The goal of this protocol is to provide a cell-based system that replicates the formation of alpha-synuclein aggregates in vivo. Intracellular alpha-synuclein inclusions are seeded in primary neurons by the internalization and propagation of exogenous administered native microsomes-associated alpha-synuclein aggregates isolated from diseased alpha-synuclein transgenic mice.
This method can help answer key questions in the alpha-synuclein field, such as elucidate the mechanism of aggregation spreading and toxicity of alpha-synuclein in vivo. The main advantage of this technique is that it does not require the time-consuming purification of alpha-synuclein preformed fibrils. Demonstrating the procedure will be Lucia Rota, a grad student from the COLA Lab.
To begin the procedure, prepare the homogenization buffer and keep it on ice. Next, homogenize the fresh or frozen tissue in a one-to-10 volume of ice-cold homogenization buffer using a Teflon pestle homogenizer with 10 to 15 strokes. Afterward, transfer all of the initial homogenate to a microcentrifuge tube, and centrifuge at 1, 000 times G for 10 minutes at four degrees celsius in order to remove nuclei and unbroken cells in the resulting pellet.
Subsequently, discard the result pellet. Next, transfer the supernatant to a clean microcentrifuge tube, and centrifuge it at 10, 000 times G for 20 minutes at four degrees celsius using a refrigerated centrifuge in order to obtain the second supernatant and the pellet. Then transfer this supernatant to a polycarbonate bottle, and centrifuge it at 100, 000 times G for one hour at four degrees celsius using an ultracentrifuge and a fixed-angle rotor.
Resuspend the P100 crude pellet with 500 microliters of the homogenization buffer. Transfer the P100 crude pellet to a clean microcentrifuge tube, and centrifuge at 10, 000 times G for 20 minutes at four degrees celsius in a refrigerated centrifuge. After 20 minutes, discard the supernatant, and resuspend P100 with 100 microliters of homogenization buffer.
Then, sonicate the sample for two seconds on ice, and store it at minus 80 degrees celsius. To perform Western Blot, cast a gradient four to 20%tris-glycine polyacrylamide gel on a vertical electrophoresis apparatus. Dissolve one microgram of microsomes-associated alpha-synuclein fractions in the denaturing sample buffer, and load it on the gel.
In a different well, load five microliters of protein-standard marker. Run the gel at 100 volt in a tris-glycine SDS running buffer until protein marker reaches the end of the gel. Then, transfer the proteins to a nitrocellulose membrane using a basic carbonate buffer.
Keep the sample overnight in the cold room with a constant power set at 200 milliamps. The next day, block the membrane with PBS-T with 5%nonfat dry milk on an orbital shaker for 30 minutes at room temperature. Afterward, briefly rinse the membrane with PBS-T.
Incubate the membrane with SIN-1 at one to 5, 000, or P-Ser129 alpha-synuclein antibody at one to 5, 000 in 2.5%nonfat dry milk in PBS-T overnight in the cold room or refrigerator at four degrees celsius on an orbital shaker. The next day, wash the membrane for 10 minutes at room temperature with PBS-T on an orbital shaker. Incubate the membrane with anti-mouse or anti-rabbit HRP-conjugated secondary antibody at one to 3, 000 in 2.5%nonfat dry milk in PBS-T for one hour at room temperature on an orbital shaker.
Then, wash the membrane for 10 minutes at room temperature with PBS-T on an orbital shaker for three times. Obtain the signal through the regular chemiluminescence kit. To treat the neurons, pull the microsomes-associated alpha-synuclein aggregates obtained from the spinal cord of three different diseased transgenic mice in order to have a one-microgram-per-microliter solution diluted with the original homogenization buffer.
Next, remove one third of the medium, and replace it gently with fresh neurons medium. Add one microgram of pooled microsomes-associated alpha-s aggregates to the cell medium. Return the neurons in the incubator at 37 degrees celsius.
Add one-third of fresh medium to the sample without replacing the medium once every three days for one week. After one week of treatment, remove one-third of the medium, and replace it gently with fresh medium containing 2%B27, 10-microgram-per-milliliter gentamicin, and two millimolar glutamine. Repeat every three days for one week before fixing the cells.
To perform immunofluorescence, in a chemical fume hood fix the neurons with 2%paraformaldehyde and PBS in 5%sucrose solution for 15 minutes at room temperature without shaking. After 15 minutes, remove the fixing solution, and briefly wash the neurons with PBS three times. Next, permeabilize the neurons with 0.3%of non-ionic surfactin in PBS for five minutes at room temperature.
Then, briefly wash the neurons with PBS three times. Incubate the neurons with 3%FBS and PBS for 30 minutes at room temperature on an orbital shaker to block unspecific binding sites. Afterward, incubate the neurons with appropriate primary antibody dissolved in 3%FBS and PBS overnight in the cold room or refrigerator overnight at four degrees celsius on an orbital shaker.
The next day, remove the antibody solution, and wash the cells briefly with PBS for three times. Then, incubate the neurons with the appropriate fluorescent secondary antibody dissolved in 3%FBS and PBS for one hour at room temperature in the dark on an orbital shaker. Remove the antibody solution, and wash briefly with PBS for three times.
Next, stain the neurons with DAPI solution for 15 minutes at room temperature in the dark on an orbital shaker. After 15 minutes, remove the antibody solution, and wash briefly with PBS for three times. Mount the coverslips on the slide using anti-fade mounting medium.
In this study, administration of one microgram of pooled microsomes-associated alpha-synuclein aggregates from diseased A53T transgenic mice to the culture medium of cortical or hippocampal neurons induces a time-dependent formation of alpha-synuclein inclusions, positive for aggregate-specific alpha-synuclein antibodies. After two days of treatment, these aggregates appear as small scattered puncta that will become more abundant at later time points. After two weeks, alpha-synuclein inclusions resemble long and mature beads-like structures heavily spread throughout the neuronal cultures following a neurite pattern and partially colocalizing with presynaptic and neurites markers.
While attempting this procedure it is important to remember to adjust the ratio of micrograms of microsome-associated alpha-synuclein aggregates to the density of neurons plated and to isolate microsome-associated alpha-synuclein aggregates from tissue rich in alpha-synuclein inclusions. After watching this video you should have a good understanding of how to purify microsome-associated alpha-synuclein aggregates from diseased transgenic mice, to be used as treatment for primary neurons to induce formation of alpha-synuclein inclusions.
