In vitro models of synucleinopathies: informing on molecular mechanisms and protective strategies.

Alpha-synuclein (α-Syn) is a central player in Parkinson's disease (PD) and in a spectrum of neurodegenerative diseases collectively known as synucleinopathies. The protein was first associated with PD just over 20 years ago, when it was found to (i) be a major component of Lewy bodies and (ii) to be also associated with familial forms of PD. The characterization of α-Syn pathology has been achieved through postmortem studies of human brains. However, the identification of toxic mechanisms associated with α-Syn was only achieved through the use of experimental models. In vitro models are highly accessible, enable relatively rapid studies, and have been extensively employed to address α-Syn-associated neurodegeneration. Given the diversity of models used and the outcomes of the studies, a cumulative and comprehensive perspective emerges as indispensable to pave the way for further investigations. Here, we subdivided in vitro models of α-Syn pathology into three major types: (i) models simulating α-Syn fibrillization and the formation of different aggregated structures in vitro, (ii) models based on the intracellular expression of α-Syn, reporting on pathogenic conditions and cellular dysfunctions induced, and (iii) models using extracellular treatment with α-Syn aggregated species, reporting on sites of interaction and their downstream consequences. In summary, we review the underlying molecular mechanisms discovered and categorize protective strategies, in order to pave the way for future studies and the identification of effective therapeutic strategies. This article is part of the Special Issue "Synuclein".

The status of the terminal regions of α-synuclein in different forms of aggregates during fibrillization.

The α-synuclein (αSN) amyloid fibrillization process is known to be a crucial phenomenon associated with neuronal loss in various neurodegenerative diseases, most famously Parkinson's disease. The process involves different aggregated species and ultimately leads to formation of ß-sheet rich fibrillar structures. Despite the essential role of αSN aggregation in the pathoetiology of various neurological disorders, the characteristics of various assemblies are not fully understood. Here, we established a fluorescence-based model for studying the end-parts of αSN to decipher the structural aspects of aggregates during the fibrillization. Our model proved highly sensitive to the events at the early stage of the fibrillization process, which are hardly detectable with routine techniques. Combining fluorescent and PAGE analysis, we found different oligomeric aggregates in the nucleation phase of fibrillization with different sensitivity to SDS and different structures based on αSN termini. Moreover, we found that these oligomers are highly dynamic: after reaching peak levels during fibrillization, they decline and eventually disappear, suggesting their transformation into other αSN aggregated species. These findings shed light on the structural features of various αSN aggregates and their dynamics in synucleinopathies.

Inhibition of lysozyme fibrillation by human serum albumin nanoparticles: Possible mechanism.

Amyloid fibrillation is a prevalent phenomenon in different proteins and peptides, which results in a variety of disorders. Over the last decade, implementation of nanoparticles (NPs), with or without drugs, is considered as a promising approach to protect against the aggregation process of amyloid proteins. In this study, we investigated the effect of human serum albumin NPs (HSA NPs) on the fibrillation of Hen Egg White Lysozyme (HEWL). The results showed that HSA NPs decrease the fibrillation of HEWL in a size dependent manner. Surprisingly, despite their inhibitory effects on the formation of long fibrils, our studies revealed that the NPs do not preserve the stability of the protein's structure in denaturing conditions. In fact, different structural analysis methods revealed that in the presence of the NPs, the protein's tendency to expose hydrophobic patches increased. Therefore, it seems that HSA NPs are responsible for decrease in HEWL fibrillation by reducing its concentration and blocking hot spot regions for self-assembly via moderate interaction. Collectively, our results shed light on the impact of HSA NPs on HEWL fibrillation and open new challenges on the implications of these NPs for drug delivery purposes or direct use as therapeutic agents.

Strong interactions with polyethylenimine-coated human serum albumin nanoparticles (PEI-HSA NPs) alter α-synuclein conformation and aggregation kinetics.

The interaction between nanoparticles (NPs) and the small intrinsically disordered protein α-synuclein (αSN), whose aggregation is central in the development of Parkinson's disease, is of great relevance in biomedical applications of NPs as drug carriers. Here we showed using a combination of different techniques that αSN interacts strongly with positively charged polyethylenimine-coated human serum albumin (PEI-HSA) NPs, leading to a significant alteration in the αSN secondary structure. In contrast, the weak interactions of αSN with HSA NPs allowed αSN to remain unfolded. These different levels of interactions had different effects on αSN aggregation. While the weakly interacting HSA NPs did not alter the aggregation kinetic parameters of αSN, the rate of primary nucleation increased in the presence of PEI-HSA NPs. The aggregation rate changed in a PEI-HSA NP-concentration dependent and size independent manner and led to fibrils which were covered with small aggregates. Furthermore, PEI-HSA NPs reduced the level of membrane-perturbing oligomers and reduced oligomer toxicity in cell assays, highlighting a potential role for NPs in reducing αSN pathogenicity in vivo. Collectively, our results highlight the fact that a simple modification of NPs can strongly modulate interactions with target proteins, which may have important and positive implications in NP safety.