Amyloid Aggregation Of Α-synuclein Research Articles

The impact of hUC MSC-derived exosome-nanoliposome hybrids on α-synuclein fibrillation and neurotoxicity.

Amyloid aggregation of α-synuclein (αSN) protein amplifies the pathogenesis of neurodegenerative diseases (NDs) such as Parkinson's disease (PD). Consequently, blocking aggregation or redirecting self-assembly to less toxic aggregates could be therapeutic. Here, we improve brain-specific nanocarriers using a hybrid of exosomes (Ex) from human umbilical cord mesenchymal stem cells (hUC MSCs) and nanoliposomes containing baicalein (Ex-NLP-Ba) and oleuropein (Ex-NLP-Ole). The hybrids contained both lipid membranes, Ex proteins, and baicalein or oleuropein. Fluorescence resonance energy transfer analysis confirmed their proper integration. The hybrids reduced the extent of αSN fibrillation and interfered with secondary nucleation and disaggregation. They not only reduced αSN pathogenicity but also enhanced drug internalization into cells, surpassing the efficacy of NLP alone, and also crossed the blood-brain barrier in a cellular model. We conclude that Ex can be successfully extracted and efficiently merged with NLPs while retaining its original properties, demonstrating great potential as a theranostic drug delivery vehicle against NDs like PD.

Lipid-induced polymorphic amyloid fibril formation by α-synuclein.

Many proteins that self-assemble into amyloid and amyloid-like fibers can adopt diverse polymorphic forms. These forms have been observed both in vitro and in vivo and can arise through variations in the steric-zipper interactions between β-sheets, variations in the arrangements between protofilaments, and differences in the number of protofilaments that make up a given fiber class. Different polymorphs arising from the same precursor molecule not only exhibit different levels of toxicity, but importantly can contribute to different disease conditions. However, the factors which contribute to formation of polymorphic forms of amyloid fibrils are not known. In this work, we show that in the presence of 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine, a highly abundant lipid in the plasma membrane of neurons, the aggregation of α-synuclein is markedly accelerated and yields a diversity of polymorphic forms under identical experimental conditions. This morphological diversity includes thin and curly fibrils, helical ribbons, twisted ribbons, nanotubes, and flat sheets. Furthermore, the amyloid fibrils formed incorporate lipids into their structures, which corroborates the previous report of the presence of α-synuclein fibrils with high lipid content in Lewy bodies. Thus, the present study demonstrates that an interface, such as that provided by a lipid membrane, can not only modulate the kinetics of α-synuclein amyloid aggregation but also plays an important role in the formation of morphological variants by incorporating lipid molecules in the process of amyloid fibril formation.

Elevated concentrations cause upright alpha-synuclein conformation at lipid interfaces

The amyloid aggregation of α-synuclein (αS), related to Parkinson’s disease, can be catalyzed by lipid membranes. Despite the importance of lipid surfaces, the 3D-structure and orientation of lipid-bound αS is still not known in detail. Here, we report interface-specific vibrational sum-frequency generation (VSFG) experiments that reveal how monomeric αS binds to an anionic lipid interface over a large range of αS-lipid ratios. To interpret the experimental data, we present a frame-selection method ("ViscaSelect”) in which out-of-equilibrium molecular dynamics simulations are used to generate structural hypotheses that are compared to experimental amide-I spectra via excitonic spectral calculations. At low and physiological αS concentrations, we derive flat-lying helical structures as previously reported. However, at elevated and potentially disease-related concentrations, a transition to interface-protruding αS structures occurs. Such an upright conformation promotes lateral interactions between αS monomers and may explain how lipid membranes catalyze the formation of αS amyloids at elevated protein concentrations.

Single-Particle Analysis of the Interaction Between Molecules and Protein Aggregated Species by Dual-Color Time-Resolved Fluorescence Spectroscopy.

Amyloid protein aggregation is widely involved in a number of neurodegenerative diseases for which novel therapeutic and diagnostic strategies are still needed. Owing to the complex and heterogeneous nature of the aggregated species responsible for toxicity in these disorders, a detailed characterization of the interaction of molecules of interest with the amyloid aggregates is a challenging endeavor. Here, we present the experimental and analytical steps of a protocol which combines dual-color fluorescence cross-correlation spectroscopy and dual-color single-particle fluorescence spectroscopy to quantify the binding affinity and stoichiometryof an inhibitor of α-synuclein amyloid aggregation. This approach allows studying the interaction in detail and through two independent analytical methods, thus yielding a remarkably robust tool that could be extended to investigating the interaction of molecules of interest to other pathogenic protein aggregates as well as multi-ligand/multi-receptor complexes.

Molecular Insights into the Misfolding and Dimerization Dynamics of the Full-Length α-Synuclein from Atomistic Discrete Molecular Dynamics Simulations.

The misfolding and pathological aggregation of α-synuclein forming insoluble amyloid deposits is associated with Parkinson's disease, the second most common neurodegenerative disease in the world population. Characterizing the self-assembly mechanism of α-synuclein is critical for discovering treatments against synucleinopathies. The intrinsically disordered property, high degrees of freedom, and macroscopic timescales of conformational conversion make its characterization extremely challenging in vitro and in silico. Here, we systematically investigated the dynamics of monomer misfolding and dimerization of the full-length α-synuclein using atomistic discrete molecular dynamics simulations. Our results suggested that both α-synuclein monomers and dimers mainly adopted unstructured formations with partial helices around the N-terminus (residues 8-32) and various β-sheets spanning the residues 35-56 (N-terminal tail) and residues 61-95 (NAC region). The C-terminus mostly assumed an unstructured formation wrapping around the lateral surface and the elongation edge of the β-sheet core formed by an N-terminal tail and NAC regions. Dimerization enhanced the β-sheet formation along with a decrease in the unstructured content. The inter-peptide β-sheets were mainly formed by the N-terminal tail and NACore (residues 68-78) regions, suggesting that these two regions played critical roles in the amyloid aggregation of α-synuclein. Interactions of the C-terminus with the N-terminal tail and the NAC region were significantly suppressed in the α-synuclein dimer, indicating that the interaction of the C-terminus with the N-terminal tail and NAC regions could prevent α-synuclein aggregation. These results on the structural ensembles and early aggregation dynamics of α-synuclein will help understand the nucleation and fibrillization of α-synuclein.

Curcumin Interacts with α-Synuclein Condensates To Inhibit Amyloid Aggregation under Phase Separation.

The amyloid aggregation of α-synuclein (α-Syn) is highly associated with Parkinson’s disease (PD). Discovering α-Syn amyloid inhibitors is one of the strategies for PD therapies. Recent studies suggested that α-Syn undergoes phase separation to accelerate amyloid aggregation. Molecules modulating α-Syn phase separation or transition have the potential to regulate amyloid aggregation. Here, we discovered that curcumin, a small natural molecule, interacts with α-Syn during phase separation. Our study showed that curcumin neither affects the formation of α-Syn condensates nor influences the initial morphology of α-Syn condensates. However, curcumin decreases the fluidity of α-Syn inside the condensates and efficiently inhibits α-Syn from turning into an amyloid. It also inhibits the amyloid aggregations of PD disease-related α-Syn E46K and H50Q mutants under phase separation. Furthermore, curcumin can destabilize preformed α-Syn amyloid aggregates in the condensates. Together, our findings demonstrate that curcumin regulates α-Syn amyloid formation during protein phase separation and reveal that α-Syn amyloid aggregation under phase separation can be modulated by small molecules.

Molecular mechanism for the synchronized electrostatic coacervation and co-aggregation of alpha-synuclein and tau

Amyloid aggregation of α-synuclein (αS) is the hallmark of Parkinson’s disease and other synucleinopathies. Recently, Tau protein, generally associated with Alzheimer’s disease, has been linked to αS pathology and observed to co-localize in αS-rich disease inclusions, although the molecular mechanisms for the co-aggregation of both proteins remain elusive. We report here that αS phase-separates into liquid condensates by electrostatic complex coacervation with positively charged polypeptides such as Tau. Condensates undergo either fast gelation or coalescence followed by slow amyloid aggregation depending on the affinity of αS for the poly-cation and the rate of valence exhaustion of the condensate network. By combining a set of advanced biophysical techniques, we have been able to characterize αS/Tau liquid-liquid phase separation and identified key factors that lead to the formation of hetero-aggregates containing both proteins in the interior of the liquid protein condensates.

3,4-Dihydroxyphenylethanol and 3,4-dihydroxyphenylacetic acid affect the aggregation process of E46K variant of α-synuclein at different extent: Insights into the interplay between protein dynamics and catechol effect.

Parkinson's disease (PD) is a chronic multifactorial disease, whose etiology is not completely understood. The amyloid aggregation of α‐synuclein (Syn) is considered a major cause in the development of the disease. The presence of genetic mutations can boost the aggregation of the protein and the likelihood to develop PD. These mutations can lead to early onset (A30P, E46K, and A53T) or late‐onset (H50Q) forms of PD. The disease is also linked to an increase in oxidative stress and altered levels of dopamine metabolites. The molecular interaction of these molecules with Syn has been previously studied, while their effect on the pathological mutant structure and function is not completely clarified. By using biochemical and biophysical approaches, here we have studied the interaction of the familial variant E46K with two dopamine‐derived catechols, 3,4‐dihydroxyphenylacetic acid and 3,4‐dihydroxyphenylethanol. We show that the presence of these catechols causes a decrease in the formation of amyloid fibrils in a dose‐dependent manner. Native‐ and Hydrogen/deuterium exchange‐mass spectrometry (HDX‐MS) provide evidence that this effect is strongly conformation dependent. Indeed, these molecules interact differently with the interconverting conformers of Syn and its familial variant E46K in solution, selecting the most prone‐to‐aggregation one, confining it into an off‐pathway oligomer. These findings suggest that catechols could be a molecular scaffold for the design of compounds potentially useful in the treatment of Parkinson's disease and related conditions.

Structural Insights of Fe3+ Induced α-synuclein Fibrillation in Parkinson’s Disease

Amyloid aggregation of α-synuclein (α-syn) in Lewy bodies (LBs) is the pathological hallmark of Parkinson’s disease (PD). Iron, especially Fe3+, is accumulated in substantia nigra of PD patients and co-deposited with α-syn in LBs. However, how Fe3+ modulates α-syn fibrillation at molecular level remains unclear. In this study, we found that Fe3+ can promote α-syn fibrillation at low concentration while inhibit its fibrillation at high concentration. NMR titration study shows poor interaction between α-syn monomer and Fe3+. Instead, we found that Fe3+ binds to α-syn fibrils. By using cryo-electron microscopy (cryo-EM), we further determined the atomic structure of α-syn fibril in complex with Fe3+ at the resolution of 2.7 Å. Strikingly, two extra electron densities adjacent to His50 and Glu57 were observed as putative binding sites of Fe3+ and water molecules, suggesting that Fe3+ binds to the negative cleft of the fibril and stabilizes the fibril structure for promoting α-syn aggregation. Further mutagenesis study shows mutation of His50 abolishes the Fe3+-facilitated fibrillation of α-syn. Our work illuminates the structural basis of the interaction of Fe3+ and α-syn in both monomeric and fibrillar forms, and sheds light on understanding the pathological role of Fe3+ in α-syn aggregation in PD.

Mechanistic insights into accelerated α-synuclein aggregation mediated by human microbiome-associated functional amyloids

The gut microbiome has been shown to have key implications in the pathogenesis of Parkinson’s disease (PD). The Escherichia coli functional amyloid CsgA is known to accelerate α-synuclein aggregation in vitro and induce PD symptoms in mice. However, the mechanism governing CsgA-mediated acceleration of α-synuclein aggregation is unclear. Here, we show that CsgA can form stable homodimeric species that correlate with faster α-synuclein amyloid aggregation. Furthermore, we identify and characterize new CsgA homologs encoded by bacteria present in the human microbiome. These CsgA homologs display diverse aggregation kinetics, and they differ in their ability to modulate α-synuclein aggregation. Remarkably, we demonstrate that slowing down CsgA aggregation leads to an increased acceleration of α-synuclein aggregation, suggesting that the intrinsic amyloidogenicity of gut bacterial CsgA homologs affects their ability to accelerate α-synuclein aggregation. Finally, we identify a complex between CsgA and α-synuclein that functions as a platform to accelerate α-synuclein aggregation. Taken together, our work reveals complex interplay between bacterial amyloids and α-synuclein that better informs our understanding of PD causation.

Comparing the copper binding features of alpha and beta synucleins

Amyloid aggregation of α-synuclein (AS) is one of the hallmarks of Parkinson's disease (PD). Copper ions specifically bind at the N-terminus of AS, accelerating protein aggregation. Its protein homolog β-synuclein (BS) is also a copper binding protein, but it inhibits AS aggregation. Here, a comparative spectroscopic study of the Cu2+ binding properties of AS and BS has been performed, using electronic absorption, circular dichroism (CD) and electronic paramagnetic resonance (EPR). Our comparative spectroscopic study reveals striking similarities between the Cu2+ binding features of the two proteins. The Cu2+ binding site at the N-terminal group of BS protein, modeled by the BS (1–15) fragment is identical to that of AS; however, its rate of reduction is three times faster as compared to the AS site, consistent with BS having an additional Met residue in its Met1-Xn-Met5-Xn-Met10 motif. The latter is also evident in the cyclic voltammetry studies of the Cu-BS complex. On the other hand, the Cu2+ binding features of the His site in both proteins, as modeled by AS(45–55) and BS(60–70), are identical, indicating that the shift in the His position does not affect its coordination features. Finally, replacement of Glu46 by Ala does not alter Cu2+ binding to the His site, suggesting that the familial PD E46K mutation would not impact copper-induced aggregation. While further studies of the redox activity of copper bound to His50 in AS are required to understand the role of this site in metal-mediated aggregation, our study contributes to a better understanding of the bioinorganic chemistry of PD.

Manganese promotes α-synuclein amyloid aggregation through the induction of protein phase transition

α-Synuclein (α-Syn) is the major protein component of Lewy bodies, a key pathological feature of Parkinson’s disease (PD). The manganese ion Mn2+ has been identified as an environmental risk factor of PD. However, it remains unclear how Mn2+ regulates α-Syn aggregation. Here, we discovered that Mn2+accelerates α-Syn amyloid aggregation through the regulation of protein phase separation. We found that Mn2+ not only promotes α-Syn liquid-to-solid phase transition but also directly induces soluble α-Syn monomers to form solid-like condensates. Interestingly, the lipid membrane is integrated into condensates during Mn2+-induced α-Syn phase transition; however, the preformed Mn2+/α-syn condensates can only recruit lipids to the surface of condensates. In addition, this phase transition can largely facilitate α-Syn amyloid aggregation. Although the Mn2+-induced condensates do not fuse, our results demonstrated that they could recruit soluble α-Syn monomers into the existing condensates. Furthermore, we observed that a manganese chelator reverses Mn2+-induced α-Syn aggregation during the phase transition stage. However, after maturation, α-Syn aggregation becomes irreversible. These findings demonstrate that Mn2+ facilitates α-Syn phase transition to accelerate the formation of α-Syn aggregates and provide new insights for targeting α-Syn phase separation in PD treatment.

Physical mechanisms of amyloid nucleation on fluid membranes

Biological membranes can dramatically accelerate the aggregation of normally soluble protein molecules into amyloid fibrils and alter the fibril morphologies, yet the molecular mechanisms through which this accelerated nucleation takes place are not yet understood. Here, we develop a coarse-grained model to systematically explore the effect that the structural properties of the lipid membrane and the nature of protein-membrane interactions have on the nucleation rates of amyloid fibrils. We identify two physically distinct nucleation pathways-protein-rich and lipid-rich-and quantify how the membrane fluidity and protein-membrane affinity control the relative importance of those molecular pathways. We find that the membrane's susceptibility to reshaping and being incorporated into the fibrillar aggregates is a key determinant of its ability to promote protein aggregation. We then characterize the rates and the free-energy profile associated with this heterogeneous nucleation process, in which the surface itself participates in the aggregate structure. Finally, we compare quantitatively our data to experiments on membrane-catalyzed amyloid aggregation of α-synuclein, a protein implicated in Parkinson's disease that predominately nucleates on membranes. More generally, our results provide a framework for understanding macromolecular aggregation on lipid membranes in a broad biological and biotechnological context.

Multiplicity of α-Synuclein Aggregated Species and Their Possible Roles in Disease.

α-Synuclein amyloid aggregation is a defining molecular feature of Parkinson’s disease, Lewy body dementia, and multiple system atrophy, but can also be found in other neurodegenerative disorders such as Alzheimer’s disease. The process of α-synuclein aggregation can be initiated through alternative nucleation mechanisms and dominated by different secondary processes giving rise to multiple amyloid polymorphs and intermediate species. Some aggregated species have more inherent abilities to induce cellular stress and toxicity, while others seem to be more potent in propagating neurodegeneration. The preference for particular types of polymorphs depends on the solution conditions and the cellular microenvironment that the protein encounters, which is likely related to the distinct cellular locations of α-synuclein inclusions in different synucleinopathies, and the existence of disease-specific amyloid polymorphs. In this review, we discuss our current understanding on the nature and structure of the various types of α-synuclein aggregated species and their possible roles in pathology. Precisely defining these distinct α-synuclein species will contribute to understanding the molecular origins of these disorders, developing accurate diagnoses, and designing effective therapeutic interventions for these highly debilitating neurodegenerative diseases.

Parkinson\u2019s disease associated mutation E46K of \u03b1-synuclein triggers the formation of a distinct fibril structure

Amyloid aggregation of α-synuclein (α-syn) is closely associated with Parkinson’s disease (PD) and other synucleinopathies. Several single amino-acid mutations (e.g. E46K) of α-syn have been identified causative to the early onset of familial PD. Here, we report the cryo-EM structure of an α-syn fibril formed by N-terminally acetylated E46K mutant α-syn (Ac-E46K). The fibril structure represents a distinct fold of α-syn, which demonstrates that the E46K mutation breaks the electrostatic interactions in the wild type (WT) α-syn fibril and thus triggers the rearrangement of the overall structure. Furthermore, we show that the Ac-E46K fibril is less resistant to harsh conditions and protease cleavage, and more prone to be fragmented with an enhanced seeding capability than that of the WT fibril. Our work provides a structural view to the severe pathology of the PD familial mutation E46K of α-syn and highlights the importance of electrostatic interactions in defining the fibril polymorphs.

Rational design of small molecules able to inhibit α-synuclein amyloid aggregation for the treatment of Parkinson’s disease

Parkinson’s disease is one of the most common neurodegenerative disorders in elderly age. One of the mechanisms involved in the neurodegeneration appears related to the aggregation of the presynaptic protein alpha synuclein (α-syn) into toxic oligomers and fibrils. To date, no highly effective treatment is currently available; therefore, there is an increasing interest in the search of new therapeutic tools. The modulation of α-syn aggregation represents an emergent and promising disease-modifying strategy for reducing or blocking the neurodegenerative process. Herein, by combining in silico and in vitro screenings we initially identified 3-(cinnamylsulfanyl)-5-(4-pyridinyl)-1,2,4-triazol-4-amine (3) as α-syn aggregation inhibitor that was then considered a promising hit for the further design of a new series of small molecules. Therefore, we rationally designed new hit-derivatives that were synthesised and evaluated by biological assays. Lastly, the binding mode of the newer inhibitors was predicted by docking studies.

ZPD-2, a Small Compound That Inhibits α-Synuclein Amyloid Aggregation and Its Seeded Polymerization.

α-Synuclein (α-Syn) forms toxic intracellular protein inclusions and transmissible amyloid structures in Parkinson’s disease (PD). Preventing α-Syn self-assembly has become one of the most promising approaches in the search for disease-modifying treatments for this neurodegenerative disorder. Here, we describe the capacity of a small molecule (ZPD-2), identified after a high-throughput screening, to inhibit α-Syn aggregation. ZPD-2 inhibits the aggregation of wild-type α-Syn and the A30P and H50Q familial variants in vitro at substoichiometric compound:protein ratios. In addition, the molecule prevents the spreading of α-Syn seeds in protein misfolding cyclic amplification assays. ZPD-2 is active against different α-Syn strains and blocks their seeded polymerization. Treating with ZPD-2 two different PD Caenorhabditis elegans models that express α-Syn either in muscle or in dopaminergic (DA) neurons substantially reduces the number of α-Syn inclusions and decreases synuclein-induced DA neurons degeneration. Overall, ZPD-2 is a hit compound worth to be explored in order to develop lead molecules for therapeutic intervention in PD.

Role of N-terminal methionine residues in the redox activity of copper bound to alpha-synuclein.

Amyloid aggregation of α-synuclein (AS) is one of the hallmarks of Parkinson's disease. The interaction of copper ions with the N-terminal region of AS promotes its amyloid aggregation and metal-catalyzed oxidation has been proposed as a plausible mechanism. The AS(1-6) fragment represents the minimal sequence that models copper coordination to this intrinsically disordered protein. In this study, we evaluated the role of methionine residues Met1 and Met5 in Cu(II) coordination to the AS(1-6) fragment, and in the redox activity of the Cu-AS(1-6) complex. Spectroscopic and electronic structure calculations show that Met1 may play a role as an axial ligand in the Cu(II)-AS(1-6) complex, while Met5 does not participate in metal coordination. Cyclic voltammetry and reactivity studies demonstrate that Met residues play an important role in the reduction and reoxidation processes of this complex. However, Met1 plays a more important role than Met5, as substitution of Met1 by Ile decreases the reduction potential of the Cu-AS(1-6) complex by ~80mV, causing a significant decrease in its rate of reduction. Reoxidation of the complex by oxygen results in oxidation of the Met residues to sulfoxide, being Met1 more susceptible to copper-catalyzed oxidation than Met5. The sulfoxide species can suffer elimination of methanesulfenic acid, rendering a peptide with no thioether moiety, which would impair the ability of AS to bind Cu(I) ions. Overall, our study underscores the important roles that Met1 plays in copper coordination and the reactivity of the Cu-AS complex.

Site-specific copper-catalyzed oxidation of α-synuclein: tightening the link between metal binding and protein oxidative damage in Parkinson's disease.

Amyloid aggregation of α-synuclein (AS) has been linked to the pathological effects associated with Parkinson's disease (PD). Cu(II) binds specifically at the N-terminus of AS and triggers its aggregation. Site-specific Cu(I)-catalyzed oxidation of AS has been proposed as a plausible mechanism for metal-enhanced AS amyloid formation. In this study, Cu(I) binding to AS was probed by NMR spectroscopy, in combination with synthetic peptide models, site-directed mutagenesis, and C-terminal-truncated protein variants. Our results demonstrate that both Met residues in the motif (1)MDVFM(5) constitute key structural determinants for the high-affinity binding of Cu(I) to the N-terminal region of AS. The replacement of one Met residue by Ile causes a dramatic decrease in the binding affinity for Cu(I), whereas the removal of both Met residues results in a complete lack of binding. Moreover, these Met residues can be oxidized rapidly after air exposure of the AS-Cu(I) complex, whereas Met-116 and Met-127 in the C-terminal region remain unaffected. Met-1 displays higher susceptibility to oxidative damage compared to Met-5 because it is directly involved in both Cu(II) and Cu(I) coordination, resulting in closer exposure to the reactive oxygen species that may be generated by the redox cycling of copper. Our findings support a mechanism where the interaction of AS with copper ions leads to site-specific metal-catalyzed oxidation in the protein under physiologically relevant conditions. In light of recent biological findings, these results support a role for AS-copper interactions in neurodegeneration in PD.

Determination of Primary Nucleation Mechanisms of α-Synuclein Amyloid Aggregation

Protein conformational diseases represent a class of pathologies in which specific peptides or proteins form aberrant self-assemblies that constitute the hallmark of several neurodegenerative diseases. Specifically, the formation of intra-neuronal inclusions of the protein α-synuclein (αSyn) is associated with the pathogenesis of Parkinson's disease (PD).A great interest is in the early stages of αSyn aggregation, for which soluble monomeric proteins are converted into fibrillar nanostructures. It has been shown that at these stages many parallel and competing pathways take contemporaneously place and it is currently very difficult to address on these mechanisms by using standard techniques of molecular investigations. In order to overcome the limitations of standard approaches, we employed ensemble-averaged kinetic studies coupled with microdloplet technology in order to characterize the primary nucleation early stages of αSyn amyloid formation and therefore to elucidate the fundamental mechanisms underlying this phenomenon. Testing different aggregation conditions, we have been able to understand that the primary nucleation mechanism underlying αSyn aggregation is not homogeneous, whereas it is catalysed by different factors, including air/water surface interactions.The full characterization of all the processes involved in the aggregation mechanism of αSyn will be fundamental for devising new and innovative therapeutic strategies against PD. Indeed, based on our analysis, we expect that it will be possible to design and screen pharmacological compounds able to selectively inhibit the nucleation steps that trigger either the overall of the process or specifically the formation of the toxic aggregated species.