated by the Von Hippel-Lindau (VHL) E3 ubiquitin ligase that recognizes hydroxytated proline residues on HIFa (Kaolin. 2008). Knockout of SIRTI did not affect HIF-la hydroxylation (Figure S4D), but in the SIRTI iKO mouse and trans- genic overexpressor the levels of SIRTI correlated with VHL levels (Figures 4A-4D). VHL promoter activity was not altered by SIRTI deletion, suggesting posttranscriptional regulation (Fig- ures 4F and 4G). HIF-2a was also stabilized by SIRT1, though HIF-2a target genes were not upregulated (Figures S4E and S4F). The re-establishment of SIRTI eliminated HIF-la protein and restored levels of mitochondria' OXPHOS mRNA in SIRTI iKO myoblasts, but these effects were lost when VHL was knocked down (Figures 4H-4J: also see Figure 5F). Thus, SIRT1 is constantly required to maintain mitochondria' homeostasis by inducing VHL and by ensuring that HIF-la is degraded efficiently. SIRT1-HIF-ta Regulates Mitochondria by Modulating c-Myc's Ability to Activate TFAM These results raised the question of how HIF-la. a nuclear pro- tein, inhibits mitochondria' OXPHOS genes. Analysis of gene expression in SIRTI iKO mice identified the nuclear-encoded mitochondria! factor TFAM as a candidate (Figures 5A and SSA). Consistent with this, 7FAM promoter activity in SIRTI iKO myoblasts was greatly reduced (Figure 5B), the reintroduc- tion of TFAM into SIRTI iKO cells restored levels of mitochond- rially encoded mRNAs and ATP (Figure 5C-E), and in time course studies, TFAM levels declined 6 hr after VHL and HIF-1a (Figure 5F). Knockdown of ARNT, a HIF-la transcriptional binding partner (Wang et al., 1995), had no appreciable effect on mitochondrially encoded OXPHOS genes and ATP levels (Figures S5B-S5D), indicating that HIF-la acts via a different mechanism. In cancer cells, metabolic reprogramming is mediated by crosstalk be- tween HIF-1 a and c-Myc (Gordan et al.. 2007), raising the possibility that c-Myc was the missing factor. In fact, c-Myc DNA-binding sites are found at mitochondria! biogenesis genes (Kim et al.. 2008: U et al., 2005). Deletion of SIRTI in primary myoblasts kicreased the binding between HIF-1 a and c-Myc and reduced c-Myc reporter activity (Figures 5G and S5E). Similarly, knockdown of c-Myc completely blocked the ablity of SIRT1 to induce mitochondrially encoded mRNAs and mtDNA (Figures S5F-S5H). Conversely, overexpression of c-Myc in myoblasts treated with a SIRTI inhibitor, EX-527, prevented loss of mtDNA, mitochondrially encoded mRNA, and cellular ATP levels (Figures 551-S5L). We tested whether c-Myc directly controls TFAM promoter in myoblasts and is modulated by SIRT1-HIF-1 a. TFAM is known to be regulated by PGC-1 a, which interacts with NRF 1/2 bound at positions -311 and -154 in the TFAM promoter (Figure 514). Knockdown of c-Myc reduced TFAM promoter activity (Figure 51), consistent with a study in cancer cells (U et al.. 2005). We identified a putative c-Myc consensus sequence, CACGTG, 1,028 bp upstream of the ATG site—the mutation of which decreased promoter activity by about half without affecting PGC-1 a-mediated induction (Figures 5J and 5K). Overexpression of SIRTI also induced the TFAM promoter Figure 3. Loss of SIRTI Induces a Pseudohypoxic State that Disrupts Mitochondrial-Encoded Genes and Mitochondrial Homeostasis (A and 8) HK2. PFKM. PKM. and LDHA mRNA (A) and lactate levels 03) of WT and SIRTI iK0 mice (n = 5.'p < 0.05 versus WI). (C) Immunoblot for HIF-tor and tubulin in WT and SIRTI iKO mice and in SAT! floc/flox Cre-ERT2 primary myoblasts treated with vehicle or OHT to ncluoeSIR71 excision for 24 hr (SIRT1 iK0). (D)Immunoblot for HIF-Ix and tubule) in primary myoblasts transduced with NMNATI or nontargeting shRNA. (E) Immunoblot of HIF-tor and tubule) n primary myoblasts treated with pyruvate. lactate. cr vehicle for 24 M. (F) ImmurioNot for HIF-1a and tubulin in WT and EON? KO mice. (G) Expression of nuclear- and mitochondrially encoded genes of WT and EON, l<0 mice (n = 5.'p < 0.05 versus WT). (H) Matochondrial DNA content of WT and EgINI KO mice (n = 5. 'p < 0.05 versus WI). (I) Expression of mitochondrially encoded genes in POC-1,41 KO myotubes treated with adenovirus overexpressing SIRT1 treated with OMS0 or the HIF- stabilizing compound DMOG (n = 4.13 <0.05 versus empty DMSO: Hp <0.05 versus SIRTI OE DMSO). Immunoblot for HA tag and tubulin in control and C2C12 cells overexpressing either HIP-tor or HIF-2a with the proline residues mutated NIF-1x DPA and HIF-2x DPA). 09 Expression of nuclear- versus milochcodriaIN encoded genes in HIF-1: DPA or HIF-2x DPA C2C12 cells (n = 6.13 < 0.05 versus empty vector). (L) Expression of mitochondrially encoded genes in HIF-1x DPAcr HIF-2x DPAC2C12 cells with adenovirus overexpressing SAT! = 4. <0.05venue empty vector. sip < 0.05 versus SIRTI OE). (M) Immunoblot for HIF-I x and tubulin in &ATI flox/flox Cre-ERT2 primary myoblasts transduced with HIF-1 a or nontargeting shRNA and treated with °MOO. N) Mitochcadrial DNA in SIRTI flox/flox Cre-ERT2 primary myodasts transduced with HIF-1: or nontargeting shRNA. treated with vehicle or OHT to induce SIRTI excision (SAT? iKO) (n = 4. 'p < 0.05 versus shill vehicle: lip < 0.05 versus shNT SIRTI *OD). (0) Expression of mitochondrially encoded genes kr SAT/ oxlflox Cre-ERT2 primary myoblasts transduced with HIF-la or nontargeting shRNA and treated with OHT to induce SIRTI excision (SIRT1 iKO) (n = 4.13 < 0.05 versus shNT vehicle: lip < 0.05 versus shNT SIRTI iK0). (P) ATP content in S1R71 flox/flox Cre-ERT2 primary myoblasts transduced with HtF-1a or nontargeting shRNA and treated with vehicle or OHT to induce Stan excision (n = 5. 'p <0.05 versus shNT vehicle: Op < 0.05 versus shill SIRTI IK0). Nuclear- and mitochondrially encoded genes were ND1. Cytb. COXI. A7P6 and NDUFS8. SDHb. Uqorl. COXSb. ATP5a? . respectively. Tissue samples are gastrocnemius unless otherwise stated. values are expressed as mean x SEM. See also Fig we S3. 1630 Cell 155, 1624-1638, December 19, 2013 02013 Elsevier Inc. EFTA00611142 Cell A B C 1.2 1.0 D 5.0 4.0 WT SIRT1 IK0 WT SIRT1-Tg 0.8 a e CC a 3.0 • VHL VHL E E 0.6 -J E 11 2 2.0 0.4 Tubulin Tubulin 0.2 ! 1.0 • 0.0 0.0 H WT SIRT1 iKG WT SIRT1-Tg E 6 mo 22 mo VHL HIF-la Tubulin F 1.2 1.0 SP 0.8 g 8.0 0.6 s OA > 0.2 _L G 1.4 1.2 12s 1.0 1 -g 0.8 81 0 6 > 2 OA 0.2 0.0 0.0 .4% 47 40 4 Q.' & e 4 • S VHL Vehicle SIRT1 iK0 SIRT1 HIF-la Tubulin Tubulin Vehicle SIRT1 IK0 shNT shVHL/01 shVHL#2 + + + + + + + + + + + + J < Emz .0 Em mi x ; c 9) co 5.0 4.0 3.0 2.0 1.0 0.0 Empty SIRT1 OE • shNT Empty ■ shNT SIRT1 OE 0 shVHL#1 SIRT1 OE ■ ShVHLN2 SIRT1 OE Figure 4. SIRT1 Regulates HIF-1.x Stabilization in Skeletal Muscle through Regulation of VHL Expression (A and B) Immisrobtot fce VI-ft. and tubule) in gastrocnemius of WT and SIRT1 'KO mice (A) and WT and SIRT1-Tg mice (8). (C and 0) VHf. mRNA in WT and SAT! 'KO mice (C) and WT and SIR'"? -Tg mice (D). Values normalized to WT mice (n = 5. 'p < 0.05 versus W1). (E) Immunoblot for VI-IL. HIF- Ix, and tubulin in gastrocnemius of 6- and 22-month-old mice. (F) VHL promoter activity in SIRT1 flortflox Cre-ERT2 primary myoblasts treated with vehicle or OHT for 24 hr to induce SAT; excision (SIR'"? iK0). Luciferase values normalized to vehicle cells (n = 5). (8) VHL promoter activity in primary myoblasts with adenavirus expressing SIRT1 or empty vector. Luciferase values normalized to empty vector cells (n = 5). (H) Immunoblot for VHL and tubulin in SIR'"? flox/flox Cre-ERT2 Omani myoblasts transduced with VHL or nontargeting shRNA. (I) Representative immunoblot for HIF-1x in SAT? flox/flox Cre-ERT2 primary myoblasts transduced with VHL or nontargeterg shRNA and treated with OHT for 24 hr (SAT? 'KG). after which SIRT1 was added back by adenoviral infection. (J) Expression of mitochondrially encoded genes in wintery myoblasts transduced with VHL or nontargeterg shRNA and treated with adencivius expressing SIRT7 or empty vector (n = 5. 'p <0.05 venzus shNT empty: tip< 0.05 versus shNT SIRT1 OE). Mitochcodnaly encoded genes were ND; .Cytb.COX1 and ATP6. Values are expressed as mean x SEM. See also Egure 54. reporter, and mutation of the c-Myc binding site blocks this effect (Figure 5L). Chromatin IP experiments detected an inter- action between c-Myc and the TFAM promoter, which was markedly reduced when SIRT1 was deleted, but not when HIF-12 was also knocked down (Figures 5M-0). We did not detect direct HIF-12 binding to TFAM (Figures 5M and 5N). with LONA as a positive control (Figures S5M and S5N). Together, these data provide the first direct link between HIF-12 and the regulation of mitochondrially encoded genes in skeletal muscle and identify a mechanism of PGC-1 2/6-inde- pendent regulation of mitochondrial function. AMPK Functions as a Switch between PGC- 1,x-Dependent and -Independent Pathways Driven by SIRT1 Next, we determined the mechanisms that determine whether SIRT1 utilizes the PGC-1 2-dependent or -independent path- ways. Under conditions of low energy. AMPK-mediated Cell 155. 1624-1638. December 19. 2013 02013 Elsevier Inc. 1631 EFTA00611143 Cell A 1.2 1.0 cc g z - 0.8 2 x 0.6 ° am u- .8 0.4 I- 3 g 0.2 E 0.0 ATP (pmollmg protein) • WT SIRT1 KO 0.25 0.20 0.15 0.10 0.05 0.00 Vehicle SIRT1 1KO(h) TFAM (h) H 5=. El B 1.2 1.0 Si: t 0.8 i 0.6 0.4 LL 1-2 - 0.2 24 c-Myc binding site (consensus sequence) CACGTG -1340 0.0 Vehicle SIRT1 iKO C Vehicle SIRT1 IKO (h) TEAM (h1 SIRTI TFAM Tubulin + 24 24 48 48 48 D 1.4 1.2 0 to 1.0 0.8 x cc e a& t, 0.4 0.2 0.0 Vehicle nc-encoded mt-encoded SIRT1 - - 24 24 48 48 iKO (h) TFAM (h) 48 G Time ot SIRT1 excision (hours) IP: Vehicle SIRTI 24h + 0 6 12 24 SIRT1 IgG HIF-la + + + + SIRT1 HIF-lo VI-IL WS it 4-14° HIF-lo TFAM Input 24 48 48 PatYc 24 - 48 Tubulin NRF.1 NRF.2r O I I TFAM I J 1.2 2.5 1.0 • i . 0 0.6 a ..5. ▪ 1.5 0.8 • e a a • i l 1.0 r S 0.2 U. I- To '!" 0.5 LL OA 0.0 0.0 S$ 2.0 shNT shMyc shMyc #1 #2 L M N TFAM Promoter 2.0 Vehicle SIRT1 iKO 300 250 1.5 ChIP: c•Myc 200 150 #' 8.; 1.0 ChIP: HIF-lo !!9 o ° E 0.5 ChIP: IgG 6 = 100 u. 50 Input 0 0.0 Chip: Full Ac-Myc length K O Empty 2.5 . • c-Myc OE 2.0 • 1.5 • 21 1 0 • n .xx_ FL' 0.5 • TFAM promoter O Vehicle I • SIRT1 iKO c-Myc HIF-lo 0 ChIP: c-Myc ChIP: IgG Input 0.0 Full Ac-Myc length TFAM Promoter shNT shHIF-to Vehicle SIRT1 IKO O Empty '• PGC-1a OE ••• • • • wain (legend on next page) 1632 Cell 155. 1624-1638. December 19. 2013 ;)2013 Elsevier Inc. EFTA00611144 Cell phosphorylation of PGC-la allows it to be deacetylated and activated by SIRT1 (Canto et al.. 2009; Gerhart-Hines et al.. 2007: Rodgers et al.. 2005), whereas under basal conditions, acetylation status is primarily regulated by the acetyltransferase GCN5 (Femandez-Marcos and Auwerx. 2011). We speculated that the biphasic decline in OXPHOS subunits (in Figure 1L) might be due to AMPK. In time course experiments following SIRT1 deletion, AMPK activation occurred after 48 hr, well after the decline in VHL-TFAM and mitochondria! genes (Figures 1L- 1M and 6A) but coincident with the decline in nuclear-encoded OXPHOS genes and mitochondria! mass (see Figures 1L-1M). An AMPK dominant-negative adenovirus (AMPK-DN) prevented the decline of nuclear OXPHOS mRNAs at 48 hr (Figures 6B and 6O), whereas forced maintenance of TEAM prevented AMPK activation (Figures 6D, 5D, and 5E). Together. these results strongly suggest that AMPK is the switch between the PGC-la-dependent and -independent pathways. In this model. AMPK activation occurs in the absence of SIRT1 only when ATP levels fall below a threshold. Consistent with this, AMPK was un- changed under fed conditions in the SIRT1 iKO mice and 22- month-old wild-type mice but was markedly increased in fasting animals. when we observe changes in both nuclear- and mito- chondrially encoded OXPHOS genes (Figure 6E and 69. Increasing NAD' Levels Restores Mitochondrial Homeostasis through the SIRT1-HIF-12-c-Myc Pathway CR is known to delay numerous diseases of aging in mammals, including cancer and type 2 diabetes. Interestingly, CR (30%- 40% instituted at 6 weeks) completely prevented the decline in VHL and the increase in HIF-la that occurs in ad-libitum (AL)- fed 22-month-old mice (Figure 7A). The observed decreases in HAD. and ATP levels. COX activity, mtDNA. and mitochondrially encoded OXPHOS components with age were also prevented by CR (Figures 7B-7D, S6A, and S6B). Unlike the accumulation of mutations in mtDNA, the pathway that we describe here should be rapidly reversible. Treatment of 22-month-old mice for 1 week with NMN, a precursor to NAD' that increases NAD' levels in vivo (Yoshino et al.. 2011), reversed the decline in VHL and accumulation of HIF-la (Figures 7E and 79; reduced lactate levels: and increased ATP, COX activity, and mitochond- rially encoded OXPHOS transcripts (Figures 7G-71 and S6D). In EgIN1 and SIRT1 iKO mice, however, NMN failed to induce mito- chondrially encoded genes or to raise ATP levels (Figures 7J-7L). Knockdown of NMNAT1 also prevented NMN from inducing mitochondrially encoded OXPHOS genes (Figure 7M), consis- tent with nuclear HAD* being a key regulatory molecule. The SIRT1 iKO and the 22-month-old mice had increased levels of markers of muscle atrophy and inflammation compared to young WT mice, along with impaired insulin signaling and insulin-stim- ulated glucose uptake (Figures S1G-SIJ and S6E-S6H). Strik- ingly, treatment of old mice with NMN reversed all of these biochemical aspects of aging and switched gastrocnemius muscle to a more oxidative fiber type (Figures S6E-56H). How- ever, we did not observe an improvement in muscle strength (data not shown). indicating that 1 week of treatment might not be sufficient to reverse whole-organism aging and that longer treatments might be required. DISCUSSION Impairment in mitochondria' homeostasis is one of the hallmarks of aging that may underlie common age-related diseases (Lanza and Nair. 2010; Wallace, 2010). Despite its importance, there is still controversy as to why mitochondria' homeostasis is dis- rupted with age and whether this process can be slowed or even reversed. Here, we present evidence fora PGC-1/./6-inde- pendent pathway that ensures OXPHOS function and mainte- nance of mitochondria! homeostasis (Figure 7f1). During aging, however. decline in nuclear energetic state or NAD' levels re- duces the activity of SIRT1 in the nucleus, causing VHL levels to decline and HIF-la to be stabilized. This program, which likely evolved to modulate mitochondrial metabolism in response to Figure 5. SIRT1 Regulates Mitochondria! Homeostasis by Modulation of the TFAM Promoter through HIF-1a/c-Myc (A) TFAM mRNA analyzed by qPCR in gastrocnemius of WT and SIRT1 iKO animals. Values were normalized to WT mice (n = 5.'p < 0.05 versus WI). (B) TFAM promoter activity in smn flox/flox Cre-ERT2 primary myoblasts treated with vehicle or OHT to induce SAT, excision for 24 hr (MT! IKO). Relative luciferase values were normalized to vehicle cells (n = 6. 'p < 0.06 versus vehicle). (C) Immunoblot for SIRTI. TFAM. and tubulin in SAT! flox/flox Cre-ERT2 primary myoblasts treated with vehicle or OM to induce SAT! excision (SIRTI IKO) for 24 ce 48 hr. after which cells were infected with control or TFAM adenovirus. (0) Expression of nuclear- versus mitochondrially encoded genes in SAT! flox/flox Cre-ERT2 primary myoblasts treated with OHT to educe SIRT1 excision (SIRT1 iKO) for 24 0r48 M. after which cells were elected with TFAMadenovina. Values were normalized to vehicle cells (n =4. *p <0.05 versusvehicle: Np <0.05 versus SAT! iKO 24 hr. 8p < 0.05 versus SIRTI iKO 48 hr). (E)ATP content in SIRTI flox/flox Cre-ERT2 primary myoblasts treated with vehicle or OM for 24 or 48 Has in (D) (n = 4.'p <0.05 versus veNcle: Np < 0.05versus SIRTI iKO 24 hr. 8p < 0.05 versus SIRTI iKO 48 hr). (F) Immunoblot of SIRT1. VHL. TFAM. and tubulin in SIRTI flox/flox Cre-ERT2 primary myoblasts treated with vehicle or OHT to induce SIRT1 excision and in cells treated with OHT for 24 Iv. after which SIRT1 was added back by adenoviral infection. (G) Interaction of HIF-1a and c-Myc determined by immunopeecipitaticer of HIF- I a in SIRT1 flox/flox Cre-ERT2 primary myoblasts treated with OHT to excise SIRT1. (H) The c-Myc-binding site on the TFAM promoter. (I) TFAM promoter actMty in primary myoblasts transduced with c-Myc or nontargeting shRNA (n = 4.'p < 0.05 versus shNT). (J-L) TFAM promoter full-length activity ce with mutatica of c-Myc-bindirg site (il c-Myc) in primary myoblasts overexpressing c-Myc (J). PGC-1= (K). &ATI (L). or empty vector (n = 4. 'p < 0.05 versus empty: Np <0.05 versus a c-Myc empty). (M and N) Chromatin immunoprectitation (ChIP) (M) and respective quantification by qPCR (N) of c-Myc and HIF-1a to the TFAM promoter nSIRT7 flox/flox Cre- ERT2 primary myoblasts treated with vehicle or OHT to induce SIRT1 excision (n = 3. 'p < 0.05 versus vehicle). (O) ChIP of c-Myc to the TFAM promoter in SAT! flothlox Cre-ERT2 primary myoblasts transduced with HIF-12 or nontargeting shRNA treated with vehicle or OHT to induce SIRT? excision for 24 hr (SIRT1 IKO). Mitochondrialty encoded genes were ND!. Cytb. COXI. ATMS. Values are expressed as mean SEM. See also Figure S5. Cell 155. 1624-1638. December 19.2013 02013 Elsevier Inc. 1633 EFTA00611145 Cell A lime of SIRTI excision (hours) 6 12 24 48 • lime *ma p-AMPK AMPK SIRT1 0(0 4811 Vehicle SIRTI IKO + 48h AMPK-DN p-ACC ACC C 1A 1 1.2 1.0 g a. 0.8 E m 0.6 0.4 0.2 0.0 0 nc-encoded U ni-encoded V hide SIRTI iK0 S RT1 ilt0 48h 48h AMPK-ON D E F Vehicle • • SIRTI 24 24 48 48 Fed Fasted SIRTI SIRT1 KO (h) WT WT 6 mo 22 mo TFAM (h) p-AMPK 24 - - 48 IKO p-AMPK p-AMPK MEIr e a e o AMPK AMPK AMPK Figure 6. AMPK Activity Regulates Switch between PSC-Ix-Dependent and -Independent Mechanisms of Mitochondrial Regulation by SIRT1 (A) Immunoblot for p-AMPK (Thr172) and AMPK in SAP floxMox Cre-ERT2 pinery myoblasts treated with vehicle (0 hr) a OHT to induce SIRTI excision. (B) lmmunoblot for p-ACC (Serie) and ACC in SIRTI floxMox Cre-ERT2 primary myoblasts treated with vehicle or OliT for 48 lv (SIRT1 'KO) and infected with AMPK-DN adenovirus. (C) Expression of nuclear- and mitochondrialy encoded genes as in SIRTI flox/flox Cre-ERT2 primary myoblasts treated with vehicle or OHT (SIRT1 iK0) and infected with empty or AMPK-DN adenovirus for the same period of tine (n = 4. V <0.05 versus vehicle: tip < 0.05 versus SIRTI 'KO). (D) Immunoblot for p-AMPK (1hr172) and AMPK in SIRTIfloxiflox Cre-ERT2 prenery myoblasts treated with vehicle or OHT for 24 or 48 hr. after which cells were infected with control or TFAM adenovirus. (E) Immunoblot for p-AMPK (M172) and AMPK n gastrocnemius of WT and SIRTI iK0 mice under fed and fasted conditions. (F) Representative inmunoblot for p-AMPK (TIv172) and AMPK in gastrocnemius of 6- and 22-month-old mice. Nuclear- and mitochondrially encoded genes were NDI . Cytb. COX1. ATP6. and NDUFS8. SDHb. Uqcrc1. COX5b. and ATP5a I. respectively. Values are expressed as mean * SEM. changes in energy supply, becomes chronically activated in old mice, inducing a pseudohypoxic state that disrupts OXPHOS, a phenomenon that is consistent with antagonistic pleiotropy (Wil- liams and Day. 2003). One of the more surprising findings is the existence of a SIRT1- mediated pathway that regulates mitochondria independently of PGC-12/6 The data indicate that SIRT1 can regulate these two pathways in response to the energetic state of the cell. Which one predominates depends on AMPK activity and the phosphor- ylation status of PGC-12 (Canto et al., 2009). This study shows that HIF-12-induced metabolic reprogram- ming occurs in normal tissue and that it disrupts mitochondria' homeostasis. We consider the metabolic state of the old mice as pseudohypoxic because the downstream effects are similar to hypoxia but occur even when oxygen is abundant, as previ- ously in type 2 diabetes and cancer (Ido and Williamson. 1997: Sanders. 2012: Williamson et al., 1993). An interesting implication is that reprogramming of normal tissue toward a Warburg-like state may increase ROS and establish a milieu for subsequent mutations to initiate carcinogenesis, a possibility that may help explain why cancer risk increases exponentially with age. All of the main players in the nuclear NAD+-SIRT1-HIF- 1 2-0XPHOS pathway are present in lower eukaryotes, indicating that the pathway evolved early in life's history. This pathway may have evolved to coordinate nuclear-mitochondrial synchrony in response to changes in energy supplies and oxygen levels, and its decline may be a conserved cause of aging. In C. elegans. HIF-12 is known to be a key determinant of lifespan, though its precise role is still a matter of debate (Leiser and Kaeberlein, 2010). HIF-1 2 modulation may have differential effects on lifespan depending on the animal's diet or whether the mtUPR is activated (Dillin et al., 2002: Durieux et al.. 2011; Houtkooper et al.. 2013). Though we did not detect mUPR in skeletal muscle, we do not exclude the possibility that mtUPR plays a role in other tissues or under different conditions. Additional studies will be required to elucidate complex feed- back loops that likely regulate the SIRT1-HIF-12-Myc-TFAM pathway. For example, in cancer cells, SIRT1 directly regulates c-Myc transcriptional activity, either by deacetylation of c-Myc (Menssen et al.. 2012) or by binding c-Myc and promoting its association with Max (Mao et al., 2011). Given that SIRT3 and SIRT6 also regulate HIF-la and compromise respiration (Bell et al.. 2011; Finley et al.. 2011; Zhong et al.. 2010), it will be inter- esting to test whether a decline in the activity of other sirtuins causes a similar loss of TFAM and mitochondrially encoded OXPHOS components. How broadly applicable might these findings be? High-fat diet feeding increases levels of HIF-12 in liver (Carabelli et al.. 2011) 1634 Cell 155. 1624-1638, December 19, 2013 ©2013 Elsevier Inc. EFTA00611146 Cell A VEIL HIF-lo Tubulin E 6 mo AL 22 moAL 22 mo CR • ga — 400 000 I 200 - O 100 - Z 0 2 • 0 M 0 PBS • NMN 6 22 Age (months) 0 PBS • NMN 6 22 Age (months) 2.0 F VHL HIF-lo Tubulin B — 500 '§ 400 E 300 200 ccb 100 z 0 6 22 22 Age (months) C 0 AL IN CR < Z2 as E :3 -I 3 6 months 22 months PBS NMN PBS NMN s uommoopimps. aSell. J 2.0 cif 1.5 E t 1.o E a° 0.5 D Vehicle • NMN shNT shNMNAT1 0.0 0 PBS MI MAN WT EgIN1 KO N CR I NMN K 4.0 a -§ 3.0 oh 2.0 to C 0.0 High energy supply 2.5 2.0 1.5 1.0 0.5 0.0 G 6 22 22 Age (months) — 0.6 -8 • 0.5 a E cr, 0.4 - , 0.3 — • 0.2 0.1 CO 0 PBS 0 PBS 6 22 Age (months) L ■ NMN WT EgIN I KO { Nuclear NAD HIF to Stabilization D • AL • CR ■ NMN 1.0 0.8 II 0.6 61 0.4 3.0 2 .2 2.5 z et se E se, 2.0 Ei 0.5 0.0 0.2 0.0 • 6 22 22 Age (months) H 0 PBS ■ NMN 2.0 < 2 .9 1.5 CC ta E n 1.0 E EE 0.0 6 22 Age (months) 0 PBS • NMN WT SIRT1 KO Aging Low energy supply Promoter TFAM 4 1O Mitochondria' Biogenesis Loss of Mitochondria' Homeostasis Deficiency in mItochondrially-encoded genes ()Vend on next page) Cell 155. 1624-1638. December 19. 2013 02013 Elsevier Inc. 1635 EFTA00611147 Cell and white adipose tissue, the latter of which correlated with a decline in mitochondria' gene expression (Krishnan et al.. 2012). Moreover, insulin-resistant human skeletal muscle has a signature reminiscent of hypoxia (Ptitsyn et al.. 2006). In SIRT1 iKO mice. specific dysregulation of mitochondria' OXPHOS genes is also observed in the heart, demonstrating that the pathway is relevant not only to skeletal muscle (Figure 51K- SOO, but not in liver, WAT, or brain. In these tissues, other factors such as SIRT3 or SIRT6 may be responsible for regula- tion of HIF-1x, or the metabolic status of the tissue at the time of harvest may also be critical. Current dogma is that aging is irreversible. Our data show that 1 week of treatment with a compound that boosts NAD' levels is sufficient to restore the mitochondria' homeostasis and key biochemical markers of muscle health in a 22-month-old mouse to levels similar to a 6-month-old mouse. Although further work is necessary, this study suggests that increasing NAD' levels and/or small compounds that prevent HIF-la stabilization or promote its degradation might be an effective therapy for organismal decline with age. In summary, these findings provide evidence for a new pathway that controls carbon utilization and OXPHOS independently of PGC-1,1, a pathway that goes awry over time but is readily reversible, with implications for treating aging and age-related diseases. EXPERIMENTAL PROCEDURES Aging Cohorts, SIRT1 -180, EgINI KO, and SIRTI-Tg Mice and NMNATI Electroporation Wild-type C5781.J6J race were from the National Institutes of Agng. NIH. EgNill KO. SIRTI-E0. and SIRTI-Tg mice were described previously (Mina- mishima et al.. 2006: Price et al.. 2012). For NMN experiments. mice were given IP injections of 500 mg NMN/kg body weight pee day or the equivalent volume of PBS for 7 consecutive days at 6 PM and 8 MI on day 8 and were sacrificed 4 Iv after last injection. All animal care followed the guidelines and was approved by Institutional Animal Care and Use Committees (1ACUCs). Adenovirus Generation and Mutagenesis Adenoviruses were generated as described before (Rodgers et al.. 2005) and were used to infect cells for the time points described in the figures. HIF-la and TFAM promoter mutants were generated using a commercially available kit (Stratagene) accordng to the manufacturer's instructions. Details about these methodologies and the prenera used for the mutagenesis can be found in the Extended Experimental Procedures. Generation of Primary Myoblasts, Rho° Cells, Cell Culture Treatments, Adenoviral Infections, and Gene Silencing Primary myoblasts were isolated from PGC-17.18 KO. SIRT1 iKO. and FCC-Ix null mice as described before (Price et al.. 2012). Fttro0 cells were generated by culturing the cells in media supplemented with 4 gA glucose and 2 mhl pyruvate. 50 ng/ml ethidium bromide (Ma aesar). and 100 pg/ml uridne (Sigma-Aldrich) for 4 weeks to deplete mitochondrial DNA. Cels were treated with the NMN (Sigma-Aldrich) and DMOG (Cayman) as described in the figtre legends. and details can be found in the Exterdec zxpenmental Procedures. Gene silencing was achieved using pLX0.1 shRNAs for the genes of interest. as described before (Comes et al.. 2012). and was used after selection with eu- karyotic mancer of the vector. Details about these methodologies can also been found in the Extended Expenrnental Procedures. Mitochondrial Function Cytochrome c oxidase and succinate dehydrogenase measurements were determned pollarographically as described before (Comes et al.. 2012). Cyto- chrome c oxidase was also measured spectrophotometrically using a commercialy available Mil (Sigma-Aldrich) according to the manufacturer's instructions. ATP content was measured with a commercial Mil Oloche) according to the manufacturer's instructions. Mitochondria] membrane poten- tial and reactive oxygen species were measured by FACS as described (Sell et al.. 2011: Price et al.. 2012). Electron microscopy was also deterrnned as described (Price et al.. 2012). Details about these methodologies can be found in the Extended Experimental Procedures. Gene Expression and mtDNA Analysis Total RNA and genornic DNA were isolated using a commercially available kit ((DAGEN) according to the manufact.rer's instructions. cDNA was generated using the iScript kit (BioRad). Gene expression and mtDNA were determined by qPCR as described (Puce et a[.. 2012). Details and primer sequences can be found n the Extended Experimental Procedures. Coimmunoprecipltation Proteins from primary myoblasts were crosslinked using DSP (1 mM. Pierce). after which the cells were !pied n a low-strivency IP buffer (0.05% NP-40. 50 mM NaCI. 0.5 mM EDTA. 50 mM Tris-HCI (pH 7.4l) supplemented with protease inibitcr cocIdail (Racheland 25 U/mlendcnuclease(Pierce). Endog- enous HF-12 protein was immunoprecipitated using anti-H1F-12 antibody Figure 7. Increasing NAD' Levels Rescues Age-Related Pseudohypoxia and OXPHOS Dysfunction through a SIRT1-HIF-12 Pathway (A) Immunoblot for H IF-12 and tubulin of 6- and 22-month-old AL and 22-month-old CR mice. (3) NAD' levels in the same cohorts as ri (A) (n = 5. 'p < 0.05 versus 6-month-old animals: rip < 0.05 versus 22-month-old AL). (C) Expression of rritochondrially encoded genes of the same cohorts as in (A). Values normalized to 6-month-old mice (n = 5,'p <0.05 versus 6-mcnth-old mice: Sp < 0.05 versus 22-month-old AL). (D)Cylochrome c oxidase (COX) activity (n = 4. 'p < 0.05 versus 6-month-old animals: Np <0.05 versus 22-month-old AL). (E)NAD' levels n 6- and 22-month-old mice treated with vehicle (P136) or NMN (n = 6. 'p <0.05 versus 6-month-old PBS: Np <0.05 versus 22-month-old PBS). (F) Immunoblot for VHL. HIF-1x. and tubulin of same cohorts as in p. (G) Lactate levels of same cohorts as in p (n = 6. 'p <0.05 versus 6-month-old PBS: Np <0.05 versus 22-month-old PBS). (H) Expression of mitochondrially encoded genes of same cohorts as n(E). Values normalized to 6-month-old PBS mice (n = 6. "p <0.05 versus 6-month-old PBS: Np < 0.05 versus 22-month-old PBS). (I) ATP content of same cohorts as in (E) (n = 6. 'p < 0.05 versus 6-month.old PBS: Np < 0.05 versus 22-month-old PBS). (J) Expression of mitochondrialy encoded genes in WT and EgINI KO mice heated with either vehicle (PBS) or NMN (n = 6, 'p <0.05 versus WT PBS). 09 ATP content of same cohorts as n (J) (n = 5. 'p < 0.05 versus WT PBS). (.) Expression of mitochondrially encoded genes of WT and SIRTI iK0 mice treated with either vehicle (PBS) or NMN (n = 4. 'p < 0.05 versus WT PBS). (M) Expression of mitochondrially encoded genes in primary myoblasts transduced with NMNATI or nontargeting shRNA heated with either PBS or NMN (n = 4. 'p < 0.05 versus shNT vehicle). N)Nuclear-nvtochondnal communication and its decline during aging. Nuclear NAD' levels regulate mitochondria via a PGC-1 a-independent pathway that ensures the correct stoichometry of OXPHOS subunits. but over time. a chronic pseudohypoxic response s activated. inhdaiting OXPHOS. Mitochondrially encoded genes were ND?, cob. COX). and AIRE. Values are expressed as meant SBA. See also Fqure S6. 1636 Cell 155,1624-1638, December 19,2013 02013 Elsevier Inc. EFTA00611148 Cell (Cayman) coated NG magnetic beads (Pierce). and anti4g0 was used as control. Imunoprecipitated proteins and input were run on SOS-PAGE and were revealed with anti-HIF-1 (Cayman) and anti-c-Myc (Abeam) antibodies. Details can be found in the Extended Experimental Procedures. Chromatin Immunoprecipitation and Immunobtots Chin:mate) immunoprecipitation was performed using a commercially avail- able kit (Millipore) according to the manufacturer's instructions and using anti-HIFI: (Cayman). anti-c-Myc (Abeam). and anti-IgG as control. Immuno- blots were performed as described (Price et al.. 2012). and details can be found in the Extended Experimental Procedures. TFAM Promoter, VHL Promoter, HRE, and c-Myc Activity TEAM promoter. Wit promoter. HRE. and c-Myc activity were determined using a luciferase-based system. Luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega) with Renate as the refer- ence, and details about the constructs can be found in the Extended Experi- mental Procedures. HAD' Measurement NAD' from skeletal muscle was quantified with a commercially available kit (BioVision) according to the manufacturer's instructions and as described before (Comes et al.. 2012). Statistical Analysis Data were analyzed by a two-laded Student's t test. Statistical analysts was performed using Excel software. SUPPLEMENTAL INFORMATION Supplemental Information ncludes Extended Experimental Procedures, six Ogees. and one table and can be found with this article online at http://dx. doi.org/10.10161.cell.2013.11.037. ACKNOWLEDGMENTS The Sinclair lab es supported by the NIH/NIA. the Glenn Foundation for Medical Research. the United Mitochondrial Disease Foundation. the Juvenile Dia- betes Research Foundation. and a gift from the Schulak family. M. was supported by the Portuguese Foundation for Science and Technology (SFRH/80/44674/ 2008) and M. by an NSERC PGS-D fellowship. M. es supported by an Australian Research Council Future Fellowship. We are grateful to Michael Bonkowski. Carlos Daniel de Magarees Frlho. Meghan Rego. Nikolna Clouts. and David Zhang for technical advice and experi- mental assistance: William Kasai) Jr. for kndly providing the EON! 1(0 mice: Daniel Kelly. John Fturrisay. and Teresa Leone for unpublished PGC-1,10 KO myoblasts and advice: Bruce Spiege&nan for PGC-1: null myoblasts and advice: and Pere Repave( and Zachary Gerhart-Hines for a SIRT1 adenovirus. M. is a consultant to Cotter. OvaScience. HorizonScience. Segterra. Metro8iotech. and GiaxoSmithKline. Cohbar. MetroBiotech. and GiaxoSmitN0ine work en mitoctiondrially derived peptides. NADI. and sirtuin modulation. respectively. Received: September 18.2012 Revised: October 25. 2013 Accepted: November 21.2013 Published: December 19.2013 REFERENCES 8aur. JA, Pearson. K.J.. Price.... Jamieson. H.A. Lorin. C.. Kalra. A.. Prabhu. V.V.. Mud. J.S.. Lopez-Uuch. G.. Lewis. K.. et al. (2006). Resveratrol improves health and survival of mice on a high-calorie diet. Nature 444. 337-342. Bell. E.L.. Klimova. TA. EisenbarL J.. 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