CO-mediated cytoprotection is dependent on cell metabolism modulation

This work was supported by Fundação para a Ciência e Tecnologia (FCT) grant UID/Multi/04462/2013, I&D 2015–2020 iNOVA4Health - Programme in Translacional Medicine. FCT provided individual financial support to CFP (SFRH/BD/106057/2015), DDP (PD/BD/128338/2017), NLS (PD/BD/127819/2016) and HLAV (IF/00185/2012).Carbon monoxide (CO) is a gasotransmitter endogenously produced by the activity of heme oxygenase, which is a stress-response enzyme. Endogenous CO or low concentrations of exogenous CO have been described to present several cytoprotective functions: anti-apoptosis, anti-inflammatory, vasomodulation, maintenance of homeostasis, stimulation of preconditioning and modulation of cell differentiation. The present review revises and discuss how CO regulates cell metabolism and how it is involved in the distinct cytoprotective roles of CO. The first found metabolic effect of CO was its increase on cellular ATP production, and since then much data have been generated. Mitochondria are the most described and studied cellular targets of CO. Mitochondria exposure to this gasotransmitter leads several consequences: ROS generation, stimulation of mitochondrial biogenesis, increased oxidative phosphorylation or mild uncoupling effect. Likewise, CO negatively regulates glycolysis and improves pentose phosphate pathway. More recently, CO has also been disclosed as a regulating molecule for metabolic diseases, such as obesity and diabetes with promising results.publishersversionpublishe

The mito-QC reporter for quantitative mitophagy assessment in primary retinal ganglion cells and experimental glaucoma models

This research was funded by Ministerio de Ciencia, Innovación y Universidades (MCIU), Agencia Estatal de Investigación (AEI) and Fondo Europeo de Desarrollo Regional (FEDER) PGC2018-098557-B-I00 and European Union’s Horizon 2020 research and innovation programme under grant agreement No 765912. BVZ is a recipient of PhD contract from the Fundación Tatiana Pérez de Guzmán el Bueno (Spain), PT from H2020-MSCA-ITN-2017, NRM a Juan de la Cierva Grant from Ministerio Ciencia e Innovación (Spain) and KB from DFG (Deutsche Forschungsgemeinschaft, Germany, 6619/1-1).Mitochondrial damage plays a prominent role in glaucoma. The only way cells can degrade whole mitochondria is via autophagy, in a process called mitophagy. Thus, studying mitophagy in the context of glaucoma is essential to understand the disease. Up to date limited tools are available for analyzing mitophagy in vivo. We have taken advantage of the mito-QC reporter, a recently generated mouse model that allows an accurate mitophagy assessment to fill this gap. We used primary RGCs and retinal explants derived from mito-QC mice to quantify mitophagy activation in vitro and ex vivo. We also analyzed mitophagy in retinal ganglion cells (RGCs), in vivo, using different mitophagy inducers, as well as after optic nerve crush (ONC) in mice, a commonly used surgical procedure to model glaucoma. Using mito-QC reporter we quantified mitophagy induced by several known inducers in primary RGCs in vitro, ex vivo and in vivo. We also found that RGCs were rescued from some glaucoma relevant stress factors by incubation with the iron chelator deferiprone (DFP). Thus, the mito-QC reporter-based model is a valuable tool for accurately analyzing mitophagy in the context of glaucoma.publishersversionpublishe

Control of Mitochondrial Membrane Permeabilization by Adenine Nucleotide Translocator Interacting with HIV-1 Viral Protein R and Bcl-2

Viral protein R (Vpr), an apoptogenic accessory protein encoded by HIV-1, induces mitochondrial membrane permeabilization (MMP) via a specific interaction with the permeability transition pore complex, which comprises the voltage-dependent anion channel (VDAC) in the outer membrane (OM) and the adenine nucleotide translocator (ANT) in the inner membrane. Here, we demonstrate that a synthetic Vpr-derived peptide (Vpr52-96) specifically binds to the intermembrane face of the ANT with an affinity in the nanomolar range. Taking advantage of this specific interaction, we determined the role of ANT in the control of MMP. In planar lipid bilayers, Vpr52-96 and purified ANT cooperatively form large conductance channels. This cooperative channel formation relies on a direct protein–protein interaction since it is abolished by the addition of a peptide corresponding to the Vpr binding site of ANT. When added to isolated mitochondria, Vpr52-96 uncouples the respiratory chain and induces a rapid inner MMP to protons and NADH. This inner MMP precedes outer MMP to cytochrome c. Vpr52-96–induced matrix swelling and inner MMP both are prevented by preincubation of purified mitochondria with recombinant Bcl-2 protein. In contrast to König's polyanion (PA10), a specific inhibitor of the VDAC, Bcl-2 fails to prevent Vpr52-96 from crossing the mitochondrial OM. Rather, Bcl-2 reduces the ANT–Vpr interaction, as determined by affinity purification and plasmon resonance studies. Concomitantly, Bcl-2 suppresses channel formation by the ANT–Vpr complex in synthetic membranes. In conclusion, both Vpr and Bcl-2 modulate MMP through a direct interaction with ANT

Effect of the morphogene bolA on the permeability of the Escherichia coli outer membrane

Escherichia coli bolA is a morphogene involved in stress response and cell division. Overexpression of bolA induces biofilm formation and affects the levels of carboxypeptidases PBP5, PBP6 and β-lactamase AmpC. In this study, we have shown that changes in the expression of bolA result in alterations in the properties of the outer membrane. The sensitivity to detergents and vancomycin was reduced when bolA was overexpressed and fluorescent probes indicated that different levels of bolA had an effect on outer membrane protein accessibility. Moreover, bolA was shown to be involved in the modulation of the OmpF/OmpC balance.publishersversionpublishe

Improvement of neuronal differentiation by carbon monoxide: Role of pentose phosphate pathway

Over the last decades, the silent-killer carbon monoxide (CO) has been shown to also be an endogenous cytoprotective molecule able to inhibit cell death and modulate mitochondrial metabolism. Neuronal metabolism is mostly oxidative and neurons also use glucose for maintaining their anti-oxidant status by generation of reduced glutathione (GSH) via the pentose-phosphate pathway (PPP). It is established that neuronal differentiation depends on reactive oxygen species (ROS) generation and signalling, however there is a lack of information about modulation of the PPP during adult neurogenesis. Thus, the main goal of this study was to unravel the role of CO on cell metabolism during neuronal differentiation, particularly by targeting PPP flux and GSH levels as anti-oxidant system.A human neuroblastoma SH-S5Y5 cell line was used, which differentiates into post-mitotic neurons by treatment with retinoic acid (RA), supplemented or not with CO-releasing molecule-A1 (CORM-A1). SH-SY5Y cell differentiation supplemented with CORM-A1 prompted an increase in neuronal yield production. It did, however, not alter glycolytic metabolism, but increased the PPP. In fact, CORM-A1 treatment stimulated (i) mRNA expression of 6-phosphogluconate dehydrogenase (PGDH) and transketolase (TKT), which are enzymes for oxidative and non-oxidative phases of the PPP, respectively and (ii) protein expression and activity of glucose 6-phosphate dehydrogenase (G6PD) the rate-limiting enzyme of the PPP. Likewise, whenever G6PD was knocked-down CO-induced improvement on neuronal differentiation was reverted, while pharmacological inhibition of GSH synthesis did not change CO's effect on the improvement of neuronal differentiation. Both results indicate the key role of PPP in CO-modulation of neuronal differentiation. Furthermore, at the end of SH-SY5Y neuronal differentiation process, CORM-A1 supplementation increased the ratio of reduced and oxidized glutathione (GSH/GSSG) without alteration of GSH metabolism. These data corroborate with PPP stimulation. In conclusion, CO improves neuronal differentiation of SH-S5Y5 cells by stimulating the PPP and modulating the GSH system. Keywords: Carbon monoxide, Neuronal differentiation, Glycolysis, Pentose phosphate pathway, Glutathion

Weaning from mechanical ventilation in intensive care units across 50 countries (WEAN SAFE): a multicentre, prospective, observational cohort study

International audienceBackground: Current management practices and outcomes in weaning from invasive mechanical ventilation are poorly understood. We aimed to describe the epidemiology, management, timings, risk for failure, and outcomes of weaning in patients requiring at least 2 days of invasive mechanical ventilation. Methods: WEAN SAFE was an international, multicentre, prospective, observational cohort study done in 481 intensive care units in 50 countries. Eligible participants were older than 16 years, admitted to a participating intensive care unit, and receiving mechanical ventilation for 2 calendar days or longer. We defined weaning initiation as the first attempt to separate a patient from the ventilator, successful weaning as no reintubation or death within 7 days of extubation, and weaning eligibility criteria based on positive end-expiratory pressure, fractional concentration of oxygen in inspired air, and vasopressors. The primary outcome was the proportion of patients successfully weaned at 90 days. Key secondary outcomes included weaning duration, timing of weaning events, factors associated with weaning delay and weaning failure, and hospital outcomes. This study is registered with ClinicalTrials.gov, NCT03255109. Findings: Between Oct 4, 2017, and June 25, 2018, 10 232 patients were screened for eligibility, of whom 5869 were enrolled. 4523 (77·1%) patients underwent at least one separation attempt and 3817 (65·0%) patients were successfully weaned from ventilation at day 90. 237 (4·0%) patients were transferred before any separation attempt, 153 (2·6%) were transferred after at least one separation attempt and not successfully weaned, and 1662 (28·3%) died while invasively ventilated. The median time from fulfilling weaning eligibility criteria to first separation attempt was 1 day (IQR 0–4), and 1013 (22·4%) patients had a delay in initiating first separation of 5 or more days. Of the 4523 (77·1%) patients with separation attempts, 2927 (64·7%) had a short wean (≤1 day), 457 (10·1%) had intermediate weaning (2–6 days), 433 (9·6%) required prolonged weaning (≥7 days), and 706 (15·6%) had weaning failure. Higher sedation scores were independently associated with delayed initiation of weaning. Delayed initiation of weaning and higher sedation scores were independently associated with weaning failure. 1742 (31·8%) of 5479 patients died in the intensive care unit and 2095 (38·3%) of 5465 patients died in hospital. Interpretation: In critically ill patients receiving at least 2 days of invasive mechanical ventilation, only 65% were weaned at 90 days. A better understanding of factors that delay the weaning process, such as delays in weaning initiation or excessive sedation levels, might improve weaning success rates. Funding: European Society of Intensive Care Medicine, European Respiratory Society