MicroRNA-382 silencing induces a mitonuclear protein imbalance and activates the mitochondrial unfolded protein response in muscle cells

Proper mitochondrial function plays a central role in cellular metabolism. Various diseases as well as aging are associated with diminished mitochondrial function. Previously, we identified 19 miRNAs putatively involved in the regulation of mitochondrial metabolism in skeletal muscle, a highly metabolically active tissue. In the current study, these 19 miRNAs were individually silenced in C2C12 myotubes using antisense oligonucleotides, followed by measurement of the expression of 27 genes known to play a major role in regulating mitochondrial metabolism. Based on the outcomes, we then focused on miR-382-5p and identified pathways affected by its silencing using microarrays, investigated protein expression, and studied cellular respiration. Silencing of miRNA-382-5p significantly increased the expression of several genes involved in mitochondrial dynamics and biogenesis. Conventional microarray analysis in C2C12 myotubes silenced for miRNA-382-5p revealed a collective downregulation of mitochondrial ribosomal proteins and respiratory chain proteins. This effect was accompanied by an imbalance between mitochondrial proteins encoded by the nuclear and mitochondrial DNA (1.35-fold, p < 0.01) and an induction of HSP60 protein (1.31-fold, p < 0.05), indicating activation of the mitochondrial unfolded protein response (mtUPR). Furthermore, silencing of miR-382-5p reduced basal oxygen consumption rate by 14% (p < 0.05) without affecting mitochondrial content, pointing towards a more efficient mitochondrial function as a result of improved mitochondrial quality control. Taken together, silencing of miR-382-5p induces a mitonuclear protein imbalance and activates the mtUPR in skeletal muscle, a phenomenon that was previously associated with improved longevity.

Mitochondrial unfolded protein response, its molecular mechanism and physiological impact

Virginija Jovaisaite

Mitochondria are the main producers of ATP, the energy currency of the cell, and they perform a wide array of other fundamental functions. These organelles are essential not only for cellular metabolism, but also for organismal physiology and lifespan. A recently discovered mitochondrial unfolded protein response (UPRmt) maintains the mitochondrial proteostasis by managing protein quality control (PQC) network during proteotoxic stress. Recent findings show that activation of this repair pathway improves mitochondrial function and is associated with increased longevity. My thesis aims to expand the knowledge about the UPRmt by exploring its molecular mechanism and impact on mitochondrial stress induced longevity. We performed three studies: Identification of two epigenetic UPRmt regulators (chapter 2). In collaboration with Prof. Dillinâs laboratory (UCB, USA), we identified the conserved histone demethylases jmjd-1.2/PHF8 and jmjd-3.1/JMJD3 as positive regulators of UPRmt and longevity in response to mitochondrial electron transport chain (ETC) dysfunction across species. A systems genetics analysis of data from the BXD mouse genetic reference population (GRP) further indicates conserved roles of the mammalian orthologs of these demethylases in longevity and UPRmt signaling. These findings illustrate an evolutionary conserved epigenetic mechanism that determines the rate of aging, downstream of mitochondrial perturbations. Relation between UPRmt and TCA cycle (chapter 3). We investigated the effects of genetic perturbations of the TCA cycle on the UPRmt and lifespan. We found that downregulation of several TCA cycle enzymes induces UPRmt, but they have divergent effects on lifespan. Restricting the knockdown of these genes to early larval development stages had positive effects on longevity, which was dependent on UPRmt. We analysed the transcriptomes of C. elegans with knockdown of these TCA cycle genes and compared them with those obtained after the knockdown of other mitochondrial genes, which are known to induce the UPRmt. We identified a core set of transcripts, which change in common and which likely represent a signature of mitochondrial dysfunction associated with UPRmt induction. Bioinformatic analysis of UPRmt (chapter 4). In two collaborative studies, we investigated the UPRmt using newly collected data from the BXD mouse GRP. In the first study, we quantified the transcriptome and proteome from 40 strains of the BXD mouse GRP on two different diets. Integrated molecular profiles were used to characterize the UPRmt, which shows strikingly variant responses at the transcript and protein level that are conserved between C.elegans, mice and humans. In the second study, whole genomes of BXD GRP strains were sequenced to detect sequence variants, which were then used to find novel associations within a high coverage phenome of various traits. One of the novel associations included a missense mutation in fumarate hydratase that controls variation in the UPRmt in both mouse and C. elegans. In summary, our work characterized novel epigenetic regulators of UPRmt and associated mitochondrial stress induced longevity, described the connection between the UPRmt and the TCA cycle, and illustrated the conservation of UPRmt in a mouse GRP. These results expand the knowledge about the UPRmt and promote further investigation of its role in longevity and mitochondrial diseases.

EPFL2016

Metabolic control of muscle and muscle stem cell function

Hongbo Zhang

Skeletal muscle composed of myofibers and a small amount of muscle stem cells (MuSCs). It plays important roles in energy metabolism. Some of the key metabolites, such as NAD+ and acetyl-CoA were recently found to regulate muscle and MuSCs function. Most of these functions are related to the cellular mitochondria. In my PhD thesis projects, we aimed at exploring the link between NAD+ levels, mitochondrial function, muscle structural homeostasis and MuSCs senescence. We also investigated the impact of acetyl-CoA on MuSCs activation and proliferation, through the control of protein acetylation. These projects mainly focus on three aspects: Rejuvenation of adult stem cells by NAD+ repletion. Aging is accompanied by SCs senescence. However, the initial signaling events mediating SCs senescence are still not well defined. We demonstrate the importance of mitochondrial activity as a pivotal modulator of MuSCs senescence. MuSCs from aged mice have reduced NAD+ levels, mitochondrial content and capacity for oxidative respiration. Importantly, the induction of the mitochondrial unfolded protein response (UPRmt), subsequent to increasing cellular NAD+ with the precursor nicotinamide riboside (NR), prevents MuSCs senescence and improves muscle function and regeneration in aged mice. NR also prevents MuSCs dysfunction in the mdx mouse, a known mouse model of accelerated MuSCs senescence. Extending these observations to other SC pools and on the organism as a whole, we demonstrate that NR delays neural and melanocyte SCs senescence, while also increasing mouse lifespan. Control of MuSCs function by acetyltransferase KAT2A. MuSCs are essential for skeletal muscle homeostasis and repair. The complete mechanism controlling MuSCs maintenance and differentiation, however, remains elusive. We found that the acetyltransferase KAT2A expression is essential for myogenic differentiation of MuSCs. KAT2A loss-of-function in cells blocks MuSCs differentiation, and in mice impairs muscle regeneration after damage. The regulation of KAT2A in MuSCs function might rely on its ability to enzymatically acetylate other proteins, and in particular the transcription factor PAX7. NAD+ repletion improves muscle function in muscular dystrophy. Duchenne muscular dystrophy (DMD) is caused by dystrophin gene mutations that lead to progressive muscle degeneration. Our bioinformatics analysis in mice and DMD patients suggests a role for NAD+ in protecting the muscle from metabolic and structural degeneration. Validating these findings, we reveal that the mdx mouse is characterized by reductions in muscle NAD+ levels, concurrent to increased PARP activity and reduced expression of nicotinamide phosphoribosyltransferase (NAMPT), the key enzymes for NAD+ consumption and biosynthesis. Replenishing the NAD+ pool by dietary NR supplementation benefits muscle function in mdx and mdx/Utr-/- mice, an effect replicated in C. elegans models of DMD. The beneficial effects of NAD+ repletion in muscular dystrophy are pleiotropic and rely on the improvement in mitochondrial function, structural protein expression and on reductions in general PARylation, inflammation and fibrosis. In summary, our work demonstrates the critical role of NAD+ and Acetyl-coA in maintaining muscle and MuSCs function. Repletion of NAD+ levels, by NR administration, and/or boosting the function of acetyltransferase KAT2A might be attractive strategies to maintain muscle homeostasis and MuSCs function in aging and muscular dystrophy.

EPFL2016

Role of metabolism and mitochondria during glucose sensing and glucose-induced dysfunction in human pancreatic beta-cells

Isabelle Chareyron

Glucose sensing and regulation of insulin secretion are the two main functions performed by the pancreatic beta-cell. Together these processes contribute to the tight control of blood glucose in our body. Compromising the ability of the beta-cells to properly secrete insulin can lead to the development of Type 2 diabetes. Mitochondria play an important role in beta-cell function, by integrating nutrients signals and modulating insulin secretion. In this thesis, we aimed at better understanding the role of metabolism and mitochondria during glucose sensing and glucose-induced dysfunction in human pancreatic beta-cells. To that end, we designed three studies looking at different aspects of beta-cell mitochondrial function. In the first one, we developed an in vitro model of glucose overload in human islet. We report an over activation of the mitochondria when returned to basal conditions after a 4 day culture in elevated glucose. Additionally, these mitochondria only poorly responded to nutrient stimulation. Impaired metabolism secretion coupling and insulin secretion indicated a partial loss of beta-cell function, despite the increased mitochondrial activity. After 4 days of elevated glucose treatment, unusually high levels of glycerol-3-phosphate and pyruvate, even in sub-stimulatory extracellular glucose concentrations, were observed. We propose that the accumulation of these metabolites renders beta-cells blind to subsequent glucose stimulation. After removing the glucose stress for 24 hours the mitochondrial energy metabolism was fully restored and metabolism-secretion coupling was normalized. In the second approach, we measured for the first time the matrix pH in primary human pancreatic beta-cells. We showed that the mitochondrial pH in beta-cell is more acidic than in other cell types. Furthermore, in response to glucose, the beta-cell mitochondrial matrix slightly acidifies, possibly due to uptake and metabolism of pyruvate. Lastly, we observed that adenoviruses used to deliver the genetically encoded pH sensor can have an intrinsic impact on mitochondrial pH and calcium signaling. It must be used at low infection units per cell in the study of primary beta-cells. In the third project, we tried to modify the mitochondrial energy metabolism in beta-cells by inhibiting mitochondrial protein synthesis. We used different antibiotics to achieve this, in particular chloramphenicol, known to interfere with the activity of the mitochondrial ribosome. Surprisingly, we observe that when mitochondrial protein synthesis is inhibited, the respiration of insulin-secreting cells (INS-1E cells, human islets) is not affected. Expression of mtDNA encoded respiratory chain subunits is reduced and a mitochondrial unfolded protein response initiated without reducing respiratory rates. This raises the possibility that limited activation of unfolded protein response has a positive effect on the cells. This doctoral thesis provides novel insights into various aspects of mitochondrial behavior in pancreatic beta-cells, both during physiological and pathophysiological conditions. Additional work is needed to better understand the molecular mechanisms underlying the mitochondrial adaptation to nutrient overload and the associated beta-cell dysfunction leading to the accelerated impairment of glucose homeostasis during the development of Type 2 diabetes.

EPFL2019

Metabolic control of muscle and muscle stem cell function

Hongbo Zhang

EPFL2016

Role of metabolism and mitochondria during glucose sensing and glucose-induced dysfunction in human pancreatic beta-cells

Isabelle Chareyron

EPFL2019

Mitochondrial unfolded protein response, its molecular mechanism and physiological impact

Virginija Jovaisaite

EPFL2016