Alpha-synuclein amyloid fibres incubated with chaperones (DNAJB1, Hsc70, +/- Apg2)
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ORGANISM(S): Homo sapiens (human)
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PROVIDER: S-BIAD415 | bioimages |
REPOSITORIES: bioimages
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Project description:α-Synuclein (α-Syn) aggregation into fibrils with prion-like features is intimately associated with Lewy pathology and various synucleinopathies. Emerging studies suggest that α-Syn could form liquid condensates through phase separation. The role of these condensates in aggregation and disease remains elusive and the interplay between α-Syn fibrils and α-Syn condensates remains unexplored, possibly due to difficulties in triggering the formation of α-Syn condensates in cells. To address this gap, we developed an assay allowing the controlled assembly/disassembly of α-Syn condensates in cells and studied them upon exposure to preformed α-Syn fibrillar polymorphs. Fibrils triggered the evolution of liquid α-Syn condensates into solid-like structures displaying growing needle-like extensions and exhibiting pathological amyloid hallmarks. No such changes were elicited on α-Syn that did not undergo phase separation. We, therefore, propose a model where α-Syn within condensates fuels exogenous fibrillar seeds growth, thus speeding up the prion-like propagation of pathogenic aggregates.
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Project description:Parkinson's disease (PD) is an increasingly prevalent and currently incurable neurodegenerative disorder linked to the accumulation of α-synuclein (αS) protein aggregates in the nervous system. While αS binding to membranes in its monomeric state is correlated to its physiological role, αS oligomerization and subsequent aberrant interactions with lipid bilayers have emerged as key steps in PD-associated neurotoxicity. However, little is known of the mechanisms that govern the interactions of oligomeric αS (OαS) with lipid membranes and the factors that modulate such interactions. This is in large part due to experimental challenges underlying studies of OαS-membrane interactions due to their dynamic and transient nature. Here, we address this challenge by using a suite of microfluidics-based assays that enable in-solution quantification of OαS-membrane interactions. We find that OαS bind more strongly to highly curved, rather than flat, lipid membranes. By comparing the membrane-binding properties of OαS and monomeric αS (MαS), we further demonstrate that OαS bind to membranes with up to 150-fold higher affinity than their monomeric counterparts. Moreover, OαS compete with and displace bound MαS from the membrane surface, suggesting that disruption to the functional binding of MαS to membranes may provide an additional toxicity mechanism in PD. These findings present a binding mechanism of oligomers to model membranes, which can potentially be targeted to inhibit the progression of PD.
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Project description:Parkinson´s disease (PD) pathology progresses throughout the nervous system. Whereas motor symptoms are always present, there is a high variability in the prevalence of non-motor symptoms. It has been postulated that the progression of the pathology is based on a prion-like disease mechanism partly due to the seeding effect of endocytosed-alpha-synuclein (ASYN) on the endogenous ASYN. Here, we analyzed the role of endogenous ASYN in the progression of PD-like pathology in vivo and in vitro and compared the effect of endocytosed-ASYN as well as paraquat and rotenone on primary enteric, dopaminergic and cortical neurons from wild-type and ASYN-KO mice. Our results show that, in vivo, pathology progression did not occur in the absence of endogenous ASYN. Remarkably, the damage caused by endocytosed-ASYN, rotenone or paraquat was independent from endogenous ASYN and related to the alteration of the host´s mitochondrial membrane potential. Dopaminergic neurons were very sensitive to these noxae compared to other neuronal subtypes. These results suggest that ASYN-mitochondrial interactions play a major role in initiating the pathological process in the host neuron and endogenous ASYN is essential for the transsynaptical transmission of the pathology. Our results also suggest that protecting mitochondrial function is a valid primary therapeutic target.
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Project description:Emerging evidence supports that altered α-tubulin acetylation occurs in Parkinson's disease (PD), a neurodegenerative disorder characterized by the deposition of α-synuclein fibrillary aggregates within Lewy bodies and nigrostriatal neuron degeneration. Nevertheless, studies addressing the interplay between α-tubulin acetylation and α-synuclein are lacking. Here, we investigated the relationship between α-synuclein and microtubules in primary midbrain murine neurons and the substantia nigra of post-mortem human brains. Taking advantage of immunofluorescence and Proximity Ligation Assay (PLA), a method allowing us to visualize protein-protein interactions in situ, combined with confocal and super-resolution microscopy, we found that α-synuclein and acetylated α-tubulin colocalized and were in close proximity. Next, we employed an α-synuclein overexpressing cellular model and tested the role of α-tubulin acetylation in α-synuclein oligomer formation. We used the α-tubulin deacetylase HDAC6 inhibitor Tubacin to modulate α-tubulin acetylation, and we evaluated the presence of α-synuclein oligomers by PLA. We found that the increase in acetylated α-tubulin significantly induced α-synuclein oligomerization. In conclusion, we unraveled the link between acetylated α-tubulin and α-synuclein and demonstrated that α-tubulin acetylation could trigger the early step of α-synuclein aggregation. These data suggest that the proper regulation of α-tubulin acetylation might be considered a therapeutic strategy to take on PD.
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Project description:AimsWe previously found that a decoy peptide derived from the C-terminal sequence of α-Synuclein (αSyn) prevents cytotoxic αSyn aggregation caused by fatty acid-binding protein 3 (FABP3) in vitro. In this study, we continued to utilize αSyn-derived peptides to further validate their effects on αSyn neurotoxicity and behavioral impairments in αSyn preformed fibrils (PFFs)-injected mouse model of Parkinson's disease (PD).MethodsMice were injected with αSyn PFFs in the bilateral olfactory bulb (OB) and then were subjected to behavioral analysis at 2-week intervals post-injection. Peptides nasal administration was initiated one week after injection. Changes in phosphorylation of αSyn and neuronal damage in the OB were measured using immunostaining at week 4. The effect of peptides on the interaction between αSyn and FABP3 was examined using co-immunoprecipitation.ResultsαSyn PFF-injected mice showed significant memory loss but no motor function impairment. Long-term nasal treatment with peptides effectively prevented memory impairment. In peptide-treated αSyn PFF-injected mice, the peptides entered the OB smoothly through the nasal cavity and were mainly concentrated in neurons in the mitral cell layer, significantly suppressing the excessive phosphorylation of αSyn and reducing the formation of αSyn-FABP3 oligomers, thereby preventing neuronal death. The addition of peptides also blocked the interaction of αSyn and FABP3 at the recombinant protein level, and its effect was strongest at molar concentrations comparable to those of αSyn and FABP3.ConclusionsOur findings suggest that the αSyn decoy peptide represents a novel therapeutic approach for reducing the accumulation of toxic αSyn-FABP3 oligomers in the brain, thereby preventing the progression of synucleinopathies.
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Project description: Not available
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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 (α-syn) aggregation in the both laboratory animal and human. In this study, exposure to high METH doses increased the expression of α-syn and the small ubiquitin-related modifier 1 (SUMO-1). Therefore, we hypothesized that SUMOylation of α-syn is involved in high-dose METH-induced α-syn aggregation. We measured the levels of α-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 α-syn SUMOylation on α-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 α-syn by transfecting a lentivirus containing the sequence of WT α-syn or K96/102R α-syn into SH-SY5Y cells and injecting an adenovirus containing the sequence of WT α-syn or K96/102R α-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 α-syn, although the expression of α-syn and SUMO-1 are increased. One possible cause is the reduction of UBC9 level. The increase in α-syn SUMOylation by UBC9 overexpression relieves METH-induced α-syn overexpression and aggregation, whereas the decrease in α-syn SUMOylation by SUMO-1 silencing exacerbates the same pathology. Furthermore, mutations in the major SUMOylation acceptor sites of α-syn also aggravate α-syn overexpression and aggregation by impairing degradation through the UPS and the ALP in vitro and in vivo. These results suggest that SUMOylation of α-syn plays a fundamental part in α-syn overexpression and aggregation induced by METH and could be a suitable target for the treatment of neurodegenerative diseases.
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Project description:BackgroundAlpha-synuclein (α-syn) is involved in pathology of Parkinson's disease, and 90% of α-syn in Lewy bodies is phosphorylated at serine 129 (pS129 α-syn).ObjectiveTo assess behavior impairments and brain levels of α-syn and pS129 α-syn in mice overexpressing human α-syn under Thy1 promoter (Thy1-α-syn) and wild type (wt) littermates.MethodsMotor and non-motor behaviors were monitored, brain human α-syn levels measured by ELISA, and α-syn and pS129 α-syn mapped by immunohistochemistry.ResultsMale and female wt littermates did not show differences in the behavioral tests. Male Thy1-α-syn mice displayed more severe impairments than female counterparts in cotton nesting, pole tests, adhesive removal, finding buried food, and marble burying. Concentrations of human α-syn in the olfactory regions, cortex, nigrostriatal system, and dorsal medulla were significantly increased in Thy1-α-syn mice, higher in males than females. Immunoreactivity of α-syn was not simply increased in Thy1-α-syn mice but had altered localization in somas and fibers in a few brain areas. Abundant pS129 α-syn existed in many brain areas of Thy1-α-syn mice, while there was none or only a small amount in a few brain regions of wt mice. The substantia nigra, olfactory regions, amygdala, lateral parabrachial nucleus, and dorsal vagal complex displayed different distribution patterns between wt and transgenic mice, but not between sexes.ConclusionThe severer abnormal behaviors in male than female Thy1-α-syn mice may be related to higher brain levels of human α-syn, in the absence of sex differences in the altered brain immunoreactivity patterns of α-syn and pS129 α-syn.
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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:Abnormal α-synuclein (αSyn), including an oligomeric form of αSyn, accumulates and causes neuronal dysfunction in the brains of patients with multiple system atrophy. Neuroprotective drugs that target abnormal αSyn aggregation have not been developed for the treatment of multiple system atrophy. In addition, treating diseases at an early stage is crucial to halting the progress of neuronal damage in neurodegeneration. In this study, using early-stage multiple system atrophy mouse model and in vitro kinetic analysis, we investigated how intranasal and oral administration of trehalose can improve multiple system atrophy pathology and clinical symptoms. The multiple system atrophy model showed memory impairment at least four weeks after αSyn induction. Behavioural and physiological analyses showed that intranasal and oral administration of trehalose reversed memory impairments to near-normal levels. Notably, trehalose treatment reduced the amount of toxic αSyn and increased the aggregated form of αSyn in the multiple system atrophy model brain. In vitro kinetic analysis confirmed that trehalose accelerated the aggregate formation of αSyn. Based on our findings, we propose a novel strategy whereby accelerated αSyn aggregate formation leads to reduced exposure to toxic αSyn oligomers, particularly during the early phase of disease progression.
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