Jun 18, 2020
Generation and Sonication of α-synuclein Fibrils
- Vijay Singh1,
- Marta Castellana-Cruz2,
- Nunilo Cremades3,
- Laura Volpicelli-Daley1
- 1Department of Neurology, Center for Neurodegeneration and Experimental Therapeutics, University of Alabama at Birmingham, Birmingham, AL, 35294, USA.;
- 2Centre for Misfolding Diseases, Department of Chemistry, University of Cambridge, CB2 1EW, UK.;
- 3Institute for Biocomputation and Physics of Complex Systems (BIFI)-Joint Unit BIFI-IQFR (CSIC), University of Zaragoza, Zaragoza 50018, Spain.
- Neurodegeneration Method Development CommunityTech. support email: ndcn-help@chanzuckerberg.com
Protocol Citation: Vijay Singh, Marta Castellana-Cruz, Nunilo Cremades, Laura Volpicelli-Daley 2020. Generation and Sonication of α-synuclein Fibrils. protocols.io https://dx.doi.org/10.17504/protocols.io.bhhrj356
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: June 13, 2020
Last Modified: June 18, 2020
Protocol Integer ID: 38161
Keywords: monomeric α-synuclein fibrils, in vitro primary neuron, human recombinant, iPSCs,
Abstract
Animal models that accurately recapitulate the accumulation of alpha-synuclein (α-syn) inclusions, progressive neurodegeneration of the nigrostriatal system and motor deficits can be useful tools for Parkinson's disease (PD) research. The preformed fibril (PFF) synucleinopathy model in rodents generally displays these PD-relevant features, however, the magnitude and predictability of these events is far from established. We therefore have optimized the synthesis generation of α-syn fibrils to ensure reliable, robust results. These fibrils can be added to neurons in culture, differentiated iPSCs, or injected into mice or rats. The protocol includes steps for fibril synthesis as well as sonication for fibril fragmentaion which is a critical step for inducing formation of α-syn inclusions.
Attachments
Guidelines
This protocol is a modification from previously published manuscripts (Patterson et al., 2019; Polinski et al., 2018; Stoyka et al., 2020; Volpicelli-Daley, Luk, & Lee, 2014).
For safe handling of fibrils please read Bousset L et al. (2016) An Efficient Procedure for Removal and Inactivation of alpha-Synuclein Assemblies from Laboratory Materials J Parkinsons Dis.6:143-51 https://pubmed.ncbi.nlm.nih.gov/26639448/
When opening tubes and pipetting, perform in a BSL2 safety hood to prevent contamination.
References:
CITATION
LINK
10.3791/59758CITATION
CITATION
CITATION
Materials
Equipment:
- Spectrophotometer
- Shaker at 37 °C
- -80 °C freezer
- Dynamic light scattering detector such as Dynapro Nanostar (WDPN; Wyatt Technology) or access to transmission electron microscopy
- Benchtop centrifuge
- Qsonica 700W cup horn sonicator with chiller at 15 °C (other labs use Bioruptor Plus (Diagenode; Denville, NJ) with success)
OR
Probe tip sonicator (our lab uses Fisher FB12011).**
Note
**This is not recommended for in vivo work. If it is used for cell culture, use it in the BSL2 hood. Wear disposable lab sleeves over lab coat, Filtering Facepiece Respirator (Fisher 19-002-711), goggles. Clean hood with 1% SDS followed by water followed by 70% ethanol.
- 1.5 mL sonication tube (cat# NC0869649 Fisher Scientific)
Materials:
- Monomeric α-synuclein
Note
Monomeric α-synuclein. For in vitro primary neuron experiments, fibrils made using mouse or human recombinant α-synuclein will work. For differentiated human iPSCs, use human α-synuclein. For in vivo mouse models in which α-synuclein is endogenously expressed, use recombinant mouse α-synuclein because human α-synuclein is not as efficient in seeding α-synuclein inclusions from endogenously expressed mouse α-synuclein.
We recommend expressing and purifying α-synuclein from Escherichia coli as described (Volpicelli-Daley et al., 2014) or obtaining purified α-synuclein. For best results the α-synuclein should not have a tag (His, HA, GFP etc.). Our lab has not had success making fibrils from commercial sources.
Remove endotoxin using Pierce High Capacity Endotoxin Removal Spin columns (PI88276). Most other endotoxin removal kits have detergent which can be toxic in neuron assays.
Determine endotoxin levels using LAL endotoxin assay kit (GenScript catalog number L00350). Our values are <0.05 endotoxin units per 1 μg of protein.
Store purified α-synuclein in 10 mM Tris, 7.5 at >10 mg/mL at -80 °C .
- 8M guanidinium chloride
- 1.5 mL sterile LoBind microcentrifuge tubes
- 500 mM KCl; sterile filtered
- 500 mM Tris, 7.5 ; sterile filtered
- Ice
- LAL endotoxin assay kit (GenScript catalog number L00350)
- PBS
- Uranyl acetate solution
- Deionized water
- Cuvettes
- 1% SDS
Safety warnings
Please see the Safety Data Sheet (SDS) for safety warnings and hazards before start.
When opening tubes and pipetting, perform in a BSL2 safety hood to prevent contamination.
Before start
Sonicating Fibril
Proper sonication is a key step for the fibril model to work. For all in vivo work which involved injecting fibrils into mice or rats, we use the QSonica 700 sonicator with cup horn and tube rack for 1.5 mL polypropylene tubes with a chiller at 16°C. The cup horn sonication produces short fragments which maintain their morphology for 6-8 hours (at least) and can be stored in dry ice overnight, thawed and maintained at room temperature, and therefore remain active after overnight shipments.We found that over time, the heat generated by a probe tip sonicator causes the fibrils to form amorphous aggregates (Figure 1). This is a problem because stereotaxic surgeries can take several hours and the amorphous aggregates that form while the fibrils sit on the bench causes variability and reduces the concentration of seeding competent fragments. Another advantage of using the cup horn sonicator over probe tip is that 25 μL of fibrils can be sonicated, reducing the volume needed. This is also a closed tube system which increases safety. For neuron or cell culture work in which the fibrils are added to media and then the cells immediately after sonication, a probe tip sonicator is okay. Again, this should be performed in a BSL2 hood with all proper PPE (nanoparticle respirator, goggles, gloves etc.). The volume of fibrils to be sonicated cannot be less than 100 μL.
In all cases, we wear PPE when working with fibrils. We clean any spills with 1% SDS.
Figure 1: Transmission electron microscopy of α-synuclein fibrils. Immediately after probe tip or cup horn sonication, long fibrils are broken into small fragments. However, after six hours at room temperature, probe tip sonicated fibrils begin to form amorphous aggregates. With cup horn sonication performed at 16°C, the fragments after 6 hours appear similar in morphology compared to immediately after sonication. When the sonicated fibrils are placed in dry ice overnight, thawed and left at room temperature for 6 hours, the fragments appear similar to immediately after sonication, indicating that overnight shipments will maintain active fragments.
Making fibrils
Making fibrils
Thaw monomer On ice .
Spin monomer for 00:30:00 at max speed on a benchtop centrifuge (15000 x g - 20000 x g depending on centrifuge) at 4 °C .
Note
This step removes large fibrils or aggregates that may have been generated when storing and thawing high concentration of α-synuclein.
Keep monomer On ice at all times to prevent fibrillization.
Note
We always check concentration of monomer after thawing. We have found that after a few freeze/thaws, the protein concentration becomes lower, likely because of oligomer or fibril formation (we assume path length =1). Use the spectrophotometer, A280, and Beer-Lambert law (A = ɛ *c) to determine concentration of monomer. To measure the concentration of synuclein, ɛ for synuclein is 5960 M–1 cm–1 for human synuclein and 7450 M–1 cm–1 for mouse synuclein. Use 14.5 kDa for molecular weight.
Note
Example from our lab:
Protein concentration written on tube says 13 mg/mL
Use 10 mM Tris, pH7.5, as blank (what the monomer is in)
A280 = 3.6
Calculate (3.6/5.96)*14.5
= 8.7 mg/mL protein
Dilute monomer to 5 mg /ml in 50 millimolar (mM) Tris-HCL , 150 millimolar (mM) KCL , 7.5 , final volume 500 µL .
Shake at 37 °C for 168:00:00 (1 week); check for turbidity at end of week.
Note
If it is not turbid, it did not work.
Spin fibrils for 00:10:00 at max speed in benchtop centrifuge at Room temperature .
Discard supernatant and resuspend in approximately 50% of initial volume in 50 millimolar (mM) Tris-HCL , 150 millimolar (mM) KCL , 7.5 (for example, if making at 500 µL , resuspend in 250 µL ).
Measure the concentration of fibrils.
Mix 5 µL of fibril with 5 µL of 8 Molarity (M) guanidinium chloride (GnCl) (SAMPLE) and 5 µL of 50 millimolar (mM) Tris-HCL , 150 millimolar (mM) KCL , 7.5 buffer with 5 µL GnCl (BLANK) .
Incubate for 01:00:00 on the bench at Room temperature .
Use the spectrophotometer to determine concentration of fibrils using BLANK as the blank and SAMPLE to determine concentration.
Note
Note that the fibrils are diluted in GnCl, so the actual concentration will be 2X the readout.
Use 50 millimolar (mM) Tris-HCL , 150 millimolar (mM) KCL , 7.5 to bring fibrils to 5 mg /mL .
Aliquot and store at -80 °C
Sonicating Fibrils
Sonicating Fibrils
Pipet 25 µL fibrils (5 mg/mL) in 1.5 mL sonication tube.
Fill Qsonica water reservoir with about 900 mL water .
Note
Make sure reservoir water level height is 7 cm.
Attach cooling system to Qsonica and set the temperature at 15 °C .
Place 1.5 mL , therefore,sonication tube with fibrils in Qsonica tube holder.
Note
The gap between tube bottom surface and Qsonica probe upper surface should be 1 cm.
Figure 2. Diagram of Qsonica700 with multi-tube holder
Note
Sonication cycle parameters:
We initially spent time optimizing the parameters for sonication using our Qsonica system. The following parameters may not be ideal depending on the sonicator in an individual lab, and we therefore recommend personally optimizing conditions first. The goal is to consistently obtain fragments of α-synuclein that are on average 50 nm in length (Figure 1, 3). If the fibrils are not sufficiently fragmented, the abundance of α-synuclein inclusions produced can be low and highly variable.
Total sonication time: 00:15:00 .
Sonication pulse on for 00:00:03 and off for 00:00:02 .
Amplitude 30%.
Note
After sonication sometimes, there are few droplets inside the sample tube and sometimes not. If droplets are there take out tube and spin it at 1000 rpm, 00:00:10 . Take the sample tube in safety hood and mix the PFF sample with pipette 5 times in and out (avoid introducing bubbles while pipetting).
Sonicate sample for another 07:30:00 .
Note
After sonication PFF sample can be used that day or stored in -80 °C or on dry ice (for shipping) for one day, but a sample in -80 °C should be fine for longer. After taking out from-80 °C sample is good at least for 08:00:00 at Room temperature .
Confirming Fragmentation
Confirming Fragmentation
Note
Before injecting fibrils into a large cohort of mice and waiting several months for results, researchers should ensure that their sonication protocol results in sufficient fibril fragmentation. We use a dynamic light scattering detector as a quick and reliable method to check for fragmentation. Transmission electron microscopy is another method that can be used.
Dynamic Light Scattering:
Pipet 1 µL sonicated PFF sample and dilute with 4 µL 1x PBS (filtered with 0.22 μm filter) .
Put 5 µL diluted PFF sample in disposable microcuvette (WYATT Technology) for DLS.
Figure 3. Example of DLS profiles of fibrils before and after sonication. A radius of 50-70nm with minimal variability is optimal.
When done, fill cuvette with 2% SDS to decontaminate fibrils (using a squirt bottle).
Let sit for at least 00:30:00 .
Use squirt bottle to rinse several times with DI water.
Use filtered water for last rinse.
Make sure dry before next use.
Note
The important point is to make sure the cuvette remains dust free.
Note
In the next step we perform our transmission electron microscopy with the help of our High Resolution Core facility. Many universities offer an EM core.
Transmission Electron Microscopy
Transmission Electron Microscopy
Immediately after sonication, dilute 2 µL of sonicated fibrils with 98 µL of PBS .
Spot 5 µL of diluted fibrils on glow discharged EM grid.
Wait for 00:00:20 and remove extra buffer by allowing it to wick onto a kimwipe.
Add 8 µL uranyl acetate solution .
Remove extra uranyl acetate solution after 00:00:10 .
Let dry and image.
Citations
Patterson, J. R., Polinski, N. K., Duffy, M. F., Kemp, C. J., Luk, K. C., Volpicelli-Daley, L. A., . . . Sortwell, C. E. (2019). Generation of Alpha-Synuclein Preformed Fibrils from Monomers and Use In Vivo
10.3791/59758Polinski, N. K., Volpicelli-Daley, L. A., Sortwell, C. E., Luk, K. C., Cremades, N., Gottler, L. M., . . . Dave, K. D. (2018). Best Practices for Generating and Using Alpha-Synuclein Pre-Formed Fibrils to Model Parkinson's Disease in Rodents
10.3233/JPD-171248Stoyka, L. E., Arrant, A. E., Thrasher, D. R., Russell, D. L., Freire, J., Mahoney, C. L., . . . Volpicelli-Daley, L. A. (2020). Behavioral defects associated with amygdala and cortical dysfunction in mice with seeded alpha-synuclein inclusions
10.1016/j.nbd.2019.104708Volpicelli-Daley, L. A., Luk, K. C., & 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
10.1038/nprot.2014.143