PGC-1α affects aging-related changes in muscle and motor function by modulating specific exercise-mediated changes in old mice

The age-related impairment in muscle function results in a drastic decline in motor coordination and mobility in elderly individuals. Regular physical activity is the only efficient intervention to prevent and treat this age-associated degeneration. However, the mechanisms that underlie the therapeutic effect of exercise in this context remain unclear. We assessed whether endurance exercise training in old age is sufficient to affect muscle and motor function. Moreover, as muscle peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) is a key regulatory hub in endurance exercise adaptation with decreased expression in old muscle, we studied the involvement of PGC-1α in the therapeutic effect of exercise in aging. Intriguingly, PGC-1α muscle-specific knockout and overexpression, respectively, precipitated and alleviated specific aspects of aging-related deterioration of muscle function in old mice, while other muscle dysfunctions remained unchanged upon PGC-1α modulation. Surprisingly, we discovered that muscle PGC-1α was not only involved in improving muscle endurance and mitochondrial remodeling, but also phenocopied endurance exercise training in advanced age by contributing to maintaining balance and motor coordination in old animals. Our data therefore suggest that the benefits of exercise, even when performed at old age, extend beyond skeletal muscle and are at least in part mediated by PGC-1α

CB1 receptor blockade counters age-induced insulin resistance and metabolic dysfunction

Funding This work was supported by the BBSRC and Diabetes UK.Peer reviewedPublisher PD

Blunted angiogenesis and hypertrophy are associated with increased fatigue resistance and unchanged aerobic capacity in old overloaded mouse muscle.

We hypothesize that the attenuated hypertrophic response in old mouse muscle is (1) partly due to a reduced capillarization and angiogenesis, which is (2) accompanied by a reduced oxidative capacity and fatigue resistance in old control and overloaded muscles, that (3) can be rescued by the antioxidant resveratrol. To investigate this, the hypertrophic response, capillarization, oxidative capacity, and fatigue resistance of m. plantaris were compared in 9- and 25-month-old non-treated and 25-month-old resveratrol-treated mice. Overload increased the local capillary-to-fiber ratio less in old (15 %) than in adult (59 %) muscle (P < 0.05). Although muscles of old mice had a higher succinate dehydrogenase (SDH) activity (P < 0.05) and a slower fiber type profile (P < 0.05), the isometric fatigue resistance was similar in 9- and 25-month-old mice. In both age groups, the fatigue resistance was increased to the same extent after overload (P < 0.01), without a significant change in SDH activity, but an increased capillary density (P < 0.05). Attenuated angiogenesis during overload may contribute to the attenuated hypertrophic response in old age. Neither was rescued by resveratrol supplementation. Changes in fatigue resistance with overload and aging were dissociated from changes in SDH activity, but paralleled those in capillarization. This suggests that capillarization plays a more important role in fatigue resistance than oxidative capacity

Understanding the function of PGC-1α Isoforms in ß-cell survival and diabetes

Peroxisome proliferator-activated receptor gamma (PPARγ) co-activator 1 alpha (PGC-1α) is a transcriptional co-activator responsible for mitochondrial biogenesis and oxidative metabolism. Many isoforms of PGC-1α have been described in the literature, most of which are shown to function similarly to the canonical PGC-1α protein. Recently, however, a novel isoform of PGC-1α was identified, PGC-1α4. It was shown to have a different, yet complementary function to canonical PGC-1α (PGC-1α1) in muscle. It is also expressed in other metabolically active tissues; however, it is unknown whether it has additional distinct tissue-specific functions. Furthermore, PGC-1α plays an important role in controlling metabolism in pancreatic β-cells and expression of the co-activator is decreased in diabetic islets; however, the role of PGC-1α isoforms in diabetes is unknown. Our objective is to determine whether PGC-1α4 has a unique function in β-cells and whether it plays a role in the pathogenesis of diabetes. We show that stimulation with forskolin, exendin-4 and a cytokine cocktail of TNFalpha, IL-1beta and IFNgamma, induced specific PGC-1α isoforms in β-cells. Following over-expression of these isoforms in INS-1 cells, PGC-1α4 prevented the cleavage of caspase-3 in response to cytokines, suggesting that the novel isoform is uniquely anti-apoptotic. To assess whether PGC-1α isoforms play a role in β-cell survival in vivo, mice with a β-cell specific PGC-1α knockout of all isoforms were subjected to low-dose streptozotocin (STZ) treatment to induce β-cell apoptosis. Unexpectedly, knockout mice were protected from STZ induced hyperglycemia. However, there was no difference in percentage of cleaved caspase-3 positive cells in control versus knockout mice, suggesting no difference in apoptosis. Therefore, PGC-1α4 could be a novel factor important for β-cell survival and over-expression of this unique isoform may protect against the pathogenesis of diabetes.Peroxisome proliferator-activated receptor gamma (PPARγ) co-activator 1 alpha (PGC-1α) est un co-activateur transcriptionnel responsable de la biogenèse mitochondriale et du métabolisme oxydatif. De nombreux isoformes de PGC-1α ont été décrits dans la littérature, dont la plupart ont été démontrés comme fonctionnant de manière similaire à la protéine PGC-1α canonique. Récemment, cependant, un nouvel isoforme de PGC-1α a été identifié, PGC-1α4, possédant une fonction différente mais complémentaire de la protéine canonique (PGC-1α1) dans le muscle. PGC-1α4 est également exprimé dans d'autres tissus métaboliquement actifs, mais il n'est pas connu s'il démontre de nouvelles fonctions dépendantes du tissu. PGC-1α joue un rôle important dans la regulation métabolique des cellules β du pancreas; de plus l'expression du co-activateur est dérégulée dans les îlots de Langerhans de patients diabétiques. Notre objectif est de déterminer si PGC-1α4 a une fonction unique dans les cellules β du pancréas et s'il joue un rôle dans la pathogenèse du diabète. Nous montrons que la stimulation avec la forskoline, l'exendine-4 et un cocktail de cytokines, TNFalpha, IL-1beta et IFNgamma, induit les isoformes de PGC-1α dans les cellules β. Suite à la surexpression des isoformes dans les cellules INS-1, PGC-1α4 empêche le clivage de la caspase-3 en réponse à des cytokines, suggérant qu'uniquement le nouvel isoforme est anti-apoptotique. Pour déterminer si les isoformes de PGC-1α jouent un rôle dans la survie des cellules β in vivo, les souris déficientes de tous les isoforms de PGC-1α, spécifiquequement dans les cellules β, ont été soumises à une faible dose de streptozotocine (STZ) provoquant l'apoptose des cellules β. De façon inattendue, les souris déficientes en PGC-1α ont été protégées contre l'hyperglycémie induite par la STZ. Cependant, il n'y avait pas de différence du pourcentage de cellules en apoptose chez les souris témoins par rapport aux souris déficientes. Donc, PGC-1α4 pourrait être un nouveau facteur important pour la survie des cellules β du pancreas et la surexpression de cette isoform unique, peut protéger contre la pathogenèse du diabète

Identifying Novel Candidate Biomarkers of Early Epithelial Ovarian Cancer

Ovarian cancer (OC) is the fifth leading cause of cancer deaths in women, and is the most lethal gynaecological cancer worldwide, with the most common subtype being epithelial OC (EOC). The asymptomatic nature and limited understanding of early disease hampers research into early-stage OC and culminates in diagnostic methods only capable of detecting malignant markers of OC. Therefore, there is an urgent need for biomarkers of early-stage OC to be identified. The first aim of this thesis was to validate a unique mouse model of early OC development. The homozygous Fanconi anemia complementation group D2 knock-out mouse (Fancd2-/-) develops multiple ovarian tumor phenotypes in a sequential manner as they age. Our group previously identified by immunohistochemistry the purported initiating precursor cells, termed ‘sex cords’, that are hypothesised to progress into EOC in this model. To genetically validate this hypothesis, the sex cords, a late-stage tumor phenotype (tubulostromal adenoma), and Fancd2 wild-type (Fancd2+/+) granulosa cell and stromal controls, were isolated by laser-capture microdissection for downstream genetic investigation. Multiplexed gene expression analyses of ~ 90 selected genes were performed using the Genome Lab GeXP Genetic Analysis System (GeXP). Principal component analyses and unbiased hierarchical clustering of the resultant gene expression data determined that the gene signature profiles within sex cords and tubulostromal adenoma samples indeed clustered together genetically confirming the identity of a precursor lesion in this mouse model. Furthermore, key epithelial OC markers included in the GeXP assay (Pax8, Cdh1 and Muc16) were expressed in both the sex cords and tubulostromal adenoma demonstrating neoplastic transformation was already occurring in the precursor lesion. Despite confirmation of the identity and developmental origin of the precursor lesion (sex cords), an in-depth understanding of the model was limited due to restricted numbers of genes able to be investigated using the GeXP assay, thus unique markers of the precursor lesion were not identified. Therefore, the second aim of this thesis was to comprehensively investigate the genetic signature of the Fancd2-/- model via total and small RNA sequencing of micro-dissected cells of the sex cords, tubulostromal adenomas and the equivalent Fancd2+/+ controls. Sequencing results of matched messenger (m) RNAs and micro (mi) RNA expression in micro-dissected tissue of the phenotypes of interest provided novel information about the development of early EOC and unique transcriptional markers, namely upregulated Brinp2, Slc6a7, Smpd3, Grin2b, Grik4, AW55198, Lrp11 and Mro mRNAs and downregulated miR-122/b-5p, miR-129-5p, miR-138-5p, miR-465a/b/-3p, miR-465c-3p/5p, miR-500-3p and miR-129b-3p miRNAs of the sex cord phenotype through differential gene expression analysis. Whilst tissue markers of EOC are relevant as biomarkers of EOC, the location of the ovaries within the peritoneum prevents their diagnostic implementation without surgical intervention. To circumvent this limitation, liquid biopsy has become paramount for OC diagnosis. However, the detection of early biomarkers of EOC in humans is ultimately hampered by their low systemic concentration and the presence of circulating factors from all other organ systems. Therefore, to bypass detection limitations within systemic circulation, the next aim of this thesis was to leverage the established Fancd2-/- model of early EOC to provide proof of concept that secreted miRNA encapsulated into extracellular vesicles (EV) secreted from the precursor lesion (i.e., sex cords) could be detected in conditioned media from ovary explant culture. Performing RNA sequencing from EV-derived small RNAs isolated from conditioned culture medium resulted in the identification of five common upregulated secreted EV miRNAs in Fancd2-/- mouse ovaries that had sex cords or tubulostromal adenomas present, compared to their age-matched Fancd2+/+ counterparts. Additionally, the small RNA sequencing data of EVs was compared to the sequencing data of the micro-dissected sex cords and tubulostromal adenomas and these identical five miRNAs were also significantly upregulated as compared to the control group. This suggests that these five secreted EV miRNAs (miR-200a-3p, miR-200b-3p, miR-200c-3p, miR-429-3p and miR-375-3p) originate from the sex cord and tubulostromal adenoma cells and therefore provided proof-of-concept that small transformational changes such as the development of a precursor lesion can be detected within secreted EVs. The final aim of this thesis was to determine whether the methodologies adopted for early candidate biomarker discovery in a murine model can be applied to human biopsy specimens and subsequently the relevance of the Fancd2-/- model for identifying novel biomarkers for human EOC. Two serous subtypes of human EOC, early low-grade serous borderline tumours (SBT; as an early phenotype) and high-grade serous ovarian cancer (HGSOC; as a late malignant phenotype) were selected for study. SBT and HGSOC specimens were prepared for matched micro-dissected neoplastic epithelial cells and explant cultures. Total and small RNA sequencing of micro-dissected tissue and small RNA sequencing of EVs from explant cultures were performed as per the mouse studies. This sequencing data resulted in the identification of SBT tissue-specific mRNA and miRNA markers as well as SBT-secreted miRNA markers. Remarkably, comparing the human and mouse sequencing data revealed an overlap in differentially expressed genes between the SBT, HGSOC, sex cords, and tubulostromal adenomas, pertaining to key epithelial markers of EOC (Muc16, Cdh1, Krt7, Krt19, Epcam) and the five secreted EV miRNAs firstly described in the murine studies. This provides strong evidence that these novel candidate biomarkers are relevant to early human EOC. Overall, the results from this thesis confirmed the relevance of the Fancd2 model for human OC investigation and identified novel candidate biomarkers of early EOC that may be applied to both the mouse model and human ovarian cancer.</p

The Validation of a Precursor Lesion of Epithelial Ovarian Cancer in <i>Fancd2-</i>KO Mice

Ovarian cancer (OC) has the highest mortality rate of all gynaecological malignancies. The asymptomatic nature and limited understanding of early disease hamper research into early-stage OC. Therefore, there is an urgent need for models of early-stage OC to be characterised to improve the understanding of early neoplastic transformations. This study sought to validate a unique mouse model for early OC development. The homozygous Fanconi anaemia complementation group D2 knock-out mice (Fancd2−/−) develop multiple ovarian tumour phenotypes in a sequential manner as they age. Using immunohistochemistry, our group previously identified purported initiating precursor cells, termed ‘sex cords’, that are hypothesised to progress into epithelial OC in this model. To validate this hypothesis, the sex cords, tubulostromal adenomas and equivalent controls were isolated using laser capture microdissection for downstream multiplexed gene expression analyses using the Genome Lab GeXP Genetic Analysis System. Principal component analysis and unbiased hierarchical clustering of the resultant expression data from approximately 90 OC-related genes determined that cells from the sex cords and late-stage tumours clustered together, confirming the identity of the precursor lesion in this model. This study, therefore, provides a novel model for the investigation of initiating neoplastic events that can accelerate progress in understanding early OC

The Validation of a Precursor Lesion of Epithelial Ovarian Cancer in Fancd2-KO Mice

No description supplie

PGC-1&alpha;4 Interacts with REST to Upregulate Neuronal Genes and Augment Energy Consumption in Developing Cardiomyocytes

Transcriptional coactivator PGC-1&alpha; is a main regulator of cardiac energy metabolism. In addition to canonical PGC-1&alpha;1, other PGC-1&alpha; isoforms have been found to exert specific biological functions in a variety of tissues. We investigated the expression patterns and the biological effects of the non-canonical isoforms in the heart. We used RNA sequencing data to identify the expression patterns of PGC-1&alpha; isoforms in the heart. To evaluate the biological effects of the alternative isoform expression, we generated a transgenic mouse with cardiac-specific overexpression of PGC-1&alpha;4 and analysed the cardiac phenotype with a wide spectrum of physiological and biophysical tools. Our results show that non-canonical isoforms are expressed in the heart, and that the main variant PGC-1&alpha;4 is induced by &beta;-adrenergic signalling in adult cardiomyocytes. Cardiomyocyte specific PGC-1&alpha;4 overexpression in mice relieves the RE1-Silencing Transcription factor (REST)-mediated suppression of neuronal genes during foetal heart development. The resulting de-repression of REST target genes induces a cardiac phenotype with increased cellular energy consumption, resulting in postnatal dilated cardiomyopathy. These results propose a new concept for actions of the PGC-1&alpha; protein family where activation of the Pgc-1&alpha; gene, through its isoforms, induces a phenotype with concurrent supply and demand for cellular energy. These data highlight the biological roles of the different PGC-1&alpha; isoforms, which should be considered when future therapies are developed

Molecular forms of BMP15 and GDF9 in mammalian species that differ in litter size

Bone morphogenetic protein (BMP15) and growth differentiation factor (GDF9) are critical for ovarian follicular development and fertility and are associated with litter size in mammals. These proteins initially exist as pre-pro-mature proteins, that are subsequently cleaved into biologically active forms. Thus, the molecular forms of GDF9 and BMP15 may provide the key to understanding the differences in litter size determination in mammals. Herein, we compared GDF9 and BMP15 forms in mammals with high (pigs) and low to moderate (sheep) and low (red deer) ovulation-rate. In all species, oocyte lysates and secretions contained both promature and mature forms of BMP15 and GDF9. Whilst promature and mature GDF9 levels were similar between species, deer produced more BMP15 and exhibited, together with sheep, a higher promature:mature BMP15 ratio. N-linked glycosylation was prominant in proregion and mature GDF9 and in proregion BMP15 of pigs, and present in proregion GDF9 of sheep. There was no evidence of secreted native homo- or hetero-dimers although a GDF9 dimer in red deer oocyte lysate was detected. In summary, GDF9 appeared to be equally important in all species regardless of litter size, whilst BMP15 levels were highest in strict monovulatory species