A blood marker for Parkinson’s Disease: Neuronal exosome-derived α-synuclein
Summary (2 min read)
- Therefore, the correct diagnosis and appropriate therapy is still highly dependent on the professional experience of the examiner, and many epidemiological or post-mortem studies found high rates of misdiagnoses in PD     .
- An earlier detection of the disease, ideally in the prodromal phase before motor symptoms occur, is of utmost importance for the development and application of disease-modifying therapies.
- Finally, the assessment of clinical symptoms by scales is still used as primary outcome parameter in most clinical trials.
- Many studies have focused on the identification of α-syn in accessible peripheral tissues for instance biopsies of the gastrointestinal tract, skin or salivary glands     .
- Which are released extracellularly and can be isolate from plasma samples.
- There was no age distribution difference among the groups (p = 0.49).
- Mean disease duration of PD patients was 3 (1-13) years and mean clinical motor symptom score (Movement Disorder Society Unified Parkinson's Disease Rating Scale Part 3, MDS-UPDRS-III) was 26.7.
Isolation and detection of EVs from peripheral blood
- After gradual centrifugation and exosome precipitation (Fig. 1a ), the successful isolation of EVs was confirmed through immunoblotting, dynamic light scattering (DLS) and transmission electron microscopy (TEM).
- The isolation of EVs was confirmed through dot blot analyses using native plasma samples and comparing them to isolated EVs (PD#1-#2, Ctrl#1-#2) (Fig. 1b , Extended Data Fig. 1a ).
- The exosomal marker CD63 was significantly enriched in samples of EVs after normalization to total protein (Fig. 1c ).
- Further characterization of the diameter and morphology of EVs was gained by negative stain TEM and showed a homogenous preparation of EVs (Fig. 1f ).
- Analyzed samples of PD patients and controls showed no differences in mean radius distribution according to the TEM-based size distribution.
Identification of NEs from peripheral blood
- The purification of NEs (Fig. 2a ) led to a significantly increased signal of L1 cell adhesion molecule (NCAM-L1) when compared to native plasma samples and plasma-derived exosomes (PD#4-#5, Ctrl#4-#5) (Fig. 2b-c ; Extended Data Fig. 2b ).
- Unspecific binding to the used beads and/or the anti-NCAM-L1 antibody were excluded using an immunoblot approach (Extended Data Fig. 2a ).
- In addition, TEM and DLS studies of NEs revealed no significant differences between both groups.
- NEs from PD patients showed significantly increased signal intensity in dot blot analyses utilizing the structure-specific α-syn antibody (MJFR) in comparison to control NEs (PD#5-#6, Ctrl#5-#6) (Fig. 3d, e ; Extended Data Fig. 3n ).
Amplification of pathological plasma exosomal α-syn
- Seeding capacity of the detected soluble α-syn species was tested using a PMCA assay optimized for α-syn 28, 29 .
- In addition, the authors analyzed the signal intensity after incubation with the MJFR antibody of the untreated plasma samples before and after PMCA by dot blot analysis (PD#8-#9, Ctrl#8-#9) (Fig. 4c ).
- For both time points no significant signal differences between PD and control plasma samples were detected (Fig. 4c-e ).
- Individual ThioT signal curves of all analyzed samples (native plasma, EVs, NEs) of PD patients (red, n=15) and controls (grey, n=15) subjected to the sixth PMCA round are depicted in Extended Data Fig. 3g-i .
- To visualize formed α-syn structures, TEM imaging was applied on amplified α-syn conformers after six rounds of PMCA and fibrillary structures/aggregates were observed in NEs derived from PD plasma (PD#1) (Fig. 4r ).
- The results of their study clearly demonstrate that a pathological α-syn form can be extracted and amplified from NEs derived from blood plasma of PD patients.
- Interestingly, some studies detected a higher level of α-syn in NEs in PD patients by ELISA and Luminex assays or mass spectrometry and multiplexed electrochemiluminescence compared to healthy controls, which was not seen in their experiments using common standard methods like immunoblotting 20, 47, 53 .
- Taken together, their findings are based on a strict sequence and essential combination of experimental steps containing the isolation of NEs and subsequent analysis of the soluble fraction under native conditions with an antibody that detects pathological α-syn species.
- The authors could show incipient differences in MJFR antibody signal intensities between controls and PD patients for EVs after six rounds of PMCA and a substantial increase of the difference after analyzing NEs.
- In summary, the authors demonstrate for the first time that pathological α-syn detected in plasma-derived NEs can serve as a biomarker to differentiate PD patients from healthy controls.
- Further confirmation of the presence of pathological α-syn was reached by amplification and visualizing of the aggregates.
- Clinical parameters of analyzed cohorts Statistics were determined by unpaired two-tailed Student´s t-test and Fisher´s exact test, also known as Table 1.
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Q1. What are the contributions in "A blood marker for parkinson’s disease: neuronal exosome-derived α-synuclein" ?
In this paper, the potential of pathological α-synuclein originating from 20 neuron-derived exosomes from blood plasma as a possible biomarker was assessed.