Dataset Information

CNS-derived extracellular vesicles from superoxide dismutase 1 (SOD1)G93A ALS mice originate from astrocytes and neurons and carry misfolded SOD1.

ABSTRACT: Extracellular vesicles (EVs) are secreted by myriad cells in culture and also by unicellular organisms, and their identification in mammalian fluids suggests that EV release also occurs at the organism level. However, although it is clearly important to better understand EVs' roles in organismal biology, EVs in solid tissues have received little attention. Here, we modified a protocol for EV isolation from primary neural cell culture to collect EVs from frozen whole murine and human neural tissues by serial centrifugation and purification on a sucrose gradient. Quantitative proteomics comparing brain-derived EVs from nontransgenic (NTg) and a transgenic amyotrophic lateral sclerosis (ALS) mouse model, superoxide dismutase 1 (SOD1)G93A, revealed that these EVs contain canonical exosomal markers and are enriched in synaptic and RNA-binding proteins. The compiled brain EV proteome contained numerous proteins implicated in ALS, and EVs from SOD1G93A mice were significantly depleted in myelin-oligodendrocyte glycoprotein compared with those from NTg animals. We observed that brain- and spinal cord-derived EVs, from NTg and SOD1G93A mice, are positive for the astrocyte marker GLAST and the synaptic marker SNAP25, whereas CD11b, a microglial marker, was largely absent. EVs from brains and spinal cords of the SOD1G93A ALS mouse model, as well as from human SOD1 familial ALS patient spinal cord, contained abundant misfolded and nonnative disulfide-cross-linked aggregated SOD1. Our results indicate that CNS-derived EVs from an ALS animal model contain pathogenic disease-causing proteins and suggest that brain astrocytes and neurons, but not microglia, are the main EV source.

SUBMITTER: Silverman JM

PROVIDER: S-EPMC6416428 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

CNS-derived extracellular vesicles from superoxide dismutase 1 (SOD1)<sup>G93A</sup> ALS mice originate from astrocytes and neurons and carry misfolded SOD1.

Silverman Judith M JM Christy Darren D Shyu Chih Cheih CC Moon Kyung-Mee KM Fernando Sarah S Gidden Zoe Z Cowan Catherine M CM Ban Yuxin Y Stacey R Greg RG Grad Leslie I LI McAlary Luke L Mackenzie Ian R IR Foster Leonard J LJ Cashman Neil R NR

The Journal of biological chemistry 20190111 10

Extracellular vesicles (EVs) are secreted by myriad cells in culture and also by unicellular organisms, and their identification in mammalian fluids suggests that EV release also occurs at the organism level. However, although it is clearly important to better understand EVs' roles in organismal biology, EVs in solid tissues have received little attention. Here, we modified a protocol for EV isolation from primary neural cell culture to collect EVs from frozen whole murine and human neural tissu ...[more]

PMID: 30635404

Similar Datasets

Project description:Mutations in superoxide dismutase 1 (SOD1), which are one cause of familial amyotrophic lateral sclerosis (fALS), induce misfolding and aggregation of the protein. Misfolding can be detected by the binding of antibodies raised against peptide epitopes that are normally buried in the native conformation, shifts in solubility in non-ionic detergents, and the formation of macromolecular inclusions. In the present study, we investigate the relationship between detergent-insoluble and sedimentable forms of mutant SOD1, forms of mutant SOD1 with aberrantly accessible epitopes, and mutant protein in inclusions with the goal of defining the spectrum of misfolded states that mutant SOD1 can adopt.Using combined approaches in cultured cell models, we demonstrate that a substantial fraction of mutant SOD1 adopts a non-native conformation that remains soluble and freely mobile. We also show that mutant SOD1 can produce multimeric assemblies of which some are insoluble in detergent and large enough to sediment by ultracentrifugation and some are large enough to detect visually. Three conformationally restricted antibodies were found to be useful in discriminating mal-folded forms of mutant SOD1. An antibody termed C4F6 displays properties consistent with recognition of soluble, freely mobile, mal-folded mutant SOD1. An antibody termed SEDI, which recognizes C-terminal residues, detects larger inclusion structures as well as soluble misfolded entities. An antibody termed hSOD1, which recognizes aa 24-36, detects an epitope shared by soluble non-natively folded WT and mutant SOD1. This epitope becomes inaccessible in aggregates of mutant SOD1.Our studies demonstrate how different methods of detecting misfolding and aggregation of mutant SOD1 reveal different forms of aberrantly folded protein. Immunological and biochemical methods can be used in combination to detect soluble and insoluble misfolded forms of mutant SOD1. Our findings support the view that mutant SOD1 can adopt multiple misfolded conformations with the potential that different structural variants mediate different aspects of fALS.

Dataset Information

CNS-derived extracellular vesicles from superoxide dismutase 1 (SOD1)G93A ALS mice originate from astrocytes and neurons and carry misfolded SOD1.

Publications

CNS-derived extracellular vesicles from superoxide dismutase 1 (SOD1)<sup>G93A</sup> ALS mice originate from astrocytes and neurons and carry misfolded SOD1.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets