scholarly journals Alpha-synuclein from patient Lewy bodies exhibits distinct pathological activity that can be propagated in vitro

2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Nicholas P. Marotta ◽  
Jahan Ara ◽  
Norihito Uemura ◽  
Marshall G. Lougee ◽  
Emily S. Meymand ◽  
...  
Keyword(s):  
Cellular Response ◽  
Amyloid Fibrils ◽  
Cultured Cells ◽  
Biological Activities ◽  
Lewy Bodies ◽  
Alpha Synuclein ◽  
Biological Behavior ◽  
Motor Deficits ◽  
Original Material

AbstractLewy bodies (LBs) are complex, intracellular inclusions that are common pathological features of many neurodegenerative diseases. They consist largely of aggregated forms of the protein alpha-Synuclein (α-Syn), which misfolds to give rise to beta-sheet rich amyloid fibrils. The aggregation of monomers into fibrils occurs readily in vitro and pre-formed fibrils (PFFs) generated from recombinant α-Syn monomers are the basis of many models of LB diseases. These α-Syn PFFs recapitulate many pathological phenotypes in both cultured cells and animal models including the formation of α-Syn rich, insoluble aggregates, neuron loss, and motor deficits. However, it is not clear how closely α-Syn PFFs recapitulate the biological behavior of LB aggregates isolated directly from patients. Direct interrogation of the cellular response to LB-derived α-Syn has thus far been limited. Here we demonstrate that α-Syn aggregates derived from LB disease patients induce pathology characterized by a prevalence of large somatic inclusions that is distinct from the primarily neuritic pathology induced by α-Syn PFFs in our cultured neuron model. Moreover, these LB-derived aggregates can be amplified in vitro using recombinant α-Syn to generate aggregates that maintain the unique, somatic pathological phenotype of the original material. Amplified LB aggregates also showed greater uptake in cultured neurons and greater pathological burden and more rapid pathological spread in injected mouse brains, compared to α-Syn PFFs. Our work indicates that LB-derived α-Syn from diseased brains represents a distinct conformation species with unique biological activities that has not been previously observed in fully recombinant α-Syn aggregates and demonstrate a new strategy for improving upon α-Syn PFF models of synucleinopathies using amplified LBs.

scholarly journals Observation of an α-synuclein liquid droplet state and its maturation into Lewy body-like assemblies

2021 ◽  
Author(s):  
Maarten C Hardenberg ◽  
Tessa Sinnige ◽  
Sam Casford ◽  
Samuel Dada ◽  
Chetan Poudel ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Self Assembly ◽  
Lewy Body ◽  
Amyloid Fibrils ◽  
Liquid Droplet ◽  
Large Body ◽  
Lewy Bodies ◽  
Cellular Components

Abstract Misfolded α-synuclein is a major component of Lewy bodies, which are a hallmark of Parkinson’s disease. A large body of evidence shows that α-synuclein can aggregate into amyloid fibrils, but the relationship between α-synuclein self-assembly and Lewy body formation remains unclear. Here we show, both in vitro and in a Caenorhabditis elegans model of Parkinson’s disease, that α-synuclein undergoes liquid‒liquid phase separation by forming a liquid droplet state, which converts into an amyloid-rich hydrogel with Lewy-body-like properties. This maturation process towards the amyloid state is delayed in the presence of model synaptic vesicles in vitro. Taken together, these results suggest that the formation of Lewy bodies may be linked to the arrested maturation of α-synuclein condensates in the presence of lipids and other cellular components.

scholarly journals (De)stabilization of Alpha-Synuclein Fibrillary Aggregation by Charged and Uncharged Surfactants

2021 ◽  
Vol 22 (22) ◽  
pp. 12509
Author(s):  
Joana Angélica Loureiro ◽  
Stéphanie Andrade ◽  
Lies Goderis ◽  
Ruben Gomez-Gutierrez ◽  
Claudio Soto ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Hydrophobic Interactions ◽  
Neurodegenerative Disorder ◽  
Sodium Dodecyl ◽  
Lewy Bodies ◽  
Alpha Synuclein ◽  
Cetyltrimethylammonium Chloride ◽  
Α Helix ◽  
Β Sheet

Parkinson’s disease (PD) is the second most common neurodegenerative disorder. An important hallmark of PD involves the pathological aggregation of proteins in structures known as Lewy bodies. The major component of these proteinaceous inclusions is alpha (α)-synuclein. In different conditions, α-synuclein can assume conformations rich in either α-helix or β-sheets. The mechanisms of α-synuclein misfolding, aggregation, and fibrillation remain unknown, but it is thought that β-sheet conformation of α-synuclein is responsible for its associated toxic mechanisms. To gain fundamental insights into the process of α-synuclein misfolding and aggregation, the secondary structure of this protein in the presence of charged and non-charged surfactant solutions was characterized. The selected surfactants were (anionic) sodium dodecyl sulphate (SDS), (cationic) cetyltrimethylammonium chloride (CTAC), and (uncharged) octyl β-D-glucopyranoside (OG). The effect of surfactants in α-synuclein misfolding was assessed by ultra-structural analyses, in vitro aggregation assays, and secondary structure analyses. The α-synuclein aggregation in the presence of negatively charged SDS suggests that SDS-monomer complexes stimulate the aggregation process. A reduction in the electrostatic repulsion between N- and C-terminal and in the hydrophobic interactions between the NAC (non-amyloid beta component) region and the C-terminal seems to be important to undergo aggregation. Fourier transform infrared spectroscopy (FTIR) measurements show that β-sheet structures comprise the assembly of the fibrils.

scholarly journals Parkinson’s disease is a type of amyloidosis featuring accumulation of amyloid fibrils of α-synuclein

2019 ◽  
Vol 116 (36) ◽  
pp. 17963-17969 ◽  
Author(s):  
Katsuya Araki ◽  
Naoto Yagi ◽  
Koki Aoyama ◽  
Chi-Jing Choong ◽  
Hideki Hayakawa ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Amyloid Fibrils ◽  
Lewy Bodies ◽  
X Ray Diffraction ◽  
Thin Sections ◽  
X Ray ◽  
Β Sheet ◽  
The Brain

Many neurodegenerative diseases are characterized by the accumulation of abnormal protein aggregates in the brain. In Parkinson’s disease (PD), α-synuclein (α-syn) forms such aggregates called Lewy bodies (LBs). Recently, it has been reported that aggregates of α-syn with a cross-β structure are capable of propagating within the brain in a prionlike manner. However, the presence of cross-β sheet-rich aggregates in LBs has not been experimentally demonstrated so far. Here, we examined LBs in thin sections of autopsy brains of patients with PD using microbeam X-ray diffraction (XRD) and found that some of them gave a diffraction pattern typical of a cross-β structure. This result confirms that LBs in the brain of PD patients contain amyloid fibrils with a cross-β structure and supports the validity of in vitro propagation experiments using artificially formed amyloid fibrils of α-syn. Notably, our finding supports the concept that PD is a type of amyloidosis, a disease featuring the accumulation of amyloid fibrils of α-syn.

scholarly journals Simultaneous Occurrence of Bioactive Compounds (Melatonin, Protocatechuic Acid and Hydroxytyrosol) Increases Their Neuroprotective Effects Against Alpha-Synuclein-Induced Proteotoxicity

2018 ◽  
Author(s):  
Marta Gallardo-Fernández ◽  
Ruth Hornedo-Ortega ◽  
Ana B Cerezo ◽  
Ana M Troncoso ◽  
Mª Carmen Garcia-Parrilla
Keyword(s):  
Bioactive Compounds ◽  
Protocatechuic Acid ◽  
Lewy Bodies ◽  
Initial Step ◽  
Inhibitory Effect ◽  
Alpha Synuclein ◽  
Simultaneous Occurrence ◽  
Neuroprotective Effects ◽  
In Vitro Techniques

The abnormal assembly of α-synuclein (α-Syn) is an initial step in the formation of Lewy bodies in the brain, which finally causes the neuronal death, being considered as a pathological hallmark in Parkinson’s disease (PD). Certain food bioactives or their metabolites at very low concentrations can trespass the blood brain barrier (BBB) that might, thereafter, act simultaneously. The aim of this work was to evaluate the inhibitory and destabilising capacities on α-Syn kinetics and the neuroprotective effects of three well-known bioactive compounds able to cross the BBB and present in foods; melatonin (MEL), protocatechuic acid (PCA) and hydroxytyrosol (HT), and their combinations. For this purpose, different in vitro techniques (Thioflavin T (ThT), Transmission Electronic Microscopy (TEM), electrophoresis and MTT assay) were used. All tested compounds and their combinations were able to abolish the toxicity induced by α-Syn. In addition, the combination of PCA (100 µM) +HT (100 µM) showed the highest inhibitory effect against α-Syn fibril formation and destabilises α-Syn fibrils (88 and 62%, respectively). This is the first time that MEL, PCA and HT prove a joint effect against α-Syn aggregation and toxicity when they are tested together.

scholarly journals Pathogenicity of Vibrio alginolyticusfor Cultured Gilt-Head Sea Bream (Sparus aurataL.)

1998 ◽  
Vol 64 (11) ◽  
pp. 4269-4275 ◽  
Author(s):  
M. Carmen Balebona ◽  
Manuel J. Andreu ◽  
M. Angeles Bordas ◽  
Irene Zorrilla ◽  
Miguel A. Moriñigo ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Biological Activities ◽  
Embryo Cell ◽  
Sea Bream ◽  
Fish Cell ◽  
Whole Cells ◽  
Hydrolytic Activities ◽  
Extracellular Products

ABSTRACT The in vivo and in vitro pathogenic activities of whole cells and extracellular products of Vibrio alginolyticus for cultured gilt-head sea bream were evaluated. The 50% lethal doses ranged from 5.4 × 104 to 1.0 × 106 CFU/g of body weight. The strains examined had the ability to adhere to skin, gill, and intestinal mucus of sea bream and to cultured cells of a chinook salmon embryo cell line. In addition, the in vitro ability ofV. alginolyticus to adhere to mucus and skin cells of sea bream was demonstrated by scanning electron microscopy. The biological activities of extracellular products of V. alginolyticus were hydrolytic activities; the products were able to degrade sea bream mucus. V. alginolyticus was cytotoxic for fish cell lines and lethal for sea bream. Moreover, the extracellular products could degrade sea bream tissues. However, experiments performed with the bath immersion inoculation technique demonstrated that V. alginolyticus should be considered a pathogen for sea bream only when the mucus layer is removed and the skin is damaged.

A cullin-RING ubiquitin ligase targets exogenous α-synuclein and inhibits Lewy body–like pathology

2019 ◽  
Vol 11 (495) ◽  
pp. eaau6722 ◽  
Author(s):  
Juan A. Gerez ◽  
Natalia C. Prymaczok ◽  
Edward Rockenstein ◽  
Uli S. Herrmann ◽  
Petra Schwarz ◽  
...  
Keyword(s):  
Lewy Body ◽  
Cultured Cells ◽  
Neuroblastoma Cell ◽  
Lewy Bodies ◽  
Neuroblastoma Cell Line ◽  
Loss Of Function ◽  
Intracerebral Administration ◽  
F Box Protein

Parkinson’s disease (PD) is a neurological disorder characterized by the progressive accumulation of neuronal α-synuclein (αSyn) inclusions called Lewy bodies. It is believed that Lewy bodies spread throughout the nervous system due to the cell-to-cell propagation of αSyn via cycles of secretion and uptake. Here, we investigated the internalization and intracellular accumulation of exogenous αSyn, two key steps of Lewy body pathogenesis, amplification and spreading. We found that stable αSyn fibrils substantially accumulate in different cell lines upon internalization, whereas αSyn monomers, oligomers, and dissociable fibrils do not. Our data indicate that the uptake-mediated accumulation of αSyn in a human-derived neuroblastoma cell line triggered an adaptive response that involved proteins linked to ubiquitin ligases of the S-phase kinase-associated protein 1 (SKP1), cullin-1 (Cul1), and F-box domain–containing protein (SCF) family. We found that SKP1, Cul1, and the F-box/LRR repeat protein 5 (FBXL5) colocalized and physically interacted with internalized αSyn in cultured cells. Moreover, the SCF containing the F-box protein FBXL5 (SCFFBXL5) catalyzed αSyn ubiquitination in reconstitution experiments in vitro using recombinant proteins and in cultured cells. In the human brain, SKP1 and Cul1 were recruited into Lewy bodies from brainstem and neocortex of patients with PD and related neurological disorders. In both transgenic and nontransgenic mice, intracerebral administration of exogenous αSyn fibrils triggered a Lewy body–like pathology, which was amplified by SKP1 or FBXL5 loss of function. Our data thus indicate that SCFFXBL5 regulates αSyn in vivo and that SCF ligases may constitute targets for the treatment of PD and other α-synucleinopathies.

scholarly journals Reducing C-terminal truncation mitigates synucleinopathy and neurodegeneration in a transgenic model of multiple system atrophy

2016 ◽  
Vol 113 (34) ◽  
pp. 9593-9598 ◽  
Author(s):  
Fares Bassil ◽  
Pierre-Olivier Fernagut ◽  
Erwan Bezard ◽  
Alain Pruvost ◽  
Thierry Leste-Lasserre ◽  
...  
Keyword(s):  
Multiple System Atrophy ◽  
Neurodegenerative Disorder ◽  
Disease Onset ◽  
Neuronal Cell ◽  
Alpha Synuclein ◽  
Motor Deficits ◽  
Neuroprotective Effects ◽  
Caspase 1

Multiple system atrophy (MSA) is a sporadic orphan neurodegenerative disorder. No treatment is currently available to slow down the aggressive neurodegenerative process, and patients die within a few years after disease onset. The cytopathological hallmark of MSA is the accumulation of alpha-synuclein (α-syn) aggregates in affected oligodendrocytes. Several studies point to α-syn oligomerization and aggregation as a mediator of neurotoxicity in synucleinopathies including MSA. C-terminal truncation by the inflammatory protease caspase-1 has recently been implicated in the mechanisms that promote aggregation of α-syn in vitro and in neuronal cell models of α-syn toxicity. We present here an in vivo proof of concept of the ability of the caspase-1 inhibitor prodrug VX-765 to mitigate α-syn pathology and to mediate neuroprotection in proteolipid protein α-syn (PLP-SYN) mice, a transgenic mouse model of MSA. PLP-SYN and age-matched wild-type mice were treated for a period of 11 wk with VX-765 or placebo. VX-765 prevented motor deficits in PLP-SYN mice compared with placebo controls. More importantly, VX-765 was able to limit the progressive toxicity of α-syn aggregation by reducing its load in the striatum of PLP-SYN mice. Not only did VX-765 reduce truncated α-syn, but it also decreased its monomeric and oligomeric forms. Finally, VX-765 showed neuroprotective effects by preserving tyrosine hydroxylase-positive neurons in the substantia nigra of PLP-SYN mice. In conclusion, our results suggest that VX-765, a drug that was well tolerated in a 6 wk-long phase II trial in patients with epilepsy, is a promising candidate to achieve disease modification in synucleinopathies by limiting α-syn accumulation.

scholarly journals Rab27 GTPases regulate alpha-synuclein uptake, cell-to-cell transmission, and toxicity

2020 ◽  
Author(s):  
Rachel Underwood ◽  
Bing Wang ◽  
Aneesh Pathak ◽  
Laura Volpicelli-Daley ◽  
Talene A. Yacoubian
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Lewy Bodies ◽  
Neuronal Loss ◽  
Alpha Synuclein ◽  
Rab Gtpases ◽  
Double Knockout ◽  
The Impact

SUMMARYParkinson’s disease and Dementia with Lewy Bodies are two common neurodegenerative disorders marked by proteinaceous aggregates composed primarily of the protein α-synuclein. α-Synuclein is hypothesized to have prion-like properties, by which misfolded α-synuclein induces the pathological aggregation of endogenous α-synuclein and neuronal loss. Rab27a and Rab27b are two highly homologous Rab GTPases that regulate α-synuclein secretion, clearance, and toxicity in vitro. In this study, we tested the impact of Rab27a/b on the transmission of pathogenic α-synuclein. Double knockout of both Rab27 isoforms eliminated α-synuclein aggregation and neuronal toxicity in primary cultured neurons exposed to fibrillary α-synuclein. In vivo, Rab27 double knockout mice lacked fibril-induced α-synuclein inclusions, dopaminergic neuron loss, and behavioral deficits seen in wildtype mice with fibril-induced inclusions. Studies using AlexaFluor488-labeled α-synuclein fibrils revealed that Rab27a/b knockout prevented α-synuclein internalization without affecting bulk endocytosis. Rab27a/b knockout also blocked the cell-to-cell spread of α-synuclein pathology in multifluidic, multichambered devices. This study provides critical insight into the role of Rab GTPases in Parkinson’s disease and identifies Rab27s as key players in the progression of synucleinopathies.

scholarly journals A simple, versatile and robust centrifugation-based filtration protocol for the isolation and quantification of α-synuclein monomers, oligomers and fibrils: towards improving experimental reproducibility in α-synuclein research

2019 ◽  
Author(s):  
Senthil T. Kumar ◽  
Sonia Donzelli ◽  
Anass Chiki ◽  
Muhammed Muazzam Kamil Syed ◽  
Hilal A. Lashuel
Keyword(s):  
Amyloid Fibrils ◽  
Lewy Bodies ◽  
Biological Properties ◽  
Alpha Synuclein ◽  
Structural Basis ◽  
Experimental Conditions ◽  
Toxic Species ◽  
Slow Progress ◽  
Therapeutic Compounds ◽  
Multiple Samples

AbstractIncreasing evidence suggests that the process of alpha-synuclein (aSyn) aggregation from monomers into amyloid fibrils via oligomeric intermediates plays an essential role in the pathogenesis of different synucleinopathies, including Parkinson’s disease (PD), multiple system atrophy and dementia with Lewy bodies. However, the nature of the toxic species and the mechanisms by which they contribute to neurotoxicity and disease progression remain elusive. Over the past two decades, significant efforts and resources have been invested in studies aimed at identifying the putative toxic species along the pathway of aSyn fibrillization, and to develop small molecule drugs or antibodies that target toxic aSyn oligomeric intermediates. Although this approach has helped to advance the field and provide insights into the biological properties and toxicity of different aSyn species, many of the fundamental questions regarding the role of aSyn aggregation in PD remain unanswered, and no therapeutic compounds targeting aSyn oligomers have passed clinical trials. Several factors have contributed to this slow progress, including the complexity of the aggregation pathways and the heterogeneity and dynamic nature of aSyn aggregates. In the majority of experiment, the aSyn samples used contain mixtures of aSyn species that exist in an equilibrium and their ratio changes upon modifying experimental conditions. The failure to quantitatively account for the distribution of different aSyn species in different studies has contributed not only to experimental irreproducibility but also to misinterpretation of results and misdirection of valuable resources. Towards addressing these challenges and improving experimental reproducibility in Parkinson’s research, we describe here a simple centrifugation-based filtration protocol for the isolation, quantification and assessment of the distribution of of aSyn monomers, oligomers and fibrils, in heterogeneous aSyn samples of increasing complexity. The protocol is simple, does not require any special instrumentation and can be performed rapidly on multiple samples using small volumes. Here, we present and discuss several examples that illustrate the applications of this protocol and how it could contribute to improving the reproducibility of experiments aimed at elucidating the structural basis of aSyn aggregation, seeding activity, toxicity and pathology spreading. This protocol is applicable, with slight modifications, to other amyloid-forming proteins.Table of Content Figure

scholarly journals NTH1 Is a New Target for Ubiquitylation-Dependent Regulation by TRIM26 Required for the Cellular Response to Oxidative Stress

2018 ◽  
Vol 38 (12) ◽  
Author(s):  
Sarah C. Williams ◽  
Jason L. Parsons
Keyword(s):  
Oxidative Stress ◽  
Cellular Response ◽  
Cultured Cells ◽  
Excision Repair ◽  
Thymine Glycol ◽  
Endonuclease Iii ◽  
Hydrogen Peroxide Treatment ◽  
Base Excision ◽  
The Stability

ABSTRACT Endonuclease III-like protein 1 (NTH1) is a DNA glycosylase required for the repair of oxidized bases, such as thymine glycol, within the base excision repair pathway. We examined regulation of NTH1 protein by the ubiquitin proteasome pathway and identified the E3 ubiquitin ligase tripartite motif 26 (TRIM26) as the major enzyme targeting NTH1 for polyubiquitylation. We demonstrate that TRIM26 catalyzes ubiquitylation of NTH1 predominantly on lysine 67 present within the N terminus of the protein in vitro . In addition, the stability of a ubiquitylation-deficient protein mutant of NTH1 (lysine to arginine) at this specific residue was significantly increased in comparison to the wild-type protein when transiently expressed in cultured cells. We also demonstrate that cellular NTH1 protein is induced in response to oxidative stress following hydrogen peroxide treatment of cells and that accumulation of NTH1 on chromatin is exacerbated in the absence of TRIM26 through small interfering RNA (siRNA) depletion. Stabilization of NTH1 following TRIM26 siRNA also causes significant acceleration in the kinetics of DNA damage repair and cellular resistance to oxidative stress, which can be recapitulated by moderate overexpression of NTH1. This demonstrates the importance of TRIM26 in regulating the cellular levels of NTH1, particularly under conditions of oxidative stress.

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