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Funder: EC | EC Seventh Framework Programm | FP7 Ideas: European Research Council (FP7-IDEAS-ERC - Specific Programme: "Ideas" Implementing the Seventh Framework Programme of the European Community for Research, Technological Development and Demonstration Activities (2007 to 2013)); doi: https://doi.org/10.13039/100011199, Funder: EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council); doi: https://doi.org/10.13039/100010663, Molecular chaperones contribute to the maintenance of cellular protein homoeostasis through assisting de novo protein folding and preventing amyloid formation. Chaperones of the Hsp70 family can further disaggregate otherwise irreversible aggregate species such as α-synuclein fibrils, which accumulate in Parkinson’s disease. However, the mechanisms and kinetics of this key functionality are only partially understood. Here, we combine microfluidic measurements with chemical kinetics to study α-synuclein disaggregation. We show that Hsc70 together with its co-chaperones DnaJB1 and Apg2 can completely reverse α-synuclein aggregation back to its soluble monomeric state. This reaction proceeds through first-order kinetics where monomer units are removed directly from the fibril ends with little contribution from intermediate fibril fragmentation steps. These findings extend our mechanistic understanding of the role of chaperones in the suppression of amyloid proliferation and in aggregate clearance, and inform on possibilities and limitations of this strategy in the development of therapeutics against synucleinopathies.

Microcapsules and microgels consisting of macromolecular networks have received increasing attention due to their biomedical and pharmaceutical applications. Protein microgels and in particular silk-based microcapsules have desirable properties due to their biocompatibility and lack of toxicity. Typically such structures formed through emulsion templating are spherical in geometry due to interfacial tension. However, approaches to synthesis particles with more complex and non-spherical geometries are sought due to their packing properties and cargo release characteristics. Here, we describe a droplet-microfluidic strategy for generating asymmetric tubular-like microgels from reconstituted silk fibroin; a major component of native silk. It was determined using fluorescence microscopy, that the shear stress within the microchannel promotes surface protein aggregation, resulting in the asymmetric morphology of the microgels. Moreover, the structural transition that the protein undergoes was confirmed using FTIR. Crucially, the core of the microgels remains liquid, while the surface has fully aggregated into a fibrillar network. Additionally, we show that microgel morphology could be controlled by varying the dispersed to continuous phase flow rates, while it was determined that the radius of curvature of the asymmetric microgels is correlated to the wall shear stress. By comparing the surface fluorescence intensity of the microgels as a function of radius of curvature, the effect of the shear stress on the amount of aggregation could be quantified. Finally, the potential use of these asymmetric microgels as carriers of cargo molecules is showcased. As the core of the microgel remains liquid but the shell has gelled, this approach is highly suitable for the storage of bio-active cargo molecules such as antibodies, making such a delivery system attractive in the context of biomedical and pharmaceutical applications.

Funder: RCUK | Medical Research Council (MRC); doi: https://doi.org/10.13039/501100000265, Funder: Alzheimer's Society; doi: https://doi.org/10.13039/501100000320, Funder: Alzheimer's Research UK (ARUK); doi: https://doi.org/10.13039/501100002283, Soluble aggregates of the microtubule-associated protein tau have been challenging to assemble and characterize, despite their important role in the development of tauopathies. We found that sequential hyperphosphorylation by protein kinase A in conjugation with either glycogen synthase kinase 3β or stress activated protein kinase 4 enabled recombinant wild-type tau of isoform 0N4R to spontaneously polymerize into small amorphous aggregates in vitro. We employed tandem mass spectrometry to determine the phosphorylation sites, high-resolution native mass spectrometry to measure the degree of phosphorylation, and super-resolution microscopy and electron microscopy to characterize the morphology of aggregates formed. Functionally, compared with the unmodified aggregates, which require heparin induction to assemble, these self-assembled hyperphosphorylated tau aggregates more efficiently disrupt membrane bilayers and induce Toll-like receptor 4-dependent responses in human macrophages. Together, our results demonstrate that hyperphosphorylated tau aggregates are potentially damaging to cells, suggesting a mechanism for how hyperphosphorylation could drive neuroinflammation in tauopathies.

Funder: Royal Society; doi: https://doi.org/10.13039/501100000288, Soluble α-synuclein aggregates varying in size, structure, and morphology have been closely linked to neuronal death in Parkinson’s disease. However, the heterogeneity of different co-existing aggregate species makes it hard to isolate and study their individual toxic properties. Here, we show a reliable non-perturbative method to separate a heterogeneous mixture of protein aggregates by size. We find that aggregates of wild-type α-synuclein smaller than 200 nm in length, formed during an in vitro aggregation reaction, cause inflammation and permeabilization of single-liposome membranes and that larger aggregates are less toxic. Studying soluble aggregates extracted from post-mortem human brains also reveals that these aggregates are similar in size and structure to the smaller aggregates formed in aggregation reactions in the test tube. Furthermore, we find that the soluble aggregates present in Parkinson’s disease brains are smaller, largely less than 100 nm, and more inflammatory compared to the larger aggregates present in control brains. This study suggests that the small non-fibrillar α-synuclein aggregates are the critical species driving neuroinflammation and disease progression.

Significant efforts have been devoted in the last twenty years to developing compounds that can interfere with the aggregation pathways of proteins related to misfolding disorders, including Alzheimer’s and Parkinson’s diseases. However, no disease-modifying drug has become available for clinical use to date for these conditions. One of the main reasons for this failure is the incomplete knowledge of the molecular mechanisms underlying the process by which small molecules interact with protein aggregates and interfere with their aggregation pathways. Here, we leverage the single molecule morphological and chemical sensitivity of infrared nanospectroscopy to provide the first direct measurement of the structure and interaction between single Aβ42 oligomeric and fibrillar species and an aggregation inhibitor, bexarotene, which is able to prevent Aβ42 aggregation in vitro and reverses its neurotoxicity in cell and animal models of Alzheimer’s disease. Our results demonstrate that the carboxyl group of this compound interacts with Aβ42 aggregates through a single hydrogen bond. These results establish infrared nanospectroscopy as a powerful tool in structure-based drug discovery for protein misfolding diseases., Our understanding of the molecular mechanisms underlying pathological protein aggregation remains incomplete. Here, single molecule infrared nanospectroscopy (AFM-IR) offers insight into the structure of Aβ42 oligomeric and fibrillar species and their interaction with an aggregation inhibitor, paving the way for single molecule drug discovery studies.

16 pags., 6 figs., The self-assembly of α-synuclein (αS) into intraneuronal inclusion bodies is a key characteristic of Parkinson’s disease. To define the nature of the species giving rise to neuronal damage, we have investigated the mechanism of action of the main αS populations that have been observed to form progressively during fibril growth. The αS fibrils release soluble prefibrillar oligomeric species with cross-β structure and solvent-exposed hydrophobic clusters. αS prefibrillar oligomers are efficient in crossing and permeabilize neuronal membranes, causing cellular insults. Short fibrils are more neurotoxic than long fibrils due to the higher proportion of fibrillar ends, resulting in a rapid release of oligomers. The kinetics of released αS oligomers match the observed kinetics of toxicity in cellular systems. In addition to previous evidence that αS fibrils can spread in different brain areas, our in vitro results reveal that αS fibrils can also release oligomeric species responsible for an immediate dysfunction of the neurons in the vicinity of these species., This research was supported by the University of Florence (Fondi Ateneo to F.C. and C.C.), the Ministry of Education, Universities and Research of Italy (Progetto Dipartimento di Eccellenza “Gender Medicine” to C.C.), Parkinson’s UK (G-1508 to S.W.C. and C.M.D.), the Center for Misfolding Diseases of the University of Cambridge (S.W.C. and C.M.D.), the UK Medical Research Council (MR/N000676/1 to C.M.D.), the Agency of Science, Technology and Research of Singapore (to S.W.C.), and the Ministry of Economy and Competitiveness of Spain (MINECO RYC-2012-12068 and MINECO/FEDER EU BFU2015-64119-P to N.C.).

The aggregation of α-synuclein (αS), a protein abundant at presynaptic terminals, is associated with a range of highly debilitating neurodegenerative conditions, including Parkinson’s disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). Emerging evidence indicates that the interaction of αS with lipid membranes defines both its physiological function and pathological effects. The characterisation of the modes of membrane binding by αS is therefore crucial to clarify the balance between normal and aberrant behaviour of this protein. Here we used solid-state nuclear magnetic resonance (ssNMR) spectroscopy to probe the nature of the N-terminally acetylated form of αS (NTAc-αS) bound to synaptic-like lipid vesicles. This post-translational modification is prevalent for the physiological form of αS and modulates the binding to lipid bilayers. By probing the structure, dynamics and membrane topology of NTAc-αS, we found that N-terminal acetylation does not alter significantly the conformational and topological properties of the membrane-bound state of αS, despite increasing its propensity for binding. Taken together, our data and previous characterisations of the cytosolic state of NTAc-αS clarify that the role of the N-terminal acetylation is to regulate the binding affinity of αS for synaptic vesicles without altering the structural properties of the bound state.

Funder: Centre for Misfolding Diseases, Funder: Parkinson's UK, Funder: Cambridge University | St. John's College, University of Cambridge Centre for Misfolding Diseases, The aggregation of α-synuclein (αS), a protein abundant at presynaptic terminals, is associated with a range of highly debilitating neurodegenerative conditions, including Parkinson’s disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). Emerging evidence indicates that the interaction of αS with lipid membranes defines both its physiological function and pathological effects. The characterisation of the modes of membrane binding by αS is therefore crucial to clarify the balance between normal and aberrant behaviour of this protein. Here we used solid-state nuclear magnetic resonance (ssNMR) spectroscopy to probe the nature of the N-terminally acetylated form of αS (NTAc-αS) bound to synaptic-like lipid vesicles. This post-translational modification is prevalent for the physiological form of αS and modulates the binding to lipid bilayers. By probing the structure, dynamics and membrane topology of NTAc-αS, we found that N-terminal acetylation does not alter significantly the conformational and topological properties of the membrane-bound state of αS, despite increasing its propensity for binding. Taken together, our data and previous characterisations of the cytosolic state of NTAc-αS clarify that the role of the N-terminal acetylation is to regulate the binding affinity of αS for synaptic vesicles without altering the structural properties of the bound state.