EFTA00611136

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 and Nair. 2010: Wallace et al., 2010). There is considerable debate, however, about why mitochondria' homeostasis is Ever since eukaryotes subsumed the bacterial disrupted in the first place. The original idea of Hannan, that ancestor of mitochondria, the nuclear and mitochon- reactive oxygen species (ROS) from mitochondria are a primary drial genomes have had to closely coordinate their cause of aging (Harman. 1972), has been challenged by recent activities, as each encode different subunits of the studies of long-lived species and genetically altered animals (La- oxidative phosphorylation (OXPHOS) system. Mito- pointe and Hekimi, 2010). Though most mitochondrial genes have been transferred chondria] dysfunction is a hallmark of aging, but its to the nuclear genome, 13 subunits of the oxidative phos- causes are debated. We show that, during aging, phorylation (OXPHOS) system remain, demanding functional there is a specific loss of mitochondrial, but not communication between the nucleus and mitochondria to form nuclear, encoded OXPHOS subunits. We trace the stoichiometric OXPHOS complexes. This is mediated in large cause to an alternate PGC-1a/frindependent path- part by the peroxisome proliferator-activated receptor-y coacti- way of nuclear-mitochondrial communication that is vators ct and p (PGC-1x and PGC1-1p), which along with NRF-1 induced by a decline in nuclear HAD' and the accu- and -2, induce nuclear-encoded proteins, such as TFAM (mito- mulation of HIF-1a under normoxic conditions, with chondria] transcription factor A), that carry out the replication, parallels to Warburg reprogramming. Deleting transcription, and translation of mitochondrial DNA (mtDNA) SIRT1 accelerates this process, whereas raising (Larsson. 2010). Mammalian sirtuins (SIRT1-7) are a conserved family of NAD- - HAD' levels in old mice restores mitochondrial dependent lysine-modifying acylases that control physiological function to that of a young mouse in a SIRT1-depen- responses to diet and exercise (Haigis and Sinclair. 2010). The dent manner. Thus, a pseudohypoxic state that expression of SIRT1, an NAD•-dependent deacetylase, is disrupts PGC-1O3-independent nuclear-mitochon- elevated in a number of tissues following calorie restriction (CR) drial communication contributes to the decline in (Cohen et al.. 2004), an intervention that extends lifespan in mitochondrial function with age, a process that is diverse species. Overexpression or pharmacological activation apparently reversible. of SIRT1 reproduces many of the health benefits of CR, including protection from metabolic decline, cardiovascular disease, can- INTRODUCTION cer. and neurodegeneration (Haigis and Sinclair, 2010: Libert and Guarente. 2013). Some of the health benefits of SIRT1 have also One of the most conserved and robust phenomena in biology is been linkedto improved mitochondria' function (Baur et al., 2006: a progressive decline in mitochondrial function with age, leading Gerhart-Hines et al.. 2007: Price et al.. 2012: Rodgers et al., to a loss of cellular homeostasis and organismal health (Lanza 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 under these conditions there was no evidence of a mtUPR at lower organisms has been challenged (Burnett et al., 2011). 22 months of age (Figure SIA available online). A hallmark of cancer is a shift away from OXPHOS toward anaerobic glycolysis that provides cells with sufficient substrates Knockout of SIRT1 Mimics Aging by Decreasing for biomass. This metabolic reprogramming, known as the Mitochondria!, but Not Nuclear-Encoded, OXPHOS Warburg effect (Warburg. 1956), is driven by several different Components pathways, including mTOR, c-Myc, and hypoxia-inducible factor We wondered whether the specific decline in mitochondrially 1 (HIF-1a) (Deng, 2012). Interestingly, SIRT1 increases HIF-1a encoded OXPHOS components in aged mice might be due. in transcriptional activity (Lim et al.. 2010), SIRT3 destabilizes part, to a loss of SIRT1 activity. To test this, we utilized an HIF-la protein (Bell et al.. 2011; Finley et al., 2011), and SIRT6 adult-inducible SIRT1 knockout mouse (SIRTHKO) (Price functions as a HIF-la corepressor (Thong et al.. 2010), raising et al.. 2012), which circumvents the developmental abnormal- the possibility that HIF-1a may also be relevant to aging. Consis- ities of germline SIRT1 knockouts. VAT, was deleted at tent with this, in C. slogans, Hif-1 regulates lifespan and the 2-4 months of age, and skeletal muscle was analyzed response to CR (Leiser and Kaeberlein. 2010). A role for HIF-la 2-6 months later. As expected, the mRNA levels of all 13 mito- in mammalian aging, however, has not been explored. chondrially encoded OXPHOS genes and the two rRNAs were In this study, we provide evidence for a PGC-1a/11-indepen- reduced in the SIRT1 iKO mice compared to wild-type controls dent pathway of mitochondria' regulation that plays a role in (Figures 1G and SIB). Strikingly. there was no decrease in the the aging process. Activity of this pathway declines during aging expression of any of the nuclear-encoded components under due to changes in nuclear NAD' levels, causing a pseudo- fed conditions (Figure 1G). Again, protein levels of mitochondri- hypoxia-driven imbalance between nuclear- and mitochondrially ally encoded COX2 were significantly decreased, whereas the encoded OXPHOS subunits —a process that is prevented by CR nuclear-encoded COX4 was unaltered (Figure 1I-1), coincident and is reversed by raising NAD', with implications for treating with a decline in complex IV (CO)Q, but not complex II (SDH). age-related diseases. including cancer. activity (Figures S1I3 and S1E). Similar to old mice, cellular ATP levels and mtDNA content were reduced (Figures 11 and 1J). RESULTS with no apparent induction of mtUPR (Figure S1C). Given that SIRT1 maintains mitochondrial mass by increasing Aging Leads to a Specific Decline in Mitochondrially PGC-1a activity, we were surprised to see that, under these Encoded Genes basal conditions 0.e., the fed state), there was no effect of Aging is associated with disruption of mitochondrial homeosta- SIRT1 deletion on mitochondria] mass (Figure 1K). To under- sis. but the underlying mechanisms are unclear. As in previous stand why, we cultured SIRT1 iKO primary myoblasts and reports (Lanza and Nair. 2010), we observed a progressive. induced Cre-mediated deletion of the SIRT1 catalytic core age-dependent decline in OXPHOS efficiency with age in skel- ex vivo. After 12 hr, only the mitochondrially encoded OXPHOS etal muscle (Figures 1A and 1B). By 22 months of age. ATP mRNAs decreased (Figure 11..). Again, mtDNA content and mito- content and complex IV (CO4 activity were decreased, even chondria! membrane potential declined, with no change in mito- more so by 30 months of age. Although mtDNA content declined chondria! mass (Figures 1M, S2A, and S2B). By 48 hr, mRNA at both ages. the integrity of mtDNA was only lower in the from both the nuclear- and mitochondrially encoded genes had 30 month olds (Figures 1C and 1D). Together with previous decreased, with a loss of mitochondria' mass and a further reports (Lapointe and Hekimi. 2010), this suggested an aging decrease in membrane potential (Figures 1L, 1M, S2A, and mechanism that disrupts OXPHOS prior to the accumulation of S2B). These data suggested that loss of SIRT1 results in a significant mtDNA damage. biphasic disruption of mitochondria' homeostasis. A clue came from observations that the activity of OXPHOS complexes I, Ill, and IV decline with age, but complex II, the Nuclear NAD' Levels Regulate Mitochondrially Encoded only complex composed exclusively of nuclear-encoded sub- Genes units, does not (Kwong and Sohal. 2000). Thus, we tested Because there was no decline in SIRT1 protein with age (Fig- whether OXPHOS decline might be due to the specific loss of ure S2E), we hypothesized that SIRT1 activity might be com- mitochondrially encoded transcripts. Mitochondrially encoded promised in old mice due to a paucity of NAD'. Recent studies OXPHOS mRNAs (ND1, Cylb, COX1, AW6) were all significantly show that NAD' levels are regulated independently in different lower at 22 months relative to 6 month olds, whereas those cell compartments and that overall NAD' levels decline during encoded by the nuclear genome (NDUFS8, SDHb, Uqcrcl, aging (Braidy et al.. 2011; Massudi et al., 2012; Yang et al.. COX5,ATP5a) remained unchanged: but by 30 months of age, 2007). However, it is not clear in which cellular compartment(s) both the nuclear- and the mitochondrially encoded mRNAs is NAD` relevant to aging (Canto and Auwenc. 2011). Consistent were lower (Figures 1E). Protein levels of the mitochondrially en- with other reports (Braidy et al., 2011; Massudi et al.. 2012), coded COX2 gene were decreased at 22 months, but COX4, a there was less total NAD' in the skeletal muscle of elderly nuclear-encoded subunit, was only slightly lower. By 30 months, mice (Figure 2A). To determine which compartment might be both proteins were reduced relative to young mice (Figure 1F). responsible, we manipulated NAD' levels in the different com- The mitochondrial unfolded protein response (mtUPR) has partments by independently knocking down isoforms of nico- been recently linked to longevity (Durieux et al., 2011: tinamide mononucleotide adenylyltransferase, which regulate Houtkooper et al., 2013: Mouchiroud et al.. 2013); however, NAD' levels in the nucleus (NMNAT1). golgi/cytoplasm Cell 155.1624-1638. December 19. 2013 *2013 Elsevier Inc. 1625 EFTA00611137 Cell A C 10 1.2 1.2 ATP (pmolimg protein) 8 - E 1.0 1.0 - E Er'd 1.0 0.8 g0.8- 6 - 0.8 • E Z.' 0.6 Z ci o 0.6 4 0.4 is 0.4 2 •O 0.2 0.2 0.2 0 0.0 0.0 0.0 6 30 6 22 30 6 22 30 6 22 30 Age (months) Age (months) Age (months) Age (months) E F G H 1.2 6 mo 22 mo O WT 30 mo 2.0 • SIRT1 iKO 1.0 WT SIRT I iKO COX 2 1.5 0.8 win COX 2 M.M110111,1= (mt) 0.6 COX4 COX4 0.4 0.5 (nc) 0.2 enc-encoded emt-encoded Tubulin Tubulin 0.0 0.0 10 20 30 40 nc-encoded mt-encoded Age (months) J K 6.0 1.2 WT SIRT1 iKO 1.4 .5 so 1.2 ra 4.0 To — 1.0 3.0 9 is 0.6 - 0.8 E E .? a 2.0 .g 0.6 a 0.4 a. 0.4 l— 1.0 0.2 0.2 0.0 0.0 WT SIRT1 iKO WT SIRT1 iKO 0.0 WT SIRT1 iKO L M 1.2 enc-encoded 60 emt-encoded i 0.8 1.0 ca 1 50 •a Fl 3 rn 40 I. 1 L6) cc E co 0.6 I i 30 i 0.4 10 20 rp- 0.2 E1 10 0.0 0 0 20 40 60 12 24 48 Time of SIRT1 excision (hours) Time of SIRT1 excision (hours) 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). target genes were considerably higher in the SIRT1 iKO (Figures Knockdown of NMNAT2 or NMNAT3 had no effect on OXPHOS 3C and S3A). Despite being cultured under normoxic conditions, genes. whereas knockdown of NMNAT1 resulted in a specific primary myoblasts deleted for SIRT1 also had increased HIF-12 reduction in the expression of mitochondrially encoded protein levels and activity of a HIF-1a reporter (Figures 3C and OXPHOS. mtDNA content, and ATP levels (Figures 2B-29. S3B). Reducing NAD' levels. either by knocking down NMNAT1 These results indicated that increasing the production of NAD' or by treating cells with lactate (which decreases the NAD'/ within the nuclear pool might stimulate mitochondria. Overex- NADH ratio), also caused HIF-12 protein stabilization (Figures pression of NMNAT1 in skeletal muscle of 10- to 12-month-old 3D, 3E, and S3C). mice dramatically increased the expression of mitochondrially HIF-12 has been studied extensively in cancer and during encoded OXPHOS genes (Figure 2G). Overexpression of hypoxia; however, its role in normal physiology remains largely NMNAT1 in primary myoblasts produced a similar effect that unknown. To better understand this, HIF-la was stabilized was SIRT1 dependent (Figure 2H). Together, these data indi- ectopically in vivo by deleting the EgIN1 gene encoding HIF cated that mitochondria are regulated by nuclear NAD' and prolyl hydroxylase 2 (PHD2) (Minamishima et al.. 2008). Upon that the impairment in OXPHOS function during aging may be EgIN1 deletion and HIF-la stabilization in muscle. there was a precipitated by depletion of the nuclear NAD pool. specific decline in mtDNA content and decreased levels of mitochondrially encoded, but not nuclear-encoded, OXPHOS SIRTI Can Regulate Mitochondria through a PGC-1a/ mRNA, paralleling the effects of SIRTI deletion and normal aging (3-Independent Pathway (Figures 3F-3H). Pharmacological stabilization of HIF-la inPGC- A central dogma in the sirtuin field is that SIRTI promotes mito- 1a/I3 knockout myotubes reduced expression of mitochondrially chondria' function in response to fasting and CR by deacetylat- encoded genes (Figures 31 and S3D), whereas treating PGC- ing PGC-la (Gerhart-Hines et al., 2007: Rodgers et al.. 2005). /gip KO cells with pyruvate (to increase NAD' levels) up- Consistent with this, SIRTI iKO animals failed to upregulate regulated mitochondrially encoded genes, an effect that was both nuclear- and mitochondrially encoded OXPHOS genes in prevented by stabilization of HIF-1a (Figure S3E). Stabilization response to fasting (Figure S2C). However, our findings in fed of HIF-la in primary cells and transgenic mice blocked the ability animals (see Figure 1) indicated that SIRTI can regulate mito- of SIRT1 to upregulate mitochondrially encoded genes and chondria' genes independently of PGC-12. To test this. we increase ATP levels, with a specific loss of mitochondrially examined primary myotubes from PGC-1a/II knockout (KO) encoded mRNAs (Figures 31-3L and S3F-SFI). Overexpression mice (Zechner et al., 2010) and from PGC-1a muscle-specific of a stabilized mutant version of the related factor HIF-2a did null mice (Handschin et al.. 2007), and we saw no defect in the not have the same effect (Figures 3J-3L and S3I), demonstrating ability of SIRT1 and NMNAT1 to induce mitochondrially encoded that the inhibition of OXPHOS and mitochondrially encoded OXPHOS genes (Figures 21 and 24 Thus. SIRT1 can induce genes is HIF-1a specific. In primary myoblasts lacking HIF-la, OXPHOS genes in the absence of PGC-12/p (Figure S2D). deletion of WATT had no effect on mtDNA content, mitochond- rially encoded gene expression, or ATP levels (Figures 3M-3P). SIRTI Regulates Mitothondrially Encoded Genes Together, our results show that HIF-12, but not HIF-2a, regulates through HIF-1 mitochondria in response to SIRT1 activity, which is under the Next. we sought to understand how SIRT1 regulates mitochon- control of nuclear NAD' levels. dria independently of PGC-17.43. Analysis of SIRTI iKO animals indicated that genes involved in glycolysis were upregulated, SIRT1 Stabilizes HIF-1a via VHL with increased lactate levels (Figures 3A and 3B) and a switch HIF-la can be stabilized by ROS originating from complex ill from slow-twitch oxidative fibers (MyHCIla) to fast-twitch glyco- of the ETC as part of retrograde response (Bell et al., 2007). lytic fibers (MyHCllb) (Figure S1F). These metabolic changes Six hours after inducing SIRTI deletion in primary myoblasts, were reminiscent of Warburg remodeling of metabolism in HIF-la levels increased (Figure 59, and by 12 hr, mitochondria] cancer cells, which is known to be mediated, in part, by the homeostasis was impaired (Figures 1L, S2A, and S2B). Yet, ROS stabilization of the transcription factor HIF-12 (Majmundar levels did not increase until the 24 hr time point (Figure 54A). et al.. 2010). The levels of HIF-12 and the expression of HIF-1a 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 WOI. 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 riSIRT7 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 C NANAT2 1.4 Tubtin 250 3.0 •••••• 2 1.2 yi 2.5 S 200 .2 2 .2 S 10 g 2.0 ge 150 • < ■ shNT 0.8 O shNT 1.5 • shNMNAT1 #1 re ro 0.6 ❑ shNMNAT2 x1 E e Eg E 100 • Ea 1.0 O shNMNAT1 #2 O shNMNAT2 P*2 a 11! 0 .4 .2 50 - as 0 0.0 0.0 6 22 nc-encoded mt-encoded nc-e coded mt-en oded Age (months) D E F O shNT 9 shNMNAT1 #1 shNMNAT1 N2 1.6 1.2 1.4 _ 1.0 1.2 1.0 ... 0.8 0 0.10 a shNT a 0.8 Z (12 0.6 E a shNMNAT3 #1 0 m 0.6 O shNMNAT3 #2 t ia OA 10.05 0.4 a. 2 0.2 0.2 I 0.0 0.0 0.00 nc-encoded mt-encoded G H EmOy MfflaTIOE • WT Empty • PGC-1a/6 KO Empty MINATI ▪ WT SIRT1 OE • PGC-le) KO SIRTI OE PGC-1aIS KO myotubes LI Taal, 2.0 O Empty 2.5 20 2.5 • NMNATI OE <2 2.0 - < 2.0 z 2 15 re r, E .,.° 1.5 • E o v a x 10 N. to • 1 1.5 - v o c., c. .17.5 1105 1.0 - 5 05 • 0.5 • E E •e 0.5 0.0 r. 0.0 0 0.0 Vehicle NMNAT1 OE Vehide SIRT1 KO nc-encoded mt-e coded 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 withNMIVAT1(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 C Skeletal muscle WT • SIRT1 KO WT SIRT1 iKO 5.0 O WT • SIRTI iKO HIF-la E" 4.0 2 3.0 Tubulin 1 2.0 Primary myoblasts E -E- 1.0 HIF-la 0.0 Tubulin D shNT sh•MNAT1 F G H wr • EgIN1 KO VVT t4 1.2 ■ EgIN1 KO HIF-la 1.2 - - 1.0 WT EgIN1 KO Tubulin 1.0 • HIF-lo Ea 2 0.8 • g 02 0.6 E Vehicle Pyruvate Lactate E 0 0.6 - Ea 0.4 • i 0.4 Tubulin ; -IIF-1a 0.2 - 0.2 0.0 1 0.0 Tubulin no -encoded mt-e coded J K L PGC-1a/p KO myotubes • Empty • HIF-la DPA O I-IIF-2o DPA 3.5 1.4 7 • D DMSO o oQP g é 3.0- .2r7 03 1.2 0 <2 6 y • DMOG sN 4, É 2.5 4 11- • Empty i I 5 •2 9. 2.0 - g I. 0.8 rc 0 -1 4 • SIRT1 OE ° 1s - HA E >CD 0.6 — O HIF-la DPA §i 41 m 1.0 - :§" OA .§ : 3 (7) f 5 2 + SIRTI OE Tubulin O HIF-2a DPA É$ ED, -7- 0.2 E .?_. 1 + SIRT1 OE 0.0 Iì i 0.0 • 0 Empty SIRT1 OE nc-encoded mt-encoded M N P • shNT + Vehide • shNT + SIRTI iKO O shHIF-lo + SIRTI iKO 1.4 1.2 12 1.2 rg . 1.0 1.0 ••••••. shNT shHIF-1a 1.0 eta o E e 0.8 a 0.8 0.8 HIF-1a 0.6 ,p 0.6 o 0.6 Tubulin 0.4 0.4 ci 0.4 0.2 0.2 u- 0.2 0.0 0.0 00 (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 mitochondria! factor TFAM as a candidate (Figures 5A and Schumacker. 1999), were similar to the parental control cells SSA). Consistent with this, 7FAM promoter activity in SIRTI (Figure S4B). indicating that ROS and retrograde signaling are iKO myoblasts was greatly reduced (Figure 5B), the reintroduc- not the cause of HIF-1a stabilization. tion of TFAM into SIRTI iKO cells restored levels of mitochond- HIF-1a stability has been previously reported to be regulated rially encoded mRNAs and ATP (Figure 5C-E), and in time course by acetylation of lysine 709 (Geng et al.. 2011). To test whether studies, TFAM levels declined 6 hr after VHL and HIF-1a SIRT1-mediated deacetytation was the mechanism, we mutated (Figure 5F). K709 to glutamine (an acetylation mimetic) or to arginine Knockdown of ARNT, a HIF-la transcriptional binding partner (nonacetylated mimetic), with K674 serving as a negative control (Wang et al., 1995), had no appreciable effect on mitochondrially (Um et al.. 2010). Neither of the K709 substitutions stabilized encoded OXPHOS genes and ATP levels (Figures S5B-S5D), HIF-la, nor were they affected by SIRT1 deletion (Figure S4C), indicating that HIF-la acts via a different mechanism. In cancer indicating that SIRT1 does not regulate HIF-la protein stability cells, metabolic reprogramming is mediated by crosstalk be- by deacetylating K709. tween HIF-1a and c-Myc (Gordan et al.. 2007), raising the HIFa proteins are regulated by a proteasomal degradation possibility that c-Myc was the missing factor. In fact, c-Myc mechanism mediated by the Von Hippel-Lindau (VHL) E3 DNA-binding sites are found at mitochondria! biogenesis genes ubiquitin ligase that recognizes hydroxytated proline residues (Kim et al.. 2008: U et al., 2005). Deletion of SIRTI in primary on HIFa (Kaolin. 2008). Knockout of SIRTI did not affect HIF-la myoblasts kicreased the binding between HIF-1a and c-Myc hydroxylation (Figure S4D), but in the SIRTI iKO mouse and trans- and reduced c-Myc reporter activity (Figures 5G and S5E). genic overexpressor the levels of SIRTI correlated with VHL Similarly, knockdown of c-Myc completely blocked the ablity levels (Figures 4A-4D). VHL promoter activity was not altered by of SIRT1 to induce mitochondrially encoded mRNAs and mtDNA SIRTI deletion, suggesting posttranscriptional regulation (Fig- (Figures S5F-S5H). Conversely, overexpression of c-Myc in ures 4F and 4G). HIF-2a was also stabilized by SIRT1, though myoblasts treated with a SIRTI inhibitor, EX-527, prevented HIF-2a target genes were not upregulated (Figures S4E and loss of mtDNA, mitochondrially encoded mRNA, and cellular S4F). The re-establishment of SIRTI eliminated HIF-la protein ATP levels (Figures 551-S5L). and restored levels of mitochondria' OXPHOS mRNA in SIRTI We tested whether c-Myc directly controls TFAM promoter iKO myoblasts, but these effects were lost when VHL was in myoblasts and is modulated by SIRT1-HIF-1a. TFAM is knocked down (Figures 4H-4J: also see Figure 5F). Thus, SIRT1 known to be regulated by PGC-1a, which interacts with NRF is constantly required to maintain mitochondria' homeostasis by 1/2 bound at positions -311 and -154 in the TFAM promoter inducing VHL and by ensuring that HIF-la is degraded efficiently. (Figure 514). Knockdown of c-Myc reduced TFAM promoter activity (Figure 51), consistent with a study in cancer cells (U SIRT1-HIF-ta Regulates Mitochondria by Modulating et al.. 2005). We identified a putative c-Myc consensus c-Myc's Ability to Activate TFAM sequence, CACGTG, 1,028 bp upstream of the ATG site—the These results raised the question of how HIF-la. a nuclear pro- mutation of which decreased promoter activity by about half tein, inhibits mitochondria' OXPHOS genes. Analysis of gene without affecting PGC-1a-mediated induction (Figures 5J and expression in SIRTI iKO mice identified the nuclear-encoded 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-1a 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 withHIF-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 D 5.0 1.0 4.0 WT SIRT1 IK0 WT SIRT1-Tg 0.8 e 3.0 • CC a VHL VHL E E a 0.6 E -J 11 2 2.0 0.4 Tubulin Tubulin ! 1.0 • 0.2 0.0 0.0 WT SIRT1 iKG WT SIRT1-Tg E F G 1.2 1.4 6 mo 22 mo 1.0 _L 1.2 SP 12s 1.0 VHL 0.8 g 1 -g 0.8 8.0 0.6 81 0 6 HIF-la s OA > > 2 OA Tubulin 0.2 0.2 0.0 0.0 Vehicle SIRT1 IK0 Empty SIRT1 OE H J 5.0 shNT shVHL/01 shVHL#2 < Vehicle + + + Emz .0 4.0 • shNT Empty .4% Q.' e& 4 4 47 SIRT1 iK0 + + + + + + Em mi x 3.0 ■ shNT SIRT1 OE 40 • SIRT1 + + + 0 shVHL#1 SIRT1 OE ; 2.0 ■ ShVHLN2 SIRT1 OE VHL c HIF-la 9) co 1.0 S Tubulin Tubulin 0.0 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 skeletal muscle and identify a mechanism of PGC-12/6-inde- effect (Figure 5L). Chromatin IP experiments detected an inter- pendent regulation of mitochondrial function. action between c-Myc and the TFAM promoter, which was markedly reduced when SIRT1 was deleted, but not when AMPK Functions as a Switch between PGC- HIF-12 was also knocked down (Figures 5M-0). We did not 1,x-Dependent and -Independent Pathways Driven by detect direct HIF-12 binding to TFAM (Figures 5M and 5N). SIRT1 with LONA as a positive control (Figures S5M and S5N). Next, we determined the mechanisms that determine whether Together, these data provide the first direct link between SIRT1 utilizes the PGC-12-dependent or -independent path- HIF-12 and the regulation of mitochondrially encoded genes in ways. Under conditions of low energy. AMPK-mediated Cell 155. 1624-1638. December 19. 2013 02013 Elsevier Inc. 1631 EFTA00611143 Cell A B C D Vehicle + nc-encoded mt-encoded 1.2 1.2 1.4 SIRT1 24 24 48 48 1.0 1.0 IKO (h) 0 1.2 Si: to 1.0 z -g 0.8 cc t 0.8 TEAM (h1 48 0.8 2 ° x 0.6 • i 0.6 SIRTI cc ex a& am I- 3 0.4 u- .8 LL 0.4 t, 0.4 1-2 g 0.2 - 0.2 TFAM 0.2 0.0 0.0 Tubulin 0.0 WT SIRT1 Vehicle SIRT1 Vehicle KO iKO SIRT1 - - 24 24 48 48 iKO (h) TFAM (h) 48 E G Time ot SIRT1 excision (hours) 0.25 ATP (pmollmg protein) IP: Vehicle SIRTI 24h + 0.20 0 6 12 24 SIRT1 IgG 5=. + + 0.15 HIF-la + + SIRT1 0.10 HIF-lo VI-IL WS 0.05 0.00 Vehicle El it 4-14° HIF-lo TFAM Input SIRT1 24 24 48 48 1KO(h) PatYc Tubulin TFAM (h) 24 - 48 H J K 1.2 2.5 O Empty 2.5 O Empty . • c-Myc OE '• PGC-1a OE c-Myc binding site (consensus sequence) 1.0 • i S$ 2.0 2.0 • CACGTG 0.8 • ..5. 1.5 ▪ 1.5 • NRF.1 NRF.2r . 0 0.6 ae -1340 O I I TFAM I LL a a r S OA 0.2 • i l 1.0 U. To I- '!" 0.5 21 1 0 • n.xx_ FL' 0.5 • 0.0 0.0 0.0 shNT shMyc shMyc Full Ac-Myc Full Ac-Myc #1 #2 length length L M N 0 TFAM Promoter TFAM Promoter TFAM promoter Vehicle SIRT1 iKO 300 shNT shHIF-to 2.0 O Vehicle Vehicle SIRT1 IKO 1.5 ChIP: c•Myc !! 9o 200 250 I • SIRT1 iKO ChIP: c-Myc ••• • • • #' 8.; 1.0 ChIP: HIF-lo 150 ° 6 = 100 ChIP: IgG ChIP: IgG E 0.5 u. 50 0.0 Input Chip: 0 c-Myc HIF-lo Input 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 for 1 week with NMN, a precursor to NAD' that increases activated by SIRT1 (Canto et al.. 2009; Gerhart-Hines et al.. NAD' levels in vivo (Yoshino et al.. 2011), reversed the decline 2007: Rodgers et al.. 2005), whereas under basal conditions, in VHL and accumulation of HIF-la (Figures 7E and 79; reduced acetylation status is primarily regulated by the acetyltransferase lactate levels: and increased ATP, COX activity, and mitochond- GCN5 (Femandez-Marcos and Auwerx. 2011). We speculated rially encoded OXPHOS transcripts (Figures 7G-71 and S6D). In that the biphasic decline in OXPHOS subunits (in Figure 1L) EgIN1 and SIRT1 iKO mice, however, NMN failed to induce mito- might be due to AMPK. In time course experiments following chondrially encoded genes or to raise ATP levels (Figures 7J-7L). SIRT1 deletion, AMPK activation occurred after 48 hr, well after Knockdown of NMNAT1 also prevented NMN from inducing the decline in VHL-TFAM and mitochondria! genes (Figures 1L- mitochondrially encoded OXPHOS genes (Figure 7M), consis- 1M and 6A) but coincident with the decline in nuclear-encoded tent with nuclear HAD* being a key regulatory molecule. The OXPHOS genes and mitochondria! mass (see Figures 1L-1M). SIRT1 iKO and the 22-month-old mice had increased levels of An AMPK dominant-negative adenovirus (AMPK-DN) prevented markers of muscle atrophy and inflammation compared to young the decline of nuclear OXPHOS mRNAs at 48 hr (Figures 6B and WT mice, along with impaired insulin signaling and insulin-stim- 6O), whereas forced maintenance of TEAM prevented AMPK ulated glucose uptake (Figures S1G-SIJ and S6E-S6H). Strik- activation (Figures 6D, 5D, and 5E). Together. these results ingly, treatment of old mice with NMN reversed all of these strongly suggest that AMPK is the switch between the biochemical aspects of aging and switched gastrocnemius PGC-la-dependent and -independent pathways. In this model. muscle to a more oxidative fiber type (Figures S6E-56H). How- AMPK activation occurs in the absence of SIRT1 only when ATP ever, we did not observe an improvement in muscle strength levels fall below a threshold. Consistent with this, AMPK was un- (data not shown). indicating that 1 week of treatment might not changed under fed conditions in the SIRT1 iKO mice and 22- be sufficient to reverse whole-organism aging and that longer month-old wild-type mice but was markedly increased in fasting treatments might be required. animals. when we observe changes in both nuclear- and mito- chondrially encoded OXPHOS genes (Figure 6E and 69. DISCUSSION Increasing NAD' Levels Restores Mitochondrial Impairment in mitochondria' homeostasis is one of the hallmarks Homeostasis through the SIRT1-HIF-12-c-Myc Pathway of aging that may underlie common age-related diseases (Lanza CR is known to delay numerous diseases of aging in mammals, and Nair. 2010; Wallace, 2010). Despite its importance, there is including cancer and type 2 diabetes. Interestingly, CR (30%- still controversy as to why mitochondria' homeostasis is dis- 40% instituted at 6 weeks) completely prevented the decline in rupted with age and whether this process can be slowed or VHL and the increase in HIF-la that occurs in ad-libitum (AL)- even reversed. Here, we present evidence fora PGC-1/./6-inde- fed 22-month-old mice (Figure 7A). The observed decreases in pendent pathway that ensures OXPHOS function and mainte- HAD. and ATP levels. COX activity, mtDNA. and mitochondrially nance of mitochondria! homeostasis (Figure 7f1). During aging, encoded OXPHOS components with age were also prevented however. decline in nuclear energetic state or NAD' levels re- by CR (Figures 7B-7D, S6A, and S6B). Unlike the accumulation duces the activity of SIRT1 in the nucleus, causing VHL levels of mutations in mtDNA, the pathway that we describe here to decline and HIF-la to be stabilized. This program, which likely should be rapidly reversible. Treatment of 22-month-old mice 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 0r 48 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 inSIRTI 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 C 0 nc-encoded U ni-encoded 1A 1 1.2 lime of SIRTI excision (hours) SIRT1 0(0 4811 1.0 Vehicle SIRTI IKO + 6 12 24 48 48h AMPK-DN g a. 0.8 E m 0.6 • lime *ma p-AMPK p-ACC 0.4 0.2 AMPK ACC 0.0 V hide SIRTI iK0 S RT1 ilt0 48h 48h AMPK-ON D E F Vehicle • • Fed Fasted SIRTI 24 24 48 48 KO (h) SIRTI SIRT1 WT IKO WT 6 mo 22 mo TFAM (h) 24 - - 48 p-AMPK MEIr eaeo p-AMPK p-AMPK 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 have evolved to coordinate nuclear-mitochondrial synchrony in mice, inducing a pseudohypoxic state that disrupts OXPHOS, a response to changes in energy supplies and oxygen levels, phenomenon that is consistent with antagonistic pleiotropy (Wil- and its decline may be a conserved cause of aging. In liams and Day. 2003). C. elegans. HIF-12 is known to be a key determinant of lifespan, One of the more surprising findings is the existence of a SIRT1- though its precise role is still a matter of debate (Leiser and mediated pathway that regulates mitochondria independently of Kaeberlein, 2010). HIF-12 modulation may have differential PGC-12/6 The data indicate that SIRT1 can regulate these two effects on lifespan depending on the animal's diet or whether pathways in response to the energetic state of the cell. Which the mtUPR is activated (Dillin et al., 2002: Durieux et al.. 2011; one predominates depends on AMPK activity and the phosphor- Houtkooper et al.. 2013). Though we did not detect mUPR in ylation status of PGC-12 (Canto et al., 2009). skeletal muscle, we do not exclude the possibility that mtUPR This study shows that HIF-12-induced metabolic reprogram- plays a role in other tissues or under different conditions. ming occurs in normal tissue and that it disrupts mitochondria' Additional studies will be required to elucidate complex feed- homeostasis. We consider the metabolic state of the old mice back loops that likely regulate the SIRT1-HIF-12-Myc-TFAM as pseudohypoxic because the downstream effects are similar pathway. For example, in cancer cells, SIRT1 directly regulates to hypoxia but occur even when oxygen is abundant, as previ- c-Myc transcriptional activity, either by deacetylation of c-Myc ously in type 2 diabetes and cancer (Ido and Williamson. 1997: (Menssen et al.. 2012) or by binding c-Myc and promoting its Sanders. 2012: Williamson et al., 1993). An interesting implication association with Max (Mao et al., 2011). Given that SIRT3 and is that reprogramming of normal tissue toward a Warburg-like SIRT6 also regulate HIF-la and compromise respiration (Bell state may increase ROS and establish a milieu for subsequent et al.. 2011; Finley et al.. 2011; Zhong et al.. 2010), it will be inter- mutations to initiate carcinogenesis, a possibility that may help esting to test whether a decline in the activity of other sirtuins explain why cancer risk increases exponentially with age. causes a similar loss of TFAM and mitochondrially encoded All of the main players in the nuclear NAD+-SIRT1-HIF- OXPHOS components. 12-0XPHOS pathway are present in lower eukaryotes, indicating How broadly applicable might these findings be? High-fat diet that the pathway evolved early in life's history. This pathway may 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 B C D • AL — 500 0 AL • CR 1.0 2.5 6 mo AL 22 moAL 22 mo CR IN CR < '§ 400 Z2 0.8 II as 2.0 VEIL E E 300 -I 1.5 0.6 HIF-lo • ga 200 :33 1.0 61 0.4 b cc 100 0.5 0.2 • Tubulin z 0 0.0 0.0 6 22 22 6 22 22 6 22 22 Age (months) Age (months) Age (months) E F G H 0 PBS • NMN 6 months 22 months 0 PBS ■ NMN 0 PBS ■ NMN — 400 — 0.6 2.0 PBS NMN PBS NMN •-8 0.5 <2 000 a .9 1.5 E cr, 0.4 - CC ta VHL E I 200 - , 0.3 n 1.0 HIF-lo — 0.2 • O 100 - E EE Z s 0.1 Tubulin uommoopimps. CO aSell. 0 0.0 6 22 6 22 6 22 Age (months) Age (months) Age (months) J K L 0 PBS • NMN 0 PBS MI MAN 0 PBS ■ NMN 0 PBS • NMN 2.0 4.0 3.0 cif E 1.5 a -§ 3.0 2 2.5 z .2 et se E se, 2.0 oh t 1.o 2.0 a° 0.5 to 2 • E C Ei 0.5 0 0.0 0.0 0.0 6 22 WT EgIN1 KO WT EgIN I KO WT SIRT1 KO Age (months) M D Vehicle • NMN N CRI NMN { Nuclear NAD Aging 2.0 Low energy supply High energy HIF to supply Stabilization Mitochondria' Biogenesis shNT shNMNAT1 Loss of Promoter TFAM 4 1O 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 TFAM promoter mutants were generated using a commercially available kit decline in mitochondria' gene expression (Krishnan et al.. (Stratagene) accordng to the manufacturer's instructions. Details about these methodologies and the prenera used for the mutagenesis can be found in the 2012). Moreover, insulin-resistant human skeletal muscle has a Extended Experimental Procedures. signature reminiscent of hypoxia (Ptitsyn et al.. 2006). In SIRT1 iKO mice. specific dysregulation of mitochondria' OXPHOS Generation of Primary Myoblasts, Rho° Cells, Cell Culture genes is also observed in the heart, demonstrating that the Treatments, Adenoviral Infections, and Gene Silencing pathway is relevant not only to skeletal muscle (Figure 51K- Primary myoblasts were isolated from PGC-17.18 KO. SIRT1 iKO. and FCC-Ix SOO, but not in liver, WAT, or brain. In these tissues, other null mice as described before (Price et al.. 2012). Fttro0 cells were generated factors such as SIRT3 or SIRT6 may be responsible for regula- 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 tion of HIF-1x, or the metabolic status of the tissue at the time (Sigma-Aldrich) for 4 weeks to deplete mitochondrial DNA. Cels were treated of harvest may also be critical. Current dogma is that aging is with the NMN (Sigma-Aldrich) and DMOG (Cayman) as described in the figtre irreversible. Our data show that 1 week of treatment with a legends. and details can be found in the Exterdec zxpenmental Procedures. compound that boosts NAD' levels is sufficient to restore the Gene silencing was achieved using pLX0.1 shRNAs for the genes of interest. mitochondria' homeostasis and key biochemical markers of as described before (Comes et al.. 2012). and was used after selection with eu- muscle health in a 22-month-old mouse to levels similar to a karyotic mancer of the vector. Details about these methodologies can also been found in the Extended Expenrnental Procedures. 6-month-old mouse. Although further work is necessary, this study suggests that increasing NAD' levels and/or small Mitochondrial Function compounds that prevent HIF-la stabilization or promote its Cytochrome c oxidase and succinate dehydrogenase measurements were degradation might be an effective therapy for organismal decline determned pollarographically as described before (Comes et al.. 2012). Cyto- with age. In summary, these findings provide evidence for a chrome c oxidase was also measured spectrophotometrically using a new pathway that controls carbon utilization and OXPHOS commercialy available Mil (Sigma-Aldrich) according to the manufacturer's independently of PGC-1,1, a pathway that goes awry over time instructions. ATP content was measured with a commercial Mil Oloche) according to the manufacturer's instructions. Mitochondria] membrane poten- but is readily reversible, with implications for treating aging and tial and reactive oxygen species were measured by FACS as described (Sell age-related diseases. 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 EXPERIMENTAL PROCEDURES in the Extended Experimental Procedures. Aging Cohorts, SIRT1-180, EgINI KO, and SIRTI-Tg Mice and Gene Expression and mtDNA Analysis NMNATI Electroporation Total RNA and genornic DNA were isolated using a commercially available kit Wild-type C5781.J6J race were from the National Institutes of Agng. NIH. ((DAGEN) according to the manufact.rer's instructions. cDNA was generated EgNill KO. SIRTI-E0. and SIRTI-Tg mice were described previously (Mina- using the iScript kit (BioRad). Gene expression and mtDNA were determined mishima et al.. 2006: Price et al.. 2012). For NMN experiments. mice were by qPCR as described (Puce et a[.. 2012). Details and primer sequences can given IP injections of 500 mg NMN/kg body weight pee day or the equivalent be found n the Extended Experimental Procedures. 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 Coimmunoprecipltation was approved by Institutional Animal Care and Use Committees (1ACUCs). 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. Adenovirus Generation and Mutagenesis 50 mM NaCI. 0.5 mM EDTA. 50 mM Tris-HCI (pH 7.4l) supplemented with Adenoviruses were generated as described before (Rodgers et al.. 2005) and protease inibitcr cocIdail(Racheland 25 U/mlendcnuclease(Pierce). Endog- were used to infect cells for the time points described in the figures. HIF-la and 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 HIF-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-1a-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 factor-dependent extension of the replicative He span during hypoxia. Mol. control. Imunoprecipitated proteins and input were run on SOS-PAGE and Cell. Biol. 27. 5737-5745. were revealed with anti-HIF-1 (Cayman) and anti-c-Myc (Abeam) antibodies. Bel. E.L. Emerling. S. Rican, S.J.. and Guarente. L (2011). SirT3 Details can be found in the Extended Experimental Procedures. suppresses hypoxia inducible factor Is and tumor growth by inhibiting mito- chendrial ROS production. Oncogene 30.2986-2996. Chromatin Immunoprecipitation and Immunobtots Berger. F.. Lau. C.. Dahlman. M.. and Ziegler. M. (2005). Subcellular compart- Chin:mate) immunoprecipitation was performed using a commercially avail- mentation and differential catalytic properties of the three human nicotinamide able kit (Millipore) according to the manufacturer's instructions and using mononucleotide adenylybransfenase doh:rms. J. Biol. Chem. 280. 36334- anti-HIFI: (Cayman). anti-c-Myc (Abeam). and anti-IgG as control. Immuno- 36341. blots were performed as described (Price et al.. 2012). and details can be found in the Extended Experimental Procedures. Brady, N.. Guillemin. O.J.. Mansour. H.. Chan-Lng, T.. Pollak. A.. and Grant. R. (2011). 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Supplemental Information ncludes Extended Experimental Procedures, six Ogees. and one table and can be found with this article online at http://dx. Cohen. H.Y.. Miller. C.. Merman. K.J.. liekkrig. B.. Kessler. 8.. doi.org/10.10161.cell.2013.11.037. Howie. K.T.. Gorospe. M.. de Cabo. R.. and Srclair. (2004). Calorie restriction promotes mammalian cell sunrrval by riducirg the SIRT1 deacety- ACKNOWLEDGMENTS lase. Science 305.390-392. Dang. C.V. (2012). Links between metabolism and cancer. Genes Dev. 26. The Sinclair lab es supported by the NIH/NIA. the Glenn Foundation for Medical 877-890. Research. the United Mitochondrial Disease Foundation. the Juvenile Dia- Dillin. A.. Hsu. A.L. Arantes-Olivera. N.. Lehrer-Graiwer. J.. Hs.). H.. Fraser. betes Research Foundation. and a gift from the Schulak family. M. was A.G.. Kamath. R.S.. Ahringer. J.. and Kenya). C. (2002). Rates of behavior supported by the Portuguese Foundation for Science and Technology and aging specified by mitochondria! function during development. Science (SFRH/80/44674/ 2008) and M. by an NSERC PGS-D fellowship. M. es 298.2398-2401. supported by an Australian Research Council Future Fellowship. We are Durieux. J.. Wolff. S.. and Dillin. A. (2011). The cell-non-autonomous nature of grateful to Michael Bonkowski. Carlos Daniel de Magarees Frlho. Meghan electron transport chain-mediated longevity. Cell 144. 79-91. Rego. Nikolna Clouts. and David Zhang for technical advice and experi- mental assistance: William Kasai) Jr. for kndly providing the EON! 1(0 mice: Fernandez-Marcos. and Auwerx. J. (2011). Regulation of PC1C-1:. a Daniel Kelly. John Fturrisay. and Teresa Leone for unpublished PGC-1,10 KO nodal regulate.' of mitochondrial biogenesis. Am. J. Gin. Nutt 93. 884S-90. myoblasts and advice: Bruce Spiege&nan for PGC-1: null myoblasts and Finley. L.W.. Carraceclo. A. Lee. J.. Souza. A..Egia. A.. Zhang. J.. Teruya-Feld- advice: and Pere Repave( and Zachary Gerhart-Hines for a SIRT1 stein. J.. Mortara... Cardoso. M. Chsh.C.B.. et al. (2011). SIRT3 opposes adenovirus. M. is a consultant to Cotter. OvaScience. HorizonScience. reprogramme); of cancer cell metabolism through HIFIit destabilization. Segterra. Metro8iotech. and GiaxoSmithKline. Cohbar. MetroBiotech. and Cancer Cell 19.416-428. GiaxoSmitN0ine work en mitoctiondrially derived peptides. NADI. and sirtuin Geng. H.. Harvey. C.T.. Pittsenbarger. J.. Liu.0.. Xue. C..and Qian. modulation. respectively. (2011). HDAC4 protein regulates HIFI% protein lysine acetylation and cancer cell response to hypoxia. J. Biol. Chem. 286.38095-38102. Received: September 18.2012 Revised: October 25. 2013 Gerhart-Hines. 2. Rodgers. J.T.. Bare. O.. Lorin. C.. Kim. S.H.. Mostoslaysky. Accepted: November 21.2013 R.. Alt. F.W.. Wu. Z.. and Puigserver. P. (2007). 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