Identification of Tmem10/Opalin as a novel marker for oligodendrocytes using gene expression profiling

<p>Abstract</p> <p>Background</p> <p>During the development of the central nervous system, oligodendrocytes generate large amounts of myelin, a multilayered insulating membrane that ensheathes axons, thereby allowing the fast conduction of the action potential and maintaining axonal integrity. Differentiation of oligodendrocytes to myelin-forming cells requires the downregulation of RhoA GTPase activity.</p> <p>Results</p> <p>To gain insights into the molecular mechanisms of oligodendrocyte differentiation, we performed microarray expression profiling of the oligodendroglial cell line, Oli-neu, treated with the Rho kinase (ROCK) inhibitor, Y-27632 or with conditioned neuronal medium. This resulted in the identification of the transmembrane protein 10 (Tmem10/Opalin), a novel type I transmembrane protein enriched in differentiating oligodendrocytes. In primary cultures, Tmem10 was abundantly expressed in O4-positive oligodendrocytes, but not in oligodendroglial precursor cells, astrocytes, microglia or neurons. In mature oligodendrocytes Tmem10 was enriched in the rims and processes of the cells and was only found to a lesser extent in the membrane sheets.</p> <p>Conclusion</p> <p>Together, our results demonstrate that Tmem10 is a novel marker for in vitro generated oligodendrocytes.</p

Neuron to glia signaling triggers myelin membrane exocytosis from endosomal storage sites

During vertebrate brain development, axons are enwrapped by myelin, an insulating membrane produced by oligodendrocytes. Neuron-derived signaling molecules are temporally and spatially required to coordinate oligodendrocyte differentiation. In this study, we show that neurons regulate myelin membrane trafficking in oligodendrocytes. In the absence of neurons, the major myelin membrane protein, the proteolipid protein (PLP), is internalized and stored in late endosomes/lysosomes (LEs/Ls) by a cholesterol-dependent and clathrin-independent endocytosis pathway that requires actin and the RhoA guanosine triphosphatase. Upon maturation, the rate of endocytosis is reduced, and a cAMP-dependent neuronal signal triggers the transport of PLP from LEs/Ls to the plasma membrane. These findings reveal a fundamental and novel role of LEs/Ls in oligodendrocytes: to store and release PLP in a regulated fashion. The release of myelin membrane from LEs/Ls by neuronal signals may represent a mechanism to control myelin membrane growth

Molekulare Mechanismen der Biogenese der Myelin-Membran

Während der Entwicklung des Gehirnes von Vertebraten werden Axone durch Oligodendrozyten mit Myelin, einer isolierenden Membranschicht, umhüllt. Unser Ziel ist es die zelluläre Maschinerie, die zum Aufbau dieser Membran benötigt wird, zu verstehen. Da die Entstehung der Myelinscheide von axonalen Signalen abhängig ist, analysierten wir, wie neuronale Faktoren die Produktion dieser Membran regulieren. Unter Verwendung primärer Oligodendrozyten-Kulturen und oligodendroglialer Zelllinien untersuchten wir die Regulation des intrazellulären Transports der Myelinmembran durch Neurone. In der Abwesenheit löslicher neuronaler Signale wird das Proteolipid-Protein (PLP), ein Transmembranprotein, das den größten Anteil der Proteine des Myelins ausmacht, über einem Cholesterol-abhängigen und Clathrin-unabhängigen Weg endozytiert, der das Vorhandensein eines funktionsfähigen Actin-Zytoskeletts und des kleinen G-Proteins RhoA voraussetzt. Anschließend wird PLP in späte Endosomen transportiert, in welchen es sowohl in der äußeren begrenzenden Membran als auch in intraluminalen Vesikeln zu finden ist. Das Protein liegt hierbei in distinkten Mikrodomänen getrennt vom EGF-Rezeptor, einem Marker für Degradation, vor. Wir beobachteten, dass der Transport von PLP ins Innere des späten Endosoms im Gegensatz zu dem des EGFR (Epidermal Growth Factor Receptor) vom ESCRT (Endosomal Sorting Complex Required for Transport) unabhängig ist. PLP wird im späten Endosom bis zum Eintreffen neuronaler Signale gespeichert. Erst beim Vorliegen dieser Signale verändert sich die Verteilung des Proteins entscheidend. Die Endozytose-Rate ist dann herabgesetzt und der Transport von PLP aus dem späten Endosom zur Plasmamembran wird eingeleitet. Unsere Untersuchungen zeigen, dass der Übergang von vorwiegender Endozytose zur Exozytose hin mit einer Reduktion der Aktivität einzelner kleiner GTPasen der Rho-Familie einhergeht. Wir konnten zeigen, dass lösliche neuronale Faktoren diese GTPasen in Oligodendrozyten inaktivieren, was zum vermindert Transport vo n PLP zum späten Endosom und einer erhöhten Dynamik dieser Zellorganelle führt. Weiterhin konnten wir demonstrieren, dass die Regulation des intrazellulären Transportes der Myelinmembran von der Tyrosinkinase c-src koordiniert wird. Die Ergebnisse dieser Untersuchungen zeigen eine bedeutende und neuartige Funktion später Endosomen in Oligodendrozyten, die PLP unter neuronaler Regulation entweder speichern oder aber freigeben. Dieser Mechanismus erlaubt eine zeitlich abgestimmte kontrollierte Entwicklung der Myelinmembran.During vertebrate brain development, axons are ensheathed by myelin, an insulating membrane generated by oligodendrocytes. Our aim is to elucidate the cellular machinery that is required for the formation of this membrane. Since the development of myelin depends on axonal cues, we analyzed how neuronal signals regulate the generation of myelin. Using primary oligodendrocytes and oligodendroglial precursor cell lines as cell culture models, we have investigated the regulation of myelin membrane trafficking in oligodendrocytes by neurons. In the absence of soluble neuronal signals, the major myelin membrane protein, the proteolipid protein (PLP), is endocytosed via a cholesterol-dependent and clathrin-independent pathway, which requires functional actin cytoskeleton and the small guanosine triphosphatase RhoA. Subsequently, PLP is targeted to late endosomes (LE), where it resides both within intraluminal vesicles (ILVs) and on the limiting membrane, and it segregates from the epidermal growth factor receptor (EGFR), a degradation marker, into distinct microdomains. We found that the sorting of PLP into the lumen of LE does not require ESCRT (endosomal sorting complex required for transport), in contrast to EGFR, which is sorted into ILVs via the ESCRT-dependent pathway. PLP is stored in LE until the arrival of neuronal signals. After receiving signals from neurons, the distribution of PLP changes dramatically. The rate of endocytosis is reduced, and the transport of PLP from LE to the plasma membrane is triggered. Our study shows that the molecular mechanisms of this switch from endocytosis to exocytosis involve a decrease of Rho GTPases activity. We found that soluble neuron-derived factors inactivate Rho in oligodendrocytes, leading to a reduced transport of PLP to LE and increased dynamics of LE. In addition, we demonstrate that the regulation of myelin membrane trafficking is coordinated by the tyrosine kinase, c-src. These findings reveal a fundamental and novel role of LE in oligodendrocytes: to store and release PLP in a regulated fashion. We propose that this mechanism ensures the proper timing and controls the growth of the myelin membrane

Fluctuations in cell density alter protein markers of multiple cellular compartments, confounding experimental outcomes.

The life cycle of cultured proliferating cells is characterized by fluctuations in cell population density induced by periodic subculturing. This leads to corresponding changes in micro- and macroenvironment of the cells, accompanied by altered cellular metabolism, growth rate and locomotion. Studying cell density-dependent morphological, physiological and biochemical fluctuations is relevant for understanding basic cellular mechanisms and for uncovering the intrinsic variation of commonly used tissue culture experimental models. Using multiple cell lines, we found that expression levels of the autophagic markers p62 and LC3II, and lysosomal enzyme cathepsin D were altered in highly confluent cells as a consequence of nutrient depletion and cell crowding, which led to inactivation of the mTOR signaling pathway. Furthermore, both Lamp1 and active focal adhesion kinase (FAK) were reduced in high-density cells, while chemical inhibition or deletion of FAK led to alterations in lysosomal and autophagic proteins, as well as in the mTOR signaling. This was accompanied by alterations in the Hippo signaling pathway, while cell cycle checkpoint regulator p-cdc2 remained unaffected in at least one studied cell line. On the other hand, allometric scaling of cellular compartments in growing cell populations resulted in biochemically detectable changes in the plasma membrane proteins Na+K+-ATPase and cadherin, and nuclear proteins HDAC1 and Lamin B1. Finally, we demonstrate how treatment-induced changes in cell density and corresponding modulation of susceptible proteins may lead to ambiguous experimental outcomes, or erroneous interpretation of cell culture data. Together, our data emphasize the need to recognize cell density as an important experimental variable in order to improve scientific rigor of cell culture-based studies

Nanotechnology in Tumor Biomarker Detection: The Potential of Liganded Nanoclusters as Nonlinear Optical Contrast Agents for Molecular Diagnostics of Cancer

Cancer is one of the leading causes of premature death, and, as such, it can be prevented by developing strategies for early and accurate diagnosis. Cancer diagnostics has evolved from the macroscopic detection of malignant tissues to the fine analysis of tumor biomarkers using personalized medicine approaches. Recently, various nanomaterials have been introduced into the molecular diagnostics of cancer. This has resulted in a number of tumor biomarkers that have been detected in vitro and in vivo using nanodevices and corresponding imaging techniques. Atomically precise ligand-protected noble metal quantum nanoclusters represent an interesting class of nanomaterials with a great potential for the detection of tumor biomarkers. They are characterized by high biocompatibility, low toxicity, and suitability for controlled functionalization with moieties specifically recognizing tumor biomarkers. Their non-linear optical properties are of particular importance as they enable the visualization of nanocluster-labeled tumor biomarkers using non-linear optical techniques such as two-photon-excited fluorescence and second harmonic generation. This article reviews liganded nanoclusters among the different nanomaterials used for molecular cancer diagnosis and the relevance of this new class of nanomaterials as non-linear optical probe and contrast agents.Project STIM-REI, European Regional Development Fund—the Operational Programme Competitiveness and Cohesion 2014–2020Peer Reviewe

Inhibition of mTOR-dependent autophagy sensitizes leukemic cells to cytarabine-induced apoptotic death.

The present study investigated the role of autophagy, a cellular self-digestion process, in the cytotoxicity of antileukemic drug cytarabine towards human leukemic cell lines (REH, HL-60, MOLT-4) and peripheral blood mononuclear cells from leukemic patients. The induction of autophagy was confirmed by acridine orange staining of intracellular acidic vesicles, electron microscopy visualization of autophagic vacuoles, as well as by the increase in autophagic proteolysis and autophagic flux, demonstrated by immunoblot analysis of p62 downregulation and LC3-I conversion to autophagosome-associated LC3-II in the presence of proteolysis inhibitors, respectively. Moreover, the expression of autophagy-related genes Atg4, Atg5 and Atg7 was stimulated by cytarabine in REH cells. Cytarabine reduced the phosphorylation of the major negative regulator of autophagy, mammalian target of rapamycin (mTOR), and its downstream target p70S6 kinase in REH cells, which was associated with downregulation of mTOR activator Akt and activation of extracellular signal- regulated kinase. Cytarabine had no effect on the activation of mTOR inhibitor AMP-activated protein kinase. Leucine, an mTOR activator, reduced both cytarabine-induced autophagy and cytotoxicity. Accordingly, pharmacological downregulation of autophagy with bafilomycin A1 and chloroquine, or RNA interference-mediated knockdown of LC3β or p62, markedly increased oxidative stress, mitochondrial depolarization, caspase activation and subsequent DNA fragmentation and apoptotic death in cytarabine-treated REH cells. Cytarabine also induced mTOR-dependent cytoprotective autophagy in HL-60 and MOLT-4 leukemic cell lines, as well as primary leukemic cells, but not normal leukocytes. These data suggest that the therapeutic efficiency of cytarabine in leukemic patients could be increased by the inhibition of the mTOR-dependent autophagic response

Les interruptions volontaires de grossesse depuis la loi du 4 juillet 2001 (premiers résultats à partir d'une étude rétrospective, données épidémiologiques et techniques)

LILLE2-BU Santé-Recherche (593502101) / SudocPARIS-BIUM (751062103) / SudocSudocFranceF

Cytarabine induces Atg expression, inhibits mTOR and modulates Akt/ERK signaling in REH cells.

<p>(A, B) REH cells were incubated for the indicated time periods with cytarabine (3.2 μM) and the levels of phosphorylated/total mTOR, p70S6K, AMPK, Akt and ERK were determined by immunoblotting. The representative blots from three independent experiments are shown (A), while the densitometry data are mean ± SD values (*p<0.05 compared to untreated cells) (B). (C, D) REH cells were incubated for 16h (C) or indicated time periods (D) with cytarabine (3.2 μM) in the absence or presence of leucine (2 mM) (C). Intracellular acidification in acridine orange-stained cells was determined by flow cytometry (C), while the amounts of Atg4, Atg5, Atg7, Atg12 and p62 mRNA were analyzed by RT-PCR (D). The data are mean ± SD values from three independent experiments (C) or mean ± SD values of triplicates from a representative of three experiments (D) (*p<0.05 compared to untreated cells).</p