Generation of Alpha-Synuclein Preformed Fibrils from Monomers and Use In Vivo

The Alpha-Synuclein Preformed Fibril model is being used by labs worldwide to study synucleinopathies. Although the model's been used successfully by many labs, some groups have experienced inconsistencies generating fibrils and producing consistent alpha-synuclein pathology. We hope that this protocol, which details the generation of fibrils from alpha-synuclein monomers and the use of pre-formed fibrils in vivo, can answer questions that researchers have about the use of the model.

The Alpha-Synuclein Preformed Fibril model recapitulates key features of Parkinson's disease, such as alpha-synuclein pathology and neurodegeneration. And it's been shown to lead to modest motor impairments in some animal models. The formation of inclusions containing phosphorylated alpha-synuclein and protracted time course towards neurodegeneration offers researchers different stages throughout the progression of the synucleinopathy, to study and target for potential therapeutic interventions.

Demonstrating the preparation of custom glass needles, is our lab technician, Christopher Kemp. To begin this procedure, thaw alpha-synuclein monomers on ice. Re-suspened the thawed monomers by gently flicking on the tube.

And centrifuge at 15, 000 g and four degrees Celsius for 10 minutes. Then, transfer the supernatant to a clean 1.5 mL micro centrifuge tube and record the amount transferred. Dilute the monomers with 1x dPBS to a final concentration of 5 mg per mL.

Briefly vortex to mix, and briefly centrifuge to collect all of the liquid at the bottom of the tube. Next, use adhesive film to secure the micro centrifuge tube lid closed. Place the tube into a orbital thermo mixer with a lid for seven days at 37 degrees Celsius while shaking at 1, 000 rpm.

At the end of seven days, the contents of the tube should appear turbid. First, don all necessary personal protective equipment including gloves, a lab coat, and a face shield. Then, in a cell culture hood, attach a 3.2 mm diameter probe to the converter.

Set the sonicators amplitude to 30%and pulse to one second on and one second off and the time to one minute. Thaw the fibrils at room temperature, and while still in the culture hood, dilute the fibrils with sterile dPBS. Next, dampen a lab tissue with 70%ethanol and use this to wipe the probe of the sonicator to clean it.

Submerge the tip of the probe in double distiled water and pulse 10 times to clean the probe further. After this, wipe the probe dry with a lab tissue. Place the cleaned probe tip into the tube of diluted fibrils and position the the tip at the bottom of the tube.

Sonicate the diluted fibrils using the previously set parameters while moving the probe up and down during each pulse to ensure that all of the fibrils in the liquid are sonicated. After sonication, briefly centrifuge for one second at 2, 000 g to collect all the liquid from the sides of the tube. Submerge the probe tip in 1%SDS and pulse 10 times to clean the probe.

Then, remove the tip from the SDS, and submerge it in double distilled water, and pulse 10 times. Wipe the probe with a lab tissue dampened with 70%ethanol and wipe it dry with a dry lab tissue. Detach the probe from the converter and store.

After this, wipe down all surfaces in the hood with 1%SDS followed by 70%ethanol. To begin, place a siliconized glass capillary tube into a glass needle puller. Turn on the heating element, and allow the attached weights to stretch the heated glass capillary tube.

Next, use scissors to cut the pulled glass capillary tube at the thinnest point in the middle and remove the glass needle from the glass needle puller. Then, use scissors to cut a length of shrink wrap tubing to approximately 40 mm. Slide the shrink wrap over the metal needle of a 10 microliter beveled syringe.

Use an open flame to heat and adhere the shrink wrap to the needle, while making sure to rotate the needle to apply heat evenly. Slide the larger end of the pulled glass needle carefully over the metal needle of the syringe. After this, cut a length of shrink wrap tubing to approximately 40 mm and carefully slide it over the glass needle to overlap the base of the glass needle and the metal needle of the syringe.

Use an open flame to heat the shrink wrap to secure the glass needle to the metal needle. To further secure the needle and prevent potential leaks, cut an additional length of shrink wrap tubing to approximately 40 mm, and carefully slide it over the glass needle to overlap the base of the glass needle and the metal needle of the syringe. Use an open flame to heat the shrink wrap to secure the glass needle to the metal needle.

Then, use scissors to trim the glass needle so that the tip is approximately 8 mm long. To test the needle, fill a 1 mL syringe with an attached 26 gauge needle with distilled water. Remove the metal plunger from the custom glass needle syringe and insert the needle of the water-filled syringe into the base of the custom syringe.

Inspect the interface of the glass needle and the metal needle for leaks and to confirm a steady flow of water. Then, fill a micro centrifuge tube with distilled water. Use the custom glass needle syringe to draw in the distilled water.

Inspect the needle to confirm liquid is being taken into the syringe and that there are no bubbles. If there are bubbles, or if the water is not being drawn up into the needle, trimming the needle can help alleviate the pressure. After this, carefully store the syringe with the attached glass needles, in the syringe boxes until needed for surgeries.

Examination with transmission electron microscopy confirms the presence of long fibrils. In comparison, alpha-synuclein monomers are barley visible and have no discernible shape. Analoid confirmation of the fibrils is then confirmed using a Thioflavin T Assay.

A representative assay shows the dPBS and mouse monomers produce a lower signal in relative flouresent units compared to mouse PFFs. Human alpha-synuclein monomer produces a similar signal to mouse monomer. While the human PFFs likewise produce a higher signal than monomers.

To asses the presence of pelitible fibrils, a sedimentation assay is performed. In both the mouse and human PFF samples, the supernate infraction should have more protein in the pellet than the supernatant. In contrast, the majority of the protein from the mouse and human monomers is present in the supernatant with little present in the pellet.

Both mouse and human PFFs are sonicated to produce PFFs of appropriate lengths for seeding alpha synuclein inclusions. A comprehensive examination of approximately 500 fibrils reveals that the average length of mouse fibrils is approximately 44 nanometers. With 86.6%of the PFFs measuring 60 nanometers or less.

Human PFFs, however, have an average length of approximately 55.9 nanometers with 69.6%of the PFFs measuring 60 nanometers or less. In vivo examination of PFF efficacy is confirmed using immunohistochemistry. Inclusions formed in the substantia nigra, share similar properties to Lewy bodies in that they contain phosphorylated alpha synuclein, contain aminloyed structures, and stain with thioflavin S, and are resistant to Proteinase K digestion.

Sonication can expose the individual sonicating to aerosolized preformed fibrils. As such, proper safety attire should be worn and sonication performed in a hood to minimize the risk of exposure.