Alpha-Synuclein-Specific Naturally Occurring Antibodies Inhibit Aggregation In Vitro and In Vivo

Abstract

Parkinson's disease (PD) is associated with motor and non-motor symptoms and characterized by aggregates of alpha-synuclein (αSyn). Naturally occurring antibodies (nAbs) are part of the innate immune system, produced without prior contact to their specific antigen, and polyreactive. The abundance of nAbs against αSyn is altered in patients with PD. In this work, we biophysically characterized nAbs against αSyn (nAbs-αSyn) and determined their biological effects. nAbs-αSyn were isolated from commercial intravenous immunoglobulins using column affinity purification. Biophysical properties were characterized using a battery of established in vitro assays. Biological effects were characterized in HEK293T cells transiently transfected with fluorescently tagged αSyn. Specific binding of nAbs-αSyn to monomeric αSyn was demonstrated by Dot blot, ELISA, and Surface Plasmon Resonance. nAbs-αSyn did not affect viability of HEK293T cells as reported by Cell Titer Blue and LDH Assays. nAbs-αSyn inhibited fibrillation of αSyn reported by the Thioflavin T aggregation assay. Altered fibril formation was confirmed with atomic force microscopy. In cells transfected with EGFP-tagged αSyn we observed reduced formation of aggresomes, perinuclear accumulations of αSyn aggregates. The results demonstrate that serum of healthy individuals contains nAbs that specifically bind αSyn and inhibit aggregation of αSyn in vitro. The addition of nAbs-αSyn to cultured cells affects intracellular αSyn aggregates. These findings help understanding the role of the innate immune systems for the pathogenesis of PD and suggest that systemic αSyn binding agents could potentially affect neuronal αSyn pathology.

Keywords: Parkinson’s disease; aggregation; alpha-synuclein; intravenous immunoglobulins (IVIG); naturally occurring antibodies.

Figure 1

Binding characteristics of nAbs-αSyn in dot blot, ELISA, and SPR. (a) nAbs-αSyn recognize αSyn on a dot blot: αSyn at different concentrations were spotted on a nitrocellulose membrane and then probed with flow through and nAbs-αSyn. Anti-αSyn antibody was used as a control. Although the control antibody (211) is a mouse antibody, FT and nAbs-αSyn are of human origin (see sheme). Each dot blot requires a different secondary antibody (either anti-mouse or anti-human) (b) nAbs-αSyn recognize αSyn in an ELISA: nAbs-αSyn from three different project sites were tested in an ELISA, to determine their capacity to bind to αSyn in vitro. (c–f) nAbs-αSyn recognize αSyn in SPR: SPR curves of nAbs-αSyn (e) binding to recombinant αSyn. IVIG (c) and FT (d) were used as controls. A concentration range of 39 nM–5 µM of nAbs-αSyn was applied in SPR. The binding affinity was determined from three replicates.

Figure 2

ThT fibrillation assay and atomic force microscopy. De novo fibrillation of 25 µM αSyn in the absence (a) and presence of 0.1 µM IVIG (b), FT (c) and nAbs-αSyn (d) followed by ThT fluorescence. Three replicates are shown per condition. At the end of the 60 h time course, samples were imaged via AFM (e–l). Representative overview images of the samples showing 10 × 10 µm areas (e–h) and 2 × 22 µM close-ups (i–l).

Figure 3

Effect of nAbs-αSyn on GFP-tagged αSyn in HEK293T cells. HEK293T cells were transfected with αSyn flexibily tagged by GFP, grown for 24 h and then treated with nAbs-αSyn for another 24 h. (a–d) First row, example images of cells in the following categories into which cells were manually classified: Unhealthy cells (a), cells with homogenous GFP fluorescence (b), cells with aggresomes and aggregates (c), cells with aggresomes (d). Scale bar represents 5 µm. (a–d) Second row, quantification of n = 3 experiments with at least 100 cells classified for each condition. FT: flow through. Bars represent p values from three replicates as calculated with one-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001.