Analysis of the mechanisms of interaction of alpha-synuclein and membranes in cellular models of Parkinson´s Disease

Abstract

Alpha-synuclein (aSyn) plays a crucial role in Parkinson’s disease (PD) and other synucleinopathies, since it misfolds and accumulates in typical proteinaceous inclusions known as Lewy bodies. The physiological function of aSyn is thought to be related to vesicle binding and trafficking, but the precise molecular mechanisms leading to aSyn pathogenicity are still obscure. Based on studies using patient-derived brain tissue, animal models, and in vitro experiments, it has been hypothesized that aSyn pathology can spread in a prion-like manner and be transferred between interconnected neurons, contributing to the propagation of the disease. This hypothesis assumes that pathogenic aSyn may act as template to seed the aggregation of non-pathogenic protein. In the present study we investigated the molecular mechanisms involved in the interaction of aSyn with membranes and the trafficking machinery in cellular models of PD. First, we demonstrated that different species of aSyn can enter cells and form high molecular weight species. By screening a pool of small GTPases family proteins, the RABs, we found that aSyn partially colocalizes with Rab 5A and Rab 7, suggesting the involvement of the endocytic pathway and of the autophagy-lysosomal pathway in the internalization and processing of aSyn monomers. Additionally, Rab 4A seems to play an important and active role in the internalization of aSyn, although further data are needed in order to clarify the molecular mechanism and the effectors involved. We also demonstrated that membrane binding is essential for the internalization of aSyn and the consequent interaction with the selected pathways. Taken together, our results suggest that the uptake of aSyn monomers might be sufficient to initiate the spreading of aSyn pathology. We also investigated the structural features of a variant of aSyn, known as SynT, which renders aSyn more prone to aggregation in cell models. Using Nuclear Magnetic Resonance (NMR) we performed a detailed structural characterization of SynT through a systematic comparison with the unmodified aSyn. We found that the conformations adopted by SynT resemble those described for the unmodified protein. However, subtle differences were observed at the N-terminal region involving transient intra and/or intermolecular interactions. Our results indicate that disturbances in the N-terminal region of SynT, and the consequent decrease in membrane binding of the modified protein, might contribute to the pathobiology of aSyn. This work emphasizes the importance of membrane binding properties in the physiological and pathological function of aSyn, and the fundamental role of RAB proteins in the modulation of aSyn processing, clearance and spreading. Taken together, our results suggest that targeting the activity of RAB proteins may hold important therapeutic value in PD.