Lipidomic Analysis of Extracellular Vesicles from the Pathogenic Phase of Paracoccidioides brasiliensis

Background: Fungal extracellular vesicles are able to cross the cell wall and transport molecules that help in nutrient acquisition, cell defense, and modulation of the host defense machinery.Methodology/Principal Findings: Here we present a detailed lipidomic analysis of extracellular vesicles released by Paracoccidioides brasiliensis at the yeast pathogenic phase. We compared data of two representative isolates, Pb3 and Pb18, which have distinct virulence profiles and phylogenetic background. Vesicle lipids were fractionated into different classes and analyzed by either electrospray ionization- or gas chromatography-mass spectrometry. We found two species of monohexosylceramide and 33 phospholipid species, including phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, phosphatidylserine, phosphatidylinositol, and phosphatidylglycerol. Among the phospholipid-bound fatty acids in extracellular vesicles, C181 predominated in Pb3, whereas C18:2 prevailed in Pb18. the prevalent sterol in Pb3 and Pb18 vesicles was brassicasterol, followed by ergosterol and lanosterol. Inter-isolate differences in sterol composition were observed, and also between extracellular vesicles and whole cells.Conclusions/Significance: the extensive lipidomic analysis of extracellular vesicles from two P. brasiliensis isolates will help to understand the composition of these fungal components/organelles and will hopefully be useful to study their biogenesis and role in host-pathogen interactions.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)National Institutes of Health (NIH)Universidade Federal de São Paulo, UNIFESP, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilUniv Texas El Paso, Dept Biol Sci, Border Biomed Res Ctr, El Paso, TX 79968 USAUniversidade Federal de São Paulo, UNIFESP, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilFAPESP: 06/05095-6FAPESP: 07/04757-8FAPESP: 07/59768-4CNPq: 301666/2010-5National Institutes of Health (NIH): 5G12RR008124-16A1National Institutes of Health (NIH): 5G12RR008124-16A1S1National Institutes of Health (NIH): G12MD007592Web of Scienc

Cyclodextrin Induces Calcium-Dependent Lysosomal Exocytosis

Cyclodextrins (CDs) have long been used to manipulate cellular cholesterol levels both in vitro and in vivo, but their direct effects at a cellular level are not well characterized. Recently, CDs have garnered much interest because of their ability to clear stored cholesterol from Niemann Pick Type C (NPC) cells and markedly prolong the life of NPC1 disease mice. Here, we investigate the hypothesis that treatment with 2-hydroxypropyl- β-cyclodextrin (HPB-CD) stimulates lysosomal exocytosis in a calcium-enhanced manner. We propose that this exocytosis is the mechanism by which HPB-CD ameliorates the endolysosomal cholesterol storage phenotype in NPC cells. These findings have significant implications for the use of HPB-CD in biochemical assays and data interpretation as well as for their use for the treatment for NPC and other disorders

Plasmalogen enrichment in exosomes secreted by a nematode parasite versus those derived from its mouse host: implications for exosome stability and biology

Extracellular vesicles (EVs) mediate communication between cells and organisms across all 3 kingdoms of life. Several reports have demonstrated that EVs can transfer molecules between phylogenetically diverse species and can be used by parasites to alter the properties of the host environment. Whilst the concept of vesicle secretion and uptake is broad reaching, the molecular composition of these complexes is expected to be diverse based on the physiology and environmental niche of different organisms. Exosomes are one class of EVs originally defined based on their endocytic origin, as these derive from multivesicular bodies that then fuse with the plasma membrane releasing them into the extracellular environment. The term exosome has also been used to describe any small EVs recovered by high-speed ultracentrifugation, irrespective of origin since this is not always well characterized. Here, we use comparative global lipidomic analysis to examine the composition of EVs, which we term exosomes, that are secreted by the gastrointestinal nematode, Heligmosomoides polygyrus, in relation to exosomes secreted by cells of its murine host. Ultra-performance liquid chromatography – tandem mass spectrometry (UPLC-MS/MS) analysis reveals a 9- to 62-fold enrichment of plasmalogens, as well as other classes of ether glycerophospholipids, along with a relative lack of cholesterol and sphingomyelin (SM) in the nematode exosomes compared with those secreted by murine cells. Biophysical analyses of the membrane dynamics of these exosomes demonstrate increased rigidity in those from the nematode, and parallel studies with synthetic vesicles support a role of plasmalogens in stabilizing the membrane structure. These results suggest that nematodes can maintain exosome membrane structure and integrity through increased plasmalogens, compensating for diminished levels of other lipids, including cholesterol and SM. This work also illuminates the prevalence of plasmalogens in some EVs, which has not been widely reported and could have implications for the biochemical or immunomodulatory properties of EVs. Further comparative analyses such as those described here will shed light on diversity in the molecular properties of EVs that enable them to function in cross-species communication

Neuronal lysosomal dysfunction releases exosomes harboring APP C-terminal fragments and unique lipid signatures

Defects in endolysosomal and autophagic functions are increasingly viewed as key pathological features of neurodegenerative disorders. A master regulator of these functions is phosphatidylinositol-3-phosphate (PI3P), a phospholipid synthesized primarily by class III PI 3-kinase Vps34. Here we report that disruption of neuronal Vps34 function in vitro and in vivo impairs autophagy, lysosomal degradation as well as lipid metabolism, causing endolysosomal membrane damage. PI3P deficiency also promotes secretion of unique exosomes enriched for undigested lysosomal substrates, including amyloid precursor protein C-terminal fragments (APP-CTFs), specific sphingolipids, and the phospholipid bis(monoacylglycero)phosphate (BMP), which normally resides in the internal vesicles of endolysosomes. Secretion of these exosomes requires neutral sphingomyelinase 2 and sphingolipid synthesis. Our results reveal a homeostatic response counteracting lysosomal dysfunction via secretion of atypical exosomes eliminating lysosomal waste and define exosomal APP-CTFs and BMP as candidate biomarkers for endolysosomal dysfunction associated with neurodegenerative disorders.Fan Wang for the kind gift of the Pi3kc3flox/flox mice. We thank Basant Abdulrahman and Hermann Schaetzl for providing the gene-edited Atg5 KO N2a cells. We are also grateful to Zhenyu Yue, Ralph Nixon, and Jean Gruenberg for the kind gift of anti-Atg14L, Cathepsin D, and BMP antibodies, respectively. We thank Thomas Südhof for sharing Cre recombinase lentiviruses. We thank the OCS Microscopy Core of New York University Langone Medical Center for the support of the EM work and Rocio Perez-Gonzalez and Efrat Levy of New York University for their support during optimization of the brain exosome isolation technique. We thank Elizabeta Micevska for the maintenance and genotyping of the animal colony and Bowen Zhou for the preliminary lipidomic analysis of conditional Pi3kc3 cKO mice. We also thank Rebecca Williams and Catherine Marquer for critically reading the manuscript. This work was supported by grants from the Fundação para a Ciência e Tecnologia (PD/BD/105915/2014 to A.M.M.); the National Institute of Health (R01 NS056049 to G.D.P., transferred to Ron Liem, Columbia University; T32-MH015174 to Rene Hen (Z.M.L.)). Z.M.L. and R.B.C. received pilot grants from ADRC grant P50 AG008702 to S.A.S.info:eu-repo/semantics/publishedVersio

Nanostructural and Transcriptomic Analyses of Human Saliva Derived Exosomes

Exosomes, derived from endocytic membrane vesicles are thought to participate in cell-cell communication and protein and RNA delivery. They are ubiquitous in most body fluids (breast milk, saliva, blood, urine, malignant ascites, amniotic, bronchoalveolar lavage, and synovial fluids). In particular, exosomes secreted in human saliva contain proteins and nucleic acids that could be exploited for diagnostic purposes. To investigate this potential use, we isolated exosomes from human saliva and characterized their structural and transcriptome contents.Exosomes were purified by differential ultracentrifugation and identified by immunoelectron microscopy (EM), flow cytometry, and Western blot with CD63 and Alix antibodies. We then described the morphology, shape, size distribution, and density using atomic force microscopy (AFM). Microarray analysis revealed that 509 mRNA core transcripts are relatively stable and present in the exosomes. Exosomal mRNA stability was determined by detergent lysis with RNase A treatment. In vitro, fluorescently labeled saliva exosomes could communicate with human keratinocytes, transferring their genetic information to human oral keratinocytes to alter gene expression at a new location.Our findings are consistent with the hypothesis that exosomes shuttle RNA between cells and that the RNAs present in the exosomes may be a possible resource for disease diagnostics

Membrane vesicles, current state-of-the-art: emerging role of extracellular vesicles

Release of membrane vesicles, a process conserved in both prokaryotes and eukaryotes, represents an evolutionary link, and suggests essential functions of a dynamic extracellular vesicular compartment (including exosomes, microparticles or microvesicles and apoptotic bodies). Compelling evidence supports the significance of this compartment in a broad range of physiological and pathological processes. However, classification of membrane vesicles, protocols of their isolation and detection, molecular details of vesicular release, clearance and biological functions are still under intense investigation. Here, we give a comprehensive overview of extracellular vesicles. After discussing the technical pitfalls and potential artifacts of the rapidly emerging field, we compare results from meta-analyses of published proteomic studies on membrane vesicles. We also summarize clinical implications of membrane vesicles. Lessons from this compartment challenge current paradigms concerning the mechanisms of intercellular communication and immune regulation. Furthermore, its clinical implementation may open new perspectives in translational medicine both in diagnostics and therapy

Characterization of brain‐derived extracellular vesicle lipids in Alzheimer's disease

Lipid dyshomeostasis is associated with the most common form of dementia, Alzheimer's disease (AD). Substantial progress has been made in identifying positron emission tomography and cerebrospinal fluid biomarkers for AD, but they have limited use as front-line diagnostic tools. Extracellular vesicles (EVs) are released by all cells and contain a subset of their parental cell composition, including lipids. EVs are released from the brain into the periphery, providing a potential source of tissue and disease specific lipid biomarkers. However, the EV lipidome of the central nervous system is currently unknown and the potential of brain-derived EVs (BDEVs) to inform on lipid dyshomeostasis in AD remains unclear. The aim of this study was to reveal the lipid composition of BDEVs in human frontal cortex, and to determine whether BDEVs have an altered lipid profile in AD. Using semi-quantitative mass spectrometry, we describe the BDEV lipidome, covering four lipid categories, 17 lipid classes and 692 lipid molecules. BDEVs were enriched in glycerophosphoserine (PS) lipids, a characteristic of small EVs. Here we further report that BDEVs are enriched in ether-containing PS lipids, a finding that further establishes ether lipids as a feature of EVs. BDEVs in the AD frontal cortex offered improved detection of dysregulated lipids in AD over global lipid profiling of this brain region.  AD BDEVs had significantly altered glycerophospholipid and sphingolipid levels, specifically increased plasmalogen glycerophosphoethanolamine and decreased polyunsaturated fatty acyl containing lipids, and altered amide-linked acyl chain content in sphingomyelin and ceramide lipids relative to CTL. The most prominent alteration was a two-fold decrease in lipid species containing anti-inflammatory/pro-resolving docosahexaenoic acid. The in-depth lipidome analysis provided in this study highlights the advantage of EVs over more complex tissues for improved detection of dysregulated lipids that may serve as potential biomarkers in the periphery