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

Summary

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.

Abstract

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'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.

Introduction

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 acids^1,2,3,4,5,6. Indeed, a recent genome-wide study demonstrated that Tau binds genic and intergenic DNA sequences in vivo⁷. A role in nucleolar organization has been suggested^8,9,10,11. Moreover, Tau has been proposed to be involved in DNA protection from oxidative and hyperthermic stress^5,10,12,13, whereas mutated Tau has been linked to chromosome instability and aneuploidy^14,15,16.

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 protection^{11,12,17,18,19} 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 expression^20,21. 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.

Protocol

1. Cell Culture

1. Culture SH-SY5Y cells (human neuroblastoma cell line, CRL-2266) in complete medium (Dulbecco'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 µg/mL streptomycin). Maintain the cells in an incubator at 37 °C and 5% CO₂. Grow cells in 10 cm plates and split when confluent.

2. Cell Differentiation

1. To differentiate SH-SY5Y cells, the day after the plating, add 10 µM retinoic acid (RA) to complete medium for 5 days.
2. 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.
3. Grow the cells in differentiation medium for 3 days.

3. Chimeric Constructs Cloning

1. Generate Tau-NLS construct by cloning by restriction enzyme digestion in frame at the 3' end of Tau sequence 0N4R (383aa) the 3xNLS(SV40NLS):5'-CCAAAAAAGAAGAGAAAGGTAGATCCAAAAAAGAAGAGAAAGGTAGATCCAAAAAAGAAGAGAAAGGTA-3'.
   NOTE: The 3xNLS at the 3' 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).
2. Generate Tau-NES construct by cloning by restriction enzyme digestion in frame at the 3' end of Tau sequence 0N4R (383aa) the NES sequence: 5'-AGTGAGCTGCAGAACAAGCTGGAAGAGTTGGATCTGGACTCGTACAA-3'.
   NOTE: The NES at the 3' end of Tau is cloned into the pCMV-Tau plasmid for mammalian expression exploiting the EcoRI and BamHI restriction sites into the MCS.
3. 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 °C.
4. Pick a single colony and spike the cells into 5 mL of LB with ampicillin. Let the cells grow at 37 °C in agitation overnight.
5. Extract plasmid with a DNA miniprep (Table of Materials) and sequence to verify the constructs.
6. Transform DH5alpha E. coli strain with the sequence verified constructs and plate cells on LB-Agar plates with ampicillin. Let grow overnight at 37 °C.
7. Pick a single colony and spike the cells into 5 mL of LB with ampicillin. Let the cells grow at 37 °C in agitation for 2 h.
8. Put the cells from step 3.7 in 200 mL of LB with ampicillin. Let grow overnight at 37 °C.
9. Pellet the cells at 3,500 x g for 10 min at 4 °C.
10. Extract plasmid with a DNA maxiprep (Table of Materials).

4. Cell Transfection

1. 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.
2. 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's instructions.
   1. Incubate the DNA and the cationic lipids separately in 250 µL (for 6-well plates) or 25 µL (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.
   2. Replace the culture medium with 2 mL (for 6-well plates) or 250 µL (for 8-well chamber slides) of fresh complete culture medium. Add the DNA-lipid complex to the cells and incubate at 37 °C overnight.
3. Alternatively, transfect DNA with the cationic reagent polyethylenimine (PEI).
   1. Mix 2 µg of DNA and 6 µL of PEI with 200 µL of complete culture medium (for each well in 6-well plates), or 1 µg of DNA and 3 µL of PEI with 100 µL of complete culture medium (for each well in 8-well chamber slides), vortex and incubate for 10 min at RT.
   2. Add the mix to the cells and add 1.8 mL of complete culture medium per well in 6-well plates or 150 µL of complete culture medium per well in 8-well chamber slides to reach plating volume.
4. Change the medium the day after transfection and add the differentiation media as described in step 2.2.

5. Immunofluorescence

1. 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.
2. Permeabilize with 0.1% non-ionic surfactant in 1x PBS for 5 min at room temperature (RT). Briefly, wash with 1x PBS, 3 times.
3. Incubate cells with blocking buffer (0.1% Tween 20 and 1% BSA in PBS) for 30 min at RT on an orbital shaker.
4. Incubate with appropriate primary antibodies (e.g., mouse monoclonal anti-Tau13 antibody) diluted 1:500 in blocking buffer overnight at 4 °C on an orbital shaker. Remove the antibody solution and wash, briefly, with 1x PBS.
5. 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.
6. 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.

6. Western Blot

1. To collect the cell pellet from step 4.4, remove the medium, and wash cells with PBS. Incubate with 500 µL of 0.1% trypsin for 4 min at 37 °C. Add an equal volume of complete medium and resuspend cells.
2. 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 °C for long-term storage.
3. 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 µL to 100 µL of lysis buffer.
   1. 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 µg of proteins with 5 µL of 4x Laemmli buffer in a total volume of 20 µL and boil at 100 °C for 5 min.
      NOTE: The sample can be stored at -20 °C.
4. 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.
   1. 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/µL 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.
      1. To isolate the cytosolic fraction, incubate cell pellets in 100 µL of ice-cold cytoplasmic extraction buffer supplemented with protease inhibitors at 4 °C with gentle mixing for 10 min. Centrifuge at 500 x g at 4 °C for 5 min and transfer the supernatant to pre-chilled tubes.
      2. Add 100 µL of ice-cold membrane extraction buffer supplemented with protease inhibitors to the pellet from step 6.4.1.1, and incubate at 4 °C with gentle mixing for 10 min. Centrifuge at 3,000 x g at 4 °C for 5 min and collect the supernatant.
      3. For the soluble nuclear fraction, add 50 µL of nuclear extraction buffer supplemented with protease inhibitors to the pellet from step 6.4.1.2, and vortex. Incubate at 4 °C for 30 min, centrifuge at 5,000 x g at 4 °C for 5 min, and collect the supernatant.
      4. For the insoluble nuclear fraction, add 50 µL 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 °C for 5 min, and then vortex again. Centrifuge at 16,000 x g at RT for 5 min and collect the supernatant.
      5. For the cytoskeletal fraction, add 50 µL 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 temperature^{22,23,24,25,26,27,28,29}. 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 °C.
   2. For the SDS-PAGE, add 7 µL of 4x Laemmli buffer to 20 µL of subcellular fractions obtained from steps 6.4.1.1−6.4.1.5, boil at 100 °C for 5 min.
5. 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.
6. 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.
7. 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.
8. Hybridize the membrane with the primary antibody in blocking solution (1% milk in TBST) overnight at 4 °C. Wash 3 times with TBST for 5 min.
9. 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.
10. 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.

Results

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.

Discussion

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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Alexa Fluor 633 goat anti-mouse IgG	Life Technologies	A21050	IF 1:500
anti Actin Antibody	BETHYL LABORATORIE	A300-485A	anti-rabbit WB 1:10,000
anti GAPDH Antibody	Fitzgerald Industries International	10R-G109a	anti-mouse WB 1:10,000
anti H2B Antibody	Abcam	ab1790	anti-rabbit WB 1:15,000
anti Tau-13 Antibody	Santa Cruz Biotechnology	sc-21796	anti-mouse WB 1:1,000; IF 1:500
anti Tubulin alpha Antibody	Thermo Fisher Scientific	PA5-16891	anti-mouse WB 1:5,000
anti VGluT1 Antibody	Sigma-Aldrich	AMAb91041	anti-mouse WB 1:500
BCA Protein Assay Kit	Euroclone	EMPO14500
BDNF	Alomone Labs	B-250
Blotting-Grade Blocker	Biorad	1706404	Non-fat dry milk
BOVIN SERUM ALBUMIN	Sigma-Aldrich	A4503-50g
cOmplete Mini	Roche	11836170001	protease inhibitor
Criterion TGX 4-20% Stain Free, 10 well	Biorad	5678093
DAPI	Thermo Fisher Scientific	62247
DMEM/F-12	GIBCO	21331-020
Dulbecco's Modified Eagle's Medium Low Glucose	Euroclone	ECM0060L
EDTA	Sigma-Aldrich	0390-100ml	pH = 8, 0.5 M
Foetal Bovine Serum	Euroclone	EC50182L
Glycerol	Sigma-Aldrich	G5516-500ml
Goat anti-mouse IgG-HPR	Santa Cruz Biotechnology	sc-2005	WB 1:1,000
Goat anti-rabbit IgG-HPR	Santa Cruz Biotechnology	sc-2004	WB 1:1,000
IGEPAL CA-630	Sigma-Aldrich	I8896-50ml	Octylphenoxy poly(ethyleneoxy)ethanol
Immobilon Western	MERCK	WBKLS0500
Lab-Tech Chamber slide 8 well glass slide	nunc	177402
L-glutamine	Euroclone	ECB3000D	100X
Lipofectamine 2000 transfection reagent	Thermo Fisher Scientific	12566014	cationic lipid
Methanol	Sigma-Aldrich	322415-6X1L
MgCl2	Sigma-Aldrich	M8266-100G
NaCl	Sigma-Aldrich	S3014-1kg
Opti-MEM reduced serum medium	Gibco	31985070
PEI	Sigma-Aldrich	40,872-7
Penicillin/Streptomycin	Thermo Fisher Scientific	15140122	10,000 U/mL, 100 mL
Phosphate Buffered Saline (Dulbecco A)	OXOID	BR0014G
PhosStop	Roche	4906837001	phosphatase inhibitor
QIAGEN Plasmid Maxi Kit	Qiagen	12163	Step 3.10
Retinoic acid	Sigma-Aldrich	R2625-100mg
Subcellular Protein Fractionation Kit for cultured cells	Thermo Fisher Scientific	78840
Supported Nitrocellulose membrane	Biorad	1620097
TC-Plate 6well	SARSTEDT	833,920
TCS SP2 laser scanning confocal microscope	Leica	N/A
Triton x-100	Sigma-Aldrich	X100-500ml	Non-ionic surfactant
Trypsin-EDTA	Thermo Fisher Scientific	15400054	0.50%
Tween-20	Sigma-Aldrich	P9416-100ml
VECTASHIELD antifade mounting medium	Vector Laboratories	H-1000
Wizard Plus SV Minipreps DNA Purification Systems	Promega	A1330	Step 3.5