Protein aggregation is a complex process resulting in the formation of heterogeneous mixtures of aggregate populations that are closely linked to neurodegenerative conditions, such as Alzheimer’s disease. Here, we find that soluble aggregates formed at different stages of the aggregation process of amyloid beta (Aβ42) induce the disruption of lipid bilayers and an inflammatory response to different extents. Further, by using gradient ultracentrifugation assay, we show that the smaller aggregates are those most potent at inducing membrane permeability and most effectively inhibited by antibodies binding to the C-terminal region of Aβ42. By contrast, we find that the larger soluble aggregates are those most effective at causing an inflammatory response in microglia cells and more effectively inhibited by antibodies targeting the N-terminal region of Aβ42. These findings suggest that different toxic mechanisms driven by different soluble aggregated species of Aβ42 may contribute to the onset and progression of Alzheimer’s disease.

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 α-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 α-synuclein oligomers into membranes, leading to altered membrane conductance and abnormal calcium influx. α-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 β-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.

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.

Soluble aggregates of amyloid-β (Aβ) have been associated with neuronal and synaptic loss in Alzheimer’s disease (AD). However, despite significant recent progress, the mechanisms by which these aggregated species contribute to disease progression are not fully determined. As the analysis of human cerebrospinal fluid (CSF) provides an accessible window into the molecular changes associated with the disease progression, we characterised soluble aggregates present in CSF samples from individuals with AD, mild cognitive impairment (MCI) and healthy controls using a range of sensitive biophysical methods. We used super-resolution imaging and atomic force microscopy to characterise the size and structure of the aggregates present in CSF and correlate this with their ability to permeabilise lipid membranes and induce an inflammatory response. We found that these aggregates are extremely heterogeneous and exist in a range of sizes, varying both structurally and in their mechanisms of toxicity during the disease progression. A higher proportion of small aggregates of Aβ that can cause membrane permeabilization are found in MCI CSF; in established AD, a higher proportion of the aggregates were larger and more prone to elicit a pro-inflammatory response in glial cells, while there was no detectable change in aggregate concentration. These results show that large aggregates, some longer than 100 nm, are present in the CSF of AD patients and suggest that different neurotoxic mechanisms are prevalent at different stages of AD. Electronic supplementary material The online version of this article (10.1186/s40478-019-0777-4) contains supplementary material, which is available to authorized users.

The aberrant misfolding and subsequent conversion of monomeric protein into amyloid aggregates characterises many neurodegenerative disorders, including Parkinson's and Alzheimer's diseases. These aggregates are highly heterogeneous in structure, generally of low abundance and typically smaller than the diffraction limit of light (≈250 nm). To overcome the challenges these characteristics pose to the study of endogenous aggregates formed in cells, we have developed a method to characterise them at the nanometre scale without the need for a conjugated fluorophore. Using a combination of DNA PAINT and an amyloid‐specific aptamer, we demonstrate that this technique is able to detect and super‐resolve a range of aggregated species, including those formed by α‐synuclein and amyloid‐β. Additionally, this method enables endogenous protein aggregates within cells to be characterised. We found that neuronal cells derived from patients with Parkinson's disease contain a larger number of protein aggregates than those from healthy controls.

To quantify and characterize the potentially toxic protein aggregates associated with neurodegenerative diseases, a high‐throughput assay based on measuring the extent of aggregate‐induced Ca2+ entry into individual lipid vesicles has been developed. This approach was implemented by tethering vesicles containing a Ca2+ sensitive fluorescent dye to a passivated surface and measuring changes in the fluorescence as a result of membrane disruption using total internal reflection microscopy. Picomolar concentrations of Aβ42 oligomers could be observed to induce Ca2+ influx, which could be inhibited by the addition of a naturally occurring chaperone and a nanobody designed to bind to the Aβ peptide. We show that the assay can be used to study aggregates from other proteins, such as α‐synuclein, and to probe the effects of complex biofluids, such as cerebrospinal fluid, and thus has wide applicability.

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.

SummaryThe 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.