Extracorporeal Membrane Oxygenation for Severe Acute Respiratory Distress Syndrome associated with COVID-19: An Emulated Target Trial Analysis.

RATIONALE: Whether COVID patients may benefit from extracorporeal membrane oxygenation (ECMO) compared with conventional invasive mechanical ventilation (IMV) remains unknown. OBJECTIVES: To estimate the effect of ECMO on 90-Day mortality vs IMV only Methods: Among 4,244 critically ill adult patients with COVID-19 included in a multicenter cohort study, we emulated a target trial comparing the treatment strategies of initiating ECMO vs. no ECMO within 7 days of IMV in patients with severe acute respiratory distress syndrome (PaO2/FiO2 <80 or PaCO2 ≥60 mmHg). We controlled for confounding using a multivariable Cox model based on predefined variables. MAIN RESULTS: 1,235 patients met the full eligibility criteria for the emulated trial, among whom 164 patients initiated ECMO. The ECMO strategy had a higher survival probability at Day-7 from the onset of eligibility criteria (87% vs 83%, risk difference: 4%, 95% CI 0;9%) which decreased during follow-up (survival at Day-90: 63% vs 65%, risk difference: -2%, 95% CI -10;5%). However, ECMO was associated with higher survival when performed in high-volume ECMO centers or in regions where a specific ECMO network organization was set up to handle high demand, and when initiated within the first 4 days of MV and in profoundly hypoxemic patients. CONCLUSIONS: In an emulated trial based on a nationwide COVID-19 cohort, we found differential survival over time of an ECMO compared with a no-ECMO strategy. However, ECMO was consistently associated with better outcomes when performed in high-volume centers and in regions with ECMO capacities specifically organized to handle high demand. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Régulation de l’homéostasie cellulaire du Mn : rôles insoupçonnés de TMEM165, SERCA et SPCA1

Glycosylation is a universal cellular process in all living organisms where monosaccharides are added one by one onto an acceptor molecule, most of the time a protein, a lipid or another monosaccharide. In eukaryotes, many glycosylation pathways occur simultaneously, resulting in the biosynthesis of a broad variety of glycan structures with different functions. In humans, if one -or more- glycosylation reactions are genetically impaired, Congenital Disorders of Glycosylation (CDG) appear. One of them, TMEM165-CDG, was identified in 2012 by our group and is at the heart of this work. Pathogenic mutations in TMEM165 gene cause severe glycosylation defects mainly characterized by hypo-galactosylated N-glycan structures. While characterizing these glycosylation abnormalities, a link has rapidly been established by the team between TMEM165 deficiency and Golgi manganese (Mn2+) homeostasis disruption. Therefore, and based on previous work, TMEM165 was assumed to act as a Ca2+/Mn2+ antiporter, allowing the import of Mn2+ into the Golgi lumen in order to sustain an adequate ionic environment, required for all glycosylation reactions. Interestingly, we also found that exogenous addition of Mn2+ in the culture medium of TMEM165 deficient cells completely rescues the N-glycosylation defects observed in these cells. Moreover, TMEM165, like Gdt1p its yeast ortholog, is a protein highly sensitive to Mn2+, being rapidly degraded via the lysosomal pathway in the presence of high Mn2+ concentrations. All in all, a close link exists between TMEM165/Gdt1p, Golgi Mn2+ homeostasis and Golgi glycosylation; the three major aspects focused in my PhD research. More precisely, my thesis focuses on (i) understanding the mechanisms of Mn2+-induced glycosylation rescue in TMEM165 deficient cells and (ii) the potential links between different key players acting in the regulation of the secretory pathway ionic homeostasis which are the Sarco/Endoplasmic Reticulum calcium (Ca2+)-ATPase SERCA2, TMEM165 and SPCA1 (Secretory Pathway Ca2+/Mn2+-ATPase), the only pump of the Golgi apparatus known to import both Ca2+ and Mn2+ in the Golgi lumen. Through the use of isogenic human cell lines knockout for either TMEM165 or ATP2C1 and yeasts lacking Gdt1p and/or Pmr1p, we highlighted three main concepts that closely link these proteins: TMEM165 (Gdt1p), SPCA1 (Pmr1p) and SERCA2. On the one hand, we demonstrated that the activity of SERCA pumps is crucial to sustain Golgi glycosylation reactions in absence of TMEM165 by their contribution in Mn2+ pumping and redistribution into the Golgi lumen. On the other hand, TMEM165 was found essential for maintaining Golgi glycosylation reactions in absence of both SPCA1 and when SERCA2 are inhibited by pharmacological agents. Moreover, we also shed light on the fact that expression and stability of TMEM165 (in humans) and Gdt1p (in yeast) were directly linked to the capacities of SPCA1 and Pmr1p to import Mn2+ into the Golgi lumen. Although differences exist between humans and yeast Saccharomyces cerevisiae, all of our work illustrates the crucial importance of the ionic homeostasis of the Golgi apparatus to sustain Golgi glycosylation reactions.La glycosylation est un processus cellulaire universel chez tous les organismes vivants visant aux transferts successifs de monosaccharides sur une molécule acceptrice, le plus souvent une protéine, un lipide ou un autre monosaccharide. Chez les eucaryotes, différentes voies de glycosylation coexistent, aboutissant à la biosynthèse d’une grande diversité de structures glycanniques aux fonctions diverses. Chez l’homme, des perturbations au cours d’une ou plusieurs réactions de glycosylation sont à l’origine de glycopathologies génétiques rares appelées Congenital Disorders of Glycosylation (CDG). L’une d’entre elles, TMEM165-CDG, a été identifiée en 2012 par notre équipe et est au cœur de ces travaux. Des mutations pathogéniques dans le gène TMEM165 sont en effet responsables de l’apparition de sévères défauts de glycosylation caractérisés par la présence de structures N-glycanniques principalement sous-galactosylées. Lors de la caractérisation de ces anomalies de glycosylation, les travaux de l’équipe ont rapidement établi un lien entre déficience en TMEM165 et dérégulation de l’homéostasie du manganèse (Mn2+) de l’appareil de Golgi. Dès lors, et au regard de précédents résultats de l’équipe, une fonction d’antiport Ca2+/Mn2+ fut assignée à TMEM165, permettant l’import d’ions Mn2+ dans l’appareil de Golgi afin d’assurer un environnement ionique adéquat et nécessaire au bon déroulement des réactions de glycosylation. De façon extrêmement intéressante, il s’avère qu’un apport exogène de Mn2+ dans le milieu de culture de cellules déficientes en TMEM165 corrige complètement les défauts de N-glycosylation observés dans ces cellules. Par ailleurs, TMEM165, tout comme Gdt1p son orthologue chez la levure Saccharomyces cerevisiae, est une protéine extrêmement sensible aux ions Mn2+ étant rapidement dégradée via la voie lysosomale en présence de fortes concentrations de Mn2+. Un lien étroit s’établit donc entre fonctions de TMEM165/Gdt1p, homéostasie du Mn2+ de l’appareil de Golgi et glycosylation golgienne ; trois aspects qui furent au centre de mes travaux. Plus particulièrement, ma thèse porte sur (i) la compréhension des mécanismes de correction des défauts de glycosylation observés dans les cellules déficientes en TMEM165 et induits par le Mn2+ et (ii) les liens potentiels entre différents acteurs essentiels au maintien de l’homéostasie ionique de la voie de sécrétion que sont les pompes calciques (Ca2+) réticulaires SERCA2, TMEM165 et SPCA1, seule pompe ATPasique de l’appareil de Golgi connue à ce jour pour importer à la fois des ions Ca2+ et Mn2+. A travers l’utilisation de lignées cellulaires humaines génétiquement invalidées pour TMEM165 ou ATP2C1 et de levures déficientes en Gdt1p et/ou Pmr1p, notre étude a conduit à l’élaboration de différents concepts reliant intimement ces protéines. D’une part, nous avons démontré que l’activité des pompes SERCA était cruciale au maintien des réactions de glycosylation golgiennes en absence de TMEM165 par leur contribution dans le pompage et la redistribution des ions Mn2+ depuis le cytosol vers l’appareil de Golgi. D’autre part, TMEM165 est indispensable au maintien des réactions de glycosylation golgiennes en absence de SPCA1 et lorsque SERCA2 est inhibée par des agents pharmacologiques. Parallèlement, nos travaux ont mis en évidence que l’expression et la stabilité des protéines TMEM165, chez l’homme et Gdt1p, chez la levure étaient directement liées aux capacités de SPCA1 et Pmr1p à importer des ions Mn2+ dans l’appareil de Golgi. Bien que des différences s’observent entre l’homme et la levure Saccharomyces cerevisiae, l’ensemble de mes travaux illustre l’importance de l’homéostasie ionique de l’appareil de Golgi dans le maintien du processus de glycosylation golgien

Anomalies congénitales de la glycosylation (CDG)

La glycosylation est un processus cellulaire complexe conduisant à des transferts successifs de monosaccharides sur une molécule acceptrice, le plus souvent une protéine ou un lipide. Ce processus est universel chez tous les organismes vivants et est très conservé au cours de l’évolution. Chez l’homme, des perturbations survenant au cours d’une ou plusieurs réactions de glycosylation sont à l’origine de glycopathologies génétiques rares, appelées anomalies congénitales de la glycosylation ou congenital disorders of glycosylation (CDG). Cette revue propose de revisiter ces CDG, de 1980 à aujourd’hui, en présentant leurs découvertes, leurs diagnostics, leurs causes biochimiques et les traitements actuellement disponibles

Fetal bovine serum impacts the observed N‐glycosylation defects in TMEM165 KO HEK cells

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Towards understanding the extensive diversity of protein N‐glycan structures in eukaryotes

International audienceN-glycosylation is an important post-translational modification of proteins that has been highly conserved during evolution and is found in Eukaryota, Bacteria and Archaea. In eukaryotes, N-glycan processing is sequential, involving multiple specific steps within the secretory pathway as proteins travel through the endoplasmic reticulum and the Golgi apparatus. In this review, we first summarize the different steps of the N-glycan processing and further describe recent findings regarding the diversity of N-glycan structures in eukaryotic clades. This comparison allows us to explore the different regulation mechanisms of N-glycan processing among eukaryotic clades. Recent findings regarding the regulation of protein N-glycosylation are highlighted, especially the regulation of the biosynthesis of complex-type N-glycans through manganese and calcium homeostasis and the specific role of transmembrane protein 165 (TMEM165) for which homologous sequences have been identified in several eukaryotic clades. Further research will be required to characterize the function of TMEM165 homologous sequences in different eukaryotic clades

Towards understanding the regulation of the N-glycosylation pathway in regards to the huge diversity of the N-glycan structures identified in Eukaryotes

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Investigating the functional link between TMEM165 and SPCA1

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Protein N-glycosylation alteration and glycolysis inhibition both contribute to the antiproliferative action of 2-deoxyglucose in breast cancer cells

International audiencePurpose : Cancer cells often elicit a higher glycolytic rate than normal cells, supporting the development of glycolysis inhibitors as therapeutic agents. 2-Deoxyglucose (2-DG) is used in this context due to its ability to compete with glucose. However, many studies do not take into account that 2-DG inhibits not only glycolysis but also N-glycosylation. Since there are limited publications on 2-DG mechanism of action in breast cancer, we studied its effects in breast cancer cell lines to determine the part played by glycolysis inhibition and N-linked glycosylation interference.Methods and Results : 2-Deoxyglucose behaved as an anticancer agent with a similar efficiency on cell number decrease between the hormone-dependent MCF-7 and hormone-independent MDA-MB-231 breast cancer cells. It also interfered with the N-linked glycosylation process in both cell lines as illustrated by the migration profile of the lysosomal-associated membrane protein 2 and calumenin. These results are reinforced by the appearance of an abnormal Man7GlcNAc2 structure both on lipid-linked oligosaccharides and N-linked glycoproteins of 2-DG incubated MDA-MB-231 cells. Besides, 2-DG-induced a transient endoplasmic reticulum stress that was more sustained in MDA-MB-231 cells. Both changes were abrogated by mannose. 2-DG, even in the presence of mannose, decreased glycolysis in both cell lines. Mannose partially reversed the effects of 2-DG on cell numbers with N-linked glycosylation interference accounting for 37 and 47% of 2-DG anti-cancerous effects in MDA-MB-231 and MCF-7 cells, respectively.Conclusion : N-linked glycosylation interference and glycolysis disruption both contribute to the anticancer properties of 2-DG in breast cancer cells

Involvement of thapsigargin– and cyclopiazonic acid–sensitive pumps in the rescue of TMEM165-associated glycosylation defects by Mn 2+

International audienceCongenital disorders of glycosylation are severe inherited diseases in which aberrant protein glycosylation is a hallmark. Transmembrane protein 165 (TMEM165) is a novel Golgi transmembrane protein involved in type II congenital disorders of glycosylation. Although its biologic function is still a controversial issue, we have demonstrated that the Golgi glycosylation defect due to TMEM165 deficiency resulted from a Golgi Mn2+ homeostasis defect. The goal of this study was to delineate the cellular pathway by which extracellular Mn2+ rescues N-glycosylation in TMEM165 knockout (KO) cells. We first demonstrated that after extracellular exposure, Mn2+ uptake by HEK293 cells at the plasma membrane did not rely on endocytosis but was likely done by plasma membrane transporters. Second, we showed that the secretory pathway Ca2+-ATPase 1, also known to mediate the influx of cytosolic Mn2+ into the lumen of the Golgi apparatus, is not crucial for the Mn2+-induced rescue glycosylation of lysosomal-associated membrane protein 2 (LAMP2). In contrast, our results demonstrate the involvement of cyclopiazonic acid- and thapsigargin (Tg)-sensitive pumps in the rescue of TMEM165-associated glycosylation defects by Mn2+. Interestingly, overexpression of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) 2b isoform in TMEM165 KO cells partially rescues the observed LAMP2 glycosylation defect. Overall, this study indicates that the rescue of Golgi N-glycosylation defects in TMEM165 KO cells by extracellular Mn2+ involves the activity of Tg and cyclopiazonic acid-sensitive pumps, probably the SERCA pumps.-Houdou, M., Lebredonchel, E., Garat, A., Duvet, S., Legrand, D., Decool, V., Klein, A., Ouzzine, M., Gasnier, B., Potelle, S., Foulquier, F. Involvement of thapsigargin- and cyclopiazonic acid-sensitive pumps in the rescue of TMEM165-associated glycosylation defects by Mn2+

Dissection of TMEM165 function in Golgi glycosylation and its Mn2+ sensitivity

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