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

Abstract

Edited by Phyllis I. Hanson 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 SOD1 G93A 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 SOD1 G93A 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 SOD1 G93A 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. This work was supported by a Bernice Ramsay ALS Canada grant, along with funding from the Paul Heller Memorial Fund (to J. M. S.). N. R. C. is the Chief Scientific Officer of ProMIS Neurosciences, which has licensed the 3H1 misfolded SOD1-specific antibody technology. This article contains Figs. S1 and S2 and Tables S1-S9.

Proteomic and biological profiling of extracellular vesicles from Alzheimer's disease human brain tissues

“…The EV samples were isolated from 20 AD and 18 sex‐ and age‐matched cognitively unimpaired controls using the discontinuous sucrose gradient ultracentrifugation as previously described with modifications (see Materials and Methods). This technique of EV isolation has been successfully used to isolate EVs from frozen mouse brain tissues 21,27 . In addition, we performed the quantitative proteomics analysis of human brain tissue homogenates and purified EV samples.…”

Characterization of brain‐derived extracellular vesicles reveals changes in cellular origin after stroke and enrichment of the prion protein with a potential role in cellular uptake

“…Relative quantification of EVs in brain is not an easy issue. In a recent paper, Silverman et al [89] have also characterized the relative proportion of EVs derived from different brain Under physiological conditions, microglia appear to be the main contributor to the sEV pool, followed by oligodendrocytes and astrocytes, while neurons contribute relatively little (contribution indicated by thickness of solid arrows). Upon experimental stroke (indicated by the yellow thunderbolt), astrocytic release of sEVs is upregulated and astrocytes appear to be the main contributor 24 h after reperfusion (indicated by the bold dotted arrow).…”

Extracellular vesicles and amyotrophic lateral sclerosis: from misfolded protein vehicles to promising clinical biomarkers

“…These findings strongly support the role of EVs in misfolded protein propagation following a prion-like fashion. Recently, a study performed in SOD1 transgenic mouse model has shown that misfolded SOD1 protein is enriched in EVs released by neurons and astrocytes [49]. TARDBP encodes for mainly nuclear RNA-binding protein (RBP) involved in splicing and RNA metabolism.…”