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MicroRNA‑34a‑5p expression in the plasma and in its extracellular vesicle fractions in subjects with Parkinson's disease: An exploratory study.

Author(s): Grossi I, Radeghieri A, Paolini L, Porrini V, Pilotto A, Padovani A, Marengoni A, Barbon A, Bellucci A, Pizzi M, Salvi A, De Petro G

Publication: Int J Mol Med, 2021, Vol. 47, Page 533-546

PubMed ID: 33416118 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared the size, morphology, protein and small RNA content, and miR-341-5p expression of extracellular vesicles fractions isolated by serial centrifugation from the plasma of control patients and those with Parkinson’s disease. EDTA blood was collected from 15 Parkinson’s disease patients and 14 control subjects. Plasma was obtained within 2 h of blood collection by room temperature centrifugation at 800 x g for 10 min followed by recentrifugation of the plasma twice at 2,500 x g for 15 min with low acceleration and avoiding use of brake. Plasma was then aliquoted and stored at -80°C. Aliquots from each patient were thawed and stored at 4°C or centrifuged at 800 x g for 30 min to obtain LEVs followed by centrifugation of the supernatant at 16,000 x g for 45 min to obtain MEVs and then recentrifugation of the supernatant at 100,000 x g for 2 h to obtain SEVs. SEVS were further processed by sucrose gradient. EV morphology and size were evaluated by atomic force microscopy. Protein expression of Alix, ADAM10, Actinin-4, Flotiiiin-1, TSG-101, ESCRT-1, Annexin V, and CD63 were evaluated by Western blot analysis. Small RNA and miRNA yields were evaluated by bioanalyzer and miR-341-5p was quantified by real-time RT-PCR.

   Summary of Findings:

   The LEVs, MEVs, SEVs, and sucrose gradient-purified SEVs were all round-shaped but the diameters were 200-600 nm, 50-400 nm, 30-200 nm, and 50-100 nm, respectively. Importantly, exogenous protein contaminant levels were generally low, but the SEVs had higher and more variable exogenous protein levels. As expected, the SEVs had higher levels of Alix, ADAM10, and Actinin4 compared to LEVs and MEVs but Flotillin‑1, TSG101, Annexin V, CD 63, and CD 81 were present in LEVs, MEVs, and SEVs. Importantly, ApoA1 was only found in fractions 4-5 of the SEV sucrose gradient fractions whereas the majority of the SEV markers Alix, CD63, and ADAM10 were in gradients 6-7, indicating that the HDLs and SEVs do not co-purify.

   The yield of small RNA increased with further purification of plasma from control and PD patients with the lowest yield from plasma (2.26±1.27 ng and 8.37±4.32, respectively) followed by LEVs (4.32±1.04 ng and 13.15±2.15 ng, respectively), MEVs (4.90±0.95 ng and 16.18±3.94 ng, respectively), SEVs (10.34±3.51 ng and 11.76±3.94 ng, respectively), and sucrose gradient-purified SEVs (14.51±8.03 ng and 19.51±4.10 ng, respectively). Similarly, the yield of miRNA increased with further purification of plasma from control and PD patients with the lowest miRNA yield from plasma (0.82±0.49 ng and 4.03±2.14, respectively) followed by LEVs 1.70±0.45 ng and 5.38±0.96 ng, respectively), MEVs (2.12±0.26 ng and 7.00±1.48 ng, respectively), SEVs (4.16±1.17 ng and 4.74±1.48 ng, respectively), and sucrose gradient-purified SEVs (9.87±6.72 ng and 19.51±4.10 ng, respectively). miR‑34a‑5p was detected in plasma, LEVs, MEVs, SEVs, and pure SEVs. While miR‑34a‑5p levels were comparable in plasma, LEVs, MEVs, and SEVs between PD patients and controls, they were higher in the purified SEVs from PD patients than controls (P<0.05). SEVs from patients diagnosed with PD ≤5 years ago or with Hoer & Yahr Grade 3 disease had higher levels of miR-34a-5p compared to SEVs from control patients, but no difference was found between SEVs from control patients and patients diagnosed >5 years ago or with 1.5-2.5 Grade disease. Levels of miR-34a-5p were modestly correlated with the BDI score (r=0.572, P=0.041).

   Biospecimens

   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen

   Diagnoses:

   • Parkinson's Disease
   • Not specified

   Platform:

   Analyte	Technology Platform
   RNA	Automated electrophoresis/Bioanalyzer
   Protein	Western blot
   RNA	Real-time qRT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Diagnosis/ patient condition	Control Parkinson’s Disease
   Preaquisition	Prognostic factor	H&Y grade 1.5-2.5 Beck Depression Inventory 0-25 Diagnosed <5 years ago Diagnosed >5 years ago H&Y Grade 3
   Biospecimen Aliquots and Components	Centrifugation	Different number of centrifugation steps compared
   Biospecimen Aliquots and Components	Blood and blood products	Extracellular vesicles Plasma

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