Mechanistic insights into accelerated α-synuclein aggregation mediated by human microbiome-associated functional amyloids.
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ABSTRACT:
SUBMITTER: Bhoite SS
PROVIDER: S-EPMC9253359 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Aggregated α-synuclein (α-syn) is the main constituent of Lewy bodies, which are a pathological hallmark of Parkinson's disease (PD). Environmental factors are thought to be potential triggers capable of initiating the aggregation of the otherwise monomeric α-syn. Braak's seminal work redirected attention to the intestine and recent reports of dysbiosis have highlighted the potential causative role of the microbiome in the initiation of pathology of PD. Staphylococcus aureus is a bacterium carried by 30-70% of the general population. It has been shown to produce functional amyloids, called phenol soluble modulins (PSMαs). Here, we studied the kinetics of α-syn aggregation under quiescent conditions in the presence or absence of four different PSMα peptides and observed a remarkable shortening of the lag phase in their presence. Whereas pure α-syn monomer did not aggregate up to 450 h after initiation of the experiment in neither neutral nor mildly acidic buffer, the addition of different PSMα peptides resulted in an almost immediate increase in the Thioflavin T (ThT) fluorescence. Despite similar peptide sequences, the different PSMα peptides displayed distinct effects on the kinetics of α-syn aggregation. Kinetic analyses of the data suggest that all four peptides catalyze α-syn aggregation through heterogeneous primary nucleation. The immunogold electron microscopic analyses showed that the aggregates were fibrillar and composed of α-syn. In addition of the co-aggregated materials to a cell model expressing the A53T α-syn variant fused to GFP was found to catalyze α-syn aggregation and phosphorylation in the cells. Our results provide evidence of a potential trigger of synucleinopathies and could have implications for the prevention of the diseases.
| S-EPMC8584152 | biostudies-literature
Project description:α-Synuclein (aSyn) aggregation is an attractive target for therapeutic development for a range of neurodegenerative conditions, collectively termed synucleinopathies. Here, we probe the mechanism of action of a peptide 4554W, (KDGIVNGVKA), previously identified through intracellular library screening, to prevent aSyn aggregation and associated toxicity. We utilize NMR to probe association and identify that 4554W associates with a "partially aggregated" form of aSyn, with enhanced association occurring over time. We also report the ability of 4554W to undergo modification through deamidation of the central asparagine residue, occurring on the same timescale as aSyn aggregation in vitro, with peptide modification enhancing its association with aSyn. Additionally, we report that 4554W can act to reduce fibril formation of five Parkinson's disease associated aSyn mutants. Inhibitory peptide binding to partially aggregated forms of aSyn, as identified here, is particularly attractive from a therapeutic perspective, as it would eliminate the need to administer the therapy at pre-aggregation stages, which are difficult to diagnose. Taken together the data suggest that 4554W could be a suitable candidate for future therapeutic development against wild-type, and most mutant aSyn aggregation.
| S-EPMC7594713 | biostudies-literature
Project description:The aggregation of the amyloidogenic protein α-synuclein (α-Syn) into toxic oligomers and mature fibrils is the major pathological hallmark of Parkinson's disease (PD). Small molecules that inhibit α-Syn aggregation thus may be useful therapeutics for PD. Mannitol and naphthoquinone-tryptophan (NQTrp) have been shown in the past to inhibit α-Syn aggregation by different mechanisms. Herein, we tested whether the conjugation of Mannitol and NQTrp may result in enhance efficacy toward α-Syn. The molecules were conjugated either by a click linker or via a PEG linker. The effect of the conjugate molecules on α-Syn aggregation in vitro was monitored using Thioflavin T fluorescence assay, circular dichroism, transmission electron microscopy, and Congo red birefringence assay. One of the conjugate molecules was found to be more effective than the two parent molecules and as effective as a mixture of the two. The conjugate molecules attenuated the disruptive effect of α-Syn on artificial membrane of Large Unilamellar Vesicles as monitored by dye leakage assay. The conjugates were found to be have low cytotoxicity and reduced toxicity of α-Syn toward SH-SY5Y neuroblastoma cells. These novel designed entities can be attractive scaffold for the development of therapeutic agents for PD.
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Project description:N-α-acetylation is a frequently occurring post-translational modification in eukaryotic proteins. It has manifold physiological consequences on the regulation and function of several proteins, with emerging studies suggesting that it is a global regulator of stress responses. For decades, in vitro biochemical investigations into the precise role of the intrinsically disordered protein alpha-synuclein (αS) in the etiology of Parkinson's disease (PD) were performed using non-acetylated αS. The N-terminus of α-synuclein is now unequivocally known to be acetylated in vivo, however, there are many aspects of this post-translational modifications that are not understood well. Is N-α-acetylation of αS a constitutive modification akin to most cellular proteins, or is it spatio-temporally regulated? Is N-α-acetylation of αS relevant to the as yet elusive function of αS? How does the N-α-acetylation of αS influence the aggregation of αS into amyloids? Here, we provide an overview of the current knowledge and discuss prevailing hypotheses on the impact of N-α-acetylation of αS on its conformational, oligomeric, and fibrillar states. The extent to which N-α-acetylation of αS is vital for its function, membrane binding, and aggregation into amyloids is also explored here. We further discuss the overall significance of N-α-acetylation of αS for its functional and pathogenic implications in Lewy body formation and synucleinopathies.
| S-EPMC9709446 | biostudies-literature
Project description:α-Synuclein misfolding and aggregation play an important role in the pathogenesis of Parkinson's disease (PD). Loss of function and mutation of the PARK7/DJ-1 gene cause early-onset familial PD. DJ-1 can inhibit α-synuclein aggregation, and may function at an early step in the aggregation process. Soluble wild-type (WT) α-synuclein is mainly degraded by chaperone-mediated autophagy (CMA), and impairment of CMA is closely related to the pathogenesis of PD. Here, we investigated whether DJ-1 could reduce α-synuclein accumulation and aggregation by CMA. DJ-1 knockout mice and DJ-1 siRNA knockdown SH-SY5Y cells were used to investigate the potential mechanisms underlying the relationship between DJ-1 deficiency and α-synuclein aggregation. First, we confirmed that DJ-1 deficiency increased the accumulation and aggregation of α-synuclein in both SH-SY5Y cells and PD animal models, and overexpression of DJ-1 in vitro effectively decreased α-synuclein levels. α-Synuclein overexpression activated CMA by elevating the levels of lysosome-associated membrane protein type-2A (LAMP2A), but DJ-1 deficiency suppressed upregulation of LAMP2A. DJ-1 deficiency downregulated the level of lysosomal 70 kDa heat-shock cognate protein (HSC70) but not the levels of that in homogenates. Further studies showed that DJ-1 deficiency accelerated the degradation of LAMP2A in lysosomes, leading to the aggregation of α-synuclein. Our study suggests that DJ-1 deficiency aggravates α-synuclein aggregation by inhibiting the activation of CMA and provides further evidence of the molecular interaction between PD-related proteins via the CMA pathway.
| S-EPMC5623690 | biostudies-literature
Project description:Methamphetamine (METH) is an illegal and widely abused psychoactive stimulant. METH abusers are at high risk of neurodegenerative disorders, including Parkinson's disease (PD). Previous studies have demonstrated that METH causes alpha-synuclein (?-syn) aggregation in the both laboratory animal and human. In this study, exposure to high METH doses increased the expression of ?-syn and the small ubiquitin-related modifier 1 (SUMO-1). Therefore, we hypothesized that SUMOylation of ?-syn is involved in high-dose METH-induced ?-syn aggregation. We measured the levels of ?-syn SUMOylation and these enzymes involved in the SUMOylation cycle in SH-SY5Y human neuroblastoma cells (SH-SY5Y cells), in cultures of C57 BL/6 primary mouse neurons and in brain tissues of mice exposure to METH. We also demonstrated the effect of ?-syn SUMOylation on ?-syn aggregation after METH exposure by overexpressing the key enzyme of the SUMOylation cycle or silencing SUMO-1 expression in vitro. Then, we make introduced mutations in the major SUMOylation acceptor sites of ?-syn by transfecting a lentivirus containing the sequence of WT ?-syn or K96/102R ?-syn into SH-SY5Y cells and injecting an adenovirus containing the sequence of WT ?-syn or K96/102R ?-syn into the mouse striatum. Levels of the ubiquitin-proteasome system (UPS)-related makers ubiquitin (Ub) and UbE1, as well as the autophagy-lysosome pathway (ALP)-related markers LC3, P62 and lysosomal associated membrane protein 2A (LAMP2A), were also measured in SH-SY5Y cells transfected with lentivirus and mice injected with adenovirus. The results showed that METH exposure decreases the SUMOylation level of ?-syn, although the expression of ?-syn and SUMO-1 are increased. One possible cause is the reduction of UBC9 level. The increase in ?-syn SUMOylation by UBC9 overexpression relieves METH-induced ?-syn overexpression and aggregation, whereas the decrease in ?-syn SUMOylation by SUMO-1 silencing exacerbates the same pathology. Furthermore, mutations in the major SUMOylation acceptor sites of ?-syn also aggravate ?-syn overexpression and aggregation by impairing degradation through the UPS and the ALP in vitro and in vivo. These results suggest that SUMOylation of ?-syn plays a fundamental part in ?-syn overexpression and aggregation induced by METH and could be a suitable target for the treatment of neurodegenerative diseases.
| S-EPMC6117395 | biostudies-literature
Project description:Parkinson's disease (PD), a neurodegenerative disorder characterized by distinct aging-independent loss of dopaminergic neurons in substantia nigra pars compacta (SNpc) region urging toward neuronal loss. Over the decade, various key findings from clinical perspective to molecular pathogenesis have aided in understanding the genetics with assorted genes related with PD. Subsequently, several pathways have been incriminated in the pathogenesis of PD, involving mitochondrial dysfunction, protein aggregation, and misfolding. On the other hand, the sporadic form of PD cases is found with no genetic linkage, which still remain an unanswered question? The exertion in ascertaining vulnerability factors in PD considering the genetic factors are to be further dissevered in the forthcoming decades with advancement in research studies. One of the major proponents behind the prognosis of PD is the pathogenic transmutation of aberrant alpha-synuclein protein into amyloid fibrillar structures, which actuates neurodegeneration. Alpha-synuclein, transcribed by SNCA gene is a neuroprotein found predominantly in brain. It is implicated in the modulation of synaptic vesicle transport and eventual release of neurotransmitters. Due to genetic mutations and other elusive factors, the alpha-synuclein misfolds into its amyloid form. Therefore, this review aims in briefing the molecular understanding of the alpha-synuclein associated with PD.
| S-EPMC8558257 | biostudies-literature
Project description:The protein alpha-synuclein (?S) self-assembles into small oligomeric species and subsequently into amyloid fibrils that accumulate and proliferate during the development of Parkinson's disease. However, the quantitative characterization of the aggregation and spreading of ?S remains challenging to achieve. Previously, we identified a conformational conversion step leading from the initially formed oligomers to more compact oligomers preceding fibril formation. Here, by a combination of single-molecule fluorescence measurements and kinetic analysis, we find that the reaction in solution involves two unimolecular structural conversion steps, from the disordered to more compact oligomers and then to fibrils, which can elongate by further monomer addition. We have obtained individual rate constants for these key microscopic steps by applying a global kinetic analysis to both the decrease in the concentration of monomeric protein molecules and the increase in oligomer concentrations over a 0.5-140-µM range of ?S. The resulting explicit kinetic model of ?S aggregation has been used to quantitatively explore seeding the reaction by either the compact oligomers or fibrils. Our predictions reveal that, although fibrils are more effective at seeding than oligomers, very high numbers of seeds of either type, of the order of 10(4), are required to achieve efficient seeding and bypass the slow generation of aggregates through primary nucleation. Complementary cellular experiments demonstrated that two orders of magnitude lower numbers of oligomers were sufficient to generate high levels of reactive oxygen species, suggesting that effective templated seeding is likely to require both the presence of template aggregates and conditions of cellular stress.
| S-EPMC4780632 | biostudies-literature
Project description:Endoplasmic reticulum (ER) dysfunction is important for alpha-synuclein (αS) acquired toxicity. When targeted to the ER in SH-SY5Y cells, transient or stable expression of αS resulted in the formation of compact αS-positive structures in a small subpopulation of cells, resembling αS inclusions. Thus, because of the limitations of immunofluorescence, we developed a set of αS FRET biosensors (AFBs) able to track αS conformation in cells. In native conditions, expression in i36, a stable cell line for ER αS, of intermolecular AFBs, reporters in which CFP or YFP has been fused with the C-terminal of αS (αS-CFP/αS-YFP), resulted in no Förster resonance energy transfer (FRET), whereas expression of the intramolecular AFB, a probe obtained by fusing YFP and CFP with αS N- or C- termini (YFP-αS-CFP), showed a positive FRET signal. These data confirmed that αS has a predominantly globular, monomeric conformation in native conditions. Differently, under pro-aggregating conditions, the intermolecular AFB was able to sense significantly formation of αS oligomers, when AFB was expressed in the ER rather than ubiquitously, suggesting that the ER can favor changes in αS conformation when aggregation is stimulated. These results show the potential of AFBs as a new, valuable tool to track αS conformational changes in vivo.
| S-EPMC7460339 | biostudies-literature
Project description:Aggregation of alpha-synuclein (αSyn) is a crucial event underlying the pathophysiology of synucleinopathies. The existence of various intracellular and extracellular αSyn species, including cleaved αSyn, complicates the quest for an appropriate therapeutic target. Hence, to develop efficient disease-modifying strategies, it is fundamental to achieve a deeper understanding of the relevant spreading and toxic αSyn species. Here, we describe comparative and proof-of-principle approaches to determine the involvement of αSyn fragments in intercellular spreading. We demonstrate that two different αSyn fragments (1-95 and 61-140) fulfill the criteria of spreading species. They efficiently instigate formation of proteinase-K-resistant aggregates from cell-endogenous full-length αSyn, and drive it into different aggregation pathways. The resulting aggregates induce cellular toxicity. Strikingly, these aggregates are only detectable by specific antibodies. Our results suggest that αSyn fragments might be relevant not only for spreading, but also for aggregation-fate determination and differential strain formation.
| S-EPMC6997403 | biostudies-literature