Characterization of Pairs of Toxic and Nontoxic Misfolded Protein Oligomers Elucidates the Structural Determinants of Oligomer Toxicity in Protein Misfolding Diseases.

The aberrant misfolding and aggregation of peptides and proteins into amyloid aggregates occurs in over 50 largely incurable protein misfolding diseases. These pathologies include Alzheimer's and Parkinson's diseases, which are global medical emergencies owing to their prevalence in increasingly aging populations worldwide. Although the presence of mature amyloid aggregates is a hallmark of such neurodegenerative diseases, misfolded protein oligomers are increasingly recognized as of central importance in the pathogenesis of many of these maladies.

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 stoichiometry of an inhibitor of α-synuclein amyloid aggregation.

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.

Effects of oligomer toxicity, fibril toxicity and fibril spreading in synucleinopathies.

Protein misfolding is a general hallmark of protein deposition diseases, such as Alzheimer's disease or Parkinson's disease, in which different types of aggregated species (oligomers, protofibrils and fibrils) are generated by the cells. Despite widespread interest, the relationship between oligomers and fibrils in the aggregation process and spreading remains elusive. A large variety of experimental evidences supported the idea that soluble oligomeric species of different proteins might be more toxic than the larger fibrillar forms.

The Pathological G51D Mutation in Alpha-Synuclein Oligomers Confers Distinct Structural Attributes and Cellular Toxicity.

A wide variety of oligomeric structures are formed during the aggregation of proteins associated with neurodegenerative diseases. Such soluble oligomers are believed to be key toxic species in the related disorders; therefore, identification of the structural determinants of toxicity is of upmost importance. Here, we analysed toxic oligomers of α-synuclein and its pathological variants in order to identify structural features that could be related to toxicity and found a novel structural polymorphism within G51D oligomers.

All-or-none amyloid disassembly via chaperone-triggered fibril unzipping favors clearance of α-synuclein toxic species.

α-synuclein aggregation is present in Parkinson's disease and other neuropathologies. Among the assemblies that populate the amyloid formation process, oligomers and short fibrils are the most cytotoxic. The human Hsc70-based disaggregase system can resolve α-synuclein fibrils, but its ability to target other toxic assemblies has not been studied.

α-Helical peptidic scaffolds to target α-synuclein toxic species with nanomolar affinity.

α-Synuclein aggregation is a key driver of neurodegeneration in Parkinson's disease and related syndromes. Accordingly, obtaining a molecule that targets α-synuclein toxic assemblies with high affinity is a long-pursued objective. Here, we exploit the biophysical properties of toxic oligomers and amyloid fibrils to identify a family of α-helical peptides that bind to these α-synuclein species with low nanomolar affinity, without interfering with the monomeric functional protein.

The release of toxic oligomers from α-synuclein fibrils induces dysfunction in neuronal cells.

The self-assembly of α-synuclein (αS) into intraneuronal inclusion bodies is a key characteristic of Parkinson's disease. To define the nature of the species giving rise to neuronal damage, we have investigated the mechanism of action of the main αS populations that have been observed to form progressively during fibril growth. The αS fibrils release soluble prefibrillar oligomeric species with cross-β structure and solvent-exposed hydrophobic clusters.

The role of water in the primary nucleation of protein amyloid aggregation.

The understanding of the complex conformational landscape of amyloid aggregation and its modulation by relevant physicochemical and cellular factors is a prerequisite for elucidating some of the molecular basis of pathology in amyloid related diseases, and for developing and evaluating effective disease-specific therapeutics to reduce or eliminate the underlying sources of toxicity in these diseases. Interactions of proteins with solvating water have been long considered to be fundamental in mediating their function and folding; however, the relevance of water in the process of protein amyloid aggregation has been largely overlooked. Here, we provide a perspective on the role water plays in triggering primary amyloid nucleation of intrinsically disordered proteins (IDPs) based on recent experimental evidences.

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.

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-β (Aβ) 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, Aβ 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.

Novel Small Molecules Targeting the Intrinsically Disordered Structural Ensemble of α-Synuclein Protect Against Diverse α-Synuclein Mediated Dysfunctions.

The over-expression and aggregation of α-synuclein (αSyn) are linked to the onset and pathology of Parkinson's disease. Native monomeric αSyn exists in an intrinsically disordered ensemble of interconverting conformations, which has made its therapeutic targeting by small molecules highly challenging. Nonetheless, here we successfully target the monomeric structural ensemble of αSyn and thereby identify novel drug-like small molecules that impact multiple pathogenic processes.

Aberrant expression of N-glycolyl GM3 ganglioside is associated with the aggressive biological behavior of human sarcomas.

Background: The aberrant expression of N-glycolyl GM3 ganglioside (NeuGcGM3) in patients with sarcomas was reevaluated by assessing the relation of this molecule with some clinicopathological features and overall survival (OS) of patients.

Methods: Fifty formalin-fixed and paraffin-embedded specimens from patients diagnosed with sarcomas were included. For the evaluation of NeuGcGM3, the 14F7 monoclonal antibody followed by a peroxidase avidin-biotin system was used.

Defining α-synuclein species responsible for Parkinson's disease phenotypes in mice.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by fibrillar neuronal inclusions composed of aggregated α-synuclein (α-syn). These inclusions are associated with behavioral and pathological PD phenotypes. One strategy for therapeutic interventions is to prevent the formation of these inclusions to halt disease progression.

Multistep Inhibition of α-Synuclein Aggregation and Toxicity in Vitro and in Vivo by Trodusquemine.

The aggregation of α-synuclein, an intrinsically disordered protein that is highly abundant in neurons, is closely associated with the onset and progression of Parkinson's disease. We have shown previously that the aminosterol squalamine can inhibit the lipid induced initiation process in the aggregation of α-synuclein, and we report here that the related compound trodusquemine is capable of inhibiting not only this process but also the fibril-dependent secondary pathways in the aggregation reaction. We further demonstrate that trodusquemine can effectively suppress the toxicity of α-synuclein oligomers in neuronal cells, and that its administration, even after the initial growth phase, leads to a dramatic reduction in the number of α-synuclein inclusions in a Caenorhabditis elegans model of Parkinson's disease, eliminates the related muscle paralysis, and increases lifespan.

Preparation of α-Synuclein Amyloid Assemblies for Toxicity Experiments.

Amyloid assemblies of certain proteins, including the Parkinson disease-related protein α-synuclein, are commonly associated with the development and spreading of neurodegenerative diseases, although the nature of the most toxic forms and the mechanisms by which they trigger neurodegeneration remain largely unknown. This is at least in part due to the inherent challenges involved in the preparation of stable and structurally homogeneous samples of amyloid assemblies that could be used in toxicity experiments. Here, we describe the preparation of two different types of stable α-synuclein amyloid assemblies, namely a kinetically trapped oligomeric species and a propagating-competent fibrillar polymorph.

Best Practices for Generating and Using Alpha-Synuclein Pre-Formed Fibrils to Model Parkinson's Disease in Rodents.

Parkinson's disease (PD) is the second most common neurodegenerative disease, affecting approximately one-percent of the population over the age of sixty. Although many animal models have been developed to study this disease, each model presents its own advantages and caveats. A unique model has arisen to study the role of alpha-synuclein (aSyn) in the pathogenesis of PD.

Structural basis of membrane disruption and cellular toxicity by α-synuclein oligomers.

Oligomeric species populated during the aggregation process of α-synuclein have been linked to neuronal impairment in Parkinson's disease and related neurodegenerative disorders. By using solution and solid-state nuclear magnetic resonance techniques in conjunction with other structural methods, we identified the fundamental characteristics that enable toxic α-synuclein oligomers to perturb biological membranes and disrupt cellular function; these include a highly lipophilic element that promotes strong membrane interactions and a structured region that inserts into lipid bilayers and disrupts their integrity. In support of these conclusions, mutations that target the region that promotes strong membrane interactions by α-synuclein oligomers suppressed their toxicity in neuroblastoma cells and primary cortical neurons.

The contribution of biophysical and structural studies of protein self-assembly to the design of therapeutic strategies for amyloid diseases.

Many neurodegenerative disorders, including Alzheimer's, Parkinson's and the prion diseases, are characterized by a conformational conversion of normally soluble proteins or peptides into pathological species, by a process of misfolding and self-assembly that leads ultimately to the formation of amyloid fibrils. Recent studies support the idea that multiple intermediate species with a wide variety of degrees of neuronal toxicity are generated during such processes. The development of a high level of knowledge of the nature and structure of the pathogenic amyloid species would significantly enhance efforts to underline the molecular origins of these disorders and also to develop both accurate diagnoses and effective therapeutic interventions for these types of conditions.

Inhibition of α-Synuclein Fibril Elongation by Hsp70 Is Governed by a Kinetic Binding Competition between α-Synuclein Species.

The Hsp70 family of chaperones plays an essential role in suppressing protein aggregation in the cell. Here we investigate the factors controlling the intrinsic ability of human Hsp70 to inhibit the elongation of amyloid fibrils formed by the Parkinson's disease-related protein α-synuclein. Using kinetic analysis, we show that Hsp70 binds preferentially to α-synuclein fibrils as a consequence of variations in the association and dissociation rate constants of binding to the different aggregated states of the protein.

Structural Characteristics of α-Synuclein Oligomers.

Oligomeric forms of amyloid aggregates have been detected in the brains and tissues of patients suffering from neurodegenerative disorders such as Parkinson's disease, and it is widely thought that such species are key pathogenic agents in the development and spreading of the disease; however, the study of these species has been proven to be extremely challenging, primarily as a result of their intrinsically transient nature and high levels of heterogeneity. Identifying the structural nature and the details of the mechanisms of formation and interconversion of individual oligomeric species, particularly those with high toxicity, is of fundamental importance not only for understanding the mechanisms of protein misfolding and amyloid aggregation but also for the identification of diagnostic and therapeutic targets. In this review, we will focus on the current knowledge of the multitude of oligomeric forms of α-synuclein that have been reported to date, with particular emphasis on their structural features and possible relationship to other amyloid species, in order to build a clearer understanding of the types of oligomeric species that accumulate during the aggregation of α-synuclein and to develop a comprehensive picture of the misfolding behavior of the protein.

A natural product inhibits the initiation of α-synuclein aggregation and suppresses its toxicity.

The self-assembly of α-synuclein is closely associated with Parkinson's disease and related syndromes. We show that squalamine, a natural product with known anticancer and antiviral activity, dramatically affects α-synuclein aggregation in vitro and in vivo. We elucidate the mechanism of action of squalamine by investigating its interaction with lipid vesicles, which are known to stimulate nucleation, and find that this compound displaces α-synuclein from the surfaces of such vesicles, thereby blocking the first steps in its aggregation process.