Misfolding of Tau protein in its self-assembly into paired helical filaments is a pathological hallmark of Alzheimer disease and other Tauopathies. Here we present a robust Tau aggregation assay that could help elucidate the Tau pathogenesis process. The conversion follows all the steps of a nucleation dependent polymerization process and is highly reproducible, allowing drug screening in a more rigorous format that met OBSI level to date.
The quality of reagents is crucial. The recombinant Tau needs to be highly pure and devoid of aggregates and of fragments. First turn on the computer and the multimode microplate reader.
After allowing the equipment to stabilize, start the software and select Standard Protocol as the protocol type. Set the temperature to 37 degrees Celsius and select Preheatment before continuing the protocol. Next, set the kinetic run time to 50 hours and the measurement interval to 15 minutes.
Set the orbital shaking to 425 cycles per minute in continuous mode. Following this, select the read method as fluorescence intensity endpoint kinetic momochromators. Set the excitation wavelength to 440 nanometers and the emission wavelength to 485 nanometers, the optics position to top, and the gain to 80.
Select normal read speed and set the read height to 4.50 millimeters. Then validate and save the protocol. Start the run using the created protocol.
Name the experiment, select the destination of the newly created file, and allow the instrument to pre-equilibrate to the desired temperature. For a spontaneous recombinant Tau conversion experiment, prepare a 1, 000 microliter reaction sample with 800 microliters for thioflavin-T fluorescence, and 130 microliters for SEC-MALS analysis by first mixing the protein with the reaction buffer in a 1.5 milliliter tube. Then add heparin and thioflavin-T and mix well by pipetting up and down five times.
It's important to execute the protocol precisely to ensure high reproducibility, mix reagents in the same order, and keep within timeframes. Spin the sample at 12, 000 x g and 25 degrees Celsius for five minutes to eliminate air bubbles. Following centrifugation dispense 200 microliters of reaction sample per well in a 96-well microplate, avoiding the formation of air bubbles.
Then seal the microplate to avoid evaporation. Place the microplate in a multimode microplate reader. Select full plate, or if the plate is not full, select the wells to be read, and start the measurement.
Export the data to data processing software. Then remove the microplate from the equipment. Next, remove the sealer from the microplate.
Mix the aggregated sample in the wells by pipetting up and down two times, and pool the different replicates in 1.5 milliliter tubes. Mix each sample thoroughly by pipetting up and down five times, and dispense 10 to 20 microliters on a mica surface for analyzing the aggregates by atomic force microscopy. Following this, harvest the aggregates by spinning each one 1.5 milliliter tube at 20, 000 x g and 4 degrees Celsius for one hour.
Analyze the supernatant by SEC-MALS to confirm the absence of monomeric Tau in the sample, indicating a successful conversion into aggregates. Next, remove all remaining supernatant and label the 1.5 milliliter tube containing the remaining aggregates with the initial recombinant Tau protein concentration and sample volume. Snap freeze the aggregates and store at 80 degrees Celsius.
For a seeded experiment, remove the recombinant Tau aggregates from the freezer, add the volume of reaction buffer indicated on the label, and allow the tube to stabilize to room temperature. Then resuspend the aggregates by pipetting up and down five to eight times. Sonicate the 200 microliter aggregated sample on ice using a 1/8 inch microtip for a total period of 15 seconds with pulses of one second on and two seconds off at 30 percent amplitude.
Next, prepare an 800 microliter reaction sample by first mixing the protein with the reaction buffer in a 1.5 milliliter tube. Then add heparin and thioflavin-T and mix well by pipetting up and down five times. Spin the samples at 12, 000 x g and 25 degrees Celsius for five minutes to eliminate air bubbles.
Following centrifugation, dispense 198 microliters of reaction sample per well in a 96-well microplate, avoiding the formation of air bubbles. Homogenize the pre-formed fibral sample by pipetting up and down three times. Add the amount corresponding to the desired percentage of seeds to each well and mix by pipetting up and down three times.
Then seal the microplate to avoid evaporation. Place the microplate in the multimode microplate reader and start the measurement. After the experiment is complete, remove the microplate from the equipment.
Then export the data to a spreadsheet. Recombinant Tau containing the C291A and C322A mutations and n terminal his and c terminal c-tags are highly pure as visualized on SDS page and virtually 100 percent monomeric as assessed by SEC-MALS. The heparin induced recombinant Tau aggregation was followed by thioflavin-T fluorescence using an excitation wavelength of 440 nanometers and an emission wavelength of 485 nanometers.
The assay is highly reproducible with results from 10 individual wells being virtually indistinguishable. The recombinant Tau aggregates are a homogenous mixture of fibular structures of different lengths, similar to reported ex-vivo morphologies. The final reaction mixture does not contain monomer, suggesting a full conversion into aggregates, as shown by SEC-MALS measurements.
The kinetics of recombinant Tau aggregation in independent experimental runs are similar, as emphasized by similar sigmoidal curves and indistinguishable lag and growth phases. The high level of reproducibility is maintained when different batches of protein are used. Pre-formed recombinant Tau aggregates are efficient in recruiting Tau monomer and inducing formation of de novo Tau aggregates, and the efficiency of the process is directly proportional with the percentage of seeds.
Amounts as low as 0.0025 percent of pre-formed Tau aggregates expressed in volume are capable of bypassing recombinant Tau nucleation and trigger de novo fibral generation. The current assay mimics what is believed to be the in vivo misfolding and aggregation of Tau and enables mechanistic studies that could shed light on the pathogenesis process and constitutes a valuable tool for drug screening and studying the interference of drug candidates with different stages of the process. The high reproducibility of the assay will allow scientists to implement it with relative ease in the laboratory.
Although the current assay focuses only on the longest Tau isoform, human Tau-441, the application can be adapted to study different Tau variants. And do not forget, selecting the right protein reagents and producing them at high quality standards are essential in setting up a robust and highly reproducible assay.
