Abstract
α-Synuclein fibrils are considered a hallmark of Parkinson’s disease and other synucleinopathies. However, small oligomers that formed during the early stages of α-synuclein aggregation are thought to be the main toxic species causing disease. The formation of α-synuclein oligomers has proven difficult to follow, because of the heterogeneity and transient nature of the species formed. Here, a novel bead-based aggregation assay for monitoring the earliest stages of α-synuclein oligomerization, α-Synuclein–Confocal Nanoscanning (ASYN-CONA), is presented. The α-synuclein A91C single cysteine mutant is modified with a trifunctional chemical tag, which allows simultaneous fluorescent labeling with a green dye (tetramethylrhodamine, TMR) and attachment to microbeads. Beads with bound TMR-labeled α-synuclein are then incubated with a red dye (Cy5)-labeled variant of α-synuclein A91C, and EtOH (20%) to induce aggregation. Aggregation is detected by confocal scanning imaging, below the equatorial plane of the beads, which is known as the CONA technique. On-bead TMR-labeled α-synuclein and aggregated Cy5-labeled α-synuclein from the solution are quantitatively monitored in parallel by detection of fluorescent halos or “rings”. α-Synuclein on-bead oligomerization results in a linear increase of red bead ring fluorescence intensity over a period of 5 h. Total internal reflection fluorescence microscopy was performed on oligomers cleaved from the beads, and it revealed that (i) oligomers are sufficiently stable in solution to investigate their composition, consisting of 6 ± 1 monomer units, and (ii) oligomers containing a mean of 15 monomers bind Thioflavin-T. Various known inhibitors of α-synuclein aggregation were used to validate the ASYN-CONA assay for drug screening. Baicalein, curcumin, and rifampicin showed concentration-dependent inhibition of the α-synuclein aggregation and the IC50 (the concentration of the compound at which the maxiumum intensity was reduced by one-half) were calculated.
Highlights
Parkinson’s disease and other synucleinopathies are characterized by the misfolding and aggregation of αsynuclein. α-Synuclein is a small presynaptic protein whose main function is believed to occur at the presynaptic terminals and may play a role in regulating synaptic transmission.[1]
This region is found to be unstructured in α-synuclein fibrils.[4] α-Synuclein exists primarily as an unfolded monomer in equilibrium alongside some partially folded monomers and multimers, depending on the local environments of the protein.[5−8] In disease, α-synuclein is believed to misfold, acquiring a conformation prone to aggregation that leads to the formation of highly organized fibrils.[9]
The kinetics of fibril formation are classically detected by fluorescence emission intensity changes of solvatochromic dyes such as thioflavinT, ThT, which exhibit enhanced fluorescence upon binding to β-sheet-rich fibrils. αSynuclein fibrils are the species found to be the main components of Lewy bodies and Lewy neurites in the brains of patients.[11,12]
Summary
Parkinson’s disease and other synucleinopathies are characterized by the misfolding and aggregation of αsynuclein. α-Synuclein is a small presynaptic protein whose main function is believed to occur at the presynaptic terminals and may play a role in regulating synaptic transmission.[1]. The N-terminus, which is defined by residues 1−60, contains an imperfect repeated sequence (KTKEGV) involved in the amphiphilic α-helical structure adopted when bound to lipids.[2] The nonamyloid component (NAC), region, which is defined by residues 61−95, is highly hydrophobic and forms the core of the highly organized fibril structures.[3] The C-terminus, which is defined by residues 96−140, is negatively charged and contains several proline residues, making it very flexible This region is found to be unstructured in α-synuclein fibrils.[4] α-Synuclein exists primarily as an unfolded monomer in equilibrium alongside some partially folded monomers and multimers, depending on the local environments of the protein.[5−8] In disease, α-synuclein is believed to misfold, acquiring a conformation prone to aggregation that leads to the formation of highly organized fibrils.[9] The structure of mature fibrils have recently been established in detail by solid-state NMR4 and cryo-EM10 to be Greek key-like. It appears that no universal standard detection method for oligomer formation has been established.[15]
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