DYSO
Our group has a long experience in the study of organelles, and particularly mitochondria, under different aspects:
1. Mitochondrial biogenesis during hepatogenic differentiation of human stem cells
A more recently developed research axis is devoted to investigate the link between mitochondria and cell differentiation, as mitochondrial biogenesis and metabolism have recently emerged as important actors of stemness and differentiation (Wanet et al, Stem Cells Dev 2015). We have first characterized the mitochondrial changes occurring during the hepatogenic differentiation of human bone marrow-mesenchymal stem cells (hBM-MSCs) and highlighted a strong mitochondrial biogenesis characterized by an increase in mitochondrial content, in mtDNA abundance, in several proteins of the ETC, and in OXPHOS activity, capacity and efficiency, accompanied by a net fragmentation of the mitochondrial network (Wanet et al, Int J Biochem Cell Biol 2014).
We then investigated the molecular mechanisms underlying the interplay between mitochondrial biogenesis and hepatogenic differentiation. A new regulatory axis between Wnt-TCF7L2 and PGC1-a, the master regulator of mitochondrial gene expression was demonstrated (Wanet : The Transcription Factor 7-Like 2-Peroxisome Proliferator-Activated Receptor Gamma Coactivator-1 Alpha Axis Connects Mitochondrial Biogenesis and Metabolic Shift with Stem Cell Commitment to Hepatic Differentiation et al, 2017 ).Translational regulatory mechanisms of hiPSCs hepatogenic differentiation are currently under investigation (M. Caruso et al, submitted).
2. Encapsulation of iPSC differentiated into beta cells for type I diabetes therapy
A multidisciplinary and collaborative (Prof. Bao Lian Su, CMI, UNamur) research is currently ongoing to set up a potential cell therapy for Type I diabetes. The purpose is to combine human iPSCs in vitro differentiation into beta-like cells with customized encapsulation. Specifically, the two-layers capsules must encounter specific properties of porosity, mechanical resistance, and biocompatibility (Leroux G et al., ACS Appl Mater Interfaces. 2018; Leroux G et al., Colloids Surf B Biointerfaces. 2021)
3. Study of the role of mitochondria in the regulation of pluripotency states
Two different states of pluripotent stem cells have been described: the naïve embryonic stem cells (ESC), found in the inner cell mass (ICM) of the pre-implantation embryo, and the primed ESC, located in the epiblast of the post-implantation stage. Although closely related chronologically speaking, these two stages are fundamentally different in terms of gene expression, epigenetics and metabolism, among others. Taking advantage of recently published functional CRISPR-Cas9 screens, we studied the role of the heme biosynthesis pathway (partially mitochondrial) in the naïve-to-primed transition (D. Detraux, submitted manuscript).
4. Study of the mitochondrial co-translational import in mammals
Most of mitochondrial proteins are synthetized in the cytosol before being imported into mitochondria following a post-translational import mechanism. Evidence mostly acquired in yeast suggest the existence of a mitochondrial co-translational import involving Tom20. Using BioID proximity labelling, we have characterized the Tom20 proxisome in human cells, and the results support the existence of a mammalian co-translational import mechanism (with the identification of ribosomal proteins in the close vicinity of Tom20, among others). Using MTS-DHFR reporter constructs, we are currently investigating a potential contribution of several candidates of the BioID to the mitochondrial co-translational import.
5. Mitochondria and intracellular bacteria
Brucella, once in its host cells, traffics inside a vacuole called BCV (Brucella Containing Vacuole) that is able to interact with several components of the endosomal pathway. Afterwards, Brucella reaches a “permissive niche” sharing some properties and markers with the endoplasmic reticulum and in which bacteria multiply massively. As the role of mitochondria in innate immune response is now recognized (Lobet el al., Biochem. Phamarcol. 2015), we hypothesized that mitochondria might be affected and/or play a role during Brucella infection, intracellular trafficking and/or virulence, we thus study the effects of the bacteria on mitochondria in infected cells and demonstrated a strong fragmentation of the mitochondrial population in infected myeloid and non-myeloid cells (Lobet et al., Sci. Rep., 2018).
6. Mitochondria, obesity and insulin resistance: role of SIRT3
Obesity is associated with increased adiposity and low-grade inflammation and oxidative stress in the white adipose tissues (WAT) in which TNFa and down-regulation of Sirtuin 3 (SIRT3), a mitochondrial deacetylase, plays a major role, respectively. These conditions predispose to insulin resistance, type II diabetes (mellitus), hypertension and cardiovascular complications that are hallmarks of the metabolic syndrome. The pro-inflammatory cytokine is known to control gene expression by post-transcriptional mechanisms through the regulation of several microRNAs (miRNAs) that are short 20-22 nucleotide non-coding RNAs. The aim of our ongoing study is to identify the role of miRNAs that are differentially regulated in adipocytes stimulated with TNFα and to characterize their function in the biology of adipocytes exposed to inflammatory cytokine. The effects of TNFa and SIRT3 in the control of adipogenesis and adipocyte dedifferentiation were also analyzed.
7. Organelle cross-talk : a non lethal endoplasmic reticulum stress or a lysosomal dysfunction triggers mitochondria fragmentation
Alterations of these crosstalks might be involved in several biological processes including the regulation of mitochondrial bioenergetics, control of cell death pathways and more and more seem to play a role in metabolic disorders, cancers and neurodegenerative diseases. We study the mechanisms by which a sublethal ER stress/UPR induced by thapsigargin (a SERCA pump inhibitor) or brefeldin A (an inhibitor of protein transport from ER to Golgi) triggers the fragmentation of mitochondria and the consequences of these changes on the biology of the cell in the adaptive response to the ER stress (Vannuvel et al., J. Cell Physiol. 2013; J. Cell Physiol. 2016).
In addition, other organelle crosstalks between mitochondria and lysosomes were also investigated as recent data suggests that the pathogenesis of NCL (neuronal ceroid lipofuscinoses) is also associated with the appearance of fragmented mitochondria. We showed that fibroblasts that are deficient for the TPP-1 (tripeptidyl peptidase-1), a lysosomal hydrolase encoded by the gene mutated in the LINCL (late infantile NCL, CLN2 form), also exhibit a fragmented mitochondrial network (Van Beersel et al., Biosci. Rep., 2013). While maturation and trafficking of lysosomal enzymes are also affected in cells in mtDNA-depleted cells (Hamer et al., Biol Cell, 2009).
8. Study of the effects of mitochondria dysfunction
To study the impact of mitochondria dysfunction on cultured mammalian cells, we use different cell models presenting a mitochondrial dysfunction caused by chemical inhibitors of the electron transport chain (ETC), by uncoupling agents, by mitochondrial DNA (mtDNA) depletion (rho° cells) or by point mutations in mtDNA (such as MERRF or MELAS mutations) that impair the mitochondrial protein synthesis. We have mainly studied the impact of mitochondrial dysfunction on 3 types of cell responses: the retrograde signalling pathways, the apoptotic response and the metabolism in (pre)adipocytes.
9. Mitochondria and cell irradiation
Cancer as a disease becomes increasingly important in our society and it is estimated that about 50 % of patients will receive radiotherapy sessions. When cells are exposed to radiations, one of the downstream effects is an oxidative stress due to an increase of free radical production (ROS). This state is even stronger for cells preincubated with gold nanoparticles (GNPs), known for their radiosensitizing properties. These ROS engender lesions ranging from DNA to mitochondria. While the mechanisms related to the development and repair of the DNA damages are widely studied, the ones concerning mitochondria remain poorly investigated. Nonetheless, mitochondria are organelles involved in plethora of key cell functions and are known to be harmed by ROS. Defective mitochondria are recycled by mitophagy, a type of autophagy specifically degrading mitochondria. Therefore, in collaboration with Professor Anne-Catherine Heuskin (LARN-NARILIS, UNamur), we study the putative role of mitophagy in cancer cells exposed to GNPs and/or ionising radiations (herein protons). Interferential strategies potentiating the GNPs effect will be explored. The long-term outcome of this research may potentially lead to the reduction of the total dose planned in protontherapy treatment and thereby provide a better quality of life for patients.
10. Autophagy and in the fatty kidney
Obesity became these last decades one of the major health problem worldwide. Indeed, last data from WHO estimates than in 2050, half of the population will be obese. Obesity may be associated with other affections such as type-2 diabetes, cardiovascular disease and the development of chronic kidney disease (CKD). Obesity-induced CKD is characterized by glomerular and tubular lesions which induce, in parallel with a pro-inflammatory and pro-fibrotic environment, a decline in the kidney function. In this regard, proximal tubules are of particular interest because it accumulates lipid droplets specifically leading to their dysfunction. At the cell level, this lipid overload was associated with mitochondrial alterations and perturbation of the autophagic flux. In addition, these features were concomitant with a decline in AMPK activity, a major energetic sensor of eukaryotic cells.
In view of these considerations, in the PhD thesis of Louise Pierre, we study the putative role of the AMPK - autophagy axis in obesity-induced CKD with a particular focus on vacuolated proximal tubules in collaboration with Anne-Emilie Declèves (Molecular Biology Laboratory, University of Mons). The thesis involves the use of mouse primary PTEC cells treated with palmitic acid to mimic lipid overload along with AMPK-KO renal tubule specific mice fed with high fat diet. The outcomes of this research will likely clarify the role of the autophagy pathway in the catabolism of lipid droplets in these cells as well as the contribution of AMPK in this metabolism.
Autophagy and in the fatty kidney