A Method for Isolation of Extracellular Vesicles and Characterization of Exosomes from Brain Extracellular Space

Pérez‐González, Rocío; Gauthier, Sébastien A.; Kumar, Asok; Shimada, Mitsuo; Saito, Mariko; Levy, Efrat

doi:10.1007/978-1-4939-6728-5_10

Cited by 77 publications

(70 citation statements)

References 16 publications

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“…Brain EVs were isolated as previously described [40, 41] from DS patients and age-matched normal controls (Table 1), and from the right hemibrain of 3-, 8-, 12- and 24-month-old Ts2 mice and 2N littermates. Separation of the EVs on a sucrose gradient resulted in 7 fractions, from a, the least dense, to g, the densest fraction, and Western-blot analysis showed that fractions with densities higher than 1.07 and lower than 1.17 (fractions b, c and d) were immunoreactive to Flotillin-1 and Flotillin-2, lipid raft proteins found in EVs, and established exosomal markers (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…In each experiment, EVs were simultaneously isolated from a brain of either a DS patient or a Ts2 mouse and from an age-matched 2N control. Brain EVs were isolated and purified as we have previously described [40, 41]. Briefly, frozen brain tissues were treated with 20 units/ml papain (Worthington, Lakewood, NJ) in Hibernate A solution (HA, 3.5 ml/sample; BrainBits, Springfield, IL) for 15 min at 37 °C.…”

Section: Methodsmentioning

confidence: 99%

“…Acetylcholine esterase (AChE) activity assay, commonly used to quantify exosomes, was also performed on fractions b, c, and d, as we have previously described [40, 41]. Total EVs protein levels and EVs AChE activity were normalized to total brain sample protein content.…”

Section: Methodsmentioning

confidence: 99%

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Enhanced exosome secretion in Down syndrome brain - a protective mechanism to alleviate neuronal endosomal abnormalities

Gauthier

Pérez‐González

Sharma

et al. 2017

acta neuropathol commun

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100

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A dysfunctional endosomal pathway and abnormally enlarged early endosomes in neurons are an early characteristic of Down syndrome (DS) and Alzheimer’s disease (AD). We have hypothesized that endosomal material can be released by endosomal multivesicular bodies (MVBs) into the extracellular space via exosomes to relieve neurons of accumulated endosomal contents when endosomal pathway function is compromised. Supporting this, we found that exosome secretion is enhanced in the brains of DS patients and a mouse model of the disease, and by DS fibroblasts. Furthermore, increased levels of the tetraspanin CD63, a regulator of exosome biogenesis, were observed in DS brains. Importantly, CD63 knockdown diminished exosome release and worsened endosomal pathology in DS fibroblasts. Taken together, these data suggest that increased CD63 expression enhances exosome release as an endogenous mechanism mitigating endosomal abnormalities in DS. Thus, the upregulation of exosome release represents a potential therapeutic goal for neurodegenerative disorders with endosomal pathology.Electronic supplementary materialThe online version of this article (doi:10.1186/s40478-017-0466-0) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Enhanced exosome secretion in Down syndrome brain - a protective mechanism to alleviate neuronal endosomal abnormalities

Gauthier

Pérez‐González

Sharma

et al. 2017

acta neuropathol commun

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100

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“…On the contrary, the role of Alix, an accessory ESCRT-associated protein, is more complex (Bissig & Gruenberg, 2014). Although it is commonly found in brain exosome preparations (Perez-Gonzalez et al, 2017), its contribution to ILVs formation depends on the cell type (Baietti et al, 2012;Colombo et al, 2013), can be dispensable for exosome biogenesis (Trajkovic et al, 2008), and it does not correlate with TSG101 levels in the brain (Peng et al, 2019). Moreover, in contrast to TSG101, Alix downregulation does not alter MVBs functionality and lysosomal sorting (Bowers et al, 2006;Luyet et al, 2008), suggesting that it may act more upstream in the pathway, probably at the level of the cargo loading into MVBs (Bissig & Gruenberg, 2014).…”

Section: Discussion/conclusionmentioning

confidence: 99%

Enhanced generation of intraluminal vesicles in neuronal late endosomes in the brain of a Down syndrome mouse model with endosomal dysfunction

D’Acunzo

Hargash

Pawlik

et al. 2019

Developmental Neurobiology

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Down syndrome (DS) is a human genetic disease caused by trisomy of chromosome 21 and characterized by early developmental brain abnormalities. Dysfunctional endosomal pathway in neurons is an early event of DS and Alzheimer's disease. Recently, we have demonstrated that exosome secretion is upregulated in human DS postmortem brains, in the brain of the trisomic mouse model Ts[Rb(12.1716)]2Cje (Ts2) and by DS fibroblasts as compared with disomic controls. High levels of the tetraspanin CD63, a regulator of exosome biogenesis, were observed in DS brains. Partially blocking exosome secretion by DS fibroblasts exacerbated a pre‐existing early endosomal pathology. We thus hypothesized that enhanced CD63 expression induces generation of intraluminal vesicles (ILVs) in late endosomes/multivesicular bodies (MVBs), increasing exosome release as an endogenous mechanism to mitigate endosomal abnormalities in DS. Herein, we show a high‐resolution electron microscopy analysis of MVBs in neurons of the frontal cortex of 12‐month‐old Ts2 mice and littermate diploid controls. Our quantitative analysis revealed that Ts2 MVBs are larger, more abundant, and contain a higher number of ILVs per neuron compared to controls. These findings were further corroborated biochemically by Western blot analysis of purified endosomal fractions showing higher levels of ILVs proteins in the same fractions containing endosomal markers in the brain of Ts2 mice compared to controls. These data suggest that upregulation of ILVs production may be a key homeostatic mechanism to alleviate endosomal dysregulation via the endosomal–exosomal pathway.

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“…Extracellular vesicles (EVs) enriched with exosomes were isolated and purified from frozen mouse hemibrains, as described by Perez-Gonzalez et al (31). In brief, frozen hemibrains were treated with 20 U/ml papain (Worthington, Lakewood, NJ, USA) in Hibernate A solution (HA, 3.5 ml/sample; BrainBits, Springfield, IL, USA) for 15 min at 37°C.…”

Section: Extracellular Vesicle Isolationmentioning

confidence: 99%

Intracellular metalloprotease activity controls intraneuronal Aβ aggregation and limits secretion of Aβviaexosomes

et al. 2018

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Accumulating evidence suggests that the abnormal aggregation of amyloid‐β (Aβ) peptide in Alzheimer's disease (AD) begins intraneuronally, within vesicles of the endosomal‐lysosomal pathway where Aβ is both generated and degraded. Metalloproteases, including endothelin‐converting enzyme (ECE)‐1 and ‐2, reside within these vesicles and normally limit the accumulation of intraneuronally produced Aβ. In this study, we determined whether disruption of Aβ catabolism could trigger Aβ aggregation within neurons and increase the amount of Aβ associated with exosomes, small extracellular vesicles derived from endosomal multivesicular bodies. Using cultured cell lines, primary neurons, and organotypic brain slices from an AD mouse model, we found that pharmacological inhibition of the ECE family of metalloproteases increased intracellular and extracellular Aβ levels and promoted the intracellular formation of Aβ oligomers, a process that did not require internalization of secreted Aβ. In vivo, the accumulation of intraneuronal Aβ aggregates was accompanied by increased levels of both extracellular and exosome‐associated Aβ, including oligomeric species. Neuronal exosomes were found to contain both ECE‐1 and ‐2 activities, suggesting that multivesicular bodies are intracellular sites of Aβ degradation by these enzymes. ECE dysfunction could lead to the accumulation of intraneuronal Aβ aggregates and their subsequent release into the extracellular space via exosomes.—Pacheco‐Quinto, J., Clausen, D., Pérez‐González, R., Peng, H., Meszaros, A., Eckman, C. B., Levy, E., Eckman, E. A. Intracellular metalloprotease activity controls intraneuronal Aβ aggregation and limits secretion of Aβ via exosomes. FASEB J. 33, 3758–3771 (2019). http://www.fasebj.org

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A Method for Isolation of Extracellular Vesicles and Characterization of Exosomes from Brain Extracellular Space

Cited by 77 publications

References 16 publications

Enhanced exosome secretion in Down syndrome brain - a protective mechanism to alleviate neuronal endosomal abnormalities

Enhanced exosome secretion in Down syndrome brain - a protective mechanism to alleviate neuronal endosomal abnormalities

Enhanced generation of intraluminal vesicles in neuronal late endosomes in the brain of a Down syndrome mouse model with endosomal dysfunction

Intracellular metalloprotease activity controls intraneuronal Aβ aggregation and limits secretion of Aβviaexosomes

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