Evaluation of α‐synuclein in CNS‐originating extracellular vesicles for Parkinsonian disorders: A systematic review and meta‐analysis

Abstract Background & Aims Parkinsonian disorders, such as Parkinson's disease (PD), multiple system atrophy (MSA), dementia with Lewy bodies (DLB), progressive supranuclear palsy (PSP) and corticobasal syndrome (CBS), share early motor symptoms but have distinct pathophysiology. As a result, accurate premortem diagnosis is challenging for neurologists, hindering efforts for disease‐modifying therapeutic discovery. Extracellular vesicles (EVs) contain cell‐state‐specific biomolecules and can cross the blood‐brain barrier to the peripheral circulation, providing a unique central nervous system (CNS) insight. This meta‐analysis evaluated blood‐isolated neuronal and oligodendroglial EVs (nEVs and oEVs) α‐synuclein levels in Parkinsonian disorders. Methods Following PRISMA guidelines, the meta‐analysis included 13 studies. An inverse‐variance random‐effects model quantified effect size (SMD), QUADAS‐2 assessed risk of bias and publication bias was evaluated. Demographic and clinical variables were collected for meta‐regression. Results The meta‐analysis included 1,565 patients with PD, 206 with MSA, 21 with DLB, 172 with PSP, 152 with CBS and 967 healthy controls (HCs). Findings suggest that combined concentrations of nEVs and oEVs α‐syn is higher in patients with PD compared to HCs (SMD = 0.21, p = 0.021), while nEVs α‐syn is lower in patients with PSP and CBS compared to patients with PD (SMD = ‐1.04, p = 0.0017) or HCs (SMD = ‐0.41, p < 0.001). Additionally, α‐syn in nEVs and/or oEVs did not significantly differ in patients with PD vs. MSA, contradicting the literature. Meta‐regressions show that demographic and clinical factors were not significant predictors of nEVs or oEVs α‐syn concentrations. Conclusion The results highlight the need for standardized procedures and independent validations in biomarker studies and the development of improved biomarkers for distinguishing Parkinsonian disorders.


| INTRODUC TI ON
Parkinsonian disorders encompass a group of neurodegenerative conditions characterized by motor symptoms such as bradykinesia, rigidity, and tremor, among which Parkinson's disease (PD) is the most prevalent. 1Other less common but clinically significant Parkinsonian disorders include multiple system atrophy (MSA), dementia with Lewy body (DLB), progressive supranuclear palsy (PSP), and corticobasal degeneration (CBD).Despite the common prevalence of shared symptoms, these disorders differ in the types of cells affected and the specific regions of the brain involved and do not respond to the same treatments. 2, MSA and DLB share a common pathological hallmark in the form of abnormal α-synuclein (α-syn) aggregation.In PD, α-syn aggregates primarily form in dopaminergic neurons of the substantia nigra, causing their degeneration and the resulting motor symptoms. 3,4In contrast, DLB presents with α-syn inclusions predominantly in cortical neurons, leading to cognitive and psychiatric manifestations. 5MSA, on the other hand, features α-syn accumulation in both neurons and oligodendrocytes, known as glial cytoplasmic inclusions (GCIs), which affect various brain regions, including the basal ganglia, cerebellum, and brainstem. 6ditionally, PSP and CBD share the presence of tau protein pathology, but their regional distribution differs.PSP is characterized by the accumulation of hyperphosphorylated tau proteins in both neurons and glial cells, primarily affecting the brainstem, basal ganglia, and cerebellum, leading to a range of motor, cognitive, and oculomotor deficits. 7In CBD, the abnormal deposition of tau proteins is more localized, predominantly affecting the frontoparietal cortex and basal ganglia asymmetrically, causing varied motor, cognitive and sensory symptoms. 8In this review, we utilize the term corticobasal syndrome (CBS) instead of CBD based on clinical assessments or diagnostic criteria employed in the included studies, as definitive postmortem confirmation of the diagnosis was not available.These differences in cellular and regional involvement of α-syn and tau proteoforms, different molecular variants of the protein such as post-translationally modified oligomers, underscore the distinct pathophysiological mechanisms at play in PD, DLB, MSA, PSP, and CBD/CBS.Despite these differences, they are frequently misdiagnosed due to overlapping clinical features. 9Recognizing these nuances is crucial for accurate diagnosis and tailored therapeutic approaches in managing these complex neurodegenerative conditions.
Extracellular vesicles (EVs) are small membrane-bound structures released by cells that play critical roles in intercellular communication and the regulation of various physiological processes.
These vesicles encapsulate a wide range of biomolecules, including proteins, lipids, and nucleic acids, which reflect the state of the parent cell. 102][13] By carrying cellstate-specific messages from the CNS to the peripheral circulation, EVs have emerged as an avenue for minimally invasive diagnostic and therapeutic approaches in neurodegenerative diseases 14 and Parkinsonian disorders. 12,15,16,17,18,19,20,21,22,23,24,25,26nce 2014, many studies have employed the approach of measuring α-syn in neuronal and oligodendroglial extracellular vesicles (nEVs and oEVs) to differentially diagnose Parkinsonian disorders from one another or from healthy controls (HCs).Two recent meta-analyses compared α-syn levels in putative "exosomes" in patients with PD, 27,28 but they had different objectives than the current meta-analysis.These meta-analyses grouped α-syn concentrations from EVs isolated from various sources, which are known to be incomparable, omitted at least six studies with CNSoriginating EV α-syn measurements in PD, and did not conduct separate analyses for patients with PD against other Parkinsonian disorders or patients with other Parkinsonian disorders against HCs.Additionally, they did not take into consideration key aspects in the literature known to affect CNS-originating EVs' downstream protein analyses. 29,30erefore, this systematic review and meta-analysis aimed to compile the evidence regarding α-syn proteoform levels in isolated nEVs and oEVs from patients with Parkinsonian disorders or HCs.Furthermore, we assessed whether demographic or clinical variables influenced nEVs and/or oEVs α-syn using meta-regressions.

| ME THODOLOGY
We conducted a systematic review, meta-analysis and metaregression according to the protocol recommended by the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocols (PRISMA).Our study only involved anonymized data, and we did not collect any personal information or perform any procedures on human subjects.Therefore, ethical approval was not necessary.We did not register the study's protocol.

| Eligibilitycriteria
The eligible studies had to assess the levels of α-syn in CNSoriginating EVs obtained from plasma, serum, or the cerebrospinal fluid in patients with PD along with at least one of the following diseases: MSA, DLB, PSP, CBS, or HCs.We excluded studies that used animals or cell lines, studies that did not include the specified diseases and studies that did not report the sample size.If α-syn levels were not included in the study, we reached out to the authors to obtain the mean ± standard deviation (SD).For studies that included longitudinal measurements or treatment interventions, we only considered the baseline assessments.For studies that included more than one cohort, all cohorts were averaged.For studies that included subgroup analyses for the diseases (e.g., PD + dementia, PD + mild-cognitive impairment, etc.,), all values were averaged and included together.

| Dataextraction
Data from eligible studies were extracted by two independent researchers (HBT and SSA) using a standardized form.Both authors checked the database for accuracy and completeness, and the extracted data included the following information: first au- TA B L E 1 Summary of demographic characteristics of patients with a Parkinsonian disorder or healthy controls (HC) in studies included in the meta-analysis and meta-regression.(Continued) obtain the information.In one study, 21 the values were given as median (IQR), and the mean and SD were estimated using established statistical formulas, 36,37 "Mean ≈ Median + 1.35 * (IQR/2)" and "SD ≈ IQR/1.35".

| Riskofbiasassessment
We assessed the quality and risk of bias of all eligible studies using the Quality Assessment for Diagnostic Accuracy Studies (QUADAS-2) criteria. 38Independent researchers (HBT and SSA) conducted the quality assessment and any discrepancies were resolved through discussion until a consensus was reached.More information about the quality assessment is available in Table S2.

| Datasynthesisandstatistics
Meta-analyses were performed in R software (version 2022.12.0+353).We examined the differences of nEVs and oEVs α-syn among patients with PD, MSA, DLB, PSP and CBS versus HCs together and separately.Pooled standardized mean differences and 95% CIs were calculated based on Cohen's d 36 using the below formula: Funnel plots, Begg's rank correlation and Egger's regression tests were used to evaluate publication bias. 39Meta-analyses were based on a random effects model with inverse-sample weighting.In cases with high heterogeneity, sensitivity analyses were conducted to repeat the analysis with the exclusion of highly influential studies.
Meta-regressions were used to investigate whether age, gender, disease duration, motor impairment and cognition severity were acting as covariates.We excluded disease stage from the meta-regression due to a significantly high correlation with motor impairment severity (r = 0.66, p < 0.001).

| RE SULTS
The systematic and hand search identified 164 articles of which 17 duplicated articles were removed.After title and abstract screening of 147 articles, 15 articles were considered potentially eligible (Figure 1).One article was removed 40 because it included only preliminary data.Three articles did not include nEVs or oEVs α-syn mean ± SD, 12,20,25 while the mean ± SD in one article was estimated using established statistical formulas (see above). 21All authors were contacted to obtain the missing information.One study with high bias quantified nEVs α-syn using dot blots without a reference to a standard calibrator and did not estimate the α-syn mean ± SD concentrations. 20As such, it was removed from the meta-analysis.In total, thirteen articles 12,15,16,17,18,19,21,22,23,24,25,26,41 were included in the meta-analysis.A detailed description of each individual study is provided in Table 1.

| Studycharacteristics
In total, the meta-analysis included 1,565 patients with PD, 206 with MSA, 21 with DLB, 172 with PSP, 152 with CBS and 967 HCs.
All patients included measurements of at least one single proteoform of α-syn (non-modified, oligomeric or phosphorylated).
Three studies quantified nEVs oligomeric α-syn in a total of 140 patients with PD and 58 HCs.Two studies quantified oEVs 17,25 α-syn in 138 patients with PD, 112 patients with MSA and 132 HCs.One study quantified 24 nEVs and oEVs phosphorylated αsyn at Ser 129 (pS129α-syn) in 46 patients with PD, 30 patients with MSA and 32 HCs.The publication year ranged from 2014 to 2023.Most studies quantified proteoforms of α-syn using electrochemiluminescence (ECLIA) or Sandwich ELISA, while two studies used a Luminex assay 12,25 and one used a Simoa assay. 41Two articles divided the PD population into subgroups of non-cognitive  The overall quality of the studies included was high (see Table S3).The risk of bias in patient selection for all included studies was unclear, as the sampling method was not reported.No study participant exclusion was determined by any of the studies.As measurement of α-syn in nEVs and oEVs is an objective measure, the index test domain was deemed to be low bias because prior knowledge of the clinical status (patients with a Parkinsonian disorder or HCs) should not influence the objective measurement of α-syn.
Most of the articles (n = 10, 76.9%) had low bias regarding Reference Standard, while three studies 12,24,25 that have used an in-house test were deemed as high risk of bias.With respect to Flow and Timing domain, all studies were deemed low risk of bias because the time interval from clinical diagnosis to the index test (i.e., α-syn proteoforms measurement) could be estimated.
It is important to note that EVs' purity, content, size and number are known to be dependent on a variety of preanalytical factors including the choice of anticoagulation agent mixed with plasma, the time of preparation, centrifugation methodology, the nature of transport, number of freeze/thaw cycles, storage conditions, temperature, and the type of collection tube, [50][51][52][53][54][55][56] which are not standardized across biobanks or clinical laboratory collection methodologies.Further, the anti-L1CAM antibody clone UJ127 has come under scrutiny for its ability to cross-react with α-syn antibodies. 57erefore, when applicable, for each of the sections below, we conducted subgroup analyses by (1) medium (plasma vs. serum), (2)   antibody clone used (i.e., anti-L1CAM UJ127 vs. 5G3), (3) analytical method, (4) center/cohort and (5) subgroup diagnoses and their variable combinations.To avoid redundancy, subgroup analyses are discussed in detail if they are applicable and show contrasting results to grouped analyses.
As heterogeneity was high in most analyses, we conducted all meta-analyses using an inverse-variance random effect model with the restricted maximum likelihood method and performed sensitivity analyses to explore the exclusion of influential studies driving the effect.

| nEVs&oEVsα-syn:Parkinsoniandisorders versushealthycontrols
We first asked whether nEVs α-syn is different in all Parkinsonian disorders versus HCs.The meta-analysis suggests that nEVs α-syn is not different in Parkinsonian disorders vs. HCs (k = 20, SMD = 0.15; 95% CI −0.15, 0.99; p = 0.047; Figure 2).Due to high heterogeneity (97.6%), we conducted sensitivity analyses to identify influential studies.Two studies were identified to be influential. 15,19Removal of the studies from the analysis did not alter the results and heterogeneity continued to be high (97.5%).As described above, the medium used for the isolation of EVs and the antibody used for the enrichment of nEVs are known to impact downstream analyses.Separating the analyses by medium and antibody did not alter the results.Lastly, and surprisingly, a meta-analysis revealed that nEVs αsyn was found to be lower in the Parkinsonian tauopathies (PSP and CBS) versus HCs (k = 4, SMD = −0.41;95% CI −0.53, 0.28; p < 0.001; Figure 4) with low heterogeneity (9.7%).
Though inspection of Funnel plots in all analyses suggested the possibility of publication bias, Begg's rank correlational and Egger's regression tests revealed no such significance (p > 0.05).In all analyses, except for PSP and CBS versus HCs, heterogeneity was deemed to be high (≥88.0%),and sensitivity analyses were conducted to exclude studies with high influence.However, removal of any such studies did not influence the significance, direction of the results or decrease the heterogeneity by a large margin, likely indicating that the results are robust.
Meta-regression analyses revealed that age, gender, disease duration, motor impairment severity and cognition were not predictors of nEVs and/or oEVs α-syn concentrations (p > 0.05).The above results suggest that combined nEVs and oEVs α-syn may be a good marker for patients with PD versus HCs.It is also possible that nEVs α-syn is a good marker for PSP and CBS versus HCs, though the interpretation is limited as the meta-analyses included only two studies from the same research group.

| nEVsα-syn:Parkinsoniansynucleinopathy versustauopathy
As α-syn pathology mainly affects one of three synucleinopathies: PD, DLB or MSA, we further investigated whether nEVs α-syn found in patients with a synucleinopathy differed from those who have an atypical Parkinsonian tauopathy (PSP or CBS).All studies included in this analysis used a similar antibody clone (anti-L1CAM clone UJ127).

F I G U R E 4
Meta-analysis for neuronal EVs (nEVs) α-synuclein in CBS and PSP versus HCs.A negative SMD indicates lower nEVs αsynuclein concentrations in CBS and PSP versus HCs.CBS, corticobasal syndrome; CI, confidence intervals; HC, healthy controls; PSP, progressive supranuclear palsy; SMD, standardized mean difference.
The meta-analysis revealed that nEVs α-syn is higher only in PD and DLB versus PSP (k = 6, SMD = 1.04; 95% CI 0.39, 1.69; p = 0.0017; Figure 6A) and CBS (k = 6, SMD = 0.87; 95% CI 0.27, 1.47; p = 0.0020; Figure 6B).This is to be expected as the above meta-analyses suggest that nEVs and oEVs α-syn is significantly higher in PD versus HCs but nEVs α-syn is significantly lower in PSP and CBS versus HCs.The heterogeneity was high, but sensitivity analyses revealed that no study was influential, indicating that the results are robust.

| oEVsα-syn:Parkinson'sdisease,multiple systematrophyandhealthycontrols
As oligodendroglial pathology and α-syn aggregates (i.e., GCIs) are exclusive to MSA, three studies have quantified the concentration of α-syn in oEVs in patients with PD, MSA or HCs in hopes of differentiating PD or HCs from MSA. 17 likely explanation for this contradiction is that two studies from one research group 17,24 reported higher oEVs α-syn concentrations in patients with MSA while one study from another research group 25 reported lower oEVs α-syn concentrations in patients with MSA.
Further, because MSA is more a severe disease than PD, and is usually distinguishable from HCs, while the accurate diagnosis of PD in early stages from HCs is more challenging, we asked whether oEVs α-syn in patients with MSA or HCs is different than patients with PD.The meta-analysis revealed no such differences when MSA and HC were combined (k = 3, SMD = 0.17; 95% CI −0.42, 0.76; p = 0.57; Figure S5).
Two studies have suggested that a combination of nEVs and oEVs α-syn is more useful than either measurement alone in separating patients with PD and HCs from MSA. 17,24 A meta-analysis combining nEVs and oEVs α-syn suggest that this is not the case (k = 8, SMD = 0.25; 95% CI −0.21, 0.71; p = 0.28; Figure 8) even when combined with Yu et al. 25 We did not perform sensitivity analyses or meta-regressions due to the small number of studies (n = 3).

| DISCUSS ION
The Third, early diagnosis provides patients and their families with the information necessary to make informed decisions about end-of-life care and to plan accordingly.Therefore the use of non-invasive or minimally invasive diagnostic approaches, such as biomarkers found in blood CNS-originating EVs, has been popular as it contains the potential to reduce patient discomfort and minimize the risks associated with more invasive procedures by providing a window into the brain's biochemistry. 12,15,17,18,19,21,23,24 this systematic review and meta-analysis, we aimed to gather conclusive evidence regarding blood-isolated nEVs and oEVsassociated α-syn proteoform levels in patients with a Parkinsonian disorder (PD, MSA, DLB, PSP or CBS) and HCs.In the 13 studies included, the results suggest that combined nEVs and oEVs α-syn in patients with PD is higher than HCs (SMD = 0.21; p = 0.021; Figure 3B), though oligodendrocytes are not known to be affected in PD and a plausible mechanism is not known.In contrast, patients with PSP and CBS had lower nEVs α-syn versus HCs (SMD = −0.41;p < 0.001; Figure 4).It is important to note, however, that the analyses with PSP and CBS versus HCs included only two studies from the same research group, 18,19 and as such, the reproducibility and generalizability are limited.Further, nEVs α-syn was found to be higher in patients with PD versus PSP (SMD = 1.04; p = 0.0017; Figure 6A) and CBS (SMD = 0.87; p = 0.0020; Figure 6B), which is supported by the findings of individual studies. 18,19,21However, the analysis included only three studies and two of those studies were from the same research group. 18,19ough oligodendrocytes α-syn pathophysiology is believed to be exclusive to MSA, the results indicate that oEVs α-syn is not different in patients with MSA versus patients with PD or HCs (Figure 7B,C).This is to be expected as only two studies from one research group 17,24 reported higher oEVs α-syn concentrations in patients with MSA while one study from another research group 25 reported lower oEVs α-syn concentrations in patients with MSA.The results suggest that the measurement of α-syn in oEVs is not reliable and is contradictory to known pathophysiological mechanisms.Lastly, these two studies from the same research group 17,24 have suggested that combining α-syn measurement in nEVs and oEVs distinguishes PD and HCs from MSA with high accuracy.However, this is contradicted by the present metaanalyses, which show that this is not the case (Figure 8).ders. 12,15,16,17,18,19,21,22,23,24,25,26,41,58,59Instead of spending time, effort and money on these measurements, investigators should be aiming at standardizing critical preanalytical factors before the isolation of CNS-originating EVs including the choice of anticoagulation agent mixed with plasma, the time of preparation, centrifugation methodology, the nature of transport, number of freeze/thaw cycles, storage conditions, temperature, and the type of collection tube [50][51][52][53][54][55][56] as well as after enrichment of CNS-originating EVs.A systematic review for L1CAM + EVs (i.e., putative nEVs) by Gomes & Witwer 30 highlighted major limitations in these studies in terms of rigorous reporting of preanalytical factors before isolation of EVs as well as issues with reproducibility across the studies.The present meta-analysis further supports these findings for the majority of analyses.
Two recent meta-analyses evaluated the levels of putative "exosomal" α-syn in patients with PD. 27,28 Nila et al. 27 suggest that α-syn is higher in patients with PD compared to HCs; however, as seen in their Figure 2., the effect is driven by a single study, 60 which is the only study that evaluated α-syn in EVs isolated from saliva.
Furthermore, this study grouped together α-syn concentrations found in bulk EV preparations with those originating from the CNS, which is problematic because they are not comparable. 29The purity, content, size, and number of EVs isolated from similar mediums (i.e., plasma vs. serum) are known to be dependent on various preanalytical factors.Lastly, their analyses for α-syn in CNS-originating EVs seen in Figure 4A include six studies, 15,17,23,26,41,61 one of which did not appear to enrich for nEVs. 61On the other hand, Valencia et al. 28 conducted a much smaller study and omitted crucial analyses for αsyn in CNS-originating EVs.
The present meta-analysis offers several advantages over previous studies conducted by Nila et al. 27 and Valencia et al. 28  Lastly, the current meta-analysis takes into account key aspects known to influence α-syn concentrations, such as the medium used for isolation (i.e., plasma vs. serum) and the antibody used for immunocapture of putative CNS-originating EVs, further enhancing the robustness of the findings.

| CON CLUS ION
The need to establish reliable and minimally invasive diagnostic biomarkers for Parkinsonian disorders is crucial for targeted interventions, improved clinical trial patient stratification and informed end-of-life care planning.Biomarkers found in blood-isolated CNS-originating EVs may provide insight into the brain's biochemistry, and thus, have become a popular source for biomarker discovery.The present metaanalysis, comprising 13 studies, suggests that nEVs α-syn may have value for differentiating patients with PD from HCs only when combined with oEVs α-syn, which contradicts the existing literature. 17,24,25rthermore, the levels of nEVs α-syn demonstrate higher values in patients with PD compared to those with PSP and CBS.It is important to note, however, that these findings are based on three studies, two of which were conducted by the same research group. 18,19On the other hand, nEVs and oEVs α-syn have been suggested to differentiate patients with PD from MSA with high accuracy, yet the present metaanalysis contradicted this particular finding.
Ultimately, this meta-analysis highlights the necessity for standardized blood collection and EV isolation techniques/methodologies and underscores the importance of exploring additional biomarkers using the same approach such as Clusterin 19 or a specific microRNA such as Linc-POU3F3, 41 as well as considering other biofluids such as cerebrospinal fluid, 62 urine, 63 skin 64 stool 65 or tears. 66 also suggests the exploration of other techniques for biomarker discovery such as protein seeding assays, 20,67 measurements of neurophysiological factors 68 or kinematic fluctuations, 69 among other potential approaches.
thor's surname, publication year, medium (plasma or serum), EV isolation methodology, antibody marker and clone for enrichment nEVs or oEVs, EV characterization and quantification methodology, analytical method, mean ± SD of α-syn proteoforms, number of patients in each group, age, female percentage, disease duration, disease stage (Hoehn & Yahr scale), motor severity (UPDRSIII or UMSARS), and cognitive score (MMSE or MoCA).If any information was missing, we contacted the corresponding author to F I G U R E 1 PRISMA flow diagram for study inclusion or exclusion strategy.

F I G U R E 6
Meta-analysis for neuronal EVs (nEVs) α-synuclein in (A) PD, MSA and DLB vs PSP and (B) PD, MSA and DLB versus CBS.A positive or negative SMD indicates higher or lower concentrations of α-synuclein, respectively.CBS, corticobasal syndrome; CI, confidence intervals; DLB, dementia with Lewy body; MSA, multiple system atrophy; PD, Parkinson's disease; PSP, progressive supranuclear palsy; SMD, standardized mean difference.F I G U R E 5 Meta-analysis for neuronal EVs (nEVs) α-synuclein in PD and DLB vs. MSA.A positive or negative SMD indicates higher or lower nEVs α-synuclein concentrations in PD or DLB versus MSA, respectively.CI, confidence intervals; DLB, dementia with Lewy body; MSA, multiple system atrophy; PD, Parkinson's disease; SMD, standardized mean difference.
accurate early diagnosis of Parkinsonian synucleinopathies: PD, MSA, and DLB and tauopathies: PSP and CBS, is crucial for several reasons.First, these disorders present with overlapping clinical symptoms despite clear differences in the pathophysiology of diseases, making accurate diagnosis challenging.This hinders the timely and accurate initiation of targeted therapeutic interventions, which may slow disease progression and improve patients' quality of life.Second, the ability to distinguish between these disorders allows for better patient stratification in clinical trials, leading to the development of more effective and tailored treatment strategies.

F I G U R E 8
Meta-analysis for neuronal and oligodendroglial EVs (oEVs) α-synuclein in MSA and HC versus PD.A positive and negative SMD indicates higher or lower concentrations of α-synuclein, respectively.CI, confidence intervals; DLB, dementia with Lewy body; HC, healthy controls; MSA, multiple system atrophy; PD, Parkinson's disease; SMD, standardized mean difference.The above results clearly illustrate the need for more standardized and rigorous methodologies across CNS-originating EVs biomarkers for the differential diagnosis of Parkinsonian disorders from one another or from HCs.Currently, the consistent failure of independent validation/replication plagues the field of biomarkers in CNS-originating EVs for Parkinsonian disor- Firstly, it includes at least six studies not evaluated or included in the earlier analyses, providing unique insights into α-syn in CNS-originating EVs.Secondly, it examines EVs from different cell types (nEVs and oEVs) across a range of Parkinsonian disorders (PD, MSA, DLB, PSP, and CBS) and HCs, offering a more comprehensive understanding.