TITLE:  Sonication of α-synuclein Fibrils for injection into the mouse brain
AUTHORS: Vijay Singh, Marta Castellana-Cruz, Nunilo Cremades, Laura Volpicelli-Daley

[DESCRIPTION]
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 sonication protocol of α-syn fibrils to ensure reliable, robust results. This protocol includes steps for sonication of PFFs on the day of injection into mice.

[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: 
 

 

 

 


[STEPS]
SECTION: Sonicating α-synuclein Fibrils (PFFs)
3. After the temperature inside Q700 Sonicator water reservoir is equilibrated to 10 °C, thaw and pipette 25-22 µL PFFs stored at -80 °C and transfer to polystyrene sonication tube (Active Motif, Cat No. 53071) inside a biosafety cabinet. Screw the cap of the polystyrene sonication tube tightly after transferring, but avoid overtightening. 

 

SECTION: Sonicating α-synuclein Fibrils (PFFs)
1. Fill Qsonica Sonicator (Q700 Sonicator) water reservoir with about 900 mL deionized water.  
SECTION: Sonicating α-synuclein Fibrils (PFFs)
2. Turn on the heating system of the Q700 Sonicator and set the temperature at 10 °C.

SECTION: Sonicating α-synuclein Fibrils (PFFs)
4. Fit the polystyrene sonication tube containing PFFs to the Q700 Sonicator tube holder and transfer it into the Q700 Sonicator water reservoir. Ensure the reservoir water level is maintained up to the level of the upper meniscus of the PFFs (PFFs: green in the figure below). Operate the sonication for  15 min at an amplitude of 45%, with pulse on and off durations of 3 s and 3 s respectively, which should generate a power of around 110 watts for each pulse.
 

 

SECTION: Sonicating α-synuclein Fibrils (PFFs)
5. Take the sample tube after sonication and wipe it to remove any water from the reservoir. Spin the tube at 1000 rpm for a few seconds to settle PFF droplets inside. Inside the biosafety hood, remix the PFF sample by pipetting up and down 5 times, ensuring to avoid introducing bubbles. After remixing, re-sonicate the sample for 450 s  using the same sonication parameters as described above in step 4.
 


SECTION: PFFs Injection
6. Inject the sonicated PFFs in the mouse brain as soon as possible within 480 min . PFFs should be kept at room temperature until all mice are injected. The monomer, serving as a control, should be spun down at °C at 20000 x g for 15 min to obtain supernatant for injection and then kept on ice until all mice are injected. Any leftover PFFs should be properly disposed of after SDS treatment as indicated earlier.