Mitochondrial Signatures in Circulating Extracellular Vesicles of Older Adults with Parkinson's Disease: Results from the EXosomes in PArkiNson's Disease (EXPAND) Study

Anna Picca, Flora Guerra, Riccardo Calvani, Federico Marini, Alessandra Biancolillo, Giovanni Landi, Raffaella Beli, Francesco Landi, Roberto Bernabei, Anna Rita Bentivoglio, Maria Rita Lo Monaco, Cecilia Bucci, Emanuele Marzetti, Anna Picca, Flora Guerra, Riccardo Calvani, Federico Marini, Alessandra Biancolillo, Giovanni Landi, Raffaella Beli, Francesco Landi, Roberto Bernabei, Anna Rita Bentivoglio, Maria Rita Lo Monaco, Cecilia Bucci, Emanuele Marzetti

Abstract

Systemic inflammation and mitochondrial dysfunction are involved in neurodegeneration in Parkinson's disease (PD). Extracellular vesicle (EV) trafficking may link inflammation and mitochondrial dysfunction. In the present study, circulating small EVs (sEVs) from 16 older adults with PD and 12 non-PD controls were purified and characterized. A panel of serum inflammatory biomolecules was measured by multiplex immunoassay. Protein levels of three tetraspanins (CD9, CD63, and CD81) and selected mitochondrial markers (adenosine triphosphate 5A (ATP5A), mitochondrial cytochrome C oxidase subunit I (MTCOI), nicotinamide adenine dinucleotide reduced form (NADH):ubiquinone oxidoreductase subunit B8 (NDUFB8), NADH:ubiquinone oxidoreductase subunit S3 (NDUFS3), succinate dehydrogenase complex iron sulfur subunit B (SDHB), and ubiquinol-cytochrome C reductase core protein 2 (UQCRC2)) were quantified in purified sEVs by immunoblotting. Relative to controls, PD participants showed a greater amount of circulating sEVs. Levels of CD9 and CD63 were lower in the sEV fraction of PD participants, whereas those of CD81 were similar between groups. Lower levels of ATP5A, NDUFS3, and SDHB were detected in sEVs from PD participants. No signal was retrieved for UQCRC2, MTCOI, or NDUFB8 in either participant group. To identify a molecular signature in circulating sEVs in relationship to systemic inflammation, a low level-fused (multi-platform) partial least squares discriminant analysis was applied. The model correctly classified 94.2% ± 6.1% PD participants and 66.7% ± 5.4% controls, and identified seven biomolecules as relevant (CD9, NDUFS3, C-reactive protein, fibroblast growth factor 21, interleukin 9, macrophage inflammatory protein 1β, and tumor necrosis factor alpha). In conclusion, a mitochondrial signature was identified in circulating sEVs from older adults with PD, in association with a specific inflammatory profile. In-depth characterization of sEV trafficking may allow identifying new biomarkers for PD and possible targets for personalized interventions.

Keywords: aging; biomarkers; exosomes; mitochondrial dynamics; mitochondrial quality control; mitochondrial-derived vesicles; mitochondrial-lysosomal axis; mitophagy.

Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Figures

Figure 1
Figure 1
Representative blots of preliminary characterization of small extracellular vesicles (sEVs). (A) Blots of the cytosolic protein flotilin and ribonucleoprotein (HNRNPA1) as positive and negative markers, respectively, in purified sEVs obtained by ultracentrifugation from controls and participants with Parkinson’s disease (PD). MCF-7 cell extract was used as the positive control for the anti-HNRNPA1 antibody. (B) Blots of tetraspanins CD9 and CD63, flotilin, and HNRNPA1 in purified sEVs obtained from one control and one PD participant using a commercial precipitation kit.
Figure 2
Figure 2
Levels of purified small extracellular vesicles (sEVs) in serum of controls (n = 12) and participants with Parkinson’s disease (PD; n = 16). Data were normalized for the amount of total serum protein and are shown as percentage of the control group set at 100%. Bars represent mean values (± standard deviation of the mean). * p < 0.0001 vs. controls.
Figure 3
Figure 3
Protein expression of (A) CD9, (B) CD63, and (C) CD81 in purified small extracellular vesicles (sEVs) from controls (n = 12) and participants with Parkinson’s disease (PD; n = 16). Data were normalized for the amount of sEV total proteins and are shown as percentage of the control group set at 100%. Bars represent mean values (± standard deviation of the mean). Representative blots are shown in Figure S1. * p = 0.0001 vs. controls.
Figure 4
Figure 4
Protein expression of adenosine triphosphate 5A (ATP5A), succinate dehydrogenase complex iron sulfur subunit (SDHB), and nicotinamide adenine dinucleotide reduced form (NADH):ubiquinone oxidoreductase subunit S3 (NDUFS3) in purified small extracellular vesicles (sEVs) from controls (n = 12) and participants with Parkinson’s disease (PD; n = 16). Data were normalized for the amount of sEV total proteins and are shown as percentage of the control group set at 100%. Bars represent mean values (± standard deviation of the mean). Representative blots are shown in Figure S1. * p < 0.0001 vs. controls.
Figure 5
Figure 5
Soft independent modeling of class analogies modeling of Parkinson’s disease (PD) showing the projection of samples onto the spaces described by the statistical variables Tred2 and Qred. The dashed line indicates the threshold for acceptance d=2.

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