CNS-Derived Blood Exosomes as a Promising Source of Biomarkers: Opportunities and Challenges

Abstract

Eukaryotic cells release different types of extracellular vesicles (EVs) including exosomes, ectosomes, and microvesicles. Exosomes are nanovesicles, 30-200 nm in diameter, that carry cell- and cell-state-specific cargo of proteins, lipids, and nucleic acids, including mRNA and miRNA. Recent studies have shown that central nervous system (CNS)-derived exosomes may carry amyloidogenic proteins and facilitate their cell-to-cell transfer, thus playing a critical role in the progression of neurodegenerative diseases, such as tauopathies and synucleinopathies. CNS-derived exosomes also have been shown to cross the blood-brain-barrier into the bloodstream and therefore have drawn substantial attention as a source of biomarkers for various neurodegenerative diseases as they can be isolated via a minimally invasive blood draw and report on the biochemical status of the CNS. However, although isolating specific brain-cell-derived exosomes from the blood is theoretically simple and the approach has great promise, practical details are of crucial importance and may compromise the reproducibility and utility of this approach, especially when different laboratories use different protocols. In this review we discuss the role of exosomes in neurodegenerative diseases, the usefulness of CNS-derived blood exosomes as a source of biomarkers for these diseases, and practical challenges associated with the methodology of CNS-derived blood exosomes and subsequent biomarker analysis.

Keywords: ALS; Alzheimer' disease; Parkinson's and related diseases; biomarker; exosome; extracellular vesicle (EV); neurodegenerative diseases.

Figure 1

Biogenesis, secretion, and uptake of exosomes and their cargo. (1) Invagination of the cell membrane leads to the formation of early endosomes. (2) ESCRT-0 recognizes and binds ubiquitynated proteins and further recruits ESCRT-I (including the exosomal marker TSG101) and ESCRT-II to this complex. (3) ESCRT-III is a transient protein complex that plugs the inward budding vesicle to avoid the escape of the cargo during scission. Alix recruits deubiquitinases to ESCRT-III, which remove ubiquitin from cargo proteins (Budnik et al., 2016). ESCRT proteins detach from the membrane and are released into the cytoplasm. (4) The formed intralumenal vesicle contains the cargo protein and can either be (5) degraded in lysosomes or (6) secreted into the extracellular space. (7) The formation of intralumenal vesicles can occur via ESCRT-independent pathways and is promoted by higher levels of ceramides in the lipid membrane (Budnik et al., 2016). Exosomes released into the extracellular space can be taken up by recipient cells by endocytosis (8) or fusion with the plasma membrane (9) allowing the transport of cargo between different cells and body parts.

Figure 2

Electron micrographs of exosomes. Exosomes were isolated from cultured primary endothelial cells. Left: exosomes were stained with uranyl acetate and embedded as whole mount preparations in methylcellulose. The image shows a cup-shaped morphology and heterogeneous sizes ranging from 30 to 100 nm. Right: Exosomes were analyzed by cryoelectron microscopy without chemical fixation or contrasting. Exosomes appear as round membranous structures. Adapted from panels B and C in Figure 1 of Banizs et al., © 2014, originally published in International Journal of Nanomedicine (Dovepress). https://doi.org/10.2147/IJN.S64267.

Figure 3

Isolation of CNS-derived exosomes from blood. (A) The protocol of the Zhang group relies on anti-L1CAM antibody-coupled epoxy beads, which are incubated directly with diluted plasma to bind neuronal exosomes. The following washing steps in 0.1% BSA remove unbound, non-neuronal exosomes in the sample. (B) The method described by Goetzl et al. The protocol uses first an exosome precipitation step by ExoQuick followed by capturing specifically neuronal exosomes with biotinylated anti-L1CAM antibodies and a streptavidin-conjugated resin. Subsequent washing steps remove non-neuronal exosomes as well as the antibody and resin to yield neuronal exosomes.