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. 2023 Oct;1(4):e20230016.
doi: 10.1002/INMD.20230016. Epub 2023 Aug 15.

Toward a human brain extracellular vesicle atlas: Characteristics of extracellular vesicles from different brain regions, including small RNA and protein profiles

Yiyao Huang  1 , Tanina Arab  1 , Ashley E Russell  1   2 , Emily R Mallick  1 , Rajini Nagaraj  3 , Evan Gizzie  3 , Javier Redding-Ochoa  4 , Juan C Troncoso  4   5 , Olga Pletnikova  4   6 , Andrey Turchinovich  7   8 , David A Routenberg  3 , Kenneth W Witwer  1   5   9
Affiliations

Toward a human brain extracellular vesicle atlas: Characteristics of extracellular vesicles from different brain regions, including small RNA and protein profiles

Yiyao Huang et al. Interdiscip Med. 2023 Oct.

Abstract

Extracellular vesicles (EVs) are released from different cell types in the central nervous system (CNS) and play roles in regulating physiological and pathological functions. Although brain-derived EVs (bdEVs) have been successfully collected from brain tissue, there is not yet a "bdEV Atlas" of EVs from different brain regions. To address this gap, we separated EVs from eight anatomical brain regions of a single individual and subsequently characterized them by count, size, morphology, and protein and RNA content. The greatest particle yield was from cerebellum, while the fewest particles were recovered from the orbitofrontal, postcentral gyrus, and thalamus regions. EV surface phenotyping indicated that CD81 and CD9 were more abundant than CD63 in all regions. Cell-enriched surface markers varied between brain regions. For example, putative neuronal markers NCAM, CD271, and NRCAM were more abundant in medulla, cerebellum, and occipital regions, respectively. These findings, while restricted to tissues from a single individual, suggest that additional studies are warranted to provide more insight into the links between EV heterogeneity and function in the CNS.

Keywords: brain; cerebellum; corpus callosum; ectosomes; exosomes; extracellular vesicles; hippocampus; medulla; occipital gyrus; orbitofrontal; postcentral gyrus; thalamus; tissue.

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Conflict of interest statement

RN, EG, and DAR are employed by Meso Scale Diagnostics, LLC, but are neither shareholders nor officers of the company. Prof. Kenneth W. Witwer is the member of Interdisciplinary Medicine editorial board. The authors declare no other conflict of interests.

Figures

FIGURE 1
FIGURE 1
Workflow for brain‐derived EV (bdEV) enrichment and characterization from different brain regions. bdEVs from 8 brain regions were separated by collagenase digestion, differential centrifugation, and size exclusion chromatography (SEC). After separation, bdEVs were characterized by particle count, imaging, protein phenotyping and small RNA sequencing. Created with BioRender.com.
FIGURE 2
FIGURE 2
(A) Particle concentrations of bdEVs from brain regions were measured using NFCM. The particle concentration for each region was normalized by tissue mass (per 100 mg). (B, C) bdEVs were visualized by negative staining transmission electron microscopy (TEM) at 60, 000× and 100,000× magnification, separately; scale bar = 500 nm in (B), and 100 nm in (C). TEM is representative of 10 images taken of each region. (D) Size (diameter) distributions of bdEVs from brain regions as measured by NFCM and calculated as particles in each 5 nm size bin versus total detected particles in each sample (percentage). (E) Size distributions of bdEVs from brain regions as measured in TEM images and calculated as particles in each 50 nm size bin versus total detected particles in each sample (percentage).
FIGURE 3
FIGURE 3
EV surface protein phenotyping. CD63, CD81, and CD9 were detected on the intact bdEV surface by single‐particle interferometric reflectance imaging sensor (SP‐IRIS) (A) and multiplexed ELISA (B) and normalized per 100 mg tissue input. bdEVs were captured by antibodies to EV membrane proteins and detected by a signal from a cocktail of anti‐tetraspanin antibodies (CD63, CD81, and CD9).
FIGURE 4
FIGURE 4
Cell‐of‐origin marker profile on the bdEV surface. (A) Distribution of markers by cell types: neurons, microglia, and astrocytes. Cell‐enriched markers were used as bdEV capture antibodies; EVs were then detected by a signal from a cocktail of anti‐tetraspanin antibodies (CD63, CD81, and CD9). Levels of neuron (B), microglia (C), astrocyte (D), overlapping (E) and non‐CNS cell (F) markers were then normalized to the average of tetraspanin capture spot signals.
FIGURE 5
FIGURE 5
bdEV small RNA profiles. (A) Principal component analysis (PCA) based on quantitative small RNA profiles of bdEVs from different regions. (B) Unsupervised hierarchical clustering of 15 of the most abundant bdEV miRNAs across regions.
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