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.

Human alpha-synuclein is a 140-amino acid protein of unknown function abundantly expressed in the brain and found in Lewy bodies, a characteristic feature of Parkinson's disease. Alpha-synuclein is random in water under physiological conditions, but the first approximately 100 residues interact with SDS micelles or acidic phospholipid small unilamellar vesicles and adopt an ordered conformation. The rest of the molecule remains disordered in the bulk of the solution. The conformation of the N-terminal portion of the molecule in lipids was described as an extended helix [Ramakrishnan, M., Jensen, P. H., and Marsh, D. (2003) Biochemistry 42, 12919-12926], as two distinct alpha-helices interrupted by a two-residue break [Chandra, S., Chen, X., Rizo, J., Jahn, R., and Sudhof, T. C. (2003) J. Biol. Chem. 278, 15313-15318], or as a noncanonical conformation, the alpha11/3 helix [Bussell, R., Jr., and Eliezer, D. (2003) J. Mol. Biol. 329, 763-778]. We characterized the topology of the different regions of alpha-synuclein relative to the surface of SDS micelles using spin probe-induced broadening of NMR signals, (15)N relaxation measurements, and fluorescence spectroscopy. Our results support the presence of two N-terminal helices, positioned on the surface of the micelle and separated by a flexible stretch. The region of residues 61-95 of the protein also adopts a helical conformation, but it is partially embedded in the micelle. These results could shed some light on the role of the membrane on the aggregation process of alpha-synuclein.

Parkinson’s disease is a neurodegenerative disorder characterized by the death of dopaminergic neurons and by accumulation of alpha-synuclein (aS) aggregates in the surviving neurons. The dopamine catabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL) is a highly reactive and toxic molecule that leads to aS oligomerization by covalent modifications to lysine residues. Here we show that DOPAL-induced aS oligomer formation in neurons is associated with damage of synaptic vesicles, and with alterations in the synaptic vesicles pools. To investigate the molecular mechanism that leads to synaptic impairment, we first aimed to characterize the biochemical and biophysical properties of the aS-DOPAL oligomers; heterogeneous ensembles of macromolecules able to permeabilise cholesterol-containing lipid membranes. aS-DOPAL oligomers can induce dopamine leak in an in vitro model of synaptic vesicles and in cellular models. The dopamine released, after conversion to DOPAL in the cytoplasm, could trigger a noxious cycle that further fuels the formation of aS-DOPAL oligomers, inducing neurodegeneration.

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.

Alpha-synuclein is a natively unfolded protein widely expressed in neurons at the presynaptic level. It is linked to Parkinson's disease by two lines of evidence: amyloid fibrils of the protein accumulate in patients' brains and three genetic mutants cause autosomal dominant forms of the disease. The biological role of the protein and the mechanisms involved in the etiopathogenesis of Parkinson's disease are still unknown. Membrane binding causes the formation of an amphipathic alpha-helix, which lies on the surface without crossing the bilayer. Recent observations however reported that the application of a voltage induces a pore-like activity of alpha-synuclein. This study aims to characterize the pore forming activity of the protein starting from its monomeric form. In particular, experiments with planar lipid membranes allowed recording of conductance activity bursts with a defined and reproducible fingerprint. Additional experiments with deletion mutants and covalently bound alpha-synuclein dimers were performed to understand both pore assembly and stoichiometry. The information acquired allowed formulation of a model for pore formation at different conductance levels.

The region 35-43 of human alpha-Synuclein bound to small unilamellar lipid vesicles and to sodium dodecyl sulfate micelles has been investigated by site-directed spin labeling and electron paramagnetic resonance spectroscopy. The distance distributions obtained from spectral fitting have been analyzed on the basis of the allowed rotamers of the spin-label side-chain. Very similar results have been obtained in the two environments: an unbroken helical structure of the investigated region can be ruled out. The distance distributions are rather compatible with the presence of conformational disorder, in agreement with previous findings for micelle-bound alpha-Synuclein. The propensity for helix breaking is confirmed by molecular dynamics simulations.

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