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Aggregation of alpha-synuclein (α-Syn) drives Parkinson’s disease (PD), although the initial stages of self-assembly and structural conversion have not been directly observed inside neurons. In this study, we tracked the intracellular conformational states of α-Syn using a single-molecule Förster resonance energy transfer (smFRET) biosensor, and we show here that α-Syn converts from a monomeric state into two distinct oligomeric states in neurons in a concentration-dependent and sequence-specific manner. Three-dimensional FRET-correlative light and electron microscopy (FRET-CLEM) revealed that intracellular seeding events occur preferentially on membrane surfaces, especially at mitochondrial membranes. The mitochondrial lipid cardiolipin triggers rapid oligomerization of A53T α-Syn, and cardiolipin is sequestered within aggregating lipid–protein complexes. Mitochondrial aggregates impair complex I activity and increase mitochondrial reactive oxygen species (ROS) generation, which accelerates the oligomerization of A53T α-Syn and causes permeabilization of mitochondrial membranes and cell death. These processes were also observed in induced pluripotent stem cell (iPSC)–derived neurons harboring A53T mutations from patients with PD. Our study highlights a mechanism of de novo α-Syn oligomerization at mitochondrial membranes and subsequent neuronal toxicity.

Protein aggregation and abnormal lipid homeostasis are both implicated in neurodegeneration through unknown mechanisms. Here we demonstrate that aggregate-membrane interaction is critical to induce a form of cell death called ferroptosis. Importantly, the aggregate-membrane interaction that drives ferroptosis depends both on the conformational structure of the aggregate, as well as the oxidation state of the lipid membrane. We generated human stem cell-derived models of synucleinopathy, characterized by the intracellular formation of a-synuclein aggregates that bind to membranes. In human iPSC-derived neurons with SNCA triplication, physiological concentrations of glutamate and dopamine induce abnormal calcium signaling owing to the incorporation of excess a-synuclein oligomers into membranes, leading to altered membrane conductance and abnormal calcium influx. a-synuclein oligomers further induce lipid peroxidation. Targeted inhibition of lipid peroxidation prevents the aggregate-membrane interaction, abolishes aberrant calcium fluxes, and restores physiological calcium signaling. Inhibition of lipid peroxidation, and reduction of iron-dependent accumulation of free radicals, further prevents oligomer-induced toxicity in human neurons. In summary, we report that peroxidation of polyunsaturated fatty acids underlies the incorporation of ß-sheet-rich aggregates into the membranes, and that additionally induces neuronal death. This suggests a role for ferroptosis in Parkinson’s disease, and highlights a new mechanism by which lipid peroxidation causes cell death.

α-Synuclein fibrils are considered a hallmark of Parkinson’s disease and other synucleinopathies. However, small oligomers that formed during the early stages of α-synuclein aggregation are thought to be the main toxic species causing disease. The formation of α-synuclein oligomers has proven difficult to follow, because of the heterogeneity and transient nature of the species formed. Here, a novel bead-based aggregation assay for monitoring the earliest stages of α-synuclein oligomerization, α-Synuclein–Confocal Nanoscanning (ASYN-CONA), is presented. The α-synuclein A91C single cysteine mutant is modified with a trifunctional chemical tag, which allows simultaneous fluorescent labeling with a green dye (tetramethylrhodamine, TMR) and attachment to microbeads. Beads with bound TMR-labeled α-synuclein are then incubated with a red dye (Cy5)-labeled variant of α-synuclein A91C, and EtOH (20%) to induce aggregation. Aggregation is detected by confocal scanning imaging, below the equatorial plane of the beads, which is known as the CONA technique. On-bead TMR-labeled α-synuclein and aggregated Cy5-labeled α-synuclein from the solution are quantitatively monitored in parallel by detection of fluorescent halos or “rings”. α-Synuclein on-bead oligomerization results in a linear increase of red bead ring fluorescence intensity over a period of 5 h. Total internal reflection fluorescence microscopy was performed on oligomers cleaved from the beads, and it revealed that (i) oligomers are sufficiently stable in solution to investigate their composition, consisting of 6 ± 1 monomer units, and (ii) oligomers containing a mean of 15 monomers bind Thioflavin-T. Various known inhibitors of α-synuclein aggregation were used to validate the ASYN-CONA assay for drug screening. Baicalein, curcumin, and rifampicin showed concentration-dependent inhibition of the α-synuclein aggregation and the IC50(the concentration of the compound at which the maxiumum intensity was reduced by one-half) were calculated.

Proteins fold into a single structural ensemble but can also misfold into many diverse structures including small aggregates and fibrils, which differ in their toxicity. The aggregate surface properties play an important role in how they interact with the plasma membrane and cellular organelles, potentially inducing cellular toxicity, however, these properties have not been measured to date due to the lack of suitable methods. Here, we used a spectrally resolved, super-resolution imaging method combined with an environmentally sensitive fluorescent dye to measure the surface hydrophobicity of individual aggregates formed by the protein α-synuclein (αS), whose aggregation is associated with Parkinson’s disease. We show that the surface of soluble oligomers is more hydrophobic than fibrils and populates a diverse range of coexisting states. Overall, our data show that the conversion of oligomers to fibril-like aggregates and ultimately to fibrils results in a reduction in both hydrophobicity and the variation in hydrophobicity. This funneling characteristic of the energy landscape explains many of the observed properties of αS aggregates and may be a common feature of aggregating proteins.

Protein aggregation is a key molecular feature underlying a wide array of neurodegenerative disorders, including Alzheimer’s and Parkinson’s diseases. To understand protein aggregation in molecular detail, it is crucial to be able to characterize the array of heterogeneous aggregates that are formed during the aggregation process. We present here a high-throughput method to detect single protein aggregates, in solution, from a label-free aggregation reaction, and we demonstrate the approach with the protein associated with Parkinson’s disease, α-synuclein. The method combines single-molecule confocal microscopy with a range of amyloid-binding extrinsic dyes, including thioflavin T and pentameric formylthiophene acetic acid, and we show that we can observe aggregates at low picomolar concentrations. The detection of individual aggregates allows us to quantify their numbers. Furthermore, we show that this approach also allows us to gain structural insights from the emission intensity of the extrinsic dyes that are bound to aggregates. By analyzing the time evolution of the aggregate populations on a single-molecule level, we then estimate the fragmentation rate of aggregates, a key process that underlies the multiplication of pathological aggregates. We additionally demonstrate that the method permits the detection of these aggregates in biological samples. The capability to detect individual protein aggregates in solution opens up a range of new applications, including exploiting the potential of this method for high-throughput screening of human biofluids for disease diagnosis and early detection.

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.

As a key player of the protein quality control network of the cell, the molecular chaperone Hsp70 inhibits the aggregation of the amyloid protein tau. To date, the mechanism of this inhibition and the tau species targeted by Hsp70 remain unknown. This is partly due to the inherent difficulty of studying amyloid aggregates because of their heterogeneous and transient nature. Here, we used ensemble and single-molecule fluorescence measurements to dissect how Hsp70 counteracts the self-assembly process of the K18 ΔK280 tau variant. We found that Hsp70 blocks the early stages of tau aggregation by suppressing the formation of tau nuclei. Additionally, Hsp70 sequesters oligomers and mature tau fibrils with nanomolar affinity into a protective complex, efficiently neutralizing their ability to damage membranes and seed further tau aggregation. Our results provide novel insights into the molecular mechanisms by which the chaperone Hsp70 counteracts the formation, propagation, and toxicity of tau aggregates.

Fluorescence microscopy enables specific visualization of proteins in living cells and has played an important role in our understanding of the protein subcellular location and function. Some proteins, however, show altered localization or function when labeled using direct fusions to fluorescent proteins, making them difficult to study in live cells. Additionally, the resolution of fluorescence microscopy is limited to ∼200 nm, which is 2 orders of magnitude larger than the size of most proteins. To circumvent these challenges, we previously developed LIVE-PAINT, a live-cell super-resolution approach that takes advantage of short interacting peptides to transiently bind a fluorescent protein to the protein-of-interest. Here, we successfully use LIVE-PAINT to image yeast membrane proteins that do not tolerate the direct fusion of a fluorescent protein by using peptide tags as short as 5-residues. We also demonstrate that it is possible to resolve multiple proteins at the nanoscale concurrently using orthogonal peptide interaction pairs.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.