Laura Tosatto

Kinetic model of the aggregation of alpha-synuclein provides insights into prion-like spreading

Significance Growing experimental evidence suggests that the pathological spreading of alpha-synuclein aggregates in Parkinson’s disease is mediated through a process of templated seeding whereby aggregates catalyze the conversion of soluble protein molecules into their aggregated forms. A molecular-level understanding of this process is still lacking. Here, we determine the concentrations and numbers of aggregates necessary for the effective seeding of alpha-synuclein, thus providing a quantitative framework to understand the conditions when its seeded propagation is favorable. We find that high concentrations of aggregates are needed for seeding yet that aggregates cause cytotoxicity at significantly lower concentrations. This suggests that templated seeding is unlikely to be the main mechanism of spreading in Parkinson’s disease but occurs together with oligomer-induced cellular stress. The protein alpha-synuclein (αS) self-assembles into small oligomeric species and subsequently into amyloid fibrils that accumulate and proliferate during the development of Parkinson’s disease. However, the quantitative characterization of the aggregation and spreading of αS remains challenging to achieve. Previously, we identified a conformational conversion step leading from the initially formed oligomers to more compact oligomers preceding fibril formation. Here, by a combination of single-molecule fluorescence measurements and kinetic analysis, we find that the reaction in solution involves two unimolecular structural conversion steps, from the disordered to more compact oligomers and then to fibrils, which can elongate by further monomer addition. We have obtained individual rate constants for these key microscopic steps by applying a global kinetic analysis to both the decrease in the concentration of monomeric protein molecules and the increase in oligomer concentrations over a 0.5–140-µM range of αS. The resulting explicit kinetic model of αS aggregation has been used to quantitatively explore seeding the reaction by either the compact oligomers or fibrils. Our predictions reveal that, although fibrils are more effective at seeding than oligomers, very high numbers of seeds of either type, of the order of 104, are required to achieve efficient seeding and bypass the slow generation of aggregates through primary nucleation. Complementary cellular experiments demonstrated that two orders of magnitude lower numbers of oligomers were sufficient to generate high levels of reactive oxygen species, suggesting that effective templated seeding is likely to require both the presence of template aggregates and conditions of cellular stress.

Single-molecule FRET studies on alpha-synuclein oligomerization of Parkinson’s disease genetically related mutants

Oligomers of alpha-synuclein are toxic to cells and have been proposed to play a key role in the etiopathogenesis of Parkinson’s disease. As certain missense mutations in the gene encoding for alpha-synuclein induce early-onset forms of the disease, it has been suggested that these variants might have an inherent tendency to produce high concentrations of oligomers during aggregation, although a direct experimental evidence for this is still missing. We used single-molecule Förster Resonance Energy Transfer to visualize directly the protein self-assembly process by wild-type alpha-synuclein and A53T, A30P and E46K mutants and to compare the structural properties of the ensemble of oligomers generated. We found that the kinetics of oligomer formation correlates with the natural tendency of each variant to acquire beta-sheet structure. Moreover, A53T and A30P showed significant differences in the averaged FRET efficiency of one of the two types of oligomers formed compared to the wild-type oligomers, indicating possible structural variety among the ensemble of species generated. Importantly, we found similar concentrations of oligomers during the lag-phase of the aggregation of wild-type and mutated alpha-synuclein, suggesting that the properties of the ensemble of oligomers generated during self-assembly might be more relevant than their absolute concentration for triggering neurodegeneration.

Fast Flow Microfluidics and Single-Molecule Fluorescence for the Rapid Characterization of α-Synuclein Oligomers

α-Synuclein oligomers can be toxic to cells and may be responsible for cell death in Parkinsons disease. Their typically low abundance and highly heterogeneous nature, however, make such species challenging to study using traditional biochemical techniques. By combining fast-flow microfluidics with single-molecule fluorescence, we are able to rapidly follow the process by which oligomers of αS are formed and to characterize the species themselves. We have used the technique to show that populations of oligomers with different FRET efficiencies have varying stabilities when diluted into low ionic strength solutions. Interestingly, we have found that oligomers formed early in the aggregation pathway have electrostatic repulsions that are shielded in the high ionic strength buffer and therefore dissociate when diluted into lower ionic strength solutions. This property can be used to isolate different structural groups of αS oligomers and can help to rationalize some aspects of αS amyloid fibril formation.

Single-Molecule Imaging of Individual Amyloid Protein Aggregates in Human Biofluids

The misfolding and aggregation of proteins into amyloid fibrils characterizes many neurodegenerative disorders such as Parkinson’s and Alzheimer’s diseases. We report here a method, termed SAVE (single aggregate visualization by enhancement) imaging, for the ultrasensitive detection of individual amyloid fibrils and oligomers using single-molecule fluorescence microscopy. We demonstrate that this method is able to detect the presence of amyloid aggregates of α-synuclein, tau, and amyloid-β. In addition, we show that aggregates can also be identified in human cerebrospinal fluid (CSF). Significantly, we see a twofold increase in the average aggregate concentration in CSF from Parkinson’s disease patients compared to age-matched controls. Taken together, we conclude that this method provides an opportunity to characterize the structural nature of amyloid aggregates in a key biofluid, and therefore has the potential to study disease progression in both animal models and humans to enhance our understanding of neurodegenerative disorders.

Multi-dimensional super-resolution imaging enables surface hydrophobicity mapping.

Super-resolution microscopy allows biological systems to be studied at the nanoscale, but has been restricted to providing only positional information. Here, we show that it is possible to perform multi-dimensional super-resolution imaging to determine both the position and the environmental properties of single-molecule fluorescent emitters. The method presented here exploits the solvatochromic and fluorogenic properties of nile red to extract both the emission spectrum and the position of each dye molecule simultaneously enabling mapping of the hydrophobicity of biological structures. We validated this by studying synthetic lipid vesicles of known composition. We then applied both to super-resolve the hydrophobicity of amyloid aggregates implicated in neurodegenerative diseases, and the hydrophobic changes in mammalian cell membranes. Our technique is easily implemented by inserting a transmission diffraction grating into the optical path of a localization-based super-resolution microscope, enabling all the information to be extracted simultaneously from a single image plane.

Arachidonic acid mediates the formation of abundant alpha-helical multimers of alpha-synuclein

The protein alpha-synuclein (αS) self-assembles into toxic beta-sheet aggregates in Parkinson’s disease, while it is proposed that αS forms soluble alpha-helical multimers in healthy neurons. Here, we have made αS multimers in vitro using arachidonic acid (ARA), one of the most abundant fatty acids in the brain, and characterized them by a combination of bulk experiments and single-molecule Fӧrster resonance energy transfer (sm-FRET) measurements. The data suggest that ARA-induced oligomers are alpha-helical, resistant to fibril formation, more prone to disaggregation, enzymatic digestion and degradation by the 26S proteasome, and lead to lower neuronal damage and reduced activation of microglia compared to the oligomers formed in the absence of ARA. These multimers can be formed at physiologically-relevant concentrations, and pathological mutants of αS form less multimers than wild-type αS. Our work provides strong biophysical evidence for the formation of alpha-helical multimers of αS in the presence of a biologically relevant fatty acid, which may have a protective role with respect to the generation of beta-sheet toxic structures during αS fibrillation.

Shedding light on aberrant interactions – a review of modern tools for studying protein aggregates

The link between protein aggregation and neurodegenerative disease is well established. However, given the heterogeneity of species formed during the aggregation process, it is difficult to delineate details of the molecular events involved in generating pathological aggregates from those producing soluble monomers. As aberrant aggregates are possible pharmacological targets for the treatment of neurodegenerative diseases, the need to observe and characterise soluble oligomers has pushed traditional biophysical techniques to their limits, leading to the development of a plethora of new tools capable of detecting soluble oligomers with high precision and specificity. In this review, we discuss a range of modern biophysical techniques that have been developed to study protein aggregation, and give an overview of how they have been used to understand, in detail, the aberrant aggregation of amyloidogenic proteins associated with the two most common neurodegenerative disorders, Alzheimers disease and Parkinsons disease.

Single-Molecule Characterization of the Interactions between Extracellular Chaperones and Toxic α-Synuclein Oligomers

Summary The aberrant aggregation of α-synuclein is associated with several human diseases, collectively termed the α-synucleinopathies, which includes Parkinson’s disease. The progression of these diseases is, in part, mediated by extracellular α-synuclein oligomers that may exert effects through several mechanisms, including prion-like transfer, direct cytotoxicity, and pro-inflammatory actions. In this study, we show that two abundant extracellular chaperones, clusterin and α2-macroglobulin, directly bind to exposed hydrophobic regions on the surface of α-synuclein oligomers. Using single-molecule fluorescence techniques, we found that clusterin, unlike α2-macroglobulin, exhibits differential binding to α-synuclein oligomers that may be related to structural differences between two previously described forms of αS oligomers. The binding of both chaperones reduces the ability of the oligomers to permeabilize lipid membranes and prevents an oligomer-induced increase in ROS production in cultured neuronal cells. Taken together, these data suggest a neuroprotective role for extracellular chaperones in suppressing the toxicity associated with α-synuclein oligomers.

QuantifyingCo-Oligomer Formation by α‑Synuclein

Small oligomers of the protein α-synuclein (αS) are highly cytotoxic species associated with Parkinson’s disease (PD). In addition, αS can form co-aggregates with its mutational variants and with other proteins such as amyloid-β (Aβ) and tau, which are implicated in Alzheimer’s disease. The processes of self-oligomerization and co-oligomerization of αS are, however, challenging to study quantitatively. Here, we have utilized single-molecule techniques to measure the equilibrium populations of oligomers formed in vitro by mixtures of wild-type αS with its mutational variants and with Aβ40, Aβ42, and a fragment of tau. Using a statistical mechanical model, we find that co-oligomer formation is generally more favorable than self-oligomer formation at equilibrium. Furthermore, self-oligomers more potently disrupt lipid membranes than do co-oligomers. However, this difference is sometimes outweighed by the greater formation propensity of co-oligomers when multiple proteins coexist. Our results suggest that co-oligomer formation may be important in PD and related neurodegenerative diseases.