The role of clearance mechanisms in the kinetics of pathological protein aggregation involved in neurodegenerative diseases.

The deposition of pathological protein aggregates in the brain plays a central role in cognitive decline and structural damage associated with neurodegenerative diseases. In Alzheimer's disease, the formation of amyloid-β plaques and neurofibrillary tangles of the tau protein is associated with the appearance of symptoms and pathology. Detailed models for the specific mechanisms of aggregate formation, such as nucleation and elongation, exist for aggregation in vitro where the total protein mass is conserved. However, in vivo, an additional class of mechanisms that clear pathological species is present and is believed to play an essential role in limiting the formation of aggregates and preventing or delaying the emergence of disease. A key unanswered question in the field of neuro-degeneration is how these clearance mechanisms can be modeled and how alterations in the processes of clearance or aggregation affect the stability of the system toward aggregation. Here, we generalize classical models of protein aggregation to take into account both production of monomers and the clearance of protein aggregates. We show that, depending on the specifics of the clearance process, a critical clearance value emerges above which accumulation of aggregates does not take place. Our results show that a sudden switch from a healthy to a disease state can be caused by small variations in the efficiency of the clearance process and provide a mathematical framework to explore the detailed effects of different mechanisms of clearance on the accumulation of aggregates

Lipid vesicles trigger α-synuclein aggregation by stimulating primary nucleation.

α-Synuclein (α-syn) is a 140-residue intrinsically disordered protein that is involved in neuronal and synaptic vesicle plasticity, but its aggregation to form amyloid fibrils is the hallmark of Parkinson's disease (PD). The interaction between α-syn and lipid surfaces is believed to be a key feature for mediation of its normal function, but under other circumstances it is able to modulate amyloid fibril formation. Using a combination of experimental and theoretical approaches, we identify the mechanism through which facile aggregation of α-syn is induced under conditions where it binds a lipid bilayer, and we show that the rate of primary nucleation can be enhanced by three orders of magnitude or more under such conditions. These results reveal the key role that membrane interactions can have in triggering conversion of α-syn from its soluble state to the aggregated state that is associated with neurodegeneration and to its associated disease states.This work was supported by the UK BBSRC and the Wellcome Trust (CMD, TPJK, MV), the Frances and Augustus Newman Foundation (TPJK), Magdalene College, Cambridge (AKB) , St John’s College, Cambridge (TCTM), the Cambridge Home and EU Scholarship Scheme (GM), Elan Pharmaceuticals (CMD, TPJK, MV, CG) and the Leverhulme Trust (AKB).This is the accepted manuscript. The final version is available from NPG at http://www.nature.com/nchembio/journal/v11/n3/abs/nchembio.1750.htm

In situ kinetic measurements of α-synuclein aggregation reveal large population of short-lived oligomers.

Knowledge of the mechanisms of assembly of amyloid proteins into aggregates is of central importance in building an understanding of neurodegenerative disease. Given that oligomeric intermediates formed during the aggregation reaction are believed to be the major toxic species, methods to track such intermediates are clearly needed. Here we present a method, electron paramagnetic resonance (EPR), by which the amount of intermediates can be measured over the course of the aggregation, directly in the reacting solution, without the need for separation. We use this approach to investigate the aggregation of α-synuclein (αS), a synaptic protein implicated in Parkinson's disease and find a large population of oligomeric species. Our results show that these are primary oligomers, formed directly from monomeric species, rather than oligomers formed by secondary nucleation processes, and that they are short-lived, the majority of them dissociates rather than converts to fibrils. As demonstrated here, EPR offers the means to detect such short-lived intermediate species directly in situ. As it relies only on the change in size of the detected species, it will be applicable to a wide range of self-assembling systems, making accessible the kinetics of intermediates and thus allowing the determination of their rates of formation and conversion, key processes in the self-assembly reaction

An Environmentally Sensitive Fluorescent Dye as a Multidimensional Probe of Amyloid Formation

We have explored amyloid formation using poly(amino acid) model systems in which differences in peptide secondary structure and hydrophobicity can be introduced in a controlled manner. We show that an environmentally sensitive fluorescent dye, dapoxyl, is able to identify β-sheet structure and hydrophobic surfaces, structural features likely to be related to toxicity, as a result of changes in its excitation and emission profiles and its relative quantum yield. These results show that dapoxyl is a multidimensional probe of the time dependence of amyloid aggregation, which provides information about the presence and nature of metastable aggregation intermediates that is inaccessible to the conventional probes that rely on changes in quantum yield alone.The authors acknowledge the European Research Council (ERC), Biotechnology and Biological Sciences Research Council (BBSRC), Wellcome Trust, and Winston Churchill Foundation for financial support

Direct, high-throughput linking of single cell imaging and gene expression

Single-cell technologies have transformed our ability to dissect cellular heterogeneity by enabling measurements of individual molecular modalities, from genome and transcriptome to proteome and metabolome. However, at the single-cell level, the physical properties of cells, such as size, morphology, and mechanical state, remain largely disconnected from molecular profiling, limiting our understanding of the relationship between these aspects of cellular phenotype and gene expression. We introduce im-seq, a high-throughput, flow-based platform that integrates live-cell imaging with droplet-based mRNA sequencing at the single-cell level. By optically barcoding individual cells, im-seq enables the joint capture of physical and transcriptional profiles from single cells. We demonstrate that this multimodal approach can resolve physical and molecular features across cell lines, to reveal genes associated with phenotypic properties at unprecedented resolution. Our results establish im-seq as a versatile high-throughput framework for linking genetic information to physical properties, providing the large scale, information-dense datasets needed to power the next generation of data-driven discoveries in cell biology

Amyloid Plaques Beyond Aβ: A Survey of the Diverse Modulators of Amyloid Aggregation

Aggregation of the amyloid-β (Aβ) peptide is strongly correlated with Alzheimer’s disease (AD). Recent research has improved our understanding of the kinetics of amyloid fibril assembly and revealed new details regarding different stages in plaque formation. Presently, interest is turning toward studying this process in a holistic context, focusing on cellular components which interact with the Aβ peptide at various junctures during aggregation, from monomer to cross-β amyloid fibrils. However, even in isolation, a multitude of factors including protein purity, pH, salt content, and agitation affect Aβ fibril formation and deposition, often producing complicated and conflicting results. The failure of numerous inhibitors in clinical trials for AD suggests that a detailed examination of the complex interactions that occur during plaque formation, including binding of carbohydrates, lipids, nucleic acids, and metal ions, is important for understanding the diversity of manifestations of the disease. Unraveling how a variety of key macromolecular modulators interact with the Aβ peptide and change its aggregation properties may provide opportunities for developing therapies. Since no protein acts in isolation, the interplay of these diverse molecules may differentiate disease onset, progression, and severity, and thus are worth careful consideration

Modelling of the effect of ELMs on fuel retention at the bulk W divertor of JET

Effect of ELMs on fuel retention at the bulk W target of JET ITER-Like Wall was studied with multi-scale calculations. Plasma input parameters were taken from ELMy H-mode plasma experiment. The energetic intra-ELM fuel particles get implanted and create near-surface defects up to depths of few tens of nm, which act as the main fuel trapping sites during ELMs. Clustering of implantation-induced vacancies were found to take place. The incoming flux of inter-ELM plasma particles increases the different filling levels of trapped fuel in defects. The temperature increase of the W target during the pulse increases the fuel detrapping rate. The inter-ELM fuel particle flux refills the partially emptied trapping sites and fills new sites. This leads to a competing effect on the retention and release rates of the implanted particles. At high temperatures the main retention appeared in larger vacancy clusters due to increased clustering rate

Extrinsic Amyloid-Binding Dyes for Detection of Individual Protein Aggregates in Solution

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