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Identification of distinct circulating exosomes in Parkinson's disease.

Tomlinson PR, Zheng Y, Fischer R, Heidasch R, Gardiner C, Evetts S, Hu M, Wade-Martins R, Turner MR, Morris J, Talbot K, Kessler BM, Tofaris GK - Ann Clin Transl Neurol (2015)

Bottom Line: To interrogate their biological effect, microvesicles were added to primary rat cortical neurons subjected to either nutrient deprivation or sodium arsenite.Among 1033 proteins identified, 23 exosome-associated proteins were differentially abundant in PD, including the regulator of exosome biogenesis syntenin 1.Accordingly, we showed in models of neuronal stress that Parkinson's-derived microvesicles have a protective effect.

View Article: PubMed Central - PubMed

Affiliation: Nuffield Department of Clinical Neurosciences, University of Oxford Oxford, United Kingdom.

ABSTRACT

Objective: Whether circulating microvesicles convey bioactive signals in neurodegenerative diseases remains currently unknown. In this study, we investigated the biochemical composition and biological function of exosomes isolated from sera of patients with Parkinson's disease (PD).

Methods: Proteomic analysis was performed on microvesicle preparations from grouped samples of patients with genetic and sporadic forms of PD, amyotrophic lateral sclerosis, and healthy subjects. Nanoparticle-tracking analysis was used to assess the number and size of exosomes between patient groups. To interrogate their biological effect, microvesicles were added to primary rat cortical neurons subjected to either nutrient deprivation or sodium arsenite.

Results: Among 1033 proteins identified, 23 exosome-associated proteins were differentially abundant in PD, including the regulator of exosome biogenesis syntenin 1. These protein changes were detected despite similar exosome numbers across groups suggesting that they may reflect exosome subpopulations with distinct functions. Accordingly, we showed in models of neuronal stress that Parkinson's-derived microvesicles have a protective effect.

Interpretation: Collectively, these data suggest for the first time that immunophenotyping of circulating exosome subpopulations in PD may lead to a better understanding of the systemic response to neurodegeneration and the development of novel therapeutics.

No MeSH data available.


Related in: MedlinePlus

Isolation and characterization of circulating microvesicles. (A) Flow diagram showing the extraction method used and Coomassie staining of the final microvesicle preparation. Note that an additional wash step significantly reduces the number of contaminant proteins without major changes in microvesicle numbers. (B) Nanoparticle-tracking analysis of serum microvesicles revealed a major peak at the size corresponding to exosomes isolated from NSC34-conditioned media, whereas spike-in experiments showed a further increment in microvesicles of the same size. (C) Immunoblotting confirmed the presence of the exosome markers flotillin1 and Tsg101 both in cell-conditioned media and serum preparations (D) Electron microscopy showed membrane-bound microvesicles, negatively stained with 2% aqueous uranyl acetate on a formvar film. (E) Venn diagram showing protein overlap between albumin/immunoglobulin depleted serum, serum microvesicles, and human cell lysates. Proteins were quantified using Oribtrap-Velos LC-MS/MS. The enrichment for serum-derived microvesicles enables the identification and quantitation of proteins that are not detected in routinely processed serum samples as shown by gene ontology analysis.
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fig01: Isolation and characterization of circulating microvesicles. (A) Flow diagram showing the extraction method used and Coomassie staining of the final microvesicle preparation. Note that an additional wash step significantly reduces the number of contaminant proteins without major changes in microvesicle numbers. (B) Nanoparticle-tracking analysis of serum microvesicles revealed a major peak at the size corresponding to exosomes isolated from NSC34-conditioned media, whereas spike-in experiments showed a further increment in microvesicles of the same size. (C) Immunoblotting confirmed the presence of the exosome markers flotillin1 and Tsg101 both in cell-conditioned media and serum preparations (D) Electron microscopy showed membrane-bound microvesicles, negatively stained with 2% aqueous uranyl acetate on a formvar film. (E) Venn diagram showing protein overlap between albumin/immunoglobulin depleted serum, serum microvesicles, and human cell lysates. Proteins were quantified using Oribtrap-Velos LC-MS/MS. The enrichment for serum-derived microvesicles enables the identification and quantitation of proteins that are not detected in routinely processed serum samples as shown by gene ontology analysis.

Mentions: We successfully adapted previously validated protocols based on filtration and differential centrifugation,11 to isolate circulating microvesicles from human serum. As shown in Figure1A, our protocol effectively minimized contamination with highly abundant serum proteins, thus overcoming a major challenge in the analysis of complex proteomes. We used NTA to quantitate the number of purified microvesicles and found that the mean size of the most abundant vesicles (about 120 nm) corresponded to exosomes. This conclusion was supported by the detection of the common exosomal markers Tsg101 and flotillin by immunoblotting and electron microscopy showing that the isolated microvesicles are membrane bound (Fig.1B–D). As a positive control, we spiked the serum with purified exosomes from conditioned media derived from NSC34 cells. We observed an increment in the corresponding vesicle size and abundance indicative of exosomes as well as an increase in relevant protein markers (Fig.1B and C).


Identification of distinct circulating exosomes in Parkinson's disease.

Tomlinson PR, Zheng Y, Fischer R, Heidasch R, Gardiner C, Evetts S, Hu M, Wade-Martins R, Turner MR, Morris J, Talbot K, Kessler BM, Tofaris GK - Ann Clin Transl Neurol (2015)

Isolation and characterization of circulating microvesicles. (A) Flow diagram showing the extraction method used and Coomassie staining of the final microvesicle preparation. Note that an additional wash step significantly reduces the number of contaminant proteins without major changes in microvesicle numbers. (B) Nanoparticle-tracking analysis of serum microvesicles revealed a major peak at the size corresponding to exosomes isolated from NSC34-conditioned media, whereas spike-in experiments showed a further increment in microvesicles of the same size. (C) Immunoblotting confirmed the presence of the exosome markers flotillin1 and Tsg101 both in cell-conditioned media and serum preparations (D) Electron microscopy showed membrane-bound microvesicles, negatively stained with 2% aqueous uranyl acetate on a formvar film. (E) Venn diagram showing protein overlap between albumin/immunoglobulin depleted serum, serum microvesicles, and human cell lysates. Proteins were quantified using Oribtrap-Velos LC-MS/MS. The enrichment for serum-derived microvesicles enables the identification and quantitation of proteins that are not detected in routinely processed serum samples as shown by gene ontology analysis.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4402081&req=5

fig01: Isolation and characterization of circulating microvesicles. (A) Flow diagram showing the extraction method used and Coomassie staining of the final microvesicle preparation. Note that an additional wash step significantly reduces the number of contaminant proteins without major changes in microvesicle numbers. (B) Nanoparticle-tracking analysis of serum microvesicles revealed a major peak at the size corresponding to exosomes isolated from NSC34-conditioned media, whereas spike-in experiments showed a further increment in microvesicles of the same size. (C) Immunoblotting confirmed the presence of the exosome markers flotillin1 and Tsg101 both in cell-conditioned media and serum preparations (D) Electron microscopy showed membrane-bound microvesicles, negatively stained with 2% aqueous uranyl acetate on a formvar film. (E) Venn diagram showing protein overlap between albumin/immunoglobulin depleted serum, serum microvesicles, and human cell lysates. Proteins were quantified using Oribtrap-Velos LC-MS/MS. The enrichment for serum-derived microvesicles enables the identification and quantitation of proteins that are not detected in routinely processed serum samples as shown by gene ontology analysis.
Mentions: We successfully adapted previously validated protocols based on filtration and differential centrifugation,11 to isolate circulating microvesicles from human serum. As shown in Figure1A, our protocol effectively minimized contamination with highly abundant serum proteins, thus overcoming a major challenge in the analysis of complex proteomes. We used NTA to quantitate the number of purified microvesicles and found that the mean size of the most abundant vesicles (about 120 nm) corresponded to exosomes. This conclusion was supported by the detection of the common exosomal markers Tsg101 and flotillin by immunoblotting and electron microscopy showing that the isolated microvesicles are membrane bound (Fig.1B–D). As a positive control, we spiked the serum with purified exosomes from conditioned media derived from NSC34 cells. We observed an increment in the corresponding vesicle size and abundance indicative of exosomes as well as an increase in relevant protein markers (Fig.1B and C).

Bottom Line: To interrogate their biological effect, microvesicles were added to primary rat cortical neurons subjected to either nutrient deprivation or sodium arsenite.Among 1033 proteins identified, 23 exosome-associated proteins were differentially abundant in PD, including the regulator of exosome biogenesis syntenin 1.Accordingly, we showed in models of neuronal stress that Parkinson's-derived microvesicles have a protective effect.

View Article: PubMed Central - PubMed

Affiliation: Nuffield Department of Clinical Neurosciences, University of Oxford Oxford, United Kingdom.

ABSTRACT

Objective: Whether circulating microvesicles convey bioactive signals in neurodegenerative diseases remains currently unknown. In this study, we investigated the biochemical composition and biological function of exosomes isolated from sera of patients with Parkinson's disease (PD).

Methods: Proteomic analysis was performed on microvesicle preparations from grouped samples of patients with genetic and sporadic forms of PD, amyotrophic lateral sclerosis, and healthy subjects. Nanoparticle-tracking analysis was used to assess the number and size of exosomes between patient groups. To interrogate their biological effect, microvesicles were added to primary rat cortical neurons subjected to either nutrient deprivation or sodium arsenite.

Results: Among 1033 proteins identified, 23 exosome-associated proteins were differentially abundant in PD, including the regulator of exosome biogenesis syntenin 1. These protein changes were detected despite similar exosome numbers across groups suggesting that they may reflect exosome subpopulations with distinct functions. Accordingly, we showed in models of neuronal stress that Parkinson's-derived microvesicles have a protective effect.

Interpretation: Collectively, these data suggest for the first time that immunophenotyping of circulating exosome subpopulations in PD may lead to a better understanding of the systemic response to neurodegeneration and the development of novel therapeutics.

No MeSH data available.


Related in: MedlinePlus