The Effect of Increased Mitochondrial Biogenesis and Activation of PPAR Pathways in a Mouse Model of Aging

Aging is the progressive decline in cellular, tissue and organ function. The mitochondrial theory of aging suggests that the accumulation of mitochondrial DNA (mtDNA) mutations leads to mitochondrial dysfunction, loss of organ function and consequently a decrease in lifespan. This theory is appealing as there is a correlation between age-dependent alterations in mtDNA and an increased risk for developing cardiovascular diseases, neurodegenerative disorders and myopathy. To further investigate the role of mtDNA mutations in aging, the mtDNA mutator mouse, a mouse model with a proof-reading deficient mtDNA polymerase γ (POLG) was created. These mice have a premature aging phenotype and develop hair loss, anemia, kyphosis, sarcopenia, cardiomyopathy and decreased lifespan. This phenotype was associated with an accumulation of mtDNA mutations and mitochondrial dysfunction, suggesting that there is a link between mtDNA mutations, mitochondrial dysfunction and the aging phenotype in mammals. The work presented in this dissertation demonstrates three strategies employed to compensate for mitochondrial dysfunction in aging using the mutator mouse as a model system. We illustrate that increased mitochondrial biogenesis and activation of peroxisome proliferator-activated receptor (PPAR) pathways can improve some aging phenotypes in the mutator mouse. In chapter 2, we show that increased expression of PPAR γ coactivator-1α (PGC-1α), a crucial regulator of mitochondrial biogenesis and function, in muscle of mutator mice increased mitochondrial biogenesis and function, and also improved the skeletal muscle and heart phenotypes of the mice. However, deep sequencing analysis of mtDNA showed that the increased mitochondrial biogenesis did not reduce the accumulation of mtDNA mutations in the mutator mouse but rather caused a small increase. Therefore, our results indicate that increased muscle PGC-1α expression is able to improve some premature aging phenotypes in the mutator mice without reverting the accumulation of mtDNA mutations. Bezafibrate is pharmacological agent that activates peroxisome proliferator-activated receptors (PPARs) and PGC-1α pathways that has been shown to improve mitochondrial function and energy metabolism. In chapter 3 we show that mutator mice treated with bezafibrate for 8-months had delayed hair loss and improved skin and spleen phenotypes. Bezafibrate did not induce global mitochondrial biogenesis/function in mutator mice; instead it increased mostly markers of fatty acid oxidation. Although we observed positive effects, bezafibrate induced hepatomegaly and did not slow the development of sarcopenia or increased the lifespan of the mutator mice. Our results show that despite its toxic effects, bezafibrate improved some aging phenotypes in the mutator mouse. Because increased PGC-1α expression in muscle conferred benefits to mutator mice, in chapter 4 we created wild-type and mutator mice that inducibly and ubiquitously express either PGC-1α or its family member PGC-1β. We found that increased systemic expression of PGC-1β was toxic and caused lethality, however, ubiquitous induction of PGC-1α did not appear to be deleterious. These animals are valuable tools for studying the effects of systemic increases in mitochondrial biogenesis during aging.</p

Mitochondrial DNA transcription regulation and nucleoid organization

Mitochondrial biogenesis is a complex process depending on both nuclear and mitochondrial DNA (mtDNA) transcription regulation to tightly coordinate mitochondrial levels and the cell’s energy demand. The energy requirements for a cell to support its metabolic function can change in response to varying physiological conditions, such as, proliferation and differentiation. Therefore, mitochondrial transcription regulation is constantly being modulated in order to establish efficient mitochondrial oxidative metabolism and proper cellular function. The aim of this article is to review the function of major protein factors that are directly involved in the process of mtDNA transcription regulation, as well as, the importance of mitochondrial nucleoid structure and its influence on mtDNA segregation and transcription regulation. Here, we discuss the current knowledge on the molecular mode of action of transcription factors comprising the mitochondrial transcriptional machinery, as well as the action of nuclear receptors on regulatory regions of the mtDNA

The role of PGC-1 coactivators in aging skeletal muscle and heart

Aging is the progressive decline in cellular, tissue, and organ function. This complex process often manifests as loss of muscular strength, cardiovascular function, and cognitive ability. Mitochondrial dysfunction and decreased mitochondrial biogenesis are believed to participate in metabolic abnormalities and loss of organ function, which will eventually contribute to aging and decreased lifespan. In this review, we discuss what is currently known about mitochondrial dysfunction in the aging skeletal muscle and heart. We focused our discussion on the role of PGC-1 coactivators in the regulation of mitochondrial biogenesis and function and possible therapeutic benefits of increased mitochondrial biogenesis in compensating for mitochondrial dysfunction and circumventing aging and aging-related diseases

Long-term bezafibrate treatment improves skin and spleen phenotypes of the mtDNA mutator mouse.

Pharmacological agents, such as bezafibrate, that activate peroxisome proliferator-activated receptors (PPARs) and PPAR γ coactivator-1α (PGC-1α) pathways have been shown to improve mitochondrial function and energy metabolism. The mitochondrial DNA (mtDNA) mutator mouse is a mouse model of aging that harbors a proofreading-deficient mtDNA polymerase γ. These mice develop many features of premature aging including hair loss, anemia, osteoporosis, sarcopenia and decreased lifespan. They also have increased mtDNA mutations and marked mitochondrial dysfunction. We found that mutator mice treated with bezafibrate for 8-months had delayed hair loss and improved skin and spleen aging-like phenotypes. Although we observed an increase in markers of fatty acid oxidation in these tissues, we did not detect a generalized increase in mitochondrial markers. On the other hand, there were no improvements in muscle function or lifespan of the mutator mouse, which we attributed to the rodent-specific hepatomegaly associated with fibrate treatment. These results showed that despite its secondary effects in rodent's liver, bezafibrate was able to improve some of the aging phenotypes in the mutator mouse. Because the associated hepatomegaly is not observed in primates, long-term bezafibrate treatment in humans could have beneficial effects on tissues undergoing chronic bioenergetic-related degeneration

Bezafibrate improved spleen size and structure of Mut mice.

<p>(A) Spleen weight of 10 month-old mice and (B) picture of spleen from Mut mice (n = 4–6/group). (C) Quantification of cleaved caspase-3 immunostaining in paraffin sections from the spleen of 10 month-old mice (n = 3–4/group). *, <i>P</i><0.05, one-way analysis of variance followed by Bonferroni’s multiple comparison test. (D) H&E staining of the spleen of 10 month-old mice showing the organization of white pulp (purple) and red pulp (pink) (n = 3/group). (E) Results from complete blood cell count in 10 month-old mice showing RBC (red blood cells, x10⁶µl ) (n = 5–6/group) (F) <i>PGC-1α</i> and <i>PPARγ</i> mRNA levels in the spleen of 10 month-old mice normalized to actin. (G) Quantification of western blot showing mitochondrial protein levels in total homogenate from the spleen of 10 month-old mice normalized to actin. NADH dehydrogenase (ubiquinone) 1β subcomplex subunit 8 (NDUFB8; subunit of complex I), succinate dehydrogenase subunit B (SDHB; subunits of complex II), ubiquinol-cytochrome <i>c</i> reductase core protein 2 (UQCRC2; subunit of complex III), and ATP synthase subunit 5α (ATP5A; subunit of complex V). *, <i>P</i><0.05; **, <i>P</i><0.01, Student’s <i>t</i>-test. Error bars represent the SEM.</p

Bezafibrate induces hepatomegaly and fatty acid oxidation in Mut and WT mice.

<p>(A) Liver weight of 10 month-old mice (n = 4–6/group). (B) H&E staining of the liver of 10 month-old mice showing hepatocytes and central vein (n = 4/group). (C) The level of liver enzymes alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the blood of 10 month-old mice (n = 6/group). (D) Gene expression of <i>PGC-1</i> coactivators and <i>PPARs</i> in the liver of 10 month-old mice normalized to actin (n = 4/group). (E) The mRNA level of markers of fatty acid oxidation in the liver of 10 month-old mice normalized to actin (n = 4/group). Acyl-coenzymeA oxidase 1 (<i>ACOX</i>), cluster of differentiation 36 (<i>CD36</i>), carnitine palmitoyl transferase (<i>CPT1</i>) and short-chain-acyl-coenzymeA dehydrogenase (<i>SCAD</i>). *, <i>P</i><0.05; **, <i>P</i><0.01, ***<i>P</i><0.001, Student’s <i>t</i>-test. Error bars represent the SEM.</p

Increased mitochondrial biogenesis in muscle improves aging phenotypes in the mtDNA mutator mouse

Aging is an intricate process that increases susceptibility to sarcopenia and cardiovascular diseases. The accumulation of mitochondrial DNA (mtDNA) mutations is believed to contribute to mitochondrial dysfunction, potentially shortening lifespan. The mtDNA mutator mouse, a mouse model with a proofreading-deficient mtDNA polymerase γ, was shown to develop a premature aging phenotype, including sarcopenia, cardiomyopathy and decreased lifespan. This phenotype was associated with an accumulation of mtDNA mutations and mitochondrial dysfunction. We found that increased expression of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), a crucial regulator of mitochondrial biogenesis and function, in the muscle of mutator mice increased mitochondrial biogenesis and function and also improved the skeletal muscle and heart phenotypes of the mice. Deep sequencing analysis of their mtDNA showed that the increased mitochondrial biogenesis did not reduce the accumulation of mtDNA mutations but rather caused a small increase. These results indicate that increased muscle PGC-1α expression is able to improve some premature aging phenotypes in the mutator mice without reverting the accumulation of mtDNA mutations