Sensitive Detection of Proteopathic Seeding Activity with FRET Flow Cytometry

Summary

Cell-to-cell transfer of protein aggregates, or proteopathic seeds, may underlie the progression of pathology in neurodegenerative diseases. Here, a novel FRET flow cytometry assay is described that enables specific and sensitive detection of seeding activity from recombinant or biological samples.

Abstract

Increasing evidence supports transcellular propagation of toxic protein aggregates, or proteopathic seeds, as a mechanism for the initiation and progression of pathology in several neurodegenerative diseases, including Alzheimer's disease and the related tauopathies. The potentially critical role of tau seeds in disease progression strongly supports the need for a sensitive assay that readily detects seeding activity in biological samples.

By combining the specificity of fluorescence resonance energy transfer (FRET), the sensitivity of flow cytometry, and the stability of a monoclonal cell line, an ultra-sensitive seeding assay has been engineered and is compatible with seed detection from recombinant or biological samples, including human and mouse brain homogenates. The assay employs monoclonal HEK 293T cells that stably express the aggregation-prone repeat domain (RD) of tau harboring the disease-associated P301S mutation fused to either CFP or YFP, which produce a FRET signal upon protein aggregation. The uptake of proteopathic tau seeds (but not other proteins) into the biosensor cells stimulates aggregation of RD-CFP and RD-YFP, and flow cytometry sensitively and quantitatively monitors this aggregation-induced FRET. The assay detects femtomolar concentrations (monomer equivalent) of recombinant tau seeds, has a dynamic range spanning three orders of magnitude, and is compatible with brain homogenates from tauopathy transgenic mice and human tauopathy subjects. With slight modifications, the assay can also detect seeding activity of other proteopathic seeds, such as α-synuclein, and is also compatible with primary neuronal cultures. The ease, sensitivity, and broad applicability of FRET flow cytometry makes it useful to study a wide range of protein aggregation disorders.

Introduction

The accumulation of intracellular tau amyloids defines tauopathies such as Alzheimer's disease. In early disease stages, pathology is generally localized to discrete regions of the brain, but with disease progression, pathology invariably spreads along distinct neural networks^1-5. Accumulating evidence suggests transcellular propagation of toxic protein aggregates underlies this pathology (reviewed in ^6-10). In this model, proteopathic seeds (e.g., tau) are released from donor cells and enter neighboring cells, transforming native tau protein into the misfolded form via templated conformational change^11-15. The assay described here was developed to sensitively detect such seeding activity. It is compatible with recombinant protein and biological samples and enables quantification of minute levels of proteopathic seeding activity¹⁶.

HEK 293T cells that stably express tau repeat domain (RD) containing the disease-associated P301S mutation fused to either CFP or YFP (hereafter referred to as tau-RD-CFP/YFP cells) serve as a stable biosensor of seeding activity. In the absence of proteopathic seeds, the cells maintain tau as a soluble monomer, and have no appreciable background FRET. Spontaneous uptake or liposome-mediated transduction of tau seeds into cells, however, results in RD-CFP and RD-YFP aggregation, which produces a FRET signal that is measured within single cells via flow cytometry.

Numerous components of this assay were engineered to enhance sensitivity and reduce variability. A monoclonal cell line with a 1:1 RD-CFP/YFP expression ratio was selected, as it provides optimal signal:noise. To increase sensitivity, phospholipids are used to introduce seeds directly into cells (although to study biological mechanisms of uptake, this can be omitted). Finally, flow cytometry monitors FRET at a population level and a single cell level, unlike other protein aggregation assays. The final outcome measure, integrated FRET density, is highly quantitative and accounts both for the number of cells with aggregation, and the degree to which aggregation has occurred within each cell. All of these optimized parameters enhance sensitivity and ensure reproducibility.

This system was recently employed in a comprehensive study in transgenic P301S tauopathy mice¹⁷ that evaluated the temporal onset and progression of tau seeding activity relative to other commonly used tau pathological markers (e.g., MC1, AT8, PG5, and ThioflavinS). Seeding activity is by far the earliest and most robust marker of tau pathology evaluated, preceding histological detection by at least 6 weeks. Seeding activity appears at 1.5 months and increases progressively with age, suggesting a causal role of proteopathic seeds in the onset and/or progression of neurodegeneration¹⁶.

Precise quantitation of minute levels of seed material from biological samples can facilitate studies that monitor early disease progression. By shortening trial duration and enabling use of younger animals, this could increase the efficiency and accuracy of preclinical animal trials. For example, in the P301S mouse previously described, lead compounds could be delivered as early as 4-6 weeks (immediately prior to, or at onset of seeding activity), and monitored for efficacy 2-4 weeks later. The assay should accurately quantify any reductions in seeding activity. FRET flow cytometry has in vitro screening applications as well. For example, anti-tau reagents (e.g., antibodies, small molecules, etc.) can be tested rapidly for their capacity to block seeding induction directly in culture, using either recombinant tau aggregates or brain-derived lysates as a seed source (Figure 5). With this setup, once seed material is prepared, an experiment takes just three days to complete, including data analysis. The rapid quantitation of proteopathic seeding activity can thus facilitate many studies of neurodegeneration.

Protocol

NOTE: This protocol emphasizes the use of FRET flow cytometry for detecting seeding activity from mouse biological samples. It is also compatible with recombinant fibrils and human biological samples. Mouse euthanasia and brain harvesting was performed in accordance with IACUC-approved procedures.

1. Brain Extraction

1. Following deep anesthetization with isoflurane (2%), perfuse a mouse with ice-cold PBS containing 0.03% heparin, and extract the brain following the details described in Gage et al.¹⁸
2. Place extracted tissue in a cryo-vial, and snap freeze by placing in liquid nitrogen. Alternatively, freeze tissue on dry ice. Once frozen, transfer tissue to -80 °C and store for an extended period of time, if necessary.
   NOTE: This protocol is compatible with whole brain homogenates or micro-dissected brain regions. See Hagihara et al. for a detailed micro-dissection protocol¹⁹.

2. Preparation of Biological Seed Material

1. Prepare homogenization buffer by dissolving protease inhibitors (EDTA free) into 1x TBS (1 tablet per 10 ml). Vortex vigorously, and store on ice. Protease inhibitors must be EDTA free in order to preserve biosensor cell viability.
2. Weigh frozen brain tissue in a disposable weigh boat, and transfer to a 5 ml conical tube. Add ice-cold homogenization buffer such that the final solution is 10% weight/volume. Temporarily store on ice. Tissue mass and subsequent homogenization buffer volume will vary depending on the brain size and/or regions used. Here, an adult mouse hemibrain weighing 0.2 g is suspended in 2 ml for a 10% w/vol solution.
3. Transfer samples into a cold room, and adjust a probe sonicator to the appropriate settings. For probe sonication with an Omni Ruptor (shown here), set the power to 20%, which corresponds to approximately 75 W. Use a pulse mode to ensure samples are not over-heated during sonication. Set the pulser to 30%, corresponding to approximately 500 msec.
4. Clean the tip of the probe sonicator by rinsing with water, isopropanol, and water again, wiping off the probe in between each solution. Be certain to rinse and wipe both the sides and bottom of the probe with lab-wipes.
5. Working with one sample at a time, submerge the probe tip into the homogenization buffer, and start the sonicator. Using the power and pulser settings described above, deliver 25 total pulses. Ensure that tissue is completely in suspension. Be careful to avoid foaming of the sample during this step.
   NOTE: Samples are susceptible to foaming if a) the total volume is low or b) the homogenate warms up. With this specific instrument, do not sonicate with volumes less than 250 µl. To ensure samples stay chilled, tubes may be stored on ice during this step.
6. Clean the probe by wiping away residual lysate, then deliver 10 pulses into a beaker of clean water. Rinse the sides and bottom of the probe with water, isopropanol, and water again (as in step 2.4), wiping dry in between each step. To avoid contamination, thoroughly rinse the probe in between samples.
   NOTE: With tau aggregates, we have not found that more stringent cleaning methods are necessary for preventing sample-to-sample contamination. Other amyloids, however, may require additional care which has been extensively described in literature^20,21.
7. Store homogenates on ice until all samples have been processed.
8. Spin homogenates at 21,300 x g for 15 min at 4 °C.
9. Transfer the supernatant to a clean tube, taking care not to disturb the pellet. Discard the pellet. Aliquot the supernatant, and store lysates at -80 °C for further use. Avoid freeze/thaw cycles of the homogenates, however, as this reduces seeding efficiency.

3. Replating Biosensor Cells

NOTE: Use four cell lines for this assay: HEK 293T (cell line #1), RD-P301S-CFP (cell line #2), RD-P301S-YFP (cell line #3), and RD-P301S-CFP/YFP (cell line #4). Please reference Table 1 for each cell line’s contribution to the assay.

1. In a sterile environment, aspirate culture medium. Rinse cells with warm PBS, and aspirate. Trypsinize cells (3 ml of trypsin-EDTA (0.05%)) for 3 min, quench with warm culture medium (9 ml of DMEM, 10% FBS, 1% pen/strep, 1% GlutaMax), and immediately transfer cells to a conical tube.
2. Centrifuge cells at 1,000 x g for 5 min at RT. Aspirate medium, and resuspend cell pellet in warm culture medium.
3. Using a hemocytometer, determine the cell density for each cell line. Cell density will vary depending on confluency at time of harvesting and resuspension volume. Resuspend cells harvested from a 10 cm dish at 90% confluency in 10 ml media, for cell density to be approximately 1 million cells/ml.
4. Make a master mix of cells + media such that each well of a 96 well plate will contain 35,000 cells in 130 µl media (e.g., to make a master mix for 100 wells, resuspend 3.5 million cells in 13 ml media). Modify the cell number to fit individual experimental designs and timelines. For cell treatment ~18 hr after plating, add 35,000 cells to each well of a 96-well plate.
5. Using a multi-channel pipet, slowly pipette 130 µl of master mix cell suspension into each well of a flat bottom, tissue culture-treated 96 well plate. While plating, place the pipet tip in the center of the well, not touching the bottom of the plate.
   NOTE: Though plating format can be modified, making n = 4 wells for each of cell lines 1-3 per plate is recommended. The remainder of the plate (n = 84) is reserved for cell line #4 (biosensor cells).
6. Allow the cells to settle by leaving the plate undisturbed for 10 min at RT. Incubate O/N at 37 °C, 5% CO₂, and ≥80% relative humidity.

4. Treating Cells

NOTE: The following day, when tau biosensor cells are 60-65% confluent, prepare seed transduction complexes as follows:

1. In one tube, combine transfection reagent, such as Lipofectamine, with reduced-serum media, such as Opti-MEM, to make a master mix. Per well, add 1.25 µl transfection reagent and 8.75 µl reduced-serum media. Flick tube gently to mix, briefly spin down, and incubate for 5 min at RT.
2. In another tube, combine seed material (aliquot of lysate from step 2.9) with reduced-serum media.
   NOTE: Volume of seed material will vary according to the individual experiment. When choosing seed volume, take into consideration: abundance of seeds in a sample and potential toxicity. Crude homogenates can be toxic to cells. As such, use the lowest volume possible to monitor your desired effect. Previously tested volumes of lysate/well range from 1-5 µl, corresponding to 5-20 µg total protein. Ensure that the total volume per well (seeds + reduced-serum media) is 10 µl.
3. Combine contents from the two tubes described in 4.1 and 4.2, flick gently to mix, briefly spin down (1,000 x g, 5 sec), and incubate at RT for at least 20 min and up to 2 hr.
4. Gently pipette 20 µl of transduction complex on the side of individual biosensor wells. Use three technical replicates, if possible. Return treated cells to the incubator for 24-48 hr. Incubate under the same conditions as described in step 3.6.
   NOTE: The appropriate negative control condition for this setup is biosensor cells treated with empty liposomes (i.e., liposome reagent + reduced-serum media) because application of phospholipids introduces a slight shift in the fluorescence profile relative to biosensor cells unexposed to liposome reagent.

5. Harvesting Cells for FRET Flow Cytometry

NOTE: Before harvesting cells—generally 24-48 hr post-treatment—it is possible to get a preliminary readout of seeding activity using the GFP filter on a standard inverted fluorescence microscope. Cells treated without seed material (i.e., empty liposomes) will show diffuse fluorescence, whereas cells treated with seed material will show intense punctate and reticular intracellular inclusions (Figure 1A-B).

1. Using a multi-channel pipet, aspirate all cell medium (150 µl) . Trypsinize (50 µl) cells for 5 min, and quench with 150 µl chilled culture medium. Do not include a PBS rinse prior to trypsinization at this step, as it may cause cell lifting and subsequent cell loss. Exclude any contaminating dead cells and debris during flow cytometry via gating strategies.
2. Immediately after quenching, transfer cells to a 96 well round bottom plate, and centrifuge at 1,000 x g for 5 min at RT.
3. Aspirate and discard medium, taking care to avoid disturbing the cell pellet.
4. Gently, but thoroughly, resuspend the cell pellet in 50 µl 2% paraformaldehyde, and incubate for 10 min. Alternatively, run cells live, although fixation provides cleaner and more consistent results.
5. Centrifuge cells at 1,000 x g for 5 min at RT. Aspirate and discard paraformaldehyde, taking care to avoid disturbing the cell pellet.
6. Gently, but thoroughly, resuspend the cell pellet in 200 µl chilled flow cytometry buffer (HBSS, 1% FBS, 1 mM EDTA) and run the plate as soon as possible to avoid cell clumping.

6. FRET Flow Cytometry

NOTE: Use a flow cytometer such as the MACSQuant VYB, which is equipped with FRET-compatible laser lines and filter sets (Table 2). For each step within this section, click the well of interest using the software’s 96 well template, and click “play” to begin sample uptake and flow. Make plots or statistics tables by clicking the 'new analysis window' icon. Change axis parameters on individual bivariate plots by clicking the title on either the X or Y axis and selecting the appropriate filter. To shift cell populations or fluorescence signals, increase or decrease the voltages associated with the appropriate filters. With this instrument, run <1,000 events/sec to ensure accurate single-cell monitoring.

1. Make a Side Scatter-Area (SSC-A) vs Forward Scatter-Area (FSC-A) bivariate plot, and with cell line #1 running, adjust the SSC and FSC voltages until the cell population is in the lower left quadrant. Click the polygon tool, and define the cell population (Gate P1). For all gating parameters see Figure 2.
2. Make a FSC-H (height) vs FSC-A bivariate plot, and apply Gate P1 to this plot by clicking “live” above the plot, and selecting P1. With cell line #1 running, click the polygon tool, and define single cells (Gate P2).
3. Make 3 histogram plots, one for each of the filters of interest: CFP, YFP, and FRET. Apply Gate P2 to all of these plots by clicking “live” above the plots, and selecting P2. With cell line #1 running, adjust voltages such that the median cell population in the CFP, YFP, and FRET histograms are all between 0 and 1. To measure CFP and FRET, excite cells with the 405 nm laser, and capture fluorescence with a 450/50 nm and 525/50 nm filter, respectively. To measure YFP, excite cells with a 488 nm laser and capture fluorescence with a 525/50 nm filter. Laser and filter settings are further described in Table 2.
4. Perform compensation for CFP spillover into the FRET and YFP channels.
   NOTE: Compensation is the process of fluorescence spillover correction. Fluorescence spillover occurs whenever the fluorescence emission of one fluorochrome is detected within a filter designed to measure signal from another fluorochrome. With proper compensation, the CFP spillover fluorescence can be removed from the FRET and YFP channels.
   NOTE: Compensation requires the presence of both fluorescence -positive and -negative cells. Thus, to compensate on CFP-positive cells (cell line #2), negative cells (cell line #1) must be added to the sample prior to the run.
   1. Spike in 30 µl of cell line #1 suspension from a single well into 200 µl of cell line #2 suspension immediately prior to compensating.
   2. Click the “Instrument Settings” icon, then 'compensation' tab, and check 'matrix' to open the compensation table.
   3. Make a FRET-A vs CFP-A bivariate plot, and apply gate P2, as described in step 6.3. With cell lines 1+2 running, click the 'quadrant' icon and draw quadrants such that the fluorescence -negative and -positive populations are separated by the lower two quadrants (Gates LL3 and LR3, respectively).
   4. Create a statistics table that displays the median fluorescence intensity (MFI) of FRET for the LL3 and LR3 gates. Adjust the FRET parameter in the compensation matrix until the MFI of the FRET signal is equivalent between LL3 and LR3. Following this step, the MFI of the FRET signal is equal between unstained cells and CFP-positive cells, suggesting the absence of CFP spillover into the FRET channel.
   5. Make a YFP-A vs CFP-A bivariate plot, and apply gate P2, as described in step 6.3. Draw quadrants (LL4 and LR4) similar to that done in step 6.4.3.
   6. Create a statistics table that displays the MFI of YFP for LL4 and LR4. Adjust the YFP parameter in the compensation matrix until the MFI of the YFP signal is equivalent between LL4 and LR4. Following this step, the MFI of a YFP signal is equal between unstained cells and CFP-positive cells, suggesting the absence of CFP spillover into the YFP channel.
5. Following gate setup and compensation, highlight wells of interest and run the remainder of the plate.
6. After all samples have been run, export data files by clicking 'file', then 'copy'. Select the 'data files' tab, highlight the experiment folder, and click 'copy'.

7. Data Analysis

NOTE: Change axis parameters on individual bivariate plots by clicking the title on either the X or Y axis and selecting the appropriate filter.

1. Open FCS files in the flow cytometry analysis program.
2. Based on scatter properties, use a polygonal gate to define the cell population (SSC vs FSC) and singlet population (FSC-H vs FSC-A) from cells treated with empty liposomes, similarly to that described in Section 6.
3. If CFP compensation was performed during setup, do not further adjust CFP.
4. Create a FRET vs YFP bivariate plot. Using cell line #3, define a false FRET gate by drawing a polygonal gate that runs along the slope of the FRET-positive cell population. This gate excludes YFP single-positive cells that emit a signal within the FRET filter, and is drawn similar to that previously shown by Banning et al.²²
5. Define a FRET gate by plotting empty liposome-treated CFP/YFP cells on a FRET vs CFP bivariate plot. Introduce a polygonal gate along the slope of the population that extends upward and leftward away from the population. This gate should exclude most cells (~99%), such that background FRET is ≥1% of cells. Any events that shift into this gate are considered FRET-positive.
   NOTE: Each individual gate described above is applied to all samples before constructing subsequent gates. Following analysis, four individual gates are defined (Cell population; singlet; false FRET; FRET).
6. Record analysis parameters of interest, including: percent FRET positivity and MFI of FRET-positive cells. Manually calculate the integrated FRET density by measuring the product of percent positivity and MFI.
   NOTE: If integrated FRET density is used as an outcome measure, set background FRET (as defined in step 7.5) and MFI to greater than or equal to 1 in order to avoid calculating a product that is less than its two individual factors.

Results

FRET flow cytometry enables sensitive, quantitative, and rapid detection of seeding activity from recombinant or biological samples. Assay setup is facile: monoclonal-derived stable cell lines expressing tau-RD-CFP/YFP are transduced with seed material, incubated for 24-48 hr, and subjected to flow cytometry analysis (Figure 1A). In the absence of seeds, biosensor cells maintain tau in a soluble, monomeric form (Figure 1B). In the presence of seeds, however, biosensor cells convert tau i...

Discussion

The FRET flow cytometry system described here is a powerful tool for quickly and quantitatively assessing tau seeding activity. It requires only moderate cell culture experience and a working knowledge of FRET and flow cytometry. Other seeding assays, such as Thioflavin T - which exhibits enhanced fluorescence when bound to beta sheet structure - are laborious and require a pure, recombinant protein substrate. Additionally, in vitro seeding assays for tau are only semi-quantitative and generally insensitive to s...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
TBS	Sigma	T5912
cOmplete Protease Inhibitors (EDTA-free)	Roche	4693159001
Cryo-vials	Sarstedt	72.694.006
Analytical Balance	Mettler Toledo	XSE 105DU
Weighing Boats	Fisher Scientific	13-735-743
15 ml conical tube	USA Scientific	1475-0501
Omni Sonic Ruptor Ultrasonic Homogenizer	Omni International	18-000-115
Micro-Tip for Ultrasonic Homogenizer	Omni International	OR-T-156
2-Propanol	Fisher Scientific	A451
Noise Cancelling Ear Muffs	Fisher Scientific	19-145-412
Kimwipes	Fisher Scientific	S47299
1.5 ml tubes	USA Scientific	1615-5510
Microcentrifuge	Eppendorf	5424 000.215
DPBS	Life Technologies	14190-136
DMEM	Life Technologies	11965-084
Fetal Bovine Serum	HyClone	SH30071.03
Penicillin-Streptomycin	Life Technologies	15140-122
GlutaMax	Life Technologies	35050-061
Trypsin-EDTA	Life Technologies	25300-054
50 ml Conical Tubes	Phenix Research	SS-PH15
25 ml reagent resevoirs	VWR	41428-954
Multi channel pipet	Fisher Scientific	TI13-690-049
96 well flat bottom plates	Corning	3603
Opti-MEM	Life Technologies	31985-070
Lipofectamine 2000	Invitrogen	11668019
96 well round bottom plates	Corning	3788
16% Paraformaldehyde	Electron Microscopy Sciences	RT 15710
PBS	Sigma-Aldrich	P5493
EDTA	Sigma-Aldrich	ED2SS
HBSS	Life Technologies	14185-052
Sorvall ST 40 Centrifuge	Thermo Scientific	75004509
BIOLiner Swinging Bucket Rotor	Thermo Scientific	75003796
Hemacytometer	VWR	15170-172
MACSQuant VYB Flow Cytomter	Miltenyi Biotec	130-096-116
Chill 96 Rack	Miltenyi Biotec	130-094-459
Flow Jo analysis software	Flow Jo
20 μl pipet tips	Rainin	GPS-L10
200 μl pipet tips	Rainin	GPS-250
1 ml pipet tips	Rainin	GPS-1000
200 μl pipet tips	USA Scientific	1111-1800
5 ml serological pipett	Phenix Research	SPG-606180
10 ml serological pipett	Phenix Research	SPG-607180
25 ml Serological pipett	Phenix Research	SPG-760180