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; and 5&#8242;-TAGCGG CCGCTACTTGTACAGCTCGTCCATGCC-3&#8242;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.
<ol>
	<li>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.</li>
	<li>Keep the 10-cm diameter tissue culture plate in a CO2 incubator (37 &#176;C, 5% CO2) overnight.</li>
	<li>On Day 2, prepare a DNA transfection solution containing 10 &#181;g of lentiviral vectors (pTRIP-delta U3 CMV - SOD1WT-YFP or -SOD1A4V-YFP) together with the lentiviral packaging plasmids (5 &#181;g of pVSVg; 10 &#181;g of pCMV-dR8.71) (Figure 2A)20. Add 125 &#181;L of 1 M CaCl2 and adjust the volume to 500 &#181;L using distilled water. Gently add 500 &#181;L of 0.05 M HEPES into the mixture and incubate for 10 min at room temperature.</li>
	<li>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 &#176;C, 5% CO2.</li>
	<li>On Day 3, replace the cell supernatant with fresh culture medium.</li>
	<li>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 &#181;m filter using a large 60 mL syringe. Immediately dispense into single-use aliquots (300 &#181;L), and store at -80 &#176;C. To maintain maximum product activity, avoid a freeze-thaw cycle.</li>
</ol>
2. Lentiviral transduction
<ol>
	<li>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.</li>
	<li>Keep the 6-well tissue culture plate in a 37 &#176;C incubator at 5% CO2 overnight.</li>
	<li>Remove the frozen lentivirus from the -80 &#176;C freezer and thaw an aliquot on ice before each use; do not refreeze.</li>
	<li>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.</li>
	<li>Bring up the volume in the tubes to 1 mL with reduced serum media.</li>
	<li>Add 2 &#181;L of Polybrene (at a stock of 4 &#181;g/&#181;L) 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.</li>
	<li>Remove the virus media and replace with normal DMEM media supplemented with 10% FBS. Maintain the cells at 37 &#176;C, 5% CO2.</li>
	<li>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.</li>
	<li>Once the cells have been sufficiently expanded, seed 1.5 x 104 cells per well in 50 &#181;L 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 &#8805;3, prepare cell stock for the corresponding cell line.</li>
</ol>
3. Time dependent effect of proteasome inhibitor on protein aggregation
NOTE: Perform image acquisition using an automated microscope (see Materials).
<ol>
	<li>Dispense 0.25 &#181;L of DMSO or proteasome inhibitor dissolved in 100% DMSO (ALLN, 2 mM) on 384-well flat bottom plates.</li>
	<li>Manually seed 1.5 x 104 cells per well in 50 &#181;L of DMEM media on the same assay plate. The proteasome inhibitor (ALLN) final concentration should be 10 &#181;M.</li>
	<li>Set up the microscope system environmental control unit to 37 &#176;C and 5% CO2. A screenshot of the experimental setup is shown in Figure 3.</li>
	<li>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.</li>
</ol>
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).
<ol>
	<li>Select the appropriate algorithm to segment the primary objects (cytosol and aggregates). If required, adjust background threshold and contrast parameters (Figure 4).</li>
	<li>Count cells using &#39;find cells&#39; with an individual threshold of 0.1 for the YFP intensity channel. Determine aggregates using a &#39;find spots&#39; algorithm with a relative YFP spot intensity &#62;0.2 and a splitting coefficient of 1.0.</li>
</ol>
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.
<ol>
	<li>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.</li>
	<li>Dispense 0.25 &#181;L 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.</li>
	<li>Seed 1.5 x 104 cells per well in 50 &#181;L of DMEM Media on assay plates. Incubate the assay plates at 37 &#176;C and 5% CO2 for 24 h.</li>
	<li>Add 10 &#181;L of DMEM Media (pre-warmed at 37 &#176;C) with staining solution (Hoechst 33342 at a stock of 10 mg/mL) and incubate at room temperature for 10 min.</li>
	<li>Launch the microscope operating software. A screenshot of the experimental setup is shown in Figure 6. Select the &#34;Configuration&#34; tab and select 20X&#160;objective and the correct plate type. Ensure &#34;collar&#34; is set to the correct value on the objective allowing proper focus with different plate types.</li>
	<li>Select the &#34;Microscope&#34; 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.</li>
	<li>Select exposure 1. Set focus height to 0 &#181;m. Select &#34;Focus&#34;. Once focused, expose camera 1. Adjust the focus height to optimize the exposure plane and click on &#34;Take height&#34;. Change the exposure times and laser power to give a maximum pixel intensity of ~3,000. Save exposure parameters. Repeat for exposure 2.</li>
	<li>Select &#34;Experiment Definition&#34; tab. Create a layout and sublayout. Drag and drop the relevant layout, exposure, reference image, skewcrop file, and sublayout. Save the experiment.</li>
	<li>Select &#34;Automatic Experiment&#34; tab and acquire images. At the end of each run, captured images are automatically uploaded to the server.</li>
</ol>
6. Image analysis of two channel images
<ol>
	<li>Select the software algorithm to segment the primary objects (nuclei, cytosol, and aggregates) (Figure 7).</li>
	<li>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.</li>
	<li>Count cells using the &#39;find nuclei&#39; algorithm as Hoechst-staining regions &#62;20 &#181;m2, with a split factor of 7.0, an individual threshold of 0.40, and a contrast &#62;0.10.</li>
	<li>Create the data table and determine EC50 for each of the compounds using the &#39;Non-linear regression&#39; equation in the graphing software.</li>
</ol>

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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 (C&lt;sub&gt;20&lt;/sub&gt;H&lt;sub&gt;37&lt;/sub&gt;N&lt;sub&gt;3&lt;/sub&gt;O&lt;sub&gt;4&lt;/sub&gt;)</td><td>Millipore</td><td>208719</td><td></td></tr><tr><td>MG132 (C&lt;sub&gt;26&lt;/sub&gt;H&lt;sub&gt;41&lt;/sub&gt;N&lt;sub&gt;3&lt;/sub&gt;O&lt;sub&gt;5&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>C2211</td><td></td></tr><tr><td>Epoxomicin (C&lt;sub&gt;28&lt;/sub&gt;H&lt;sub&gt;50&lt;/sub&gt;N&lt;sub&gt;4&lt;/sub&gt;O&lt;sub&gt;7&lt;/sub&gt;)</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 ...

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JoVE Journal

Neuroscience

7.2K Views.  Institut Pasteur Korea. Beschrijven we een methode om te kwantificeren van de samenvoeging van misfolded eiwitten. Onze gegevens-protocol lentivirale geïnduceerde stabiele cel lijn generatie, geautomatiseerde confocal beeldvorming en beeldanalyse van eiwit-aggregaten. Als een illustratieve toepassing onderzocht we het effect van kleine molecules biij SOD1 aggregatie op een tijd - en dosis-afhankelijke manier.

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

A Rhodopsin Transport Assay by High-Content Imaging Analysis

Rhodopsin misfolding mutations lead to rod photoreceptor death that is manifested as autosomal dominant retinitis pigmentosa (RP), a progressive blinding disease that lacks effective treatment. We hypothesize that the cytotoxicity of the misfolded rhodopsin mutant can be alleviated by pharmacologically stabilizing the mutant rhodopsin protein. The P23H mutation, among the other Class II rhodopsin mutations, encodes a structurally unstable rhodopsin mutant protein that is accumulated in the endoplasmic reticulum (ER), whereas the wild type rhodopsin is transported to the plasma membrane in mammalian cells. We previously performed a luminescence-based high-throughput screen (HTS) and identified a group of pharmacological chaperones that rescued the transport of the P23H rhodopsin from ER to the plasma membrane. Here, using an immunostaining method followed by a high-content imaging analysis, we quantified the mutant rhodopsin protein amount in the whole cell and on the plasma membrane. This method is informative and effective to identify true hits from false positives following HTS. Additionally, the high-content image analysis enabled us to quantify multiple parameters from a single experiment to evaluate the pharmacological properties of each compound. Using this assay, we analyzed the effect of 11 different compounds towards six RP associated rhodopsin mutants, obtaining a 2-D pharmacological profile for a quantitative and qualitative understanding about the structural stability of these rhodopsin mutants and efficacy of different compounds towards these mutants.

Here, we described a high-content imaging method to quantify the transport of rhodopsin mutants associated with retinitis pigmentosa. A multiple-wavelength scoring analysis was used to quantify rhodopsin protein on the cell surface or in the whole cell.

Rhodopsin misfolding mutations lead to rod photoreceptor death that is manifested as autosomal dominant retinitis pigmentosa (RP), a progressive blinding ...

Chemistry

Assay to Measure Nucleocytoplasmic Transport in Real Time within Motor Neuron-like NSC-34 Cells

Nucleocytoplasmic transport refers to the import and export of large molecules from the cell nucleus. Recently, a number of studies have shown a connection between amyotrophic lateral sclerosis (ALS) and impairments in the nucleocytoplasmic pathway. ALS is a neurodegenerative disease affecting the motor neurons and resulting in paralysis and ultimately in death, within 2-5 years on average. Most cases of ALS are sporadic, lacking any apparent genetic linkage, but 10% are inherited in a dominant manner. Recently, hexanucleotide repeat expansions (HREs) in the chromosome 9 open reading frame 72 (C9orf72) gene were identified as a genetic cause of ALS and frontotemporal dementia (FTD). Importantly, different groups have recently proposed that these mutants affect nucleocytoplasmic transport. These studies have mostly shown the final outcome and manifestations caused by HREs on nucleocytoplasmic transport, but they do not demonstrate nuclear transport dysfunction in real time. As a result, only severe nucleocytoplasmic transport deficiency can be determined, mostly due to high overexpression or exogenous protein insertion. 
This protocol describes a new and very sensitive assay to evaluate and quantify nucleocytoplasmic transport dysfunction in real time. The rate of import of a NLS-NES-GFP protein (shuttle-GFP) can be quantified in real time using fluorescent microscopy. This is performed by using an exportin inhibitor, thus allowing the shuttle GFP only to enter the nucleus. To validate the assay, the C9orf72 HRE translated dipeptide repeats, poly(GR) and poly(PR), which have been previously shown to disrupt nucleocytoplasmic transport, were used. Using the described assay, a 50% decrease in the nuclear import rate was observed compared to the control. Using this system, minute changes in nucleocytoplasmic transport can be examined and the ability of different factors to rescue (even partially) a nucleocytoplasmic transport defect can be determined.

This protocol describes a method to sensitively measure the nucleocytoplasmic transport rate within motor neuron-like NSC-34 cells by quantifying the real-time change in the nuclear import of a NLS-NES-GFP protein.

Nucleocytoplasmic transport refers to the import and export of large molecules from the cell nucleus. Recently, a number of studies have shown a ...

Monitoring Cell-to-cell Transmission of Prion-like Protein Aggregates in Drosophila Melanogaster

Protein aggregation is a central feature of most neurodegenerative diseases, including Alzheimer&#39;s disease (AD), Parkinson&#39;s disease (PD), Huntington&#39;s disease (HD), and amyotrophic lateral sclerosis (ALS). Protein aggregates are closely associated with neuropathology in these diseases, although the exact mechanism by which aberrant protein aggregation disrupts normal cellular homeostasis is not known. Emerging data provide strong support for the hypothesis that pathogenic aggregates in AD, PD, HD, and ALS have many similarities to prions, which are protein-only infectious agents responsible for the transmissible spongiform encephalopathies. Prions self-replicate by templating the conversion of natively-folded versions of the same protein, causing spread of the aggregation phenotype. How prions and prion-like proteins in AD, PD, HD, and ALS move from one cell to another is currently an area of intense investigation. Here, a Drosophila melanogaster model that permits monitoring of prion-like, cell-to-cell transmission of mutant huntingtin (Htt) aggregates associated with HD is described. This model takes advantage of powerful tools for manipulating transgene expression in many different Drosophila tissues and utilizes a fluorescently-tagged cytoplasmic protein to directly report prion-like transfer of mutant Htt aggregates. Importantly, the approach we describe here can be used to identify novel genes and pathways that mediate spreading of protein aggregates between diverse cell types in vivo. Information gained from these studies will expand the limited understanding of the pathogenic mechanisms that underlie neurodegenerative diseases and reveal new opportunities for therapeutic intervention.

Monitoring Cell-to-cell Transmission of Prion-like Protein Aggregates in Drosophila Melanogaster

Accumulating evidence supports the idea that pathogenic protein aggregates associated with neurodegenerative diseases spread between cells with prion-like properties. Here, we describe a method that enables visualization of cell-to-cell spreading of prion-like aggregates in the model organism, Drosophila melanogaster.

Protein aggregation is a central feature of most neurodegenerative diseases, including Alzheimer&#39;s disease (AD), Parkinson&#39;s disease (PD), ...

Exogenous Administration of Microsomes-associated Alpha-synuclein Aggregates to Primary Neurons As a Powerful Cell Model of Fibrils Formation

For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the study of &#945;S aggregation in Parkinson&#39;s Disease (PD). Recently, the development of new animal models through the exogenous inoculation of brain extracts from diseased &#945;S transgenic mice or PD patients has given new hopes to the possibility of creating more adequate cell models of &#945;S aggregation. Unfortunately, when it comes to cells in cultures, administration of raw brain extracts has not proven as successful as in mice and the source of choice of exogenous aggregates is still in vitro preformed &#945;S fibrils.
We have developed a method to induce the formation of intracellular &#945;S inclusions in primary neurons through the exogenous administration of native microsomes-associated &#945;S aggregates, a highly toxic &#945;S species isolated from diseased areas of transgenic mice. This fraction of &#945;S aggregates that is associated with the microsomes vesicles, is efficiently internalized and induces the formation of intracellular inclusions positive for aggregated and phosphorylated &#945;S. Compared to in vitro-preformed fibrils which are made from recombinant &#945;S, our method is faster and guarantees that the pathogenic seeding is made with authentic &#945;S aggregates extracted from diseased animal models of PD, mimicking more closely the type of inclusions obtained in vivo. As a result, availability of tissues rich in &#945;S inclusions is mandatory.
We believe that this method will provide a versatile cell-based model to study the microscopic aspects of &#945;S aggregation and the related cellular pathophysiology in vivo and will be a starting point for the creation of more accurate and sophisticated cell paradigm of PD.

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.

For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the ...

Studying Pre-formed Fibril Induced &#945;-Synuclein Accumulation in Primary Embryonic Mouse Midbrain Dopamine Neurons

The goal of this protocol is to establish a robust and reproducible model of &#945;-synuclein accumulation in primary dopamine neurons. Combined with immunostaining and unbiased automated image analysis, this model allows for the analysis of the effects of drugs and genetic manipulations on &#945;-synuclein aggregation in neuronal cultures. Primary midbrain cultures provide a reliable source of bona fide embryonic dopamine neurons. In this protocol, the hallmark histopathology of Parkinson&#8217;s disease, Lewy bodies (LB), is mimicked by the addition of &#945;-synuclein pre-formed fibrils (PFFs) directly to neuronal culture media. Accumulation of endogenous phosphorylated &#945;-synuclein in the soma of dopamine neurons is detected by immunostaining already at 7 days after the PFF addition. In vitro cell culture conditions are also suitable for the application and evaluation of treatments preventing &#945;-synuclein accumulation, such as small molecule drugs and neurotrophic factors, as well as lentivirus vectors for genetic manipulation (e.g., with CRISPR/Cas9). Culturing the neurons in 96 well plates increases the robustness and power of the experimental setups. At the end of the experiment, the cells are fixed with paraformaldehyde for immunocytochemistry and fluorescence microscopy imaging. Multispectral fluorescence images are obtained via automated microscopy of 96 well plates. These data are quantified (e.g., counting the number of phospho-&#945;-synuclein-containing dopamine neurons per well) with the use of free software that provides a platform for unbiased high-content phenotype analysis. PFF-induced modeling of phosphorylated &#945;-synuclein accumulation in primary dopamine neurons provides a reliable tool to study the underlying mechanisms mediating formation and elimination of &#945;-synuclein inclusions, with the opportunity for high-throughput drug screening and cellular phenotype analysis.

Here, we present a detailed protocol to study neuronal &#945;-synuclein accumulation in primary mouse dopamine neurons. Phosphorylated &#945;-synuclein aggregates in neurons are induced with pre-formed &#945;-synuclein fibrils. Automated imaging of immunofluorescently labeled cells and unbiased image analysis make this robust protocol suitable for medium-to-high throughput screening of drugs that inhibit &#945;-synuclein accumulation.

The goal of this protocol is to establish a robust and reproducible model of &#945;-synuclein accumulation in primary dopamine neurons. Combined with ...

Primary Cultures of Rat Astrocytes and Microglia and Their Use in the Study of Amyotrophic Lateral Sclerosis

This protocol demonstrates how to prepare primary cultures of glial cells, astrocytes, and microglia from the cortices of Sprague Dawley rats and how to use these cells for the purpose of studying the pathophysiology of amyotrophic lateral sclerosis (ALS) in the rat hSOD1G93A model. First, the protocol shows how to isolate and culture astrocytes and microglia from postnatal rat cortices, and then how to characterize and test these cultures for purity by immunocytochemistry using the glial fibrillary acidic protein (GFAP) marker of astrocytes and the ionized calcium-binding adaptor molecule 1 (Iba1) microglial marker. In the next stage, methods are described for dye-loading (calcium-sensitive Fluo 4-AM) of cultured cells and the recordings of Ca2+ changes in video imaging experiments on live cells.
The examples of video recordings consist of: (1) cases of Ca2+ imaging of cultured astrocytes acutely exposed to immunoglobulin G (IgG) isolated from ALS patients, showing a characteristic and specific response compared to the response to ATP as demonstrated in the same experiment. Examples also show a more pronounced transient rise in intracellular calcium concentration evoked by ALS IgG in hSOD1G93A astrocytes compared to non-transgenic controls; (2) Ca2+ imaging of cultured astrocytes during a depletion of calcium stores by thapsigargin (Thg), a non-competitive inhibitor of the endoplasmic reticulum Ca2+ ATPase, followed by store-operated calcium entry elicited by the addition of calcium in the recording solution, which demonstrates the difference between Ca2+ store operation in hSOD1G93A and in non-transgenic astrocytes; (3) Ca2+ imaging of the cultured microglia showing predominantly a lack of response to ALS IgG, whereas ATP application elicited a Ca2+ change. This paper also emphasizes possible caveats and cautions regarding critical cell density and purity of cultures, choosing the correct concentration of the Ca2+ dye and dye-loading techniques.

We present here a protocol on how to prepare primary cultures of glial cells, astrocytes, and microglia from rat cortices for time-lapse video imaging of intracellular Ca2+ for research on pathophysiology of amyotrophic lateral sclerosis in the hSOD1G93A rat model.

This protocol demonstrates how to prepare primary cultures of glial cells, astrocytes, and microglia from the cortices of Sprague Dawley rats and how to ...

Assays for the Degradation of Misfolded Proteins in Cells

Protein misfolding and aggregation are associated with various neurodegenerative diseases. Cellular mechanisms that recognize and degrade misfolded proteins may serve as potential therapeutic targets. To distinguish degradation of misfolding-prone proteins from other mechanisms that regulate their levels, one important method is to measure protein half-life in cells. However, this can be challenging because misfolding-prone proteins may exist in different forms, including the native form and misfolded forms of distinct characteristics. Here we describe assays to examine the half-life of misfolded proteins in mammalian cells using a highly aggregation-prone protein, Ataxin-1 with an extended polyglutamine (polyQ) stretch, and a conformationally unstable luciferase mutant as models. Cycloheximide chase is combined with cell fractionation to examine the turnover rate of misfolding-prone proteins in various cellular fractions. We further depict a fluorescence-based assay using an enhanced green fluorescence protein (EGFP)-fusion of the luciferase mutant, which can be adapted for high throughput screening on a microplate-reader.

This report describes protocols for measuring degradation rates of misfolded proteins by either western blot or fluorescence-based assays. The methods can be applied to analysis of other misfolded proteins and for high throughput screening.

Protein misfolding and aggregation are associated with various neurodegenerative diseases. Cellular mechanisms that recognize and degrade misfolded ...

Biology

Detection of Protein Aggregation using Fluorescence Correlation Spectroscopy

Protein aggregation is a hallmark of neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and so on. To detect and analyze soluble or diffuse protein oligomers or aggregates, fluorescence correlation spectroscopy (FCS), which can detect the diffusion speed and brightness of a single particle with a single molecule sensitivity, has been used. However, the proper procedure and know-how for protein aggregation detection have not been widely shared. Here, we show a standard procedure of FCS measurement for diffusion properties of aggregation-prone proteins in cell lysate and live cells: ALS-associated 25 kDa carboxyl-terminal fragment of TAR DNA/RNA-binding protein 43 kDa (TDP25) and superoxide dismutase 1 (SOD1). The representative results show that a part of aggregates of green fluorescent protein (GFP)-tagged TDP25 was slightly included in the soluble fraction of murine neuroblastoma Neuro2a cell lysate. Moreover, GFP-tagged SOD1 carrying ALS-associated mutation shows a slower diffusion in live cells. Accordingly, we here introduce the procedure to detect the protein aggregation via its diffusion property using FCS.

We here introduce a procedure to measure protein oligomers and aggregation in cell lysate and live cells using fluorescence correlation spectroscopy.

Protein aggregation is a hallmark of neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's ...

A Method to Study &#945;-Synuclein Toxicity and Aggregation Using a Humanized Yeast Model

Parkinson's disease is the second most common neurodegenerative disorder and is characterized by progressive cell death caused by the formation of Lewy bodies containing misfolded and aggregated &#945;-synuclein. &#945;-synuclein is an abundant presynaptic protein that regulates synaptic vesicle trafficking, but the accumulation of its proteinaceous inclusions results in neurotoxicity. Recent studies have revealed that various genetic factors, including bacterial chaperones, could reduce the formation of &#945;-synuclein aggregates in vitro. However, it is also important to monitor the anti-aggregation effect in the cell to apply this as a potential treatment for the patients. It would be ideal to use neuronal cells, but these cells are difficult to handle and take a long time to exhibit the anti-aggregation phenotype. Therefore, a quick and effective in vivo tool is required for the further evaluation of in vivo anti-aggregation activity. The method described here was used to monitor and analyze the anti-aggregation phenotype in the humanized yeast Saccharomyces cerevisiae, which expressed human &#945;-synuclein. This protocol demonstrates in vivo tools that could be used for monitoring &#945;-synuclein-induced cellular toxicity, as well as the formation of &#945;-synuclein aggregates in cells.

An in vivo physiological model of &#945;-synuclein is required to study and understand the pathogenesis of Parkinson's disease. We describe a method to monitor the cytotoxicity and aggregate formation of &#945;-synuclein using a humanized yeast model.

Parkinson's disease is the second most common neurodegenerative disorder and is characterized by progressive cell death caused by the formation of Lewy ...

Protein Aggregate Formation Assay: A Method to Detect and Quantify Protein Aggregation in Cultured Cells upon Induction by Proteasome Inhibitor

Source: Lee, H. et al. <a href="https://www.jove.com/v/56425">Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells.</a> J. Vis. Exp. (2017)
In this video, we demonstrate a cell-based protein aggregation assay using proteasome inhibitors, which block proteasome activity, preventing misfolded, mutant proteins, fused to a fluorescent label, from undergoing ubiquitin-dependent proteasomal degradation, leading to their accumulation within the cell cytoplasm. The protein aggregates are then visualized and quantified by fluorescence microscopy.

Source: Lee, H. et al. Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells. J. Vis. Exp. (2017)
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Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells

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