Filter-aided extracellular vesicle enrichment (FAEVEr) for proteomics DOI Open Access
Jarne Pauwels, Tessa Van de Steene,

J. Van De Velde

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2023, Volume and Issue: unknown

Published: July 7, 2023

Abstract Extracellular vesicles (EVs), membrane-delimited nanovesicles that are secreted by cells into the extracellular environment, gaining substantial interest due to their involvement in cellular homeostasis and contribution disease pathology. The latter particular has led an exponential increase EVs as they considered be circulating packages containing potential biomarkers also a possible biological means deliver drugs cell-specific manner. However, several challenges hamper straightforward proteome analysis of generally low abundant reside complex matrices. These matrices typically contain protein concentrations vastly exceed those EV proteome. Therefore, extensive isolation purification protocols imperative many have been developed, including (density) ultracentrifugation, size-exclusion precipitation methods. Here, we describe filter-aided vesicle enrichment (FAEVEr) approach based on 300 kDa MWCO filtration allows processing multiple samples parallel within reasonable timeframe at moderate cost. We demonstrate FAEVEr is capable quantitatively retaining particles filters, whilst allowing washing with mild detergent TWEEN-20 remove interfering non-EV proteins. retained directly lysed filter for complete recovery cargo towards analysis. validate optimize recombinant material apply it conditioned medium well serum. Our results indicate isolated from MCF7 cultured or without serum drastic different because nutrient deprivation.

Language: Английский

Phosphoproteomics of Cerebrospinal Fluid-Derived Extracellular Vesicles Applied to Primary Central Nervous System Lymphoma DOI
Yuanyuan Deng, Anton Iliuk, W. Andy Tao

et al.

Methods in molecular biology, Journal Year: 2025, Volume and Issue: unknown, P. 115 - 125

Published: Jan. 1, 2025

Language: Английский

Citations

0
Most L1CAM Is not Associated with Extracellular Vesicles in Human Biofluids and iPSC–Derived Neurons DOI Creative Commons

Vaibhavi Kadam,

Madeleine Wacker,

Patrick Oeckl

et al.

Molecular Neurobiology, Journal Year: 2025, Volume and Issue: unknown

Published: April 10, 2025

Abstract Transmembrane L1 cell adhesion molecule (L1CAM) is widely used as a marker to enrich for neuron-derived extracellular vesicles (EVs), especially in plasma. However, this approach lacks sufficient robust validation. This study aimed assess whether human biofluids are indeed enriched EVs, particularly by L1CAM immunoaffinity, utilizing multiple sources (plasma, CSF, conditioned media from iPSC-derived neurons [iNCM]) and different methods (mass spectrometry [MS], nanoparticle tracking analysis [NTA]). Following systematic multi-step validation approach, we confirmed isolation of generic EV populations using size-exclusion chromatography (SEC) polymer-aided precipitation (PPT)—two most commonly applied methods—from all sources. Neurofilament light (NfL) was detected both CSF blood-derived indicating their neuronal origin. immunoprecipitation did not yield enrichment fractions. Instead, it predominantly found its free-floating form. Additionally, MS-based proteomic CSF-derived EVs also show enrichment. Our validates diverse biofluid several approaches confirms that some subpopulations Thorough testing across orthogonal methods, however, does support reliably specific subpopulation

Language: Английский

Citations

0
Extracellular Vesicles for Alzheimer’s Disease and Dementia Diagnosis DOI

Hash Brown Taha

medRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: April 24, 2024

ABSTRACT Accurate differential diagnosis of dementia disorders including Alzheimer’s disease (AD), frontotemporal (FTD), with Lewy bodies (DLB), Parkinson’s (PDD), and vascular cognitive impairment (VCID), along conditions like prodromal mild (MCI) or negative controls (NCs), continues to challenge neurologists. The nuanced sometimes shared pathophysiological features underscore the need for precision in developing disease-modifying therapies. In pursuit reliable antemortem biomarkers, extracellular vesicles (EVs) have emerged as a popular tool their capacity encapsulate disease-specific signatures, particularly neurodegenerative neurological disorders. To this end, we performed comprehensive, PRISMA-guided systematic review meta-analysis, utilizing sophisticated statistical modeling determine diagnostic accuracy, explore between-study variance heterogeneity (I 2 ), investigate potential publication bias using various tests. Biomarkers derived from general EVs demonstrated superior less variance, heterogeneity, than those speculative CNS-enriched EVs. trim-and-fill method suggested overestimation effectiveness biomarkers due four hypothesized missing studies low results, but none Meta-regressions revealed that cerebrospinal fluid serum, involving non-fasting individuals, sampling afternoon, employing citrate instead EDTA blood collection, thrombin coagulation factor depletion, isolating purer methods such combined ultracentrifugation size-exclusion chromatography, not freezing post-isolation, quantifying miRNA achieved better accuracy heterogeneity. was differentiating among different However, analysis diagnosing persons AD vs. VCID highest suggesting further may focus on comparison. Additionally, highlight several limitations included recommend following: Implement use appropriate controls, thorough documentation preanalytical factors, inclusion more groups beyond AD, comprehensive reporting pharmacological treatments, consideration racial ethnic minority groups, adherence ISEV guidelines, application A-T-N framework, detailed stages, extension diagnosis, reanalysis when postmortem definitive diagnostics become available, evaluation conversion rates, commitment accurate data transparency. We hope lessons learned meta-analysis can be beneficial attempting discover related dementias through alternative approaches.

Language: Английский

Citations

3
Trends in Developing Extracellular Vesicle-Based Therapeutics DOI
Jaewook Lee

Brain Tumor Research and Treatment, Journal Year: 2024, Volume and Issue: 12(3), P. 153 - 153

Published: Jan. 1, 2024

Extracellular vesicles are nano-sized surrounded by lipid bilayers, and all cells release them to the extracellular environment for communication. consist of molecules with various biological activities can play essential roles as therapeutics, so they attract much attention next-generation modalities treat diseases. As cell-derived nanovesicles, favorable be developed but also have limitations. In addition, there a number things consider in terms manufacturing, quality control, non-clinical studies, clinical trials during development vesicle-based therapeutics. Meanwhile, has been paid potentials many biopharmaceutical companies trying develop This review will introduce advantages limitations it cover developing therapeutics cases

Language: Английский

Citations

1
In-depth mass-spectrometry reveals phospho-RAB12 as a blood biomarker of G2019S LRRK2-driven Parkinson DOI Creative Commons
Adriana Cortés, Toan K. Phung, Lorena de Mena

et al.

medRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: May 6, 2024

ABSTRACT Leucine-rich repeat kinase 2 (LRRK2) inhibition is a promising disease-modifying therapy for LRRK2-associated Parkinson’s disease (L2PD) and idiopathic PD (iPD). Yet, pharmaco-dynamic readouts progression biomarkers modification clinical trials are insufficient. Employing phospho-/proteomic analyses we assessed the impact that LRRK2 activating mutations had in peripheral blood mononuclear cells (PBMCs) from cohort Spain (n=174) encompassing G2019S L2PD patients (n=37), non-manifesting mutation carriers of G2019S, here, L2NMCs (n=27), R1441G (n=14), (n=11), iPD (n=40), controls (n=45). We identified 207 differential proteins compared to (39 up/ 168 down) 67 (10 57 down). down-regulated affected endolysosomal pathway, proteostasis mitochondria, e.g., ATIC, RAB9A, or LAMP1. At phospho-proteome level, observed increases endogenous phosphorylation levels pSer106 RAB12 carriers, which were validated by immunoblotting after 1 year follow-up (n=48). Freshly collected PBMCs 3 L2PD, iPD, 5 (n=10) showed strong diminishment in-vitro administration MLi-2 inhibitor. Using machine learning, an 18-feature phospho-/protein signature capable discriminating L2NMCs, with 96% accuracy correlated severity, i.e., UPDRS-III motor scoring. Our study as biomarker easily accessible suggests findings human such can be deployed universal readout iPD. Future work may determine whether could help patient enrichment monitoring drug efficacy trials.

Language: Английский

Citations

0
Blood and cerebrospinal fluid differences between Parkinson's disease and related diseases DOI
Jie Ma,

Zhijian Tang,

Yaqi Wu

et al.

Research Square (Research Square), Journal Year: 2024, Volume and Issue: unknown

Published: Sept. 27, 2024

Abstract Accurately diagnosing Parkinson’s disease (PD) in its early stages is difficult due to symptoms overlapping with those of various disorders, including atypical Parkinsonian syndromes, dementia Lewy bodies (DLB), and even essential tremor. This complicates the diagnostic process for PD, which traditionally heavily relies on symptomatic assessment treatment response. Recent advances have identified several biomarkers blood cerebrospinal fluid (CSF), α-synuclein, lysosomal enzymes, fatty acid-binding proteins, neurofilament light chain, that may potentially be used diagnosed PD. However, not all can effectively distinguish PD from related disorders or identify subtypes. review advocates a paradigm shift towards biomarker-based diagnosis between similar conditions categorize into These reflect differences exist among different diseases provide an effective way accurately understand their mechanisms. focused CSF differential value molecular measurement methods high performance emerging technologies.

Language: Английский

Citations

0
Most L1CAM is not associated with extracellular vesicles in human biofluids and iPSC-derived neurons DOI Creative Commons

Vaibhavi Kadam,

Madeleine Wacker,

Patrick Oeckl

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: Oct. 15, 2024

Abstract Transmembrane L1 cell adhesion molecule (L1CAM) is widely used as a marker to enrich for neuron-derived extracellular vesicles (EVs), especially in plasma. However, this approach lacks sufficient robust validation. This study aimed assess whether human biofluids are indeed enriched EVs, particularly by L1CAM immunoaffinity, utilizing multiple sources (plasma, CSF, conditioned media from iPSC-derived neurons [iNCM]) and different methods (mass spectrometry [MS], nanoparticle tracking analysis [NTA]). Following systematic multi-step validation approach, we confirmed isolation of generic EV populations using size-exclusion chromatography (SEC) polymer-aided precipitation (PPT) – two most commonly applied all sources. Neurofilament light (NfL) was detected both CSF blood-derived indicating their neuronal origin. immunoprecipitation did not yield enrichment fractions. Instead, it predominantly found its free-floating form. Additionally, MS-based proteomic CSF-derived EVs also show enrichment. Our validates diverse biofluid several approaches confirms that some subpopulations Thorough testing across orthogonal methods, however, does support reliably specific subpopulation

Language: Английский

Citations

0
Exosomes from polarized Microglia: Proteomic insights into potential mechanisms affecting intracerebral hemorrhage DOI
Yinan Zhou, Ying Zhang,

Dongchen Xu

et al.

Gene, Journal Year: 2024, Volume and Issue: 935, P. 149080 - 149080

Published: Nov. 5, 2024

Language: Английский

Citations

0
In-depth mass-spectrometry reveals phospho-RAB12 as a blood biomarker of G2019S LRRK2-driven Parkinson’s disease DOI Creative Commons
Adriana Cortés, Toan K. Phung, Lorena de Mena

et al.

Brain, Journal Year: 2024, Volume and Issue: unknown

Published: Dec. 20, 2024

Abstract Leucine-rich repeat kinase 2 (LRRK2) inhibition is a promising disease-modifying therapy for LRRK2-associated Parkinson’s disease (L2PD) and idiopathic PD (iPD). However, pharmaco-dynamic readouts progression biomarkers clinical trials aiming modification are insufficient since no endogenous marker reflecting enhanced activity of the most common LRRK2 G2019S mutation has been reported yet in L2PD patients. Employing phospho-/proteomic analyses we assessed impact that activating mutations had peripheral blood mononuclear cells (PBMCs) from cohort Spain (n=174). The groups study encompassed patients (n=37), non-manifesting carriers G2019S, here, L2NMCs (n=27), R1441G (n=14), (n=11), iPD (n=40), healthy controls (n=45). We identified 207 differential proteins compared to (39 up/168 down) 67 (10 up/57 down). down-regulated affected endolysosomal pathway, proteostasis, mitochondria, e.g., ATIC, RAB9A, or LAMP1. At phospho-proteome level, observed increases phosphorylation levels pSer106 RAB12 carriers, which were validated by immunoblotting after 1 year follow-up (n=48). Freshly collected PBMCs 3 L2PD, iPD, 5 (n=10) showed strong diminishment in-vitro administration MLi-2 inhibitor. Using machine learning, an 18-feature phospho-/protein signature discriminating L2NMCs, with 96% accuracy correlated severity, i.e., UPDRS-III motor scoring. easily accessible cohort, elevated as biomarker carriers. Our data suggest monitoring could be relevant tracking activation, particularly Future work may determine whether help patient enrichment drug efficacy trials.

Language: Английский

Citations

0
Methodological Assessment of ExoGAG for Isolation of Cerebrospinal Fluid Extracellular Vesicles as a Source of Biomarkers DOI Open Access

Nil Salvat‐Rovira,

Anna Vázquez‐Oliver,

Elisa Rivas‐Asensio

et al.

International Journal of Molecular Sciences, Journal Year: 2024, Volume and Issue: 25(24), P. 13705 - 13705

Published: Dec. 22, 2024

Extracellular vesicles (EVs) in cerebrospinal fluid (CSF) represent a valuable source of biomarkers for central nervous system (CNS) diseases, offering new pathways diagnosis and monitoring. However, existing methods isolating EVs from CSF often prove to be labor-intensive reliant on specialized equipment, hindering their clinical application. In this study, we present novel, clinically compatible method CSF. We optimized the use ExoGAG, commercially available reagent that has been tested plasma, urine semen, compared it directly with differential ultracentrifugation using Western blotting, protein quantification, nanoparticle tracking analysis, cryogenic electron microscopy. Additionally, analyzed presence specific microRNAs (miRNAs) known CSF-derived EVs. Our data demonstrate ExoGAG is an effective CSF, yielding higher amount traditional methods, comparable levels miRNAs. conclusion, setting may facilitate testing essential brain pathology CNS diseases.

Language: Английский

Citations

0