α-Synuclein Aggregation Monitored by Thioflavin T Fluorescence Assay

Materials and Reagents

1. 50 ml Falcon centrifuge tubes (TPP Techno Plastic Products, catalog number: 91050 )
   
2. Sterile syringe filter, Filtropur S, 0.2 μm (SARSTEDT, catalog number: 83.1826.001 )
   
3. Slide-A-Lyzer^TM dialysis cassettes 10 kDA MW cutoff, 3-12 ml (Thermo Fisher Scientific, catalog number: 66810 )
   
4. HiTrap Q HP IEC column, 5 ml (GE Healthcare, catalog number: 17115401 )
   
5. Superdex 75 16/60 SEC column, ~120 ml volume (GE Healthcare, catalog number: 28989333 )
   
6. Protein LoBind Tubes 1.5 ml (Eppendorf, catalog number: 0030108116 )
   
7. 96-well plates (Corning half-area, black and clear flat bottom, non-binding surface) (Corning, catalog number: 3881 )
   
8. Sealing tape, clear polyolefin (Thermo Fisher Scientific, catalog number: 232701 )
   
9. E. coli BL21DE3 competent cells (e.g., available from Merck, catalog number: 69450 )
   
10. α-Synuclein in pT7-7 vector (e.g., Addgene, catalog number: 36046 )
    We use a version of this plasmid that is codon-optimized for E. coli
11. NatB in pACYCduet vector (e.g., Addgene, catalog number: 53613 )
12. Isopropyl β-D-1-thiogalactopyranoside (IPTG), > 99% (Ubiquitin-Proteasome Biotechnologies, catalog number: P1010-100 )
13. Ampicillin sodium salt (Sigma-Aldrich, catalog number: A9518 )
    
14. Chloramphenicol (Sigma-Aldrich, catalog number: C0378 )
    
15. YT medium (2x) premixed powder (AppliChem, catalog number: A0981 )
    
16. Magnesium sulfate heptahydrate p.a. (Merck, catalog number: 1058860500 )
    
17. Glycerol bidistilled, ≥ 99.5% (VWR, catalog number: 24388 )
    
18. Tris(hydroxymethyl)aminomethane, Trizma base p.a. (Sigma-Aldrich, catalog number: 93350 )
    
19. Sodium chloride, p.a. (Carl Roth, catalog number: 3957 )
    
20. Sodium dihydrogen phosphate monohydrate, p.a. (AppliChem, catalog number: 131965.1211 )
    
21. α-Synuclein, N-terminally acetylated (from recombinant expression in E. coli [Johnson et al., 2010] and purified as described [Wördehoff et al., 2017])
    Note: We verify N-terminal acetylation by HPLC and mass spectrometry. Alternatively, acetic acid gel electrophoresis can be performed (Iyer et al., 2016). We have included a short description of our α-synuclein purification protocol in the procedure section of this protocol.
22. Thioflavin T, ultrapure grade (AnaSpec, catalog number: AS-88306 )
    
23. Sodium azide (purum p.a., Sigma-Aldrich, catalog number: 71290 )
    
24. H₂O (Milli-Q ≥ 18.2 MΩ resistivity)
    
25. Glass beads, Ø 2.85-3.45 mm (Carl Roth, catalog number: A557.1 ), stored in 70% ethanol to ensure sterility
    
26. Potassium dihydrogen phosphate (p.a., AppliChem, catalog number: A1043 )
    
27. Dipotassium hydrogen phosphate, trihydrate (p.a., Merck, catalog number: 105099 )
    
28. Potassium chloride (p.a., Carl Roth, catalog number: 6781.1 )
    
29. Hydrochloric acid, p.a. ≥ 37% (Sigma-Aldrich, catalog number: 30721-M )
    
30. Ammonium sulfate, puriss. p.a. ≥ 99% (Merck, catalog number: 31119-M )
    
31. Modified 2YT medium (see Recipes)
    
32. 10x Medium buffer, pH 7.2 (see Recipes)
    
33. IEC buffer A (see Recipes)
    
34. IEC buffer B (see Recipes)
    
35. Saturated ammonium sulfate solution (see Recipes)
    
36. SEC buffer (see Recipes)

Equipment

1. Pipettes (e.g., Eppendorf, model: Research^® Plus , catalog numbers: 3123000039, 3123000055, 3123000063)
   
2. Sterile tweezers
   
3. -80 °C freezer
   
4. Fluorescence plate reader (e.g., BMG LABTECH, model: FLUOstar Omega ), equipped with monochromators or appropriate filters for the fluorophore Thioflavin T (e.g., BMG excitation filter: 448(nm)-10, BMG emission filter: 482(nm)-10)
   Note: The plate reader should be heatable to 37 °C.
   
5. UV/VIS spectrophotometer (e.g., JASCO, model: V-650 )
   
6. Heating Block (e.g., Grant Instruments, model: UBD2 )
   
7. Magnetic stirrer (e.g., Cole-Parmer Instrument, Stuart, model: CB161 )
   
8. Centrifuge for 50 ml Falcons (e.g., Eppendorf, model: 5804 R )
   
9. ÄKTA Purifier (GE Healthcare)
   

Software

1. Microsoft Excel
   
2. AmyloFit (http://www.amylofit.ch.cam.ac.uk/)
   

Procedure

1. Purification of acetylated α-synuclein
   Here we briefly describe how we purify our acetylated α-synuclein, as described in Wördehoff et al., 2017. As this protocol focusses on the aggregation of α-synuclein monitored by Thioflavin T fluorescence, the purification is described as concisely as possible.
   1. We coexpress codon-optimized α-synuclein in the pT7-7 vector together with the vector pNatB containing the N-terminal acetylation enzyme NatB from Schizosaccharomyzes pombe as described by Johnson et al., 2010.
      
   2. Expression is conducted in E. coli BL21DE3 grown in 2YT medium supplemented with 50 mM K/Na-phosphate buffer pH 7.2, 2 mM MgSO₄ and 0.4% glycerol (v/v), with 100 μg/ml ampicillin and 35 μg/ml chloramphenicol as selection markers. We inoculate the (pre-)culture from a glycerol stock that has been prepared by overnight growth of a single colony in 2YT medium, mixed with 50% glycerol (sterile) and stored at -80 °C.
      
   3. After pregrowth to OD₆₀₀ of 1-1.2, expression is induced by addition of 1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) and incubated while shaking at 120 rpm for 4 h at 37 °C. We routinely prepare 500 ml culture, which gives us a final expression yield of ~25 mg acetylated α-synuclein.
      
   4. Cells are harvested by centrifugation at 6,000 x g for 15 min at 4 °C.
      
   5. An individual cell pellet (from 500 ml culture) is resuspended in 15 ml of pure H₂O and frozen at -20 °C in 50 ml Falcon tubes.
      
   6. The Falcon tubes with the frozen cell suspension are transferred to a heat block preheated to 98 °C and incubated for 30 min to both lyse the cells and precipitate proteins by heat denaturation. α-Synuclein is thermostable and remains soluble.
      
   7. Cell lysate is cooled down on ice for 10 min, afterward cell debris and precipitated proteins are pelleted at 15,000 x g and 4 °C for 30 min.
      
   8. The supernatant is then filtered through a 0.2 μm filter into a new 50 ml tube.
      
   9. Saturated ammonium sulfate solution (~4.3 M at room temperature) is added in 1-ml steps to the supernatant to 50% saturation (1:1) while stirring on a magnetic stirrer, thereby precipitating α-synuclein, nucleic acids largely remain soluble.
      
   10. The turbid solution is then incubated on ice for 20 min and pelleted at 15,000 x g and 4 °C for 30 min.
       
   11. Supernatant is discarded and the pellet is resuspended in 50 ml of IEC buffer A (optional: dissolve in 5 ml IEC buffer A and dialyzed overnight against 1 L of IEC buffer A).
       
   12. The solution is then loaded onto a 5 ml HiTrap Q HP ion exchange chromatography column (GE Healthcare) connected to an ÄKTA purifier system (GE Healthcare), equilibrated in IEC buffer A.
       
   13. α-Synuclein elutes at around 250 mM NaCl on a 0-500 mM NaCl gradient to IEC buffer B, run over 100 ml at 2.5 ml/min.
       
   14. Fractions containing α-synuclein are combined, again precipitated by ammonium sulfate and pelleted as described above.
       
   15. The pellet is resuspended in 4 ml SEC buffer and finally purified in two runs by size exclusion chromatography (SEC) on a Superdex 75 16/60 column (120 ml column volume, GE Healthcare) at 1 ml/min in SEC buffer.
   16. After SEC, α-synuclein is sterile-filtered through a 0.2 μm filter and the concentration is measured on a spectrophotometer, using and extinction coefficient of 5,600 M^-1 cm^-1 at 275 nm.
   17. α-Synuclein is aliquoted, flash-frozen in liquid nitrogen and stored at -80 °C.
   
   
2. General preparation
   1. Prepare stock solutions: 1 mM Thioflavin T in H₂O, 10% (w/v) sodium azide in H₂O. We use Thioflavin T solutions for a year; sodium azide solution is prepared fresh every month (both stored in the fridge at 4-8 °C).
      
   2. Buffers and protein solutions should be filter sterilized. As α-synuclein is sensitive to degradation, aliquots of α-synuclein should be stored at -80 °C and defrosted just before the assay.
      
   3. Calculate the amounts of reagents to pipette. For triplicate measurements, 400 μl sample per condition is needed (3x 120 μl, 40 μl reserve). In order to avoid that the ThT fluorescence saturates before the equilibrium of fibril formation is reached, a sufficiently high ThT concentration on the order of the applied protein concentration should be used. We typically use 10-20 μM ThT and 0.05% sodium azide (final concentrations).
      
   4. Include a negative control, which consists of buffer, salts, Thioflavin T and sodium azide.
      
   5. Preheat your plate reader to 37 °C. Aggregation assays can be performed at different temperatures, e.g., between 20 °C and 37 °C. However, we routinely use 37 °C as it is the human body temperature and α-synuclein aggregation is quite slow at lower temperatures.
      
   6. Prepare the plate: with sterile tweezers, take out as many glass beads as you need (out of the 70% ethanol solution), dry them shortly on a lint-less cloth and transfer them into the wells–choose glass beads of homogenous size. As the glass beads we use are slightly heterogeneous in terms of size and shape, we sort out glass beads with a small and large diameter, as well as glass beads that are not spherical by visual inspection. In general, the addition of glass beads is crucial to speed up aggregation and increase reproducibility of triplicates.
      
   7. We always leave the outer wells of the plate empty; they can give unexpected readings, so first well to use is B2, last well is G11.
      
   8. Optional: Pipette 120 μl water into the spaces between the wells and idle wells–this can be done to minimize possible evaporation from the sample wells.
      
   9. Prepare and label a sufficient amount of 1.5 ml protein low-bind reaction tubes and transfer tubes onto ice, do the subsequent pipetting steps on ice.
   
   
3. Sample preparation
   1. Pipette the reagents, starting with the buffer, then sodium azide, Thioflavin T and lastly the protein solution into the 1.5 ml reaction tubes. As an example, this is the pipetting scheme for 400 μl of the 50 μM α-synuclein sample shown in Figure 1, assuming stock concentrations of 200 μM for α-synuclein, 1 mM Thioflavin T, and 10% sodium azide:
      1. 294 μl of SEC buffer
         
      2. 2 μl of 10% sodium azide
         
      3. 4 μl of 1 mM Thioflavin T
         
      4. 100 μl of α-synuclein (in SEC buffer) 
   2. Shortly (1 sec) vortex the samples to mix them and put them back on ice.
      
   3. Distribute 120 μl of sample per well.
      
   4. Seal plate with the sealing tape and transfer it to the plate reader.
      
      
      
4. Plate reader settings
   1. We use a fluorescence plate reader (FLUOstar Omega by BMG LABTECH), assay temperature: 37 °C.
      
   2. Basic parameters: Microplate: Greiner 96 half area; Excitation filter: 448-10; Emission filter: 482-10; Gain: 750; Orbital Averaging–On: 3 mm, Bottom Optic, Settling time: 1 sec; Kinetic Window: 1,000 cycles, 12 flashes per well and cycle, cycle time: 900 sec.
      
   3. Layout: Mark the distribution of your samples.
      
   4. Shaking options: Orbital, 400 rpm, 30 sec before each cycle. Shaking is crucial to speed up aggregation and improve reproducibility of triplicates.
      
   5. Samples are run until fluorescence plateaus are reached.
      
   6. See also the appendix for the plate reader settings.

Data analysis

This section should guide the reader through the data analysis. We have included two exemplary aggregation assays of α-synuclein, the first showing the aggregation of α-synuclein at different concentrations (Figure 1), the second demonstrating the influence of an aggregation inhibitor (dityrosine-crosslinked α-synuclein), adapted from Wördehoff et al., 2017 (Figure 2).

1. After the assay, we first calculate the mean of the negative control triplicates, i.e., the background fluorescence of Thioflavin T in our buffer, and subtract this mean background fluorescence from each of the sample triplicates at the given time points, e.g., in Microsoft Excel. In contrast to the negative control, the samples are not averaged and displayed as triplicates in the plot.
   
2. The evaluation of the aggregation data can be done online with the AmyloFit Aggregation Fitter tool (http://www.amylofit.ch.cam.ac.uk/). AmyloFit can fit aggregation data and extract kinetic and mechanistic parameters of aggregation based on the latest mathematic models of amyloid aggregation (Meisl et al., 2016). If aggregation proceeds according to any of these models under the applied experimental conditions, and when fitted accurately and globally, parameters like primary/secondary nucleation rate constants and elongation rate constants can be extracted (Cohen et al., 2013).
   
3. As an example, we monitored the aggregation of α-synuclein at four different concentrations and evaluated the data with AmyloFit (Figure 1).
   
4. Data was exported to a tab-separated text file, with the first column being the time trace and the other ones being the fluorescence traces.
   
5. As a first step, the raw data was plotted (see Figure 1A).
   
6. The data was normalized within the Amylofit software (Figure 1B). This is done by choosing a data point (or a range of data points) in the fluorescence plateau at the end of the assay, which (or the average of which) is set to unity and all other values scaled accordingly. In the case of Figure 1B, the fluorescence intensities after 80 h (for 25 μM to 100 μM α-synuclein) or 90 h (for 10 μM α-synuclein) were used for normalization.
   
7. The next step was plotting the aggregation half-times vs. the initial monomer concentration (m0) to extract the scaling exponent, which gives insight into which aggregation mechanisms are dominant (Figure 1C). Note that non-linear behavior in the double-logarithmic plot may be observed, pointing to a change in the dominant growth mechanisms with increasing protein concentration (Meisl et al., 2016). A selection flow chart for the dominant aggregation model to choose can be found in the supplementary information of the publication by Meisl et al., 2016.
   
   
   Figure 1. Aggregation kinetics of α-synuclein studied by ThT fluorescence. Four concentrations of α-synuclein (10 μM, 25 μM, 50 μM and 100 μM) aggregated in the presence of 10 μM Thioflavin T and 0.05% sodium azide in SEC buffer, measured in triplicates. The assay has been evaluated by the AmyloFit software (http://www.amylofit.ch.cam.ac.uk). A. Raw data, absolute ThT fluorescence readings of triplicate measurements of the four concentrations; B. Normalized aggregation data of the four concentrations; C. Double logarithmic half-time plot (log(m0): initial monomer concentration vs log(τ): aggregation half-time).
   
   
8. The scaling exponent was -0.26, reflecting the low monomer concentration dependence of α-synuclein aggregation at neutral pH. This indicates that under these experimental conditions mechanisms are active that limit the monomer concentration dependence, such as saturating fibril elongation (Meisl et al., 2016; Buell et al., 2014), fibril fragmentation (Shvadchak et al., 2015), and nucleation at the air-water interface (Campioni et al., 2014), which may become saturated with protein (Pham et al., 2016).
   
9. The assay can also be used to identify molecules that interfere with α-synuclein aggregation.
   
10. As an example, we monitored the aggregation of α-synuclein in the absence and presence of dityrosine (DiY)-crosslinked α-synuclein (Wördehoff et al., 2017) (Figure 2A).
    
11. It has to be noted that a reduced ThT fluorescence intensity does not necessarily mean that aggregation is inhibited, as inhibitors can also interact with ThT, obstruct ThT binding sites on fibrils and/or change the morphology of aggregates so that ThT can no longer bind (Jameson et al., 2012).
    
12. To complement our ThT data, we therefore collected the samples with and without inhibitor after the assay and performed size exclusion chromatography to confirm that α-synuclein stays monomeric in the presence of the inhibitor (DiY α-synuclein) (Figure 2B).
    
    
    Figure 2. Aggregation kinetics of α-synuclein in presence of an inhibitor. 25 μM α-synuclein aggregated with (blue) and without (grey) 25 μM dityrosine (DiY)-crosslinked α-synuclein and 10 μM Thioflavin T and 0.05% sodium azide in SEC buffer, measured in triplicates. A. ThT fluorescence readings of triplicate measurements with and without inhibitor. B. Size exclusion chromatography of samples taken before and after the aggregation assay with and without inhibitor. Note that in presence of the inhibitor, the monomer peak after aggregation is equally high, whereas it decreased markedly without the inhibitor.
    

Recipes

1. Modified 2YT medium (1 L)
   31 g YT medium (2x) premixed powder
   2 mM MgSO₄
   0.4% Glycerol (v/v)
   Dissolve and mix with around 950 ml Milli-Q H₂O and autoclave
   After autoclaving, mix 900 ml of the medium with 100 ml 10x medium buffer
   Store at room temperature
   
2. 10x Medium buffer, pH 7.2 (1 l)

   Final concentration	For 1 L
   360 mM K2HPO4	82.16 g K2HPO4·3H2O
   140 mM NaH2PO4	19.32 g NaH2PO4·H2O

   Dissolve in Milli-Q H₂O and autoclave
   Store at room temperature
   
3. IEC buffer A (50 mM Tris-HCl pH 8, for 500 ml)
   50 mM Tris(hydroxymethyl)aminomethane
   Dissolve 3.03 g Tris(hydroxymethyl)aminomethane in 400 ml Milli-Q H₂O, titrate to pH 8 with HCl, adjust with Milli-Q H₂O to 500 ml
   Store at room temperature
   
4. IEC buffer B (50 mM Tris-HCl pH 8, 800 mM NaCl, for 500 ml)

   Final concentration	For 500 ml
   50 mM Tris(hydroxymethyl)aminomethane	3.03 g
   800 mM NaCl	23.38 g NaCl

   Dissolve in 400 ml Milli-Q H₂O, titrate to pH 8 with HCl, adjust with Milli-Q H₂O to 500 ml
   Store at room temperature
   
5. Saturated ammonium sulfate solution (250 ml)
   4.3 M Ammonium sulfate
   Dissolve 142.05 g (NH₄)₂SO₄ in Milli-Q H₂O and adjust the volume up to 250 ml with Milli-Q H₂O and store at room temperature
   
6. SEC buffer (1 L)

   Final concentration	For 1 L
   20 mM K2HPO4	4.56 g K2HPO4·3H2O
   5 mM KH2PO4	0.68 g KH2PO4
   100 mM KCl	7.46 g KCl

   Dissolve in Milli-Q H₂O and filter sterilize
   The buffer is stored at room temperature
   

Acknowledgments

This method was adapted from Wördehoff et al. (2017). The plasmid pNatB (pACYCduet-naa20-naa25) was a gift from Dan Mulvihill (Addgene plasmid No. 53613). The project has received funding from the European Research Council under the European Union's Horizon 2020 research and innovation program, grant agreement No. 726368. The authors declare that no competing interests exist.

References

1. Anderson, J. P., Walker, D. E., Goldstein, J. M., de Laat, R., Banducci, K., Caccavello, R. J., Barbour, R., Huang, J., Kling, K., Lee, M., Diep, L., Keim, P. S., Shen, X., Chataway, T., Schlossmacher, M. G., Seubert, P., Schenk, D., Sinha, S., Gai, W. P. and Chilcote, T. J. (2006). Phosphorylation of Ser-129 is the dominant pathological modification of α-synuclein in familial and sporadic Lewy body disease. J Biol Chem 281(40): 29739-29752.
   
2. Biancalana, M. and Koide, S. (2010). Molecular mechanism of Thioflavin-T binding to amyloid fibrils. Biochim Biophys Acta 1804(7): 1405-1412.
   
3. Buell, A. K., Galvagnion, C., Gaspar, R., Sparr, E., Vendruscolo, M., Knowles, T. P., Linse, S. and Dobson, C. M. (2014). Solution conditions determine the relative importance of nucleation and growth processes in α-synuclein aggregation. Proc Natl Acad Sci U S A 111(21): 7671-7676.
   
4. Campioni, S., Carret, G., Jordens, S., Nicoud, L., Mezzenga, R. and Riek, R. (2014). The presence of an air-water interface affects formation and elongation of α-synuclein fibrils. J Am Chem Soc 136(7): 2866-2875.
   
5. Cohen, S. I., Linse, S., Luheshi, L. M., Hellstrand, E., White, D. A., Rajah, L., Otzen, D. E., Vendruscolo, M., Dobson, C. M. and Knowles, T. P. (2013). Proliferation of amyloid-β42 aggregates occurs through a secondary nucleation mechanism. Proc Natl Acad Sci U S A 110(24): 9758-9763.
   
6. Giehm, L. and Otzen, D. E. (2010). Strategies to increase the reproducibility of protein fibrillization in plate reader assays. Anal Biochem 400(2): 270-281.
   
7. Hashimoto, M., Hsu, L. J., Sisk, A., Xia, Y., Takeda, A., Sundsmo, M. and Masliah, E. (1998). Human recombinant NACP/α-synuclein is aggregated and fibrillated in vitro: relevance for Lewy body disease. Brain Res 799(2): 301-306.
   
8. Iyer, A., Roeters, S. J., Schilderink, N., Hommersom, B., Heeren, R. M., Woutersen, S., Claessens, M. M. and Subramaniam, V. (2016). The impact of N-terminal acetylation of α-synuclein on phospholipid membrane binding and fibril structure. J Biol Chem 291(40): 21110-21122.
   
9. Jameson, L. P., Smith, N. W. and Dzyuba, S. V. (2012). Dye-binding assays for evaluation of the effects of small molecule inhibitors on amyloid (Aβ) self-assembly. ACS Chem Neurosci 3(11): 807-819.
   
10. Johnson, M., Coulton, A. T., Geeves, M. A. and Mulvihill, D. P. (2010). Targeted amino-terminal acetylation of recombinant proteins in E. coli. PLoS One 5(12): e15801.
    
11. Knowles, T. P., Vendruscolo, M. and Dobson, C. M. (2014). The amyloid state and its association with protein misfolding diseases. Nat Rev Mol Cell Biol 15(6): 384-396.
    
12. LeVine, H., 3rd (1993). Thioflavine T interaction with synthetic Alzheimer's disease β-amyloid peptides: detection of amyloid aggregation in solution. Protein Sci 2(3): 404-410.
    
13. Meisl, G., Kirkegaard, J. B., Arosio, P., Michaels, T. C., Vendruscolo, M., Dobson, C. M., Linse, S. and Knowles, T. P. (2016). Molecular mechanisms of protein aggregation from global fitting of kinetic models. Nat Protoc 11(2): 252-272.
    
14. Murray, A. N., Palhano, F. L., Bieschke, J. and Kelly, J. W. (2013). Surface adsorption considerations when working with amyloid fibrils in multiwell plates and Eppendorf tubes. Protein Sci 22(11): 1531-1541.
    
15. Naiki, H., Higuchi, K., Hosokawa, M. and Takeda, T. (1989). Fluorometric determination of amyloid fibrils in vitro using the fluorescent dye, thioflavin T1. Anal Biochem 177(2): 244-249.
    
16. Pham, C. L., Rey, A., Lo, V., Soules, M., Ren, Q., Meisl, G., Knowles, T. P., Kwan, A. H. and Sunde, M. (2016). Self-assembly of MPG1, a hydrophobin protein from the rice blast fungus that forms functional amyloid coatings, occurs by a surface-driven mechanism. Sci Rep 6: 25288.
    
17. Shvadchak, V. V., Claessens, M. M. and Subramaniam, V. (2015). Fibril breaking accelerates α-synuclein fibrillization. J Phys Chem B 119(5): 1912-1918.
    
18. Vassar, P. S. and Culling, C. F. (1959). Fluorescent stains, with special reference to amyloid and connective tissues. Arch Pathol 68: 487-498.
    
19. Wördehoff, M. M., Shaykhalishahi, H., Gross, L., Gremer, L., Stoldt, M., Buell, A. K., Willbold, D. and Hoyer, W. (2017). Opposed effects of dityrosine formation in soluble and aggregated α-synuclein on fibril growth. J Mol Biol 429(20): 3018-3030.