Molecular mechanism of α-synuclein aggregation on lipid membranes revealed.

The central hallmark of Parkinson's disease pathology is the aggregation of the α-synuclein protein, which, in its healthy form, is associated with lipid membranes. Purified monomeric α-synuclein is relatively stable in vitro, but its aggregation can be triggered by the presence of lipid vesicles. Despite this central importance of lipids in the context of α-synuclein aggregation, their detailed mechanistic role in this process has not been established to date. Here, we use chemical kinetics to develop a mechanistic model that is able to globally describe the aggregation behaviour of α-synuclein in the presence of DMPS lipid vesicles, across a range of lipid and protein concentrations. Through the application of our kinetic model to experimental data, we find that the reaction is a co-aggregation process involving both protein and lipids and that lipids promote aggregation as much by enabling fibril elongation as by enabling their initial formation. Moreover, we find that the primary nucleation of lipid-protein co-aggregates takes place not on the surface of lipid vesicles in bulk solution but at the air-water and/or plate interfaces, where lipids and proteins are likely adsorbed. Our model forms the basis for mechanistic insights, also in other lipid-protein co-aggregation systems, which will be crucial in the rational design of drugs that inhibit aggregate formation and act at the key points in the α-synuclein aggregation cascade.

A Relationship between the Structures and Neurotoxic Effects of Aß Oligomers Stabilized by Different Metal Ions.

Oligomeric assemblies of the amyloid ß peptide (Aß) have been investigated for over two decades as possible neurotoxic agents in Alzheimer's disease. However, due to their heterogeneous and transient nature, it is not yet fully established which of the structural features of these oligomers may generate cellular damage. Here, we study distinct oligomer species formed by Aß40 (the 40-residue form of Aß) in the presence of four different metal ions (Al3+, Cu2+, Fe2+, and Zn2+) and show that they differ in their structure and toxicity in human neuroblastoma cells. We then describe a correlation between the size of the oligomers and their neurotoxic activity, which provides a type of structure-toxicity relationship for these Aß40 oligomer species. These results provide insight into the possible role of metal ions in Alzheimer's disease by the stabilization of Aß oligomers.

Combinations of Vitamin A and Vitamin E Metabolites Confer Resilience against Amyloid-ß Aggregation.

Alzheimer's disease is characterized by the presence in the brain of amyloid plaques formed by the aberrant deposition of the amyloid-ß peptide (Aß). Since many vitamins are dysregulated in this disease, we explored whether these molecules contribute to the protein homeostasis system by modulating Aß 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 Aß aggregation both in vitro and in a Caenorhabditis elegans model of Aß 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.

Structure-Based Discovery of Small-Molecule Inhibitors of the Autocatalytic Proliferation of α-Synuclein Aggregates.

The presence of amyloid fibrils of α-synuclein is closely associated with Parkinson's disease and related synucleinopathies. It is still very challenging, however, to systematically discover small molecules that prevent the formation of these aberrant aggregates. Here, we describe a structure-based approach to identify small molecules that specifically inhibit the surface-catalyzed secondary nucleation step in the aggregation of α-synuclein by binding to the surface of the amyloid fibrils. The resulting small molecules are screened using a range of kinetic and thermodynamic assays for their ability to bind α-synuclein fibrils and prevent the further generation of α-synuclein oligomers. This study demonstrates that the combination of structure-based and kinetic-based drug discovery methods can lead to the identification of small molecules that selectively inhibit the autocatalytic proliferation of α-synuclein aggregates.

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.

Proliferation of Tau 304-380 Fragment Aggregates through Autocatalytic Secondary Nucleation.

The self-assembly of the protein tau into neurofibrillary tangles is one of the hallmarks of Alzheimer's disease and related tauopathies. Still, the molecular mechanism of tau aggregation is largely unknown. This problem may be addressed by systematically obtaining reproducible in vitro kinetics measurements under quiescent conditions in the absence of triggering substances. Here, we implement this strategy by developing protocols for obtaining an ultrapure tau fragment (residues 304-380 of tau441) and for performing spontaneous aggregation assays with reproducible kinetics under quiescent conditions. We are thus able to identify the mechanism of fibril formation of the tau 304-380 fragment at physiological pH using fluorescence spectroscopy and mass spectrometry. We find that primary nucleation is slow, and that secondary processes dominate the aggregation process once the initial aggregates are formed. Moreover, our results further show that secondary nucleation of monomers on fibril surfaces dominates over fragmentation of fibrils. Using separate isotopes in monomers and fibrils, through mass spectroscopy measurements, we verify the isotope composition of the intermediate oligomeric species, which reveals that these small aggregates are generated from monomer through secondary nucleation. Our results provide a framework for understanding the processes leading to tau aggregation in disease and for selecting possible tau forms as targets in the development of therapeutic interventions in Alzheimer's disease.

Two human metabolites rescue a C. elegans model of Alzheimer's disease via a cytosolic unfolded protein response.

Age-related changes in cellular metabolism can affect brain homeostasis, creating conditions that are permissive to the onset and progression of neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. Although the roles of metabolites have been extensively studied with regard to cellular signaling pathways, their effects on protein aggregation remain relatively unexplored. By computationally analysing the Human Metabolome Database, we identified two endogenous metabolites, carnosine and kynurenic acid, that inhibit the aggregation of the amyloid beta peptide (Aß) and rescue a C. elegans model of Alzheimer's disease. We found that these metabolites act by triggering a cytosolic unfolded protein response through the transcription factor HSF-1 and downstream chaperones HSP40/J-proteins DNJ-12 and DNJ-19. These results help rationalise previous observations regarding the possible anti-ageing benefits of these metabolites by providing a mechanism for their action. Taken together, our findings provide a link between metabolite homeostasis and protein homeostasis, which could inspire preventative interventions against neurodegenerative disorders.

Squalamine and Its Derivatives Modulate the Aggregation of Amyloid-ß and α-Synuclein and Suppress the Toxicity of Their Oligomers.

The aberrant aggregation of proteins is a key molecular event in the development and progression of a wide range of neurodegenerative disorders. We have shown previously that squalamine and trodusquemine, two natural products in the aminosterol class, can modulate the aggregation of the amyloid-ß peptide (Aß) and of α-synuclein (αS), which are associated with Alzheimer's and Parkinson's diseases. In this work, we expand our previous analyses to two squalamine derivatives, des-squalamine and α-squalamine, obtaining further insights into the mechanism by which aminosterols modulate Aß and αS aggregation. We then characterize the ability of these small molecules to alter the physicochemical properties of stabilized oligomeric species in vitro and to suppress the toxicity of these aggregates to varying degrees toward human neuroblastoma cells. We found that, despite the fact that these aminosterols exert opposing effects on Aß and αS aggregation under the conditions that we tested, the modifications that they induced to the toxicity of oligomers were similar. Our results indicate that the suppression of toxicity is mediated by the displacement of toxic oligomeric species from cellular membranes by the aminosterols. This study, thus, provides evidence that aminosterols could be rationally optimized in drug discovery programs to target oligomer toxicity in Alzheimer's and Parkinson's diseases.

A dopamine metabolite stabilizes neurotoxic amyloid-ß oligomers.

Aberrant soluble oligomers formed by the amyloid-ß peptide (Aß) are major pathogenic agents in the onset and progression of Alzheimer's disease. A variety of biomolecules can influence the formation of these oligomers in the brain, although their mechanisms of action are still largely unknown. Here, we studied the effects on Aß aggregation of DOPAL, a reactive catecholaldehyde intermediate of dopamine metabolism. We found that DOPAL is able to stabilize Aß oligomeric species, including dimers and trimers, that exert toxic effects on human neuroblastoma cells, in particular increasing cytosolic calcium levels and promoting the generation of reactive oxygen species. These results reveal an interplay between Aß aggregation and key biochemical processes regulating cellular homeostasis in the brain.

Infrared nanospectroscopy reveals the molecular interaction fingerprint of an aggregation inhibitor with single Aß42 oligomers.

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 Aß42 oligomeric and fibrillar species and an aggregation inhibitor, bexarotene, which is able to prevent Aß42 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 Aß42 aggregates through a single hydrogen bond. These results establish infrared nanospectroscopy as a powerful tool in structure-based drug discovery for protein misfolding diseases.

Quantifying misfolded protein oligomers as drug targets and biomarkers in Alzheimer and Parkinson diseases.

Protein misfolding and aggregation are characteristic of a wide range of neurodegenerative disorders, including Alzheimer and Parkinson diseases. A hallmark of these diseases is the aggregation of otherwise soluble and functional proteins into amyloid aggregates. Although for many decades such amyloid deposits have been thought to be responsible for disease progression, it is now increasingly recognized that the misfolded protein oligomers formed during aggregation are, instead, the main agents causing pathological processes. These oligomers are transient and heterogeneous, which makes it difficult to detect and quantify them, generating confusion about their exact role in disease. The lack of suitable methods to address these challenges has hampered efforts to investigate the molecular mechanisms of oligomer toxicity and to develop oligomer-based diagnostic and therapeutic tools to combat protein misfolding diseases. In this Review, we describe methods to quantify misfolded protein oligomers, with particular emphasis on diagnostic applications as disease biomarkers and on therapeutic applications as target biomarkers. The development of these methods is ongoing, and we discuss the challenges that remain to be addressed to establish measurement tools capable of overcoming existing limitations and to meet present needs.

Structural and dynamics analysis of intrinsically disordered proteins by high-speed atomic force microscopy.

Intrinsically disordered proteins (IDPs) are ubiquitous proteins that are disordered entirely or partly and play important roles in diverse biological phenomena. Their structure dynamically samples a multitude of conformational states, thus rendering their structural analysis very difficult. Here we explore the potential of high-speed atomic force microscopy (HS-AFM) for characterizing the structure and dynamics of IDPs. Successive HS-AFM images of an IDP molecule can not only identify constantly folded and constantly disordered regions in the molecule, but can also document disorder-to-order transitions. Moreover, the number of amino acids contained in these disordered regions can be roughly estimated, enabling a semiquantitative, realistic description of the dynamic structure of IDPs.

Trodusquemine displaces protein misfolded oligomers from cell membranes and abrogates their cytotoxicity through a generic mechanism.

The onset and progression of numerous protein misfolding diseases are associated with the presence of oligomers formed during the aberrant aggregation of several different proteins, including amyloid-ß (Aß) in Alzheimer's disease and α-synuclein (αS) in Parkinson's disease. These small, soluble aggregates are currently major targets for drug discovery. In this study, we show that trodusquemine, a naturally-occurring aminosterol, markedly reduces the cytotoxicity of αS, Aß and HypF-N oligomers to human neuroblastoma cells by displacing the oligomers from cell membranes in the absence of any substantial morphological and structural changes to the oligomers. These results indicate that the reduced toxicity results from a mechanism that is common to oligomers from different proteins, shed light on the origin of the toxicity of the most deleterious species associated with protein aggregation and suggest that aminosterols have the therapeutically-relevant potential to protect cells from the oligomer-induced cytotoxicity associated with numerous protein misfolding diseases.

Rationally Designed Antibodies as Research Tools to Study the Structure-Toxicity Relationship of Amyloid-ß Oligomers.

Alzheimer's disease is associated with the aggregation of the amyloid-ß peptide (Aß), resulting in the deposition of amyloid plaques in brain tissue. Recent scrutiny of the mechanisms by which Aß aggregates induce neuronal dysfunction has highlighted the importance of the Aß oligomers of this protein fragment. Because of the transient and heterogeneous nature of these oligomers, however, it has been challenging to investigate the detailed mechanisms by which these species exert cytotoxicity. To address this problem, we demonstrate here the use of rationally designed single-domain antibodies (DesAbs) to characterize the structure-toxicity relationship of Aß oligomers. For this purpose, we use Zn2+-stabilized oligomers of the 40-residue form of Aß (Aß40) as models of brain Aß oligomers and two single-domain antibodies (DesAb18-24 and DesAb34-40), designed to bind to epitopes at residues 18-24 and 34-40 of Aß40, respectively. We found that the DesAbs induce a change in structure of the Zn2+-stabilized Aß40 oligomers, generating a simultaneous increase in their size and solvent-exposed hydrophobicity. We then observed that these increments in both the size and hydrophobicity of the oligomers neutralize each other in terms of their effects on cytotoxicity, as predicted by a recently proposed general structure-toxicity relationship, and observed experimentally. These results illustrate the use of the DesAbs as research tools to investigate the biophysical and cytotoxicity properties of Aß oligomers.

Rational design of a conformation-specific antibody for the quantification of Aß oligomers.

Protein misfolding and aggregation is the hallmark of numerous human disorders, including Alzheimer's disease. This process involves the formation of transient and heterogeneous soluble oligomers, some of which are highly cytotoxic. A major challenge for the development of effective diagnostic and therapeutic tools is thus the detection and quantification of these elusive oligomers. Here, to address this problem, we develop a two-step rational design method for the discovery of oligomer-specific antibodies. The first step consists of an "antigen scanning" phase in which an initial panel of antibodies is designed to bind different epitopes covering the entire sequence of a target protein. This procedure enables the determination through in vitro assays of the regions exposed in the oligomers but not in the fibrillar deposits. The second step involves an "epitope mining" phase, in which a second panel of antibodies is designed to specifically target the regions identified during the scanning step. We illustrate this method in the case of the amyloid ß (Aß) peptide, whose oligomers are associated with Alzheimer's disease. Our results show that this approach enables the accurate detection and quantification of Aß oligomers in vitro, and in Caenorhabditis elegans and mouse hippocampal tissues.

Complexity in Lipid Membrane Composition Induces Resilience to Aß42 Aggregation.

The molecular origins of Alzheimer's disease are associated with the aggregation of the amyloid-ß peptide (Aß). This process is controlled by a complex cellular homeostasis system, which involves a variety of components, including proteins, metabolites, and lipids. It has been shown in particular that certain components of lipid membranes can speed up Aß aggregation. This observation prompts the question of whether there are protective cellular mechanisms to counterbalance this effect. Here, to address this issue, we investigate the role of the composition of lipid membranes in modulating the aggregation process of Aß. By adopting a chemical kinetics approach, we first identify a panel of lipids that affect the aggregation of the 42-residue form of Aß (Aß42), ranging from enhancement to inhibition. We then show that these effects tend to average out in mixtures of these lipids, as such mixtures buffer extreme aggregation behaviors as the number of components increases. These results indicate that a degree of quality control on protein aggregation can be achieved through a mechanism by which an increase in the molecular complexity of lipid membranes balances opposite effects and creates resilience to aggregation.

Transthyretin Inhibits Primary and Secondary Nucleations of Amyloid-ß Peptide Aggregation and Reduces the Toxicity of Its Oligomers.

Alzheimer's disease is associated with the deposition of the amyloid-ß peptide (Aß) into extracellular senile plaques in the brain. In vitro and in vivo observations have indicated that transthyretin (TTR) acts as an Aß scavenger in the brain, but the mechanism has not been fully resolved. We have monitored the aggregation process of Aß40 by thioflavin T fluorescence, in the presence or absence of different concentrations of preformed seed aggregates of Aß40, of wild-type tetrameric TTR (WT-TTR), and of a variant engineered to be stable as a monomer (M-TTR). Both WT-TTR and M-TTR were found to inhibit specific steps of the process of Aß40 fibril formation, which are primary and secondary nucleations, without affecting the elongation of the resulting fibrils. Moreover, the analysis shows that both WT-TTR and M-TTR bind to Aß40 oligomers formed in the aggregation reaction and inhibit their conversion into the shortest fibrils able to elongate. Using biophysical methods, TTR was found to change some aspects of its overall structure following such interactions with Aß40 oligomers, as well as with oligomers of Aß42, while maintaining its overall topology. Hence, it is likely that the predominant mechanism by which TTR exerts its protective role lies in the binding of TTR to the Aß oligomers and in inhibiting primary and secondary nucleation processes, which limits both the toxicity of Aß oligomers and the ability of the fibrils to proliferate.

Screening of small molecules using the inhibition of oligomer formation in α-synuclein aggregation as a selection parameter.

The aggregation of α-synuclein is a central event in Parkinsons's disease and related synucleinopathies. Since pharmacologically targeting this process, however, has not yet resulted in approved disease-modifying treatments, there is an unmet need of developing novel methods of drug discovery. In this context, the use of chemical kinetics has recently enabled accurate quantifications of the microscopic steps leading to the proliferation of protein misfolded oligomers. As these species are highly neurotoxic, effective therapeutic strategies may be aimed at reducing their numbers. Here, we exploit this quantitative approach to develop a screening strategy that uses the reactive flux toward α-synuclein oligomers as a selection parameter. Using this approach, we evaluate the efficacy of a library of flavone derivatives, identifying apigenin as a compound that simultaneously delays and reduces the formation of α-synuclein oligomers. These results demonstrate a compound selection strategy based on the inhibition of the formation of α-synuclein oligomers, which may be key in identifying small molecules in drug discovery pipelines for diseases associated with α-synuclein aggregation.