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 cells, the day after the plating, add 10 &#181;M retinoic acid (RA) to complete medium for 5 days.</li>
	<li>The sixth day replace medium with differentiation medium: DMEM/F-12 supplemented with 50 ng/mL BDNF, 2 mM L-glutamine. Do not add FBS or antibiotics.</li>
	<li>Grow the cells in differentiation medium for 3 days.</li>
</ol>
3. Chimeric Constructs Cloning
<ol>
	<li>Generate Tau-NLS construct by cloning by restriction enzyme digestion in frame at the 3&#39; end of Tau sequence 0N4R (383aa) the 3xNLS(SV40NLS):5&#39;-CCAAAAAAGAAGAGAAAGGTAGATCCAAAAAAGAAGAGAAAGGTAGATCCAAAAAAGAAGAGAAAGGTA-3&#39;. 
	NOTE: The 3xNLS at the 3&#39; end of Tau is cloned into the pCMV-Tau plasmid for mammalian expression exploiting the XhoI and BamHI restriction sites into the multicloning site (MCS).</li>
	<li>Generate Tau-NES construct by cloning by restriction enzyme digestion in frame at the 3&#39; end of Tau sequence 0N4R (383aa) the NES sequence: 5&#39;-AGTGAGCTGCAGAACAAGCTGGAAGAGTTGGATCTGGACTCGTACAA-3&#39;. 
	NOTE: The NES at the 3&#39; end of Tau is cloned into the pCMV-Tau plasmid for mammalian expression exploiting the EcoRI and BamHI restriction sites into the MCS.</li>
	<li>Transform DH5alpha E.coli strain with 100 ng of DNA from step 3.1 or 3.2 and plate cells on LB-Agar plates with 100 mg/mL ampicillin. Let grow overnight at 37 &#176;C.</li>
	<li>Pick a single colony and spike the cells into 5 mL of LB with ampicillin. Let the cells grow at 37 &#176;C in agitation overnight.</li>
	<li>Extract plasmid with a DNA miniprep (Table of Materials) and sequence to verify the constructs.</li>
	<li>Transform DH5alpha E. coli strain with the sequence verified constructs and plate cells on LB-Agar plates with ampicillin. Let grow overnight at 37 &#176;C.</li>
	<li>Pick a single colony and spike the cells into 5 mL of LB with ampicillin. Let the cells grow at 37 &#176;C in agitation for 2 h.</li>
	<li>Put the cells from step 3.7 in 200 mL of LB with ampicillin. Let grow overnight at 37 &#176;C.</li>
	<li>Pellet the cells at 3,500 x g for 10 min at 4 &#176;C.</li>
	<li>Extract plasmid with a DNA maxiprep (Table of Materials).</li>
</ol>
4. Cell Transfection
<ol>
	<li>Seed 400,000 cells from step 1.1 in 6-well plates or 20,000 cells in 8-well chamber slides. Plate four samples: control cells to be transfected with an empty vector, cells to be transfected with untagged Tau, cells to be transfected with Tau-NLS and cells to be transfected with Tau-NES.</li>
	<li>The day after seeding transfect 400 ng of DNA for each well using the cationic lipids (Table of Materials) in 6-well plates or 200 ng of DNA for each well for 8-well chamber slides, according to the manufacturer&#39;s instructions.
	<ol>
		<li>Incubate the DNA and the cationic lipids separately in 250 &#181;L (for 6-well plates) or 25 &#181;L (for 8-well chamber slides) of reduced serum medium for 5 min at RT. Then combine them to generate the DNA-lipid complex and incubate for 20 min.</li>
		<li>Replace the culture medium with 2 mL (for 6-well plates) or 250 &#181;L (for 8-well chamber slides) of fresh complete culture medium. Add the DNA-lipid complex to the cells and incubate at 37 &#176;C overnight.</li>
	</ol>
	</li>
	<li>Alternatively, transfect DNA with the cationic reagent polyethylenimine (PEI).
	<ol>
		<li>Mix 2 &#181;g of DNA and 6 &#181;L of PEI with 200 &#181;L of complete culture medium (for each well in 6-well plates), or 1 &#181;g of DNA and 3 &#181;L of PEI with 100 &#181;L of complete culture medium (for each well in 8-well chamber slides), vortex and incubate for 10 min at RT.</li>
		<li>Add the mix to the cells and add 1.8 mL of complete culture medium per well in 6-well plates or 150 &#181;L of complete culture medium per well in 8-well chamber slides to reach plating volume.</li>
	</ol>
	</li>
	<li>Change the medium the day after transfection and add the differentiation media as described in step 2.2.</li>
</ol>
5. Immunofluorescence
<ol>
	<li>Remove the culture medium and rinse cells with 1x PBS. Fix cells with 100% ice cold methanol for 3 min without shaking. Remove the fixing solution and wash briefly with 1x PBS.</li>
	<li>Permeabilize with 0.1% non-ionic surfactant in 1x PBS for 5 min at room temperature (RT). Briefly, wash with 1x PBS, 3 times.</li>
	<li>Incubate cells with blocking buffer (0.1% Tween 20 and 1% BSA in PBS) for 30 min at RT on an orbital shaker.</li>
	<li>Incubate with appropriate primary antibodies (e.g., mouse monoclonal anti-Tau13 antibody) diluted 1:500 in blocking buffer overnight at 4 &#176;C on an orbital shaker. Remove the antibody solution and wash, briefly, with 1x PBS.</li>
	<li>Incubate with secondary antibodies conjugated to fluorophore (e.g., goat anti-mouse antibodies conjugated to Alexa Fluor 633) diluted 1:500 in blocking buffer for 1 h at RT. Remove the antibody solution and wash briefly with 1x PBS 3 times.</li>
	<li>To stain nuclei, incubate with DAPI diluted 1:20,000 in blocking buffer for 10 min at RT. Wash with 1x PBS 3 times. Mount coverslips on a slide using antifade mounting medium.</li>
</ol>
6. Western Blot
<ol>
	<li>To collect the cell pellet from step 4.4, remove the medium, and wash cells with PBS. Incubate with 500 &#181;L of 0.1% trypsin for 4 min at 37 &#176;C. Add an equal volume of complete medium and resuspend cells.</li>
	<li>Collect cells in a tube and centrifuge at 500 x g for 5 min. At the end of centrifugation carefully remove the supernatant. Add 1 mL of PBS, centrifuge at 500 x g for 5 min and carefully remove the supernatant. Store cell pellets on ice for immediate use or freeze at -80 &#176;C for long-term storage.</li>
	<li>For total protein extracts, incubate the cell pellet for 30 min on ice in lysis buffer (20 mM Tris-HCl pH 8, 20 mM NaCl, 10% glycerol, 1% octylphenoxy poly(ethyleneoxy)ethanol, branched (Table of Materials), 10 mM EDTA) supplemented with protease and phosphatase inhibitors. According to the abundance of the pellet, use 50 &#181;L to 100 &#181;L of lysis buffer.
	<ol>
		<li>Centrifuge the extract at 16,000 x g for 15 min. Collect the supernatant and quantify the protein concentration by any standard quantification assay. Prepare the protein samples for the SDS-PAGE by mixing 20 &#181;g of proteins with 5 &#181;L of 4x Laemmli buffer in a total volume of 20 &#181;L and boil at 100 &#176;C for 5 min. 
		NOTE: The sample can be stored at -20 &#176;C.</li>
	</ol>
	</li>
	<li>For subcellular fractionations, resuspend cells from step 6.2 in complete medium, and collect 1 x 106 cells per each sample. Centrifuge at 500 x g for 10 min to obtain cell pellets for the following steps.
	<ol>
		<li>To isolate subcellular compartments, fractionate according to kit specifications. To isolate each fraction, incubate the cell pellet from step 6.4 with the corresponding buffer, centrifuge, collect the supernatant and add the next buffer to the pellet as described in 6.4.1.1-6.4.1.5. Add in order cytoplasmic extraction buffer, membrane extraction buffer, nuclear extraction buffer, nuclear extraction buffer supplemented with 5 mM CaCl2 and 3 U/&#181;L micrococcal nuclease and cytoskeletal extraction buffer. 
		NOTE: All buffers must be supplemented with protease inhibitors. Scale buffer volumes according to the volume of the cell pellet.
		<ol>
			<li>To isolate the cytosolic fraction, incubate cell pellets in 100 &#181;L of ice-cold cytoplasmic extraction buffer supplemented with protease inhibitors at 4 &#176;C with gentle mixing for 10 min. Centrifuge at 500 x g at 4 &#176;C for 5 min and transfer the supernatant to pre-chilled tubes.</li>
			<li>Add 100 &#181;L of ice-cold membrane extraction buffer supplemented with protease inhibitors to the pellet from step 6.4.1.1, and incubate at 4 &#176;C with gentle mixing for 10 min. Centrifuge at 3,000 x g at 4 &#176;C for 5 min and collect the supernatant.</li>
			<li>For the soluble nuclear fraction, add 50 &#181;L of nuclear extraction buffer supplemented with protease inhibitors to the pellet from step 6.4.1.2, and vortex. Incubate at 4 &#176;C for 30 min, centrifuge at 5,000 x g at 4 &#176;C for 5 min, and collect the supernatant.</li>
			<li>For the insoluble nuclear fraction, add 50 &#181;L of nuclear extraction buffer supplemented with protease inhibitors, CaCl2 and micrococcal nuclease to the pellet from step 6.4.1.3, and vortex. Incubate at 37 &#176;C for 5 min, and then vortex again. Centrifuge at 16,000 x g at RT for 5 min and collect the supernatant.</li>
			<li>For the cytoskeletal fraction, add 50 &#181;L of cytoskeletal extraction buffer supplemented with protease inhibitors to the pellet from step 6.4.1.4, and vortex. Incubate 10 min at RT. Centrifuge the tube at 16,000 x g for 5 min, collect the supernatant and discard the pellet. 
			NOTE: Scale buffer volumes according to the cell volume, as indicated in the kit protocol. Refer to the kit protocol for further details on incubation and centrifugation time and temperature22,23,24,25,26,27,28,29. Alternatively, use any standard methods30 that, by using detergents and by increasing ionic strength and centrifugation speed, separates the cytosolic, the membrane-bound, the cytoskeletal and the nuclear fractions. Separate the soluble nuclear fraction and the insoluble nuclear fraction by exploiting standard nuclear extraction buffers. The sample can be stored at -20 &#176;C.</li>
		</ol>
		</li>
		<li>For the SDS-PAGE, add 7 &#181;L of 4x Laemmli buffer to 20 &#181;L of subcellular fractions obtained from steps 6.4.1.1&#8722;6.4.1.5, boil at 100 &#176;C for 5 min.</li>
	</ol>
	</li>
	<li>Load samples on an acrylamide gel and perform electrophoresis at a constant voltage of 120 V. Transfer proteins to nitrocellulose membrane at 250 mA for 90 min.</li>
	<li>Check proper protein gel electrophoresis and successful blotting by incubating the membrane for 5 min in Ponceau staining solution. Rinse the membrane in distilled water until the background is clean. Remove the stain by continued washing with Tris buffered saline with Tween 20 (TBST) for 10 min on a shaker.</li>
	<li>Incubate the membrane with blocking solution (5% milk in TBST) for 1 h at RT on shaker. Wash 3 times with TBST for 5 min.</li>
	<li>Hybridize the membrane with the primary antibody in blocking solution (1% milk in TBST) overnight at 4 &#176;C. Wash 3 times with TBST for 5 min.</li>
	<li>Hybridize the membrane with the HRP-conjugated secondary antibody in blocking solution for 1 h at RT. Wash 3 times with TBST for 5 min.</li>
	<li>Detect the protein band using chemiluminescence. Quantify the intensity of Western Blot bands by ImageJ. Normalize protein expression onto the product of a housekeeping gene: histone H2B for the nuclear soluble and insoluble fraction, GAPDH for the cytoplasmic fraction and for total extracts.</li>
</ol>

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<li>Serdar, B. S., Koçtürk, S., Akan, P., Erkmen, T., Ergür, B. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Which+Medium+and+Ingredients+Provide+Better+Morphological+Differentiation+of+SH-SY5Y+Cells%3F%5BTitle%5D)+AND+%22Proceedings%22%5BJournal%5D)">Which Medium and Ingredients Provide Better Morphological Differentiation of SH-SY5Y Cells?</a> Proceedings. 2 (25), 1577(2018).</li>
<li>Forster, J. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+differentiated+SH-SY5Y+as+neuronal+screening+model+reveals+increased+oxidative+vulnerability%5BTitle%5D)+AND+%22Journal+of+Biomolecular+Screening%22%5BJournal%5D)">Characterization of differentiated SH-SY5Y as neuronal screening model reveals increased oxidative vulnerability.</a> Journal of Biomolecular Screening. 21 (5), 496-509 (2016).</li>
<li>Dwane, S., Durack, E., Kiely, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimising+parameters+for+the+differentiation+of+SH-SY5Y+cells+to+study+cell+adhesion+and+cell+migration%5BTitle%5D)+AND+%22BMC+Research+Notes%22%5BJournal%5D)">Optimising parameters for the differentiation of SH-SY5Y cells to study cell adhesion and cell migration.</a> BMC Research Notes. 6 (1), 1(2013).</li>
<li>Encinas, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sequential+Treatment+of+SH-SY5Y+Cells+with+Retinoic+Acid+and+Brain-Derived+Neurotrophic+Factor+Gives+Rise+to+Fully+Differentiated%2C+Neurotrophic+Factor-Dependent%2C+Human+Neuron-Like+Cells%5BTitle%5D)+AND+%22Journal+of+Neurochemistry%22%5BJournal%5D)">Sequential Treatment of SH-SY5Y Cells with Retinoic Acid and Brain-Derived Neurotrophic Factor Gives Rise to Fully Differentiated, Neurotrophic Factor-Dependent, Human Neuron-Like Cells.</a> Journal of Neurochemistry. 75 (3), 991-1003 (2002).</li>
</ol>

The authors have nothing to disclose.

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><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 1:10,000</td></tr><tr><td>anti GAPDH Antibody</td><td>Fitzgerald Industries International</td><td>10R-G109a</td><td>anti-mouse WB 1:10,000</td></tr><tr><td>anti H2B Antibody</td><td>Abcam</td><td>ab1790</td><td>anti-rabbit WB 1:15,000</td></tr><tr><td>anti Tau-13 Antibody</td><td>Santa Cruz Biotechnology</td><td>sc-21796</td><td>anti-mouse WB 1:1,000; 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 1:5,000</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&#039;s Modified Eagle&#039;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, 0.5 M</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 1:1,000</td></tr><tr><td>Goat anti-rabbit IgG-HPR</td><td>Santa Cruz Biotechnology</td><td>sc-2004</td><td>WB 1:1,000</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>MgCl&lt;sub&gt;2&lt;/sub&gt;</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, 100 mL</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 &lt;em&gt;Plus&lt;/em&gt; 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, ...

modulation-tau-subcellular-localization-as-tool-to-investigate

Research

JoVE Journal

Genetics

6.3K Views.  Scuola Normale Superiore. 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's disease-related genes. Here, we describe a method to investigate the function of nuclear Tau while excluding any interferences coming from cytoplasmic Tau.

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

Nuclear Magnetic Resonance Spectroscopy for the Identification of Multiple Phosphorylations of Intrinsically Disordered Proteins

Aggregates of the neuronal Tau protein are found inside neurons of Alzheimer's disease patients. Development of the disease is accompanied by increased, abnormal phosphorylation of Tau. In the course of the molecular investigation of Tau functions and dysfunctions in the disease, nuclear magnetic resonance (NMR) spectroscopy is used to identify the multiple phosphorylations of Tau. We present here detailed protocols of recombinant production of Tau in bacteria, with isotopic enrichment for NMR studies. Purification steps that take advantage of Tau's heat stability and high isoelectric point are described. The protocol for in vitro phosphorylation of Tau by recombinant activated ERK2 allows for generating multiple phosphorylations. The protein sample is ready for data acquisition at the issue of these steps. The parameter setup to start recording on the spectrometer is considered next. Finally, the strategy to identify phosphorylation sites of modified Tau, based on NMR data, is explained. The benefit of this methodology compared to other techniques used to identify phosphorylation sites, such as immuno-detection or mass spectrometry (MS), is discussed.

We describe here a method to identify multiple phosphorylations of an intrinsically disordered protein by Nuclear Magnetic Resonance Spectroscopy (NMR), using Tau protein as a case study. Recombinant Tau is isotopically enriched and modified in vitro by a kinase prior to data acquisition and analysis.

Aggregates of the neuronal Tau protein are found inside neurons of Alzheimer's disease patients. Development of the disease is accompanied by increased, ...

Biochemistry

Assay for Phosphorylation and Microtubule Binding Along with Localization of Tau Protein in Colorectal Cancer Cells

The microtubule-associated protein tau is a neuronal protein that localizes mostly in axons. Generally tau is essential for normal neuronal functioning because it is involved in microtubule assembly and stabilization. Besides neurons, tau is expressed in human breast, prostate, gastric, colorectal, and pancreatic cancers where it shows nearly similar structure and exerts similar functions as the neuronal tau. The amount of tau and its phosphorylation can change its function as a stabilizer of microtubules, and lead to the development of paired helical filaments in different neurodegenerative disorders, such as Alzheimer&#39;s disease. Determining the phosphorylation state of tau and its microtubule-binding characteristics is important. In addition, examining the intracellular localization of tau is important in different diseases. This manuscript details standard protocols for measuring tau phosphorylation and tau binding to microtubules in colorectal cancer cells with or without curcumin and LiCl treatment. These treatments can be used to stop cancer cell proliferation and development. Intracellular localization of tau is examined by using immunohistochemistry and confocal microscopy while using low amounts of antibodies. These assays can be used repetitively for screening compounds that affect tau hyperphosphorylation or microtubule binding. Novel therapeutics used for different tauopathies or related anticancer agents can potentially be characterized using these protocols.

This manuscript describes standard protocols for measuring tau hyperphosphorylation, measuring tau binding to microtubules, and localization of intracellular tau following drug treatments. These protocols can be used repetitively for screening drugs or other compounds that target tau hyperphosphorylation or microtubule binding.

The microtubule-associated protein tau is a neuronal protein that localizes mostly in axons. Generally tau is essential for normal neuronal functioning ...

Sensitive Detection of Proteopathic Seeding Activity with FRET Flow Cytometry

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 &#945;-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.

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.

Increasing evidence supports transcellular propagation of toxic protein aggregates, or proteopathic seeds, as a mechanism for the initiation and ...

Bioengineering

Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer's Mouse Models

Neurotransmitter disruption is often a key component of diseases of the central nervous system (CNS), playing a role in the pathology underlying Alzheimer's disease, Parkinson's disease, depression, and anxiety. Traditionally, microdialysis has been the most common (lauded) technique to examine neurotransmitter changes that occur in these disorders. But because microdialysis has the ability to measure slow 1-20 minute changes across large areas of tissue, it has the disadvantage of invasiveness, potentially destroying intrinsic connections within the brain and a slow sampling capability. A relatively newer technique, the microelectrode array (MEA), has numerous advantages for measuring specific neurotransmitter changes within discrete brain regions as they occur, making for a spatially and temporally precise approach. In addition, using MEAs is minimally invasive, allowing for measurement of neurotransmitter alterations in vivo. In our laboratory, we have been specifically interested in changes in the neurotransmitter, glutamate, related to Alzheimer's disease pathology. As such, the method described here has been used to assess potential hippocampal disruptions in glutamate in a transgenic mouse model of Alzheimer's disease. Briefly, the method used involves coating a multi-site microelectrode with an enzyme very selective for the neurotransmitter of interest and using self-referencing sites to subtract out background noise and interferents. After plating and calibration, the MEA can be constructed with a micropipette and lowered into the brain region of interest using a stereotaxic device. Here, the method described involves anesthetizing rTg(TauP301L)4510 mice and using a stereotaxic device to precisely target sub-regions (DG, CA1, and CA3) of the hippocampus.

Here, we describe the setup, software navigation, and data analysis for a spatially and temporally precise method of measuring tonic and phasic extracellular glutamate changes in vivo using enzyme-linked microelectrode arrays (MEA).

Neurotransmitter disruption is often a key component of diseases of the central nervous system (CNS), playing a role in the pathology underlying ...

Neuroscience

In Vivo Microdialysis Method to Collect Large Extracellular Proteins from Brain Interstitial Fluid with High-molecular Weight Cut-off Probes

In vivo microdialysis is a powerful technique to collect ISF from awake, freely-behaving animals based on a dialysis principle. While microdialysis is an established method that measures relatively small molecules including amino acids or neurotransmitters, it has been recently used to also assess dynamics of larger molecules in ISF using probes with high molecular weight cut off membranes. Upon using such probes, microdialysis has to be run in a push-pull mode to avoid pressure accumulated inside of the probes. This article provides step-by-step protocols including stereotaxic surgery and how to set up microdialysis lines to collect proteins from ISF. During microdialysis, drugs can be administered either systemically or by direct infusion into ISF. Reverse microdialysis is a technique to directly infuse compounds into ISF. Inclusion of drugs in the microdialysis perfusion buffer allows them to diffuse into ISF through the probes while simultaneously collecting ISF. By measuring tau protein as an example, the author shows how its levels are altered upon stimulating neuronal activity by reverse microdialysis of picrotoxin. Advantages and limitations of microdialysis are described along with the extended application by combining other in vivo methods.

In Vivo Microdialysis Method to Collect Large Extracellular Proteins from Brain Interstitial Fluid with High-molecular Weight Cut-off Probes

In vivo microdialysis has enabled collection of molecules present in brain interstitial fluid (ISF) from awake, freely-behaving animals. In order to analyze relatively large molecules in ISF, the current article specifically focuses on the microdialysis protocol using probes with high molecular weight cut off membranes.

In vivo microdialysis is a powerful technique to collect ISF from awake, freely-behaving animals based on a dialysis principle. While microdialysis is an ...

Cell-based Assay to Study Antibody-mediated Tau Clearance by Microglia

Alzheimer&#39;s disease (AD) is a progressive neurodegenerative condition in which aggregated tau and amyloid proteins accumulate in the brain causing neuronal dysfunction which eventually leads to cognitive decline. Hyperphosphorylated tau aggregates in the neuron are believed to cause most of the pathology associated with AD. These aggregates are assumed to be released into the extracellular compartment and taken up by adjacent healthy neurons where they induce further tau aggregation. This &#34;prion-like&#34; spreading can be interrupted by antibodies capable of binding and &#34;neutralizing&#34; extracellular tau aggregates as shown in preclinical mouse models of AD. One of the proposed mechanisms by which therapeutic antibodies reduce pathology is antibody-mediated uptake and clearance of pathological aggregated forms of tau by microglia. Here, we describe a quantitative cell-based assay to assess tau uptake by microglia. This assay uses the mouse microglial cell line BV-2, allows for high specificity, low variability and medium throughput. Data generated with this assay can contribute to a better characterization of anti-tau antibody effector functions.

Here we describe a cell-based assay to quantitatively assess tau uptake by microglia with the aim of creating an investigational tool to better characterize the mechanisms of action of anti-tau antibodies.

Alzheimer&#39;s disease (AD) is a progressive neurodegenerative condition in which aggregated tau and amyloid proteins accumulate in the brain causing ...

An In Vitro Model for Studying Tau Aggregation Using Lentiviral-mediated Transduction of Human Neurons

Aberrant aggregation of the protein tau is pathogenically involved in a number of neurodegenerative diseases, including Alzheimer&#8217;s disease (AD). Although mouse models of tauopathy have provided a valuable resource for investigating the neurotoxic mechanisms of aggregated tau, it is becoming increasingly apparent that, due to interspecies differences in neurophysiology, the mouse brain is unsuitable for modeling the human condition. Advances in cell culture methods have made human neuronal cultures accessible for experimental use in vitro and have aided in the development of neurotherapeutics. However, despite the adaptation of human neuronal cell cultures, in vitro models of human tauopathy are not yet widely available. This protocol describes a cellular model of tau aggregation in which human neurons are transduced with lentiviral-derived vectors that code for pathogenically mutated tau fused to a yellow fluorescent protein (YFP) reporter. Transduced cultures produce tau aggregates that stain positively for thioflavin and display markers of neurotoxicity, such as decreased axonal length and increased lysosomal volume. This procedure may be a useful and cost-effective model for studying human tauopathies.

This protocol details a procedure in which human neuronal cultures are transduced with lentiviral constructs coding for mutant human tau. Transduced cultures display tau aggregates and associated pathologies.

Aberrant aggregation of the protein tau is pathogenically involved in a number of neurodegenerative diseases, including Alzheimer&#8217;s disease (AD). ...

In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

Alzheimer's disease is one of a large group of neurodegenerative disorders known as tauopathies that are manifested by the neuronal deposits of hyperphosphorylated tau protein in the form of neurofibrillary tangles (NFTs). The density of NFT correlates well with cognitive impairment and other neurodegenerative symptoms, thus prompting the endeavor of developing tau aggregation-based therapeutics. Thus far, however, tau aggregation assays use recombinant or synthetic tau that is devoid of the pathology-related phosphorylation marks. Here we describe two assays using recombinant, hyperphosphorylated tau as the subject. These assays can be scaled up for high-throughput screens for compounds that can modulate the kinetics or stability of hyperphosphorylated tau aggregates. Novel therapeutics for Alzheimer's disease and other tauopathies can potentially be discovered using hyperphosphorylated tau isoforms.

In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

Unmodified and hyperphosphorylated tau proteins were used in two in vitro aggregation assays to reveal the hyperphosphorylation-dependent fast aggregation kinetics. These assays pave the way for future screens for compounds that can modulate the propensity of hyperphosphorylated tau to form fibrils that underlie the progression of Alzheimer&#8217;s disease.

Alzheimer's disease is one of a large group of neurodegenerative disorders known as tauopathies that are manifested by the neuronal deposits of ...

Biology

Visualizing Proteins in Primary Hippocampal Neurons by Live&#45;Cell Imaging

Using Live&#45;Cell Imaging to Measure the Effects of Pathological Proteins on Axonal Transport in Primary Hippocampal Neurons

Bidirectional transport of cargos along the axon is critical for maintaining functional synapses, neural connectivity, and healthy neurons. Axonal transport is disrupted in multiple neurodegenerative diseases, and projection neurons are particularly vulnerable because of the need to transport cellular materials over long distances and sustain substantial axonal mass. Pathological modifications of several disease-related proteins negatively affect transport, including tau, amyloid-&#946;, &#945;-synuclein, superoxide dismutase, and huntingtin, providing a potential common mechanism by which pathological proteins exert toxicity in disease. Methods to study these toxic mechanisms are necessary to understand neurodegenerative disorders and identify potential therapeutic interventions.
Here, cultured primary rodent hippocampal neurons are co-transfected with multiple plasmids to study the effects of pathological proteins on fast axonal transport using live-cell confocal imaging of fluorescently-tagged cargo proteins. We begin with the harvest, dissociation, and culturing of primary hippocampal neurons from rodents. Then, we co-transfect the neurons with plasmid DNA constructs to express fluorescent-tagged cargo protein and wild-type or mutant tau (used as an exemplar of pathological proteins). Axons are identified in live cells using an antibody that binds an extracellular&#160;domain of neurofascin, an axon initial segment protein, and an axonal region of interest is imaged to measure fluorescent cargo transport.
Using KymoAnalyzer, a freely available ImageJ macro, we extensively characterize the velocity, pause frequency, and directional cargo density of axonal transport, all of which may be affected by the presence of pathological proteins. Through this method, we identify a phenotype of increased cargo pause frequency associated with the expression of pathological tau protein. Additionally, gene-silencing shRNA constructs can be added to the transfection mix to test the role of other proteins in mediating transport disruption. This protocol is easily adaptable for use with other neurodegenerative disease-related proteins and is a reproducible method to study the mechanisms of how those proteins disrupt axonal transport.

Author Spotlight: Understanding the Impact of Pathological Proteins on Axonal Transport in Neurodegenerative Diseases

Here, we demonstrate how to combine transfection of primary hippocampal rodent neurons with live-cell confocal imaging to analyze pathological protein-induced effects on axonal transport and identify mechanistic pathways mediating these effects.

Bidirectional transport of cargos along the axon is critical for maintaining functional synapses, neural connectivity, and healthy neurons. Axonal ...

A Cell-Based Assay to Assess the Tau Protein Uptake by Microglia

Source: De Marco, D. et al., <a href="https://app.jove.com/v/58576">Cell-based Assay to Study Antibody-mediated Tau Clearance by Microglia.</a>&#160;J. Vis. Exp. (2018)
This video showcases a cell-based assay quantifying Tau uptake by murine microglia using pH-sensitive fluorescent-labeled Tau aggregates, analyzed via fluorescence-activated cell sorting for a quantitative assessment.

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

Summary

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