Amyotrophic lateral sclerosis (ALS) is a progressive adult-onset neurodegenerative disease, that affects cortical, bulbar and spinal motor neurons, and it is considered a proteinopathy, in which pathological proteins (SOD1, TDP-43, and FUS) may accumulate and interfere with neuronal functions eventually leading to cell death. These proteins can be released from cells and transported in the body fluids by extracellular vesicles (EVs). EVs are spherical vesicles, which are classified mainly in microvesicles (MVs) and exosomes (EXOs) based on their biogenesis, size and surface markers. In this study we characterized MVs and EXOs isolated from plasma of sporadic ALS patients and healthy controls and determined their number, size and SOD1, TDP-43, and FUS protein composition. No variation was found in the number of EVs between ALS patients and controls. However, the mean size both for MVs and for EXOs resulted increased in ALS patients compared to controls. MVs derived from ALS patients were enriched in SOD1, TDP-43, phospho-TDP-43, and FUS proteins compared to CTRLs. SOD1 was generally more concentrated in EXOs than in MVs, while TDP-43 and FUS protein levels were slightly higher in MVs than in EXOs. We demonstrated that MVs and EXOs size were increased in ALS patients compared to controls and that MVs of ALS patients were enriched with toxic proteins compared to CTRLs. EXOs did not show any protein changes. These data may suggest that MVs can transport toxic proteins and might play a role in prion-like propagation of ALS disease.

Urinary extracellular vesicles (ueVs) provide bio-markers for kidney and urogenital diseases.centrifugation is the most common method used to enrich ueVs. However, a majority of studies to date have focused on the ultracentrifugation pellet, potentially losing a novel source of important biomarkers that could be obtained at lower centrifugation. thus, the aim of this study is to rigorously characterize for the first time uEVs in the low speed pellet and determine the minimal volume of urine required for proteomic analysis (≥9.0 mL urine) and gene ontology classification identified 75% of the protein as extracellular exosomes. cryo-transmission electron Microscopy (≥3.0 mL urine) provided evidence of a heterogeneous population of eVs for size and morphology independent of uromodulin filaments. Western blot detected several specific uEV kidney and EV markers (≥4.5 mL urine per lane). microRNAs quantification by qPCR was possible with urine volume as low as 0.5 mL. Particle enumeration with tunable resistive pulse sensing, nano particles tracking analysis and single eV high throughput imaging flow cytometry are possible starting from 0.5 and 3.0 mL of urine respectively. this work characterizes a neglected source of ueVs and provides guidance with regard to volume of urine necessary to carry out multi-omic studies and reveals novel aspects of ueV analysis such as autofluorescence of podocyte origin.Urinary extracellular vesicles (uEVs) are a medley of exosomes, exosome-like vesicles and microparticles/ microvesicles 1-4 . Confusing nomenclature aside 5,6 , all uEVs secreted in urine transport proteins, nucleic acid and small metabolites from all epithelial cells forming the nephron and lower urinary tract 7,8 . Thus, uEVs have become a valuable source of biomarkers for identifying any changes in the physio-pathological state of their parental cell. Moreover, uEVs are also bio-activators in renal diseases 9,10 . The most common method in use to enrich uEVs is a 2 or 3 step centrifugation protocol [11][12][13] . While it has been commonly discarded, the pellet obtained at relative low centrifugation force has proved to be an additional source of uEVs 14,15 . However this pellet has not been thoroughly characterized.In addition, the concomitant presence of multiple biomarker in uEVs offers the possibility to integrate multi-omic data analysis to better understand mechanism and possibly identify key role molecules implicated in the onset and progression of the disease 16 . However, no study has reported the amount of volume of urine that is necessary to collect to support multiple analyses. Hence, this study aims to: (1) estimate the minimum volume of urine necessary to yield uEVs for characterization according to both minimal information for studies of extracellular vesicles (MISEV) 17 and downstream analysis applying a very rigorous approach using several control sets for each analysis; (2) test the limit of detection of the techniques employed for downstream analysis and EV characterization before and after eliminati...

Extracellular vesicles (EVs) have been described as novel biomarkers and bioactivators in vascular dysfunction in hypertension. However, the mechanism(s) by which EVs affect vascular function is unknown. To examine the effects of EVs on endothelial-dependent vasodilation (acetylcholine), we isolated circulating EVs from platelet-poor plasma using a low centrifugation speed (17 000 g ) and mesenteric resistance arteries from 12-week-old normotensive WKYs (Wistar-Kyoto rats) and SHRs (spontaneously hypertensive rats). Arteries were cannulated on a pressure myograph, and EVs were added to the vessel lumen and circulating bath. We found that circulating EVs from normotensive WKY reduced vasodilation of normotensive WKY arteries but had no effect on hypertensive SHR arteries. In contrast, EVs from hypertensive SHR failed to reduce vasodilation of arteries from both WKY and SHR. The restraining effect on vasodilation by EVs from normotensive WKY may be mediated by inhibition of eNOS (endothelial NO synthase), as addition of L-nitro-arginine methyl ester did not provide any additive effect. Moreover, circulating EVs from normotensive 6-week-old SHR—an age where SHRs have not yet developed hypertension—had similar restraining effect on vasodilation. In addition, delipidation of EVs did not alter the restraining effect of EVs from WKY but did restore the restraining effect of EVs from SHR. Finally, EVs from normotensive humans also restrained vasodilation of normotensive mouse arteries—an effect not observed in EVs from hypertensive humans. Taken together, our data support a vasoactive role of EVs that is altered in hypertension.

In the past, amyotrophic lateral sclerosis (ALS) has been considered a ‘neurocentric’ disease; however, new evidence suggests that it should instead be looked at from a ‘multisystemic’ or ‘non-neurocentric’ point of view. From 2006, we focused on the study of non-neural cells: ALS patients’ peripheral blood mononuclear cells (PMBCs) and lymphoblastoid cell lines (LCLs). Here, we characterize LCLs of sporadic ALS (sALS) and patients carrying SOD1, TARDBP and FUS mutations to identify an ALS biologically relevant molecular signature, and determine whether and how mutations differentially affect ALS-linked pathways. Although LCLs are different from motor neurons (MNs), in LCLs we found some features typical of degenerating MNs in ALS, i.e. protein aggregation and mitochondrial dysfunction. Moreover, different gene mutations have different effects on ALS cellular mechanisms. TARDBP and FUS mutations imbalance mitochondrial dynamism toward increased fusion, whereas sALS and SOD1 mutations mainly affect fission. With regards to protein aggregation and/or mislocalization, TARDBP and SOD1 mutations show the presence of aggregates, whereas FUS mutation does not induce protein aggregation and/or mislocalization. Finally, all LCLs, independently from mutation, are not able to work in a condition of excessive energy request, suggesting that mitochondria from ALS patients are characterized by a significant metabolic defect. Taken together, these data indicate that LCLs could be a valid cellular model in ALS research in the identification and study of specific pathological pathways.

The lack of biomarkers in Amyotrophic Lateral Sclerosis (ALS) makes it difficult to determine the stage of the disease in patients and, therefore, it delays therapeutic trials. Microvesicles (MVs) are possible biomarkers implicated in physiological and pathological functions, however, their role in ALS remains unclear. We investigated whether plasma derived microvesicles could be overrepresented in a group of 40 patients affected by ALS compared to 28 Alzheimer’s Disease (AD) patients and 36 healthy volunteers. Leukocyte derived MVs (LMVs) compared to endothelial, platelet, erythrocyte derived MVs, were mostly present in ALS patients compared to AD patients and healthy donors. Correlation analysis corrected for the presence of confounding variables (riluzole, age at onset, site of onset, gender) was tested between PRL (Progression Rate at the Last visit) and LMVs, and a statistically significant value was found (Pearson partial correlation r = 0.407, p = 0.006). We also investigated SOD1, TDP-43 intravesicular protein level in LMVs. Misfolded SOD1 was selectively transported by LMVs and its protein level was associated with the percentage of LMVs in slow progressing patients ( r = 0.545, p = 0.033). Our preliminary findings suggest that LMVs are upregulated in ALS patients and they can be considered possible markers of disease progression.