A targetable N-terminal motif orchestrates α-Synuclein oligomer to fibril conversion

Santos, Jaime; Cuéllar, Jorge; Pallarès, Irantzu; Byrd, Emily J; Lends, Alons; Moro, Fernando; Abdul-Shukkoor, Muhammed Bilal; Pujols, Jordi; Velasco-Carneros, Lorea; Sobott, Frank; Otzen, Daniel E.; Calabrese, Antonio N.; Muga, Arturo; Pedersen, Jan Skov; Loquet, Antoine; Valpuesta, José M.; Radford, Sheena E.; Ventura, Salvador

doi:10.1101/2023.02.10.527650

Cited by 3 publications

(1 citation statement)

References 40 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Aggregation of α-syn involves intermolecular contacts which are likely similar between αSOs and fibrils; both species depend particularly on the NAC region for these interactions 19 , and a recent cryoEM study of a stabilized αSO was able to thread a fibrillar backbone structure through part of the αSO 47 . Nevertheless, the major αSO species that accumulates in vitro is generally very stable and self-contained 21 , i.e.…”

Section: Introductionmentioning

confidence: 99%

Monoclonal antibodies targeting cytotoxic α-synuclein oligomers: molecular properties and diagnostic potential

Otzen,

Nielsen,

Lauritsen

et al. 2023

Preprint

Self Cite

View full text Add to dashboard Cite

Aggregation of α-synuclein (α-syn) is critical to the development of synucleinopathies such as Parkinson’s Disease (PD), yet we do not have any effective therapy against these diseases. α-syn accumulates as insoluble amyloid in intracellular Lewy Bodies (LBs) but also forms soluble αSOs (αSOs) which are thought to be even more cytotoxic than fibrils. We lack tools to detect and block the unwanted activities of these αSOs. To address this, we have raised monoclonal antibodies (mAbs) against different forms of αSOs, ranging from unmodified αSOs to species stabilized by lipid peroxidation products and polyphenols, αSOs formed by C-terminally truncated α-syn, and multivalent display of α-syn on capsid virus-like particles (cVLPs). Here we report on the general biophysical and immunohistochemical properties of 30 anti-αSO mAbs obtained in this fashion. Our analysis includes direct measurement of in-solution affinity to monomeric α-syn and αSOs using Flow-Induced Dispersion Analysis, which avoids immobilization artifacts associated with ELISA. While the mAbs generally show a preference for αSOs, they also bind fibrils, but to variable extents. Overall, we observe great diversity in the mAbs’ relative affinities for monomers and αSOs, varied requirements for the C-terminal extension of α-syn, and only a modest effect on α-syn fibrillation. Several mAbs show several orders of magnitude preference for αSOs over monomers in in-solution studies, in contrast to the commercial antibody MJF14 which only bound 10-fold more strongly to αSOs than monomeric α-syn. Gratifyingly, seven mAbs almost completely block αSO permeabilization of membrane vesicles. 16 mAbs were analyzed for binding to brain sections from rats overexpressing human α-syn in the nigro-striatal pathway, and the five most promising were further investigated for their ability to recognize α-syn aggregates in human brain tissue. All five mAbs identified α-syn-related pathologies like LBs and Lewy Neurites, as well as Glial Cytoplasmic Inclusions in postmortem brains from people diagnosed for PD, dementia with LBs and multiple system atrophy, although to different extents. We have identified three mAbs as particularly useful for pathological evaluation of postmortem brain human tissue, including the early stages of PD. Although there was no straightforward connection between the individual mAbs’ biophysical and immunohistochemical properties, it is encouraging that a large collection of mAbs able to recognize different aggregated α-syn species in vitro also hold diagnostic potential.

show abstract

Section: Introductionmentioning

confidence: 99%

Monoclonal antibodies targeting cytotoxic α-synuclein oligomers: molecular properties and diagnostic potential

Otzen,

Nielsen,

Lauritsen

et al. 2023

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

Dissecting the Self-assembly Dynamics of Imperfect Repeats in α-Synuclein

Huang,

Wang,

Zhang

et al. 2023

J. Chem. Inf. Model.

View full text Add to dashboard Cite

The pathological aggregation of α-synuclein (αS) into amyloid fibrils is the hallmark of Parkinson’s disease (PD). The self-assembly and membrane interactions of αS are mainly governed by the seven imperfect 11-residue repeats of the XKTKEGVXXXX motif around residues 1–95. However, the particular role of each repeat in αS fibrillization remains unclear. To answer this question, we studied the aggregation dynamics of each repeat with up to 10 peptides in silico by conducting multiple independent micro-second atomistic discrete molecular dynamics simulations. Our simulations revealed that only repeats R3 and R6 readily self-assembled into β-sheet-rich oligomers, while the other repeats remained as unstructured monomers with weak self-assembly and β-sheet propensities. The self-assembly process of R3 featured frequent conformational changes with β-sheet formation mainly in the non-conserved hydrophobic tail, whereas R6 spontaneously self-assembled into extended and stable cross-β structures. These results of seven repeats are consistent with their structures and organization in recently solved αS fibrils. As the primary amyloidogenic core, R6 was buried inside the central cross-β core of all αS fibrils, attracting the hydrophobic tails of adjacent R4, R5, and R7 repeats forming β-sheets around R6 in the core. Further away from R6 in the sequence but with a moderate amyloid aggregation propensity, the R3 tail could serve as a secondary amyloidogenic core and form independent β-sheets in the fibril. Overall, our results demonstrate the critical role of R3 and R6 repeats in αS amyloid aggregation and suggest their potential as targets for the peptide-based and small-molecule amyloid inhibitors.

show abstract

Molecular rotors provide insight into the mechanism of formation and conversion ofα-synuclein aggregates

Allerton,

Kuimova,

Aprile

2024

Preprint

View full text Add to dashboard Cite

ABSTRACTα-Synuclein is an intrinsically disordered protein that forms amyloids in Parkinson’s disease. Currently, detection methods predominantly report on the formation of mature amyloids but have poor sensitivity to the early-stage, toxic oligomers. Molecular rotors are fluorophores that sense changes in the viscosity of their local environment. Here, we monitorα-synuclein oligomer formation using the fluorescence lifetime of molecular rotors. We detect oligomer formation and conversion into amyloids forwild typeand twoα-synuclein variants; the pathological mutant A30P and ΔPα-synuclein, which lacks a master regulator region of aggregation (residues 36-42). We report that A30Pα-synuclein shows a similar rate of oligomer formation compared towild type α-synuclein, whereas ΔPα-synuclein shows delayed oligomer formation. Additionally, both variants demonstrate a slower conversion of oligomers to amyloids. Our method provides a quantitative approach to unveiling the complex mechanism ofα-synuclein aggregation which is key to understanding the pathology of Parkinson’s disease.

show abstract

A targetable N-terminal motif orchestrates α-Synuclein oligomer to fibril conversion

Cited by 3 publications

References 40 publications

Monoclonal antibodies targeting cytotoxic α-synuclein oligomers: molecular properties and diagnostic potential

Monoclonal antibodies targeting cytotoxic α-synuclein oligomers: molecular properties and diagnostic potential

Dissecting the Self-assembly Dynamics of Imperfect Repeats in α-Synuclein

Molecular rotors provide insight into the mechanism of formation and conversion ofα-synuclein aggregates

Contact Info

Product

Resources

About