Efficient enzyme‐free isolation of brain‐derived extracellular vesicles
Abstract Extracellular vesicles (EVs) have gained significant attention as pathology mediators and potential diagnostic tools for neurodegenerative diseases. However, isolation of brain‐derived EVs (BDEVs) from tissue remains challenging, often involving enzymatic digestion steps that may compromise...
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| Format: | Article |
| Language: | English |
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Wiley
2024-11-01
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| Series: | Journal of Extracellular Vesicles |
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| Online Access: | https://doi.org/10.1002/jev2.70011 |
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| author | Andreu Matamoros‐Angles Emina Karadjuzovic Behnam Mohammadi Feizhi Song Santra Brenna Susanne Caroline Meister Bente Siebels Hannah Voß Carolin Seuring Isidre Ferrer Hartmut Schlüter Matthias Kneussel Hermann Clemens Altmeppen Michaela Schweizer Berta Puig Mohsin Shafiq Markus Glatzel |
| author_facet | Andreu Matamoros‐Angles Emina Karadjuzovic Behnam Mohammadi Feizhi Song Santra Brenna Susanne Caroline Meister Bente Siebels Hannah Voß Carolin Seuring Isidre Ferrer Hartmut Schlüter Matthias Kneussel Hermann Clemens Altmeppen Michaela Schweizer Berta Puig Mohsin Shafiq Markus Glatzel |
| author_sort | Andreu Matamoros‐Angles |
| collection | DOAJ |
| description | Abstract Extracellular vesicles (EVs) have gained significant attention as pathology mediators and potential diagnostic tools for neurodegenerative diseases. However, isolation of brain‐derived EVs (BDEVs) from tissue remains challenging, often involving enzymatic digestion steps that may compromise the integrity of EV proteins and overall functionality. Here, we describe that collagenase digestion, commonly used for BDEV isolation, produces undesired protein cleavage of EV‐associated proteins in brain tissue homogenates and cell‐derived EVs. In order to avoid this effect, we studied the possibility of isolating BDEVs with a reduced amount of collagenase or without any protease. Characterization of the isolated BDEVs from mouse and human samples (both female and male) revealed their characteristic morphology and size distribution with both approaches. However, we show that even minor enzymatic digestion induces ‘artificial’ proteolytic processing in key BDEV markers, such as Flotillin‐1, CD81, and the cellular prion protein (PrPC), whereas avoiding enzymatic treatment completely preserves their integrity. We found no major differences in mRNA and protein content between non‐enzymatically and enzymatically isolated BDEVs, suggesting that the same BDEV populations are purified with both approaches. Intriguingly, the lack of Golgi marker GM130 signal, often referred to as contamination indicator (or negative marker) in EV preparations, seems to result from enzymatic digestion rather than from its actual absence in BDEV samples. Overall, we show that non‐enzymatic isolation of EVs from brain tissue is possible and avoids artificial pruning of proteins while achieving an overall high BDEV yield and purity. This protocol will help to understand the functions of BDEV and their associated proteins in a near‐physiological setting, thus opening new research approaches. |
| format | Article |
| id | doaj-art-31fef942ede24f21be9ef2c27cfe41c4 |
| institution | OA Journals |
| issn | 2001-3078 |
| language | English |
| publishDate | 2024-11-01 |
| publisher | Wiley |
| record_format | Article |
| series | Journal of Extracellular Vesicles |
| spelling | doaj-art-31fef942ede24f21be9ef2c27cfe41c42025-08-20T02:07:09ZengWileyJournal of Extracellular Vesicles2001-30782024-11-011311n/an/a10.1002/jev2.70011Efficient enzyme‐free isolation of brain‐derived extracellular vesiclesAndreu Matamoros‐Angles0Emina Karadjuzovic1Behnam Mohammadi2Feizhi Song3Santra Brenna4Susanne Caroline Meister5Bente Siebels6Hannah Voß7Carolin Seuring8Isidre Ferrer9Hartmut Schlüter10Matthias Kneussel11Hermann Clemens Altmeppen12Michaela Schweizer13Berta Puig14Mohsin Shafiq15Markus Glatzel16Institute of Neuropathology University Medical Center Hamburg‐Eppendorf (UKE) Hamburg GermanyInstitute of Neuropathology University Medical Center Hamburg‐Eppendorf (UKE) Hamburg GermanyInstitute of Neuropathology University Medical Center Hamburg‐Eppendorf (UKE) Hamburg GermanyInstitute of Neuropathology University Medical Center Hamburg‐Eppendorf (UKE) Hamburg GermanyDepartment of Neurology, Experimental Research in Stroke and Inflammation (ERSI) University Medical Center Hamburg‐Eppendorf (UKE) Hamburg GermanyInstitute of Neuropathology University Medical Center Hamburg‐Eppendorf (UKE) Hamburg GermanySection Mass Spectrometry and Proteomics University Medical Center Hamburg‐Eppendorf (UKE) Hamburg GermanySection Mass Spectrometry and Proteomics University Medical Center Hamburg‐Eppendorf (UKE) Hamburg GermanyMulti‐User‐CryoEM‐Facility Centre for Structural Systems Biology (CSSB) Hamburg GermanyIDIBELL University of Barcelona L'Hospitalet de Llobregat SpainSection Mass Spectrometry and Proteomics University Medical Center Hamburg‐Eppendorf (UKE) Hamburg GermanyInstitute for Molecular Neurogenetics, Center for Molecular Neurobiology Hamburg (ZMNH) University Medical Center Hamburg‐Eppendorf (UKE) Hamburg GermanyInstitute of Neuropathology University Medical Center Hamburg‐Eppendorf (UKE) Hamburg GermanyElectron Microscopy Core Facility, Center for Molecular Neurobiology (ZMNH) University Medical Center Hamburg‐Eppendorf (UKE) Hamburg GermanyDepartment of Neurology, Experimental Research in Stroke and Inflammation (ERSI) University Medical Center Hamburg‐Eppendorf (UKE) Hamburg GermanyInstitute of Neuropathology University Medical Center Hamburg‐Eppendorf (UKE) Hamburg GermanyInstitute of Neuropathology University Medical Center Hamburg‐Eppendorf (UKE) Hamburg GermanyAbstract Extracellular vesicles (EVs) have gained significant attention as pathology mediators and potential diagnostic tools for neurodegenerative diseases. However, isolation of brain‐derived EVs (BDEVs) from tissue remains challenging, often involving enzymatic digestion steps that may compromise the integrity of EV proteins and overall functionality. Here, we describe that collagenase digestion, commonly used for BDEV isolation, produces undesired protein cleavage of EV‐associated proteins in brain tissue homogenates and cell‐derived EVs. In order to avoid this effect, we studied the possibility of isolating BDEVs with a reduced amount of collagenase or without any protease. Characterization of the isolated BDEVs from mouse and human samples (both female and male) revealed their characteristic morphology and size distribution with both approaches. However, we show that even minor enzymatic digestion induces ‘artificial’ proteolytic processing in key BDEV markers, such as Flotillin‐1, CD81, and the cellular prion protein (PrPC), whereas avoiding enzymatic treatment completely preserves their integrity. We found no major differences in mRNA and protein content between non‐enzymatically and enzymatically isolated BDEVs, suggesting that the same BDEV populations are purified with both approaches. Intriguingly, the lack of Golgi marker GM130 signal, often referred to as contamination indicator (or negative marker) in EV preparations, seems to result from enzymatic digestion rather than from its actual absence in BDEV samples. Overall, we show that non‐enzymatic isolation of EVs from brain tissue is possible and avoids artificial pruning of proteins while achieving an overall high BDEV yield and purity. This protocol will help to understand the functions of BDEV and their associated proteins in a near‐physiological setting, thus opening new research approaches.https://doi.org/10.1002/jev2.70011BDEVsbrain‐derived EVscollagenase‐freeEV coronaGM130prion protein |
| spellingShingle | Andreu Matamoros‐Angles Emina Karadjuzovic Behnam Mohammadi Feizhi Song Santra Brenna Susanne Caroline Meister Bente Siebels Hannah Voß Carolin Seuring Isidre Ferrer Hartmut Schlüter Matthias Kneussel Hermann Clemens Altmeppen Michaela Schweizer Berta Puig Mohsin Shafiq Markus Glatzel Efficient enzyme‐free isolation of brain‐derived extracellular vesicles Journal of Extracellular Vesicles BDEVs brain‐derived EVs collagenase‐free EV corona GM130 prion protein |
| title | Efficient enzyme‐free isolation of brain‐derived extracellular vesicles |
| title_full | Efficient enzyme‐free isolation of brain‐derived extracellular vesicles |
| title_fullStr | Efficient enzyme‐free isolation of brain‐derived extracellular vesicles |
| title_full_unstemmed | Efficient enzyme‐free isolation of brain‐derived extracellular vesicles |
| title_short | Efficient enzyme‐free isolation of brain‐derived extracellular vesicles |
| title_sort | efficient enzyme free isolation of brain derived extracellular vesicles |
| topic | BDEVs brain‐derived EVs collagenase‐free EV corona GM130 prion protein |
| url | https://doi.org/10.1002/jev2.70011 |
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