Comprehensive proteomic profiling of plasma-derived Extracellular Vesicles from dementia with Lewy Bodies patients

Altres ajuts: Grant 201405/10 from Fundació La Marató TV3Proteins and nucleic acids contained in extracellular vesicles (EVs) are considered a feasible source of putative biomarkers for physiological and pathological conditions. Within the nervous system, not only neurons but also other brain cells are able to produce EVs, which have been involved in their physiological processes and also in the development and course of several neurodegenerative diseases. Among these, dementia with Lewy bodies (DLB) is the second cause of dementia worldwide, though most cases are missed or misdiagnosed as Alzheimer's disease (AD) due to the important clinical and pathological overlap between both diseases. In an attempt to find reliable biomarkers for DLB diagnosis, our group characterized the proteome of plasma-derived EVs from DLB patients compared to aged-matched healthy controls (HCs) using two different proteomic LC-MS/MS approaches. Remarkably, we found that gelsolin and butyrylcholinesterase were differentially identified between DLB and HCs. Further validation of these results using conventional ELISA techniques, and including an additional group of AD patients, pointed to decreased levels of gelsolin in plasma-EVs from DLB compared to HCs and to AD samples. Thus, gelsolin may be considered a possible biomarker for the differentiation between DLB and AD

Size-Exclusion Chromatography-based isolation minimally alters Extracellular Vesicles' characteristics compared to precipitating agents

Extracellular vesicles (EVs) have become an attractive field among the scientific community. Yet, a major challenge is to define a consensus method for EVs isolation. Ultracentrifugation has been the most widely used methodology but rapid methods, including Size Exclusion Chromatography (SEC) and/or precipitating agents such as Polyethylene glycol (PEG) or PRotein Organic Solvent PRecipitation (PROSPR) have emerged. To evaluate the impact of these different methods on the resulting EV preparations, plasma EVs were isolated using SEC, PEG and PROSPR, and their total protein content, NTA and Cryo-electron microscopy profiles, and EV-markers were compared. Also, their effect on recipient cells was tested. Low protein content and Cryo-EM analysis showed that SEC removed most of the overabundant soluble plasma proteins, which were not removed using PEG and partially by PROSPR. Moreover, only SEC allowed the detection of the EV-markers CD9, CD63 and CD81, LGALS3BP and CD5L, suggesting a putative interference of the precipitating agents in the structure/composition of the EVs. Furthermore, PEG and PROSPR-based EV isolation resulted in reduced cell viability in vitro. These results stress that appropriate EV-isolation method should be considered depending on the forthcoming application of the purified EVs

Exploratory study on microRNA profiles from plasma-derived extracellular vesicles in Alzheimer's disease and dementia with Lewy bodies

Altres ajuts: This work was also supported by the MaratóTV3 grant 201405/10.Because of the increasing life expectancy in our society, aging-related neurodegenerative disorders are one of the main issues in global health. Most of these diseases are characterized by the deposition of misfolded proteins and a progressive cognitive decline. Among these diseases, Alzheimer's disease (AD) and dementia with Lewy bodies (DLB) are the most common types of degenerative dementia. Although both show specific features, an important neuropathological and clinical overlap between them hampers their correct diagnosis. In this work, we identified molecular biomarkers aiming to improve the misdiagnosis between both diseases. Plasma extracellular vesicles (EVs) -from DLB, AD and healthy controls- were isolated using size-exclusion chromatography (SEC) and characterized by flow cytometry, Nanoparticle Tracking Analysis (NTA) and cryo-electron microscopy. Next Generation Sequencing (NGS) and related bibliographic search was performed and a selected group of EV-associated microRNAs (miRNAs) was analysed by qPCR. Results uncovered two miRNAs (hsa-miR-451a and hsa-miR-21-5p) significantly down-regulated in AD samples respect to DLB patients, and a set of four miRNAs (hsa-miR-23a-3p, hsa-miR-126-3p, hsa-let-7i-5p, and hsa-miR-151a-3p) significantly decreased in AD respect to controls. The two miRNAs showing decreased expression in AD in comparison to DLB provided area under the curve (AUC) values of 0.9 in ROC curve analysis, thus suggesting their possible use as biomarkers to discriminate between both diseases. Target gene analysis of these miRNAs using prediction online tools showed accumulation of phosphorylation enzymes, presence of proteasome-related proteins and genes involved in cell death among others. Our data suggest that plasma-EV associated miRNAs may reflect a differential profile for a given dementia-related disorder which, once validated in larger cohorts of patients, could help to improve the differential diagnosis of DLB versus AD. The online version of this article (10.1186/s40035-019-0169-5) contains supplementary material, which is available to authorized users

Platelet miRNA Biosignature Discriminates between Dementia with Lewy Bodies and Alzheimer’s Disease

Dementia with Lewy bodies (DLB) is one of the most common causes of degenerative dementia, after Alzheimer's disease (AD), and presents pathological and clinical overlap with both AD and Parkinson's disease (PD). Consequently, only one in three DLB cases is diagnosed correctly. Platelets, previously related to neurodegeneration, contain microRNAs (miRNAs) whose analysis may provide disease biomarkers. Here, we profiled the whole platelet miRNA transcriptome from DLB patients and healthy controls. Differentially expressed miRNAs were further validated in three consecutive studies from 2017 to 2019 enrolling 162 individuals, including DLB, AD, and PD patients, and healthy controls. Results comprised a seven-miRNA biosignature, showing the highest diagnostic potential for the differentiation between DLB and AD. Additionally, compared to controls, two miRNAs were down-regulated in DLB, four miRNAs were up-regulated in AD, and two miRNAs were down-regulated in PD. Predictive target analysis identified three disease-specific clusters of pathways as a result of platelet-miRNA deregulation. Our cross-sectional study assesses the identification of a novel, highly specific and sensitive platelet-associated miRNA-based biosignature, which distinguishes DLB from AD

Proteomics study of human cord blood reticulocyte-derived exosomes

Reticulocyte-derived exosomes (Rex), extracellular vesicles of endocytic origin, were initially discovered as a cargo-disposal mechanism of obsolete proteins in the maturation of reticulocytes into erythrocytes. In this work, we present the first mass spectrometry-based proteomics of human Rex (HuRex). HuRex were isolated from cultures of human reticulocyte-enriched cord blood using different culture conditions and exosome isolation methods. The newly described proteome consists of 367 proteins, most of them related to exosomes as revealed by gene ontology over-representation analysis and include multiple transporters as well as proteins involved in exosome biogenesis and erythrocytic disorders. Immunoelectron microscopy validated the presence of the transferrin receptor. Moreover, functional assays demonstrated active capture of HuRex by mature dendritic cells. As only seven proteins have been previously associated with HuRex, this resource will facilitate studies on the role of human reticulocyte-derived exosomes in normal and pathological conditions affecting erythropoiesis

Neuron-derived extracellular vesicles contain synaptic proteins, promote spine formation, activate TrkB-mediated signalling and preserve neuronal complexity

Extracellular vesicles (EVs) play an important role in intercellular communication as carriers of signalling molecules such as bioactive miRNAs, proteins and lipids. EVs are key players in the functioning of the central nervous system (CNS) by influencing synaptic events and modulating recipient neurons. However, the specific role of neuron-to-neuron communication via EVs is still not well understood. Here, we provide evidence that primary neurons uptake neuron-derived EVs in the soma, dendrites, and even in the dendritic spines, and carry synaptic proteins. Neuron-derived EVs increased spine density and promoted the phosphorylation of Akt and ribosomal protein S6 (RPS6), via TrkB-signalling, without impairing the neuronal network activity. Strikingly, EVs exerted a trophic effect on challenged nutrient-deprived neurons. Altogether, our results place EVs in the spotlight for synaptic plasticity modulation as well as a possible therapeutic tool to fight neurodegeneration

Sociodemographic, clinical and laboratory factors on admission associated with COVID19 mortality in hospitalized patients: A retrospective observational study

Background To identify and quantify associations between baseline characteristics on hospital admission and mortality in patients with COVID-19 at a tertiary hospital in Spain. Methods and findings This retrospective case series included 238 patients hospitalized for COVID-19 at Hospital Universitario Clı´nico San Cecilio (Granada, Spain) who were discharged or who died. Electronic medical records were reviewed to obtain information on sex, age, personal antecedents, clinical features, findings on physical examination, and laboratory results for each patient. Associations between mortality and baseline characteristics were estimated as hazard ratios (HR) calculated with Cox regression models. Series mortality was 25.6%. Among patients with dependence for basic activities of daily living, 78.7% died, and among patients residing in retirement homes, 80.8% died. The variables most clearly associated with a greater hazard of death were age (3% HR increase per 1-year increase in age; 95%CI 1–6), diabetes mellitus (HR 2.42, 95%CI 1.43–4.09), SatO2/ FiO2 ratio (43% HR reduction per 1-point increase; 95%CI 23–57), SOFA score (19% HR increase per 1-point increase, 95%CI 5–34) and CURB-65 score (76% HR increase per 1- point increase, 95%CI 23–143). Conclusions The patients residing in retirement homes showed great vulnerability. The main baseline factors that were independently associated with mortality in patients hospitalized for COVID-19 were older age, diabetes mellitus, low SatO2/FiO2 ratio, and high SOFA and CURB-65 scores.Fondos Estructurales de la Union Europea (FEDER)Unit of Excellence on Exercise and Health (UCEES), University of Granad

Minimal information for studies of extracellular vesicles 2018 (MISEV2018):a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines

The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points

Alternative Splicing of Alpha- and Beta-Synuclein Genes Plays Differential Roles in Synucleinopathies

The synuclein family is composed of three members, two of which, α- and β-synuclein, play a major role in the development of synucleinopathies, including Parkinson’s disease (PD) as most important movement disorder, dementia with Lewy bodies (DLB) as the second most frequent cause of dementia after Alzheimer’s disease and multiple system atrophy. Whereas abnormal oligomerization and fibrillation of α-synuclein are now well recognized as initial steps in the development of synucleinopathies, β-synuclein is thought to be a natural α-synuclein anti-aggregant. α-synuclein is encoded by the SNCA gene, and β-synuclein by SNCB. Both genes are homologous and undergo complex splicing events. On one hand, in-frame splicing of coding exons gives rise to at least three shorter transcripts, and the functional properties of the corresponding protein isoforms are different. Another type of alternative splicing is the alternative inclusion of at least four initial exons in the case of SNCA, and two in the case of SNCB. Finally, different lengths of 3’ untranslated regions have been also reported for both genes. SNCB only expresses in the brain, but some of the numerous SNCA transcripts are also brain-specific. With the present article, we aim to provide a systematic review of disease related changes in the differential expression of the various SNCA and SNCB transcript variants in brain, blood, and non-neuronal tissue of synucleinopathies, but especially PD and DLB as major neurodegenerative disorders

Non-Coding RNAs as Sensors of Oxidative Stress in Neurodegenerative Diseases

Oxidative stress (OS) results from an imbalance between the production of reactive oxygen species and the cellular antioxidant capacity. OS plays a central role in neurodegenerative diseases, where the progressive accumulation of reactive oxygen species induces mitochondrial dysfunction, protein aggregation and inflammation. Regulatory non-protein-coding RNAs (ncRNAs) are essential transcriptional and post-transcriptional gene expression controllers, showing a highly regulated expression in space (cell types), time (developmental and ageing processes) and response to specific stimuli. These dynamic changes shape signaling pathways that are critical for the developmental processes of the nervous system and brain cell homeostasis. Diverse classes of ncRNAs have been involved in the cell response to OS and have been targeted in therapeutic designs. The perturbed expression of ncRNAs has been shown in human neurodegenerative diseases, with these changes contributing to pathogenic mechanisms, including OS and associated toxicity. In the present review, we summarize existing literature linking OS, neurodegeneration and ncRNA function. We provide evidences for the central role of OS in age-related neurodegenerative conditions, recapitulating the main types of regulatory ncRNAs with roles in the normal function of the nervous system and summarizing up-to-date information on ncRNA deregulation with a direct impact on OS associated with major neurodegenerative conditions