Telomere dysfunction induces metabolic and mitochondrial compromise

Abstract

Telomere dysfunction activates p53-mediated cellular growth arrest, senescence and apoptosis to drive progressive atrophy and functional decline in high-turnover tissues. The broader adverse impact of telomere dysfunction across many tissues including more quiescent systems prompted transcriptomic network analyses to identify common mechanisms operative in haematopoietic stem cells, heart and liver. These unbiased studies revealed profound repression of peroxisome proliferator-activated receptor gamma, coactivator 1 alpha and beta (PGC-1α and PGC-1β, also known as Ppargc1a and Ppargc1b, respectively) and the downstream network in mice null for either telomerase reverse transcriptase (Tert) or telomerase RNA component (Terc) genes. Consistent with PGCs as master regulators of mitochondrial physiology and metabolism, telomere dysfunction is associated with impaired mitochondrial biogenesis and function, decreased gluconeogenesis, cardiomyopathy, and increased reactive oxygen species. In the setting of telomere dysfunction, enforced Tert or PGC-1α expression or germline deletion of p53 (also known as Trp53) substantially restores PGC network expression, mitochondrial respiration, cardiac function and gluconeogenesis. We demonstrate that telomere dysfunction activates p53 which in turn binds and represses PGC-1α and PGC-1β promoters, thereby forging a direct link between telomere and mitochondrial biology. We propose that this telomere-p53-PGC axis contributes to organ and metabolic failure and to diminishing organismal fitness in the setting of telomere dysfunction.

Figure 1. PGC-regulated genes and networks are repressed in telomere dysfunctional tissues

Microarray-based Ingenuity pathway analysis (IPA, left) and RT–qPCR validation of PGC-1α, PGC-1β (middle) and transcriptional targets NRF-1, ERRα, TFAM and PPARα (right) in haematopoietic stem cells (HSC), liver and heart tissues show repression of genes in the PGC network including oxidative phosphorylation (OXPHOS), mitochondrial dysfunction, gluconeogenesis, oxidative stress and Huntington Disease (HD). IPA results are expressed as −log (p-value). ΔΔCt method was used to analyse RT–qPCR data (n = 5–8 per group), t-test was used to calculate the statistical significance and error bars indicate s.e.m.

Figure 2. Telomere dysfunction is associated with reduced mitochondrial DNA content in HSC, liver and heart and impaired mitochondrial function

a, Mitochondrial DNA copy number in HSC, liver and heart (n = 5–8). b, Oxygen consumption rates (OCR) in heart and liver mitochondria in the presence of glutamate/malate (liver, n = 3–5) or pyruvate/malate (heart, n = 3–5). State III was induced by injection of ADP. State IV was induced by inhibition of the ATP synthase with oligomycin and uncoupled respiration rates were determined by injection of 2,4-dinitrophenol (DNP). Antimycin A (AA) was used to determine background, non-mitochondrial OXPHOS, OCR. c, ATP synthesis rates in isolated heart mitochondria driven by complex I (pyruvate /malate (P/M)) and complex II (succinate, S) respiration. The specificity of the measurements is verified by the effect of inhibitors (oligomycin (Olig.), rotenone (Rot.) and antimycin A (AA) (n = 3 per group, duplicate measurements per sample). Succinate-dependent ATP synthesis was determined in the presence of the complex I inhibitor rotenone. d, ATP content in liver and heart tissues was determined by HPLC (n = 5). t-test was used to calculate the statistical significance and error bars indicate s.e.m.

Figure 3. Telomere dysfunction induces cardiomyopathy, defective gluconeogenesis and reduced HSC reconstitution capacity

Telomerase and PGC-1α overexpression improves gluconeogenesis and mitochondrial respiration in G4 mice. a, Decreased fractional shortening (FS %) in 15-monthold G4 mice and signs of end-stage cardiomyopathy (left ventricular diameter (LVD) increase and thinning of left ventricular wall (LVW), n = 5–8 per genotype). b, Glucose levels in mice under fed and starved conditions (n = 10 per genotype). c, Long-term repopulation capacity in competitive transplants was determined using CD45.1- and CD45.2-specific antibodies. Shown is the percentage contribution of donors after 4 months. (n = 8 donors per group, three recipients per donor). d, Overexpression of Tert by adenovirus attenuates gluconeogenesis defect in G4 mice (n = 8 per group). e, Overexpression of PGC-1α attenuates gluconeogenesis defect in G4 mice. (n = 8–10 per group). f, PGC-1α overexpression rescues respiration defect (complex I) in G4 liver mitochondria (n = 5 per group). Student t-test was used to calculate the statistical differences in all assays described and error bars represent s.e.m.

Figure 4. p53 deficiency partially rescues the transcriptional regulation of PGC-1α/β and mitochondrial DNA copy number

a, PGC-1α and PGC-1β expression in liver and heart (n = 4). b, mtDNA quantification in liver and heart (n = 4). c, p53 represses PGC-1α and PGC-1β promoter reporters. G2 Terc p53^+/+ and G2 Terc p53^−/− MEFs were transfected with empty reporter pGL4 or PGL4 containing various reporter fragments (−2.8 and −2.6 kb fragment of mouse PGC-1α and PGC-1β). Controls included the PG13-luc plasmid (containing 13 copies of a synthetic p53 DNA binding site) and MG15-luc (containing 15 copies of a mutated p53 DNA binding site). Shown is the average luciferase value (relative light units, RLU) of three different experiments. d, Chromatin immunoprecipitation (ChIP) showing p53 binding on the promoters of PGC-1α and PGC-1β at indicated sites and at p21 site (positive control). Graphs below show quantitative results in the proximal promoter regions by qPCR (three independent experiments). ΔΔCt method was used to analyse RT–qPCR data and t-test was used to calculate the statistical significance, error bars represent s.e.m.

Figure 5. p53 deficiency rescues gluconeogenesis and doxorubicin-induced cardiomyopathy

a, Glucose levels in mice (top) and in mediumof hepatocytes (bottom, n = 5–8). b, Doxorubicin-induced cardiomyopathy (decreased fractional shortening, FS) in G4 mice can be partially rescued by p53 deficiency (n = 3–5, t-test, error bars represent s.e.m.). c, Proposed model: telomere-dysfunction-induced p53 represses PGCs and induces metabolic and mitochondrial compromise. Other pathways engaged by telomere dysfunction are also involved in mediating PGC repression and mitochondrial dysfunction. Together with classical cellular outcomes of p53 activation (senescence, apoptosis and growth arrest), metabolic and mitochondrial compromise might contribute to functional organ decline in the aged.