Serum neuronal exosomes predict and differentiate Parkinson's disease from atypical parkinsonism

Abstract

Objective: Parkinson's disease is characterised neuropathologically by α-synuclein aggregation. Currently, there is no blood test to predict the underlying pathology or distinguish Parkinson's from atypical parkinsonian syndromes. We assessed the clinical utility of serum neuronal exosomes as biomarkers across the spectrum of Parkinson's disease, multiple system atrophy and other proteinopathies.

Methods: We performed a cross-sectional study of 664 serum samples from the Oxford, Kiel and Brescia cohorts consisting of individuals with rapid eye movement sleep behavioural disorder, Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, frontotemporal dementia, progressive supranuclear palsy, corticobasal syndrome and controls. Longitudinal samples were analysed from Parkinson's and control individuals. We developed poly(carboxybetaine-methacrylate) coated beads to isolate L1 cell adhesion molecule (L1CAM)-positive extracellular vesicles with characteristics of exosomes and used mass spectrometry or multiplexed electrochemiluminescence to measure exosomal proteins.

Results: Mean neuron-derived exosomal α-synuclein was increased by twofold in prodromal and clinical Parkinson's disease when compared with multiple system atrophy, controls or other neurodegenerative diseases. With 314 subjects in the training group and 105 in the validation group, exosomal α-synuclein exhibited a consistent performance (AUC=0.86) in separating clinical Parkinson's disease from controls across populations. Exosomal clusterin was elevated in subjects with non-α-synuclein proteinopathies. Combined neuron-derived exosomal α-synuclein and clusterin measurement predicted Parkinson's disease from other proteinopathies with AUC=0.98 and from multiple system atrophy with AUC=0.94. Longitudinal sample analysis showed that exosomal α-synuclein remains stably elevated with Parkinson's disease progression.

Conclusions: Increased α-synuclein egress in serum neuronal exosomes precedes the diagnosis of Parkinson's disease, persists with disease progression and in combination with clusterin predicts and differentiates Parkinson's disease from atypical parkinsonism.

Figure 1

Neuron-derived exosomal α-synuclein is increased across the spectrum of Lewy body pathology. (A) Boxplots of mean total α-synuclein across the spectrum of conditions with Lewy body pathology (RBD, motor PD, PDD, DLB), MSA and unrelated neurodegenerative diseases (FTD, PSP, CBS) as well as age-matched and sex-matched controls. Twofold increase in the content of α-synuclein was detected in L1CAM-positive exosomes isolated from conditions characterised by Lewy body pathology. (B) At the lowest detectable concentration (0.32 pg/mL), pSer129 α-synuclein was detected in a subgroup of PD patients that were tested (55.8%). No significant correlation was seen between total exosomal α-synuclein and either UPDRS (C), r=0.0267) or MoCA (D), r=0.0621) in PD patient samples. **P<0.01, ***P<0.001, ****P<0.0001. Mean values with IQR of exosomal markers and whisker range using SD with coefficient of 1 were used in the boxplots. CBS, corticobasal syndrome; DLB, dementia with Lewy bodies; FTD, frontotemporal dementia; PD, Parkinson’s disease; PDD, Parkinson’s disease with dementia; RBD, rapid eye movement sleep behaviour disorder.

Figure 2

Estimation of cut-off values of neuronal exosome-associated α-synuclein between cohorts. Boxplots of mean exosomal α-synuclein levels and corresponding ROC curves in training (A) and validation groups (B). When an exosomal α-synuclein cut-off value ≥14.21 pg/mL estimated from the training group (Keil and Brescia) was applied to the validation group (Oxford), assay performance analysis revealed a consistent result across populations with similar area under a curve (AUC), sensitivity (senS), specificity (spec), positive (PPV) and negative (NPV) predictive values in distinguishing clinical PD from controls as shown in panel C). HC, healthy controls; PD, Parkinson’s disease; ROC, receiver operating characteristic.

Figure 3

Neuron-derived exosomal clusterin is increased in tauopathies and when combined with α-synuclein improved the differential diagnosis. (A) Clusterin (CLU) release in serum neuronal exosomes is increased in FTD, PSP and CBS but not RBD, PD, PDD, DLB, MSA or age-matched and sex-matched controls. (B) Ratio of α-synuclein to clusterin improved the separation between Lewy body pathology and alternative proteinopathies. (C) Heatmap illustration of exosome profiles using α-Syn, Clu or α-Syn/Clu differentiating between diseases. The change in the concentration of each exosome marker was normalised to the value of HC. ROC analysis of individual markers and their ratio or linear regression analysis of composite measurements revealed an additive effect of the two biomarkers in differentiating prodromal or clinical PD from alternative proteinopathies as shown in (D, F) or MSA as shown in (E, G) Clinical PD refers to the combined group of PD and PDD. **P<0.01, ***P<0.001, ****P<0.0001. AUC, area under a curve; CBS, corticobasal syndrome; DLB, dementia with Lewy bodies; FTD, frontotemporal dementia; HC, healthy controls; MSA, multiple system atrophy; PD, Parkinson’s disease; PDD, Parkinson’s disease with dementia; PSP, progressive supranuclear gaze palsy; RBD, rapid eye movement sleep behaviour disorder; ROC, receiver operating characteristic.

Figure 4

Longitudinal analysis of exosome-associated α-synuclein and clusterin. Linear mixed model of exosomal α-synuclein (A) and clusterin (B) was fitted to the longitudinal values with time from first sampling as a covariant, and patients stratified by level at initial visit in relation to median value. Persistent separation between disease subgroups and controls but no overall significant difference in the gradient from zero was identified when comparing α-synuclein in clinical PD to control samples. Clinical PD refers to the combined group of PD and PDD. Patient characteristics and p values are summarised in C. HC, healthy controls; PD, Parkinson’s disease; PDD, Parkinson’s disease with dementia.