GRAPHICAL ABSTRACT

INTRODUCTION

Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterized by the accumulation of misfolded α-synuclein aggregates, which begins years before clinical symptoms appear [1]. The pathological process follows a seeding-nucleation mechanism, in which oligomeric α-synuclein with seeding properties promotes fibril formation and intracellular accumulation [2]. This mechanism enables the spread of misfolded α-synuclein across cells, thereby accelerating disease progression [3]. Consequently, detecting misfolded α-synuclein aggregates in biological fluids represents a promising approach for early PD diagnosis.

However, α-synuclein aggregates are present at extremely low concentrations in biological fluids outside the central nervous system, making their direct quantification technically challenging. To overcome this limitation, seed amplification assays have been developed that exploit the seeding-nucleation mechanism to amplify trace amounts of pathological α-synuclein. Among these methods, real-time quaking-induced conversion (RT-QuIC) has become the most widely used method. RT-QuIC assays were initially optimized using cerebrospinal fluid (CSF), where α-synuclein aggregates are relatively enriched, and have demonstrated high diagnostic accuracy. Indeed, CSF-based α-synuclein RT-QuIC assays now serve as a key technological foundation for the biological staging of PD [4,5].

Despite their high performance, CSF-based assays are limited by the invasive nature of lumbar puncture, which restricts their widespread use, particularly for screening and longitudinal monitoring. As a result, there has been growing interest in developing RT-QuIC assays using more easily accessible peripheral samples, even though these contain lower amounts of α-synuclein aggregates. Various alternative sample types, including skin tissue, olfactory mucosa, and blood, have been explored. Among these, blood-based approaches are particularly attractive because of their minimal invasiveness and feasibility for repeated sampling. To increase detection sensitivity in blood, recent strategies have focused on enriching neuronal components, such as direct immunoprecipitation of α-synuclein or the isolation of neuronal exosomes, to increase the likelihood of detecting pathogenic α-synuclein seeds [6-11]. Nevertheless, blood-based detection of α-synuclein aggregates still requires further validation to establish robust biological and diagnostic evidence.

Several recent studies have emphasized the feasibility of using extracellular vesicles (EVs), especially neuron-derived extracellular vesicles (nEVs), as carriers of α-synuclein aggregates [12,13]. Originally recognized as mediators of intracellular communication, nEVs are now thought to play an active role in the propagation of α-synuclein pathology [14,15]. They are detectable in various biological fluids, such as CSF, blood, and saliva, offering alternative approaches for identifying α-synuclein pathology [12,16,17]. Recent reports indicate that blood-derived nEVs from patients with PD contain increased levels of aggregated α-synuclein, supporting their potential utility for early diagnosis [7,8,18]. While previous studies, including those by Kluge et al. [7], have demonstrated the feasibility of detecting α-synuclein pathology in blood-derived nEVs, further validation and methodological standardization are needed to establish robust and clinically applicable workflows.

In this context, this study aims to optimize the α-synuclein RT-QuIC assay for detecting misfolded α-synuclein in serum-derived nEVs and to evaluate its potential as a more accessible biomarker for PD and other synucleinopathies. By systematically adjusting the experimental conditions, we sought to improve the sensitivity and specificity of this diagnostic method. The development of a serum-based RT-QuIC assay could provide a valuable tool for early PD diagnosis, offering a practical, repeatable, and less invasive alternative to CSF-based approaches, thereby facilitating timely therapeutic interventions and improving patient outcomes.

MATERIALS & METHODS

Study design and subjects

For this study, we recruited 15 patients with PD, 4 patients with multiple system atrophy (MSA), 5 patients with dementia with Lewy bodies (DLB), 7 healthy volunteers from the Hallym PD registry, and [19] from a prospective database that included all individuals who presented with parkinsonism at the Movement Disorder Clinic of Hallym University Sacred Heart Hospital. The registry includes demographic data, detailed clinical assessments, and collected biospecimens. Demographic information and clinical evaluations were obtained using the Movement Disorder Society-Unified Parkinson’s Disease Rating Scale (MDS-UPDRS) [20] and Hoehn and Yahr staging system [21]. Blood samples were collected from all participants after informed consent had been obtained, and no adverse events were reported following sample collection.

PD diagnoses were confirmed on the basis of the Movement Disorder Society Clinical Diagnostic Criteria [22], as determined independently by two movement disorder specialists. To ensure the inclusion of patients with typical nigrostriatal degeneration, only those whose 18F-N-(3-fluoropropyl)-2β-carboxymethoxy-3β-(4-iodophenyl) nortropane imaging results were positive were selected.

MSA was diagnosed according to the Second Consensus Statement on the Diagnosis of MSAs [23] on the basis of comprehensive clinical data collected during follow-up visits. Four patients with probable MSA were included. DLB diagnoses were made following the Fourth Consensus Report of the DLB Consortium [24]. Healthy controls (HCs) were individuals with no history or clinical evidence of neurological disorders, including dementia and parkinsonism. Clinical and demographic data for all subjects were assessed at the time of blood sample collection.

The baseline characteristics of the enrolled participants are summarized in Supplementary Table 1. This study aimed to evaluate whether serum-derived nEVs can discriminate patients with synucleinopathy from HCs. Therefore, only participants whose clinical differentiation clearly differed between patient groups and controls were included in this study. To serve as true negative references for synucleinopathy, relatively young, neurologically healthy individuals without any history of neurological disorders were included in the control group. Participant information, including age, disease duration, and MDS-UPDRS scores, is presented accordingly. Additionally, a subset of patient samples was used to validate different stages of the study, with the number of samples used indicated in the corresponding figure legends.

Ethical statement

This study was approved by the institutional review board of Hallym University Medical Center (IRB 2020-06-106). Informed consent was obtained from all participants, and all evaluations were conducted in accordance with the ethical standards of the institutional committee, as well as the 1964 Helsinki Declaration and its later amendments.

Serum collection

Human blood samples were collected using serum-separating tubes and centrifuged at 4,000 rpm for 10 minutes at 4°C. The resulting serum was processed immediately, aliquoted, and stored at -80°C for further use. Samples with visible red discoloration—indicative of red blood cell (RBC) contamination—were discarded to avoid interference in downstream analyses.

Isolation of serum-derived nEVs

To prepare for total extracellular vesicle (tEV) isolation, serum samples were first centrifuged at 2,000×g for 10 minutes at 4°C to remove any residual RBCs and cell debris. EVs were then isolated from 250 μL of serum obtained from patients and HCs using an ExoQuick EV precipitation solution (EXOQ; System BioSciences) according to the manufacturer’s instructions.

nEVs were subsequently isolated from the tEV fraction of the serum and separated according to the protocol of Mustapic et al. [25] Briefly, the EV pellet was resuspended in 250 μL of UltraPureTM water (Invitrogen) supplemented with 3x protease inhibitor cocktail (Roche) and 3x PhosSTOP (Sigma) and rotated for 2 hours at 4°C to ensure thorough mixing.

Next, 1.5 μg of CD171 biotinylated antibody (UJ127; Invitrogen) diluted in 50 μL of 3% bovine serum albumin (BSA) (prepared by a 3.33 times dilution in DPBS from Blocker BSA 10% solution; Thermo Scientific) was added to the resuspended EV solution. This mixture was incubated with continuous rotation for 1 hour at 4°C. Afterward, 10 μL of Streptavidin Plus Ultra-Link resin (Thermo Scientific) in 40 μL of 3% BSA was added, and the mixture was incubated for an additional 30 minutes at 4°C.

After incubation, the samples were centrifuged at 200×g for 10 minutes at 4°C. The supernatant was removed, and L1-cell adhesion molecule (L1CAM)-positive EVs were detached from the bead by the addition of 100 μL of 0.1 M glycine-HCl (pH 3.0). This solution was then centrifuged again at 200×g for 10 minutes at 4°C. The supernatant containing the nEV was neutralized with 15 μL of 1 M Tris-HCl (pH 8.0). Finally, 25 μL of 10% BSA supplemented with 3x protease inhibitor cocktail and 3x PhosSTOP was added to minimize background signals in downstream experiments, and the nEVs were stored at -80°C.

EV protein concentration and quantification

To investigate the characteristics of nEVs, we first isolated EVs from the serum of patients with PD and HCs. The protein concentration of the nEVs was measured using a bicinchoninic acid assay (BCA) protein assay kit (Thermo Scientific Pierce). BSA standards or samples (50 μL) were transferred to a 96-well plate to which 200 μL of working reagent was added (working reagent 50:1 ratio of assay reagents A and B). The plate was incubated for 30 min at 37°C before being analyzed with a spectrophotometer and a 562 nm microplate reader (BioTek). EV abundance was quantified by using a FluroCet EV Quantitation Kit (System Biosciences) according to the manufacturer’s protocol. Briefly, lysed EVs were added to the quantitation buffer and then incubated for 20 minutes at room temperature in a dark room. The fluorescent signal was immediately measured using a Synergy H1 Multimode Reader (BioTek) with excitation at 530–570 nm and emission at 590–600 nm. EV abundance was calculated according to the manufacturer’s recommendations.

α-synuclein RT-QuIC assay

RT-QuIC reactions were performed in Corning black 96-well plates with clear flat bottoms preloaded with six 1.0 mm zirconia/silica beads (BioSpec). Lyophilized α-synuclein was reconstituted in UltraPure^TM water, and its concentration was measured using a BCA protein assay (Pierce). For EV samples, a total sample volume of 15 μL with 85 μL of reaction mix was used following the previous protocol of the CSF RT-QuIC assay. Both 2 μL and 4 μL of nEVs diluted with UltraPure^TM water of the indicated suspension were added to wells containing 85 μL of reaction mix composed of 40 mM phosphate buffer (pH 8.0), 500 mM NaCl (Invitrogen), 0.0015% sodium dodecyl sulfate (SDS), 10 μM thioflavin T (ThT), and 0.25 mg/mL recombinant α-synuclein. RT-QuIC plates were sealed with Nunc clear sealing film (Thermo) and incubated at 42°C in a Synergy H1 Multimode Reader (BioTek) with cycles of 1 minute of shaking (double orbital, 425 cpm) and 1 minute of rest throughout the incubation time. The ThT fluorescence signal was measured every 30 minutes (440 nm excitation and 480 nm emission; bottom read) for a maximum of 40 hours. Each sample was quadraduplicated; the results were considered positive when three or more replicates were positive. The microplate reader was set up with three levels of optic gain (50, 80, and 100) with maximal fluorescence detection such that the device could record at 100,000 relative fluorescence units (RFU). Raw RFU data were normalized to the percentage of maximal fluorescence at 100,000 RFU (defined as 100%). The threshold for RT-QuIC assay positivity was determined to be 30,000 RFU, according to previous methods.

After the assay was performed, the kinetics of the reaction were analyzed using parameters that describe the fluorescence curve. Maximum fluorescence (Fmax) and the area under the curve (AUC) were then calculated.

The detailed methods used for nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), western blotting and dot blot analysis, recombinant human α-synuclein expression and purification, and SDS–polyacrylamide gel electrophoresis analysis are provided in Supplementary Method 1 and Supplementary Figure 1.

Statistical analysis

Relative RT-QuIC fluorescence signals were converted to a percentage of maximal fluorescence by Microsoft Excel (Microsoft Corporation) and then analyzed and plotted using Graph-Pad Prism version 10 (https://www.graphpad.com/features). Continuous variables in the RT-QuIC graph are expressed as the mean of four replications±standard deviation. Group differences were compared with a double-tailed unpaired t-test (Mann–Whitney U test) for continuous variables after evaluation of data normality by the Shapiro–Wilk test and Fisher’s exact test for categorical variables. Western blot quantification results between fractions were compared using one-way analysis of variance (ANOVA) or two-way ANOVA, depending on the indication. p values <0.05 were considered to indicate statistical significance. The RT-QuIC performance was evaluated using receiver operating characteristic curve analysis and calculation of the AUC. Statistical analysis and graphic visualization were performed with GraphPad Prism (version 10) and R (version 4.2.2; R Foundation for Statistical Computing).

RESULTS

Isolation and characterization of serum nEVs

The confirmation and quantification of isolated nEVs from serum were performed (Figure 1A and B). TEM images (Figure 1A, left panel) revealed EVs with a typical round or cup-shaped morphology and membrane structures ranging from 30 to 200 nm in diameter. Treatment with radioimmunoprecipitation assay buffer disrupted nEVs, confirming successful membrane lysis (Supplementary Figure 2). nEVs analyzed by NTA showed the expected size distribution, with a peak at approximately 160 nm in diameter. The mean size was 172.06±38 nm, and the median size was 165.2 nm (Figure 1A, right panel).

The quantification of nEVs was performed using the Fluoro-Cet Exosome Vesicle Quantitation Kit to assess the abundance of EVs. The mean numbers of nEVs (6.48±0.09×10⁷ particles/mL and 6.51±0.23×10⁷ particles/mL in the HC and PD or DLB groups, respectively) were similar between the groups (p>0.999) (Figure 1B). The mean number of total EVs (tEVs; 4.49±0.43×10⁹ particles/mL in the HC group and 5.99±0.31×10⁹ particles/mL in the PD group) did not significantly differ between the groups.

Western blot analysis was performed to verify the identity of the EV fractions in both patients with PD and HCs. As shown in Figure 1C and D, the general EV markers, including CD81, Alix, and TSG101, were strongly detected in both tEVs and nEVs from HC and patients with PD, whereas these markers were nearly absent in non-EVs (tEV-depleted serum fraction), confirming the EV origin of the isolated samples. The negative marker calnexin was rarely detected in any EVs, indicating minimal contamination from cellular organelles. Neuronal markers, including L1CAM, synaptophysin, and TUBB3, were consistently enriched in nEVs from both the HC and PD groups. Notably, nEVs displayed the strongest expression of L1CAM and synaptophysin, supporting their neuronal specificity (Figure 1C and D).

Demonstration of pathological α-synuclein in serum nEVs by immunoblot

To verify the presence of phosphorylated α-synuclein (p-α-Syn) and aggregated α-synuclein in serum nEVs, western blot analysis was performed using serum-derived components. p-α-Syn at Ser129 signals were detected in tEVs and nEVs from various patient groups, including PD, DLB, and MSA (Figure 2A). Total α-synuclein and TUBB3 were used as neuronal loading markers and were consistently expressed across samples. Densitometric quantification confirmed that, compared with the non-EVs (p=0.013), the level of p-α-Syn at Ser129 was significantly higher in the nEVs, although it was not statistically significantly different between the tEV fraction and the non-EV fraction (p=0.187) (Figure 2A lower panel).

Western blot analysis comparing p-α-Syn between the PD (n=4) and HC (n=4) groups using L1CAM as loading marker, showed that, compared with those from HCs, p-α-Syn (p=0.029) were significantly elevated in nEVs from patients with PD (Supplementary Figure 3).

Next, we investigated the levels of aggregated α-synuclein protein in nEVs from the HC (n=3) and various patient groups, including the DLB (n=3), MSA (n=3), and PD (n=3) groups, using dot blotting (Figure 2B, upper panel). Quantitative analysis of aggregated α-synuclein from the nEV fraction revealed a significant difference between the HC group and the three disease groups overall (p=0.001). Among the disease groups, the PD group was significantly different from the HC group (comparisons between HCs and DLB, MSA, and PD patients; p>0.999, 0.103, and 0.030, respectively). No significant differences were observed between the disease groups themselves (p=0.533 for DLB vs. PD; otherwise, p>0.999) (Figure 2B, lower panel).

Performance of the α-synuclein RT-QuIC assay

To optimize the RT-QuIC assay using nEVs, we tested three concentrations of recombinant α-synuclein monomer (0.15, 0.25, and 0.35 mg/mL) using two representative nEVs: one from an HC and one from a patient with DLB. In the HC, all the replicates exhibited the expected negative reaction kinetics at all monomer concentrations, with the exception of a single false-positive well at a concentration of 0.35 mg/mL (Supplementary Figure 4A). In contrast, the DLB-sample yielded positive reactions at both 0.25 mg/mL and 0.35 mg/mL, whereas 0.15 mg/mL resulted in negative signals (Supplementary Figure 4B). To determine the optimal volume of nEV input, we compared RT-QuIC reactions using 2 μL and 4 μL of nEVs with pure water (within a total reaction volume of 15 μL) from five PDs and two HCs (Supplementary Figure 4C). Both nEV volumes similarly differentiated between PD and HC samples, with positive reactions for synucleinopathy samples and negative reactions for HC samples. Thus, 0.25 mg/mL of α-synuclein and 2 μL of nEVs were used for further RT-QuIC assays.

Kinetic traces of the RT-QuIC reactions for nEVs derived from patients with synucleinopathies (n=6, 4 patients with PD, 1 with DLB, 1 with MSA) and HCs (n=5) using 2 μL seed volumes are presented in Figure 3A. The demographics of the participants are described in Supplementary Table 2. All synucleinopathy groups showed positive assay results (a positive result was defined as positivity in ≥3 of 4 replicate trials; all other cases were classified as negative), and all HCs were negative. As shown in Figure 3B, field-emission scanning electron microscopy (FE-SEM) imaging revealed the formation of α-synuclein fibrils in the final product of the RT-QuIC assay using nEVs of PD. The fibrillar structures appeared as long, unbranched filaments with a typical amyloid-like morphology, which is consistent with α-synuclein aggregation. High-magnification images further demonstrated dense, intertwined fibrils, supporting the successful amplification of pathological α-synuclein seeds during the RT-QuIC reaction. The results of the quantitative analysis shown in Figure 3C revealed that, compared with the HC samples, synucleinopathy samples (PD, DLB, and MSA) exhibited significantly greater Fmax (p<0.001). This difference between groups was also observed in the AUC measurements (p<0.001).

DISCUSSION

This study investigated the diagnostic potential of serum-derived nEVs as a seed source for RT-QuIC assays in detecting pathological α-synuclein aggregates in PD and other synucleinopathies. By demonstrating the identity of nEVs and their potential as a seed source containing pathological α-synuclein, we demonstrated that nEV-based RT-QuIC assays could differentiate PD or synucleinopathy patients from HCs with high diagnostic performance.

We successfully isolated nEVs from peripheral serum using a two-step method involving tEV precipitation followed by L1CAM-based immunoaffinity capture. TEM, NTA and western blotting were used to validate the morphology and marker profiles, confirming the neuronal origin of the isolated vesicles. Importantly, elevated levels of phosphorylated and aggregated α-synuclein were detected in nEVs from patients with synucleinopathies, supporting their relevance as disease biomarkers. However, it should be noted that L1CAM-based isolation may not exclusively capture nEVs, as L1CAM is also expressed in nonneuronal tissues and can exist in a soluble form in circulation. Therefore, despite our additional validation steps, the potential inclusion of nonneuronal or nonvesicular components should be considered a limitation of this approach.

Under optimized RT-QuIC assay conditions, we achieved excellent diagnostic performance. FE-SEM imaging of postassay components confirmed the presence of α-synuclein fibrils. The kinetic parameters (Fmax and AUC) significantly differed between PD and HC samples, providing quantitative confirmation of pathological α-synuclein within nEVs, although there was no significant difference in the lag phase between the patient groups.

Previous RT-QuIC studies using CSF have shown high diagnostic accuracy but are limited by the invasiveness of lumbar puncture [12]. In contrast, our work represents one of the most detailed validations of a blood-based RT-QuIC assay using serum nEVs, offering a more accessible and scalable diagnostic approach. While other studies using blood-derived nEVs have focused primarily on total protein quantification or complex amplification protocols [7,8], our approach directly assessed seed amplification performance through RT-QuIC kinetics. A recent study by Okuzumi et al. [10] demonstrated that direct immunoprecipitation of α-synuclein from blood plasma, followed by RT-QuIC analysis, could serve as a promising method for detecting PD and related synucleinopathies. While their approach effectively circumvents the need for CSF sampling and demonstrates high sensitivity, it requires specific reagents and targeted enrichment protocols for α-synuclein aggregates. In our study, serum-derived nEVs were used as a biologically relevant and cell type-specific seed source, reflecting a naturally compartmentalized environment for pathological α-synuclein. Although further comparative validation is needed, this EV-based approach may offer a complementary pathway with potential advantages in simplicity and sample handling. This enhances its potential clinical translatability. In this context, our study provides a systematically validated workflow and extends prior findings by evaluating RT-QuIC kinetics in serum-derived nEVs across a broader spectrum of synucleinopathies. Given the importance of reproducibility in this field, our results also serve as an independent validation of previous observations. The ability to detect misfolded α-synuclein using serum-derived nEV holds significant promise for early diagnosis and disease monitoring. The minimally invasive nature of this technique allows for repeated, longitudinal sampling—a key advantage in disorders such as PD, where pathophysiological changes precede symptoms by years [1,3].

Although we included patients with synucleinopathies, including MSA and DLB, it was difficult to characterize kinetic differences between synucleinopathies because our total sample size was small. To address these limitations, future studies should include larger and more diverse patient cohorts, particularly expanding MSA and DLB cases, to enhance statistical reliability and to determine whether RT-QuIC kinetics can support differential diagnosis across synucleinopathies.

Several limitations must be acknowledged. First, while HCs intentionally included a younger demographic, this was done to ensure the lowest possible likelihood of subclinical synucleinopathy. Since the enrolled patients were not autopsy-confirmed, we chose to validate the assay performance using blood samples from individuals with a minimal risk of underlying neurodegenerative disease. This design choice ensured the robustness of the negative control but introduced an age imbalance between groups, which could be a confounding factor. In addition, the limited number of participants reduces statistical power. To address this, future research should involve age-matched HCs and patients whose diagnoses are validated through CSF RT-QuIC or other standardized biomarkers. Such studies would provide stronger validation of the assay’s diagnostic utility. Furthermore, the small sample sizes, especially in the MSA and DLB groups, limited our ability to explore diagnostic differentiation between synucleinopathies. Expanding the patient cohort will be critical for determining whether RT-QuIC kinetics are specific to particular disease subtypes.

Additionally, standardization of nEV isolation protocols and minimizing variability in antibody-based purification will be necessary to facilitate clinical implementation. Although the EV immunoprecipitation protocol referred to in our research25 yielded relatively uniform and high-quality EVs, the utilization of 0.1 M glycine-HCl at a low pH (3.0) raises concerns about potential EV damage during extraction, since such conditions are sometimes used for cell lysis [26]. Given the inherent limitations of polymer-based precipitation, a follow-up study will directly compare ExoQuick plus L1CAM capture with size exclusion chromatography plus L1CAM capture on identical patient samples. This side-by-side evaluation will examine α-synuclein background, EV purity, and RT-QuIC performance, thereby generating critical data to guide the optimization of nEV isolation methods for clinical translation.

In conclusion, this study demonstrated that serum-derived nEVs contain pathologically relevant α-synuclein aggregates and can serve as effective seed material for RT-QuIC assays. Our findings suggest that this noninvasive and reproducible method holds considerable promise for the early diagnosis and monitoring of PD and other synucleinopathies. With further validation, nEV-based RT-QuIC assays could serve as a clinically applicable biomarker platform not only for PD but also potentially for other synucleinopathies.

Supplementary Materials

Notes

Conflicts of Interest

The authors have no financial conflicts of interest.

Funding Statement

This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number: RS-2023-00265159), Basic Science Research Program through the National Research Foundation of Korea (NRF) grant funded by the Ministry of Education (RS-2023-00246655), the NRF by the Korea government (MSIT) (2022R1A2C2091254 [Bio&Medical Technology Development Program], RS-2023-00223501 and RS-2024-00356135), Hallym University Research Fund, Hallym University Mighty Hallym 4.0 Campus (MHC) project, 2025 (MHC-202503-005), and the Regional Innovation System & Education (RISE) Glocal University 30 Project program through the Gangwon RISE Center, funded by the Ministry of Education (MOE) and the Gangwon State (G.S.), Republic of Korea (2025-RISE-10-009).

Acknowledgments

None

Author Contributions

Conceptualization: Young Eun Kim. Data curation: Young Eun Kim, Hye Joung Choi, Dong Gyun Ko. Formal analysis: Young Eun Kim, Hye Joung Choi, Dong Gyun Ko, Nguyen Thi Hai Thanh. Funding acquisition: Young Eun Kim, Hye Joung Choi. Investigation: Hye Joung Choi, Dong Gyun Ko. Methodology: Hye Joung Choi, Dong Gyun Ko, JeKuk Yu. Project administration: Hye Joung Choi, Dong Gyun Ko. Resources: Hye Joung Choi, Hyeo-il Ma. Software: Hye Joung Choi, Dong Gyun Ko, JeKuk Yu. Supervision: Young Eun Kim, Hyeo-il Ma. Validation: Dong Gyun Ko, JeKuk Yu. Visualization: Dong Gyun Ko. Writing—original draft: Hye Joung Choi, Dong Gyun Ko. Writing—review & editing: all authors.

Figure 1.

Characterization of serum-derived nEVs. A: The left panel shows a representative transmission electron microscopy image of isolated nEVs, demonstrating their typical cup-shaped morphology and heterogeneous sizes ranging from 30 to 200 nm (scale bar: 200 nm), as indicated by arrows. The right panel shows that the mean particle size of nEV was 172.06±38 nm, with a mode of 165.2 nm, as measured by NTA. B: The number of tEVs was quantified using the FluoroCet Exosome Quantitation Assay Kit (n=3 per group). C: Western blot analysis confirmed the presence of nEVs using neuronal markers—L1CAM and synaptophysin—as well as EV marker CD81, thereby validating the robustness of the serum-derived nEVs isolation protocol. D: Differences among EV isolation fractions (tEVs, non-EVs, and nEVs) were compared. The expression of general EV-positive markers (CD81, Alix, TSG101), the neuronal marker (TUBB3), and the negative marker (calnexin) was analyzed in serum-derived fractions from HCs and patients with PD (Two PD and two HC samples were included). nEV, neuron-derived extracellular vesicle; NTA, nanoparticle tracking analysis; tEV, total extracellular vesicle; EV, extracellular vesicle; HC, healthy control; PD, Parkinson’s disease; ns, not significant indicates p>0.05; L1CAM, L1-cell adhesion molecule.

Figure 2.

Detection of phosphorylated and aggregated α-synuclein in serum-derived nEVs. Identification of p-α-Syn and aggregated α-synuclein in different EV fractions (A, upper panel). Western blot analysis of tEVs, non-EVs, and neuronal EVs (nEVs) showing p-α-Syn (S129) and total α-synuclein levels, with TUBB3 used as a neuronal marker (A, lower panel). A total of eight samples were analyzed, with two samples from each group (HC, DLB, MSA, and PD). Protein band intensities were quantified using ImageJ software. Statistical analysis was performed using one-way ANOVA. B: Dot blot analysis of aggregated α-synuclein in serum-derived nEVs from HC (n=3), DLB (n=3), MSA (n=3), and PD (n=3). Aggregated α-synuclein levels were quantified by densitometric scanning using ImageJ software. Bars indicate SD of n=3 samples. Statistical analysis was performed using one-way ANOVA. *p<0.050; **p<0.010. tEV, total extracellular vesicle; EV, extracellular vesicle; nEV, neuron-derived extracellular vesicle; HC, healthy control; DLB, dementia with Lewy body; MSA, multiple system atrophy; PD, Parkinson’s disease; p-α-Syn, phosphorylated α-synuclein; ANOVA, analysis of variance.

Figure 3.

Graph and kinetics of RT-QuIC assay in synucleinopathy and HC. A: Total graph of α-synuclein RT-QuIC assay in patients with synucleinopathy and HC. All four replicate reactions for each individual sample are shown separately (i.e., each subject is represented by four curves). Although occasional positive signals were observed in HC samples, no HC sample met the predefined positivity criterion (≥3 positive replicates). The dotted line represents the threshold (30% of the maximum fluorescence signal) at which replication is considered positive. B: α-synuclein fibrils formed in the RT-QuIC assay visualized by field-emission scanning electron microscopy. C: Kinetic parameters of synucleinopathy and HC groups. Fmax and AUC of RT-QuIC kinetics were calculated using the mean value of Fmax or AUC of 4 repeats test. ***p<0.001. RFU, relative fluorescence units; PD, Parkinson’s disease; DLB, dementia with Lewy body; MSA, multiple system atrophy; HC, healthy control; Fmax, maximum fluorescence; AUC, area under the curve; RT-QuIC, real-time quaking-induced conversion.

REFERENCES

• 1. Spillantini MG, Crowther RA, Jakes R, Hasegawa M, Goedert M. alpha-Synuclein in filamentous inclusions of Lewy bodies from Parkinson’s disease and dementia with Lewy bodies. Proc Natl Acad Sci U S A 1998;95:6469–6473.PubMedPMC
• 2. Baba M, Nakajo S, Tu PH, Tomita T, Nakaya K, Lee VM, et al. Aggregation of alpha-synuclein in Lewy bodies of sporadic Parkinson’s disease and dementia with Lewy bodies. Am J Pathol 1998;152:879–884.PubMedPMC
• 3. Braak H, Del Tredici K, Rüb U, de Vos RA, Jansen Steur EN, Braak E. Staging of brain pathology related to sporadic Parkinson’s disease. Neurobiol Aging 2003;24:197–211.ArticlePubMed
• 4. Simuni T, Chahine LM, Poston K, Brumm M, Buracchio T, Campbell M, et al. A biological definition of neuronal α-synuclein disease: towards an integrated staging system for research. Lancet Neurol 2024;23:178–190.ArticlePubMed
• 5. Höglinger GU, Adler CH, Berg D, Klein C, Outeiro TF, Poewe W, et al. A biological classification of Parkinson’s disease: the SynNeurGe research diagnostic criteria. Lancet Neurol 2024;23:191–204.ArticlePubMed
• 6. Kluge A, Borsche M, Streubel-Gallasch L, Gül T, Schaake S, Balck A, et al. α-Synuclein pathology in PRKN-linked Parkinson’s disease: new insights from a blood-based seed amplification assay. Ann Neurol 2024;95:1173–1177.PubMed
• 7. Kluge A, Bunk J, Schaeffer E, Drobny A, Xiang W, Knacke H, et al. Detection of neuron-derived pathological α-synuclein in blood. Brain 2022;145:3058–3071.ArticlePubMedPDF
• 8. Schaeffer E, Kluge A, Schulte C, Deuschle C, Bunk J, Welzel J, et al. Association of misfolded α-synuclein derived from neuronal exosomes in blood with Parkinson’s disease diagnosis and duration. J Parkinsons Dis 2024;14:667–679.ArticlePubMedPMCPDF
• 9. Bernhardt AM, Nemati M, Boros FA, Hopfner F, Levin J, Mollenhauer B, et al. α-Synuclein seed amplification assays from blood-based extracellular vesicles in Parkinson’s disease: an evaluation of the evidence. Mov Disord 2024;39:1269–1271.ArticlePubMed
• 10. Okuzumi A, Hatano T, Matsumoto G, Nojiri S, Ueno SI, Imamichi-Tatano Y, et al. Propagative α-synuclein seeds as serum biomarkers for synucleinopathies. Nat Med 2023;29:1448–1455.ArticlePubMedPMCPDF
• 11. Hatano T, Okuzumi A, Matsumoto G, Tsunemi T, Hattori N. α-Synuclein: a promising biomarker for Parkinson’s disease and related disorders. J Mov Disord 2024;17:127–137.ArticlePubMedPMCPDF
• 12. Stuendl A, Kunadt M, Kruse N, Bartels C, Moebius W, Danzer KM, et al. Induction of α-synuclein aggregate formation by CSF exosomes from patients with Parkinson’s disease and dementia with Lewy bodies. Brain 2016;139:481–494.ArticlePubMed
• 13. Ngolab J, Trinh I, Rockenstein E, Mante M, Florio J, Trejo M, et al. Brain-derived exosomes from dementia with Lewy bodies propagate α-synuclein pathology. Acta Neuropathol Commun 2017;5:46.PubMedPMC
• 14. Emmanouilidou E, Melachroinou K, Roumeliotis T, Garbis SD, Ntzouni M, Margaritis LH, et al. Cell-produced alpha-synuclein is secreted in a calcium-dependent manner by exosomes and impacts neuronal survival. J Neurosci 2010;30:6838–6851.ArticlePubMedPMC
• 15. Danzer KM, Kranich LR, Ruf WP, Cagsal-Getkin O, Winslow AR, Zhu L, et al. Exosomal cell-to-cell transmission of alpha synuclein oligomers. Mol Neurodegener 2012;7:42.ArticlePubMedPDF
• 16. Shi M, Liu C, Cook TJ, Bullock KM, Zhao Y, Ginghina C, et al. Plasma exosomal α-synuclein is likely CNS-derived and increased in Parkinson’s disease. Acta Neuropathol 2014;128:639–650.ArticlePMCPDF
• 17. Mavroeidi P, Vetsi M, Dionysopoulou D, Xilouri M. Exosomes in alpha-synucleinopathies: propagators of pathology or potential candidates for nanotherapeutics? Biomolecules 2022;12:957.ArticlePubMed
• 18. Kluge A, Schaeffer E, Bunk J, Sommerauer M, Röttgen S, Schulte C, et al. Detecting misfolded α-synuclein in blood years before the diagnosis of Parkinson’s disease. Mov Disord 2024;39:1289–1299.Article
• 19. Kwak IH, Kim YE, Kang SY, Lee JS, Lee J, Kim MS, et al. Comparative olfactory profiles in Parkinson’s disease and drug-induced parkinsonism. J Mov Disord 2024;17:64–70.ArticlePubMedPDF
• 20. Goetz CG, Tilley BC, Shaftman SR, Stebbins GT, Fahn S, Martinez-Martin P, et al. Movement Disorder Society-sponsored revision of the Unified Parkinson’s Disease Rating Scale (MDS-UPDRS): scale presentation and clinimetric testing results. Mov Disord 2008;23:2129–2170.PubMed
• 21. Hoehn MM, Yahr MD. Parkinsonism: onset, progression, and mortality. Neurology 1967;17:427.ArticlePubMed
• 22. Postuma RB, Berg D, Stern M, Poewe W, Olanow CW, Oertel W, et al. MDS clinical diagnostic criteria for Parkinson’s disease. Mov Disord 2015;30:1591–1601.ArticlePubMed
• 23. Gilman S, Wenning GK, Low PA, Brooks DJ, Mathias CJ, Trojanowski JQ, et al. Second consensus statement on the diagnosis of multiple system atrophy. Neurology 2008;71:670–676.ArticlePubMedPMC
• 24. McKeith IG, Boeve BF, Dickson DW, Halliday G, Taylor JP, Weintraub D, et al. Diagnosis and management of dementia with Lewy bodies: fourth consensus report of the DLB Consortium. Neurology 2017;89:88–100.PubMed
• 25. Mustapic M, Eitan E, Werner JK Jr, Berkowitz ST, Lazaropoulos MP, Tran J, et al. Plasma extracellular vesicles enriched for neuronal origin: a potential window into brain pathologic processes. Front Neurosci 2017;11:278.ArticlePubMedPMC
• 26. Pandeya A, Alegun O, Cai Y, Wei Y. Distribution of fluoroquinolones in the two aqueous compartments of Escherichia coli. Biochem Biophys Rep 2020;24:100849.ArticlePubMedPMC

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