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 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.

[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. 


  


[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 Fibrils
3. Thaw and pipet 25-22 µL in 1.5 mL polystyrene sonication tube.

SECTION: Sonicating Fibrils
1. Fill Qsonica water reservoir with about 900 mL. 
 


SECTION: Sonicating Fibrils
2. Attach cooling system to Qsonica and set the temperature at 10 °C.

SECTION: Sonicating Fibrils
4. Place 1.5 mL , therefore,sonication tube with fibrils in Qsonica tube holder. 
 

 
  
 


SECTION: Sonicating Fibrils
5. Total sonication time: 15 min.

SECTION: Sonicating Fibrils
6. Sonication pulse on for 3 s and off for 2 s.

SECTION: Sonicating Fibrils
7. Amplitude 45%.
 

SECTION: Sonicating Fibrils
8. Sonicate sample for another 450 min. 
 

SECTION: Confirming Fragmentation
9. 
 

Dynamic Light Scattering:
SECTION: Confirming Fragmentation
10. Pipet 1 µL and dilute with 4 µL. 

SECTION: Confirming Fragmentation
12. When done, fill cuvette with 2% SDS to decontaminate fibrils (using a squirt bottle).
SECTION: Confirming Fragmentation
13. Let sit for at least 30 min. 

SECTION: Confirming Fragmentation
14. Use squirt bottle to rinse several times with DI water. 
SECTION: Confirming Fragmentation
15. Use filtered water for last rinse. 
SECTION: Confirming Fragmentation
16. Make sure dry before next use. 
 

 

SECTION: Transmission Electron Microscopy
17. Immediately after sonication, dilute 2 µLwith 98 µL.

SECTION: Transmission Electron Microscopy
18. Spot 5 µL on glow discharged EM grid. 

SECTION: Transmission Electron Microscopy
19. Wait for 20 s and remove extra buffer by allowing it to wick onto a kimwipe. 

SECTION: Transmission Electron Microscopy
20. Add 8 µL. 

SECTION: Transmission Electron Microscopy
21. Remove extra uranyl acetate solution after 10 s. 

SECTION: Transmission Electron Microscopy
22. Let dry and image. 
SECTION: Confirming Fragmentation
11. Put 5 µL in disposable microcuvette (WYATT Technology) for DLS.