Thierry Arnould

miR-212/132 expression and functions: within and beyond the neuronal compartment

During the last two decades, microRNAs (miRNAs) emerged as critical regulators of gene expression. By modulating the expression of numerous target mRNAs mainly at the post-transcriptional level, these small non-coding RNAs have been involved in most, if not all, biological processes as well as in the pathogenesis of a number of diseases. miR-132 and miR-212 are tandem miRNAs whose expression is necessary for the proper development, maturation and function of neurons and whose deregulation is associated with several neurological disorders, such as Alzheimers disease and tauopathies (neurodegenerative diseases resulting from the pathological aggregation of tau protein in the human brain). Although their involvement in neuronal functions is the most described, evidences point towards a role of these miRNAs in many other biological processes, including inflammation and immune functions. Incidentally, miR-132 was recently classified as a ‘neurimmiR’, a class of miRNAs operating within and between the neural and immune compartments. In this review, we propose an outline of the current knowledge about miR-132 and miR-212 functions in neurons and immune cells, by describing the signalling pathways and transcription factors regulating their expression as well as their putative or demonstrated roles and validated mRNA targets.

Endothelial cell responses to hypoxia: initiation of a cascade of cellular interactions

The origin of several vascular pathologies involves sudden or recurrent oxygen deficiency. In this review, we examine what the biochemical and molecular responses of the endothelial cells to the lack of oxygen are and how these responses may account for the features observed in pathological situations, mainly by modifications of cell-cell interactions. Two major responses of the endothelial cells have been observed depending on the degree and duration of the oxygen deficiency. Firstly, acute hypoxia rapidly activates the endothelial cells to release inflammatory mediators and growth factors. These inflammatory mediators are able to recruit and promote the adherence of neutrophils to the endothelium where they become activated. The synthesis of platelet-activating factor plays a key role in this adherence process. Secondly, longer periods of hypoxia increase the expression of specific genes such as those encoding some cytokines as well as for the growth factors platelet-derived growth factor and vascular endothelial growth factor. The transcriptional induction of these genes is mediated through the activation of several transcription factors, the most important one being hypoxia inducible factor-1. The link between our knowledge of the signalling cascade of the cellular and molecular events initiated by hypoxia and their involvement in several vascular pathological situations, varicose veins, tumor angiogenesis and pulmonary hypertension is discussed briefly.

CREB activation induced by mitochondrial dysfunction is a new signaling pathway that impairs cell proliferation

We characterized a new signaling pathway leading to the activation of cAMP‐responsive element‐binding protein (CREB) in several cell lines affected by mitochondrial dysfunction. In vitro kinase assays, inhibitors of several kinase pathways and overexpression of a dominant‐negative mutant for calcium/calmodulin kinase IV (CaMKIV), which blocks the activation of CREB, showed that CaMKIV is activated by a mitochondrial activity impairment. A high calcium concentration leading to the disruption of the protein interaction with protein phosphatase 2A explains CaMKIV activation in these conditions. Transcrip tionally active phosphorylated CREB was also found in a ρ0 143B human osteosarcoma cell line and in a MERRF cybrid cell line mutated for tRNALys (A8344G). We also showed that phosphorylated CREB is involved in the proliferation defect induced by a mitochondrial dysfunction. Indeed, cell proliferation inhibition can be prevented by CaMKIV inhibition and CREB dominant‐negative mutants. Finally, our data suggest that phosphorylated CREB recruits p53 tumor suppressor protein, modifies its transcriptional activity and increases the expression of p21Waf1/Cip1, a p53‐regulated cyclin‐dependent kinase inhibitor.

Mitochondrial (Dys)function in Adipocyte (De)differentiation and Systemic Metabolic Alterations

In mammals, adipose tissue, composed of BAT and WAT, collaborates in energy partitioning and performs metabolic regulatory functions. It is the most flexible tissue in the body, because it is remodeled in size and shape by modifications in adipocyte cell size and/or number, depending on developmental status and energy fluxes. Although numerous reviews have focused on the differentiation program of both brown and white adipocytes as well as on the pathophysiological role of white adipose tissues, the importance of mitochondrial activity in the differentiation or the dedifferentiation programs of adipose cells and in systemic metabolic alterations has not been extensively reviewed previously. Here, we address the crucial role of mitochondrial functions during adipogenesis and in mature adipocytes and discuss the cellular responses of white adipocytes to mitochondrial activity impairment. In addition, we discuss the increase in scientific knowledge regarding mitochondrial functions in the last 10 years and the recent suspicion of mitochondrial dysfunction in several 21st century epidemics (ie, obesity and diabetes), as well as in lipodystrophy found in HIV-treated patients, which can contribute to the development of new therapeutic strategies targeting adipocyte mitochondria.

Mitochondrial dysfunction induces triglyceride accumulation in 3T3-L1 cells : role of fatty acid beta-oxidation and glucose.

Mitochondrial cytopathy has been associated with modifications of lipid metabolism in various situations, such as the acquisition of an abnormal adipocyte phenotype observed in multiple symmetrical lipomatosis or triglyceride (TG) accumulation in muscles associated with the myoclonic epilepsy with ragged red fibers syndrome. However, the molecular signaling leading to fat metabolism dysregulation in cells with impaired mitochondrial activity is still poorly understood. Here, we found that preadipocytes incubated with inhibitors of mitochondrial respiration such as antimycin A (AA) accumulate TG vesicles but do not acquire specific markers of adipocytes. Although the uptake of TG precursors is not stimulated in 3T3-L1 cells with impaired mitochondrial activity, we found a strong stimulation of glucose uptake in AA-treated cells mediated by calcium and phosphatidylinositol 3-kinase/Akt1/glycogen synthase kinase 3β, a pathway known to trigger the translocation of glucose transporter 4 to the plasma membrane in response to insulin. TG accumulation in AA-treated cells is mediated by a reduced peroxisome proliferator-activated receptor γ activity that downregulates muscle carnitine palmitoyl transferase-1 expression and fatty acid β-oxidation, and by a direct conversion of glucose into TGs accompanied by the activation of carbohydrate-responsive element binding protein, a lipogenic transcription factor. Taken together, these results could explain how mitochondrial impairment leads to the multivesicular phenotype found in some mitochondria-originating diseases associated with a dysfunction in fat metabolism.

MicroRNAs Profiling in Murine Models of Acute and Chronic Asthma: A Relationship with mRNAs Targets

Background miRNAs are now recognized as key regulator elements in gene expression. Although they have been associated with a number of human diseases, their implication in acute and chronic asthma and their association with lung remodelling have never been thoroughly investigated. Methodology/Principal Findings In order to establish a miRNAs expression profile in lung tissue, mice were sensitized and challenged with ovalbumin mimicking acute, intermediate and chronic human asthma. Levels of lung miRNAs were profiled by microarray and in silico analyses were performed to identify potential mRNA targets and to point out signalling pathways and biological processes regulated by miRNA-dependent mechanisms. Fifty-eight, 66 and 75 miRNAs were found to be significantly modulated at short-, intermediate- and long-term challenge, respectively. Inverse correlation with the expression of potential mRNA targets identified mmu-miR-146b, -223, -29b, -29c, -483, -574-5p, -672 and -690 as the best candidates for an active implication in asthma pathogenesis. A functional validation assay was performed by cotransfecting in human lung fibroblasts (WI26) synthetic miRNAs and engineered expression constructs containing the coding sequence of luciferase upstream of the 3′UTR of various potential mRNA targets. The bioinformatics analysis identified miRNA-linked regulation of several signalling pathways, as matrix metalloproteinases, inflammatory response and TGF-β signalling, and biological processes, including apoptosis and inflammation. Conclusions/Significance This study highlights that specific miRNAs are likely to be involved in asthma disease and could represent a valuable resource both for biological makers identification and for unveiling mechanisms underlying the pathogenesis of asthma.

Crosstalk between mitochondrial (dys)function and mitochondrial abundance

A controlled regulation of mitochondrial mass through either the production (biogenesis) or the degradation (mitochondrial quality control) of the organelle represents a crucial step for proper mitochondrial and cell function. Key steps of mitochondrial biogenesis and quality control are overviewed, with an emphasis on the role of mitochondrial chaperones and proteases that keep mitochondria fully functional, provided the mitochondrial activity impairment is not excessive. In this case, the whole organelle is degraded by mitochondrial autophagy or “mitophagy.” Beside the maintenance of adequate mitochondrial abundance and functions for cell homeostasis, mitochondrial biogenesis might be enhanced, through discussed signaling pathways, in response to various physiological stimuli, like contractile activity, exposure to low temperatures, caloric restriction, and stem cells differentiation. In addition, mitochondrial dysfunction might also initiate a retrograde response, enabling cell adaptation through increased mitochondrial biogenesis. J. Cell. Physiol. 227: 2297–2310, 2012.

Functional and morphological impact of ER stress on mitochondria.

Over the past years, knowledge and evidence about the existence of crosstalks between cellular organelles and their potential effects on survival or cell death have been constantly growing. More recently, evidence accumulated showing an intimate relationship between endoplasmic reticulum (ER) and mitochondria. These close contacts not only establish extensive physical links allowing exchange of lipids and calcium but they can also coordinate pathways involved in cell life and death. It is now obvious that ER dysfunction/stress and unfolded protein response (UPR) as well as mitochondria play major roles in apoptosis. However, while the effects of major ER stress on cell death have been largely studied and reviewed, it becomes more and more evident that cells might regularly deal with sublethal ER stress, a condition that does not necessarily lead to cell death but might affect the function/activity of other organelles such as mitochondria. In this review, we will particularly focus on these new, interesting and intriguing metabolic and morphological events that occur during the early adaptative phase of the ER stress, before the onset of cell death, and that remain largely unknown. Relevance and implication of these mitochondrial changes in response to ER stress conditions for human diseases such as type II diabetes and Alzheimers disease will also be considered. J. Cell. Physiol. 9999: XX–XX, 2013.

Connecting Mitochondria, Metabolism, and Stem Cell Fate

As sites of cellular respiration and energy production, mitochondria play a central role in cell metabolism. Cell differentiation is associated with an increase in mitochondrial content and activity and with a metabolic shift toward increased oxidative phosphorylation activity. The opposite occurs during reprogramming of somatic cells into induced pluripotent stem cells. Studies have provided evidence of mitochondrial and metabolic changes during the differentiation of both embryonic and somatic (or adult) stem cells (SSCs), such as hematopoietic stem cells, mesenchymal stem cells, and tissue-specific progenitor cells. We thus propose to consider those mitochondrial and metabolic changes as hallmarks of differentiation processes. We review how mitochondrial biogenesis, dynamics, and function are directly involved in embryonic and SSC differentiation and how metabolic and sensing pathways connect mitochondria and metabolism with cell fate and pluripotency. Understanding the basis of the crosstalk between mitochondria and cell fate is of critical importance, given the promising application of stem cells in regenerative medicine. In addition to the development of novel strategies to improve the in vitro lineage-directed differentiation of stem cells, understanding the molecular basis of this interplay could lead to the identification of novel targets to improve the treatment of degenerative diseases.

CREB activation induced by mitochondrial dysfunction triggers triglyceride accumulation in 3T3-L1 preadipocytes.

Several mitochondrial pathologies are characterized by lipid redistribution and microvesicular cell phenotypes resulting from triglyceride accumulation in lipid-metabolizing tissues. However, the molecular mechanisms underlying abnormal fat distribution induced by mitochondrial dysfunction remain poorly understood. In this study, we show that inhibition of respiratory complex III by antimycin A as well as inhibition of mitochondrial protein synthesis trigger the accumulation of triglyceride vesicles in 3T3-L1 fibroblasts. We also show that treatment with antimycin A triggers CREB activation in these cells. To better delineate how mitochondrial dysfunction induces triglyceride accumulation in preadipocytes, we developed a low-density DNA microarray containing 89 probes, which allows gene expression analysis for major effectors and/or markers of adipogenesis. We thus determined gene expression profiles in 3T3-L1 cells incubated with antimycin A and compared the patterns obtained with differentially expressed genes during the course of in vitro adipogenesis induced by a standard pro-adipogenic cocktail. After an 8-day treatment, a set of 39 genes was found to be differentially expressed in cells treated with antimycin A, among them CCAAT/enhancer-binding protein α (C/EBPα), C/EBP homologous protein-10 (CHOP-10), mitochondrial glycerol-3-phosphate dehydrogenase (GPDmit), and stearoyl-CoA desaturase 1 (SCD1). We also demonstrate that overexpression of two dominant negative mutants of the cAMP-response element-binding protein CREB (K-CREB and M1-CREB) and siRNA transfection, which disrupt the factor activity and expression, respectively, inhibit antimycin-A-induced triglyceride accumulation. Furthermore, CREB knockdown with siRNA also downregulates the expression of several genes that contain cAMP-response element (CRE) sites in their promoter, among them one that is potentially involved in synthesis of triglycerides such as SCD1. These results highlight a new role for CREB in the control of triglyceride metabolism during the adaptative response of preadipocytes to mitochondrial dysfunction.