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Methods to Monitor and Manipulate TFEB Activity During Autophagy
CHAPTER FIVE
Methods to Monitor and Manipulate TFEB Activity During Autophagy D.L. Medina*, C. Settembre*,†,{, A. Ballabio*,{,§,¶,1 *Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Naples, Italy † Dulbecco Telethon Institute (DTI), Naples, Italy { Medical Genetics, Federico II University, Naples, Italy § Baylor College of Medicine, Houston, TX, United States ¶ Jan and Dan Duncan Neurological Research Institute, Texas Children’s Hospital, Houston, TX, United States 1 Corresponding author: e-mail address: [email protected]
Contents 1. Introduction 2. Methods to Monitor TFEB/TFE3 Activation 2.1 Analysis of TFEB Subcellular Localization by Immunoblot 2.2 Detection of TFEB Ser142 Phosphorylation by Immunoblot 2.3 Detection of P-Ser211 TFEB by Using 14-3-3 Motif 2.4 Analysis of TFEB Subcellular Localization by Immunofluorescence 2.5 Quantitative TFEB Subcellular Localization by High-Content Imaging Analysis 2.6 Measure of Autophagy Gene Expression Levels 2.7 Measurement of TFEB Binding to the Promoter of Autophagy Genes 2.8 In Vivo Autophagy Modulation via TFEB 2.9 TFEB Overexpression in Liver 2.10 TFEB Overexpression in Muscle (Gastrocnemius) 2.11 TFEB Overexpression in Brain 2.12 Isolation of Tissue Specimens From Tissues Overexpressing TFEB 2.13 Analysis of TFEB mRNA Expression Levels in Transduced Tissues 2.14 Analysis of TFEB Protein Expression Levels in Transduced Tissues 2.15 Analysis of TFEB Protein Expression and Localization in Transduced Tissues by Immunofluorescence Acknowledgments References
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Abstract Macroautophagy is a catabolic process deputed to the turnover of intracellular components. Recent studies have revealed that transcriptional regulation is a major mechanism controlling autophagy. Currently, more than 20 transcription factors have been
Methods in Enzymology, Volume 588 ISSN 0076-6879 http://dx.doi.org/10.1016/bs.mie.2016.10.008
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shown to modulate cellular autophagy levels. Among them, the transcription factor EB (TFEB) appears to have the broadest proautophagy role, given its capacity to control the biogenesis of lysosomes and autophagosomes, the two main organelles required for the autophagy pathway. TFEB has attracted major attention owing to its ability to enhance cellular clearance of pathogenic substrates in a variety of animal models of disease, such as lysosomal storage disorders, Parkinson’s, Alzheimer’s, α1-antitrypsin, obesity as well as others, suggesting that the TFEB pathway represents an extraordinary possibility for future development of innovative therapies. Importantly, the subcellular localization and activity of TFEB are regulated by its phosphorylation status, suggesting that TFEB activity can be pharmacologically targeted. Given the growing list of common and rare diseases in which manipulation of autophagy may be beneficial, in this chapter we describe a set of validated protocols developed to modulate and analyze TFEB-mediated enhancement of autophagy both in vitro and in vivo conditions.
1. INTRODUCTION Autophagy is an evolutionary conserved catabolic process that provides energy and recycles cellular components through the targeting of intracytoplasmic cargo to lysosomes. Autophagy plays a crucial role in the cellular adaptation to environmental cues such as energy demanding conditions (i.e., nutrient deprivation). Autophagy relies on the biogenesis and cooperation of two organelles, the autophagosome and the lysosome. Recent studies have shown that the transcription factor EB (TFEB) plays an important role in autophagy. TFEB belongs to the microphthalmia family of basic helix-loop-helix–leucine-zipper (bHLH-Zip) transcription factors (MiT family) (Steingrı´msson, Copeland, & Jenkins, 2004). TFEB positively regulates lysosome biogenesis and function by enhancing the expression of lysosomal hydrolases and of lysosomal membrane proteins, such as the components of the v-ATPase proton pump complex (Medina et al., 2011; Palmieri et al., 2011; Sardiello et al., 2009). TFEB also promotes autophagosome biogenesis and autophagosome fusion with the lysosome by inducing the expression of autophagy-related genes (Settembre et al., 2011). TFEB activity is primarily regulated by phosphorylation events, mainly occurring at serine residues. Notably, the mTORC1 kinase, a main negative regulator of autophagy, is a key modulator of TFEB function (Martina, Chen, Gucek, & Puertollano, 2012; Roczniak-Ferguson et al., 2012; Settembre et al., 2012).
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In the presence of nutrients, mTORC1 phosphorylates TFEB at multiple serine residues on the lysosome surface and promotes the binding of TFEB to 14-3-3 proteins preventing its nuclear translocation and activation (Martina et al., 2012; Roczniak-Ferguson et al., 2012; Settembre et al., 2012). During starvation mTORC1 dissociates from the lysosomal surface and its activity is inhibited. In parallel, upon starvation the activity of the phosphatase calcineurin is induced by lysosomal calcium release through the activation of the lysosomal cation channel MCOLN1 (Medina et al., 2015). Together, these events lead to TFEB dephosphorylation, nuclear translocation, and activation (Fig. 1). In the nucleus TFEB interacts with the transcriptional coactivator-associated arginine methyltransferase 1 (CARM1), and binds to the CLEAR sites in the promoters of many lysosomal and autophagy genes promoting their expression during starvation (Settembre et al., 2011; Shin et al., 2016). Not surprisingly, the TFEB paralogue transcription factor E3 (TFE3) has also been shown to regulate lysosomal homeostasis by inducing the expression of genes encoding proteins involved in autophagy and lysosomal biogenesis in response to starvation and lysosomal stress. TFE3 regulation seems to share the same regulatory mechanisms as TFEB and therefore TFE3 is also regulated by mTORC1-dependent phosphorylation on the lysosomal surface as well as by the phosphatase calcineurin (Martina, Diab, Brady, & Puertollano, 2016; Martina, Diab, Li, & Puertollano, 2014; Martina, Diab, Lishu, et al., 2014). The identification of a global transcriptional regulation of lysosomal function and autophagy has been exploited to boost cellular clearance in mouse models of a variety of disease conditions. TFEB overexpression results in the clearance of accumulating substrates in cells and tissues from mouse models of several types of LSDs, as well as of Parkinson’s, mTORC1 (nutrient-rich conditions) Cytoplasmic retention with 14-3-3 protein
P-TFEB
TFEB
Nuclear localization and activation of lysosomal and autophagic genes
CaN (starvation, exercise)
Fig. 1 TFEB regulation. TFEB is negatively regulated by nutrients through mTORC1mediated phosphorylation and cytoplasmic retention by binding to 14-3-3 proteins. Conditions such as nutrient deprivation or exercise promote TFEB dephosphorylation by calcineurin (CaN). Dephosphorylated TFEB translocates into the nucleus activating lysosomal and autophagic genes.
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Alzheimer’s, Huntington’s disease, α1-anti-trypsin deficiency, and spinal bulbar muscular atrophy (Cortes, 2014; Decressac et al., 2013; Medina et al., 2011; Pastore et al., 2013; Polito et al., 2014; Song et al., 2016; Spampanato et al., 2013; Tsumeni et al., 2012; Xiao et al., 2015). In the same way, overexpression of TFE3 promotes clearance in a cellular model of a lysosomal storage disorder, Pompe disease (Martina, Diab, Li, et al., 2014; Martina, Diab, Lishu, et al., 2014). These findings have opened a novel therapeutic strategy based on the modulation of cellular clearance, through the transcriptional activation of the lysosomal/autophagic pathway, with potential applicability to many diseases. The mechanism by which TFEB promotes the clearance of stored materials needs to be further characterized. However, it is possible that TFEB-mediated cellular clearance results from a combination of the effects on lysosomal biogenesis, autophagy, and lysosomal exocytosis (Medina et al., 2011; Spampanato et al., 2013). Therefore, drug-screening approaches aimed at identifying molecules that promote TFEB nuclear translocation present an interesting path forward to treat several diseases (Moskot et al., 2014; Song et al., 2016) (Fig. 2). The important implications resulting from the study of
TFEB
Autophagy
Lysosomal biogenesis
Lysosomal exocytosis
Cellular clearance Lysosomal storage diseases
Common neurodegenerative diseases
Fig. 2 Model of TFEB-mediated cellular clearance. TFEB overexpression modulates autophagy, lysosomal biogenesis, and induces lysosomal exocytosis. The activation of these biological pathways may contribute to the clearance of pathological accumulation in various disease conditions such as lysosomal storage disorders and common neurodegenerative disease.
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TFEB-mediated function prompted us to present a comprehensive set of the major experimental procedures used to enhance TFEB/TFE3 signaling and examine the transcriptional induction of the autophagy pathway.
2. METHODS TO MONITOR TFEB/TFE3 ACTIVATION
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Under basal conditions TFEB is mainly found in the cytoplasm in an inactive, phosphorylated state (Settembre et al., 2011). However, under specific conditions, such as nutrient deprivation, exercise, or lysosomal stress, TFEB is rapidly dephosphorylated and moves to the nucleus (Fig. 3). Although phosphoproteomic studies identified at least 10 different phosphorylation sites on the TFEB protein, mutation analysis indicated that two residues (Ser211 and Ser142) are particularly critical for TFEB subcellular localization (Martina et al., 2012; Roczniak-Ferguson et al., 2012; Settembre et al., 2012). In vitro studies suggest that Ser142 may either be phosphorylated by ERK2 or mTORC1, while only the latter phosphorylates Ser211. TFEB phosphorylation favors the interaction with 14-3-3 proteins and therefore its cytoplasmic retention (Roczniak-Ferguson et al., 2012). Interestingly, TFEB phosphorylation by the mTORC1 complex occurs on the lysosomal surface. Hence, TFEB subcellular localization may be used as a marker for TFEB activation. The key procedural steps to measure endogenous or overexpressed TFEB localization by western blot
Cytosolic Fractions Nuclear
Total lysate
Fig. 3 Detection of TFEB phosphorylation and subcellular localization in HeLa cells by immunoblotting. (A) Total cellular lysate isolated from cells nutrient deprived (HBSS starvation) for the indicated time points. (B) TFEB subcellular localization. Cells were cultured in complete media (nutrients), in HBSS buffer for 1 h (starvation), or in complete media supplemented with torin 1 inhibitor. Endogenous TFEB was detected using Cell Signaling antibody (Cat n. 4240).
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and immunofluorescence are summarized below. In addition, we have included a procedure to quantify TFEB subcellular localization by using high-content imaging analysis using a cell line stably overexpressing TFEBGFP fusion protein (Medina et al., 2015).
2.1 Analysis of TFEB Subcellular Localization by Immunoblot – TFEB antibodies: To detect endogenous TFEB in human cell extracts, the anti-TFEB antibody from Cell Signaling should be used (#4240). To detect endogenous TFEB in mouse cell extracts, the anti-TFEB antibody from Bethyl Laboratories (A311-347A) should be used. – Prepare the Triton X-100 lysis buffer: 50 mM Tris–HCl (pH 7.5), 0.5% Triton X-100, 137.5 mM NaCl, 10% glycerol, 5 mM ethylenediaminetetraacetic acid (EDTA), PhosSTOP, and EDTA-free protease inhibitor tablets (Roche, Indianapolis, IN, USA). Keep on ice. – Cell lysates: After three washes with cold phosphate-buffered saline (PBS), lyse 6 million cells with 0.5 mL of 0.5% Triton X-100 lysis buffer for 15 min on ice, shaking gently. – Collect and centrifuge the cell lysate at 2000 rpm for 15 min at 4°C with a tabletop centrifuge and transfer 250 μL of the supernatant, which is composed of the cytosolic and membrane fractions, to a new Eppendorf tube. Discard the rest of the supernatant. Keep the pellet. – Rinse the nuclear pellet three times with 0.5 mL of lysis buffer, and resuspend the pellet in 0.1 mL of lysis buffer, supplemented with 0.5% sodium dodecyl sulfate (SDS). Sonicate in cold room three times for 3 s at low output to shear genomic DNA. – Preclear by centrifugation at 13,000 rpm for 15 min at 4°C with a tabletop centrifuge. Transfer the supernatant to a new tube. – Determine the protein concentration in supernatant extracts using the colorimetric bicinchoninic acid protein assay kit (Pierce Chemical Co., Boston, MA, USA), following the manufacturer’s instructions. – Add an equal amount of (2) SDS-PAGE sample buffer to each of the tubes containing the nuclear and cytoplasmic fractions and boil the samples for 10 min. – Perform western blot, using 10% SDS-PAGE gels. Transfer protein onto PVDF (polyvinyl difluoride) membrane. Block the PVDF membrane with 5% nonfat milk in TBS-T buffer (TBS containing 0.05% Tween20) for 1 h at room temperature (RT) under gentle shaking. Incubate with primary antibodies, anti-TFEB (1:1000 in 5% nonfat milk in
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TBS-T buffer) overnight (ON) at 4°C, anti-TUBULIN (Sigma; 1:2000), and anti-H3 (Cell Signaling; 1:10,000) at RT for 2 h. Detect signals with horseradish peroxidase (HRP)-conjugated goat anti-mouse or anti-rabbit IgG antibody (1:2000, Vector Laboratories) and visualize with the Super Signal West Dura substrate (Thermo Scientific, Rockford, IL), according to the manufacturer’s protocol. – The purity of the fractions is confirmed by the lack of beta-tubulin in the nuclear fraction and of histone H3 in the cytosolic fraction, as detected by western blot.
2.2 Detection of TFEB Ser142 Phosphorylation by Immunoblot Phosphorylation of TFEB at Ser142 is a key step in TFEB nuclear translocation during starvation. A custom phosphospecific-antibody, recognizing phosphorylated TFEB at the Ser142 residue, was generated by the GenScript Company. Recently, a similar antibody from EMD-MILLIPORE has become commercially available (ABE1971-Anti-phospho TFEB-Ser142 antibody). The TFEB P-Ser142 antibody is suitable for western blot; however, given the high homology between the amino acid sequences surrounding Ser142 and the other members of the MiT subfamily, it is possible that cross-reaction events may occur. This issue can be avoided by analyzing samples obtained from cells overexpressing TFEB. – Follow standard western blot procedures and incubate primary antibody diluted 1:1000 in 5% milk ON at 4°C. Avoid many cycles freeze and thaw.
2.3 Detection of P-Ser211 TFEB by Using 14-3-3 Motif The phosphorylation of the Ser211 site on TFEB protein favors the binding to the cytosolic chaperone 14-3-3, which keeps TFEB sequestered in the cytosol (Martina et al., 2012; Roczniak-Ferguson et al., 2012). Hence, the phosphorylation of TFEB on Ser211 can be determined by exploiting the selective interaction between Ser211-phosphorylated TFEB and 14-3-3 proteins. The following antibodies are required: anti-FLAG (1:1000) and anti-actin (1:4000) from Sigma-Aldrich; P-Ser-14-3-3 binding motif antibodies (1:1000) from Cell Signaling Technologies; anti-pan 14-3-3 antibodies (1:1000) from Santa Cruz Biotechnology. – Rinse TFEB-3XFLAG-expressing cells twice with ice-cold PBS and lyse in ice-cold lysis buffer (400 mM NaCl, 25 mM Tris–HCl, pH 7.4, 1 mM
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EDTA, and 1% Triton X-100) containing protease and phosphatase inhibitors. Isolate soluble fractions from cell lysates by centrifugation at 11,200 rpm for 15 min in a Microfuge. Incubate with anti-3XFLAG antibodies (1:1000, SIGMA) in binding buffer (200 mM NaCl, 25 mM Tris–HCl, pH 7.4, 1 mM EDTA) with constant rotation ON at 4°C. Add 40 μL of 50% slurry of Protein-G beads (Sigma-Aldrich) to the lysates and incubate with rotation for an additional 2 h at 4°C. Wash the resins five times with binding buffer and elute the samples in Laemmli buffer. After 7.5% acrylamide SDS-PAGE gel separation and immunoblotting, incubate with the appropriated antibodies. Use standard chemiluminescence methods (ECL Western Blotting Substrate, Pierce) and peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies (Calbiochem) to visualize proteins. Develop the membranes using a Chemidoc UVP imaging system (UltraViolet Products Ltd.). Perform densitometric quantification of protein bands images using ImageJ software (NIH).
2.4 Analysis of TFEB Subcellular Localization by Immunofluorescence TFEB antibodies: To detect endogenous TFEB in human cells, use the antiTFEB antibody from Cell Signaling (Cat n. 4240), following manufacturer’s instructions. To detect endogenous TFEB in mouse cells, use anti-TFEB antibody from MyBiosource (MBS120432) at 1:50 dilution. – Plate 300,000 cells on coated glass coverslips in 35-mm tissue culture dishes. – Rinse cells with PBS once and fix them for 15 min with 4% paraformaldehyde in PBS at RT. – Rinse the slides twice with PBS and permeabilize cells with 0.05% Triton X-100 in PBS for 5 min. – Rinse twice with PBS and incubate with primary antibody in 5% normal donkey serum ON at 4°C. – Rinse four times with PBS and incubate with secondary donkey antibodies (diluted 1:1000 in 5% normal donkey serum) for 45 min at RT in the dark.
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Fed
Starved
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Fig. 4 Detection of TFEB subcellular localization in HeLa cells by immunofluorescence. Cells were cultured in complete media (Fed) or starved (HBSS buffer) for 1 h. Endogenous TFEB was detected using specific Cell Signaling antibodies (Cat n. 4240).
– Wash four times with PBS. Slides should be mounted onto glass coverslips, using Vectashield (Vector Laboratories) and imaged with a fluorescent microscope (Fig. 4).
2.5 Quantitative TFEB Subcellular Localization by High-Content Imaging Analysis A setup of the immunofluorescence assay is needed to microscale the experiment to 96- or 384-well plates. This is particularly relevant in the case; this assay will be used for high-throughput screening and drug discovery (as the method below). We suggest the following of the recommendations from the Assay Guidance Manual (available http://assay.nih.gov/assay/index.php/ Table_of_Contents). Cell line: Stable HeLa-TFEB-GFP (we do not use cells older than 7 passages) obtained from an antibiotic selection of HeLa cells transfected with a vector carrying human TFEB fused to the GFP protein. Stock plates of HeLa-TFEB-GFP cells are maintained in RPMI (10% FBS, G418 1.25 mg/mL) at 37°C, 5% CO2. Day 1—Seeding of assay plate: HeLa cells are plated on 384-well plates (Perkin Elmer, CellCarrier TM 384-well, black clear bottom, TC treated, Cat n. 6007558) at a density of 4500 cells/well in 100 μL in RPMI with L-glutamine, penicillin, streptomycin, and 10% FBS, using a Multidrop-Combi (Thermo Scientific). Day 2—Preparation of compound plate Plate format is 384 wells. Stock compounds are 10 mM in DMSO. All manipulations are made using a Hamilton liquid handler. For each plate, 3.3 μL of 28 different molecules are loaded in 96.7 μL of RPMI free (RPMI, L-glutamine, penicillin, streptomycin, NO G418,
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NO serum) (final compound concentration 330 μM), followed by five serial dilutions by 1/3, resulting in six final different concentrations. Negative control is RPMI free, 3% DMSO. Positive control is torin 1 (TOCRIS), 3.3 μM in RPMI free. Compound plates are used as a 10 stock solution. Treatment of assay plate with compounds – Cells are treated on day 2 (18 h after seeding). – The old medium is completely removed by aspiration, using an automated plate washer (Biotek), and 90 μL RPMI free/well is added using a Multidrop-Combi (ThermoFisher). – After this, plates are moved to an automated Hamilton STARlet liquid station, where the Compound Plate is replicated on the Assay Plate, placing 10 μL of compound in each well. Incubation with compounds is performed at 37°C for 3 h. Fixation and nuclei counterstaining – Incubation medium is removed using the plate washer, and cells are fixed in 50 μL of 3.7% formaldehyde in PBS added with the Multidrop. – Following fixative removal using the plate washer, cell nuclei are stained using Hoechst 1:2000 in PBS plus 0.1% Triton X-100. – Cells are rinsed once with PBS and left in 100 μL PBS. Acquisition of plates and analysis of data using the OPERA system (Perkin Elmer) – Plates are analyzed using 20 water immersion objective. The system acquires at least 6 fields/well using two exposures (laser 405 nm for Hoechst and laser 488 nm for TFEB-GFP). – A modification of the Acapella (Perkin Elmer) script for nuclear translocation is used to analyze the experiment. The script calculates the ratio of average nuclear GFP intensity/average cytosol GFP intensity in GFPpositive cells.
2.6 Measure of Autophagy Gene Expression Levels TFEB is known to regulate the expression levels of several autophagy genes through direct binding of CLEAR sites in their promoter sequences. However, TFEB can also influence the expression of many autophagy genes indirectly (Settembre et al., 2013). Thus to evaluate the involvement of TFEB and more generally of a transcriptional regulation of autophagy, the expression levels of multiple autophagy genes should be analyzed.
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To this aim the Autophagy RT2 Profiler PCR Array (SABiosciences, Frederick, MD) can be used, following the manufacturer’s procedure. – Extract total RNA from stimulated and nonstimulated (control) cells using TRIzol (Invitrogen) and perform a further purification using RNeasy mini kit with on-column DNAse digest (Qiagen). – Synthesize cDNA using the RT2 First Strand Kit (SABiosciences) and perform real-time PCR using the array plates. – Calculate fold change using SABiosciences online data analysis Web site (http://www.sabiosciences.com/pcr/arrayanalysis.php) which uses the ΔΔCt method for RT–qPCR data analysis. Average the most stable housekeeping genes included in the plate as “normalizer” genes to calculate the ΔCt value. Next, the ΔΔCt value is calculated between the “control” group and the “experimental” group. Lastly, the fold change is calculated using 2ΔΔCt. At least three experimental replicates should be grouped to calculate the fold change.
2.7 Measurement of TFEB Binding to the Promoter of Autophagy Genes To demonstrate that the nuclear translocation of TFEB is also associated to an enhanced TFEB activity a quantitative measure of TFEB binding to the CLEAR sites in the promoter of target genes should be performed. – Cells stably expressing hTFEB-3XFLAG can be used to this purpose. Fifty million cells/treatment should be used. We generally use five 150-mm subconfluent dishes. – Cross-link cells in 1% formaldehyde in PBS for 10 min (8 mL/dish). – Quench the reaction in equal volume of 0.25 M glycine in PBS. – Lyse cell on ice for 20 min in ChIP Lysis buffer (50 mM Tris–HCl, pH 7.5, 100 mM NaCl, 1% Triton X-100, 1% Tween-20). Use 1 mL lysis buffer/dish. – Scrape and collect lysate in 2-mL tubes (Non-Stick RNase-free Microfuge Tubes, Ambion). – Pass the lysate through G21 and G26S (four time each) and incubate 20 min on ice. – Perform DNA digestion using MNAse Mix (2 U of MNAse from SigmaAldrich in 20 μL 50 mM CaCl2) at 37°C for 5 min. – Stop the reaction by addition of SDS and EDTA to a final concentration of 1% and 2 mM, respectively.
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– Precipitate the unbound SDS from the cleared lysate using 50 μL of SDSOUT (Pierce, Rockford, IL, USA). – Dilute the supernatant 1:1 with ChIP dilution buffer (50 mM Tris–HCl, pH 7.5, 100 mM NaCl, 0.5% Triton X-100, 2 mM EDTA; all from Sigma-Aldrich). – Preincubate for 30 min with 50 μL high-capacity NeutrAvidin Agarose beads (Pierce). Spin for 30 s at 4°C and remove the supernatant. Discard the beads. – Add biotinylated FLAG antibody (2 mg ANTI-FLAG BioM2 antibody from Sigma-Aldrich) and 50 μL Neutravidin Agarose beads (previously resuspended in 1 vol. of ChIP dilution buffer supplemented with 10 mg/mL BSA). Immunoprecipitate protein–DNA complexes for 4 h at 4°C under gentle rotation. – Spin for 30 s at 4°C and remove the supernatant (post-IP). Wash the beads with ChIP dilution buffer five times for 5 min under gentle rotation at RT. – Elute the DNA by addition of 200 μL of 8 mM biotin, 1% SDS in TE buffer. Incubate 10 min under gentle rotation at RT. Spin for 1 min at RT. Collect the supernatant containing the DNA. – Precipitate the DNA using 0.4 M NaCl (final concentration) at 65°C, ON. Use 1 μL of DNA for each quantitative RT-PCR.
2.8 In Vivo Autophagy Modulation via TFEB The use of animal models, both lower organisms and mammals, has been very helpful to further elucidate TFEB function. Manipulation of TFEB levels in the liver has revealed a central role for this protein in regulating liver lipid metabolism (Settembre et al., 2013). In skeleton, TFEB regulates bone mass by controlling bone resorption by osteoclasts, the bone-resorbing cells (Ferron et al., 2013). Gain- and loss-of-function experiments in mouse skeletal muscle have shown that TFEB controls energy metabolism in this tissue (M. Mansueto, unpublished). Studies in mice have also shown that TFEB represents an appealing therapeutic target for many human diseases that are associated with autophagic or lysosomal dysfunction and the accumulation of toxic aggregates. Indeed, induction of TFEB activity has already been successfully used as a therapeutic strategy in several disease models, such as lysosomal storage disorders, Parkinson’s, Alzheimer’s disease, α1-anti-trypsin deficiency, and in
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both diet or genetically induced obesity (reviewed in Napolitano & Ballabio, 2016). Thus, the modulation of the activity of TFEB represents an appealing therapeutic strategy for the broad number of diseases that may potentially benefit from promoting intracellular clearance. Below we describe the strategy we have used to overexpress TFEB in tissues such as liver, muscle, and brain of mice and the different protocols to analyze TFEB expression and activity.
2.9 TFEB Overexpression in Liver The overexpression of TFEB in the liver of mice can be efficiently obtained using adenovirus-based strategies. We have used either adeno-associated viruses (AAVs) or helper-dependent adeno viruses (HDAds). The HDAd-TFEB plasmid contained the following elements (from 50 to 30 ): a liver-restricted rat phosphoenolpyruvate carboxykinase promoter, the ApoAI intron, the human TFEB cDNA, the woodchuck hepatitis virus posttranscriptional regulatory element, the ApoE locus control region, and the human growth hormone poly(A). HDAd was produced in 116 cells with the helper virus AdNG163 as described in detail elsewhere (Pastore et al., 2013). Hepatic transduction can be achieved by intravenous administration (tail or retro-orbital injection) of approximately 400 μL corresponding to 2 1012 viral particles per adult mouse (30 g weight). Age and sex-matched control mice should be infected with a transgeneless HDAd vector. Using this strategy more than 90% of the hepatocytes will express high levels of TFEB transgene. The AAV vector serotype 8 (AAV2/8) efficiently targets the liver. To restrict transgene expression to the hepatocytes, the hepatocyte-specific thyroxine-binding globulin (TBG) promoter can be used to drive hTFEB expression in the liver of infected mice. The pAAV2.8-TBG-TFEB was produced in 293 cells as described in detail elsewhere (Doria, Ferrara, Auricchio, et al., 2013). Inject each adult mouse (30 g) with 1.25 1011 viral particles. Mice can be sacrificed starting from a few days (usually 1 week) to several months after injection with either HdAD or AAV vectors. The protocols to analyze TFEB expression and activity in liver are described below.
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2.10 TFEB Overexpression in Muscle (Gastrocnemius) The AAV serotype 1 vector can be used to efficiently transduce muscle fibers. A ubiquitous promoter (e.g., CMV or CAG) can be used to drive TFEB expression in muscle fibers. – Inject adult mice with a total dose of 1011 GC of AAV2/1-CMV-hTFEB vector preparation into three sites of the right gastrocnemius (three injections of 30 μL each) using a Hamilton syringe. Inject equivalent doses of AAV2/1CMV-EGFP or equal volumes of PBS into the contralateral muscle, which will be used as nontransduced (control) sample. Mice can be sacrificed starting from a few days (usually 1 week) to several months after injection. – Isolate gastrocnemii, free from neighboring muscles and connective tissue. The protocols to analyze TFEB expression and activity in the muscle are described below.
2.11 TFEB Overexpression in Brain The AAV2/9 serotype showed higher transduction efficiency compared to other AAV serotypes for neuronal cells. The CMV promoter can be used to drive TFEB expression in brain. – For newborn (P0) injection, each mouse can be injected into the lateral ventricles of both cerebral hemispheres with 4.2 109 total viral particles per side. Adult mice (2 months of age) can be injected to target specific brain regions according to the stereotaxic atlas of Paxinos and Franklin (2001) using the same total viral concentrations. For example, to target the cortex and hippocampus, the following coordinates can be used: AP: +1.5 mm, LAT: +1.5 mm, DV: +1.5 mm and AP: 2 mm, LAT: 1.5 mm, DV: + 1.75 mm. Mice can be analyzed starting from a few days (usually 1 week) to several months after injection. The protocols to analyze TFEB expression and activity in the brain are described below.
2.12 Isolation of Tissue Specimens From Tissues Overexpressing TFEB – Synchronize mice before killing as follows: Prefast them for 24 h, then fed for 2 h (9 a.m. to 11 a.m.) (designated as “Fed”), and either sacrifice or refast them for 4 or 20 h (designated as “Fasted”) prior to sacrifice. – Anesthetize the mice and perform intracardiac perfusion with ice-cold PBS. Isolate tissues and weigh them.
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– Cut part of the tissue into small pieces (about 0.5 cm3) and snap freeze them in liquid nitrogen. Store them at 80°C until needed. These samples will be used to analyze TFEB mRNA and protein levels. – Cut the remaining tissues into pieces of 1 cm3 and fix them ON at 4°C in cold 4% PFA in PBS (50 mL). Cryoprotect the tissues in 30% sucrose in PBS solution for 24 h at 4°C, embed in Cryomatrix (Thermo Scientific), and keep them at 80°C until needed.
2.13 Analysis of TFEB mRNA Expression Levels in Transduced Tissues – Extract total RNA from 1 piece of 0.5 cm2 tissue using 1 mL of TRIzol (Invitrogen) according to manufacturer’s protocol. Repurify RNA using RNeasy MinElute Cleanup Kit (Qiagen). – Reverse transcription can be performed using commercially available reverse transcription reagents (e.g., TaqMan Applied Biosystems) according to manufacturer’s protocols. – Real-time qPCR can be performed using primers matching both human and mouse TFEB mRNA. The expression levels (measured as fold increase) of the human TFEB in infected tissues can be compared to the levels of the endogenous TFEB in nontransduced tissues isolated from control mice. Primer sequences are:
h/m-TFEB For. 50 -aggagcggcagaagaaagac-30 h/m-TFEB Rev. 50 -caggtccttctgcatcctcc-30
The expression levels of Cyclophilin and the Ribosomal protein S16 cDNA can be used as internal reference genes, since their expression is not sensitive to starvation/refeeding conditions or to TFEB overexpression. Below are the sequences of the primers: Primer sequences are:
m-Cycloph-For. 50 -ggcaaatgctggaccaaacacaa-30 m-Cycloph-Rev. 50 -gtaaaatgcccgcaagtcaaaag-30 m-S16-F 50 -aggagcgatttgctggtgtgg-30 m-S16-R 50 -gctaccagggcctttgagatg-30
– Calculate fold change values using the ΔΔCt method.
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2.14 Analysis of TFEB Protein Expression Levels in Transduced Tissues – Lyse a 0.5 cm3 tissue sample using a TissueLyser (Qiagen) in 0.5 mL cold RIPA buffer supplemented with 0.5% SDS, PhosSTOP, and EDTA-free protease inhibitor tablets (Roche). Samples are incubated for 30 min on ice, briefly sonicated on ice, and then the soluble fraction isolated by centrifugation at 14,000 rpm for 10 min at 4°C. – Separate 50 μg of protein by SDS-PAGE (Invitrogen; reduced NuPAGE 4–12% bis–tris gel, MES SDS buffer) and transfer them onto a nitrocellulose membrane with an I-Blot (Invitrogen). – Incubate with 5% nonfat milk in TBS-T buffer (TBS containing 0.05% Tween-20) for 1 h and then with the selected primary antibody (1:1000) ON at 4°C with gentle shaking. The TFEB antibody from Bethyl Laboratories (A303-672A) detects both human and mouse TFEB proteins, allowing a relative quantification of the transgene protein expression. To detect the human TFEB transgene only the rabbit anti-hTFEB polyclonal antibody from Cell Signaling (# 4240) should be used. – Use standard chemiluminescence methods (ECL Western Blotting Substrate, Pierce) and peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies (Calbiochem) to visualize proteins.
2.15 Analysis of TFEB Protein Expression and Localization in Transduced Tissues by Immunofluorescence The human specific TFEB antibody from Cell Signaling can be efficiently used to analyze tissue distribution and localization only of the human TFEB transgene, since no cross-reactivity with the murine TFEB can be detected using this antibody. – Prepare 20-μm thick slices from cryopreserved tissue specimens. – Add blocking buffer (2.5% BSA in PBS + 0.1% Triton X-100) for 2 h at RT. – Incubate specimens for 20 h with the primary anti-TFEB antibody (Cell Signaling, Cat n. 4240) 1:100 at 4°C in a humid chamber. – Wash three times in PBS + 0.05% TX-100, and then incubate for 3 h with secondary antibodies conjugated either with Alexafluor 488 or Alexafluor 555 (Invitrogen). – Wash three times in PBS, one wash in H2O and one brief wash in ethanol 70%, and dry the slides. – Mount in Mowiol (Calbiochem) containing DAPI (0.5 μM).
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– Take images by confocal microscopy. Analyze the number of TFEBpositive cells (relative to total cell number ¼ DAPI) and the ratio of average nuclear TFEB intensity/average cytosol TFEB intensity in TFEB-positive cells.
ACKNOWLEDGMENTS We are grateful to the Fondazione Telethon; the Beyond Batten Disease Foundation; the European Research Council; the Associazione Italiana per la Ricerca sul Cancro (Italian Association for Cancer Research); and the National Institutes of Health for their generous support.
REFERENCES Cortes, C. J. (2014). Polyglutamine-expanded androgen receptor interferes with TFEB to elicit autophagy defects in SBMA. Nature Neuroscience, 17(9), 1180–1189. Decressac, M., et al. (2013). TFEB-mediated autophagy rescues midbrain dopamine neurons from α-synuclein toxicity. Proceedings of the National Academy of Sciences of the United States of America, 110(19), E1817–E1826. Doria, M., Ferrara, A., Auricchio, A., et al. (2013). AAV2/8 vectors purified from culture medium with a simple and rapid protocol transduce murine liver, muscle, and retina efficiently. Human Gene Therapy Methods, 24(6), 392–398. Ferron, M., et al. (2013). A RANKL-PKCβ-TFEB signaling cascade is necessary for lysosomal biogenesis in osteoclasts. Genes & Development, 27(8), 955–969. Martina, J. A., Chen, Y., Gucek, M., & Puertollano, R. (2012). MTORC1 functions as a transcriptional regulator of autophagy by preventing nuclear transport of TFEB. Autophagy, 8, 903–914. Martina, J. A., Diab, H. I., Brady, O. A., & Puertollano, R. (2016). TFEB and TFE3 are novel components of the integrated stress response. The EMBO Journal, 35(5), 479–495. Martina, J. A., Diab, H. I., Li, H., & Puertollano, R. (2014). Novel roles for the MiTF/TFE family of transcription factors in organelle biogenesis, nutrient sensing, and energy homeostasis. Cellular and Molecular Life Sciences, 71(13), 2483–2497. Martina, J. A., Diab, H. I., Lishu, L., Jeong, A. L., Patange, S., Raben, N., et al. (2014). The nutrient-responsive transcription factor TFE3 promotes autophagy, lysosomal biogenesis, and clearance of cellular debris. Science Signaling, 7(309), ra9. Medina, D. L., et al. (2011). Transcriptional activation of lysosomal exocytosis promotes cellular clearance. Developmental Cell, 21, 421–430. Medina, D. L., et al. (2015). Lysosomal calcium signalling regulates autophagy through calcineurin and TFEB. Nature Cell Biology, 17(3), 288–299. Moskot, M., et al. (2014). The phytoestrogen genistein modulates lysosomal metabolism and transcription factor EB (TFEB) activation. The Journal of Biological Chemistry, 289(24), 17054–17069. Napolitano, G., & Ballabio, A. (2016). TFEB at a glance. Journal of Cell Science, 129(13), 2475–2481. Palmieri, M., et al. (2011). Characterization of the CLEAR network reveals an integrated control of cellular clearance pathways. Human Molecular Genetics, 20, 3852–3866. Pastore, N., et al. (2013). Gene transfer of master autophagy regulator TFEB results in clearance of toxic protein and correction of hepatic disease in alpha-1-anti-trypsin deficiency. EMBO Molecular Medicine, 5(3), 397–412. Paxinos, G., & Franklin, K. B. J. (2001). The mouse brain in stereotaxic coordinates. New York: Academic Press.
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Polito, V. A., et al. (2014). Selective clearance of aberrant tau proteins and rescue of neurotoxicity by transcription factor EB. EMBO Molecular Medicine, 6(9), 1142–1160. Roczniak-Ferguson, A., et al. (2012). The transcription factor TFEB links mTORC1 signaling to transcriptional control of lysosome homeostasis. Science Signaling, 5, ra42. Sardiello, M., et al. (2009). A gene network regulating lysosomal biogenesis and function. Science, 325, 473–477. Settembre, C., et al. (2011). TFEB links autophagy to lysosomal biogenesis. Science, 332, 1429–1433. Settembre, C., et al. (2012). A lysosome-to-nucleus signalling mechanism senses and regulates the lysosome via mTOR and TFEB. The EMBO Journal, 31, 1095–1108. Settembre, C., et al. (2013). TFEB controls cellular lipid metabolism through a starvationinduced autoregulatory loop. Nature Cell Biology, 15(6), 647–658. Shin, H. J., et al. (2016). AMPK-SKP2-CARM1 signalling cascade in transcriptional regulation of autophagy. Nature, 534(7608), 553–557. Song, J. X., et al. (2016). A novel curcumin analog binds to and activates TFEB in vitro and in vivo independent of MTOR inhibition. Autophagy, 12(8), 1372–1389. Spampanato, C., et al. (2013). Transcription factor EB (TFEB) is a new therapeutic target for Pompe disease. EMBO Molecular Medicine, 5(5), 691–706. Steingrı´msson, E., Copeland, N. G., & Jenkins, N. A. (2004). Melanocytes and the microphthalmia transcription factor network. Annual Review of Genetics, 38, 365–411. Tsumeni, T., et al. (2012). PGC-1α rescues Huntington’s disease proteotoxicity by preventing oxidative stress and promoting TFEB function. Science Translational Medicine, 4(142), 142ra9. Xiao, Q., et al. (2015). Neuronal-targeted TFEB accelerates lysosomal degradation of APP, reducing Aβ generation and amyloid plaque pathogenesis. The Journal of Neuroscience, 35(35), 12137–12151.
Methods to Monitor and Manipulate TFEB Activity During Autophagy D.L. Medina*, C. Settembre*,†,{, A. Ballabio*,{,§,¶,1 *Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Naples, Italy † Dulbecco Telethon Institute (DTI), Naples, Italy { Medical Genetics, Federico II University, Naples, Italy § Baylor College of Medicine, Houston, TX, United States ¶ Jan and Dan Duncan Neurological Research Institute, Texas Children’s Hospital, Houston, TX, United States 1 Corresponding author: e-mail address: [email protected]
Contents 1. Introduction 2. Methods to Monitor TFEB/TFE3 Activation 2.1 Analysis of TFEB Subcellular Localization by Immunoblot 2.2 Detection of TFEB Ser142 Phosphorylation by Immunoblot 2.3 Detection of P-Ser211 TFEB by Using 14-3-3 Motif 2.4 Analysis of TFEB Subcellular Localization by Immunofluorescence 2.5 Quantitative TFEB Subcellular Localization by High-Content Imaging Analysis 2.6 Measure of Autophagy Gene Expression Levels 2.7 Measurement of TFEB Binding to the Promoter of Autophagy Genes 2.8 In Vivo Autophagy Modulation via TFEB 2.9 TFEB Overexpression in Liver 2.10 TFEB Overexpression in Muscle (Gastrocnemius) 2.11 TFEB Overexpression in Brain 2.12 Isolation of Tissue Specimens From Tissues Overexpressing TFEB 2.13 Analysis of TFEB mRNA Expression Levels in Transduced Tissues 2.14 Analysis of TFEB Protein Expression Levels in Transduced Tissues 2.15 Analysis of TFEB Protein Expression and Localization in Transduced Tissues by Immunofluorescence Acknowledgments References
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Abstract Macroautophagy is a catabolic process deputed to the turnover of intracellular components. Recent studies have revealed that transcriptional regulation is a major mechanism controlling autophagy. Currently, more than 20 transcription factors have been
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shown to modulate cellular autophagy levels. Among them, the transcription factor EB (TFEB) appears to have the broadest proautophagy role, given its capacity to control the biogenesis of lysosomes and autophagosomes, the two main organelles required for the autophagy pathway. TFEB has attracted major attention owing to its ability to enhance cellular clearance of pathogenic substrates in a variety of animal models of disease, such as lysosomal storage disorders, Parkinson’s, Alzheimer’s, α1-antitrypsin, obesity as well as others, suggesting that the TFEB pathway represents an extraordinary possibility for future development of innovative therapies. Importantly, the subcellular localization and activity of TFEB are regulated by its phosphorylation status, suggesting that TFEB activity can be pharmacologically targeted. Given the growing list of common and rare diseases in which manipulation of autophagy may be beneficial, in this chapter we describe a set of validated protocols developed to modulate and analyze TFEB-mediated enhancement of autophagy both in vitro and in vivo conditions.
1. INTRODUCTION Autophagy is an evolutionary conserved catabolic process that provides energy and recycles cellular components through the targeting of intracytoplasmic cargo to lysosomes. Autophagy plays a crucial role in the cellular adaptation to environmental cues such as energy demanding conditions (i.e., nutrient deprivation). Autophagy relies on the biogenesis and cooperation of two organelles, the autophagosome and the lysosome. Recent studies have shown that the transcription factor EB (TFEB) plays an important role in autophagy. TFEB belongs to the microphthalmia family of basic helix-loop-helix–leucine-zipper (bHLH-Zip) transcription factors (MiT family) (Steingrı´msson, Copeland, & Jenkins, 2004). TFEB positively regulates lysosome biogenesis and function by enhancing the expression of lysosomal hydrolases and of lysosomal membrane proteins, such as the components of the v-ATPase proton pump complex (Medina et al., 2011; Palmieri et al., 2011; Sardiello et al., 2009). TFEB also promotes autophagosome biogenesis and autophagosome fusion with the lysosome by inducing the expression of autophagy-related genes (Settembre et al., 2011). TFEB activity is primarily regulated by phosphorylation events, mainly occurring at serine residues. Notably, the mTORC1 kinase, a main negative regulator of autophagy, is a key modulator of TFEB function (Martina, Chen, Gucek, & Puertollano, 2012; Roczniak-Ferguson et al., 2012; Settembre et al., 2012).
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In the presence of nutrients, mTORC1 phosphorylates TFEB at multiple serine residues on the lysosome surface and promotes the binding of TFEB to 14-3-3 proteins preventing its nuclear translocation and activation (Martina et al., 2012; Roczniak-Ferguson et al., 2012; Settembre et al., 2012). During starvation mTORC1 dissociates from the lysosomal surface and its activity is inhibited. In parallel, upon starvation the activity of the phosphatase calcineurin is induced by lysosomal calcium release through the activation of the lysosomal cation channel MCOLN1 (Medina et al., 2015). Together, these events lead to TFEB dephosphorylation, nuclear translocation, and activation (Fig. 1). In the nucleus TFEB interacts with the transcriptional coactivator-associated arginine methyltransferase 1 (CARM1), and binds to the CLEAR sites in the promoters of many lysosomal and autophagy genes promoting their expression during starvation (Settembre et al., 2011; Shin et al., 2016). Not surprisingly, the TFEB paralogue transcription factor E3 (TFE3) has also been shown to regulate lysosomal homeostasis by inducing the expression of genes encoding proteins involved in autophagy and lysosomal biogenesis in response to starvation and lysosomal stress. TFE3 regulation seems to share the same regulatory mechanisms as TFEB and therefore TFE3 is also regulated by mTORC1-dependent phosphorylation on the lysosomal surface as well as by the phosphatase calcineurin (Martina, Diab, Brady, & Puertollano, 2016; Martina, Diab, Li, & Puertollano, 2014; Martina, Diab, Lishu, et al., 2014). The identification of a global transcriptional regulation of lysosomal function and autophagy has been exploited to boost cellular clearance in mouse models of a variety of disease conditions. TFEB overexpression results in the clearance of accumulating substrates in cells and tissues from mouse models of several types of LSDs, as well as of Parkinson’s, mTORC1 (nutrient-rich conditions) Cytoplasmic retention with 14-3-3 protein
P-TFEB
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Nuclear localization and activation of lysosomal and autophagic genes
CaN (starvation, exercise)
Fig. 1 TFEB regulation. TFEB is negatively regulated by nutrients through mTORC1mediated phosphorylation and cytoplasmic retention by binding to 14-3-3 proteins. Conditions such as nutrient deprivation or exercise promote TFEB dephosphorylation by calcineurin (CaN). Dephosphorylated TFEB translocates into the nucleus activating lysosomal and autophagic genes.
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Alzheimer’s, Huntington’s disease, α1-anti-trypsin deficiency, and spinal bulbar muscular atrophy (Cortes, 2014; Decressac et al., 2013; Medina et al., 2011; Pastore et al., 2013; Polito et al., 2014; Song et al., 2016; Spampanato et al., 2013; Tsumeni et al., 2012; Xiao et al., 2015). In the same way, overexpression of TFE3 promotes clearance in a cellular model of a lysosomal storage disorder, Pompe disease (Martina, Diab, Li, et al., 2014; Martina, Diab, Lishu, et al., 2014). These findings have opened a novel therapeutic strategy based on the modulation of cellular clearance, through the transcriptional activation of the lysosomal/autophagic pathway, with potential applicability to many diseases. The mechanism by which TFEB promotes the clearance of stored materials needs to be further characterized. However, it is possible that TFEB-mediated cellular clearance results from a combination of the effects on lysosomal biogenesis, autophagy, and lysosomal exocytosis (Medina et al., 2011; Spampanato et al., 2013). Therefore, drug-screening approaches aimed at identifying molecules that promote TFEB nuclear translocation present an interesting path forward to treat several diseases (Moskot et al., 2014; Song et al., 2016) (Fig. 2). The important implications resulting from the study of
TFEB
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Lysosomal biogenesis
Lysosomal exocytosis
Cellular clearance Lysosomal storage diseases
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Fig. 2 Model of TFEB-mediated cellular clearance. TFEB overexpression modulates autophagy, lysosomal biogenesis, and induces lysosomal exocytosis. The activation of these biological pathways may contribute to the clearance of pathological accumulation in various disease conditions such as lysosomal storage disorders and common neurodegenerative disease.
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TFEB-mediated function prompted us to present a comprehensive set of the major experimental procedures used to enhance TFEB/TFE3 signaling and examine the transcriptional induction of the autophagy pathway.
2. METHODS TO MONITOR TFEB/TFE3 ACTIVATION
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Under basal conditions TFEB is mainly found in the cytoplasm in an inactive, phosphorylated state (Settembre et al., 2011). However, under specific conditions, such as nutrient deprivation, exercise, or lysosomal stress, TFEB is rapidly dephosphorylated and moves to the nucleus (Fig. 3). Although phosphoproteomic studies identified at least 10 different phosphorylation sites on the TFEB protein, mutation analysis indicated that two residues (Ser211 and Ser142) are particularly critical for TFEB subcellular localization (Martina et al., 2012; Roczniak-Ferguson et al., 2012; Settembre et al., 2012). In vitro studies suggest that Ser142 may either be phosphorylated by ERK2 or mTORC1, while only the latter phosphorylates Ser211. TFEB phosphorylation favors the interaction with 14-3-3 proteins and therefore its cytoplasmic retention (Roczniak-Ferguson et al., 2012). Interestingly, TFEB phosphorylation by the mTORC1 complex occurs on the lysosomal surface. Hence, TFEB subcellular localization may be used as a marker for TFEB activation. The key procedural steps to measure endogenous or overexpressed TFEB localization by western blot
Cytosolic Fractions Nuclear
Total lysate
Fig. 3 Detection of TFEB phosphorylation and subcellular localization in HeLa cells by immunoblotting. (A) Total cellular lysate isolated from cells nutrient deprived (HBSS starvation) for the indicated time points. (B) TFEB subcellular localization. Cells were cultured in complete media (nutrients), in HBSS buffer for 1 h (starvation), or in complete media supplemented with torin 1 inhibitor. Endogenous TFEB was detected using Cell Signaling antibody (Cat n. 4240).
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and immunofluorescence are summarized below. In addition, we have included a procedure to quantify TFEB subcellular localization by using high-content imaging analysis using a cell line stably overexpressing TFEBGFP fusion protein (Medina et al., 2015).
2.1 Analysis of TFEB Subcellular Localization by Immunoblot – TFEB antibodies: To detect endogenous TFEB in human cell extracts, the anti-TFEB antibody from Cell Signaling should be used (#4240). To detect endogenous TFEB in mouse cell extracts, the anti-TFEB antibody from Bethyl Laboratories (A311-347A) should be used. – Prepare the Triton X-100 lysis buffer: 50 mM Tris–HCl (pH 7.5), 0.5% Triton X-100, 137.5 mM NaCl, 10% glycerol, 5 mM ethylenediaminetetraacetic acid (EDTA), PhosSTOP, and EDTA-free protease inhibitor tablets (Roche, Indianapolis, IN, USA). Keep on ice. – Cell lysates: After three washes with cold phosphate-buffered saline (PBS), lyse 6 million cells with 0.5 mL of 0.5% Triton X-100 lysis buffer for 15 min on ice, shaking gently. – Collect and centrifuge the cell lysate at 2000 rpm for 15 min at 4°C with a tabletop centrifuge and transfer 250 μL of the supernatant, which is composed of the cytosolic and membrane fractions, to a new Eppendorf tube. Discard the rest of the supernatant. Keep the pellet. – Rinse the nuclear pellet three times with 0.5 mL of lysis buffer, and resuspend the pellet in 0.1 mL of lysis buffer, supplemented with 0.5% sodium dodecyl sulfate (SDS). Sonicate in cold room three times for 3 s at low output to shear genomic DNA. – Preclear by centrifugation at 13,000 rpm for 15 min at 4°C with a tabletop centrifuge. Transfer the supernatant to a new tube. – Determine the protein concentration in supernatant extracts using the colorimetric bicinchoninic acid protein assay kit (Pierce Chemical Co., Boston, MA, USA), following the manufacturer’s instructions. – Add an equal amount of (2) SDS-PAGE sample buffer to each of the tubes containing the nuclear and cytoplasmic fractions and boil the samples for 10 min. – Perform western blot, using 10% SDS-PAGE gels. Transfer protein onto PVDF (polyvinyl difluoride) membrane. Block the PVDF membrane with 5% nonfat milk in TBS-T buffer (TBS containing 0.05% Tween20) for 1 h at room temperature (RT) under gentle shaking. Incubate with primary antibodies, anti-TFEB (1:1000 in 5% nonfat milk in
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TBS-T buffer) overnight (ON) at 4°C, anti-TUBULIN (Sigma; 1:2000), and anti-H3 (Cell Signaling; 1:10,000) at RT for 2 h. Detect signals with horseradish peroxidase (HRP)-conjugated goat anti-mouse or anti-rabbit IgG antibody (1:2000, Vector Laboratories) and visualize with the Super Signal West Dura substrate (Thermo Scientific, Rockford, IL), according to the manufacturer’s protocol. – The purity of the fractions is confirmed by the lack of beta-tubulin in the nuclear fraction and of histone H3 in the cytosolic fraction, as detected by western blot.
2.2 Detection of TFEB Ser142 Phosphorylation by Immunoblot Phosphorylation of TFEB at Ser142 is a key step in TFEB nuclear translocation during starvation. A custom phosphospecific-antibody, recognizing phosphorylated TFEB at the Ser142 residue, was generated by the GenScript Company. Recently, a similar antibody from EMD-MILLIPORE has become commercially available (ABE1971-Anti-phospho TFEB-Ser142 antibody). The TFEB P-Ser142 antibody is suitable for western blot; however, given the high homology between the amino acid sequences surrounding Ser142 and the other members of the MiT subfamily, it is possible that cross-reaction events may occur. This issue can be avoided by analyzing samples obtained from cells overexpressing TFEB. – Follow standard western blot procedures and incubate primary antibody diluted 1:1000 in 5% milk ON at 4°C. Avoid many cycles freeze and thaw.
2.3 Detection of P-Ser211 TFEB by Using 14-3-3 Motif The phosphorylation of the Ser211 site on TFEB protein favors the binding to the cytosolic chaperone 14-3-3, which keeps TFEB sequestered in the cytosol (Martina et al., 2012; Roczniak-Ferguson et al., 2012). Hence, the phosphorylation of TFEB on Ser211 can be determined by exploiting the selective interaction between Ser211-phosphorylated TFEB and 14-3-3 proteins. The following antibodies are required: anti-FLAG (1:1000) and anti-actin (1:4000) from Sigma-Aldrich; P-Ser-14-3-3 binding motif antibodies (1:1000) from Cell Signaling Technologies; anti-pan 14-3-3 antibodies (1:1000) from Santa Cruz Biotechnology. – Rinse TFEB-3XFLAG-expressing cells twice with ice-cold PBS and lyse in ice-cold lysis buffer (400 mM NaCl, 25 mM Tris–HCl, pH 7.4, 1 mM
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EDTA, and 1% Triton X-100) containing protease and phosphatase inhibitors. Isolate soluble fractions from cell lysates by centrifugation at 11,200 rpm for 15 min in a Microfuge. Incubate with anti-3XFLAG antibodies (1:1000, SIGMA) in binding buffer (200 mM NaCl, 25 mM Tris–HCl, pH 7.4, 1 mM EDTA) with constant rotation ON at 4°C. Add 40 μL of 50% slurry of Protein-G beads (Sigma-Aldrich) to the lysates and incubate with rotation for an additional 2 h at 4°C. Wash the resins five times with binding buffer and elute the samples in Laemmli buffer. After 7.5% acrylamide SDS-PAGE gel separation and immunoblotting, incubate with the appropriated antibodies. Use standard chemiluminescence methods (ECL Western Blotting Substrate, Pierce) and peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies (Calbiochem) to visualize proteins. Develop the membranes using a Chemidoc UVP imaging system (UltraViolet Products Ltd.). Perform densitometric quantification of protein bands images using ImageJ software (NIH).
2.4 Analysis of TFEB Subcellular Localization by Immunofluorescence TFEB antibodies: To detect endogenous TFEB in human cells, use the antiTFEB antibody from Cell Signaling (Cat n. 4240), following manufacturer’s instructions. To detect endogenous TFEB in mouse cells, use anti-TFEB antibody from MyBiosource (MBS120432) at 1:50 dilution. – Plate 300,000 cells on coated glass coverslips in 35-mm tissue culture dishes. – Rinse cells with PBS once and fix them for 15 min with 4% paraformaldehyde in PBS at RT. – Rinse the slides twice with PBS and permeabilize cells with 0.05% Triton X-100 in PBS for 5 min. – Rinse twice with PBS and incubate with primary antibody in 5% normal donkey serum ON at 4°C. – Rinse four times with PBS and incubate with secondary donkey antibodies (diluted 1:1000 in 5% normal donkey serum) for 45 min at RT in the dark.
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Fig. 4 Detection of TFEB subcellular localization in HeLa cells by immunofluorescence. Cells were cultured in complete media (Fed) or starved (HBSS buffer) for 1 h. Endogenous TFEB was detected using specific Cell Signaling antibodies (Cat n. 4240).
– Wash four times with PBS. Slides should be mounted onto glass coverslips, using Vectashield (Vector Laboratories) and imaged with a fluorescent microscope (Fig. 4).
2.5 Quantitative TFEB Subcellular Localization by High-Content Imaging Analysis A setup of the immunofluorescence assay is needed to microscale the experiment to 96- or 384-well plates. This is particularly relevant in the case; this assay will be used for high-throughput screening and drug discovery (as the method below). We suggest the following of the recommendations from the Assay Guidance Manual (available http://assay.nih.gov/assay/index.php/ Table_of_Contents). Cell line: Stable HeLa-TFEB-GFP (we do not use cells older than 7 passages) obtained from an antibiotic selection of HeLa cells transfected with a vector carrying human TFEB fused to the GFP protein. Stock plates of HeLa-TFEB-GFP cells are maintained in RPMI (10% FBS, G418 1.25 mg/mL) at 37°C, 5% CO2. Day 1—Seeding of assay plate: HeLa cells are plated on 384-well plates (Perkin Elmer, CellCarrier TM 384-well, black clear bottom, TC treated, Cat n. 6007558) at a density of 4500 cells/well in 100 μL in RPMI with L-glutamine, penicillin, streptomycin, and 10% FBS, using a Multidrop-Combi (Thermo Scientific). Day 2—Preparation of compound plate Plate format is 384 wells. Stock compounds are 10 mM in DMSO. All manipulations are made using a Hamilton liquid handler. For each plate, 3.3 μL of 28 different molecules are loaded in 96.7 μL of RPMI free (RPMI, L-glutamine, penicillin, streptomycin, NO G418,
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NO serum) (final compound concentration 330 μM), followed by five serial dilutions by 1/3, resulting in six final different concentrations. Negative control is RPMI free, 3% DMSO. Positive control is torin 1 (TOCRIS), 3.3 μM in RPMI free. Compound plates are used as a 10 stock solution. Treatment of assay plate with compounds – Cells are treated on day 2 (18 h after seeding). – The old medium is completely removed by aspiration, using an automated plate washer (Biotek), and 90 μL RPMI free/well is added using a Multidrop-Combi (ThermoFisher). – After this, plates are moved to an automated Hamilton STARlet liquid station, where the Compound Plate is replicated on the Assay Plate, placing 10 μL of compound in each well. Incubation with compounds is performed at 37°C for 3 h. Fixation and nuclei counterstaining – Incubation medium is removed using the plate washer, and cells are fixed in 50 μL of 3.7% formaldehyde in PBS added with the Multidrop. – Following fixative removal using the plate washer, cell nuclei are stained using Hoechst 1:2000 in PBS plus 0.1% Triton X-100. – Cells are rinsed once with PBS and left in 100 μL PBS. Acquisition of plates and analysis of data using the OPERA system (Perkin Elmer) – Plates are analyzed using 20 water immersion objective. The system acquires at least 6 fields/well using two exposures (laser 405 nm for Hoechst and laser 488 nm for TFEB-GFP). – A modification of the Acapella (Perkin Elmer) script for nuclear translocation is used to analyze the experiment. The script calculates the ratio of average nuclear GFP intensity/average cytosol GFP intensity in GFPpositive cells.
2.6 Measure of Autophagy Gene Expression Levels TFEB is known to regulate the expression levels of several autophagy genes through direct binding of CLEAR sites in their promoter sequences. However, TFEB can also influence the expression of many autophagy genes indirectly (Settembre et al., 2013). Thus to evaluate the involvement of TFEB and more generally of a transcriptional regulation of autophagy, the expression levels of multiple autophagy genes should be analyzed.
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To this aim the Autophagy RT2 Profiler PCR Array (SABiosciences, Frederick, MD) can be used, following the manufacturer’s procedure. – Extract total RNA from stimulated and nonstimulated (control) cells using TRIzol (Invitrogen) and perform a further purification using RNeasy mini kit with on-column DNAse digest (Qiagen). – Synthesize cDNA using the RT2 First Strand Kit (SABiosciences) and perform real-time PCR using the array plates. – Calculate fold change using SABiosciences online data analysis Web site (http://www.sabiosciences.com/pcr/arrayanalysis.php) which uses the ΔΔCt method for RT–qPCR data analysis. Average the most stable housekeeping genes included in the plate as “normalizer” genes to calculate the ΔCt value. Next, the ΔΔCt value is calculated between the “control” group and the “experimental” group. Lastly, the fold change is calculated using 2ΔΔCt. At least three experimental replicates should be grouped to calculate the fold change.
2.7 Measurement of TFEB Binding to the Promoter of Autophagy Genes To demonstrate that the nuclear translocation of TFEB is also associated to an enhanced TFEB activity a quantitative measure of TFEB binding to the CLEAR sites in the promoter of target genes should be performed. – Cells stably expressing hTFEB-3XFLAG can be used to this purpose. Fifty million cells/treatment should be used. We generally use five 150-mm subconfluent dishes. – Cross-link cells in 1% formaldehyde in PBS for 10 min (8 mL/dish). – Quench the reaction in equal volume of 0.25 M glycine in PBS. – Lyse cell on ice for 20 min in ChIP Lysis buffer (50 mM Tris–HCl, pH 7.5, 100 mM NaCl, 1% Triton X-100, 1% Tween-20). Use 1 mL lysis buffer/dish. – Scrape and collect lysate in 2-mL tubes (Non-Stick RNase-free Microfuge Tubes, Ambion). – Pass the lysate through G21 and G26S (four time each) and incubate 20 min on ice. – Perform DNA digestion using MNAse Mix (2 U of MNAse from SigmaAldrich in 20 μL 50 mM CaCl2) at 37°C for 5 min. – Stop the reaction by addition of SDS and EDTA to a final concentration of 1% and 2 mM, respectively.
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– Precipitate the unbound SDS from the cleared lysate using 50 μL of SDSOUT (Pierce, Rockford, IL, USA). – Dilute the supernatant 1:1 with ChIP dilution buffer (50 mM Tris–HCl, pH 7.5, 100 mM NaCl, 0.5% Triton X-100, 2 mM EDTA; all from Sigma-Aldrich). – Preincubate for 30 min with 50 μL high-capacity NeutrAvidin Agarose beads (Pierce). Spin for 30 s at 4°C and remove the supernatant. Discard the beads. – Add biotinylated FLAG antibody (2 mg ANTI-FLAG BioM2 antibody from Sigma-Aldrich) and 50 μL Neutravidin Agarose beads (previously resuspended in 1 vol. of ChIP dilution buffer supplemented with 10 mg/mL BSA). Immunoprecipitate protein–DNA complexes for 4 h at 4°C under gentle rotation. – Spin for 30 s at 4°C and remove the supernatant (post-IP). Wash the beads with ChIP dilution buffer five times for 5 min under gentle rotation at RT. – Elute the DNA by addition of 200 μL of 8 mM biotin, 1% SDS in TE buffer. Incubate 10 min under gentle rotation at RT. Spin for 1 min at RT. Collect the supernatant containing the DNA. – Precipitate the DNA using 0.4 M NaCl (final concentration) at 65°C, ON. Use 1 μL of DNA for each quantitative RT-PCR.
2.8 In Vivo Autophagy Modulation via TFEB The use of animal models, both lower organisms and mammals, has been very helpful to further elucidate TFEB function. Manipulation of TFEB levels in the liver has revealed a central role for this protein in regulating liver lipid metabolism (Settembre et al., 2013). In skeleton, TFEB regulates bone mass by controlling bone resorption by osteoclasts, the bone-resorbing cells (Ferron et al., 2013). Gain- and loss-of-function experiments in mouse skeletal muscle have shown that TFEB controls energy metabolism in this tissue (M. Mansueto, unpublished). Studies in mice have also shown that TFEB represents an appealing therapeutic target for many human diseases that are associated with autophagic or lysosomal dysfunction and the accumulation of toxic aggregates. Indeed, induction of TFEB activity has already been successfully used as a therapeutic strategy in several disease models, such as lysosomal storage disorders, Parkinson’s, Alzheimer’s disease, α1-anti-trypsin deficiency, and in
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both diet or genetically induced obesity (reviewed in Napolitano & Ballabio, 2016). Thus, the modulation of the activity of TFEB represents an appealing therapeutic strategy for the broad number of diseases that may potentially benefit from promoting intracellular clearance. Below we describe the strategy we have used to overexpress TFEB in tissues such as liver, muscle, and brain of mice and the different protocols to analyze TFEB expression and activity.
2.9 TFEB Overexpression in Liver The overexpression of TFEB in the liver of mice can be efficiently obtained using adenovirus-based strategies. We have used either adeno-associated viruses (AAVs) or helper-dependent adeno viruses (HDAds). The HDAd-TFEB plasmid contained the following elements (from 50 to 30 ): a liver-restricted rat phosphoenolpyruvate carboxykinase promoter, the ApoAI intron, the human TFEB cDNA, the woodchuck hepatitis virus posttranscriptional regulatory element, the ApoE locus control region, and the human growth hormone poly(A). HDAd was produced in 116 cells with the helper virus AdNG163 as described in detail elsewhere (Pastore et al., 2013). Hepatic transduction can be achieved by intravenous administration (tail or retro-orbital injection) of approximately 400 μL corresponding to 2 1012 viral particles per adult mouse (30 g weight). Age and sex-matched control mice should be infected with a transgeneless HDAd vector. Using this strategy more than 90% of the hepatocytes will express high levels of TFEB transgene. The AAV vector serotype 8 (AAV2/8) efficiently targets the liver. To restrict transgene expression to the hepatocytes, the hepatocyte-specific thyroxine-binding globulin (TBG) promoter can be used to drive hTFEB expression in the liver of infected mice. The pAAV2.8-TBG-TFEB was produced in 293 cells as described in detail elsewhere (Doria, Ferrara, Auricchio, et al., 2013). Inject each adult mouse (30 g) with 1.25 1011 viral particles. Mice can be sacrificed starting from a few days (usually 1 week) to several months after injection with either HdAD or AAV vectors. The protocols to analyze TFEB expression and activity in liver are described below.
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2.10 TFEB Overexpression in Muscle (Gastrocnemius) The AAV serotype 1 vector can be used to efficiently transduce muscle fibers. A ubiquitous promoter (e.g., CMV or CAG) can be used to drive TFEB expression in muscle fibers. – Inject adult mice with a total dose of 1011 GC of AAV2/1-CMV-hTFEB vector preparation into three sites of the right gastrocnemius (three injections of 30 μL each) using a Hamilton syringe. Inject equivalent doses of AAV2/1CMV-EGFP or equal volumes of PBS into the contralateral muscle, which will be used as nontransduced (control) sample. Mice can be sacrificed starting from a few days (usually 1 week) to several months after injection. – Isolate gastrocnemii, free from neighboring muscles and connective tissue. The protocols to analyze TFEB expression and activity in the muscle are described below.
2.11 TFEB Overexpression in Brain The AAV2/9 serotype showed higher transduction efficiency compared to other AAV serotypes for neuronal cells. The CMV promoter can be used to drive TFEB expression in brain. – For newborn (P0) injection, each mouse can be injected into the lateral ventricles of both cerebral hemispheres with 4.2 109 total viral particles per side. Adult mice (2 months of age) can be injected to target specific brain regions according to the stereotaxic atlas of Paxinos and Franklin (2001) using the same total viral concentrations. For example, to target the cortex and hippocampus, the following coordinates can be used: AP: +1.5 mm, LAT: +1.5 mm, DV: +1.5 mm and AP: 2 mm, LAT: 1.5 mm, DV: + 1.75 mm. Mice can be analyzed starting from a few days (usually 1 week) to several months after injection. The protocols to analyze TFEB expression and activity in the brain are described below.
2.12 Isolation of Tissue Specimens From Tissues Overexpressing TFEB – Synchronize mice before killing as follows: Prefast them for 24 h, then fed for 2 h (9 a.m. to 11 a.m.) (designated as “Fed”), and either sacrifice or refast them for 4 or 20 h (designated as “Fasted”) prior to sacrifice. – Anesthetize the mice and perform intracardiac perfusion with ice-cold PBS. Isolate tissues and weigh them.
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– Cut part of the tissue into small pieces (about 0.5 cm3) and snap freeze them in liquid nitrogen. Store them at 80°C until needed. These samples will be used to analyze TFEB mRNA and protein levels. – Cut the remaining tissues into pieces of 1 cm3 and fix them ON at 4°C in cold 4% PFA in PBS (50 mL). Cryoprotect the tissues in 30% sucrose in PBS solution for 24 h at 4°C, embed in Cryomatrix (Thermo Scientific), and keep them at 80°C until needed.
2.13 Analysis of TFEB mRNA Expression Levels in Transduced Tissues – Extract total RNA from 1 piece of 0.5 cm2 tissue using 1 mL of TRIzol (Invitrogen) according to manufacturer’s protocol. Repurify RNA using RNeasy MinElute Cleanup Kit (Qiagen). – Reverse transcription can be performed using commercially available reverse transcription reagents (e.g., TaqMan Applied Biosystems) according to manufacturer’s protocols. – Real-time qPCR can be performed using primers matching both human and mouse TFEB mRNA. The expression levels (measured as fold increase) of the human TFEB in infected tissues can be compared to the levels of the endogenous TFEB in nontransduced tissues isolated from control mice. Primer sequences are:
h/m-TFEB For. 50 -aggagcggcagaagaaagac-30 h/m-TFEB Rev. 50 -caggtccttctgcatcctcc-30
The expression levels of Cyclophilin and the Ribosomal protein S16 cDNA can be used as internal reference genes, since their expression is not sensitive to starvation/refeeding conditions or to TFEB overexpression. Below are the sequences of the primers: Primer sequences are:
m-Cycloph-For. 50 -ggcaaatgctggaccaaacacaa-30 m-Cycloph-Rev. 50 -gtaaaatgcccgcaagtcaaaag-30 m-S16-F 50 -aggagcgatttgctggtgtgg-30 m-S16-R 50 -gctaccagggcctttgagatg-30
– Calculate fold change values using the ΔΔCt method.
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2.14 Analysis of TFEB Protein Expression Levels in Transduced Tissues – Lyse a 0.5 cm3 tissue sample using a TissueLyser (Qiagen) in 0.5 mL cold RIPA buffer supplemented with 0.5% SDS, PhosSTOP, and EDTA-free protease inhibitor tablets (Roche). Samples are incubated for 30 min on ice, briefly sonicated on ice, and then the soluble fraction isolated by centrifugation at 14,000 rpm for 10 min at 4°C. – Separate 50 μg of protein by SDS-PAGE (Invitrogen; reduced NuPAGE 4–12% bis–tris gel, MES SDS buffer) and transfer them onto a nitrocellulose membrane with an I-Blot (Invitrogen). – Incubate with 5% nonfat milk in TBS-T buffer (TBS containing 0.05% Tween-20) for 1 h and then with the selected primary antibody (1:1000) ON at 4°C with gentle shaking. The TFEB antibody from Bethyl Laboratories (A303-672A) detects both human and mouse TFEB proteins, allowing a relative quantification of the transgene protein expression. To detect the human TFEB transgene only the rabbit anti-hTFEB polyclonal antibody from Cell Signaling (# 4240) should be used. – Use standard chemiluminescence methods (ECL Western Blotting Substrate, Pierce) and peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies (Calbiochem) to visualize proteins.
2.15 Analysis of TFEB Protein Expression and Localization in Transduced Tissues by Immunofluorescence The human specific TFEB antibody from Cell Signaling can be efficiently used to analyze tissue distribution and localization only of the human TFEB transgene, since no cross-reactivity with the murine TFEB can be detected using this antibody. – Prepare 20-μm thick slices from cryopreserved tissue specimens. – Add blocking buffer (2.5% BSA in PBS + 0.1% Triton X-100) for 2 h at RT. – Incubate specimens for 20 h with the primary anti-TFEB antibody (Cell Signaling, Cat n. 4240) 1:100 at 4°C in a humid chamber. – Wash three times in PBS + 0.05% TX-100, and then incubate for 3 h with secondary antibodies conjugated either with Alexafluor 488 or Alexafluor 555 (Invitrogen). – Wash three times in PBS, one wash in H2O and one brief wash in ethanol 70%, and dry the slides. – Mount in Mowiol (Calbiochem) containing DAPI (0.5 μM).
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– Take images by confocal microscopy. Analyze the number of TFEBpositive cells (relative to total cell number ¼ DAPI) and the ratio of average nuclear TFEB intensity/average cytosol TFEB intensity in TFEB-positive cells.
ACKNOWLEDGMENTS We are grateful to the Fondazione Telethon; the Beyond Batten Disease Foundation; the European Research Council; the Associazione Italiana per la Ricerca sul Cancro (Italian Association for Cancer Research); and the National Institutes of Health for their generous support.
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