Exosomes Derived Neuronal Markers: Immunoaffinity Isolation and Characterization.
AMPA
Exosomes
GluR2/3
Immunoaffinity
Neuronal exosomes
Stroke
Journal
Neuromolecular medicine
ISSN: 1559-1174
Titre abrégé: Neuromolecular Med
Pays: United States
ID NLM: 101135365
Informations de publication
Date de publication:
09 2022
09 2022
Historique:
received:
25
06
2021
accepted:
08
11
2021
pubmed:
24
11
2021
medline:
27
8
2022
entrez:
23
11
2021
Statut:
ppublish
Résumé
Neuronal exosomes play a crucial role in intercellular communication in the brain and represent a promising biomarker for neurological diseases, including stroke. However, limited techniques are available for isolating neuronal exosomes due to their small number in the serum exosomes. Thus, the development of efficient tools with brain-specific markers is needed. Here, we show the optimization of an immunoaffinity assay-based isolation protocol for specific exosomes or neuronally derived exosomes (NDE). Our results demonstrated that one-micron functionalized magnetic beads successfully separated CD63+ and L1CAM+ exosomes from serum. The size and shape of exosomes or exosomes pulled by beads were confirmed by Dynamic light scattering and Transmission electron microscopy; also, beads were well resolved in conventional flow cytometry analysis, which revealed that CD63-pulled serum exosomes had 5% expression of L1CAM. Furthermore, transmission electron microscopy showed that exosomes eluted from magnetic beads retained their original size, shape, and form without any damage. Furthermore, we showed isolation of NDE using GluR2/3-capturing antibody (α-Amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor) using an optimized immunoaffinity bead assay utilizing 100 µl serum of stroke patients or age-matched healthy group. GluR2/3-captured exosomes were confirmed by western blot analysis. The western blot analysis showed a significant increase in the 35KDa subunit of GluR2/3 receptor protein in the exosomes of stroke patients compared to the healthy group. In addition, the multimeric GluR2/3 receptor protein in exosomes was further validated by the presence of the GluR2 subunit. Thus, our study shows GluR3/2 may be an effective candidate to isolate neuronal exosomes.
Identifiants
pubmed: 34811658
doi: 10.1007/s12017-021-08696-6
pii: 10.1007/s12017-021-08696-6
doi:
Substances chimiques
Biomarkers
0
Neural Cell Adhesion Molecule L1
0
Proteins
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
339-351Informations de copyright
© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.
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