TITLE: Production of α-synuclein preformed fibrils (PFF)
AUTHORS: Tae-In Kam, Rong Chen, Valina L. Dawson, Ted Dawson

[DESCRIPTION]
This protocol outlines the procedure to produce preformed fibrils (PFF).
It has been adapted from Volpicelli-Daley et al., 2014

[STEPS]
SECTION: Generation of α-synuclein monomer
1. Transform α-synuclein plasmids (full length human α-synuclein cloned into pRK172 vector) into ClearColiTM BL21-competent E. coli, that have been genetically modified so that LPS does not trigger LPS-mediated immune response. From the small scale culture in LB medium, make a bacteria cell stock and keep at -80 °C.

SECTION: Generation of α-synuclein monomer
2. Prepare starter culture by adding a cell stock to LB medium.
SECTION: Generation of α-synuclein monomer
3. Add starter culture to a large culture medium with ampicillin, followed by incubation   at 37 °C with shaking.

SECTION: Generation of α-synuclein monomer
4. Resuspend the pellet in high-salt buffer (750 mM Nacl, 10 mM Tris (pH 7.6) and 1 mM EDTA with protease inhibitors including 1 mM PMSF.
SECTION: Generation of α-synuclein monomer
5. Break the bacterial cells using a high-pressure homogenizer, micro-fluidizer.
SECTION: Generation of α-synuclein monomer
6. Boil for 15 min to precipitate other proteins and then immediately incubate on on ice to cool.

SECTION: Generation of α-synuclein monomer
7. Spin at 6,000 g for 20 min at 4 °C C.

SECTION: Generation of α-synuclein monomer
8. Use the supernatant for further dialysis with 10 mM Tris (pH 7.6), 50 mM NaCl and 1 mM EDTA.
SECTION: Generation of α-synuclein monomer
9. Concentrate the protein through Amicon Ultra centrifuge filter (100 kDa cutoff).

SECTION: Generation of α-synuclein monomer
14. Apply protein to a Hi-Trap Q HP anion-exchange column (gradient ranging from 25mM NaCl to 1 M NaCl) and collect fractions, followed by SDS-PAGE and Coomasie staining.
SECTION: Generation of α-synuclein monomer
12. Collect the pure fractions with an appropriate α-synuclein bands (~15 kDa) and dialyze with 10 mM Tris (pH 7.6), 25 mM NaCl, and 1 mM EDTA. 
SECTION: Generation of α-synuclein monomer
15. Generate endotoxin-free α-synuclein: remove the bacterial endotoxins using Toxineraser endotoxin removal kit (GeneScript), and measure the level of endotoxin using ToxinSensor Chromogenic LAL Endotoxin Assay Kit (GenScript).
SECTION: Generation of α-synuclein monomer
16. Concentrate the fractions, aliquot, and store at -80 °C ºC.

SECTION: Generation of fibrils
17. Centrifuge at 4 °C C for 10 min in centrifuge at 12,000xg.

SECTION: Generation of fibrils
18. Transfer the supernatant with a pipette and measure the final protein concentration using BCA protein assay.
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19. Dilute the monomeric protein into PBS for a final concentration of 5 mg/mL.
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20. Shake for 7 days at 37 °C with 1,000 RPM (Eppendorf Thermomixer). Solution should turn turbid during this period.

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21. Make20 µL of aliquots and freeze on dry ice. Store at-80 °CC.

SECTION: Generation of fibrils
22. Validation of fibril formation before move to the next step (e.g. Thioflavin T, sedimentation assay)
SECTION: Generation of fibrils
22.1. Thioflavin T assay
1. Prepare 1 mM Thioflavin T stock in PBS. 
Add 5 µL of α-synuclein PFF into 95 µLof 25 μM Thioflavin T. (Use 5 µL of PBS alone and 5 µL of monomeric α-synuclein as a control.)
3. Incubate at room temperature for 10 min.
4. Measure the fluorescence at an excitation 450 nm and emission at 490 nm.

SECTION: Generation of fibrils
22.2. Sedimentation assay
1. Centrifuge 20 µL of PFFs at 100,000 g for30 minat room temperature.
2. Transfer the supernatant to a new tube (→ 'soluble' fraction).
3. Resuspend the pellet in 20 µL of PBS, and centrifuge it again at 100,000 g for30 min at room temperature.
4. Discard the supernatant and resuspend the pellet in 20 µL of PBS (→ 'pellet' fraction).
5. Perform SDS-PAGE, followed by Coomassie staining.
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23. NOTE:
Freeze/thawing can compromise the activity of PFF. Please prevent thawing of unused aliquots. 
Sterile components are used to assemble reactions to prevent microbial contamination. 
SECTION: Preparation of fibrils for neuronal treatment or injection
24. NOTE: All the steps here should be done in a fume hood or biosafety cabinet. 
SECTION: Preparation of fibrils for neuronal treatment or injection
25. Thaw sufficient aliquots of 5 mg/mL PFF at Room temperature immediately before use.

SECTION: Preparation of fibrils for neuronal treatment or injection
26. Dilute PFF to 100 μg/mL (for primary neuronal culture experiment) or 2 mg/ml (for intrastriatal injection) by adding PFF to a sterile microcentrifuge tube containing the appropriate volume of sterile PBS.
SECTION: Preparation of fibrils for neuronal treatment or injection
27. Seal the microcentrifuge with a parafilm and make a small hole for sonication.
SECTION: Preparation of fibrils for neuronal treatment or injection
28. Sonicate (Branson Digital Sonifier SFX 150 from Emerson) at amplitude 20% for a total of 60 pulses (0.5 seconds on/off cycle). Pause briefly between every 10-12 pulses to prevent solution from heating up excessively and to avoid frothing.
SECTION: Preparation of fibrils for neuronal treatment or injection
29. Allow sonicated PFF solution to settle for 1 min. PFF suspension is now ready for use.

SECTION: Preparation of fibrils for neuronal treatment or injection
30. Quality control testing 
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30.1. Transmission electron microscopy (TEM)
1. Adsorb α-synuclein PFF (prepare the samples before and after sonication) to glow discharged 400 mesh carbon coated copper grids for 2 min.
2. Quickly transfer the grids through three drips of Tris-HCl (50 mM pH 7.4), rinse, and then float upon two consecutive drops of 0.75% uranyl formate for 30 s each. 
3. Aspirate the stained solution and allow the grid to dry before imaging.
4. Plate on a Phillips CM 120 TEM operating at 80 kV and capture the images with an ER-80 CCD.
SECTION: Preparation of fibrils for neuronal treatment or injection
30.2. Immunofluorescence with  phosphorylated α-synuclein (Ser129) antibody
1. Add 1 μg/mL of alpha-synuclein PFF into primary cultured neurons on DIV7.
2. Incubate the neurons for a further 10-14 days with replacing a half of the fresh medium every 3 days.
3. Fix the neurons and perform double-staining immunofluorescence using 
p-α-syn (Biolegend) and MAP2 (Sigma) antibodies at 4 °C 30 s
4. Visualize p-α-syn aggregates formed from endogenous alpha-synuclein with a confocal microscope. 
SECTION: Generation of α-synuclein monomer
10. Filter the protein using a 0.22 μm syringe filter and load it onto a Superdex 200 column.
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11. Check each fraction by SD-PAGE, followed by Coomassie staining.
SECTION: Generation of α-synuclein monomer
13. Store at -80 °C until needed to generate fibrils