Cells from bacteria to human release vesicles into their extracellular environment. These extracellular vesicles (EVs) contain multiple classes of molecules, including nucleic acids, proteins, and lipids. The isolation and analysis of EV cargos from mammalian cell culture and liquid biopsy samples has become a powerful approach for uncovering the messages that are packaged into these organelles. However, this approach has not been tenable in invertebrate model systems due to lack of sufficient amounts of pure EVs. Here we report a robust and reproducible procedure to isolate EVs from Caenorhabditis elegans with yields similar to those obtained from human cell culture. Through nanoparticle tracking, transmission electron microscopy, flow cytometry, mass spectrometry, RNAseq, and immunoaffinity analysis we provide the first ever detailed characterization of C.elegans EV composition and demonstrate that C. elegans EVs share fundamentally similar properties with their mammalian counterparts. These include vesicle size, enrichment for lipid rafts, and similar types of RNA and protein cargos. This ability of isolate pure EVs on a scale amenable to multiple types of downstream analyses permits, multi-omics characterization of EV cargos in an invertebrate model system. BACKGROUNDThe cellular secretion of small membrane-bound extracellular vesicles (EVs) into the external environment is an ancient capacity conserved throughout evolution 1,2,3 . EVs range in size from 30-1000 nm in diameter and can be internalized into recipient cells via endocytosis or membrane fusion. There is growing recognition that EVs may play important roles in facilitating intercellular communication through transferring protein, lipid, and genetic cargos 4,5 . The content of EVs are influenced by the physiological state of the cells and are thought to play critical roles in diverse cellular processes as well as multiple types of pathological conditions including cancer, immunity, and neurodegenerative diseases.Many studies have characterized the composition of mammalian EVs. Such EVs are highly enriched in the lipid raft species cholesterol, and sphingomyelin, and in proteins that associate with lipid rafts, including glycosylphosphatidylinositol-anchored (GPI) proteins 6,7,8 . The two main types of EVs studied so far are exosomes, which release from the endosomal network and microvesicles which bud directly from the plasma membrane 9,10 . Mammalian exosomes commonly contain membrane proteins such as CD9, CD63, and CD81, as well as lysosomal and endosomal-marking proteins, and various amounts of extracellular matrix proteins while are largely free of nuclear proteins 11 . Microvesicles have less defined protein markers but may contain proteins of mitochondria and endoplasmic reticulum origin 11 . However, the methods utilized to purify EVs do not separate these types of vesicles, so it is currently unclear how to definitively distinguish the cargos from different types of EVs 12 . EVs from diverse species, including humans, are known to carry RNA ...

Amino acid isomerization is a spontaneous chemical modification potentially related to the underlying causes of Alzheimer's disease (AD). We demonstrate that data-independent acquisition mass spectrometry can be used to characterize isomerization in complex protein mixtures. Examination of a large cohort of brain tissue samples revealed a striking relationship between isomerization of tau and AD status. Surprisingly, isomerization was found to be more abundant in both autosomal dominant and sporadic AD samples relative to controls. We hypothesize that lower autophagic flux in AD brains accounts for these results. Additional data, including quantitative analysis of proteins related to autophagy, strongly support this hypothesis. For example, isomerization of tau is positively correlated with levels of p62, a recognized indicator of autophagic inhibition. In sum, the data suggest strong ties between isomerization and autophagic flux, which may therefore represent a promising target for future investigations into the therapy and prevention of AD.