Reconsidering α-Synuclein inclusion pathology in neurons, mice, and humans with an antibody sensing NAC engagement during α-Synuclein amyloid conversion

Abstract

Abstract The neuropathology of α-Synucleinopathies (αSP) is characterized by the spread of subcellular inclusions containing fibrils made of stacked-up α-Synuclein (α-Syn) monomers. The repetitive amyloid fold adopted by α-Syn has now been characterized at the atomic scale. However, the direct observation of amyloid α-Syn using routine immuno-histological procedures remains an issue. In particular, the widely used phosphorylated α-Syn (pS129) is only a surrogate marker of aggregation. We report here that pS129 is misleading in overexpression-based models in which it detects the overflow of soluble α-Syn while no fibrillization takes place. Further, frequent pS129-negative α-Syn inclusions are observed when seeding with preformed fibrils (PFFs) is used to force fibrillization in neurons overexpressing α-Syn. This prompted us to scrutinize a series of routine antibodies for their genuine ability to discriminate α-Syn monomers engaged or not into amyloid fibrils, irrespective of phosphorylation. We observed unexpected antibody properties and utilized these latter in neurons and brain sections to detect the loss of accessibility of interlocked NAC domains when the monomers engage into fibrils. In cultured neurons, we observed that α-Syn mutations associated with familial Parkinson’s disease (PD), or S129A which prevents α-Syn phosphorylation, are neither sufficient to trigger spontaneous α-Syn fibrillization nor aggravate the process seeded by PFFs. Further challenging the pathogenic role of fibrillization, our results also indicated that the pS129-positive α-Syn inclusions detected in the brains of mice inoculated with PFFs and of a sporadic PD patient are not exclusively amyloid. This not only points to the notion that pS129 positivity is not tantamount to amyloid α-Syn but also indicates that the experimental α-Syn inclusions seeded in mice as well as the Lewy bodies forming in PD are populated by non-amyloid species which might represent alternative proxies of the α-Syn mutations endowed with a pathogenic potential.