Natural Alkaloid Compounds as Inhibitors for Alpha-Synuclein Seeded Fibril Formation and Toxicity.
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ABSTRACT:
SUBMITTER: Ghanem SS
PROVIDER: S-EPMC8234408 | biostudies-literature |
REPOSITORIES: biostudies-literature
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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.
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Project description:The aggregation of α-synuclein (α-syn) is considered the key pathogenic event in many neurological disorders such as Parkinson's disease (PD), dementia with Lewy bodies and multiple system atrophy, giving rise to a whole category of neurodegenerative diseases known as synucleinopathies. Although the molecular basis of α-syn toxicity has not been precisely elucidated, a great deal of effort has been put into identifying compounds that could inhibit or even reverse the aggregation process. Previous reports indicated that many phenolic compounds are potent inhibitors of α-syn aggregation. The aim of the present study was to assess the anti-aggregating effect of gallic acid (GA) (3,4,5-trihydroxybenzoic acid), a benzoic acid derivative that belongs to a group of phenolic compounds known as phenolic acids. By employing an array of biophysical and biochemical techniques and a cell-viability assay, GA was shown not only to inhibit α-syn fibrillation and toxicity but also to disaggregate preformed α-syn amyloid fibrils. Interestingly, GA was found to bind to soluble, non-toxic oligomers with no β-sheet content, and to stabilize their structure. The binding of GA to the oligomers may represent a potential mechanism of action. Additionally, by using structure activity relationship data obtained from fourteen structurally similar benzoic acid derivatives, it was determined that the inhibition of α-syn fibrillation by GA is related to the number of hydroxyl moieties and their position on the phenyl ring. GA may represent the starting point for designing new molecules that could be used for the treatment of PD and related disorders.
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Project description:Aggregation of the protein α-Synuclein (αSyn) is of great interest due to its involvement in the pathology of Parkinson's disease. However, under in vitro conditions αSyn is very soluble and kinetically stable for extended time periods. As a result, most αSyn aggregation assays rely on conditions that artificially induce or enhance aggregation, often by introducing rather non-native conditions. It has been shown that αSyn interacts with membranes and conditions have been identified in which membranes can promote as well as inhibit αSyn aggregation. It has also been shown that αSyn has the intrinsic capability to assemble lipid-protein-particles, in a similar way as apolipoproteins can form lipid-bilayer nanodiscs. Here we show that these αSyn-lipid particles (αSyn-LiPs) can also effectively induce, accelerate or inhibit αSyn aggregation, depending on the applied conditions. αSyn-LiPs therefore provide a general platform and additional tool, complementary to other setups, to study various aspects of αSyn amyloid fibril formation.
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Project description:There is much interest in delineating the mechanisms by which the α-synuclein protein accumulates in brains of individuals with Parkinson's disease (PD). Preclinical studies with rodent and primate models have indicated that fibrillar forms of α-synuclein can initiate the propagation of endogenous α-synuclein pathology. However, the underlying mechanisms by which α-synuclein fibrils seed pathology remain unclear. To investigate this further, we have used exogenous fibrillar α-synuclein to seed endogenous α-synuclein pathology in human neuronal cell lines, including primary human neurons differentiated from induced pluripotent stem cells. Fluorescence microscopy and immunoblot analyses were used to monitor levels of α-synuclein and key autophagy/lysosomal proteins over time in the exogenous α-synuclein fibril-treated neurons. We observed that temporal changes in the accumulation of cytoplasmic α-synuclein inclusions were associated with changes in the key autophagy/lysosomal markers. Of note, chloroquine-mediated blockade of autophagy increased accumulation of α-synuclein inclusions, and rapamycin-induced activation of autophagy, or use of 5'-AMP-activated protein kinase (AMPK) agonists, promoted the clearance of fibril-mediated α-synuclein pathology. These results suggest a key role for autophagy in clearing fibrillar α-synuclein pathologies in human neuronal cells. We propose that our findings may help inform the development of human neural cell models for screening of potential therapeutic compounds for PD or for providing insight into the mechanisms of α-synuclein propagation. Our results further add to existing evidence that AMPK activation may be a therapeutic option for managing PD.
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Project description:Amyloid fibril formation is associated with various amyloidoses, including neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Amyloid fibrils form above the solubility of amyloidogenic proteins or peptides upon breaking supersaturation, followed by a nucleation and elongation mechanism, which is similar to the crystallization of solutes. Many additives, including salts, detergents, and natural compounds, promote or inhibit amyloid formation. However, the underlying mechanisms of the opposing effects are unclear. We examined the effects of two polyphenols, that is, epigallocatechin gallate (EGCG) and kaempferol-7─O─glycoside (KG), with high and low solubilities, respectively, on the amyloid formation of α-synuclein (αSN). EGCG and KG inhibited and promoted amyloid formation of αSN, respectively, when monitored by thioflavin T (ThT) fluorescence or transmission electron microscopy (TEM). Nuclear magnetic resonance (NMR) analysis revealed that, although interactions of αSN with soluble EGCG increased the solubility of αSN, thus inhibiting amyloid formation, interactions of αSN with insoluble KG reduced the solubility of αSN, thereby promoting amyloid formation. Our study suggests that opposing effects of polyphenols on amyloid formation of proteins and peptides can be interpreted based on the solubility of polyphenols.
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Project description:The generation of α-synuclein (α-syn) truncations from incomplete proteolysis plays a significant role in the pathogenesis of Parkinson's disease. It is well established that C-terminal truncations exhibit accelerated aggregation and serve as potent seeds in fibril propagation. In contrast, mechanistic understanding of N-terminal truncations remains ill defined. Previously, we found that disease-related C-terminal truncations resulted in increased fibrillar twist, accompanied by modest conformational changes in a more compact core, suggesting that the N-terminal region could be dictating fibril structure. Here, we examined three N-terminal truncations, in which deletions of 13-, 35-, and 40-residues in the N terminus modulated both aggregation kinetics and fibril morphologies. Cross-seeding experiments showed that out of the three variants, only ΔN13-α-syn (14‒140) fibrils were capable of accelerating full-length fibril formation, albeit slower than self-seeding. Interestingly, the reversed cross-seeding reactions with full-length seeds efficiently promoted all but ΔN40-α-syn (41-140). This behavior can be explained by the unique fibril structure that is adopted by 41-140 with two asymmetric protofilaments, which was determined by cryogenic electron microscopy. One protofilament resembles the previously characterized bent β-arch kernel, comprised of residues E46‒K96, whereas in the other protofilament, fewer residues (E61‒D98) are found, adopting an extended β-hairpin conformation that does not resemble other reported structures. An interfilament interface exists between residues K60‒F94 and Q62‒I88 with an intermolecular salt bridge between K80 and E83. Together, these results demonstrate a vital role for the N-terminal residues in α-syn fibril formation and structure, offering insights into the interplay of α-syn and its truncations.
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Project description: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.
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Project description:Alpha-synuclein (alpha-syn), a presynaptic protein implicated in Parkinson's disease, binds copper(II) ion (1:1) with submicromolar affinity in vitro. Insights on the molecular details of soluble- and fibrillar-Cu-alpha-syn are gained through X-ray absorption spectroscopy. Our results indicate that the copper coordination environment (3-to-4 N/O ligands, average Cu-ligand distance approximately 1.96 A) exhibits little structural rearrangement upon amyloid formation in spite of the overall polypeptide conformational change from disordered-to-beta-sheet. Interestingly, we find that some population of Cu(II)-alpha-syn reduces to Cu(I)-alpha-syn in the absence of O(2). This autoreduction event appears diminished in the presence of O(2) suggestive of preceding Cu(I)/O(2) chemistry. Evidence for generation of reactive oxygen species is obtained by the observation of new emission features attributed to dityrosine cross-links in fibrillar samples.
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Project description:α-Synuclein (αSyn), which forms amyloid fibrils, is linked to the neuronal pathology of Parkinson's disease, as it is the major fibrillar component of Lewy bodies, the inclusions that are characteristic of the disease. Oligomeric structures, common to many neurodegenerative disease-related proteins, may in fact be the primary toxic species, while the amyloid fibrils exist either as a less toxic dead-end species or even as a beneficial mechanism for clearing damaged proteins. To alter the progression of the aggregation and gain insights into the prefibrillar structures, we determined the effect of heme on αSyn oligomerization by several different techniques, including native (nondenaturing) polyacrylamide gel electrophoresis, thioflavin T fluorescence, transmission electron microscopy, atomic force microscopy, circular dichroism, and membrane permeation using a calcein release assay. During aggregation, heme is able to bind the αSyn in a specific fashion, stabilizing distinct oligomeric conformations and promoting the formation of αSyn into annular structures, thereby delaying and/or inhibiting the fibrillation process. These results indicate that heme may play a regulatory role in the progression of Parkinson's disease; in addition, they provide insights into how the aggregation process may be altered, which may be applicable to the understanding of many neurodegenerative diseases.
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Project description:The role of alpha-synuclein (αS) amyloid fibrillation has been recognized in various neurological diseases including Parkinson's Disease (PD). In early stages, fibrillation occurs by the structural transition from helix to extended states in monomeric αS followed by the formation of beta-sheets. This alpha-helix to beta-sheet transition (αβT) speeds up the formation of amyloid fibrils through the formation of unstable and temporary configurations of the αS. In this study, the most important regions that act as initiating nuclei and make unstable the initial configuration were identified based on sequence and structural information. In this regard, a Targeted Molecular Dynamics (TMD) simulation was employed using explicit solvent models under physiological conditions. Identified regions are those that are in the early steps of structural opening. The trajectory was clustered the structures characterized the intermediate states. The findings of this study would help us to better understanding of the mechanism of amyloid fibril formation.
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