TITLE: Generation and Sonication of α-synuclein Fibrils
AUTHORS: Vijay Singh, Marta Castellana-Cruz, Nunilo Cremades, Laura Volpicelli-Daley

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">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.</div></div>

[STEPS]
?. [Making fibrils]
Thaw monomer .
on ice
?. [Making fibrils]
Spin monomer for  at max speed on a benchtop centrifuge ( -  depending on centrifuge) at .
Centrifuge: 15000 34
Centrifuge: 20000 34
4 °C
This step removes large fibrils or aggregates that may have been generated when storing and thawing high concentration of α-synuclein.
?. [Making fibrils]
Keep monomer  at all times to prevent fibrillization.
on ice
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.
Example from our lab:Protein concentration written on tube says 13 mg/mLUse 10 mM Tris, pH7.5, as blank (what the monomer is in)A280 = 3.6Calculate (3.6/5.96)*14.5= 8.7 mg/mL protein
?. [Making fibrils]
Dilute monomer to  in  , , , final volume .
[/ml]
[Tris-HCL]
[KCL]
500 µl
?. [Making fibrils]
Spin fibrils for  at max speed in benchtop centrifuge at .
0 Room temperature
?. [Making fibrils]
Discard supernatant and resuspend in approximately 50% of initial volume in   , ,  (for example, if making at , resuspend in ).
[Tris-HCL]
[KCL]
500 µl
250 µl
?. [Making fibrils]
Measure the concentration of fibrils.
?. [Making fibrils]
Mix  with  of  and  of   , ,  buffer with .
[of fibril]
5 µl
[guanidinium chloride (GnCl) (SAMPLE) ]
5 µl
[Tris-HCL]
[KCL]
[GnCl (BLANK)]
?. [Making fibrils]
Incubate for  on the bench at .
0 Room temperature
?. [Making fibrils]
Use the spectrophotometer to determine concentration of fibrils using BLANK as the blank and SAMPLE to determine concentration.
Note that the fibrils are diluted in GnCl, so the actual concentration will be 2X the readout.
?. [Making fibrils]
Use   , ,  to bring fibrils to .
[Tris-HCL]
[KCL]
[/mL]
?. [Making fibrils]
Aliquot and store at
-80 °C
?. [Sonicating Fibrils]
Pipet  in  sonication tube.
[fibrils (5 mg/mL)]
1.5 mL
?. [Sonicating Fibrils]
Fill Qsonica water reservoir with about .
[water]
Make sure reservoir water level height is 7 cm.
?. [Sonicating Fibrils]
Attach cooling system to Qsonica and set the temperature at .
15 °C
?. [Sonicating Fibrils]
Place  , therefore,sonication tube with fibrils in Qsonica tube holder.
1.5 mL
The gap between tube bottom surface and Qsonica probe upper surface should be 1 cm.
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.
?. [Sonicating Fibrils]
Total sonication time: .
?. [Sonicating Fibrils]
Sonication pulse on for  and off for .
?. [Sonicating Fibrils]
Amplitude 30%.
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  . Take the sample tube in safety hood and mix the PFF sample with pipette 5 times in and out (avoid introducing bubbles while pipetting).
?. [Sonicating Fibrils]
Sonicate sample for another .
After sonication PFF sample can be used that day or stored in   or on dry ice (for shipping) for one day, but a sample in -80 °C should be fine for longer. After taking out from  sample is good at least for   at  .
?. [Confirming Fragmentation]
Dynamic Light Scattering:
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.
?. [Confirming Fragmentation]
Pipet  and dilute with .
[sonicated PFF sample]
[1x PBS (filtered with 0.22 μm filter)]
?. [Confirming Fragmentation]
When done, fill cuvette with 2% SDS to decontaminate fibrils (using a squirt bottle).
?. [Confirming Fragmentation]
Let sit for at least .
?. [Confirming Fragmentation]
Use squirt bottle to rinse several times with DI water.
?. [Confirming Fragmentation]
Use filtered water for last rinse.
?. [Confirming Fragmentation]
Make sure dry before next use.
The important point is to make sure the cuvette remains dust free.
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]
Immediately after sonication, dilute with .
[of sonicated fibrils]
[of PBS]
?. [Transmission Electron Microscopy]
Spot  on glow discharged EM grid.
[of diluted fibrils]
?. [Transmission Electron Microscopy]
Wait for  and remove extra buffer by allowing it to wick onto a kimwipe.
?. [Transmission Electron Microscopy]
Add .
[uranyl acetate solution]
?. [Transmission Electron Microscopy]
Remove extra uranyl acetate solution after .
?. [Transmission Electron Microscopy]
Let dry and image.
?. [Making fibrils]
Shake at  for  (1 week); check for turbidity at end of week.
37 °C
If it is not turbid, it did not work.
?. [Confirming Fragmentation]
Put  in disposable microcuvette (WYATT Technology) for DLS.
[diluted PFF sample]