Nov 07, 2023
Production of α-synuclein preformed fibrils (PFF)
- Tae-In Kam1,2,
- Rong Chen1,2,
- Valina L. Dawson1,2,3,4,
- Ted Dawson1,2,3,5
- 1Neuroregeneration and Stem Cell Programs, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA;
- 2Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA;
- 3Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA;
- 4Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA;
- 5Department of Pharmacology and Molecular Sciences, and SJ Yan and HJ Mao Laboratory of Chemical Biology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
Protocol Citation: Tae-In Kam, Rong Chen, Valina L. Dawson, Ted Dawson 2023. Production of α-synuclein preformed fibrils (PFF). protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpbw28lzp/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 24, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 57361
Keywords: ASAPCRN, alpha-synuclein, preformed fibrils, PFF, SNCA
Abstract
This protocol outlines the procedure to produce preformed fibrils (PFF).
It has been adapted from Volpicelli-Daley et al., 2014
Materials
1X PBSQuality BiologicalCatalog #114-058-101
ClearColi BL21(DE3) Electrocompetent cellsLucigenCatalog #60810
Protease Inhibitor CocktailSigma AldrichCatalog #P8340
Superdex 200 increase 10/300GGe HealthcareCatalog #45-002-570
Amicon Ultra centrifugal filterEmd MilliporeCatalog #n/a
Hitrap Q Sepharose Fast Flow anion-exchange columnsGe HealthcareCatalog #450-002-58
Ni Sepharose 6 Fast Flow Ge HealthcareCatalog #17-5318-06
ToxinSensor Chromogenic LAL Endotoxin Assay KitGenscriptCatalog # L00350
PD-10 columns Ge HealthcareCatalog #17085101
Pierce BCA protein assayThermo ScientificCatalog #23227
400 mesh carbon coated copper gridsSPI suppliesCatalog #3540C-CF
Mouse anti-pSer129-α-synucleinBioLegendCatalog #825701
Mouse anti-MAP2Sigma AldrichCatalog #M9942
Donkey polyclonal anti-mouse Alexa fluor 488Jackson ImmunoresearchCatalog # Cat#715-545-151
Donkey polyclonal anti-mouse CY3Jackson ImmunoresearchCatalog #715-165-151
Primary cultured neuron (mouse cortical neuron) on DIV 7.Catalog #n/a
High-salt buffer :750 mM Nacl, 10 mM Tris (pH 7.6) and 1 mM EDTA with protease inhibitors including 1 mM PMSF.
Coomassie stain: 0.2% (wt/vol) Coomassie Brilliant Blue R250 and 50% (vol/vol) methanol; dissolve the dye, add 10% (vol/vol) acetic acid, and then bring it to the final volume with water. This solution can be stored indefinitely at room temperature.
SDS-PAGE (12%): 4.9mL H20 (autoclaved), 2.5mL Tris HCl pH 8.8, 120uL SDS 20%, 2.5mL Bisacrylamide, 60uL APS, 5uL TEMED
Equipment
Eppendorf Thermomixer
Phillips CM 120 TEM (80 kV) with an AMT ER-80 charge-coupled device (8 megapixel).
Philips EM 410 TEM with a Soft Imaging System Megaview III digital camera.
Safety warnings
CAUTION: Because of highly neurotoxic and transmission characters of α-synuclein (α-syn) preformed fibrils (PFF), it’s strongly recommended the use of gloves, face mask, and protective goggles for all procedures involving the use of synuclein fibrils. Clean any spills with a solution of 10% SDS in water, followed by multiple successive washes in 70 % ethanol and distilled water.
Step 3. Preparation of fibrils for neuronal treatment or injection. The steps here should be done
in a fume hood or biosafety cabinet.
Generation of α-synuclein monomer
Generation of α-synuclein monomer
13h 5m
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 .
Prepare starter culture by adding a cell stock to LB medium.
Add starter culture to a large culture medium with ampicillin, followed by incubation Overnight at 37 °C with shaking.
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.
Break the bacterial cells using a high-pressure homogenizer, micro-fluidizer.
Boil for 00:15:00 to precipitate other proteins and then immediately incubate on On ice to cool.
15m
Spin at 6,000 g for 00:20:00 at 4 °C C.
20m
Use the supernatant for further dialysis with 10 mM Tris (pH 7.6), 50 mM NaCl and 1 mM EDTA.
Concentrate the protein through Amicon Ultra centrifuge filter (3.5 kDa cutoff).
Filter the protein using a 0.22 μm syringe filter and load it onto a Superdex 200 column.
Collect samples and check each fraction by SD-PAGE, followed by Coomassie staining.
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.
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.
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).
Dialyze with 10 mM Tris (pH 7.6) and 50 mM NaCl.
Concentrate the fractions, aliquot, and store at -80 °C ºC.
Generation of fibrils
Generation of fibrils
1w 0d 0h 10m
Thaw aliquot of recombinant α-synuclein monomer onOn ice .
Centrifuge at 4 °C C for 00:10:00 in centrifuge at 12,000xg.
10m
Transfer the supernatant with a pipette and measure the final protein concentration using BCA protein assay.
Dilute the monomeric protein into PBS for a final concentration of 5 mg/mL.
Vortex tubes for 00:00:03 to mix contents and seal the microcentrifuge lid with a parafilm to prevent opening of lid.
3s
Shake for 7 days at 37 °C with 1,000 RPM (Eppendorf Thermomixer). Solution should turn turbid during this period.
Make20 µL of aliquots and freeze on dry ice. Store at-80 °C C.
Validation of fibril formation before move to the next step (e.g. Thioflavin T, sedimentation assay)
Thioflavin T assay
1. Prepare 1 mM Thioflavin T stock in PBS.
Add 5 µL of α-synuclein PFF into 95 µL of 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 00:10:00 .
4. Measure the fluorescence at an excitation 450 nm and emission at 490 nm.
10m
Sedimentation assay
1. Centrifuge 20 µL of PFFs at 100,000 g for00:30:00 at 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 for00:30:00 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.
1h
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.
Preparation of fibrils for neuronal treatment or injection
Preparation of fibrils for neuronal treatment or injection
4m
NOTE: All the steps here should be done in a fume hood or biosafety cabinet.
Thaw sufficient aliquots of 5 mg/mL PFF at Room temperature immediately before use.
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.
Seal the microcentrifuge with a parafilm and make a small hole for sonication.
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.
Allow sonicated PFF solution to settle for 00:01:00 . PFF suspension is now ready for use.
1m
Quality control testing
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 00:02:00 .
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 00:00:30 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.
2m 30s
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 Overnight
4. Visualize p-α-syn aggregates formed from endogenous alpha-synuclein with a confocal microscope.
30s
Protocol references
Volpicelli-Daley, L.A., Luk, K.C., and Lee, V.M. (2014). Addition of exogenous alpha-synuclein preformed fibrils to primary neuronal cultures to seed recruitment of endogenous alpha-synuclein to Lewy body and Lewy neurite-like aggregates. Nat Protoc 9, 2135-2146. 10.1038/nprot.2014.143.