Lipids are an important factor in the disease-associated aggregation of α-synuclein. Here we develop a kinetic model that allows the determination of mechanistic details and rate constants of this process.

Objective: To develop small molecules which inhibit the key molecular mechanisms responsible for the generation of toxic alpha-synuclein oligomers in Parkinson's Disease (PD) and other synucleinopathies with precision, from in vitro to in vivo systems. Background: Oligomeric forms of alpha-synuclein have been shown to have wide-ranging neurotoxicity and underlie the onset and progression of PD. They bind to membranes, receptors and organelles, disrupt metabolic and neuronal functional pathways and ultimately cause neuronal death, but are challenging to target with conventional drug discovery approaches. Using the framework of "chemical kinetics", we mapped the mechanisms of oligomer generation: primary, lipid-induced nucleation (where lipid membranes catalyse the formation of oligomers by several monomers) and secondary nucleation (where oligomer formation is catalysed by larger alpha-synuclein aggregates). Design/Methods: Compounds were initially characterized in in vitro protein aggregation assays starting from monomeric recombinant human alphasynuclein. Compounds were then optimised for potency, oral pharmacokinetics and brain penetration. Following this optimisation, compounds were tested in a range of cellular and in vivo systems, including iPSC-derived cortical and dopaminergic neurons, and the Line-61 transgenic mouse model. Results: Our compounds were able to inhibit both of the key processes generating alpha-synuclein oligomers by >96% in vitro, and retained potency when reactions were seeded with human PD brain tissue. Efficacy was also demonstrated in iPSC derived cells treated with compound, reducing oligomers and pS129 aggregates and improving functional markers. In the Line 61 mouse model, the same compound substantially reduced pS129 aggregate levels (−44% vs vehicle) with on-target reduction of oligomers (−25% vs. vehicle) after dosing at 15 mg/kg for 10 weeks. Conclusions: Our small molecule inhibitors of alpha-synuclein oligomer generation show a robust effect across systems from in vitro, to cellular and in vivo models, demonstrating both target engagement and functional benefit in translational models. Disclosure: Dr. Thomson has received personal compensation for serving as an employee of Wren Therapeutics. Dr. Thomson has stock in Amylyx. Dr. Thomson has received intellectual property interests from a discovery or technology relating to health care. Dr. Thomson has received intellectual property interests from a discovery or technology relating to health care. Mr. Cridland has nothing to disclose. Dr. Ball has received personal compensation for serving as an employee of Wren Therapeutics Ltd. Dr. Staats has received personal compensation for serving as an employee of Wren Therapeutics Ltd. Dr. Staats has stock in Wren Therapeutics Ltd. Dr. Pandey has stock in Wren Therapeutics. Dr. Castellana Cruz has received personal compensation in the range of $5,000-$9,999 for serving as a Consultant for Wren Therapeutics. Dr. Pisani has received personal compensation in the range of $5,000-$9,999 for serving as a Consultant for Wren Therapeutics. Dr. Meisl has received personal compensation for serving as an employee of Wren therapeutics. Dr. Meisl has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Fluidic Analytics. The institution of Dr. Meisl has received research support from ERC. Dr. Meisl has received intellectual property interests from a discovery or technology relating to health care. Dr. Meisl has received publishing royalties from a publication relating to health care. Dr. Kitchen-Smith has received personal compensation for serving as an employee of Wren Therapeutics. Dr. Yang has nothing to disclose. Dr. Mannini has received personal compensation for serving as an employee of Wren therapeutics. Dr. Pollack has nothing to disclose. Dr. Rajah has received personal compensation for serving as an employee of Wren Therapeutics. Dr. Brewerton has received personal compensation for serving as an employee of Wren Therapeutics. Dr. Habchi has stock in Wren Therapeutics. Dr. Habchi has received intellectual property interests from a discovery or technology relating to health care. Dr. Plowright has received personal compensation for serving as an employee of Wren Therapeutics.

Alzheimer's disease is characterized by the presence in the brain of amyloid plaques formed by the aberrant deposition of the amyloid-β peptide (Aβ). Since many vitamins are dysregulated in this disease, we explored whether these molecules contribute to the protein homeostasis system by modulating Aβ aggregation. By screening 18 fat-soluble and water-soluble vitamin metabolites, we found that retinoic acid and α-tocopherol, two metabolites of vitamin A and vitamin E, respectively, affect Aβ aggregation both in vitro and in a Caenorhabditis elegans model of Aβ toxicity. We then show that the effects of these two vitamin metabolites in specific combinations cancel each other out, consistent with the "resilience in complexity" hypothesis, according to which the complex composition of the cellular environment could have an overall protective role against protein aggregation through the simultaneous presence of aggregation promoters and inhibitors. Taken together, these results indicate that vitamins can be added to the list of components of the protein homeostasis system that regulate protein aggregation.

Alzheimer’s disease is characterized by the presence in the brain of amyloid plaques formed by the aberrant deposition of the amyloid-β peptide (Aβ). Since many vitamins are dysregulated in this disease, we explored whether these molecules participate in protein homeostasis by modulating Aβ aggregation. By screening 18 fat-soluble and water-soluble vitamins, we found that retinoic acid and alpha-tocopherol, two metabolites of vitamin A and vitamin E, respectively, affect Aβ42 aggregation both in vitro and in a C.

Alzheimer’s disease is characterized by the presence in the brain of amyloid plaques formed by the aberrant deposition of the amyloid-β peptide (Aβ). Since many vitamins are dysregulated in this disease, we explored whether these molecules participate in protein homeostasis by modulating Aβ aggregation. By screening 18 fat-soluble and water-soluble vitamins, we found that retinoic acid and α-tocopherol, two metabolites of vitamin A and vitamin E, respectively, affect Aβ aggregation both in vitro and in a C. elegans model of Alzheimer’s disease. We also show that effects of these two vitamin metabolites in combination can cancel each other out, suggesting that the complex composition of the cellular environment could have a protective role against protein aggregation through the simultaneous presence of aggregation promoters and inhibitors. Taken together, these results indicate that vitamins and their metabolites may be added to the list of components of the quality control system that regulate protein aggregation.

The identification of effective pharmacological tools for Alzheimer's disease (AD) represents one of the main challenges for therapeutic discovery. Due to the variety of pathological processes associated with AD, a promising route for pharmacological intervention involves the development of new chemical entities that can restore cellular homeostasis. To investigate this strategy, we designed and synthetized SG2, a compound related to the thyroid hormone thyroxine, that shares a pleiotropic activity with its endogenous parent compound, including autophagic flux promotion, neuroprotection, and metabolic reprogramming. We demonstrate herein that SG2 acts in a pleiotropic manner to induce recovery in a C. elegans model of AD based on the overexpression of Aβ42 and improves learning abilities in the 5XFAD mouse model of AD. Further, in vitro ADME-Tox profiling and toxicological studies in zebrafish confirmed the low toxicity of this compound, which represents a chemical starting point for AD drug development.

The formation of amyloid deposits in human tissues is a defining feature of more than 50 medical disorders, including Alzheimer's disease. Strong genetic and histological evidence links these conditions to the process of protein aggregation, yet it has remained challenging to identify a definitive connection between aggregation and pathogenicity. Using time-resolved fluorescence microscopy of individual synthetic vesicles, we show for the Aβ42 peptide implicated in Alzheimer's disease that the disruption of lipid bilayers correlates linearly with the time course of the levels of transient oligomers generated through secondary nucleation. These findings indicate a specific role of oligomers generated through the catalytic action of fibrillar species during the protein aggregation process in driving deleterious biological function and establish a direct causative connection between amyloid formation and its pathological effects.

A thiazolidinedione or rhodanine compound or a pharmaceutically acceptable salt, tautomer, solvate, hydrate, prodrug, derivative, stereoisomer, analog or isotopically labelled derivative thereof, for use in the treatment and/or prevention of an ophthalmological condition, wherein said compound is not Pioglitazone, Rosiglitazone, Rivoglitazone, Balaglitazone or Mitoglitazone.

A thiazolidinedione or rhodanine compound or a pharmaceutically acceptable salt, tautomer, solvate, hydrate, prodrug, derivative, stereoisomer, analog or isotopically labelled derivative thereof, for use in the treatment and/or prevention of a protein misfolding disease, wherein said compound is not Pioglitazone, Rosiglitazone, Rivoglitazone, Balaglitazone or Mitoglitazone.

Nucleation of new peptide and protein aggregates on the surfaces of amyloid fibrils of the same peptide or protein has emerged in the past two decades as a major pathway for both the generation of molecular species responsible for cellular toxicity and for the autocatalytic proliferation of peptide and protein aggregates. A key question in current research is the molecular mechanism and driving forces governing such processes, known as secondary nucleation. In this context, the analogies with other self-assembling systems for which monomer-dependent secondary nucleation has been studied for more than a century provide a valuable source of inspiration. Here, we present a short overview of this background and then review recent results regarding secondary nucleation of amyloid-forming peptides and proteins, focusing in particular on the amyloid β peptide (Aβ) from Alzheimer's disease, with some examples regarding α-synuclein from Parkinson's disease. Monomer-dependent secondary nucleation of Aβ was discovered using a combination of kinetic experiments, global analysis, seeding experiments and selective isotope-enrichment, which pinpoint the monomer as the origin of new aggregates in a fibril-catalyzed reaction. Insights into driving forces are gained from variations of solution conditions, temperature and peptide sequence. Selective inhibition of secondary nucleation is explored as an effective means to limit oligomer production and toxicity. We also review experiments aimed at finding interaction partners of oligomers generated by secondary nucleation in an ongoing aggregation process. At the end of this feature article we bring forward outstanding questions and testable mechanistic hypotheses regarding monomer-dependent secondary nucleation in amyloid formation.

The aggregation of the amyloid-β (Aβ) peptide is linked to the pathogenesis of Alzheimer's disease (AD). In particular, some point mutations within Aβ are associated with early-onset familial Alzheimer's disease. Here we set out to explore how the physical properties of the altered side chains, including their sizes and charges, affect the molecular mechanisms of aggregation. We focus on Aβ42 with familial mutations-A21G (Flemish), E22K (Italian), E22G (Arctic), E22Q (Dutch), and D23N (Iowa)-which lead to similar or identical pathology with sporadic AD or severe cerebral amyloid angiopathy. Through global kinetic analysis, we find that for the E22K, E22G, E22Q, and D23N mutations, the acceleration of the overall aggregation originates primarily from the modulation of the nucleation processes, in particular secondary nucleation on the surface of existing fibrils, whereas the elongation process is not significantly affected. Remarkably, the D23 position appears to be responsible for most of the charge effects during nucleation, while the size of the side chain at the E22 position plays a more significant role than its charge. Thus, we have developed a kinetic approach to determine the nature and the magnitude of the contribution of specific residues to the rate of individual steps of the aggregation reaction, through targeted mutations and variations in ionic strength. This strategy can help rationalize the effect of some disease-related mutations as well as yield insights into the mechanism of aggregation and the transition states of the wild-type protein.

The ability to apply highly controlled electric fields within microfluidic devices is valuable as a basis for preparative and analytical processes. A challenge encountered in the context of such approaches in conductive media, including aqueous buffers, is the generation of electrolysis products at the electrode/liquid interface which can lead to contamination, perturb fluid flows and generally interfere with the measurement process. Here, we address this challenge by designing a single layer microfluidic device architecture where the electric potential is applied outside and downstream of the microfluidic device while the field is propagated back to the chip via the use of a co-flowing highly conductive electrolyte solution that forms a stable interface at the separation region of the device. The co-flowing electrolyte ensures that all the generated electrolysis products, including Joule heat and gaseous products, are flowed away from the chip without coming into contact with the analytes while the single layer fabrication process where all the structures are defined lithographically allows producing the devices in a simple yet highly reproducible manner. We demonstrate that by allowing stable and effective application of electric fields in excess of 100 V cm-1, the described platform provides the basis for rapid separation of heterogeneous mixtures of proteins and protein complexes directly in their native buffers as well as for the simultaneous quantification of their charge states. We illustrate this by probing the interactions in a mixture of an amyloid forming protein, amyloid-β, and a molecular chaperone, Brichos, known to inhibit the process of amyloid formation. The availability of a platform for applying stable electric fields and its compatibility with single-layer soft-lithography processes opens up the possibility of separating and analysing a wide range of molecules on chip, including those with similar electrophoretic mobilities.

The aggregation of the amyloid β peptide (Aβ42), which is linked to Alzheimer's disease, can be altered significantly by modulations of the peptide's intermolecular electrostatic interactions. Variations in sequence and solution conditions have been found to lead to highly variable aggregation behaviour. Here we modulate systematically the electrostatic interactions governing the aggregation kinetics by varying the ionic strength of the solution. We find that changes in the solution ionic strength induce a switch in the reaction pathway, altering the dominant mechanisms of aggregate multiplication. This strategy thereby allows us to continuously sample a large space of different reaction mechanisms and develop a minimal reaction network that unifies the experimental kinetics under a wide range of different conditions. More generally, this universal reaction network connects previously separate systems, such as charge mutants of the Aβ42 peptide, on a continuous mechanistic landscape, providing a unified picture of the aggregation mechanism of Aβ42.

Disease related mutations and environmental factors are key determinants of the aggregation mechanism of the amyloid-β peptide implicated in Alzheimer's disease. Here we present an approach to investigate these factors through acquisition of highly reproducible data and global kinetic analysis to determine the mechanistic influence of intrinsic and extrinsic factors on the Aβ aggregation network. This allows us to translate the shift in macroscopic aggregation behaviour into effects on the individual underlying microscopic steps. We apply this work-flow to the disease-associated Aβ42-A2V variant, and to a variation in pH as examples of an intrinsic and an extrinsic perturbation. In both cases, our data reveal a shift towards a mechanism in which a larger fraction of the reactive flux goes via a pathway that generates potentially toxic oligomeric species in a fibril-catalyzed reaction. This is in agreement with the finding that Aβ42-A2V leads to early-onset Alzheimer's disease and enhances neurotoxicity.

The amyloid β peptide (Aβ42), whose aggregation is associated with Alzheimer's disease, is an amphiphatic peptide with a high propensity to self-assemble. Aβ42 has a net negative charge at physiological pH and modulations of intermolecular electrostatic interactions can significantly alter its aggregation behaviour. Variations in sequence and solution conditions lead to varied macroscopic behaviour, often resulting in a number of different mechanistic explanations for the aggregation of these closely related systems. Here we alter the electrostatic interactions governing the fibril aggregation kinetics by varying the ionic strength over an order of magnitude, which allows us to sample the space of different reaction mechanisms, and develop a minimal reaction network that explains the experimental kinetics under all the different conditions. We find that an increase in the ionic strength leads to an increased rate of surface catalysed nucleation over fragmentation and eventually to a saturation of this nucleation process. More generally, this reaction network connects previously separate systems, such as mutants of Aβ42 and the wild type, on a continuous mechanistic landscape, thereby providing a unified picture of the aggregation mechanism of Aβ42 and the means of directly comparing the effects of intrinsic modifications of the peptide to those of simple electrostatic shielding.

Reaction network starting from monomer mixtures of Aβ40 and Aβ42. Interaction at the level of primary nucleation only accelerates Aβ40 fibril formation. Separate fibrils form as secondary nucleation and elongation are highly specific.

Reaction network starting from monomer mixtures of Aβ40 and Aβ42. Interaction at the level of primary nucleation only accelerates Aβ40 fibril formation. Separate fibrils form as secondary nucleation and elongation are highly specific.

Alzheimer's disease is an increasingly prevalent neurodegenerative disorder whose pathogenesis has been associated with aggregation of the amyloid-β peptide (Aβ42). Recent studies have revealed that once Aβ42 fibrils are generated, their surfaces effectively catalyze the formation of neurotoxic oligomers. Here we show that a molecular chaperone, a human Brichos domain, can specifically inhibit this catalytic cycle and limit human Aβ42 toxicity. We demonstrate in vitro that Brichos achieves this inhibition by binding to the surfaces of fibrils, thereby redirecting the aggregation reaction to a pathway that involves minimal formation of toxic oligomeric intermediates. We verify that this mechanism occurs in living mouse brain tissue by cytotoxicity and electrophysiology experiments. These results reveal that molecular chaperones can help maintain protein homeostasis by selectively suppressing critical microscopic steps within the complex reaction pathways responsible for the toxic effects of protein misfolding and aggregation.

Significance Alzheimer's disease and several related disorders are associated with the assembly of specific proteins into ordered fibrillar aggregates. In Alzheimer's disease, the key component of pathological aggregates, the Aβ peptide, is produced from a precursor protein in variable lengths: Aβ40 is more abundant and Aβ42 more aggregation-prone. To shed light on the molecular basis of disease progression, the aggregation process has been studied in vitro. New theoretical models allow us to relate kinetic measurements to the rates of the individual processes underlying the aggregation reaction. We find that the loss of two residues in Aβ40 relative to Aβ42 significantly slows nucleation of aggregates in solution, thereby shifting the mechanism yet more strongly towards nucleation on the surface of fibrils.

Aggregation of the amyloid β-protein (Aβ) is believed to be involved in Alzheimer's disease pathogenesis. The central hydrophobic region (CHR) and the Aβ42/Aβ40 ratio play key roles in Aβ aggregation. Studying intrinsic (amino acid substitutions) and extrinsic (temperature, other molecules) factors contributes to understanding the mechanisms that cause Aβ monomers to aggregate and form oligomers and fibrils. This could facilitate the development of agents that therapeutically target toxic assemblies or prevent their formation. In our studies we mainly used a highly reproducible thioflavin T assay to probe the aggregation kinetics. Substitution of phenylalanine with leucine at position 19 in the CHR significantly affected the aggregation rate. This is seen as an extended half-time and steeper concentration dependence suggesting a change in aggregation mechanism relative to the wild type protein. We also studied co-aggregation and cross-seeding between the kinetically faster Aβ42 and the slower Aβ40. The aggregation process starting from mixed monomers displays two transitions and our data imply that there is cross-reactivity between Aβ40 and Aβ42 at the level of primary nucleation only, while fibril elongation and surface-catalysed secondary nucleation are highly specific events. In contrast, co-aggregation of Aβ42 with the slower mutant F19L only displays a single sigmoidal transition and the cross-seeding is as efficient as the self-seeding. The main reason for the discrimination in the different pathways is the length at the C-terminus rather than the difference in intrinsic aggregation rates. To further investigate the relative role of intermolecular interactions we changed the temperature to provide information on energy barriers and their enthalpic and entropic components. With an analytically solved model we could do a global fitting to estimate the activation energy for Aβ42 for the primary nucleation, secondary nucleation and elongation.