This work was supported by a grant funded by the Korean government (MSIP) (NRF-2014K1A4A7A01074642), and the National Research Foundation of Korea (NRF) individual scientist support program (NRF-2013M3A9B5076486/NRF-2015R1D1A1A09057239).

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&#8242;-ATCGTCTAGACACCATGGCGACGAAGGTCGTGTGC-3&#8242; an...

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I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disulfide+bond+mediates+aggregation,+toxicity,+and+ubiquitylation+of+familial+amyotrophic+lateral+sclerosis-linked+mutant+SOD1.">Disulfide bond mediates aggregation, toxicity, and ubiquitylation of familial amyotrophic lateral sclerosis-linked mutant SOD1.</a> J Biol Chem. 282 (38), 28087-28095 (2007).</li><li>Juneja, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prognosis+in+familial+amyotrophic+lateral+sclerosis:+progression+and+survival+in+patients+with+glu100gly+and+ala4val+mutations+in+Cu,Zn+superoxide+dismutase.">Prognosis in familial amyotrophic lateral sclerosis: progression and survival in patients with glu100gly and ala4val mutations in Cu,Zn superoxide dismutase.</a> Neurology. 48 (1), 55-57 (1997).</li><li>Kaur, S. 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The authors have nothing to disclose.

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...

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 aggregate9,10. 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 inclusions11,12. 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 aggregates13,14, 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 &#945;-synuclein and &#946;-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.

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

assay development for high content quantification of sod1 mutant protein aggregate formation in living cells

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.

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</strong...

Watch this Scientific Journal Video about Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells at JoVE.com

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

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.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc ...

The overall goal of this experiment is to develop an assay for studying the effect of small molecules in modulating the aggregation of misfolded proteins in a time and dose dependent manner. This method can help answer the key questions for development of cellular assay, and it's use is for high-content screening techniques. The main advantage of this technique is to provide a simple method to detect and quantify protein aggregation occurring in several neurodegenerative disorders.The implication of this technique extends towards potential therapy of neurodegenerative diseases, such as ALS, because it proposes a simple method that allow to develop cellular assay and perform screening for small molecule moderators. To start virus production, prior to plasmid transfection split HEK-293 T-cells at 30 to 40%confluence in a 10 centimeter tissue culture dish and 10 milliliters of cell culture medium. Incubate the culture plate in a 5%carbon dioxide incubator at 37 degrees Celsius overnight.On the following day, prepare DNA transfection solution with packaging plasmids lentiviral vector, 125 microliters of one molar calcium chloride and add distilled water to adjust the volume to 500 microliters. Gently add 500 microliters of 0.05 molar HEPS buffer and incubate for 10 minutes at room temperature. After that, replace HEK-293 T-cell medium with 10 milliliters pre-warmed antibiotic-free fresh culture medium mixed with one milliliter of DNA transfection solution.Incubate at 37 degrees Celsius and 5%carbon dioxide incubator over night. Replace cell supernatent with fresh culture medium on the following day and incubate again overnight. Finally, on the following day harvest the supernatent and transfer to a 15 milliliter tube.To remove the dead cells and debris, centrifuge the tube at 500 times g for five minutes. To further purify the supernatent, pass it through a 0.45 micrometer filter using a 60-milliliter syringe. Then, immediately dispense the filtered supernatent into 300-microliter single use aliquots.To prepare the cells for transduction, split them to be infected at 60%confluence in a six-well tissue culture plate in two milliliters of DMAM supplemented with 10%FBS. Incubate the cells in a 37-degree Celsius incubator at 5%carbon dioxide overnight. On the following day, thaw one aliquot of the frozen lentivirus on ice for each use.Meanwhile, warm the cell culture medium containing the serum compatible with the cell line of interest. Once the virus is fully thawed, dilute it in DMEM in new 1.5 milliliter micro-centrifuge tubes. Then adjust the volume to one milliliter with reduced serum medium.Next, add two microliters of polybrene to one milliliter of virus per medium and mix well by pipetting. Add one milliliter of this mixture to the cells and incubate at 37 degrees Celsius for 24 hours. After incubation, remove the virus containing medium and replace it with normal DMEM supplemented with 10%FBS and return the cells to the incubator.Monitor the cell growth and change the medium every two days When the cells reach confluence, expand each well of the six-well dish into a 10-centimeter tissue culture plate. Move the plates to the incubator. Finally, save 1.5 times 10 to the 4th cells per well and 50 microliters of DMEM on 384 well assay plates.Then check the expression level of the YFP-tagged protein under the microscope. If the expression shows signal-to-noise ratio equal or greater than three, prepare cell stock for the corresponding cell line. For the compound transfer, on a 384-well storage polypropylene plate, add serial dilutions of compounds by the standard one to three dilution series.Then, using a liquid handler equipped with a 384-capillary head, distribute 0.25 microliters of proteasome inhibitors on empty, flat-bottomed black 384-well assay plates. Then seed 1.5 times 10 to the 4th cells on these plates. Incubate at 37 degrees Celsius and 5%carbon dioxide for 24 hours.Prior to imaging the cell line treated with proteasome inhibitors, add 10 microliters of pre-warmed DMEM with staining solution and incubate at room temperature for 10 minutes. After staining, start the automated microscope operating software by selecting the microscope tab. First, select the configuration tab and then select 20 times objective in the correct plate type.To allow proper focus with different plate types, make sure that the caller is set to the correct value on the objective. Next, select exposure one. Set focus height to zero micrometers, then select focus.Once it is focused, expose camera one. To optimize the exposure plane, adjust the focus height and click on take height. Change exposure times and laser power for a maximum pixel intensity of approximately 3000 and save exposure parameters.Select experiment definition tab, create a layout and sublayout. Next, drag and drop the relevant layout exposure, reference image, skew crop file, and sublayout. Then, save the experiment.Finally, select automatic experiment tab and acquire images. For two-channel images, first select the software algorithm to segment the primary objects, such as nuclei, cytosol, and aggregates. First, distribute 0.25 microliters of proteasome inhibitor dissolved in 100%DMSO or DMSO on 384-well flat-bottomed plates.On the same plate, manually seed 1.5 times 10 to the 4th cells per well in 50-microliters of DMEM. Then set up the microscope system environmental control unit to 37 degrees Celsius and 5%carbon dioxide. Operate the microscope in wide-field florescence mode and as software settings, choose LWD 20 times objective non-confocal mode, four fields per well, and capture interval of one hour.Then, select the appropriate algorithm to segment the primary object such as cytosol and aggregates. Adjust background threshold and contrast parameters if required. The tested cell lines transduced with SOD-1 wild type and SOD-1 A4V mutant remained healthy, showed high expression levels, and longterm wide phi-labeling irrespective of the SOD form.Treatment with proteasome inhibitor ALLN for 24 hours promoted the accumulation of SOD-1 A4V YFP aggregates in HEK-293 cells but not in SOD-1 Wild Type YFP transduced cells. Furthermore, there were very low levels of aggregation in U2 OS and SH-SY5Y cell lines SOD-1 A4V YFP transduced with the same vectors. After seeding the cells in the presence or absence of ALLN, they were monitored for aggregate formation for a period of 50 hours.ALLN aggregate formation reached a plateau at 24 hours and remained stable over the next 12 hours. Furthermore, cell density significantly decreased after 28 hours as a result of the cytotoxic effect of ALLN, while cell number increased in the DMSO-treated negative control. SOD-1 A4V YFP cells cultured in the presence of proteasome inhibitors for 24 hours were stained with Hoechst solution.The relative toxicity for three different proteasome inhibitors was quatified by measuring the number of adherent cell lines remaining in the well labeled with Hoechst dye. The total cell number decreased in response to increased dose of protease inhibitors. Inversely, the percentage of cells expressing SOD-1 A4V aggregates increased with the concentration of protease inhibitor.When doing this process it is important to remember that the cell quality will drastically influence your result. So it's important to determine that the cells are microplasma free and are healthy all along the steps. Following this process, another imaging method can be used in order to answer additional questions regarding the degradation of protein or the interaction of the protein with others using resonance energy transfer.After watching this video, you should have a good understanding of how to generate the tools to test the fate of protein in multiple living cells using high-content image analysis. So this method can provide insight into protein aggregation as it occurs in simple cellular models. It can also be applied to other systems such as neuron differentiated from patient specific induced pluripotent stem cells.

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7.1K Görüntüleme.  Institut Pasteur Korea. The overall goal of this experiment is to develop an assay for studying the effect of small molecules in modulating the aggregation of misfolded proteins in a time and dose dependent manner. This method can help answer the key questions for development of cellular assay, and it's use is for high-content screening techniques. The main advantage of this technique is to provide a simple method to detect and quantify protein aggregation occurring in several neurodegenerative disorders.The ...