Formation and Characterization of α-Synuclein Oligomers

Paslawski, Wojciech; Lorenzen, Nikolai; Otzen, Daniel E.

doi:10.1007/978-1-4939-2978-8_9

Cited by 41 publications

(37 citation statements)

References 30 publications

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“…Cells were lysed using an osmotic shock protocol modified from Paslawski et al [ 28 ]. Using a 25 mL serological pipette, the cell pellets were gently resuspended in 10% volume of room temperature osmotic shock buffer, (25 mL per 250 mL of cell culture before centrifugation) and incubated at room temperature for 10 min.…”

Section: Methodsmentioning

confidence: 99%

Rapid and ultra-sensitive quantitation of disease-associated α-synuclein seeds in brain and cerebrospinal fluid by αSyn RT-QuIC

Groveman

Orrú

Hughson

et al. 2018

acta neuropathol commun

282

371

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The diagnosis and treatment of synucleinopathies such as Parkinson disease and dementia with Lewy bodies would be aided by the availability of assays for the pathogenic disease-associated forms of α-synuclein (αSynD) that are sufficiently sensitive, specific, and practical for analysis of accessible diagnostic specimens. Two recent αSynD seed amplification tests have provided the first prototypes for ultrasensitive and specific detection of αSynD in patients’ cerebrospinal fluid. These prototypic assays require 5–13 days to perform. Here, we describe an improved α-synuclein real time quaking-induced conversion (αSyn RT-QuIC) assay that has similar sensitivity and specificity to the prior assays, but can be performed in 1–2 days with quantitation. Blinded analysis of cerebrospinal fluid from 29 synucleinopathy cases [12 Parkinson’s and 17 dementia with Lewy bodies] and 31 non-synucleinopathy controls, including 16 Alzheimer’s cases, yielded 93% diagnostic sensitivity and 100% specificity for this test so far. End-point dilution analyses allowed quantitation of relative amounts of αSynD seeding activity in cerebrospinal fluid samples, and detection in as little as 0.2 μL. These results confirm that αSynD seeding activity is present in cerebrospinal fluid. We also demonstrate that it can be rapidly detected, and quantitated, even in early symptomatic stages of synucleinopathy.Electronic supplementary materialThe online version of this article (10.1186/s40478-018-0508-2) contains supplementary material, which is available to authorized users.

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Section: Methodsmentioning

confidence: 99%

Rapid and ultra-sensitive quantitation of disease-associated α-synuclein seeds in brain and cerebrospinal fluid by αSyn RT-QuIC

Groveman

Orrú

Hughson

et al. 2018

acta neuropathol commun

282

371

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“…), or 30 monomers (Paslawski et al . ). However, many studies define oligomers as any arrangement larger than a α‐Syn monomer.…”

Section: Discussionmentioning

confidence: 97%

“…This term is ambiguous and is used to refer to very different types of species in different laboratories. a-Syn oligomers may range between a couple of monomers to 10 monomers (Cremades et al 2017), or 30 monomers (Paslawski et al 2016). However, many studies define oligomers as any arrangement larger than a a-Syn monomer.…”

Section: Discussionmentioning

confidence: 99%

In vitro models of synucleinopathies: informing on molecular mechanisms and protective strategies

Marvian

Koss

Aliakbari

et al. 2019

Journal of Neurochemistry

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Alpha‐synuclein (α‐Syn) is a central player in Parkinson's disease (PD) and in a spectrum of neurodegenerative diseases collectively known as synucleinopathies. The protein was first associated with PD just over 20 years ago, when it was found to (i) be a major component of Lewy bodies and (ii) to be also associated with familial forms of PD. The characterization of α‐Syn pathology has been achieved through postmortem studies of human brains. However, the identification of toxic mechanisms associated with α‐Syn was only achieved through the use of experimental models. In vitro models are highly accessible, enable relatively rapid studies, and have been extensively employed to address α‐Syn‐associated neurodegeneration. Given the diversity of models used and the outcomes of the studies, a cumulative and comprehensive perspective emerges as indispensable to pave the way for further investigations. Here, we subdivided in vitro models of α‐Syn pathology into three major types: (i) models simulating α‐Syn fibrillization and the formation of different aggregated structures in vitro, (ii) models based on the intracellular expression of α‐Syn, reporting on pathogenic conditions and cellular dysfunctions induced, and (iii) models using extracellular treatment with α‐Syn aggregated species, reporting on sites of interaction and their downstream consequences. In summary, we review the underlying molecular mechanisms discovered and categorize protective strategies, in order to pave the way for future studies and the identification of effective therapeutic strategies. This article is part of the Special Issue “Synuclein”.

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“…The latter objects could correspond to the oligomers of E46K, H50Q or A53T that we detect. Starting from purified recombinant α-synuclein, production of oligomeric species of α-synuclein is faster, with oligomers being obtained for example by incubation of low millimolar solution at either room temperature for 24 h2854 or 5 h at 37 °C5255. Oligomerization was actually detected in the first 20 minutes after solubilization of freeze-thawed monomeric α-synuclein52.…”

Section: Discussionmentioning

confidence: 99%

Nanomolar oligomerization and selective co-aggregation of α-synuclein pathogenic mutants revealed by single-molecule fluorescence

Sierecki

Giles

Bowden

et al. 2016

Sci Rep

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Protein aggregation is a hallmark of many neurodegenerative diseases, notably Alzheimer’s and Parkinson’s disease. Parkinson’s disease is characterized by the presence of Lewy bodies, abnormal aggregates mainly composed of α-synuclein. Moreover, cases of familial Parkinson’s disease have been linked to mutations in α-synuclein. In this study, we compared the behavior of wild-type (WT) α-synuclein and five of its pathological mutants (A30P, E46K, H50Q, G51D and A53T). To this end, single-molecule fluorescence detection was coupled to cell-free protein expression to measure precisely the oligomerization of proteins without purification, denaturation or labelling steps. In these conditions, we could detect the formation of oligomeric and pre-fibrillar species at very short time scale and low micromolar concentrations. The pathogenic mutants surprisingly segregated into two classes: one group forming large aggregates and fibrils while the other tending to form mostly oligomers. Strikingly, co-expression experiments reveal that members from the different groups do not generally interact with each other, both at the fibril and monomer levels. Together, this data paints a completely different picture of α-synuclein aggregation, with two possible pathways leading to the development of fibrils.

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Formation and Characterization of α-Synuclein Oligomers

Cited by 41 publications

References 30 publications

Rapid and ultra-sensitive quantitation of disease-associated α-synuclein seeds in brain and cerebrospinal fluid by αSyn RT-QuIC

Rapid and ultra-sensitive quantitation of disease-associated α-synuclein seeds in brain and cerebrospinal fluid by αSyn RT-QuIC

In vitro models of synucleinopathies: informing on molecular mechanisms and protective strategies

Nanomolar oligomerization and selective co-aggregation of α-synuclein pathogenic mutants revealed by single-molecule fluorescence

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