Oligomers are transient toxic species in amyloidoses such as Alzheimer's disease. The binding of oligomers by human chaperone proteins has been inferred from the lack of detectable interactions with monomeric amyloid proteins and delay of fibril formation at sub-stoichiometric chaperone to monomer molar ratios. In this study, we provide direct experimental evidence for the binding of the human chaperone DNAJB6b (JB6) to amyloid peptide oligomers formed during an ongoing fibril formation process leading to the stabilization of these transient species. JB6 is a potent inhibitor of the aggregation of multiple amyloid peptides and here we observe the inhibition of the model amyloid-β (Aβ) 20-34 peptide at an astounding sub-stoichiometric 1 : 100 000 ratio of chaperone to amyloid peptide. Through microfluidic diffusional sizing, we detect an increase in the average hydrodynamic radius of JB6 when added to the supernatant of samples withdrawn from an ongoing fibril formation process, implying an interaction with transient non-monomeric Aβ20-34 and Aβ42 species, which we interpret as oligomers. Furthermore, the oligomer stability towards dissociation was studied using the same method. The results imply that JB6 stabilizes the oligomers against dissociation.

The aggregation of amyloid proteins into fibrillar and oligomeric aggregates is linked to a number of neurodegenerative diseases. While the disease onset remains elusive in many cases, an understanding of the driving forces for the aggregation may help finding possible causes. While effects on amyloid formation kinetics are more commonly studied, gaining insights into these driving forces require a thermodynamic approach with equilibrium measurements. Here we investigate the temperature dependence of the solubility of the amyloid beta peptide, A β 42, related to Alzheimer’s disease, using high-performance liquid chromatography coupled to a mass spectrometer and circular dichroism spectroscopy. Samples of 8 to 50 μM A β 42 were incubated for up to 168 h at pH 8.0 and moderate ionic strength at multiple temperatures in the range of 5 to 80 ° C. The remaining monomer concentration was measured after 48 to 168 h, with little change in between suggesting that equilibrium is approached at 168 h. The lowest solubility of 20 ± 10 nM is found around body temperature, with higher solubility at both lower and higher temperatures. This nonmonotonic temperature dependence is indicative of the hydrophobic effect being a major driving force for the fibril formation of this peptide.

Formation of new amyloid fibrils and oligomers from monomeric protein on the surfaces of existing fibrils is an important driver of many disorders such as Alzheimer's and Parkinson's diseases. The structural basis of this secondary nucleation process, however, is poorly understood. Here, we ask whether secondary nucleation sites are found predominantly at rare growth defects: irregularities in the fibril core structure incorporated during their original assembly. We first demonstrate using the specific inhibitor of secondary nucleation, Brichos, that secondary nucleation sites on Alzheimer's disease-associated fibrils composed of Aβ40 and Aβ42 peptides are rare compared to the number of protein molecules they contain. We then grow Aβ40 fibrils under conditions designed to eliminate most growth defects while leaving the regular fibril morphology unchanged, and confirm the latter using cryo-electron microscopy. We measure both the ability of these annealed fibrils to promote secondary nucleation and the stoichiometry of their secondary nucleation sites, finding that both are greatly reduced as predicted. Re-analysis of published data for other proteins suggests that fibril growth defects may also drive secondary nucleation generally across most amyloids. These findings could unlock structure-based drug design of therapeutics that aim to halt amyloid disorders by inhibiting secondary nucleation sites.

Abstract The aggregation of the tau protein into intraneuronal fibrillar tangles is closely associated with the pathology of Alzheimer’s disease. The endogenous defense system against this process includes molecular chaperones, among which DNAJB6b has emerged as a key component. Using a tau model system comprising the tau fragment 304-380C322S, which spans the amyloidogenic core of ex vivo Alzheimer’s disease fibrils, we investigated the impact of DNAJB6b on tau fibril formation. Here, we show that DNAJB6b potently delays tau aggregation by co-assembling with small tau aggregates and by binding to mature fibrils, thereby reducing their ability to catalyze further fibril growth. This interplay between tau and the chaperone results in greatly reduced fibril formation rate and a lower final fibril mass, which we interpret as increased tau solubility. Moreover, solution-state NMR spectroscopy confirms that DNAJB6b does not interact with tau monomers.

The molecular chaperone αB-crystallin is a small heat shock protein that inhibits the aggregation of, among others, Aβ42 and α-synuclein. These proteins are major hallmarks of Alzheimer's and Parkinson's disease, respectively. In order to understand the mechanism with which αB-crystallin performs its chaperone function it is essential to characterize its self-assembly in terms of aggregate size distribution, structure, and critical concentration. The size distribution of the assemblies has been widely discussed and they have been suggested to be monodisperse or polydisperse with varying size distributions covering a range of 10-40 monomers per assembly. Here, the size distribution was studied using dynamic and static light scattering, microfluidic diffusional sizing (MDS), as well as small-angle X-ray scattering (SAXS). Findings indicate that αB-crystallin has a preference toward forming spherical assemblies consisting of 18 monomers with a hydrodynamic radius of ≈7 nm after one week. SAXS data were modelled using a homogeneous sphere model with a radius of 6 nm, which is comparable to the light scattering and MDS results. 2D classes built from negative stain transmission electron microscopy images suggest that the spherical aggregates contain several smaller globular units. Furthermore, the findings show that the size of the assemblies is independent of protein concentration, supporting a strong preference for specific assembly constellations.

Aducanumab, a human IgG1 antibody with plaque-clearing effects and modest clinical benefit, binds selectively to aggregated Aβ via the N-terminal region. Yet, the molecular details of how the antibody engages Aβ1-42 fibrils remain unresolved. Using magic-angle spinning NMR, we show that binding of aducanumab preserves the overall architecture of the Aβ1-42 fibril core while inducing significant structural and dynamic perturbations in the N-terminal region. Antibody binding markedly reduces flexibility in this domain, with the appearance of side-chain resonances from residues D1, E3, and histidine (likely H6) in dipolar-based experiments. These side chains-previously observed in scalar-coupling spectra of the unbound state-indicate rigidification of residues that were dynamic. The interaction extends to S8 and Y10, indicating broader fibril engagement than the minimal epitope (residues 3 to 7) defined in fragment-based studies. Perturbations in the C-terminal segment (G37-A42) are consistent with its spatial proximity to the antibody-bound N termini of neighboring monomers. Cryo-TEM images reveal fibrils bundling in the presence of aducanumab, consistent with lateral association via antibody cross-linking, supporting a model where surface coating and steric hindrance suppress secondary nucleation. This mode of action restricts monomer access to catalytic sites on the fibril surface, resulting in partial inhibition (~threefold reduction) of secondary nucleation. The effect depends on high avidity and relatively high stoichiometry but is ultimately limited by antibody size relative to N-terminal spacing along the fibril. These findings provide atomic-level insights into aducanumab's binding mode and supply a structural framework for understanding antibody-mediated fibril recognition and for guiding next-generation therapies targeting Aβ aggregates in Alzheimer's disease.

Chaperones may retard the aggregation of other proteins and increase their solubility. An important goal is a thermodynamic understanding of such an action. Here, the chaperone DNAJB6b (JB6) is found to suppress amyloid formation of the protein α-synuclein (α-syn) leading to a reduced rate of fibril formation and an increase in apparent solubility of α-syn. These findings were reached at mildly acidic pH and with light seeding under conditions where the effect on secondary nucleation is visible. Cryo-transmission electron microscopy (cryo-TEM) imaging reveals that coaggregates of α-syn and JB6 are formed with significantly altered ultrastructure compared to both pure protein fibrils and pure chaperone aggregates. This is further supported by the formation of ThT-negative aggregates and by the depletion of JB6 from solution in the presence of α-syn. The identification of such coaggregates provides a plausible thermodynamic explanation for an increase in α-syn solubility in the presence of JB6; the reduced chemical potential of the chaperone upon formation of coaggregates can compensate for an increased chemical potential of α-syn, and the system as a whole can lower its free energy to sustain an increased free α-syn concentration.

Oligomeric assemblies consisting of only a few protein subunits are key species in the cytotoxicity of neurodegenerative disorders, such as Alzheimer's and Parkinson's diseases. Their lifetime in solution and abundance, governed by the balance of their sources and sinks, are thus important determinants of disease. While significant advances have been made in elucidating the processes that govern oligomer production, the mechanisms behind their dissociation are still poorly understood. Here, we use chemical kinetic modeling to determine the fate of oligomers formed in vitro and discuss the implications for their abundance in vivo. We discover that oligomeric species formed predominantly on fibril surfaces, a broad class which includes the bulk of oligomers formed by the key Alzheimer's disease-associated Aβ peptides, also dissociate overwhelmingly on fibril surfaces, not in solution as had previously been assumed. We monitor this "secondary nucleation in reverse" by measuring the dissociation of Aβ42 oligomers in the presence and absence of fibrils via two distinct experimental methods. Our findings imply that drugs that bind fibril surfaces to inhibit oligomer formation may also inhibit their dissociation, with important implications for rational design of therapeutic strategies for Alzheimer's and other amyloid diseases.

Shear forces affect self-assembly processes ranging from crystallization to fiber formation. Here, the effect of mild agitation on amyloid fibril formation was explored for four peptides and investigated in detail for A β 42, which is associated with Alzheimer’s disease. To gain mechanistic insights into the effect of mild agitation, nonseeded and seeded aggregation reactions were set up at various peptide concentrations with and without an inhibitor. First, an effect on fibril fragmentation was excluded by comparing the monomer-concentration dependence of aggregation kinetics under idle and agitated conditions. Second, using a secondary nucleation inhibitor, Brichos, the agitation effect on primary nucleation was decoupled from secondary nucleation. Third, an effect on secondary nucleation was established in the absence of inhibitor. Fourth, an effect on elongation was excluded by comparing the seeding potency of fibrils formed under idle or agitated conditions. We find that both primary and secondary nucleation steps are accelerated by gentle agitation. The increased shear forces facilitate both the detachment of newly formed aggregates from catalytic surfaces and the rate at which molecules are transported in the bulk solution to encounter nucleation sites on the fibril and other surfaces. Ultrastructural evidence obtained with cryogenic transmission electron microscopy and free-flow electrophoresis in microfluidics devices imply that agitation speeds up the detachment of nucleated species from the fibril surface. Our findings shed light on the aggregation mechanism and the role of detachment for efficient secondary nucleation. The results inform on how to modulate the relative importance of different microscopic steps in drug discovery and investigations.

Amyloid formation by α-synuclein (αSyn) occurs in Parkinson’s disease, multiple system atrophy, and dementia with Lewy bodies. Deciphering the residues that regulate αSyn amyloid fibril formation will not only provide mechanistic insight but may also reveal targets to prevent and treat disease. Previous investigations have identified several regions of αSyn to be important in the regulation of amyloid formation, including the non-amyloid-β component (NAC), P1 region (residues 36 to 42), and residues in the C-terminal domain. Recent studies have also indicated the importance of the N-terminal region of αSyn for both its physiological and pathological roles. Here, the role of residues 2 to 7 in the N-terminal region of αSyn is investigated in terms of their ability to regulate amyloid fibril formation in vitro and in vivo. Deletion of these residues (αSynΔN7) slows the rate of fibril formation in vitro and reduces the capacity of the protein to be recruited by wild-type (αSynWT) fibril seeds, despite cryo-EM showing a fibril structure consistent with those of full-length αSyn. Strikingly, fibril formation of αSynΔN7 is not induced by liposomes, despite the protein binding to liposomes with similar affinity to αSynWT. A Caenorhabditis elegans model also showed that αSynΔN7::YFP forms few puncta and lacks motility and lifespan defects typified by expression of αSynWT::YFP. Together, the results demonstrate the involvement of residues 2 to 7 of αSyn in amyloid formation, revealing a target for the design of amyloid inhibitors that may leave the functional role of the protein in membrane binding unperturbed.

The DNAJB6 chaperone inhibits fibril formation of aggregation-prone client peptides through interaction with aggregated and oligomeric forms of the amyloid peptides. Here, we studied the role of its C-terminal domain (CTD) using constructs comprising either the entire CTD or the first two or all four of the CTD n-strands grafted onto a scaffold protein. Each construct was expressed as WT and as a variant with alanines replacing five highly conserved and functionally important serine and threonine residues in the first n-strand. We investigated the stability, oligomerization, antiamyloid activity, and affinity for amyloid-n (An42) species using optical spectroscopy, native mass spectrometry, chemical crosslinking, and surface plasmon resonance technology. While DNAJB6 forms large and polydisperse oligomers, CTD was found to form only monomers, dimers, and tetramers of low affinity. Kinetic analyses showed a shift in inhibition mechanism. Whereas full-length DNAJB6 activity is dependent on the serine and threonine residues and efficiently inhibits primary and secondary nucleation, all CTD constructs inhibit secondary nucleation only, independently of the serine and threonine residues, although their dimerization and thermal stabilities are reduced by alanine substitution. While the full-length DNAJB6 inhibition of primary nucleation is related to its propensity to form coaggregates with An, the CTD constructs instead bind to An42 fibrils, which affects the nucleation events at the fibril surface. The retardation of secondary nucleation by DNAJB6 can thus be ascribed to the first two n-strands of its CTD, whereas the inhibition of primary nucleation is dependent on the entire protein or regions outside the CTD.

Alzheimer's disease is a neurodegenerative condition which involves heavy neuronal cell death linked to oligomers formed during the aggregation process of the amyloid β peptide 42 (Aβ42). The aggregation of Aβ42 involves both primary and secondary nucleation. Secondary nucleation dominates the generation of oligomers and involves the formation of new aggregates from monomers on catalytic fibril surfaces. Understanding the molecular mechanism of secondary nucleation may be crucial in developing a targeted cure. Here, the self-seeded aggregation of WT Aβ42 is studied using direct stochastic optical reconstruction microscopy (dSTORM) with separate fluorophores in seed fibrils and monomers. Seeded aggregation proceeds faster than nonseeded reactions because the fibrils act as catalysts. The dSTORM experiments show that monomers grow into relatively large aggregates on fibril surfaces along the length of fibrils before detaching, thus providing a direct observation of secondary nucleation and growth along the sides of fibrils. The experiments were repeated for cross-seeded reactions of the WT Aβ42 monomer with mutant Aβ42 fibrils that do not catalyze the nucleation of WT monomers. While the monomers are observed by dSTORM to interact with noncognate fibril surfaces, we fail to notice any growth along such fibril surfaces. This implies that the failure to nucleate on the cognate seeds is not a lack of monomer association but more likely a lack of structural conversion. Our findings support a templating role for secondary nucleation, which can only take place if the monomers can copy the underlying parent structure without steric clashes or other repulsive interactions between nucleating monomers.

Cryoelectron microscopy (cryo-EM) has provided unprecedented insights into amyloid fibril structures, including those associated with disease. However, these structures represent the endpoints of long assembly processes, and their relationship to fibrils formed early in assembly is unknown. Consequently, whether different fibril architectures, with potentially different pathological properties, form during assembly remains unknown. Here, we used cryo-EM to determine structures of amyloid fibrils at different times during in vitro fibrillation of a disease-related variant of human islet amyloid polypeptide (IAPP-S20G). Strikingly, the fibrils formed in the lag, growth, and plateau phases have different structures, with new forms appearing and others disappearing as fibrillation proceeds. A time course with wild-type hIAPP also shows fibrils changing with time, suggesting that this is a general property of IAPP amyloid assembly. The observation of transiently populated fibril structures has implications for understanding amyloid assembly mechanisms with potential new insights into amyloid progression in disease.

The self-assembly of the amyloid beta 42 (A beta 42) peptide is linked to Alzheimer's disease, and oligomeric intermediates are linked to neuronal cell death during the pathology of the disease. These oligomers are produced prolifically during secondary nucleation, by which the aggregation of monomers is catalyzed on fibril surfaces. Significant progress has been made in understanding the aggregation mechanism of A beta 42; still, a detailed molecular-level understanding of secondary nucleation is lacking. Here, we explore the role of four hydrophobic residues on the unstructured N-terminal region of A beta 42 in secondary nucleation. We create eight mutants with single Secondary nuclestion at flexible Nrernenal region Secondary nucleation at the WM core substitutions at one of the four positions-Ala2, Phe4, Tyr10, and Val12-to decrease the hydrophobicity at respective positions (A2T, A2S, F4A, F4S, Y10A, Y10S, V12A, and V12S) and one mutant (Y10F) to remove the polar nature of Tyr10. Kinetic analyses of aggregation data reveal that the hydrophobicity at the Nterminal region of A beta 42, especially at positions 10 and 12, affects the rate of fibril mass generated via secondary nucleation. Cryoelectron micrographs reveal that most of the mutants with lower hydrophobicity form fibrils that are markedly longer than WT A beta 42, in line with the reduced secondary nucleation rates for these peptides. The dominance of secondary nucleation, however, is still retained in the aggregation mechanism of these mutants because the rate of primary nucleation is even more reduced. This highlights that secondary nucleation is a general phenomenon that is not dependent on any one particular feature of the peptide and is rather robust to sequence perturbations.

The aggregation of the amyloid β (Aβ) peptide is one of the molecular hallmarks of Alzheimer's disease (AD). Although Aβ deposits have mostly been observed extracellularly, various studies have also reported the presence of intracellular Aβ assemblies. Because these intracellular Aβ aggregates might play a role in the onset and progression of AD, it is important to investigate their possible origins at different locations of the cell along the secretory pathway of the amyloid precursor protein, from which Aβ is derived by proteolytic cleavage. Senile plaques found in AD are largely composed of the 42-residue form of Aβ (Aβ42). Intracellularly, Aβ42 is produced in the endoplasmatic reticulum (ER) and Golgi apparatus. Since lipid bilayers have been shown to promote the aggregation of Aβ, in this study, we measure the effects of the lipid membrane composition on the in vitro aggregation kinetics of Aβ42. By using large unilamellar vesicles to model cellular membranes at different locations, including the inner and outer leaflets of the plasma membrane, late endosomes, the ER, and the Golgi apparatus, we show that Aβ42 aggregation is inhibited by the ER and Golgi model membranes. These results provide a preliminary map of the possible effects of the membrane composition in different cellular locations on Aβ aggregation and suggest the presence of an evolutionary optimization of the lipid composition to prevent the intracellular aggregation of Aβ.

Fluorescence-based single molecule techniques provide important tools towards understanding the molecular mechanism of complex neurodegenerative diseases. This requires efficient covalent attachment of fluorophores. Here we create a series of cysteine mutants (S8C, Y10C, S26C, V40C, and A42C) of Aβ42, involved in Alzheimer’s disease, based on exposed positions in the fibril structure and label them with the Alexa-fluorophores using maleimide chemistry. Direct stochastic optical reconstruction microscopy imaging shows that all the labelled mutants form fibrils that can be detected by virtue of Alexa fluorescence. Aggregation assays and cryo-electron micrographs establish that the careful choice of labelling position minimizes the perturbation of the aggregation process and fibril structure. Peptides labelled at the N-terminal region, S8C and Y10C, form fibrils independently and with wild-type. Peptides labelled at the fibril core surface, S26C, V40C and A42C, form fibrils only in mixture with wild-type peptide. This can be understood on the basis of a recent fibril model, in which S26, V40 and A42 are surface exposed in two out of four monomers per fibril plane. We provide a palette of fluorescently labelled Aβ42 peptides that can be used to gain understanding of the complex mechanisms of Aβ42 self-assembly and help to develop a more targeted approach to cure the disease.

Significance Alzheimer’s disease affects a rapidly growing number of individuals worldwide. Key unresolved questions relate to the onset and propagation of the disease, linked to the self-assembly of amyloid β peptide into fibrillar and smaller aggregates. This study investigates the propagation of aggregates of amyloid β peptide and asks whether hydrophobic molecular features observed on the fibril surface correlate with its ability to catalyze the formation of new aggregates. This question is motivated by the associated formation of intermediate forms that are toxic to neuronal cells. The results imply that surface catalysis is independent of surface details but requires that the monomers that form the new aggregate can adopt the structure of the parent aggregate without steric clashes.