Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells

Summary

We describe a method to quantify the aggregation of misfolded proteins. Our protocol details lentiviral induced stable cell line generation, automated confocal imaging, and image analysis of protein aggregates. As an illustrative application, we studied the effect of small molecules in promoting SOD1 aggregation in a time- and dose-dependent manner.

Abstract

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc superoxide dismutase 1 (SOD1). The structural instability of SOD1 and the detection of SOD1-positive inclusions in familial-ALS patients supports a potential causal role for misfolded and/or aggregated SOD1 in ALS pathology. In this study, we describe the development of a cell-based assay designed to quantify the dynamics of SOD1 aggregation in living cells by high content screening approaches. Using lentiviral vectors, we generated stable cell lines expressing wild-type and mutant A4V SOD1 tagged with yellow fluorescent protein and found that both proteins were expressed in the cytosol without any sign of aggregation. Interestingly, only SOD1 A4V stably expressed in HEK-293, but not in U2OS or SH-SY5Y cell lines, formed aggregates upon proteasome inhibitor treatment. We show that it is possible to quantify aggregation based on dose-response analysis of various proteasome inhibitors, and to track aggregate-formation kinetics by time-lapse microscopy. Our approach introduces the possibility of quantifying the effect of ALS mutations on the role of SOD1 in aggregate formation as well as screening for small molecules that prevent SOD1 A4V aggregation.

Introduction

Protein aggregation is a biological process by which misfolded proteins group up and may act as causative agents in neurodegenerative diseases (amyloidosis). Characterizing protein aggregation is essential in understanding the role of aggregates in cellular dysfunction as well as in facilitating the discovery of new factors that influence the onset of the pathology. The visualization of fluorescence-tagged proteins in living cells is a powerful method which may aid in the development of assays applicable to high content screening (HCS)^1,2,3,4.

Amyotrophic lateral sclerosis (ALS) is regarded as a proteopathic disease caused by the presence of misfolded proteins with the propensity to aggregate and accumulate in motor neurons in both familial ALS (fALS) and sporadic ALS (sALS)^5,6. A subset of ~20% of fALS cases are associated with dominant mutations in the gene encoding the cytosolic antioxidant enzyme copper-zinc superoxide dismutase type 1 (SOD1)^7,8. Several potential causes for this genetically derived dysfunction have been proposed, including changes in the structure and function of SOD1 variants, such as aberrant stability, increased unfolding rate, and propensity to aggregate^9,10. Notably, the only verified and potentially toxic property shared by both ALS-linked SOD1 variants and wild-type (WT) SOD1 is an increased propensity to form compartmentalized protein aggregates or proteinaceous inclusions^11,12. Misfolded mutant SOD1, is persistently polyubiquitinated and degraded by the ubiquitin-proteasome system. As a result, a low level of inhibition of proteasome activity leads to the accumulation of mutant SOD1 aggregates^13,14, which form amorphous structures composed of soluble components that can exchange with soluble mutant SOD1 in the cytosol¹⁵. In particular, SOD1 mutant A4V (alanine at codon 4 changed to valine) is the most common ALS-causing mutation, and leads to rapid neurodegeneration with an average survival time of less than 2 years after disease onset¹⁶. Biochemically, SOD1 A4V has an increased tendency to monomerize, aggregate, and form amyloid pores; its pore-like aggregates are similar to amyloid pores of other disease-linked mutant forms, such as α-synuclein and β-amyloid protein¹⁷. To study the dynamics of SOD1-aggregate accumulation, methods for monitoring soluble and insoluble SOD1 aggregate forms remain to be developed.

We have previously shown, using live-cell imaging and HEK-293 cells transiently transfected with fluorescent protein-tagged SOD1, that ALS-associated mutations impair SOD1 dimerization and aggregation¹¹. Although transient expression systems can provide useful information about the biological outcome of short-term gene overexpression, methods providing stable integration of desired genes may be preferred for assay development. As such, lentiviral vectors offer the ability to confer long-term and regulated gene expression on mammalian cells ¹⁸. In this study, we focused on the generation of stable cell lines transduced with recombinant lentivirus bearing WT and mutant SOD1 tagged with yellow fluorescent protein (YFP). Using live-cell imaging microscopy and automated quantification of SOD1 aggregation, we triggered and quantified SOD1 aggregation events upon inhibition of the proteasome.

Protocol

1. Lentivirus production

NOTE: The production and manipulation of lentiviral vectors was carried out according to the National Institutes of Health (NIH) guidelines for research involving recombinant DNA. The plasmid encoding the wild-type and A4V mutant SOD1 tagged with enhanced YFP (SOD1WT-YFP and SOD1A4V-YFP) are described in Kim et al.¹¹ Both gene fusion products were amplified using the PCR primer pair 5′-ATCGTCTAGACACCATGGCGACGAAGGTCGTGTGC-3′ and 5′-TAGCGG CCGCTACTTGTACAGCTCGTCCATGCC-3′and inserted into the pTRIP-delta U3 CMV plasmid¹⁹ using XhoI and BsrGI restriction sites. In prior experiments, the HEK-293T cells were split at a ratio of 1:4 to 1:6 every two days. Avoidance of more than 20 passages and maintenance of cells at less 60% confluency helps to ensure good transfection efficiencies.

1. On Day 1, split HEK-293T cells at 30 - 40% confluence in a 10-cm diameter tissue culture plate (3 x 10⁶ cells/dish) in 10 mL of cell culture medium (high-glucose DMEM with 10% FBS and 4 mM L-glutamine) for virus production.
2. Keep the 10-cm diameter tissue culture plate in a CO₂ incubator (37 °C, 5% CO₂) overnight.
3. On Day 2, prepare a DNA transfection solution containing 10 µg of lentiviral vectors (pTRIP-delta U3 CMV - SOD1WT-YFP or -SOD1A4V-YFP) together with the lentiviral packaging plasmids (5 µg of pVSVg; 10 µg of pCMV-dR8.71) (Figure 2A)²⁰. Add 125 µL of 1 M CaCl₂ and adjust the volume to 500 µL using distilled water. Gently add 500 µL of 0.05 M HEPES into the mixture and incubate for 10 min at room temperature.
4. Replace HEK-293T cells medium with 10 mL pre-warmed fresh culture medium antibiotic-free mixed with 1 mL DNA transfection solution and incubate overnight at 37 °C, 5% CO₂.
5. On Day 3, replace the cell supernatant with fresh culture medium.
6. On Day 4, harvest the supernatant in a 15 mL tube and centrifuge for 5 min at 500 x g to remove the dead cells and debris. Further purify the supernatant by passing it through a 0.45 µm filter using a large 60 mL syringe. Immediately dispense into single-use aliquots (300 µL), and store at -80 °C. To maintain maximum product activity, avoid a freeze-thaw cycle.

2. Lentiviral transduction

1. Split the cell line to be infected at 60% confluence (HEK-293 cells and SH-SY5Y; 5 x 10⁵ cells per well and U2OS; 1 x 10⁵ cells per well) in a 6-well tissue culture plate in 2 mL of DMEM media supplemented with 10% FBS.
2. Keep the 6-well tissue culture plate in a 37 °C incubator at 5% CO₂ overnight.
3. Remove the frozen lentivirus from the -80 °C freezer and thaw an aliquot on ice before each use; do not refreeze.
4. While the virus is thawing, warm the cell culture medium containing the serum compatible with the cell line of interest. Once the virus is fully thawed, prepare a range of dilutions (1:3, 1:10, 1:30, and 1:100) in DMEM in a fresh 1.5 mL microfuge tube.
5. Bring up the volume in the tubes to 1 mL with reduced serum media.
6. Add 2 µL of Polybrene (at a stock of 4 µg/µL) to 1 mL of virus/media. Mix well by pipetting and add 1 mL of mixture to the cells. Incubate the cells with the virus for 24 h.
7. Remove the virus media and replace with normal DMEM media supplemented with 10% FBS. Maintain the cells at 37 °C, 5% CO₂.
8. Monitor the growth of the cells and change the culture media every two days. At confluence, expand the 6-well dish into a 10-cm diameter tissue culture plate.
9. Once the cells have been sufficiently expanded, seed 1.5 x 10⁴ cells per well in 50 µL of DMEM media on 384-well assay plates to check the expression level of the YFP tagged protein by microscopy. If the YFP tagged protein cell expression shows a signal-to-noise ratio ≥3, prepare cell stock for the corresponding cell line.

3. Time dependent effect of proteasome inhibitor on protein aggregation

NOTE: Perform image acquisition using an automated microscope (see Materials).

1. Dispense 0.25 µL of DMSO or proteasome inhibitor dissolved in 100% DMSO (ALLN, 2 mM) on 384-well flat bottom plates.
2. Manually seed 1.5 x 10⁴ cells per well in 50 µL of DMEM media on the same assay plate. The proteasome inhibitor (ALLN) final concentration should be 10 µM.
3. Set up the microscope system environmental control unit to 37 °C and 5% CO₂. A screenshot of the experimental setup is shown in Figure 3.
4. Operate the microscope in wide field fluorescence mode, using the software with the following settings: LWD 20X objective, non-confocal mode, 4 fields/well, with a capture interval of an hour. At the end of each run, captured images are automatically uploaded to the server.

4. Time lapse imaging for YFP expression in living cells, and image analysis (single channel)

NOTE: The following steps describe application of the software (e.g., Columbus).

1. Select the appropriate algorithm to segment the primary objects (cytosol and aggregates). If required, adjust background threshold and contrast parameters (Figure 4).
2. Count cells using 'find cells' with an individual threshold of 0.1 for the YFP intensity channel. Determine aggregates using a 'find spots' algorithm with a relative YFP spot intensity >0.2 and a splitting coefficient of 1.0.

5. Dose response effect of proteasome inhibitors on protein aggregation on living cells stained for their nuclei (two channels)

NOTE: Perform image acquisition using an automated microscope. Determine the concentration range of proteasome inhibitors (ALLN, Epoxomicin, and MG132) based on the expected IC₅₀ value to ensure an optimal curve fit.

1. Prepare serial dilutions of compounds on a 384-well storage polypropylene plate by the standard 1:3 dilution series. Make sure to mix the compound dilutions well to ensure that the compound concentrations are accurate.
2. Dispense 0.25 µL of proteasome inhibitors on empty flat-bottom black 384-well assay plates. We use a liquid handler equipped with a 384-capillary head capable of transferring volumes.
3. Seed 1.5 x 10⁴ cells per well in 50 µL of DMEM Media on assay plates. Incubate the assay plates at 37 °C and 5% CO₂ for 24 h.
4. Add 10 µL of DMEM Media (pre-warmed at 37 °C) with staining solution (Hoechst 33342 at a stock of 10 mg/mL) and incubate at room temperature for 10 min.
5. Launch the microscope operating software. A screenshot of the experimental setup is shown in Figure 6. Select the "Configuration" tab and select 20X objective and the correct plate type. Ensure "collar" is set to the correct value on the objective allowing proper focus with different plate types.
6. Select the "Microscope" tab. Define exposure 1 as YFP (488 laser) and exposure 2 as Hoechst (405 laser). Activate the filter on both exposures and assign exposure 1 to camera 1 and exposure 2 to camera 2. Set exposure times to ~800 ms for exposure 1 and ~40 ms for exposure 2.
7. Select exposure 1. Set focus height to 0 µm. Select "Focus". Once focused, expose camera 1. Adjust the focus height to optimize the exposure plane and click on "Take height". Change the exposure times and laser power to give a maximum pixel intensity of ~3,000. Save exposure parameters. Repeat for exposure 2.
8. Select "Experiment Definition" tab. Create a layout and sublayout. Drag and drop the relevant layout, exposure, reference image, skewcrop file, and sublayout. Save the experiment.
9. Select "Automatic Experiment" tab and acquire images. At the end of each run, captured images are automatically uploaded to the server.

6. Image analysis of two channel images

1. Select the software algorithm to segment the primary objects (nuclei, cytosol, and aggregates) (Figure 7).
2. Select the method that segments nuclei accurately by visual inspection of the segmented objects. Hoechst-stained objects in channel 1 (Ch1) will be used to determine the number of cells. The YFP staining from the mutant SOD1-A4V in channel 2 (Ch2) will be used to determine the amount of aggregates.
3. Count cells using the 'find nuclei' algorithm as Hoechst-staining regions >20 µm², with a split factor of 7.0, an individual threshold of 0.40, and a contrast >0.10.
4. Create the data table and determine EC₅₀ for each of the compounds using the 'Non-linear regression' equation in the graphing software.

Results

Generating stable cell line using lentivirus: The overall strategy for monitoring SOD1 protein aggregates is illustrated in Figure 1. In a first step, we generated a lentiviral expression vector for SOD1 stable gene delivery into cell lines (Step 1). Two lentiviral vectors encoding YFP-tagged SOD1 WT and SOD1 A4V (SOD1WT-YFP and SOD1A4V-YFP, respectively) with packing and envelope plasmids were prepared (Figure 2A

Discussion

There are two main approaches for generating stable cell lines. The first takes several weeks and requires transient transfection and resistance selection of the genomic integrated plasmid DNA vectors. The second takes a matter of hours through the use of lentivirus, making this protocol amenable to the effective expression of target protein in multiple cell lines with limited effort. The TRIP-CMV vector¹⁹ was a sustained vector to use, with conserved transduction efficiency and stable transgene e...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
ALLN (C20H37N3O4)	Millipore	208719
MG132 (C26H41N3O5)	Sigma-Aldrich	C2211
Epoxomicin (C28H50N4O7)	Sigma-Aldrich	E3652
Hoechst 33342	Invitrogen	H-3570
Opera	Perkin Elmer	OP-QEHS-01
Opera EvoShell software	Perkin Elmer	Ver 1.8.1
Operetta	Perkin Elmer	OPRT1288
Harmony Imaging software	Perkin Elmer	Ver 3.0.0
Columbus Image analysis software	Perkin Elmer	Ver 2.3.2
CyBi Hummingwell liquid handling	CyBio AG	OL 3387 3 0110