Mitochondrial Matrix Calcium Is an Activating Signal for Hormone Secretion

Mitochondrial Ca2+ signals have been proposed to accelerate oxidative metabolism and ATP production to match Ca2+-activated energy-consuming processes. Efforts to understand the signaling role of mitochondrial Ca2+ have been hampered by the inability to manipulate matrix Ca2+ without directly altering cytosolic Ca2+. We were able to selectively buffer mitochondrial Ca2+ rises by targeting the Ca2+-binding protein SING to the matrix. We find that matrix Ca2+ controls signal-dependent NAD(P)H formation, respiration, and ATP changes in intact cells. Furthermore, we demonstrate that matrix Ca2+ increases are necessary for the amplification of sustained glucose-dependent insulin secretion in cells. Through the regulation of NAD(P)H in adrenal glomerulosa cells, matrix Ca2+ also acts as a positive signal in reductive biosynthesis, which stimulates aldosterone secretion. Our dissection of cytosolic and mitochondrial Ca2+ signals reveals the physiological importance of matrix Ca2+ in energy metabolism required for signal-dependent hormone secretion.

Metabolic and microenvironmental strategies to accelerate hematopoietic recovery post-aplasia

Vasco Ledebur de Antas de Campos

The procedure-associated mortality rate of bone marrow transplantations (BMT) remains close to 25%. The BMT is therefore only indicated for life-threatening diseases, with no replacement of this technology to be expected in the foreseeable future. Modest improvements such as the use of G-CSF to accelerate hematopoietic recovery significantly reduce mortality. Hematopoietic stem cells (HSC) reside in the bone marrow (BM) and are mainly quiescent, dividing only to self-renew. Via extracellular signals and interactions with other cells of the BM (microenvironment), a stable HSC pool is maintained, while hematopoietic progenitor cells (HPC) proliferate and differentiate to mature blood cell types. Marrow adipocytes (BMA) have recently been recognized as a hematopoietic regulatory entity, although the mechanisms by which it interacts with HSCs and HPCs (HSPC) in vivo, remain unknown. During the hematopoietic recovery period post-BMT, HSPCs increase their metabolism as a response to hematopoietic stress, partly modulated by the microenvironment. In this work we identified novel pharmacological approaches to accelerate hematopoietic recovery in the post-BMT period, addressed via two strategies: (i) by directly modulating HSC metabolism, and (ii) by regulating the differentiation of BMA. Increasing oxidative phosphorylation, combined with an impaired mitochondrial stress response, can severely compromise the regenerative capacity of HSCs. Here we demonstrate that the NAD+-boosting agent Nicotinamide Riboside (NR) reduces mitochondrial activity within HSCs through increased mitophagy, the recycling mechanism of damaged mitochondria. We observed in vitro NR treatment of HSCs to lead to increased asymmetric HSC divisions, which resulted in a significantly enlarged pool of progenitor cells, without HSC exhaustion. Dietary supplementation of NR to mice undergoing BMT accelerated blood recovery and improved survival. We therefore established a novel link between HSC mitochondrial stress, mitophagy and stem cell fate decision. Recent studies have suggested BMA maintain HSCs quiescent, which, may be detrimental to the increased expansion of HPCs in the recovery phase post-BMT. BM stromal cells (BMSC) are the cellular precursors of both adipocytes and osteoblasts, and they have been strongly implicated in supporting hematopoiesis by secreting cytokines such as SCF and Cxcl12. We observed that adipocytes quickly accumulate the marrow space of mice undergoing BMT, followed by an expansion hematopoiesis-supportive multipotent Sca1+/CD24+ stromal cells, coinciding with hematopoietic recovery. We developed a novel screening platform using Digital Holographic Microscopy (DHM), quantifying in vitro adipocytic differentiation non-invasively, allowing for follow-up co-cultures with HSPCs. Using the OP9 BMSC-line, we modulated adipocyte differentiation prior to co-culture using a pharmacological approach and observed that high adipocytic content was associated to a decrease in HPC expansion. We perfomed a screening of over 4000 FDA-approved drugs and natural compounds and identified one to efficiently prevent adipocytic differentiation and maintain the hematopoietic supportive capacity of OP9 stroma. Altogether, this work opens the door to alternative strategies accelerating hematopoietic reconstitution and unveils the potential to improve recovery of patients undergoing BMT.

EPFL2019

Semisynthetic Biosensors for the Quantification of Nicotinamide Adenine Dinucleotides in Live Cells

Olivier Vincent Sallin

Nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) are crucial redox cofactors regulating energy metabolism, reductive biosynthesis and the antioxidant defense system. Besides their role as redox cofactors, NAD(P)s have more recently also been described as important signaling molecules, acting for example as cosubstrate of different enzymes such as the sirtuins and poly(ADP-ribose) polymerases (PARPs), involved in the control of important cellular functions. Highlighting their important role, an imbalance of their intracellular concentration has been associated with aging and different pathologies. Therefore, the development of methods enabling the accurate subcellular quantification of NAD(P) in live cells is of great importance. This thesis describes the development and application of two FRET-based semisynthetic biosensors for NADPH/NADP+ and NAD+ in live cells. These sensors, named NAD(P)-Snifits, possess a high dynamic range, high specificity, are pH insensitive and can be excited at long wavelenghts. By using ratiometric imaging and fluorescence lifetime imaging, we quantified free NADPH/NADP+ and NAD+ in different subcellular compartments in live cells. We report here, for the first time, that free NADPH/NADP+ is significantly higher in the mitochondria than in the cytosol and observed moderately higher NAD+ level in the mitochondrial matrix consistent with previous report. We have also demonstrated that NADP-Snifit can be used to monitor the dynamics of NADPH/NADP+ caused by oxidative stress using conventional widefield fluorescence microscopy. The easy handing and use of these sensors make it possible to study not only the kinetics of cellular H2O2 scavenging and NADPH regeneration rate, but also to monitor drug-related side effects. Finally, we measured the effect of different conditions and drug treatment mimicking calorie restriction on free cytosolic and mitochondrial NADPH/NADP+ and NAD+ by flow cytometry. We observed that all these treatments increased free cytosolic and mitochondrial NAD+ concentrations with various levels of efficiency, but had divergent effects on the different subcellular NADPH/NADP+. NAD(P)-Snifits have the potential of becoming valuable tools to study the NAD(P) metabolism in physiological relevant conditions. Their wide applicability, in particular with high-throughput methods such as flow cytometry opens the possibilities for their use in metabolic and drug screening of large libraries.

EPFL2018

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 and microenvironmental strategies to accelerate hematopoietic recovery post-aplasia

Vasco Ledebur de Antas de Campos

EPFL2019

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

Isabelle Chareyron

EPFL2019

Semisynthetic Biosensors for the Quantification of Nicotinamide Adenine Dinucleotides in Live Cells

Olivier Vincent Sallin

EPFL2018