Monoclonal antibodies targeting cytotoxic α-synuclein oligomers: molecular properties and diagnostic potential

Abstract

Aggregation of α-synuclein (α-syn) is critical to the development of synucleinopathies such as Parkinson’s Disease (PD), yet we do not have any effective therapy against these diseases. α-syn accumulates as insoluble amyloid in intracellular Lewy Bodies (LBs) but also forms soluble αSOs (αSOs) which are thought to be even more cytotoxic than fibrils. We lack tools to detect and block the unwanted activities of these αSOs. To address this, we have raised monoclonal antibodies (mAbs) against different forms of αSOs, ranging from unmodified αSOs to species stabilized by lipid peroxidation products and polyphenols, αSOs formed by C-terminally truncated α-syn, and multivalent display of α-syn on capsid virus-like particles (cVLPs). Here we report on the general biophysical and immunohistochemical properties of 30 anti-αSO mAbs obtained in this fashion. Our analysis includes direct measurement of in-solution affinity to monomeric α-syn and αSOs using Flow-Induced Dispersion Analysis, which avoids immobilization artifacts associated with ELISA. While the mAbs generally show a preference for αSOs, they also bind fibrils, but to variable extents. Overall, we observe great diversity in the mAbs’ relative affinities for monomers and αSOs, varied requirements for the C-terminal extension of α-syn, and only a modest effect on α-syn fibrillation. Several mAbs show several orders of magnitude preference for αSOs over monomers in in-solution studies, in contrast to the commercial antibody MJF14 which only bound 10-fold more strongly to αSOs than monomeric α-syn. Gratifyingly, seven mAbs almost completely block αSO permeabilization of membrane vesicles. 16 mAbs were analyzed for binding to brain sections from rats overexpressing human α-syn in the nigro-striatal pathway, and the five most promising were further investigated for their ability to recognize α-syn aggregates in human brain tissue. All five mAbs identified α-syn-related pathologies like LBs and Lewy Neurites, as well as Glial Cytoplasmic Inclusions in postmortem brains from people diagnosed for PD, dementia with LBs and multiple system atrophy, although to different extents. We have identified three mAbs as particularly useful for pathological evaluation of postmortem brain human tissue, including the early stages of PD. Although there was no straightforward connection between the individual mAbs’ biophysical and immunohistochemical properties, it is encouraging that a large collection of mAbs able to recognize different aggregated α-syn species in vitro also hold diagnostic potential.