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On-surface aggregation of α-synuclein at nanomolar concentrations results in two distinct growth mechanisms - PubMed

  • ️Tue Jan 01 2013

. 2013 Mar 20;4(3):408-17.

doi: 10.1021/cn3001312. Epub 2013 Jan 22.

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On-surface aggregation of α-synuclein at nanomolar concentrations results in two distinct growth mechanisms

Michael Rabe et al. ACS Chem Neurosci. 2013.

Abstract

The aggregation of α-synuclein (α-Syn) is believed to be one of the key steps driving the pathology of Parkinson's disease and related neurodegenerative disorders. One of the present hypotheses is that the onset of such pathologies is related to the rise of α-Syn levels above a critical concentration at which toxic oligomers or mature fibrils are formed. In the present study, we find that α-Syn aggregation in vitro is a spontaneous process arising at bulk concentrations as low as 1 nM and below in the presence of both hydrophilic glass surfaces and cell membrane mimicking supported lipid bilayers (SLBs). Using three-dimensional supercritical angle fluorescence (3D-SAF) microscopy, we observed the process of α-Syn aggregation in situ. As soon as α-Syn monomers were exposed to the surface, they started to adsorb and aggregate along the surface plane without a prior lag phase. However, at a later stage of the aggregation process, a second type of aggregate was observed. In contrast to the first type, these aggregates showed an extended structure being tethered with one end to the surface and being mobile at the other end, which protruded into the solution. While both types of α-Syn aggregates were found to contain amyloid structures, their growing mechanisms turned out to be significantly different. Given the clear evidence that surface-induced α-Syn aggregation in vitro can be triggered at bulk concentrations far below physiological concentrations, the concept of a critical concentration initiating aggregation in vivo needs to be reconsidered.

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Figures

Figure 1
Figure 1

α-Syn aggregation on the SLB. Donor (D), acceptor (A), and FRET images of the same scanned areas acquired after 1 h (top row) and 16 h (bottom row) of exposure to a mixture of donor and acceptor labeled α-Syn (0.5 nM each) on the SLB. The section marked by a dashed line is magnified in the boxes underneath each image. FRET signals arise solely from aggregated α-Syn molecules.

Figure 2
Figure 2

Monitoring the α-Syn aggregation on the SLB. First, only donor (D) labeled α-Syn was applied to the surface followed by addition of only acceptor (A) labeled α-Syn. Light blue color indicates no energy transfer, that is, only donor fluorescence. The appearance of energy transfer (yellow to red color) results from aggregation events. The time-resolved energy transfer of each of the three aggregates indicated by a, b, and c is shown on the right as images. In addition, the graphs labeled accordingly show the integrated energy transfers of each of the three aggregates versus time (bottom).

Figure 3
Figure 3

Supercritical angle fluorescence (SAF) and undercritical angle fluorescence (UAF) detection of protein aggregation. (a) The α-Syn concentration is 1 nM in a buffer composed of 0.1 × PBS (having an ionic strength of 16.6 mM). The UAF-to-SAF-ratio shows the protrusion of the aggregates into the bulk as highlighted by the color code in the z-axis from green (close to the SLB) to red (1–3 μm away from the SLB). In the insets, magnifications of single selected aggregates are highlighted. Images shown in b and c were recorded 1 and 3.5 h after increasing the concentration to 10 nM (0.1 × PBS).

Figure 4
Figure 4

Epifluorescence microscopy images of α-Syn amyloid fibrils grown at 1 nM, 0.1 × PBS on a SLB. Extended curly structures (a, b) and short elongated surface bound aggregates (c) grown for 20 h respond to ThT staining. Images e and f present the same section (magnification from image d) with the focal plane being set on the surface and a few micrometers inside the bulk solution, respectively.

Figure 5
Figure 5

Large aggregates protrude deep into the bulk solution. The aggregates were grown at bulk concentrations of 1 nM on a SLB (a) or 10 nM on a glass surface (b, c). UAF detection shows an image of the structures up to 2.5 μm deep into the solution (left column). SAF detection shows images of these fibrils in close vicinity (∼100–200 nm) to the surface (middle column). The UAF-to-SAF-ratio images (right column) visualize the anchoring points of the amyloid fibrils (white arrows).

Figure 6
Figure 6

Bending of extended amyloid fibrils. Scan images were recorded with the buffer flowing from right to left (up) and from left to right (middle), which moves the fibrils protruding deep into the solution. The images in the third row represent overlays of the marked regions with the orange color referring to a buffer flow from right to left and the violet color referring to a buffer flow from left to right. The surface anchoring points (green) do not move. Refer to Supporting Information, Figure S7, for an analogous experiment conducted on the bare glass surface.

Figure 7
Figure 7

α-Syn aggregation process on a glass and a SLB surface. Aggregates presented in columns a and b were grown in 0.1 × PBS on a SLB at a concentration of 10 nM. Aggregates presented in column c and d were grown in (0.1 × PBS) on the glass surface at a concentration of only 0.2 nM. Kinetic plots (middle) present α-Syn aggregation in the z-direction (monitored by UAF) and α-Syn aggregation along the surface plane (monitored by SAF). Images on the right show the surface sections evaluated for the kinetic analyses.

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