PI3KCIIα-Dependent Autophagy Program Protects From Endothelial Dysfunction and Atherosclerosis in Response to Low Shear Stress in Mice.

BACKGROUND: The ability to respond to mechanical forces is a basic requirement for maintaining endothelial cell (ECs) homeostasis, which is continuously subjected to low shear stress (LSS) and high shear stress (HSS). In arteries, LSS and HSS have a differential impact on EC autophagy processes. However, it is still unclear whether LSS and HSS differently tune unique autophagic machinery or trigger specific autophagic responses in ECs. METHODS: Using fluid flow system to generate forces on EC and multiscale imaging analyses on ApoE-/- mice whole arteries, we studied the cellular and molecular mechanism involved in autophagic response to LSS or HSS on the endothelium. RESULTS: We found that LSS and HSS trigger autophagy activation by mobilizing specific autophagic signaling modules. Indeed, LSS-induced autophagy in endothelium was independent of the class III PI3K (phosphoinositide 3-kinase) VPS34 (vacuolar sorting protein 34) but controlled by the α isoform of class II PI3K (phosphoinositide 3-kinase class II α [PI3KCIIα]). Accordingly, reduced PI3KCIIα expression in ApoE-/- mice (ApoE-/-PI3KCIIα+/-) led to EC dysfunctions associated with increased plaque deposition in the LSS regions. Mechanistically, we revealed that PI3KCIIα inhibits mTORC1 (mammalian target of rapamycin complex 1) activation and that rapamycin treatment in ApoE-/-PI3KCIIα+/- mice specifically rescue autophagy in arterial LSS regions. Finally, we demonstrated that absence of PI3KCIIα led to decreased endothelial primary cilium biogenesis in response to LSS and that ablation of primary cilium mimics PI3KCIIα-decreased expression in EC dysfunction, suggesting that this organelle could be the mechanosensor linking PI3KCIIα and EC homeostasis. CONCLUSIONS: Our data reveal that mechanical forces variability within the arterial system determines EC autophagic response and supports a central role of PI3KCIIα/mTORC1 axis to prevent EC dysfunction in LSS regions.

Correction: Mitochondrial morphodynamics alteration induced by influenza virus infection as a new antiviral strategy.

[This corrects the article DOI: 10.1371/journal.ppat.1009340.].

Mitochondrial morphodynamics alteration induced by influenza virus infection as a new antiviral strategy.

Influenza virus infections are major public health threats due to their high rates of morbidity and mortality. Upon influenza virus entry, host cells experience modifications of endomembranes, including those used for virus trafficking and replication. Here we report that influenza virus infection modifies mitochondrial morphodynamics by promoting mitochondria elongation and altering endoplasmic reticulum-mitochondria tethering in host cells. Expression of the viral RNA recapitulates these modifications inside cells. Virus induced mitochondria hyper-elongation was promoted by fission associated protein DRP1 relocalization to the cytosol, enhancing a pro-fusion status. We show that altering mitochondrial hyper-fusion with Mito-C, a novel pro-fission compound, not only restores mitochondrial morphodynamics and endoplasmic reticulum-mitochondria contact sites but also dramatically reduces influenza replication. Finally, we demonstrate that the observed Mito-C antiviral property is directly connected with the innate immunity signaling RIG-I complex at mitochondria. Our data highlight the importance of a functional interchange between mitochondrial morphodynamics and innate immunity machineries in the context of influenza viral infection.

Chemical targeting of NEET proteins reveals their function in mitochondrial morphodynamics.

Several human pathologies including neurological, cardiac, infectious, cancerous, and metabolic diseases have been associated with altered mitochondria morphodynamics. Here, we identify a small organic molecule, which we named Mito-C. Mito-C is targeted to mitochondria and rapidly provokes mitochondrial network fragmentation. Biochemical analyses reveal that Mito-C is a member of a new class of heterocyclic compounds that target the NEET protein family, previously reported to regulate mitochondrial iron and ROS homeostasis. One of the NEET proteins, NAF-1, is identified as an important regulator of mitochondria morphodynamics that facilitates recruitment of DRP1 to the ER-mitochondria interface. Consistent with the observation that certain viruses modulate mitochondrial morphogenesis as a necessary part of their replication cycle, Mito-C counteracts dengue virus-induced mitochondrial network hyperfusion and represses viral replication. The newly identified chemical class including Mito-C is of therapeutic relevance for pathologies where altered mitochondria dynamics is part of disease etiology and NEET proteins are highlighted as important therapeutic targets in anti-viral research.

ER-plasma membrane contact sites contribute to autophagosome biogenesis by regulation of local PI3P synthesis.

The double-membrane-bound autophagosome is formed by the closure of a structure called the phagophore, origin of which is still unclear. The endoplasmic reticulum (ER) is clearly implicated in autophagosome biogenesis due to the presence of the omegasome subdomain positive for DFCP1, a phosphatidyl-inositol-3-phosphate (PI3P) binding protein. Contribution of other membrane sources, like the plasma membrane (PM), is still difficult to integrate in a global picture. Here we show that ER-plasma membrane contact sites are mobilized for autophagosome biogenesis, by direct implication of the tethering extended synaptotagmins (E-Syts) proteins. Imaging data revealed that early autophagic markers are recruited to E-Syt-containing domains during autophagy and that inhibition of E-Syts expression leads to a reduction in autophagosome biogenesis. Furthermore, we demonstrate that E-Syts are essential for autophagy-associated PI3P synthesis at the cortical ER membrane via the recruitment of VMP1, the stabilizing ER partner of the PI3KC3 complex. These results highlight the contribution of ER-plasma membrane tethers to autophagosome biogenesis regulation and support the importance of membrane contact sites in autophagy.

Primary cilium-dependent autophagy in the response to shear stress.

Mechanical forces, such as compression, shear stress and stretching, play major roles during development, tissue homeostasis and immune processes. These forces are translated into a wide panel of biological responses, ranging from changes in cell morphology, membrane transport, metabolism, energy production and gene expression. Recent studies demonstrate the role of autophagy in the integration of these physical constraints. Here we focus on the role of autophagy in the integration of shear stress induced by blood and urine flows in the circulatory system and the kidney, respectively. Many studies highlight the involvement of the primary cilium, a microtubule-based antenna present at the surface of many cell types, in the integration of extracellular stimuli. The cross-talk between the molecular machinery of autophagy and that of the primary cilium in the context of shear stress is revealed to be an important dialog in cell biology.

Autophagy: A Druggable Process.

Macroautophagy (hereafter called autophagy) is a vacuolar, lysosomal pathway for catabolism of intracellular material that is conserved among eukaryotic cells. Autophagy plays a crucial role in tissue homeostasis, adaptation to stress situations, immune responses, and the regulation of the inflammatory response. Blockade or uncontrolled activation of autophagy is associated with cancer, diabetes, obesity, cardiovascular disease, neurodegenerative disease, autoimmune disease, infection, and chronic inflammatory disease. During the past decade, researchers have made major progress in understanding the three levels of regulation of autophagy in mammalian cells: signaling, autophagosome formation, and autophagosome maturation and lysosomal degradation. As we discuss in this review, each of these levels is potentially druggable, and, depending on the indication, may be able to stimulate or inhibit autophagy. We also summarize the different modulators of autophagy and their potential and limitations in the treatment of life-threatening diseases.

Cholesterol trafficking and raft-like membrane domain composition mediate scavenger receptor class B type 1-dependent lipid sensing in intestinal epithelial cells.

Scavenger receptor Class B type 1 (SR-B1) is a lipid transporter and sensor. In intestinal epithelial cells, SR-B1-dependent lipid sensing is associated with SR-B1 recruitment in raft-like/ detergent-resistant membrane domains and interaction of its C-terminal transmembrane domain with plasma membrane cholesterol. To clarify the initiating events occurring during lipid sensing by SR-B1, we analyzed cholesterol trafficking and raft-like domain composition in intestinal epithelial cells expressing wild-type SR-B1 or the mutated form SR-B1-Q445A, defective in membrane cholesterol binding and signal initiation. These features of SR-B1 were found to influence both apical cholesterol efflux and intracellular cholesterol trafficking from plasma membrane to lipid droplets, and the lipid composition of raft-like domains. Lipidomic analysis revealed likely participation of d18:0/16:0 sphingomyelin and 16:0/0:0 lysophosphatidylethanolamine in lipid sensing by SR-B1. Proteomic analysis identified proteins, whose abundance changed in raft-like domains during lipid sensing, and these included molecules linked to lipid raft dynamics and signal transduction. These findings provide new insights into the role of SR-B1 in cellular cholesterol homeostasis and suggest molecular links between SR-B1-dependent lipid sensing and cell cholesterol and lipid droplet dynamics.

Mitochondrial Dynamics in Basal and Stressful Conditions.

The historical role of mitochondria resides in converting the energy released during the oxidation of macromolecules (carbohydrates, lipids and proteins) into adenosine tri-phosphate, a major form of chemically stored energy which sustains cell growth and homeostasis. Beyond this role in bioenergetics regulation, mitochondria play a role in several other cellular processes including lipid metabolism, cellular calcium homeostasis, autophagy and immune responses. Furthermore, mitochondria are highly dynamic organelles: as all other cellular endomembranes, they are continuously moving along cytoskeleton, and, most importantly, they constantly interact one with each other by membrane tethering, fusion and fission. This review aims to highlight the tight correlation between the morphodynamics of mitochondria and their biological function(s), in physiological as well as stress conditions, in particular nutrient deprivation, pathogen attack and some human diseases. Finally, we emphasize some crosstalk between the fusion/fission machinery and the autophagy pathway to ending on some speculative hypothesis to inspire future research in the field.

Fine-tuning autophagy: from transcriptional to posttranslational regulation.

Macroautophagy (hereafter called autophagy) is a vacuolar lysosomal pathway for degradation of intracellular material in eukaryotic cells. Autophagy plays crucial roles in tissue homeostasis, in adaptation to stress situations, and in immune and inflammatory responses. Alteration of autophagy is associated with cancer, diabetes and obesity, cardiovascular disease, neurodegenerative disease, autoimmune disease, infection, and chronic inflammatory disease. Autophagy is controlled by autophagy-related (ATG) proteins that act in a coordinated manner to build up the initial autophagic vacuole named the autophagosome. It is now known that the activities of ATG proteins are modulated by posttranslational modifications such as phosphorylation, ubiquitination, and acetylation. Moreover, transcriptional and epigenetic controls are involved in the regulation of autophagy in stress situations. Here we summarize and discuss how posttranslational modifications and transcriptional and epigenetic controls regulate the involvement of autophagy in the proteostasis network.

Scavenger receptor class B type I is a plasma membrane cholesterol sensor.

RATIONALE: Signal initiation by the high-density lipoprotein (HDL) receptor scavenger receptor class B, type I (SR-BI), which is important to actions of HDL on endothelium and other processes, requires cholesterol efflux and the C-terminal transmembrane domain. The C-terminal transmembrane domain uniquely interacts with plasma membrane (PM) cholesterol. OBJECTIVE: The molecular basis and functional significance of SR-BI interaction with PM cholesterol are unknown. We tested the hypotheses that the interaction is required for SR-BI signaling, and that it enables SR-BI to serve as a PM cholesterol sensor. METHODS AND RESULTS: In studies performed in COS-M6 cells, mutation of a highly conserved C-terminal transmembrane domain glutamine to alanine (SR-BI-Q445A) decreased PM cholesterol interaction with the receptor by 71% without altering HDL binding or cholesterol uptake or efflux, and it yielded a receptor incapable of HDL-induced signaling. Signaling prompted by cholesterol efflux to methyl-ß-cyclodextrin also was prevented, indicating that PM cholesterol interaction with the receptor enables it to serve as a PM cholesterol sensor. Using SR-BI-Q445A, we further demonstrated that PM cholesterol sensing by SR-BI does not influence SR-BI-mediated reverse cholesterol transport to the liver in mice. However, the PM cholesterol sensing does underlie apolipoprotein B intracellular trafficking in response to postprandial micelles or methyl-ß-cyclodextrin in cultured enterocytes, and it is required for HDL activation of endothelial NO synthase and migration in cultured endothelial cells and HDL-induced angiogenesis in vivo. CONCLUSIONS: Through interaction with PM cholesterol, SR-BI serves as a PM cholesterol sensor, and the resulting intracellular signaling governs processes in both enterocytes and endothelial cells.

Morphodynamical adaptation of the endolysosomal system to stress.

In eukaryotes, the spatiotemporal control of endolysosomal organelles is central to the maintenance of homeostasis. By providing an interface between the cytoplasm and external environment, the endolysosomal system is placed at the forefront of the response to a wide range of stresses faced by cells. Endosomes are equipped with a dedicated set of membrane-associated proteins that ensure endosomal functions as well as crosstalk with the secretory or the autophagy pathways. Morphodynamical processes operate through local spatialization of subdomains, enabling specific remodeling and membrane contact capabilities. Consequently, the plasticity of endolysosomal organelles can be considered a robust and flexible tool exploited by cells to cope with homeostatic deviations. In this review, we provide insights into how the cellular responses to various stresses (osmotic, UV, nutrient deprivation, or pathogen infections) rely on the adaptation of the endolysosomal system morphodynamics.

Canonical and Non-Canonical Roles of SNX1 and SNX2 in Endosomal Membrane Dynamics.

Sorting nexins (SNXs) are a family of membrane-binding proteins known to play a critical role in regulating endocytic pathway sorting and endosomal membrane trafficking. Among them, SNX1 and SNX2 are members of the SNX-BAR subfamily and possess a membrane-curvature domain and a phosphoinositide-binding domain, which enables their stabilization at the phosphatidylinositol-3-phosphate (PI3P)-positive surface of endosomes. While their binding to PI3P-positive platforms facilitates interaction with endosomal partners and stabilization at the endosomal membrane, their SNX-BAR region is pivotal for generating membrane tubulation from endosomal compartments. In this context, their primary identified biological roles-and their partnership-are tightly associated with the retromer and endosomal SNX-BAR sorting complex for promoting exit 1 complex trafficking, facilitating the transport of cargoes from early endosomes to the secretory pathway. However, recent literature indicates that these proteins also possess biological functions in other aspects of endosomal features and sorting processes. Notably, SNX2 has been found to regulate endosome-endoplasmic reticulum (ER) contact sites through its interaction with VAP proteins at the ER membrane. Furthermore, data from our laboratory show that SNX1 and SNX2 are involved in the tubulation of early endosomes toward ER sites associated with autophagy initiation during starvation. These findings shed light on a novel role of SNXs in inter-organelle tethering and communication. In this concise review, we will explore the non-retromer functions of SNX1 and SNX2, specifically focusing on their involvement in endosomal membrane dynamics during stress sensing and autophagy-associated processes.

A SNX1-SNX2-VAPB partnership regulates endosomal membrane rewiring in response to nutritional stress.

Nutrient deprivation ("starvation") is a major catabolic stress faced by mammalian cells in both pathological and physiological situations. Starvation induces autophagosome biogenesis in the immediate vicinity of ER and leads to lysosome spatial repositioning, but little is known about the consequences of nutritional stress on endosomes. Here, we report that starvation induces tethering of endosomal tubules to ER subregions, fostering autophagosome assembly. We show that this endosomal membrane generation is regulated by sorting nexin 1 (SNX1) protein and is important for the autophagic response. These newly formed SNX1 endosomal tubules establish connections with ER subdomains engaged in early autophagic machinery mobilization. Such endosome-ER transient tethers are regulated by a local dialog between SNX2, an endosomal partner of SNX1, and VAPB, an ER protein associated with autophagy initiation stage regulation. We propose that in a very early response to starvation, SNX1 and SNX2 cooperation induces and regulates endosomal membrane tubulation towards VAPB-positive ER subdomains involved in autophagosome biogenesis, highlighting the contribution of early endosomes in the cellular response to nutritional stress.

The AMPK-Sirtuin 1-YAP axis is regulated by fluid flow intensity and controls autophagy flux in kidney epithelial cells.

Shear stress generated by urinary fluid flow is an important regulator of renal function. Its dysregulation is observed in various chronic and acute kidney diseases. Previously, we demonstrated that primary cilium-dependent autophagy allows kidney epithelial cells to adapt their metabolism in response to fluid flow. Here, we show that nuclear YAP/TAZ negatively regulates autophagy flux in kidney epithelial cells subjected to fluid flow. This crosstalk is supported by a primary cilium-dependent activation of AMPK and SIRT1, independently of the Hippo pathway. We confirm the relevance of the YAP/TAZ-autophagy molecular dialog in vivo using a zebrafish model of kidney development and a unilateral ureteral obstruction mouse model. In addition, an in vitro assay simulating pathological accelerated flow observed at early stages of chronic kidney disease (CKD) activates YAP, leading to a primary cilium-dependent inhibition of autophagic flux. We confirm this YAP/autophagy relationship in renal biopsies from patients suffering from diabetic kidney disease (DKD), the leading cause of CKD. Our findings demonstrate the importance of YAP/TAZ and autophagy in the translation of fluid flow into cellular and physiological responses. Dysregulation of this pathway is associated with the early onset of CKD.

The location and trafficking routes of the neuronal retromer and its role in amyloid precursor protein transport.

The retromer complex plays an important role in intracellular transport, is highly expressed in the hippocampus, and has been implicated in the trafficking of the amyloid precursor protein (APP). Nevertheless, the trafficking routes of the neuronal retromer and the role it plays in APP transport in neuronal processes remain unknown. Here we use hippocampal neuronal cultures to address these issues. Using fluorescence microscopy, we find that Vps35, the core element of the retromer complex, is in dendrites and axons, is enriched in endosomes and trans-Golgi network, and is found in APP-positive vesicles. Next, to identify the role the neuronal retromer plays in cargo transport, we infected hippocampal neurons with a lentivirus expressing shRNA to silence Vps35. By live fluorescence imaging, Vps35 deficiency was found to reduce the frequency, but not the kinetics, of long-range APP transport within neuronal processes. Supporting the interpretation that retromer promotes long-range transport, Vps35 deficiency led to increased APP in the early endosomes, in processes but not the soma. Finally, Vps35 deficiency was associated with increased levels of Aß, a cleaved product of APP, increased colocalization of APP with its cleaving enzyme BACE1 in processes, and caused an enlargement of early endosomes. Taken together, our studies clarify the function of the neuronal retromer, and suggest specific mechanisms for how retromer dysfunction observed in Alzheimer's disease affects APP transport and processing.

The proteome of cytosolic lipid droplets isolated from differentiated Caco-2/TC7 enterocytes reveals cell-specific characteristics.

BACKGROUND INFORMATION: Intestinal absorption of alimentary lipids is a complex process ensured by enterocytes and leading to TRL [TAG (triacylglycerol)-rich lipoprotein] assembly and secretion. The accumulation of circulating intestine-derived TRL is associated with atherosclerosis, stressing the importance of the control of postprandial hypertriglyceridaemia. During the postprandial period, TAGs are also transiently stored as CLDs (cytosolic lipid droplets) in enterocytes. As a first step for determining whether CLDs could play a role in the control of enterocyte TRL secretion, we analysed the protein endowment of CLDs isolated by sucrose-gradient centrifugation from differentiated Caco-2/TC7 enterocytes, the only human model able to secrete TRL in culture and to store transiently TAGs as CLDs when supplied with lipids. Cells were analysed after a 24 h incubation with lipid micelles and thus in a state of CLD-associated TAG mobilization. RESULTS: Among the 105 proteins identified in the CLD fraction by LC-MS/MS (liquid chromatography coupled with tandem MS), 27 were directly involved in lipid metabolism pathways potentially relevant to enterocyte-specific functions. The transient feature of CLDs was consistent with the presence of proteins necessary for fatty acid activation (acyl-CoA synthetases) and for TAG hydrolysis. In differentiated Caco-2/TC7 enterocytes, we identified for the first time LPCAT2 (lysophosphatidylcholine acyltransferase 2), involved in PC (phosphatidylcholine) synthesis, and 3BHS1 (3-ß-hydroxysteroid dehydrogenase 1), involved in steroid metabolism, and confirmed their partial CLD localization by immunofluorescence. In enterocytes, LPCAT2 may provide an economical source of PC, necessary for membrane synthesis and lipoprotein assembly, from the lysoPC present in the intestinal lumen. We also identified proteins involved in lipoprotein metabolism, such as ApoA-IV (apolipoprotein A-IV), which is specifically expressed by enterocytes and has been proposed to play many functions in vivo, including the formation of lipoproteins and the control of their size. The association of ApoA-IV with CLD was confirmed by confocal and immunoelectron microscopy and validated in vivo in the jejunum of mice fed with a high-fat diet. CONCLUSIONS: We report for the first time the protein endowment of Caco-2/TC7 enterocyte CLDs. Our results suggest that their formation and mobilization may participate in the control of enterocyte TRL secretion in a cell-specific manner.

[Architecture, budget and dignity]. / Architecture, budget et dignité.

Drawing on its dynamic strengths, a psychiatric unit develops various projects and care techniques. In this framework, the institute director must make a number of choices with regard to architecture. Why renovate the psychiatry building? What financial investments are required? What criteria should be followed? What if the major argument was based on the respect of the patient's dignity?

You shall not pass! Protective role of autophagic machinery in response to plasma membrane damage triggered by Candida albicans invasion.

Candida albicans (C. albicans) is an opportunistic pathogen causing infections ranging from superficial to life-threatening dissemination, in which C. albicans is able to translocate through the gut barrier into deeper organs. In its filamentous form (hyphae), C. albicans can invade epithelial cells by two mechanisms: epithelial cell-driven endocytosis and C. albicans-driven active penetration of host cell plasma membrane (PM). Autophagic machinery is known to be involved in the epithelial barrier maintenance, especially the intestinal barrier that is continuously challenged by exposure to the gut microbiota or to xenobiotics. The protective role of autophagy during C. albicans infection has been investigated in myeloid cells, however, far less was known regarding its role during infection of epithelial cells. Here, we demonstrated that key proteins of the autophagic machinery and vesicles presenting features of autophagosomes are recruited at C. albicans invasion sites. These events are associated with host PM damage caused by the active penetration of C. albicans. We showed that ATG5 and ATG16L1 proteins contribute to PM repair mediated by lysosomal membrane exocytosis and participate in protection of epithelial cells' integrity against C. albicans-induced cell death. Our findings extend the knowledge on emerging roles of the autophagic machinery in stress-related membrane dynamics.