Serine phosphorylation mimics of Aß form distinct, non-cross-seeding fibril morphs.

The self-assembly of amyloid-ß peptide (Aß) into fibrils and oligomers is linked to Alzheimer's disease (AD). Fibrillar aggregates in AD patient's brains contain several post-translational modifications, including phosphorylation at positions 8 and 26. These play a key role in modifying the aggregation propensity of Aß, yet how they affect the mechanism of aggregation is only poorly understood. Here we elucidate the aggregation mechanism of Aß42 peptides with phosphomimic mutations at these positions, with glutamine mimicking the size, and glutamate mimicking both the size and charge effect. We find that all variants are less aggregation-prone than wild-type Aß42 with the glutamate mutants showing the largest reduction. Secondary nucleation is the dominant nucleation route for all variants, as confirmed using seeding experiments; however, its rate is reduced by about an order of magnitude or more for all variants relative to wild-type. S26Q and S26E fibrils fail to catalyse nucleation of wild-type monomers and vice versa, while the S8 variants co-aggregate more readily with wild-type. Ultrastructural analyses by cryo-electron microscopy and small angle X-ray scattering reveal an altered structure with longer node-to-node distance and smaller cross-section dimensions of S26Q fibrils. These results imply that structural compatibility between fibrils and monomer is a key determinant in secondary nucleation, and that small modifications can alter the preferred fibril structure, and thus its potential to induce aggregation of other variants. Overall, our results indicate that phosphorylation could play a key role in controlling aggregation propensity and may lead to the formation of distinct, non-cross-seeding fibril populations.

Amyloid ß 42 fibril structure based on small-angle scattering.

Amyloid fibrils are associated with a number of neurodegenerative diseases, including fibrils of amyloid ß42 peptide (Aß42) in Alzheimer's disease. These fibrils are a source of toxicity to neuronal cells through surface-catalyzed generation of toxic oligomers. Detailed knowledge of the fibril structure may thus facilitate therapeutic development. We use small-angle scattering to provide information on the fibril cross-section dimension and shape for Aß42 fibrils prepared in aqueous phosphate buffer at pH = 7.4 and pH 8.0 under quiescent conditions at 37 °C from pure recombinant Aß42 peptide. Fitting the data using a continuum model reveals an elliptical cross-section and a peptide mass-per-unit length compatible with two filaments of two monomers, four monomers per plane. To provide a more detailed atomistic model, the data were fitted using as a starting state a high-resolution structure of the two-monomer arrangement in filaments from solid-state NMR (Protein Data Bank ID 5kk3). First, a twofold symmetric model including residues 11 to 42 of two monomers in the filament was optimized in terms of twist angle and local packing using Rosetta. A two-filament model was then built and optimized through fitting to the scattering data allowing the two N-termini in each filament to take different conformations, with the same conformation in each of the two filaments. This provides an atomistic model of the fibril with twofold rotation symmetry around the fibril axis. Intriguingly, no polydispersity as regards the number of filaments was observed in our system over separate samples, suggesting that the two-filament arrangement represents a free energy minimum for the Aß42 fibril.

On the Cluster Formation of α-Synuclein Fibrils.

The dense accumulation of α-Synuclein fibrils in neurons is considered to be strongly associated with Parkinson's disease. These intracellular inclusions, called Lewy bodies, also contain significant amounts of lipids. To better understand such accumulations, it should be important to study α-Synuclein fibril formation under conditions where the fibrils lump together, mimicking what is observed in Lewy bodies. In the present study, we have therefore investigated the overall structural arrangements of α-synuclein fibrils, formed under mildly acidic conditions, pH = 5.5, in pure buffer or in the presence of various model membrane systems, by means of small-angle neutron scattering (SANS). At this pH, α-synuclein fibrils are colloidally unstable and aggregate further into dense clusters. SANS intensities show a power law dependence on the scattering vector, q, indicating that the clusters can be described as mass fractal aggregates. The experimentally observed fractal dimension was d = 2.6 ± 0.3. We further show that this fractal dimension can be reproduced using a simple model of rigid-rod clusters. The effect of dominatingly attractive fibril-fibril interactions is discussed within the context of fibril clustering in Lewy body formation.

Surface-Catalyzed Secondary Nucleation Dominates the Generation of Toxic IAPP Aggregates.

The aggregation of the human islet amyloid polypeptide (IAPP) is associated with diabetes type II. A quantitative understanding of this connection at the molecular level requires that the aggregation mechanism of IAPP is resolved in terms of the underlying microscopic steps. Here we have systematically studied recombinant IAPP, with amidated C-terminus in oxidised form with a disulphide bond between residues 3 and 7, using thioflavin T fluorescence to monitor the formation of amyloid fibrils as a function of time and IAPP concentration. We used global kinetic analyses to connect the macroscopic measurements of aggregation to the microscopic mechanisms, and show that the generation of new aggregates is dominated by the secondary nucleation of monomers on the fibril surface. We then exposed insulinoma cells to aliquots extracted from different time points of the aggregation process, finding the highest toxicity at the midpoint of the reaction, when the secondary nucleation rate reaches its maximum. These results identify IAPP oligomers as the most cytotoxic species generated during IAPP aggregation, and suggest that compounds that target secondary nucleation of IAPP could be most effective as therapeutic candidates for diabetes type II.

Monomeric and fibrillar α-synuclein exert opposite effects on the catalytic cycle that promotes the proliferation of Aß42 aggregates.

The coaggregation of the amyloid-ß peptide (Aß) and α-synuclein is commonly observed in a range of neurodegenerative disorders, including Alzheimer's and Parkinson's diseases. The complex interplay between Aß and α-synuclein has led to seemingly contradictory results on whether α-synuclein promotes or inhibits Aß aggregation. Here, we show how these conflicts can be rationalized and resolved by demonstrating that different structural forms of α-synuclein exert different effects on Aß aggregation. Our results demonstrate that whereas monomeric α-synuclein blocks the autocatalytic proliferation of Aß42 (the 42-residue form of Aß) fibrils, fibrillar α-synuclein catalyses the heterogeneous nucleation of Aß42 aggregates. It is thus the specific balance between the concentrations of monomeric and fibrillar α-synuclein that determines the outcome of the Aß42 aggregation reaction.