Stabilization of ?-Synuclein Fibril Clusters Prevents Fragmentation and Reduces Seeding Activity and Toxicity.
ABSTRACT: Protein misfolding results in the accumulation of aggregated ?-sheet-rich structures in Parkinson's disease (PD) and Alzheimer's disease. The toxic oligomer hypothesis stipulates that prefibrillar assemblies, such as soluble oligomers or protofibrils, are responsible for the poor prognosis of these diseases. Previous studies demonstrated that a small molecule related to the natural compound orcein, O4, directly binds to amyloid-? fibrils and stabilizes them, accelerating the formation of end-stage mature fibrils. Here we demonstrate a similar phenomenon during O4 treatment of ?-synuclein (?syn) aggregates, the protein responsible for PD pathology. While the drug did not change the kinetics of aggregate formation as measured by the amyloidophilic dye thioflavin T, O4 depleted ?syn oligomers and promoted the formation of sodium dodecyl sulfate and proteinase K resistant aggregates consisting of large fibril clusters. These fibril clusters exhibited reduced toxicity to human neuronal model cells and reduced seeding activity in vitro. The effectiveness of O4 decreased when it was added at later points in the ?syn aggregation pathway, which suggests that the incorporation of O4 into fibril assemblies stabilizes them against chemical, enzymatic, and mechanic degradation. These findings suggest that small molecules, which stabilize amyloid fibrils, can prevent fibril fragmentation and seeding and consequently prevent prion-like replication of misfolded ?syn. Inhibiting prion replication by fibril stabilization could thus be a therapeutic strategy for PD.
Project description:We investigated ?-synuclein's (?Syn) seeding activity in tissue from the brain and enteric nervous system. Specifically, we assessed the seeding propensity of pathogenic ?Syn in formalin-fixed tissue from the gastric cardia and five brain regions of 29 individuals (12 Parkinson's disease, 8 incidental Lewy body disease, 9 controls) using a protein misfolding cyclic amplification assay. The structural characteristics of the resultant ?Syn assemblies were determined by limited proteolysis and transmission electron microscopy. We show that fixed tissue from Parkinson's disease (PD) and incidental Lewy body disease (ILBD) seeds the aggregation of monomeric ?Syn into fibrillar assemblies. Significant variations in the characteristics of fibrillar assemblies derived from different regions even within the same individual were observed. This finding suggests that fixation stabilizes seeds with an otherwise limited seeding propensity, that yield assemblies with different intrinsic structures (i.e., strains). The lag phase preceding fibril assembly for patients ?80 was significantly shorter than in other age groups, suggesting the existence of increased numbers of seeds or a higher seeding potential of pathogenic ?Syn with time. Seeding activity did not diminish in late-stage disease. No statistically significant difference in the seeding efficiency of specific regions was found, nor was there a relationship between seeding efficiency and the load of pathogenic ?Syn in a particular region at a given neuropathological stage.
Project description:Alpha-synuclein (α-syn) fibrils, a major constituent of the neurotoxic Lewy Bodies in Parkinson's disease, form via nucleation dependent polymerization and can replicate by a seeding mechanism. Brazilin, a small molecule derived from red cedarwood trees in Brazil, has been shown to inhibit the fibrillogenesis of amyloid-beta (Aβ) and α-syn as well as remodel mature fibrils and reduce cytotoxicity. Here we test the effects of Brazilin on both seeded and unseeded α-syn fibril formation and show that the natural polyphenol inhibits fibrillogenesis of α-syn by a unique mechanism that alters conformational equilibria in two separate points of the assembly mechanism: Brazilin preserves the natively unfolded state of α-syn by specifically binding to the compact conformation of the α-syn monomer. Brazilin also eliminates seeding competence of α-syn assemblies from Parkinson's disease patient brain tissue, and reduces toxicity of pre-formed assemblies in primary neurons by inducing the formation of large fibril clusters. Molecular docking of Brazilin shows the molecule to interact both with unfolded α-syn monomers and with the cross-β sheet structure of α-syn fibrils. Our findings suggest that Brazilin has substantial potential as a neuroprotective and therapeutic agent for Parkinson's disease.
Project description:Seeding, in the context of amyloid disease, is the sequential transfer of pathogenic protein aggregates from cell-to-cell within affected tissues. The structure of pathogenic seeds provides the molecular basis and enables rapid conversion of soluble protein into fibrils. To date, there are no inhibitors that specifically target seeding of Parkinson's disease (PD)-associated ?-synuclein (?-syn) fibrils, in part, due to lack of information of the structural properties of pathological seeds. Here we design small peptidic inhibitors based on the atomic structure of the core of ?-syn fibrils. The inhibitors prevent ?-syn aggregation in vitro and in cell culture models with binding affinities of 0.5 ?M to ?-syn fibril seeds. The inhibitors also show efficacy in preventing seeding by human patient-derived ?-syn fibrils. Our results suggest that pathogenic seeds of ?-syn contain steric zippers and suggest a therapeutic approach targeted at the spread and progression that may be applicable for PD and related synucleinopathies.
Project description:BACKGROUND:Recent studies indicated that seeded fibril formation and toxicity of ?-synuclein (?-syn) play a main role in the pathogenesis of certain diseases including Parkinson's disease (PD), multiple system atrophy, and dementia with Lewy bodies. Therefore, examination of compounds that abolish the process of seeding is considered a key step towards therapy of several synucleinopathies. METHODS:Using biophysical, biochemical and cell-culture-based assays, assessment of eleven compounds, extracted from Chinese medicinal herbs, was performed in this study for their effect on ?-syn fibril formation and toxicity caused by the seeding process. RESULTS:Salvianolic acid B and dihydromyricetin were the two compounds that strongly inhibited the fibril growth and neurotoxicity of ?-syn. In an in-vitro cell model, these compounds decreased the insoluble phosphorylated ?-syn and aggregation. Also, in primary neuronal cells, these compounds showed a reduction in ?-syn aggregates. Both compounds inhibited the seeded fibril growth with dihydromyricetin having the ability to disaggregate preformed ?-syn fibrils. In order to investigate the inhibitory mechanisms of these two compounds towards fibril formation, we demonstrated that salvianolic acid B binds predominantly to monomers, while dihydromyricetin binds to oligomeric species and to a lower extent to monomers. Remarkably, these two compounds stabilized the soluble non-toxic oligomers lacking ?-sheet content after subjecting them to proteinase K digestion. CONCLUSIONS:Eleven compounds were tested but only two showed inhibition of ?-syn aggregation, seeded fibril formation and toxicity in vitro. These findings highlight an essential beginning for development of new molecules in the field of synucleinopathies treatment.
Project description:The aggregation of proteins into oligomers and amyloid fibrils is characteristic of several neurodegenerative diseases, including Parkinson disease (PD). In PD, the process of aggregation of α-synuclein (α-syn) from monomers, via oligomeric intermediates, into amyloid fibrils is considered the disease-causative toxic mechanism. We developed α-syn mutants that promote oligomer or fibril formation and tested the toxicity of these mutants by using a rat lentivirus system to investigate loss of dopaminergic neurons in the substantia nigra. The most severe dopaminergic loss in the substantia nigra is observed in animals with the α-syn variants that form oligomers (i.e., E57K and E35K), whereas the α-syn variants that form fibrils very quickly are less toxic. We show that α-syn oligomers are toxic in vivo and that α-syn oligomers might interact with and potentially disrupt membranes.
Project description:Synucleinopathies including Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA) are characterized by pathological accumulation of ?-synuclein (?-syn). Amongst the various approaches attempting to tackle the pathological features of synucleinopathies, antibody-based immunotherapy holds much promise. However, the large size of antibodies and corresponding difficulty in crossing the blood-brain barrier has limited development in this area. To overcome this issue, we engineered single-chain variable fragments (scFvs) against fibrillar ?-syn, a putative disease-relevant form of ?-syn. The purified scFvs showed specific activity towards ?-syn fibrils and oligomers in comparison to monomers and recognized intracellular inclusions in human post-mortem brain tissue of Lewy body disease cases, but not aged controls. In vitro studies indicated scFvs inhibit the seeding of ?-syn aggregation in a time-dependent manner, decreased ?-syn seed-induced toxicity in a cell model of PD, and reduced the production of insoluble ?-syn phosphorylated at Ser-129 (pS129-?-syn). These results suggest that our ?-syn fibril-specific scFvs recognize ?-syn pathology and can inhibit the aggregation of ?-syn in vitro and prevent seeding-dependent toxicity. Therefore, the scFvs described here have considerable potential to be utilized towards immunotherapy in synucleinopathies and may also have applications in ante-mortem imaging modalities.
Project description:The deposition of fibrillar alpha-synuclein (α-syn) within inclusions (Lewy bodies and Lewy neurites) in neurons and glial cells is a hallmark of synucleinopathies. α-syn populates a variety of assemblies ranging from prefibrillar oligomeric species to fibrils whose specific contribution to neurodegeneration is still unclear. Here, we compare the specific structural and biological properties of distinct soluble prefibrillar α-syn oligomers formed either spontaneously or in the presence of dopamine and glutaraldehyde. We show that both on-fibrillar assembly pathway and distinct dopamine-mediated and glutaraldehyde-cross-linked α-syn oligomers are only slightly effective in perturbing cell membrane integrity and inducing cytotoxicity, while mature fibrils exhibit the highest toxicity. In contrast to low-molecular weight and unstable oligomers, large stable α-syn oligomers seed the aggregation of soluble α-syn within reporter cells although to a lesser extent than mature α-syn fibrils. These oligomers appear elongated in shape. Our findings suggest that α-syn oligomers represent a continuum of species ranging from unstable low molecular weight particles to mature fibrils via stable elongated oligomers composed of more than 15 α-syn monomers that possess seeding capacity.
Project description:Oligomerization of ?-Synuclein is known to have implications for both neurodegeneration and cancer. Although it is known to co-exist with the fibrillar deposits of ?-Synuclein (Lewy bodies), a hallmark in Parkinson's disease (PD), the effect of potential therapeutic modulators on the fibrillation pathway of ?-Syn remains unexplored. By a combined use of various biophysical tools and cytotoxicity assays we demonstrate that the flavonoid epigallocatechin-3-gallate (EGCG) significantly suppresses ?-Syn fibrillation by affecting its nucleation and binds with the unstructured, nucleus forming oligomers of ?-Syn to modulate the pathway to form ?-helical containing higher-order oligomers (~158 kDa and ~ 670 kDa) that are SDS-resistant and conformationally restrained in nature. Seeding studies reveal that these oligomers although "on-pathway" in nature, are kinetically retarded and rate-limiting species that slows down fibril elongation. We observe that EGCG also disaggregates the protofibrils and mature ?-Syn fibrils into similar SDS-resistant oligomers. Steady-state and time-resolved fluorescence spectroscopy and isothermal titration calorimetry (ITC) reveal a weak non-covalent interaction between EGCG and ?-Syn with the dissociation constant in the mM range (K<sub>d</sub> ~ 2-10 mM). Interestingly, while EGCG-generated oligomers completely rescue the breast cancer (MCF-7) cells from ?-Syn toxicity, it reduces the viability of neuroblastoma (SH-SY5Y) cells. However, the disaggregated oligomers of ?-Syn are more toxic than the disaggregated fibrils for MCF-7cells. These findings throw light on EGCG-mediated modulation of ?-Syn fibrillation and suggest that investigation on the effects of such modulators on ?-Syn fibrillation is critical in identifying effective therapeutic strategies using small molecule modulators of synucleopathies.
Project description:Protein misfolding disorders are associated with conformational changes in specific proteins, leading to the formation of potentially neurotoxic amyloid fibrils. During pathogenesis of prion disease, the prion protein misfolds into ?-sheet rich, protease-resistant isoforms. A key, hydrophobic domain within the prion protein, comprising residues 109-122, recapitulates many properties of the full protein, such as helix-to-sheet structural transition, formation of fibrils and cytotoxicity of the misfolded isoform. Using all-atom, molecular simulations, it is demonstrated that the monomeric 109-122 peptide has a preference for ?-helical conformations, but that this peptide can also form ?-hairpin structures resulting from turns around specific glycine residues of the peptide. Altering a single amino acid within the 109-122 peptide (A117V, associated with familial prion disease) increases the prevalence of ?-hairpin formation and these observations are replicated in a longer peptide, comprising residues 106-126. Multi-molecule simulations of aggregation yield different assemblies of peptide molecules composed of conformationally-distinct monomer units. Small molecular assemblies, consistent with oligomers, comprise peptide monomers in a ?-hairpin-like conformation and in many simulations appear to exist only transiently. Conversely, larger assemblies are comprised of extended peptides in predominately antiparallel ?-sheets and are stable relative to the length of the simulations. These larger assemblies are consistent with amyloid fibrils, show cross-? structure and can form through elongation of monomer units within pre-existing oligomers. In some simulations, assemblies containing both ?-hairpin and linear peptides are evident. Thus, in this work oligomers are on pathway to fibril formation and a preference for ?-hairpin structure should enhance oligomer formation whilst inhibiting maturation into fibrils. These simulations provide an important new atomic-level model for the formation of oligomers and fibrils of the prion protein and suggest that stabilization of ?-hairpin structure may enhance cellular toxicity by altering the balance between oligomeric and fibrillar protein assemblies.
Project description:The aggregation of ?-synuclein (?-syn) into amyloid fibrils is a major pathological hallmark of Parkinson's disease (PD) and other synucleinopathies. The mechanisms underlying the structural transition of soluble and innocuous ?-syn to aggregated neurotoxic forms remains largely unknown. The disordered nature of ?-syn has hampered the use of structure-based protein engineering approaches to elucidate the molecular determinants of this transition. The recent 3D structure of a pathogenic ?-syn fibril provides a template for this kind of studies. The structure supports the NAC domain being a critical element in fibril formation, since it constitutes the core of the fibril, delineating a Greek-key motif. Here, we stapled the ends of this motif with a designed disulfide bond and evaluated its impact on the conformation, aggregation and toxicity of ?-syn in different environments. The new covalent link biases the native structural ensemble of ?-syn toward compact conformations, reducing the population of fully unfolded species. This conformational bias results in a strongly reduced fibril formation propensity both in the absence and in the presence of lipids and impedes the formation of neurotoxic oligomers. Our study does not support the Greek-key motif being already imprinted in early ?-syn assemblies, discarding it as a druggable interface to prevent the initiation of fibrillation. In contrast, it suggests the stabilization of native, compact ensembles as a potential therapeutic strategy to avoid the formation of toxic species and to target the early stages of PD.