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Published in final edited form as: Cell Rep. 2015 Aug 20;12(9):1391–1399. doi: 10.1016/j.celrep.2015.07.047

PGC-1α Modulates Telomere Function and DNA Damage in Protecting against Aging-Related Chronic Diseases

Shiqin Xiong 1, Nikolay Patrushev 1, Farshad Forouzandeh 1, Lula Hilenski 1, R Wayne Alexander 1
PMCID: PMC4549794  NIHMSID: NIHMS711805  PMID: 26299964

SUMMARY

Cellular senescence and organismal aging predispose age-related chronic diseases such as neurodegenerative, metabolic and cardiovascular disorders. These diseases emerge coincidently from elevated oxidative/electrophilic stress, inflammation, mitochondrial dysfunction, DNA damage and telomere dysfunction and shortening. Mechanistic linkages are incompletely understood. Herein, we show that ablation of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) accelerates vascular aging and atherosclerosis coinciding with telomere dysfunction and shortening and DNA damage. PGC-1α deletion reduces expression and activity of telomerase reverse transcriptase (TERT), and increases p53 levels. Ectopic expression of PGC-1α coactivates TERT transcription, and reverses telomere malfunction and DNA damage. Furthermore, alpha lipoic acid (ALA), a non-dispensable mitochondrial cofactor, up-regulates PGC-1α-dependent TERT and the cytoprotective Nrf-2-mediated antioxidant/electrophile-responsive element (ARE/ERE) signaling cascades, and counteracts high-fat diet-induced, age-dependent arteriopathy. These results illustrate the pivotal importance of PGC-1α in ameliorating senescence, aging and associated chronic diseases, and may inform novel therapeutic approaches involving electrophilic specificity.

Graphical Abstract

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INTRODUCTION

Cellular senescence and organismal aging are implicated in the onset of chronic diseases of disparate phenotypes with multi-organ dysfunction (Alexander, 2010; Guarente, 2014). Accumulation of senescent cells appears to drive development of multiple age-related metabolic and cardiovascular diseases. Chronic diseases generally share immune, inflammatory, and metabolic abnormalities associated with conditions inducing senescence including oxidative/electrophilic stress, insulin resistance, genomic instability, DNA damage and dysfunction of mitochondria and telomeres (Bachschmid et al., 2013). Vascular aging is an early stage of the development and progression of atherosclerosis, which is correlated with DNA damage and telomere dysfunction and shortening (Wang and Bennett, 2012). The notion that vascular senescence predisposes atherosclerosis informs the possibility that preventing accelerated cellular senescence through pharmacological interventions would be a promising therapeutic strategy by improving cardiovascular health and outcomes generally for other age-related chronic diseases.

Telomere dysfunction and DNA damage are important hallmarks of cellular senescence and organismal aging, which are correlated with development of multiple chronic inflammatory diseases including diabetes, atherosclerosis and sarcopenia (Armanios, 2013; Khan et al., 2012). TERT deficiency in mice exhibits dysfunction and shortening of telomeres, DNA damage, mitochondrial dysfunction and elevated oxidative stress (Sahin et al., 2011). Telomere shortening causes DNA damage during the development of atherosclerosis (Huzen et al., 2011). However, enforced expression of TERT protects mitochondria from oxidative DNA damage (Haendeler et al., 2009). Conditional inactivation of TERT in mice is associated with age-related, multi-organ dysfunction (Jaskelioff et al., 2011). Thus, augmentation of telomerase activity and mitigation of DNA damage via inducing TERT expression are posited to be potential targets for therapeutic interventions in age-related diseases including atherosclerosis.

PGC-1α enables multiple salutary pathways regulating metabolism, oxidative stress resistance, inflammation and mitochondrial biogenesis and function (Eisele et al., 2013; Patten and Arany, 2012; Spiegelman, 2013). PGC-1α deficiency has been implicated in the onset and progression of age-related neurodegenerative disorders (Tellone et al., 2015). Ectopic expression of PGC-1α Drosophila homologue delays aging and prolongs lifespan (Rera et al., 2011). However, the actions of PGC-1α and the mechanisms involved in mitigating mammalian cellular senescence, aging, and related chronic diseases are incompletely understood. We recently described a novel function for PGC-1α as a negative regulator of vascular senescence (Xiong et al., 2013). Functional PGC-1α prevents senescence by enabling transcription of the FoxO1-regulated longevity gene sirtuin 1 (SIRT1) (Xiong et al., 2011). Downregulation of PGC-1α triggered by TERT deficiency results in mitochondrial compromise, revealing a link between nuclear telomere dysfunction and mitochondrial compromise (Sahin et al., 2011). The roles, however, of PGC-1α in governing aging-related nuclear telomere dysfunction and DNA damage are largely unknown.

Here we show that ablation of PGC-1α functionality in ApoE knockout mice results in oxidative/electrophilic stress, telomere dysfunction and shortening as well as DNA damage coinciding with accelerated vascular aging and atherosclerosis. The developed telomere compromise and DNA damage are mainly ascribed to the positive feed-back signaling interplay between TERT reduction and elevated oxidative stress. Further, ectopic expression of PGC-1α as well as its induction by ALA increases expression of TERT, and augments the electrophilic nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf-2)-mediated cytoprotective ARE/ERE signaling, while reducing expression of the DNA damage marker p53. These results are consistent with the notion that PGC-1α is a primary and general negative regulator of conditions associated with aging, and imply a protective role for PGC-1α against aging-related or mimicked arteriopathy. Enabling PGC-1α signaling cascades by ALA or other molecules with similar electrophilic profiles may inform, generally, novel therapeutic strategies for aging-related chronic diseases.

RESULTS

Deletion of PGC-1α in ApoE Knock-out Mice Augments Vascular Aging and Atherosclerosis in the Context of Telomere Dysfunction and Shortening as Well as DNA Damage

Vascular senescence is an early onset of atherosclerosis but causality is not established (Wang et al., 2014). PGC-1α expression is decreased in human atherosclerotic plaque (McCarthy et al., 2013). Others and we demonstrated recently that PGC-1α-deficient mice developed vascular senescence, which occurred mainly in the vascular smooth muscle cells (VSMCs) (Kroller-Schon et al., 2013; Xiong et al., 2013). The findings infer that PGC-1α may protect against aging-related atherosclerosis. The apolipoprotein E (ApoE)-deficient mouse has been the most widely used animal model of atherosclerosis because it exhibits increased sensitivity to oxidative stress and inflammation, and rapidly develops spontaneous atherosclerotic lesions similar to those observed in humans. To investigate the role of PGC-1α in atherosclerosis progression, we therefore developed the PGC-1α−/−/ApoE−/− model. The PGC-1α−/−ApoE−/− mice that were fed a standard chow diet for 18–20 months developed dramatically more severe vascular senescence (Figures 1A and S1A) and atherosclerotic lesions (Figures 1B and S1B) than control PGC-1α+/+ApoE−/− mice. There was no significant difference in atherosclerosis phenotypes in 6-month old PGC-1α−/−ApoE−/− and control mice (Figure S1C). PGC-1α disruption in young (2-month) ApoE−/− mice did not exhibit increased atherosclerotic lesions compared with littermate controls (Stein et al., 2010). Thus, the athero-protective role of PGC-1α appears to be age-dependent. We posited that PGC-1α deletion impacts the function and length of telomeres and DNA damage. As predicted, expression and activity of TERT were significantly reduced in aortas from PGC-1α−/−ApoE−/− mice compared with controls (Figure 1C). PGC-1α deficiency was also associated with enhanced senescence as reflected in elevated levels of p53, a marker of senescence and DNA damage (Figure 1C). Expression of PGC-1α and TERT was markedly decreased in the aortas from 20 month-old mice compared with 12 month-old mice, while p53 levels was elevated in the older group (Figure 1D). The decreased telomerase activity is attributable to reduced TERT expression. Further, vascular senescence progressed with age and inversely correlated with the expression levels of PGC-1α (Figure S1D). Specificity of TERT antibody was corroborated by knockdown efficiency of TERT using increasing doses of SMARTpool TERT siRNA in primary mouse aortic smooth muscle cells MASMs (Figure S1E). Southern blot analysis of terminal restriction fragments (TRF) indicated that telomere length was significantly shortened in PGC-1α-deficient aortas (Figures 1E and S1F) and MASMs isolated from these aortas (Figures 1F and S1G) compared with controls. These data indicate that PGC-1α regulates telomere function and length.

Figure 1. PGC-1α Deficiency Augments Vascular Aging and Atherosclerosis Coinciding with Telomere Dysfunction and Shortening and DNA Damage through TERT Downregulation.

Figure 1

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(A) The aortas from PGC-1α+/+ApoE−/− and PGC-1α−/−ApoE−/− mice (18-month old males, standard diet, n=5) were excised for SA-βG staining.

(B) The aortic arch from PGC-1α−/−ApoE−/− and control mice (18-month old males, n=5) was dissected for examination of atherosclerotic lesion formation.

(C) The aortas from PGC-1α−/−ApoE−/− and control mice (12-month old, n=4) were analyzed for protein expression by western blot (above) or for telomerase activity by TRAP assay (bottom), respectively.

(D) The aortas from PGC-1α+/+ApoE−/− mice of various ages (12 months vs 20 months old, n=4) were isolated for analysis of protein expression and telomerase activity.

(E and F) Telomere lengths of aortas (E) isolated from PGC-1+/+ or PGC-1−/− ApoE+/+ mice or MASMs (F) from PGC-1+/+ or PGC-1−/− ApoE−/− mice were measured by the Southern blot analysis of terminal restriction fragments.

(G and H) Oxidative DNA damage in mice aortas (G) or MASMs (H) was measured by competitive ELISA.

(I and J) DNA strand breaks in individual aortic cells isolated from PGC-1α−/− ApoE+/+ mice aortas (I) or PGC-1α−/−ApoE−/− MASMs (J) were detected by alkaline single cell gel electrophoresis (comet assays). DNA damage extent was shown as the visual scoring units and tail moment.

(K–P) PGC-1α−/−ApoE−/− MASMs were infected with Ad. PGC-1α constitutive active form (PGC-1α-CA) or TERT or control Lac Z for 1 day. Quantitative analysis of senescence was performed using FDG (K). Telomerase activity (L) and telomere length (M) were determined as described in Methods. DNA strand breaks (N) in individual cells were analyzed by comet assays. Genomic DNA was used for analysis of oxidative DNA damage by measuring 8-OH-dG production (O). Protein expression was determined by western blot (P). *P<0.05, **P<0.01, vs. control or as indicated, ns denotes no statistically significant difference. All data represent mean ± SEM. Also see Figure S1.

We speculated that elevated oxidative/electrophilic stress and DNA damage were present in PGC-1α−/−ApoE−/− groups. Primary PGC-1α deficiency resulted in moderate increases of intracellular and mitochondrial reactive oxygen species (ROS) levels (Figures S1H and S1I). 8-hydroxydeoxyguanosine (8-OH-dG) is one of the oxidative DNA damage byproducts and a ubiquitous marker of oxidative stress. We observed that 8-OH-dG production was augmented in PGC-1α-deficient aortas (Figure 1G) and MASMs (Figure 1H) compared with controls. To examine if PGC-1α disruption causes DNA strand breaks, we performed alkaline comet assays, which detect single and double-strand DNA breaks under alkaline conditions. PGC-1α-deficient aortic cells (Figure 1I) and MASMs (Figure 1J) exhibited enhanced DNA strand breaks compared with controls by measurement of comet tail moment. Collectively, these results inform an important role for PGC-1α in mitigating oxidative DNA damage and strand breaks.

Reduction in TERT Expression Is Required for PGC-1α Deficiency-Triggered Telomere Dysfunction and Shortening as Well as DNA Damage

To validate whether TERT reduction directly contributes to effect of PGC-1α ablation, we performed rescue experiments in PGC-1α-deficient MASMs. Enforced expression of a PGC-1α constitutive active form (PGC-1α-CA: PGC-1α-S570A) (Li et al., 2007; Xiong et al., 2013; Xiong et al., 2010) or TERT reversed the established senescence phenotype (Figure 1K) and restored telomerase activities (Figure 1L) and telomere length (Figures 1M and S1J). Further, accumulated DNA damage (Figure 1N) and 8-OH-dG production (Figure 1O) were entirely blocked. Consistently, enforced expression of PGC-1α-CA restored reduced TERT levels and prevented increase of p53 protein levels (Figure 1P). Ectopic expression of TERT blocked p53 upregulation but had no effects on PGC-1α levels (Figure 1P). These findings support the notion that PGC-1α disruption-induced telomere dysfunction and DNA damage are mainly due to TERT downregulation.

Augmentation of Oxidative Stress Is Implicated in PGC-1α Ablation-Triggered Telomere Dysfunction and Shortening as Well as DNA Damage

Levels of oxidative stress are generally opposite directions to telomere dysfunction and DNA damage in PGC-1α-deficient systems. To investigate whether and how elevated oxidative stress per se contributes to the developed phenotypes, we analyzed the effects of PGC-1α depletion under the conditions of normal or quasi-normal levels of ROS. One mM and 5 mM N-acetylcysteine (NAC) treatments were sufficient to fix the oxidative stress to normal or quasi-normal levels in PGC-1α−/−ApoE−/− MASMs (Figure 2A). Even under normal ROS conditions, ablation of PGC-1α enabled senescence, while NAC ameliorated it (Figure 2B). Notably, NAC repression of oxidative stress increased TERT protein levels (Figure 2C), suggesting that PGC-1α disruption-induced oxidative stress may reduce TERT levels possibly via modulating protein stability by a post-translational modification mechanism. Moreover, PGC-1α depletion significantly reduced TERT expression compared with wild type control under conditions of NAC-mediated ROS inhibition (Figure 2C), implying that a ROS-independent transcriptional mechanism is also involved. However, NAC-mediated suppression of oxidative stress in PGC-1α−/−ApoE−/− MASMs did not decrease p53 levels (Figure 2C), implying that the elevated oxidative stress is not the cause of p53 upregulation. PGC-1α loss decreases telomerase activity via a reduction in TERT expression, while NAC-mediated ROS inhibition augments TERT activity due to increased TERT expression (Figure 2D). Further, PGC-1α disruption-induced 8-OH-dG production (Figure 2E) and DNA strand breaks (Figure 2F) are suppressed but not prevented by NAC treatment, indicating the elevated oxidative stress is partially implicated in the accumulation of DNA damage. Under normal ROS conditions, PGC-1α loss still promoted DNA damage (Figure 2F). Phosphorylation of histone H2AX is an indicator of DNA double-strand breaks (DSBs) which lead to development of senescence and aging (Sedelnikova et al., 2004). Phosphorylated H2AX (γ-H2AX) forms γ-foci adjacent to DNA DSB sites (Kinner et al., 2008). Immuno-staining assay revealed that loss of PGC-1α markedly increased numbers of γ-H2AX foci compared with control (Figure 2G) in the absence of NAC. The accumulation of γ-H2AX foci in PGC-1α−/−ApoE−/− MASMs was moderately attenuated by NAC treatment, implying that PGC-1α disruption-triggered DSBs partially depends on the elevated oxidative stress.

Figure 2. PGC-1α Deficiency Leads to Telomere Dysfunction and Shortening as Well as DNA Damage under Normal Levels of Oxidative Stress.

Figure 2

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(A) MASMs from PGC-1α−/−ApoE−/− mice in 12-well culture plates were treated with 1 and 5 mM NAC or control for two days. Measurement of ROS was performed.

(B–D) MASMs from PGC-1α−/−ApoE−/− mice in 6-well culture plates were treated with 1 and 5 mM NAC or control for two days. SA-β-gal activity was analyzed using FDG probes (B). Protein expression was analyzed (C). Telomerase activity was determined (D).

(E and F) MASMs from PGC-1α−/−ApoE−/− mice were treated with 1 and 5 mM NAC or control for two days. 8-OHdG production of genomic DNA was determined by ELISA (E). DNA strand breaks were analyzed by comet assays (F); DNA damage extent was quantified by tail moment.

(G) PGC-1α−/−ApoE−/− MASMs were treated with 1 and 5 mM NAC or control for two days. Immunostaining with anti-γ-H2AX antibody was performed. Representative images show endogenous γ-H2AX foci (green). DAPI staining (blue) indicates nucleic DNA. Scale bar= 20 μm.

(H and I) PGC-1α+/+ApoE−/− and PGC-1α−/−ApoE−/− MASMs were treated with 5 mM NAC or control for two days. Combined γ-H2AX Immuno-staining and telomere FISH experiments were performed. White arrows on the representative images (H) indicate colocalization of γ-H2AX foci (green) and telomeric DNA (red). Colocalized foci are amplified in the right panel. Scale bar= 20 μm. (I) Mean number of both telomeric foci and non-telomeric foci in each group was calculated.

(J) PGC-1α−/−ApoE−/− MASMs were treated with 5 mM NAC or control for two days. Telomere-FISH was performed as described in Experimental Procedures. *P<0.05, **P<0.01, vs. control or as indicated, ns denotes no statistically significant difference. Values are mean ± SEM of n=300.

To analyze whether the developed DNA damage foci occur at telomeres, we performed combined γ-H2AX immune-staining and telomere fluorescence in Situ Hybridization (FISH) experiments using anti-γ-H2AX antibody and Cy3-labelled telomere-specific peptide nucleic acid (PNA) probes. Colocalization between γ-H2AX foci and telomeres indicates that PGC-1α loss in PGC-1α−/−ApoE−/− MASMs induced both telomeric and non-telomeric DNA damage (Figures 2H and 2I). Interestingly, the accumulation of γ-H2AX foci in the presence of 5 mM NAC is mainly telomeric (Figures 2H and 2I), implying that the non-telomeric random γ-H2AX foci caused by PGC-1α deletion are likely ascribed to the elevated oxidative stress. To corroborate whether telomeric DNA damage triggered by PGC-1α loss affected telomere length, we next performed the quantitative telomere FISH analysis. PGC-1α deletion resulted in telomere shortening compared with control in the absence and presence of NAC (Figure 2J). The extent of telomere shortening in PGC-1α−/−ApoE−/− MASMs is attenuated by NAC treatment, suggesting that oxidative stress moderately contributes to telomere shortening. These data indicates that ablation of PGC-1α leads to telomere dysfunction and shortening as well as telomeric and non-telomeric DNA damage partially through the augmented oxidative stress.

ALA Enables PGC-1α-Modulated TERT and ARE/ERE Signaling Cascades

Ectopic expression of TERT in adult and old mice increases health span and life span without increasing cancer incidence (Bernardes de Jesus et al., 2012). We posited that PGC-1α directly modulates TERT expression at a transcriptional level. To test this hypothesis, we analyzed the 3 kb promoter region of the rat, mice and human TERT homologs and found that putative DNA-binding elements of transcription factors including PPAR-gamma, FoxO1, Nrf-2, p53, CREB, ER-α/ERR-α, and YY-1 (Yin and Yang 1) are present and highly and evolutionarily conserved (Figure S2A). Expression of these transcription factors is regulated generally by PGC-1α (Patten and Arany, 2012), implying that PGC-1α could directly enable expression and activities of TERT. In fact, overexpression of PGC-1α-CA in rat aortic smooth muscle cells (RASMs) dose-dependently increased TERT expression and activities, while reducing p53 levels (Figures 3A–3C). The enabled telomerase activity is likely due to elevation of TERT expression. Further, enforced expression of PGC-1α-CA upregulated multiple salutary pathways including Nrf-1, Nrf-2, mitochondrial transcription factor A (TFAM), SIRT1, Heme oxygenase-1 (HO-1), and MnSOD (Figure S2B). Many of these molecules mitigate oxidative/electrophilic stress, inflammation, and metabolic and mitochondrial dysfunction. In particular, Nrf-2 and HO-1 are pivotal for ARE/ERE adaptive electrophilic responses(Vriend and Reiter, 2015). These results intuitively suggest that saving or rescuing PGC-1α expression could have salutary effects on general health.

Figure 3. ALA Enables PGC-1α-Dependent TERT and ARE/ERE Signaling.

Figure 3

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(A–C) Increasing amounts of Ad.PGC-1α-CA or Ad.Lac Z (control) were transduced into RASMs for 24 hours. Protein expression was determined by western blot (A). Telomerase activity was analyzed by TRAP methods (B). Amplification products of telomeric repeats were subjected to electrophoresis on a 10% nondenaturing polyacrylamide gel (C).

(D) RASMs were treated with increasing doses of ALA for 1 day. Protein expression was determined by western blot. Telomerase activity was analyzed.

(E) ApoE−/− mice (6-month old males, n=4~6) were treated by an IP injection of increasing doses of ALA for 7 days. Protein expression and telomerase activity of aortas were analyzed as described before.

(F) MASMs from PGC-1α−/−ApoE−/− and control mice were treated with or without ALA for 1 day. Protein expression and telomerase activity were analyzed. Also see Figure S2.

ALA, an indispensible mitochondrial cofactor (Packer and Cadenas, 2011), upregulates PGC-1α expression, and mitigates development of atherosclerosis (Zhang et al., 2008). The detailed mechanisms are not well understood. We posited that ALA is a potent activator of PGC-1α-modulated TERT and ARE/ERE signaling cascades, thereby inhibiting oxidative/electrophilic stress and DNA damage, while improving telomere function. To test this hypothesis, we examined effects of ALA on PGC-1α-TERT signaling and telomerase activity. We found that 100 μM ALA treatment increased expression of PGC-1α and TERT, while decreasing p53 levels in dose and time-dependent manners in VSMCs (Figures 3D and S2C). ALA treatment exhibited a prolonged stimulatory effect on telomerase activity (Figure S2D), which is consistent with augmented TERT expression. In support of this notion, ALA robustly augmented expression of ARE/ERE signaling molecules including Nrf-2, HO-1 and catalase (Figures S2C and S2E). These data were reproducible in vivo. Intraperitoneal (i.p.) injection of increasing doses of ALA elevated expression of PGC-1α-modulated TERT and ARE/ERE signaling molecules, while decreasing p53 expression (Figures 3E and S2F). Importantly, the augmentation in expression and activity of TERT and ARE/ERE molecules and the reduction in p53 levels were abolished in PGC-1α−/− MASMs, indicating that PGC-1α is necessary for the salutary electrophilic-like effects of ALA (Figures 3F and S2G). These results suggest that ALA exhibits electrophile-like function in its induction of PGC-1α-modulated TERT and Nrf-2-mediated ARE/ERE signaling, which has been known to antagonize electrophilic/oxidative stress, inflammation, aging and cancer (Levonen et al., 2014).

Enabling PGC-1α-Modulated TERT Signaling by ALA Blocks High-Fat Diet (HFD)-Induced Telomere Dysfunction and DNA Damage, and Ameliorates Vascular Aging and Atherosclerosis

The cyclic AMP response element-binding protein (CREB) is an important vasculoprotective transcription factor that generally enables PGC-1α transcription (St-Pierre et al., 2006). Vascular content of CREB is decreased in numerous rodent aging models of atherosclerosis (Reusch and Klemm, 2003). We found that ALA robustly stimulated cAMP production in VSMCs and mice aortas (Figures S3A and 3B). Further, ALA treatment stimulated CREB phosphorylation at Ser133, an activation site for CREB activity, and had a prolonged effect for two days (Figure S3C). However, ALA did not alter CREB expression. The results were reproducible in vivo (Figure S3D). Altogether, ALA enabled PGC-1α transcription possibly via CREB activation.

HFD is a major risk factor for vascular aging and atherosclerotic diseases (Wang and Bennett, 2012). We posited that HFD decreases CREB activity and PGC-1α-TERT signaling, and accelerates vascular aging and atherosclerosis. Feeding a HFD for 2 and 4 weeks decreased CREB phosphorylation but not protein expression, implying that HFD reduces CREB function only (Figures 4A and S3E). However, HFD reduced PGC-1α and TERT expression, while upregulating p53 levels (Figures 4A and S3E). Reduction in expression of PGC-1α, TERT and SIRT1 by HFD was prevented by ALA (Figures 4B–4E). HFD-induced p53 expression was also blocked (Figure 4F). Further, HFD-induced reduction in telomerase activity was prevented by ALA (Figure 4G), which is consistent with changes of TERT expression levels. Elevation of 8-OHdG production by HFD was dramatically blocked in aortas, inferring that ALA mitigated DNA damage. (Figure 4H). Comet assays revealed that the accumulated DNA strand breaks were decreased (Figures 4I and S3F). We next investigated whether ALA ameliorates HFD-induced vascular aging and atherosclerosis. As shown in Figures 4J and 4K, increased vascular aging and atherosclerosis were remarkably inhibited by ALA treatment. Collectively, these results show that ALA ameliorates the accelerated vascular aging and atherosclerosis possibly by blocking HFD-induced reduction of the CREB-PGC-1α-TERT signaling cascade.

Figure 4. ALA-Induced Inhibition of Telomere Dysfunction and DNA Damage Ameliorates HFD-Induced Vascular Aging and Atherosclerosis.

Figure 4

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(A) PGC-1α+/+ApoE−/− mice were fed a HFD or a LFD for 2 and 4 weeks. CREB phosphorylation and protein expression in aortas were determined by western blot.

(B–F) PGC-1α+/+ApoE−/− mice (5~6-month old males, n=3~5) were pretreated with ALA by oral administration for 5 days and fed a low-fat diet containing 4% fat or an atherogenic diet containing 16% fat for 2 weeks. Protein expression was determined by western blot.

(G–I) PGC-1α+/+ApoE−/− mice (7~8-month old males, n=3~5) fed a low-fat diet or an atherogenic diet were orally administrated with ALA for 7 days. Telomerase activity was determined (G). 8-OHdG production was analyzed by ELISA (H). DNA damage extent was analyzed by comet assays, and quantified by the mean tail moment (I).

(J and K) PGC-1α+/+ApoE−/− mice (5~6-month old males, n=6~7) were pretreated with ALA by i.p. injection for 5 days and fed a low-fat diet or an atherogenic diet for 4 weeks. Vascular aging was quantified by FDG assay (J). Atherosclerotic lesions was quantified by ImageJ (K). All data represent mean ± SEM. *P<0.05, **P<0.01, ns denotes no significant difference. Also see Figure S3.

DISCUSSION

PGC-1α ablation in ApoE knockout mice drives telomere malfunction and shortening as well as telomeric and non-telomeric DNA damage, thereby promoting vascular aging and atherosclerosis. The effects of PGC-1α ablation are mediated by TERT reduction and elevated ROS. The developed telomere malfunction and shortening as well as DNA damage in PGC-1α−/−ApoE−/− MASMs were moderately ameliorated by NAC-mediated ROS inhibition, indicating that these outcomes are partially ascribed to the augmented oxidative stress (Figure 2). Moreover, even under conditions of normal ROS levels, the impact of PGC-1α ablation continues to be manifested by extent of TERT downregulation, telomere dysfunction, and telomeric DNA damage. These results indicate that TERT reduction and augmented oxidative stress are the dominant mechanisms underlying PGC-1α deletion-induced telomere malfunction, DNA damage, cellular senescence, and related pathological conditions. In fact, our data demonstrate that NAC inhibition of the elevated oxidative stress increases TERT protein levels (Figure 2C), suggesting that PGC-1α loss-induced ROS also drives TERT downregulation possibly via modulating protein stability. Conversely, depletion of TERT further promotes PGC-1α reduction and ROS production via a positive feed-back loop mechanism (Sahin et al., 2011). These results suggest that a conundrum of chicken-and-egg exists between the feed-back loop interplay between PGC-1α and TERT signaling in protecting against senescence and aging processes.

Here, our data demonstrate that PGC-1α directly increases TERT expression (Figure 3A). Further, we found that there are multiple conserved PGC-1α-coactivated DNA binding elements of transcriptional factors (PPAR-gamma, FoxO1, Nrf-2, CREB, p53, and others) within the rat, mouse and human TERT promoter regions (Figure S2A), informing that PGC-1α modulates TERT expression at a transcriptional level. The detailed identification of the specific transcriptional factors mediating PGC-1α-modulated TERT expression will provide valuable information on the linkage between PGC-1α and TERT signaling. Conversely, ectopic expression of TERT does not affect PGC-1α protein levels (Sahin et al., 2011). These observations suggest that PGC-1α may be a primary and general modulator of TERT expression and aging conditions during development and progression of organismal aging and related pathologies.

We have shown, as have others, that ALA stimulates PGC1-α co-transcriptional function in a broad spectrum of genes associated with health and resistance to disease and aging in animal models of diverse phenotypes. Results of efforts to apply this potential therapeutic strategy in man have been generally unsuccessful. The inability to translate the approach is multifactorial and includes issues concerning patent protection, bioavailability, perceived pharmacokinetic limitations, and particularly, lack of mechanistic insights in models that permit a coherent understanding of the organization of the signaling pathways and endpoints involved, the major problem addressed here. We show that ALA behaves like an electrophile in induction of PGC-1α-modulated TERT and ARE/ERE signaling cascades, and protects against telomere malfunction, oxidative stress and DNA damage, thereby ameliorating HFD-exacerbated vascular aging and atherosclerosis. Taken together, the findings here support the notion that PGC-1α in an appropriate electrophilic environment organizes specific signaling pathways that inform new drug targets and that mitigate premature aging and related chronic diseases.

EXPERIMENTAL PROCEDURES

Extensive experimental details are described in the Supplemental Information.

Mice

ApoE−/− mice (C57BL/6 background) were purchased from Jackson Laboratory (Bar Harbor, ME). PGC-1α−/−ApoE−/− mice were generated and genotyped by crossing PGC-1α−/− mice and ApoE−/− mice. Genotyping of PGC-1α−/−ApoE−/− mice was performed by PCR using tail DNA. The third and fourth generations of offspring were used for senescence and atherosclerosis studies. All studies were done in male mice. All experimental male mice were older than 3 months. Animals with gender- and age-matched littermates were randomly included in experiments. No animals had to be excluded attributed to illness after experiments. Animal experiments and euthanasia protocols were performed in strict compliance with the National Institutes of Health guidelines for humane treatment and approved by the Institutional Animal Care and Use Committee of Emory University.

Detection of Telomerase Activity

Telomerase activity was determined by TRAP (Telomeric Repeat Amplification Protocol) assay (Wright et al., 1995). Briefly, cell pellets were lysed in CHAPS lysis buffer containing ribonuclease inhibitor and incubated for 30 minutes at 4°C. Cell extract (1μg) was mixed with TRAPeze reaction mix containing TS primer, fluorescein-labelled RP (Reverse) primer, control template and sulforhodamine labelled control K2 primer. Extension of TS primers was performed at 30°C for 30 minutes, and followed by PCR amplification. TRAP products from the same experiment were analyzed for quantification of telomerase activity by measuring fluorescence on a fluorescence plate reader. The relative telomerase activity was normalized by the ratio of the net fluorescein to sulforhodamine (internal control) and expressed as a percentage of wild type value. Mean values ± SEM were calculated from three independent assays.

Statistical Analysis

Data were analyzed by two-tailed unpaired Student’s t test for comparisons of two groups or one-way ANOVA of the repeated experiments followed by the Tukey’s post hoc pairwise multiple comparisons when appropriate with Prism 5 (GraphPad Software Inc.). A P value of <0.05 was considered significant. For all bar graphs, the mean ± SEM is plotted. All in vitro experiments were repeated at least 3 times unless otherwise indicated.

Supplementary Material

1
2

Highlights.

  • PGC-1α disruption promotes vascular aging and atherosclerosis

  • PGC-1α modulates telomere function and length as well as DNA damage

  • High-fat diet reduces PGC-1α-TERT signaling to drive vascular aging and arteriopathy

  • Enabling PGC-1α-modulated TERT and ARE/ERE signaling obviates age-related pathology

Acknowledgments

We are grateful to Dr. Z. Arany for providing PGC-1α knockout mice and Dr. D. P. Kelly for PGC-1α antibody. This work was supported in part by the National Heart Lung and Blood Institute of the NIH as a Program of Excellence in Nanotechnology (HHSN268201000043C).

Footnotes

Competing Financial Interests:

The authors declare no competing financial interests.

SUPPLEMENTAL INFORMATION

Supplemental information includes 3 figures and figure legends.

AUTHOR CONTRIBUTIONS

S.X. designed and performed most of the experiments, analyzed the data and wrote the manuscript. N.P. and F.F. performed experiments (Figures 1A, 1B; 4J and 4K) and analyzed data. L.H. analyzed data and helped in proofreading. R.W.A. conceived and supervised the study, analyzed data, critically reviewed, and wrote the manuscript. All authors read and approved the final version of this manuscript.

The authors declare no competing financial interests.

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Associated Data

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Supplementary Materials

1
NIHMS711805-supplement-1.doc (38.5KB, doc)
2
NIHMS711805-supplement-2.pdf (1.3MB, pdf)

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