Parkinson’s disease (PD) is one of the most common neurodegenerative diseases. It is characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta of the brain. Another feature is represented by the formation in these cells of inclusions called Lewy bodies (LB), principally constituted by fibrillar α-synuclein (αSyn). This protein is considered a key element in the aetiology of a group of neurodegenerative disorders termed synucleinopathies, which include PD, but the cellular and molecular mechanisms involved are not completely clear. It is established that the inflammatory process plays a crucial role in the pathogenesis and/or progression of PD; moreover, it is known that aggregated αSyn, released by neurons, activates microglia cells to produce pro-inflammatory mediators, such as IL-1β. IL-1β is one of the strongest pro-inflammatory cytokines; it is produced as an inactive mediator, and its maturation and activation requires inflammasome activation. In particular, the NLRP3 inflammasome is activated by a wide variety of stimuli, among which are crystallized and particulate material. In this work, we investigated the possibility that IL-1β production, induced by fibrillar αSyn, is involved the inflammasome activation. We demonstrated the competence of monomeric and fibrillar αSyn to induce synthesis of IL-1β, through TLR2 interaction; we found that the secretion of the mature cytokine was a peculiarity of the fibrillated protein. Moreover, we observed that the secretion of IL-1β involves NLRP3 inflammasome activation. The latter relies on the phagocytosis of fibrillar αSyn, followed by increased ROS production and cathepsin B release into the cytosol. Taken together, our data support the notion that fibrillar αSyn, likely released by neuronal degeneration, acts as an endogenous trigger inducing a strong inflammatory response in PD.

Human α-Synuclein (αSyn) is a natively unfolded protein whose aggregation into amyloid fibrils is involved in the pathology of Parkinson disease. A full comprehension of the structure and dynamics of early intermediates leading to the aggregated states is an unsolved problem of essential importance to researchers attempting to decipher the molecular mechanisms of αSyn aggregation and formation of fibrils. Traditional bulk techniques used so far to solve this problem point to a direct correlation between αSyn's unique conformational properties and its propensity to aggregate, but these techniques can only provide ensemble-averaged information for monomers and oligomers alike. They therefore cannot characterize the full complexity of the conformational equilibria that trigger the aggregation process. We applied atomic force microscopy–based single-molecule mechanical unfolding methodology to study the conformational equilibrium of human wild-type and mutant αSyn. The conformational heterogeneity of monomeric αSyn was characterized at the single-molecule level. Three main classes of conformations, including disordered and “β-like” structures, were directly observed and quantified without any interference from oligomeric soluble forms. The relative abundance of the “β-like” structures significantly increased in different conditions promoting the aggregation of αSyn: the presence of Cu2+, the pathogenic A30P mutation, and high ionic strength. This methodology can explore the full conformational space of a protein at the single-molecule level, detecting even poorly populated conformers and measuring their distribution in a variety of biologically important conditions. To the best of our knowledge, we present for the first time evidence of a conformational equilibrium that controls the population of a specific class of monomeric αSyn conformers, positively correlated with conditions known to promote the formation of aggregates. A new tool is thus made available to test directly the influence of mutations and pharmacological strategies on the conformational equilibrium of monomeric αSyn.

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Alpha-synuclein (alpha-Syn) is an abundant brain protein whose mutations have been linked to early-onset Parkinson's disease (PD). We recently demonstrated, by means of a single-molecule force spectroscopy (SMFS) methodology, that the conformational equilibrium of monomeric wild-type (WT) alpha-Syn shifts toward beta-containing structures in several unrelated conditions linked to PD pathogenicity. Herein, we follow the same methodology previously employed for WT alpha-Syn to characterize the conformational heterogeneity of pathological alpha-Syn mutants A30P, A53T, and E46K. Contrary to the bulk ensemble-averaged spectroscopies so far employed to this end by different authors, our single-molecule methodology monitored marked differences in the conformational behaviors of the mutants with respect to the WT sequence. We found that all the mutants have a much higher propensity than the WT to adopt a monomeric compact conformation that is compatible with the acquiring of beta structure. Mutants A30P and A53T show a similar conformational equilibrium that is significantly different from that of E46K. Another class of conformations, stabilized by mechanically weak interactions (MWI), shows a higher variety in the mutants than in the WT protein. In the A30P mutant these interactions are relatively stronger, and therefore the corresponding conformations are possibly more structured. The more structured and globular conformations of the mutants can explain their higher propensity to aggregate with respect to the WT.

We herein describe an Atomic Force Microscopy (AFM)-based experimental procedure which allows the simultaneous mechanical and morphological characterization of several hundred individual nanosized vesicles within the hour timescale. When deposited on a flat rigid surface from aqueous solution, vesicles are deformed by adhesion forces into oblate spheroids whose geometry is a direct consequence of their mechanical stiffness. AFM image analysis can be used to quantitatively measure the contact angle of individual vesicles, which is a sizeindependent descriptor of their deformation and, consequently, of their stiffness. The same geometrical measurements can be used to infer vesicle diameter in its original, spherical shape. We demonstrate the applicability of the proposed approach to natural vesicles obtained from different sources, recovering their size and stiffness distributions by simple AFM imaging in liquid. We show how the combined EV stiffness/size readout is able to discriminate between subpopulations of vesicular and nonvesicular objects in the same sample, and between populations of vesicles with similar sizes but different .

Schützende Osmolyte spielen eine wesentliche Rolle dabei, die Denaturierung von Proteinen lebender Organismen unter harschen Umweltbedingungen zu verhindern. Experimentelle Hinweise sprechen für einen Mechanismus der Proteinfaltungs‐Stabilisierung durch diese Moleküle, der in Einklang mit der Hypothese eines Rückgrat‐basierten osmophoben Effekts ist (ΔG=freie Enthalpie, [O]=Osmolytkonzentration, χ=Koordinate der Entfaltungsreaktion).

Familial and idiopathic Parkinson's disease (PD) is associated with the abnormal neuronal accumulation of α-synuclein (aS) leading to β-sheet-rich aggregates called Lewy Bodies (LBs). Moreover, single point mutation in aS gene and gene multiplication lead to autosomal dominant forms of PD. A connection between PD and the 14-3-3 chaperone-like proteins was recently proposed, based on the fact that some of the 14-3-3 isoforms can interact with genetic PD-associated proteins such as parkin, LRRK2 and aS and were found as components of LBs in human PD. In particular, a direct interaction between 14-3-3η and aS was reported when probed by co-immunoprecipitation from cell models, from parkinsonian brains and by surface plasmon resonance in vitro. However, the mechanisms through which 14-3-3η and aS interact in PD brains remain unclear. Herein, we show that while 14-3-3η is unable to bind monomeric aS, it interacts with aS oligomers which occur during the early stages of aS aggregation. This interaction diverts the aggregation process even when 14-3-3η is present in sub-stoichiometric amounts relative to aS. When aS level is overwhelmingly higher than that of 14-3-3η, the fibrillation process becomes a sequestration mechanism for 14-3-3η, undermining all processes governed by this protein. Using a panel of complementary techniques, we single out the stage of aggregation at which the aS/14-3-3η interaction occurs, characterize the products of the resulting processes, and show how the processes elucidated in vitro are relevant in cell models. Our findings constitute a first step in elucidating the molecular mechanism of aS/14-3-3η interaction and in understanding the critical aggregation step at which 14-3-3η has the potential to rescue aS-induced cellular toxicity.

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