Red Blood Cells Are the Major Source of Alpha-Synuclein in Blood

Abstract

Background: α-Synuclein has been directly linked to Parkinson’s disease etiology by mutations in and multiplication of its gene that result in a familial form of Parkinson’s disease. α-Synuclein has been detected in blood, and was found to be elevated in the blood of those individuals with the α-synuclein gene multiplication. Objective: A complete analysis of the level of α-synuclein in blood has not been performed. In this report, we determine the quantitative distribution of α-synuclein in the plasma and different cellular fractions of human blood. The levels of α-synuclein in human and mouse blood are compared. Methods: α-Synuclein levels in the different fractions of blood were quantified by a sandwich ELISA with purified recombinant α-synuclein as an assay standard. Samples were further characterized by Western immunoblot analysis. Results: More than 99% of the α-synuclein resides in the red blood cells (RBCs) with less than 1% of the total detected in the plasma, platelets and peripheral blood mononuclear cells. Conclusions: More than 99% of the α-synuclein in human blood is present in the peripheral blood cells, with the remainder in plasma. Fractionation of peripheral blood cells from human blood and quantification of α-synuclein revealed that only a very small amount of the total α-synuclein is present in peripheral blood mononuclear cells, and platelets, with the majority of α-synuclein in blood being present in RBCs. Considering the abundance and fragility of RBCs, α-synuclein levels in these other blood fractions or other bodily fluids such as cerebrospinal fluid may be artificially elevated by contamination with intact or lysed RBCs.

In Vivo Cross-linking Reveals Principally Oligomeric Forms of α-Synuclein and β-Synuclein in Neurons and Non-neural Cells

“…Common solutions for the detection problem such as overexpressing ␣Syn were avoided here whenever possible, as the natural steady-state condition of ␣Syn in such cells might be altered. Fresh erythrocytes, an abundant source of endogenous ␣Syn (13,14), express high levels of hemoglobin that can interfere with ␣Syn detection by immunoblotting due to similar gel migration. We reasoned that if erythrocytes are rich in ␣Syn, their progenitor cells may also be, and we thus identified the human erythroleukemia cell line HEL (15) as our initial system for development of a cross-linking protocol.…”

Alpha-synuclein Post-translational Modifications as Potential Biomarkers for Parkinson Disease and Other Synucleinopathies

“…Limitations of Immunoassay-based Techniques-Several factors influence the sensitivity and reproducibility of the different assays described above, among which are nonstandardized CSF and blood plasma collection and handling protocols (for example, Barbour and others have demonstrated that such samples can easily be contaminated by ␣-syn originating from lysed red blood cells and platelets (48)), the small number of samples in some studies (37), and, most important, the great diversity of capture and detection antibodies and variability in the quality and purity of the molecular standards used by different groups.…”

Potential Modes of Intercellular Alpha-Synuclein Transmission

“…Alpha synuclein (α-syn) is a 140 amino acid, soluble protein found predominantly within the central nervous system (CNS), and is enriched in the peripheral nervous system and circulating erythrocytes (Barbour et al, 2008;Theillet et al, 2016;Weinreb et al, 1996). The normal role of α-syn remains unclear, however high concentrations of the protein exist within neuronal presynaptic terminals, indicating potential function(s) in synaptic transmission (Cheng, Vivacqua and Yu, 2011).…”