Optimizing amyloid fibril size for neuronal cell seeding with Zetasizer Advance

Executive summary

Dynamic Light Scattering (DLS) on the Zetasizer Advance was used not just to characterize α-synuclein (aSyn) pre-formed fibrils (PFF), but to optimize the ultrasonication process that generates ~50 nm fibrils suitable for cellular uptake. By reading out z-average (z-avg) and polydispersity index (PDI) in minutes, DLS rapidly converged on sonication settings that produced a tight, target-size population. These DLS-guided conditions translated to successful uptake and seeding in SH-SY5Y cells.

Introduction

α-Synuclein (aSyn) fibrils are a hallmark of synucleinopathies such as Parkinson’s disease, where they contribute to neurodegeneration through the formation of intracellular inclusions¹. Pre-formed fibrils (PFF) of aSyn serve as critical tools in studying the mechanisms of fibril propagation, cell seeding, and toxicity. The ability to generate, characterize, and validate aSyn PFF with consistent size and structural properties is essential for experimental reproducibility.

aSyn PFF can be prepared directly from monomeric protein or purchased from various commercial sources. The presence of amyloid fibrils is often confirmed by Thioflavin-T (ThT) fluorescence, while fibril length and diameter are measured by high-resolution techniques such as Transmission Electron Microscopy (TEM) or Atomic Force Microscopy (AFM). These technologies allow for the high-resolution evaluation of fibril size and sample homogeneity, which is crucial for ensuring that PFF are of an appropriate length to effectively seed and internalize within neuronal cells. 

This application note explores the use of Dynamic Light Scattering (DLS) as a complementary technique to enable the rapid determination of aSyn PFF size distributions. Using the Zetasizer Advance, we compare commercially sourced aSyn PFF to samples prepared in-house from monomeric aSyn. We also use DLS to measure particle size distribution (PSD) as an effect of various ultrasonication methods on in-house generated aSyn PFFs and use SH-SY5Y cells validate sample suitability for downstream neurobiological investigations. 

Results and Discussion

aSyn pre-formed fibrils (PFF) can be purchased from vendors directly, or they can be generated from monomeric aSyn. Thioflavin-T (ThT), which binds to β-sheet-rich structures, is commonly used to detect amyloid fibrils². It was therefore used to confirm the presence of PFFs generated via an in-house preparation method. Compared to the monomeric protein (gray bar), a 100-fold ThT-fluorescence signal increase was observed for the aSyn PFF (green bar), indicating the successful generation of the fibrillar protein species (Fig 1a). The intensity-weighted particle size distribution (PSD) of the fibril sample was subsequently compared with aSyn PFF from two different commercial sources using DLS (Fig 1b). In-house PFF (green) demonstrated modest overlay with those from Vendor 1 (purple) and exceptional overlay with those from Vendor 2 (blue). These results collectively provide evidence that the in-house PFF method was sufficient to proceed with additional experiments.

For aSyn PFF to effectively seed neuronal cells, they need to be small enough to incorporate into the cell. An average PFF length of roughly 50 nm has been previously reported as effective in permeating cells and seeding amyloid inclusions³. Ultrasonication has been shown to be an effective method in reorganizing other amyloid PFF into different sizes based on buffer ionic strength and protein concentration⁴. In-house PFF were subjected to various ultrasonication cycle parameters to determine the optimal method for generating aSyn PFF approximately 50 nm in size (Fig 2). Sonication times, resting times, and the number of sonication cycles were varied according to Table 1, and these samples were compared to the untreated starting material.

The intensity-weighted PSD (Fig 2a) indicates a positive correlation between a monomodal size distribution and the number of sonication cycles and sonication time, as indicated by the polydispersity index (PDI) calculations (Table 1). Longer resting times, where the sonicator is off, resulted in a larger PDI. Since larger molecules scatter more light, the intensity-weighted PSD is heavily influenced by the presence of larger populations within the sample mixture⁵. The volume-weighted PDS can be used to assess population size by volume occupied in the sample rather than by the intensity of scattered light (Fig 2b). This transformation presents much better agreement between all sonicated aSyn PFF samples and indicates that the majority species within all sonicated samples are comprised of mostly PFF within the desired size range. Overall, Sonication 2 (orange trace) gave the most promising results with a z-avg of 55.1 nm and a PDI of 0.167. The PSD profile of Sonication 3 (blue trace) was very similar to Sonication 2, with the addition of a small percentage of large (>5000 nm) particles reflected in the larger PDI value. Whereas Sonication 1 (red trace) proved to be least effective, with a z-avg of 93.6 nm and a PDI of 0.211.

To validate that the aSyn PFF size determined by DLS was suitable for cellular uptake, SH-SY5Y cells were treated with sonicated aSyn fibrils from various vendors (Fig 3). Staining cells for total and aggregated aSyn showed that untreated aSyn-overexpressing SH-SY5Y cells display a uniform cytoplasmic distribution of total aSyn, with no detectable aSyn aggregates. By contrast, PFF-treated cells display discrete intracellular accumulations formed from synuclein aggregates. These results indicate successful cellular uptake of both in-house and vendor supplied aSyn PFFs.

Conclusions

In this study, we demonstrated the utility of the Zetasizer Advance for rapid and reliable characterization of aSyn PFF. DLS measurements provided detailed insights into the size distribution of both in-house prepared and commercially sourced PFF. Optimization of ultrasonication parameters enabled the generation of PFF approximately 50 nm in length, enabling cellular uptake studies using SH-SY5Y human neuroblastoma cells. Overall, DLS offers a fast and robust workflow for the evaluation of amyloid fibril preparation.

Materials and Methods

Generation of α-Synuclein Pre-formed Fibrils

Concentrated PBS (5x PBS diluted from DPBS 10X, Gibco, Cat#14200075) was added to aSyn monomer (Proteos Inc., Cat3 RP-003) for a final concentration of 5 mg/mL and a total volume of 200 µL. All samples were incubated in low attachment 1.5 mL sonication tubes on a shaker at 1000 rpm at 37°C for 7 days. At the end of the incubation, samples were checked for turbidity to indicate potential fibril formation. Samples were then diluted to a concentration of 1 mg/mL using 1x PBS (without Mg²⁺ or Ca²⁺) and stored at -80C.

Sample Preparation for Sonication and DLS

Prior to sonication, aSyn samples were diluted to 0.1 mg/mL using 1x PBS (without Mg²⁺ or Ca²⁺) to a total volume of 200 µL. Samples were sonicated (BioRuptor Plus, Diagenode) using various cycle numbers and time settings at a constant temperature of 15 °C. Following sonication, samples were further diluted to a final concentration of 0.01 mg/mL using 1x PBS (without Mg²⁺ or Ca²⁺). DLS scattering traces were collected using back scatter mode (Zetasizer Advance Ultra, Malvern Panalytical) from 15 µL of sample in an ultra-low volume quartz cuvette (ZEN2112, Malvern Panalytical).

Thioflavin-T Staining

A 25 µM Thioflavin-T solution was prepared in PBS, and 95 µL was added to each sample well in a 96-well plate. Triplicate samples were prepared by adding a volume of 2.5 µL of either 0.1 mg/mL aSyn fibrils or monomeric aSyn was added to each sample well. The plate was incubated for 30-60 minutes at room temperature before being read on a plate reader (SpectraMax iD3, Molecular Devices) at excitation 450 nm and emission 500 nm.

SH-SY5Y Cell Culture and Staining

Human neuroblastoma SH-SY5Y (ECACC) cells, overexpressing a green fluorescent protein(GFP)-aSyn fusion chimera, were thawed and cultured in DMEM/F12 media containing 10% FBS for 24 h. The next day, sonicated aSyn PFF were added to cell culture media at a final concentration of 5 µg/ml. After 48 h of incubation, cells were fixed with a 4% PFA (in PBS) solution for 10 minutes at room temperature and washed three times with PBS. Cells were then permeabilized with Blocking Buffer 1 (0.1% Triton X-100, 2.5% normal goat serum in PBS) for 30 minutes at room temperature. Primary antibodies (total aSyn: Abcam ab138501; aggregated aSyn: Abcam MJFR14), were diluted (1:500) in Blocking Buffer 1, and 50 µL of the primary antibody solution was added per well and incubated overnight at 4 °C. The next day, cells were washed three times with PBS. Cy5-tagged secondary antibodies (1:600) and DAPI (1:10000) were diluted in Blocking Buffer 2 (0.1% Triton X-100, 5% normal goat serum in PBS) and added at 50 µL per well for 2 hours at room temperature in the dark. Finally, cells were washed three times with PBS before imaging with a confocal microscope (ImageXpress Micro Confocal, Molecular Devices). 

References

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