Name: modulation of tau subcellular localization as a tool to investigate the expression of disease-related genes
Uploaded: 2019-12-20T12:00:00
Description: Watch this Scientific Journal Video about Modulation of Tau Subcellular Localization as a Tool to Investigate the Expression of Disease-related Genes at JoVE.com

This work was supported by grants from Scuola Normale Superiore (SNS14_B_DIPRIMIO; SNS16_B_DIPRIMIO).

1. Cell Culture
<ol>
	<li>Culture SH-SY5Y cells (human neuroblastoma cell line, CRL-2266) in complete medium (Dulbecco&#39;s modified Eagle medium:nutrient mixture F12 [DMEM/F-12] supplemented with 10% fetal bovine serum [FBS], 2 mM L-glutamine, 100 U/mL penicillin and 100 &#181;g/mL streptomycin). Maintain the cells in an incubator at 37 &#176;C and 5% CO2. Grow cells in 10 cm plates and split when confluent.</li>
</ol>
2. Cell Differentiation
<ol>
	<li>To differentiate SH-SY5Y cel...

<ol>
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Q., Bartlett, P. F., G&#246;tz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+the+microtubule-associated+protein+tau+in+the+nucleus.">Visualizing the microtubule-associated protein tau in the nucleus.</a> Science China Life Sciences. 57 (4), 422-431 (2014).</li><li>Sultan, A., 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=Nuclear+Tau,+a+key+player+in+neuronal+DNA+protection.">Nuclear Tau, a key player in neuronal DNA protection.</a> Journal of Biological Chemistry. 286 (6), 4566-4575 (2011).</li><li>Sj&#246;berg, M. K., Shestakova, E., Mansuroglu, Z., Maccioni, R. 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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+specific+tau+exons+in+normal+and+tumoral+pancreatic+acinar+cells.">Expression of specific tau exons in normal and tumoral pancreatic acinar cells.</a> Journal of Cell Science. 111 (1), 1419-1432 (1998).</li><li>Liao, A., 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=Therapeutic+efficacy+of+FTY720+in+a+rat+model+of+NK-cell+leukemia.">Therapeutic efficacy of FTY720 in a rat model of NK-cell leukemia.</a> Blood. 118 (10), 2793-2800 (2011).</li><li>Cascio, S., Zhang, L., Finn, O. 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We describe a method to measure the impact of nuclear Tau protein on gene expression. With this protocol the contribution of cytoplasmic Tau is strongly limited. Critical steps of this protocol are the following: the differentiation of human neuroblastoma SH-SY5Y cells, the subcellular fractionation and the localization of Tau protein in the nuclear compartment.
First, as shown in the representative results section, the differentiation of SH-SY5Y cells by adding RA and BDNF is crucial to obtai...

The functions of Tau protein in the nucleus have garnered significant interest in recent years, as it has been shown to be closely associated with nucleic acids1,2,3,4,5,6. Indeed, a recent genome-wide study demonstrated that Tau binds genic and intergenic DNA sequences in vivo7. A role in nucleolar organization has been suggested8,9,10,11. Moreover, Tau has been proposed to be involved in DNA protection from oxidative and hyperthermic stress5,10,12,13, whereas mutated Tau has been linked to chromosome instability and aneuploidy14,15,16.
Until now, the challenges in studying the functions of Tau in the nuclear compartment remained almost unsolved due to the difficulties in dissecting the specific contribution of nuclear Tau from the contribution of cytoplasmic Tau. Moreover, the functions attributed to Tau molecules in the nuclear compartment, up to now, are only correlative because they lack an unequivocal demonstration of the direct involvement of nuclear Tau proteins. Indeed, the involvement of Tau in the mobility of retrotransposons or in the rRNA transcription or in DNA protection11,12,17,18,19 might be also explained by the contribution of cytoplasmic Tau or by the effect of other downstream factors not related to nuclear Tau.
Here, we provide a method that can solve this issue by exploiting a classical procedure to isolate the nuclear compartment combined with the use of Tau constructs 0N4R tagged with nuclear localization (NLS) or nuclear export signals (NES). This approach eliminates the complex issues related to possible artefacts due to the spillover of Tau molecules from the cytoplasmic compartment. Moreover, Tau-NLS and Tau-NES constructs induce the enrichment or the exclusion of Tau molecules from the nuclear compartment, respectively, providing positive and negative controls for the involvement of nuclear Tau molecules in a specific function. The protocol is technically easy and it is composed of classic and reliable methods that are broadly applicable to study the nuclear function of Tau in other cell types, differentiated or not, such as cancer cells that reactivate Tau expression20,21. Moreover, it might be applied also to other proteins that are present in both the cytoplasm and the nucleus in order to dissect biological functions related to different compartments.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alexa Fluor 633 goat anti-mouse IgG</td>
			<td>Life Technologies</td>
			<td>A21050</td>
			<td>IF 1:500</td>
		</tr>
		<tr>
			<td>anti Actin Antibody</td>
			<td>BETHYL LABORATORIE</td>
			<td>A300-485A</td>
			<td>anti-rabbit WB 1:10000</td>
		</tr>
		<tr>
			<td>anti GAPDH Antibody</td>
			<td>Fitzgerald Industries International</td>
			<td>10R-G109a</td>
			<td>anti-mouse WB 1:10000</td>
		</tr>
		<tr>
			<td>anti H2B Antibody</td>
			<td>Abcam</td>
			<td>ab1790</td>
			<td>anti-rabbit WB 1:15000</td>
		</tr>
		<tr>
			<td>anti Tau-13 Antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-21796</td>
			<td>anti-mouse WB 1:1000; IF 1:500</td>
		</tr>
		<tr>
			<td>anti Tubulin alpha Antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>PA5-16891</td>
			<td>anti-mouse WB 1:5000</td>
		</tr>
		<tr>
			<td>anti VGluT1 Antibody</td>
			<td>Sigma-Aldrich</td>
			<td>AMAb91041</td>
			<td>anti-mouse WB 1:500</td>
		</tr>
		<tr>
			<td>BCA Protein Assay Kit</td>
			<td>Euroclone</td>
			<td>EMPO14500</td>
			<td></td>
		</tr>
		<tr>
			<td>BDNF</td>
			<td>Alomone Labs</td>
			<td>B-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Blotting-Grade Blocker</td>
			<td>Biorad</td>
			<td>1706404</td>
			<td>Non-fat dry milk</td>
		</tr>
		<tr>
			<td>BOVIN SERUM ALBUMIN</td>
			<td>Sigma-Aldrich</td>
			<td>A4503-50g</td>
			<td></td>
		</tr>
		<tr>
			<td>cOmplete Mini</td>
			<td>Roche</td>
			<td>11836170001</td>
			<td>protease inhibitor</td>
		</tr>
		<tr>
			<td>Criterion TGX 4-20% Stain Free, 10 well</td>
			<td>Biorad</td>
			<td>5678093</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Thermo Fisher Scientific</td>
			<td>62247</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F-12</td>
			<td>GIBCO</td>
			<td>21331-020</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium Low Glucose</td>
			<td>Euroclone</td>
			<td>ECM0060L</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>0390-100ml</td>
			<td>pH=8 0.5M</td>
		</tr>
		<tr>
			<td>Foetal Bovine Serum</td>
			<td>Euroclone</td>
			<td>EC50182L</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516-500ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgG-HPR</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-2005</td>
			<td>WB 1:1000</td>
		</tr>
		<tr>
			<td>Goat anti-rabbit IgG-HPR</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-2004</td>
			<td>WB 1:1000</td>
		</tr>
		<tr>
			<td>IGEPAL CA-630</td>
			<td>Sigma-Aldrich</td>
			<td>I8896-50ml</td>
			<td>Octylphenoxy poly(ethyleneoxy)ethanol</td>
		</tr>
		<tr>
			<td>Immobilon Western</td>
			<td>MERCK</td>
			<td>WBKLS0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Lab-Tech Chamber slide 8 well glass slide</td>
			<td>nunc</td>
			<td>177402</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Euroclone</td>
			<td>ECB3000D</td>
			<td>100X</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000 transfection reagent</td>
			<td>Thermo Fisher Scientific</td>
			<td>12566014</td>
			<td>cationic lipid</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma-Aldrich</td>
			<td>322415-6X1L</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma-Aldrich</td>
			<td>M8266-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S3014-1kg</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM reduced serum medium</td>
			<td>Gibco</td>
			<td>31985070</td>
			<td></td>
		</tr>
		<tr>
			<td>PEI</td>
			<td>Sigma-Aldrich</td>
			<td>40,872-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122</td>
			<td>10,000 U/ml, 100ml</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (Dulbecco A)</td>
			<td>OXOID</td>
			<td>BR0014G</td>
			<td></td>
		</tr>
		<tr>
			<td>PhosStop</td>
			<td>Roche</td>
			<td>4906837001</td>
			<td>phosphatase inhibitor</td>
		</tr>
		<tr>
			<td>QIAGEN Plasmid Maxi Kit</td>
			<td>Qiagen</td>
			<td>12163</td>
			<td>Step 3.10</td>
		</tr>
		<tr>
			<td>Retinoic acid</td>
			<td>Sigma-Aldrich</td>
			<td>R2625-100mg</td>
			<td></td>
		</tr>
		<tr>
			<td>Subcellular Protein Fractionation Kit for cultured cells</td>
			<td>Thermo Fisher Scientific</td>
			<td>78840</td>
			<td></td>
		</tr>
		<tr>
			<td>Supported Nitrocellulose membrane</td>
			<td>Biorad</td>
			<td>1620097</td>
			<td></td>
		</tr>
		<tr>
			<td>TC-Plate 6well</td>
			<td>SARSTEDT</td>
			<td>833,920</td>
			<td></td>
		</tr>
		<tr>
			<td>TCS SP2 laser scanning confocal microscope</td>
			<td>Leica</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton x-100</td>
			<td>Sigma-Aldrich</td>
			<td>X100-500ml</td>
			<td>Non-ionic surfactant</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Thermo Fisher Scientific</td>
			<td>15400054</td>
			<td>0.50%</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma-Aldrich</td>
			<td>P9416-100ml</td>
			<td></td>
		</tr>
		<tr>
			<td>VECTASHIELD antifade mounting medium</td>
			<td>Vector Laboratories</td>
			<td>H-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Wizard Plus SV Minipreps DNA Purification Systems</td>
			<td>Promega</td>
			<td>A1330</td>
			<td>Step 3.5</td>
		</tr>
	</tbody>
</table>

modulation of tau subcellular localization as a tool to investigate the expression of disease-related genes

Tau is a microtubule binding protein expressed in neurons and its main known function is related to the maintenance of cytoskeletal stability. However, recent evidence indicated that Tau is present also in other subcellular compartments including the nucleus where it is implicated in DNA protection, in rRNA transcription, in the mobility of retrotransposons and in the structural organization of the nucleolus. We have recently demonstrated that nuclear Tau is involved in the expression of the VGluT1 gene, suggesting a molecular mechanism that could explain the pathological increase of glutamate release in the early stages of Alzheimer&#39;s disease. Until recently, the involvement of nuclear Tau in modulating the expression of target genes has been relatively uncertain and ambiguous due to technical limitations that prevented the exclusion of the contribution of cytoplasmic Tau or the effect of other downstream factors not related to nuclear Tau. To overcome this uncertainty, we developed a method to study the expression of target genes specifically modulated by the nuclear Tau protein. We employed a protocol that couples the use of localization signals and the subcellular fractionation, allowing the exclusion of the interference from the cytoplasmic Tau molecules. Most notably, the protocol is easy and is composed of classic and reliable methods that are broadly applicable to study the nuclear function of Tau in other cell types and cellular conditions.

The strategy used to dissect the impact of nuclear Tau in gene expression avoiding the contribution of cytoplasmic Tau proteins has been depicted in Figure 1. Briefly, Tau proteins tagged with NLS or NES are accumulated in or excluded from the nuclear compartment, respectively. The functional effect of this unbalance is the alteration of the gene expression measured as the product of the VGluT1 gene.
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Watch this Scientific Journal Video about Modulation of Tau Subcellular Localization as a Tool to Investigate the Expression of Disease-related Genes at JoVE.com

Modulation of Tau Subcellular Localization as a Tool to Investigate the Expression of Disease-related Genes

Tau is a neuronal protein present both in the cytoplasm, where it binds microtubules, and in the nucleus, where it exerts unconventional functions including the modulation of Alzheimer&#39;s disease-related genes. Here, we describe a method to investigate the function of nuclear Tau while excluding any interferences coming from cytoplasmic Tau.

Tau is a microtubule binding protein expressed in neurons and its main known function is related to the maintenance of cytoskeletal stability. However, ...

This method can help answer key questions about the direct role of tau in the nuclear compartment while excluding any potential cytoplasmic tau contamination. The main advantage of this technique is that it is broadly applicable to the study on the nuclear function of tau in other cell types and under different cellular conditions. Demonstrating the procedure will be Giacomo Siano, a post-doc from my laboratory.Begin by plating four times 10 to the five SH-SY5Y human neuroblastoma cell line cells to be transfected with the empty control vector, four times 10 to the five cells to be transfected with untagged tau, four times 10 to the five cells to be transfected with tau-NLS, and four times 10 to the five cells to be transfected with tau-NES into individual wells of a six-well plate. The day after plating, separately incubate 400 nanograms of DNA and the appropriate volume of cationic lipids into one tube of 250 microliters of reduced serum per well. After five minutes at room temperature, combine each vector with one volume of cationic lipid and incubate the DNA lipid complexes for 20 minutes at room temperature.At the end of the incubation, replace the supernatants in each well with two milliliters of fresh complete culture medium and transfect each well with the appropriate DNA lipid complex solution for an overnight incubation at 37 degrees Celsius. The next day, replace the supernatants with fresh differentiation medium. For Western Blot analysis, wash the transfected and differentiated cells in each well with PBS and incubate the cells with 500 microliters of 0.1%Trypsin per well for four minutes at 37 degrees Celsius.When the cells have detached, stop the reaction with an equal volume of complete medium and transfer the cell suspension from each well into individual tubes for centrifugation. After carefully removing the supernatants, resuspend the pellets in one milliliter of PBS per tube for a second centrifugation and remove the supernatants again. For total protein extracts, incubate the pellets for 30 minutes on ice in 50 to 100 microliters of lysis buffer supplemented with protease and phosphatase inhibitors.At the end of the incubation, centrifuge the extract at 16, 000 times g for 15 minutes and quantify the protein concentration by any standard quantification assay. Then mix 20 micrograms of each protein sample with five microliters of 4X Laemmli buffer to a total volume of 20 microliters and boil the samples at 100 degrees Celsius for five minutes. For subcellular fractionation, resuspend the cells in complete medium for counting and spin down one times 10 to the six cells per sample.To isolate the cytosolic fraction, incubate the pellets in 100 microliters of ice cold cytoplasmic extraction buffer supplemented with protease inhibitors at four degrees Celsius with gentle mixing for 10 minutes. At the end of the incubation, collect the samples by centrifugation and transfer the supernatants into pre-chilled tubes. Add 100 microliters of ice cold membrane extraction buffer supplemented with protease inhibitors to the pellet for a 10 minute incubation at four degrees Celsius with gentle mixing.Then centrifuge the pellets for five minutes at four degrees Celsius and 3, 000 times g and collect the supernatants into new tubes. To collect the soluble nuclear fraction, add 50 microliters of nuclear extraction buffer supplemented with protease inhibitors to the pellets with vortexing before incubating the samples at four degrees Celsius for 30 minutes. At the end of the incubation, centrifuge the samples and collect the supernatants.To collect the insoluble nuclear fraction, add 50 microliters of nuclear extraction buffer supplemented with protease inhibitors, calcium chloride and micrococcal nuclease to the pellets with vortexing. Incubate the samples for five minutes at 37 degrees Celsius before vortexing and centrifuging. Then collect the supernatants.To collect the cytoskeletal fraction, add 50 microliters of cytoskeletal extraction buffer supplemented with protease inhibitors to the pellets with vortexing. After a 10 minute incubation at room temperature, centrifuge the samples and collect the supernatants. For SDS-PAGE analysis, add seven microliters of 4X Laemmli buffer to 20 microliters of each of the collected subcellular fraction samples and boil the samples at 100 degrees Celsius for five minutes.At the end of the incubation, load the samples onto an acrylamide gel and perform the electrophoresis at a constant voltage of 120 volts. Then transfer the proteins to a nitrocellulose membrane at 250 milliamps for 90 minutes. Incubate the membrane for five minutes in Ponceau staining solution to check for proper protein gel electrophoresis and a successful blotting.At the end of the incubation, rinse the membrane in distilled water until the background is clean and remove the stain with continuous washing with Tris-buffered saline supplemented with Tween 20 for 10 minutes on a shaker. Next, incubate the membrane with blocking solution for one hour at room temperature with shaking before washing three times with Tris-buffered saline plus Tween 20 for five minutes per wash. After the last wash, hybridize the membrane with the primary antibody of interest in blocking solution overnight at four degrees Celsius followed by three washes with Tris-buffered saline plus Tween 20 for five minutes.After the last wash, hybridize the membrane with an appropriate horseradish peroxidase conjugated secondary antibody in blocking solution for one hour at room temperature before washing the membrane with Tris-buffered saline plus Tween 20 three times for five minutes per wash. After the last wash, detect the protein band using chemiluminescence and quantify the intensity of Western Blot bands by ImageJ. In the absence of retinoic acid and brain-derived neurotrophic factor, undifferentiated SH-SY5Y cells assume a rounder morphology and form cell clumps.As expected, after five days of treatment with retinoic acid, the clumps unwind and the cells spread out. After three additional days of treatment with brain-derived neurotrophic factor, the cells appear uniformly distributed and interconnected via a network of branched neurites. After tau-NLS or tau-NES transfection, tau subcellular localization can be detected by immunofluorescence with anti-tau antibodies.Depending on the efficiency of the transfection, the cells display a strong nuclear staining merging with the DAPI signal after tau-NLS transfection or a cytoplasmic staining with the empty nuclei after tau-NES. Western Blot analysis reveals the enrichment of actin within the cytoskeletal fraction, tubulin within the cytoplasmic and cytoskeletal fractions, and H2B within the nuclear fractions. Western Blot analysis also confirms the expression of tagged and untagged tau within the nuclear compartment and vesicular glutamate transporter one expression in the total extract.Indeed, the ratio of tau in the soluble nuclear and cytoplasmic fractions highlights the fact that tau-NLS is highly enriched in the soluble nuclear fraction while tau-NES is decreased. Moreover, tau-NLS is enriched in the chromatin-bound fraction with respect to the cytoplasmic fraction while tau-NES is decreased. Be sure to carefully check the number of cells used for the fractionation of each sample and to verify the enrichment of the housekeeping genes in the proper fractions.After watching this video, you should be able to isolate cellular compartments and to evaluate the functional role of tau in the nucleus.

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