TDP ‐43 loss of function increases TFEB activity and blocks autophagosome–lysosome fusion
2015; Springer Nature; Volume: 35; Issue: 2 Linguagem: Inglês
10.15252/embj.201591998
ISSN1460-2075
AutoresXia Qin, Hongfeng Wang, Zongbing Hao, Cheng Fu, Qingsong Hu, Feng Gao, Haigang Ren, Chen Dong, Junhai Han, Zheng Ying, Guanghui Wang,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoArticle23 December 2015free access Source Data TDP-43 loss of function increases TFEB activity and blocks autophagosome–lysosome fusion Qin Xia Qin Xia Laboratory of Molecular Neuropathology, Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and College of Pharmaceutical Sciences, Soochow University, Suzhou, Jiangsu, China Search for more papers by this author Hongfeng Wang Hongfeng Wang Laboratory of Molecular Neuropathology, Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and College of Pharmaceutical Sciences, Soochow University, Suzhou, Jiangsu, China Search for more papers by this author Zongbing Hao Zongbing Hao Laboratory of Molecular Neuropathology, Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and College of Pharmaceutical Sciences, Soochow University, Suzhou, Jiangsu, China Search for more papers by this author Cheng Fu Cheng Fu Key Laboratory of Brain Function and Disease, School of Life Sciences, University of Science & Technology of China, Chinese Academy of Sciences, Hefei, Anhui, China Search for more papers by this author Qingsong Hu Qingsong Hu Laboratory of Molecular Neuropathology, Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and College of Pharmaceutical Sciences, Soochow University, Suzhou, Jiangsu, China Search for more papers by this author Feng Gao Feng Gao Laboratory of Molecular Neuropathology, Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and College of Pharmaceutical Sciences, Soochow University, Suzhou, Jiangsu, China Search for more papers by this author Haigang Ren Haigang Ren Laboratory of Molecular Neuropathology, Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and College of Pharmaceutical Sciences, Soochow University, Suzhou, Jiangsu, China Search for more papers by this author Dong Chen Dong Chen Laboratory of Molecular Neuropathology, Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and College of Pharmaceutical Sciences, Soochow University, Suzhou, Jiangsu, China Search for more papers by this author Junhai Han Junhai Han Key Laboratory of Developmental Genes and Human Disease, Institute of Life Sciences, Southeast University, Nanjing, Jiangsu, China Search for more papers by this author Zheng Ying Corresponding Author Zheng Ying Laboratory of Molecular Neuropathology, Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and College of Pharmaceutical Sciences, Soochow University, Suzhou, Jiangsu, China Jiangsu Key Laboratory of Preventive and Translational Medicine for Geriatric Diseases, College of Pharmaceutical Sciences, Soochow University, Suzhou, Jiangsu, China Search for more papers by this author Guanghui Wang Corresponding Author Guanghui Wang Laboratory of Molecular Neuropathology, Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and College of Pharmaceutical Sciences, Soochow University, Suzhou, Jiangsu, China Key Laboratory of Brain Function and Disease, School of Life Sciences, University of Science & Technology of China, Chinese Academy of Sciences, Hefei, Anhui, China Search for more papers by this author Qin Xia Qin Xia Laboratory of Molecular Neuropathology, Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and College of Pharmaceutical Sciences, Soochow University, Suzhou, Jiangsu, China Search for more papers by this author Hongfeng Wang Hongfeng Wang Laboratory of Molecular Neuropathology, Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and College of Pharmaceutical Sciences, Soochow University, Suzhou, Jiangsu, China Search for more papers by this author Zongbing Hao Zongbing Hao Laboratory of Molecular Neuropathology, Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and College of Pharmaceutical Sciences, Soochow University, Suzhou, Jiangsu, China Search for more papers by this author Cheng Fu Cheng Fu Key Laboratory of Brain Function and Disease, School of Life Sciences, University of Science & Technology of China, Chinese Academy of Sciences, Hefei, Anhui, China Search for more papers by this author Qingsong Hu Qingsong Hu Laboratory of Molecular Neuropathology, Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and College of Pharmaceutical Sciences, Soochow University, Suzhou, Jiangsu, China Search for more papers by this author Feng Gao Feng Gao Laboratory of Molecular Neuropathology, Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and College of Pharmaceutical Sciences, Soochow University, Suzhou, Jiangsu, China Search for more papers by this author Haigang Ren Haigang Ren Laboratory of Molecular Neuropathology, Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and College of Pharmaceutical Sciences, Soochow University, Suzhou, Jiangsu, China Search for more papers by this author Dong Chen Dong Chen Laboratory of Molecular Neuropathology, Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and College of Pharmaceutical Sciences, Soochow University, Suzhou, Jiangsu, China Search for more papers by this author Junhai Han Junhai Han Key Laboratory of Developmental Genes and Human Disease, Institute of Life Sciences, Southeast University, Nanjing, Jiangsu, China Search for more papers by this author Zheng Ying Corresponding Author Zheng Ying Laboratory of Molecular Neuropathology, Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and College of Pharmaceutical Sciences, Soochow University, Suzhou, Jiangsu, China Jiangsu Key Laboratory of Preventive and Translational Medicine for Geriatric Diseases, College of Pharmaceutical Sciences, Soochow University, Suzhou, Jiangsu, China Search for more papers by this author Guanghui Wang Corresponding Author Guanghui Wang Laboratory of Molecular Neuropathology, Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and College of Pharmaceutical Sciences, Soochow University, Suzhou, Jiangsu, China Key Laboratory of Brain Function and Disease, School of Life Sciences, University of Science & Technology of China, Chinese Academy of Sciences, Hefei, Anhui, China Search for more papers by this author Author Information Qin Xia1, Hongfeng Wang1, Zongbing Hao1, Cheng Fu2, Qingsong Hu1, Feng Gao1, Haigang Ren1, Dong Chen1, Junhai Han3, Zheng Ying 1,4 and Guanghui Wang 1,2 1Laboratory of Molecular Neuropathology, Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and College of Pharmaceutical Sciences, Soochow University, Suzhou, Jiangsu, China 2Key Laboratory of Brain Function and Disease, School of Life Sciences, University of Science & Technology of China, Chinese Academy of Sciences, Hefei, Anhui, China 3Key Laboratory of Developmental Genes and Human Disease, Institute of Life Sciences, Southeast University, Nanjing, Jiangsu, China 4Jiangsu Key Laboratory of Preventive and Translational Medicine for Geriatric Diseases, College of Pharmaceutical Sciences, Soochow University, Suzhou, Jiangsu, China *Corresponding author. Tel: +86 512 65884845; Fax: +86 512 65884845; E-mail: [email protected] *Corresponding author. Tel: +86 512 65884845; Fax: +86 512 65884845; E-mail: [email protected] The EMBO Journal (2016)35:121-142https://doi.org/10.15252/embj.201591998 See also: N Skoko et al (January 2016) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that is characterized by selective loss of motor neurons in brain and spinal cord. TAR DNA-binding protein 43 (TDP-43) was identified as a major component of disease pathogenesis in ALS, frontotemporal lobar degeneration (FTLD), and other neurodegenerative disease. Despite the fact that TDP-43 is a multi-functional protein involved in RNA processing and a large number of TDP-43 RNA targets have been discovered, the initial toxic effect and the pathogenic mechanism underlying TDP-43-linked neurodegeneration remain elusive. In this study, we found that loss of TDP-43 strongly induced a nuclear translocation of TFEB, the master regulator of lysosomal biogenesis and autophagy, through targeting the mTORC1 key component raptor. This regulation in turn enhanced global gene expressions in the autophagy–lysosome pathway (ALP) and increased autophagosomal and lysosomal biogenesis. However, loss of TDP-43 also impaired the fusion of autophagosomes with lysosomes through dynactin 1 downregulation, leading to accumulation of immature autophagic vesicles and overwhelmed ALP function. Importantly, inhibition of mTORC1 signaling by rapamycin treatment aggravated the neurodegenerative phenotype in a TDP-43-depleted Drosophila model, whereas activation of mTORC1 signaling by PA treatment ameliorated the neurodegenerative phenotype. Taken together, our data indicate that impaired mTORC1 signaling and influenced ALP may contribute to TDP-43-mediated neurodegeneration. Synopsis RNA binding protein TDP-43 aggregates are linked to ALS. TDP-43 deficiency leads to decreased dynactin 1 expression, blocking autophagic flux; it also reduces mTORC1 activity, leading to increased autophagy and lysosome gene expression via TFEB. Abnormal regulation of autophagy contributes to TDP-43-associated neurotoxicity. TDP-43 is required for raptor mRNA stability and thus for mTORC1 activity. Loss of TDP-43 results in TFEB nuclear translocation and in increased autophagosomal and lysosomal biogenesis. Loss of TDP-43 leads to impaired autophagosome–lysosome fusion due to reduced dynactin 1 levels. The neurotoxicity of TDP-43 loss of function can be rescued by activation of mTORC1 signaling in Drosophila. Introduction Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder that is characterized by degeneration of motor neurons, leading to progressive muscle weakness, atrophy, and eventually fatal paralysis and respiratory failure. Approximately 90% of ALS cases are sporadic and the remaining 10% are familial. SOD1 gene that encodes superoxide dismutase 1 (SOD1) was the first discovered causative gene of ALS, and a breakthrough of ALS research began with the discovery of TAR DNA-binding protein-43 (TDP-43) that is encoded by TARDBP gene (Ling et al, 2013). TDP-43 was identified as a key component of the ubiquitin-positive inclusions in both ALS and frontotemporal lobar degeneration (FTLD) patients (Neumann et al, 2006), and the clinical and pathological overlap between ALS and FTLD indicated a central role for TDP-43 in diverse disease pathogenesis. TDP-43 is a RNA-binding protein, which contains two RNA recognition motifs (RRM1 and RRM2) that allow TDP-43 to bind to nucleic acids and a C-terminal glycine-rich domain (GRD) that mediates protein–protein interactions. TDP-43 functions in multiple steps of RNA processing and homeostasis including transcription, splicing, and transport of target mRNAs, such as CFTR, HDAC6, SMN, and Nefl (Bose et al, 2008; Volkening et al, 2009; Buratti & Baralle, 2010; Fiesel et al, 2010; Polymenidou et al, 2011; Alami et al, 2014). It is therefore conceivable that defects in RNA processing, due to loss of nuclear TDP-43, could be linked to neurodegeneration (Xu, 2012; Diaper et al, 2013; Vanden Broeck et al, 2013, 2014). Autophagy–lysosome pathway (ALP) is a critical cellular quality control system that is tightly associated with various neurodegenerative diseases especially in ALS, since mutations in ALP-associated genes, such as p62/SQSTM1, OPTN, and FIG 4, are the genetic cause of ALS, indicating a critical linkage between ALP and ALS disease pathogenesis (Ling et al, 2013). Recently, the transcription factor EB (TFEB) was identified as a master regulator of ALP that controls ALP by driving the expressions of autophagic gene products such as ATG5, Beclin-1, and ATG9B, as well as lysosomal gene products such as LAMP1, cathepsins, and subunits of vacuolar ATPases (Sardiello et al, 2009; Settembre et al, 2011). The cellular localization and activity of TFEB are mainly controlled by Ragulator-Rag-mTORC1 (mTOR complex 1) (Pena-Llopis et al, 2011; Martina et al, 2012; Roczniak-Ferguson et al, 2012; Settembre et al, 2012; Martina & Puertollano, 2013), an important amino acid-sensing complex on the lysosomal surface that controls cell growth, proliferation, and autophagy (Zoncu et al, 2011b; Bar-Peled & Sabatini, 2014). It has been shown that ALP and TFEB functions are affected by proteins in association with neurodegenerative diseases. For examples, presenilins in Alzheimer's disease, α-synuclein in Parkinson's disease, and polyglutamine expanded androgen receptor in spinal and bulbar muscular atrophy could affect cargo recognition, autophagosome–lysosome fusion, and TFEB nuclear localization in ALP (Wong & Cuervo, 2010; Decressac et al, 2013; Chua et al, 2014; Cortes et al, 2014). In addition, autophagic vesicle accumulation is a common feature of neurodegenerative diseases including ALS (Iguchi et al, 2013; Ling et al, 2013), indicating that ALP is involved in neurodegeneration. However, it remains unclear whether the accumulation of autophagic vesicles reflects a compensatory neuroprotective response or results from a failure of autophagic degradation. To investigate the molecular mechanism of ALP in association with loss of TDP-43 function, we comprehensively studied the effects of TDP-43 on ALP and TFEB in cellular and fly models with TDP-43 loss of function. We found that loss of TDP-43 strongly induced a nuclear translocation of TFEB by directly targeting raptor, a key component of mTORC1. TDP-43-mediated mTORC1 dysregulation could affect the expression of ALP genes and the neurotoxicity in cells and in vivo. The current study reveals a role of mTORC1 dysfunction, abnormal altered autophagy, and lysosomal biogenesis in TDP-43-mediated neurodegeneration. Results TFEB nuclear translocation in TDP-43-depleted cells Given that ALP, the critical cellular quality control system, is involved in ALS (Ling et al, 2013) and that TFEB is a master regulator of ALP and is functionally associated with neurodegenerative diseases as recently reported (Sardiello et al, 2009; Settembre et al, 2011; Tsunemi et al, 2012; Decressac et al, 2013; Spampanato et al, 2013), we wonder whether TDP-43 is able to regulate TFEB. We knocked TDP-43 down in HeLa (non-neuronal) and SH-SY5Y (neuronal) cells and examined TFEB nuclear translocation, a process that is associated with autophagy activation and lysosomal biogenesis. We observed that knockdown of TDP-43 resulted in a dramatic increase of nuclear and a decrease of cytoplasmic GFP-tagged TFEB in those cells (Fig 1A and B). Similar results were obtained using non-GFP-tagged TFEB in other types of cells (Appendix Fig S1). Consistently, similar results were obtained using subcellular fractionation assays (Fig 1C). Figure 1. Effect of TDP-43 on TFEB nuclear translocation HeLa cells were transfected with the indicated siRNAs for 48 h, and then were re-transfected with TFEB-EGFP. Twenty-four hours after transfection, the cells were fixed. DAPI (blue) was used for nuclear staining. Cells were visualized using confocal microscopy. Scale bar, 5 μm. The quantification of TFEB localization in cytoplasm and nucleus is shown on the right side. Data are from three independent experiments, means ± S.E.M.; **, P < 0.01; one-way ANOVA. Similar experiments as in (A) were performed in SH-SY5Y cells. Scale bar, 5 μm. The quantification data are shown on the right side. Data from three independent experiments represented as means ± S.E.M.; **, P < 0.01; one-way ANOVA. White arrowheads indicate nuclear localization of TFEB. HEK 293 cells were similarly transfected as in (A). Lysates from the cells were separated into cytoplasmic and nuclear fractions and then subjected to immunoblot analysis using anti-GFP, -GAPDH, and -histone 2B antibodies. The relative densities are shown on the right side. The data from three independent experiments are presented as means ± S.E.M.; **, P < 0.01; one-way ANOVA. Source data are available online for this figure. Source Data for Figure 1 [embj201591998-sup-0003-SDataFig1.pdf] Download figure Download PowerPoint TDP-43 is required for mTOR lysosomal localization and mTORC1 activity Based on the observations that mTORC1 plays an important role in the regulation of TFEB (Pena-Llopis et al, 2011; Martina et al, 2012; Roczniak-Ferguson et al, 2012; Settembre et al, 2012; Martina & Puertollano, 2013), we hypothesized that TDP-43 may regulate TFEB nuclear translocation in an mTORC1-dependent manner. To test this possibility, we knocked TDP-43 down and checked the cellular localization of mTOR, which reflects the activity of mTORC1. TDP-43-depleted cells showed a dramatic reduction of the punctate mTOR distribution that could indicate its lysosomal localization according to the previous reports (Fig 2A). Furthermore, the activity of mTORC1 was suppressed after knockdown of TDP-43, indicated by the phosphorylation of mTORC1 substrates p70S6K (Fig 2B). Given that amino acid is a well-established regulator of mTORC1 and promotes mTOR translocation to the lysosomal surface, we next examined the intracellular localization of mTOR under starvation or re-addition of amino acids in cells after starvation with or without TDP-43 knockdown. Under normal conditions, mTOR was co-localized with a lysosomal-specific marker LAMP1, indicating that mTOR is indeed localized to the lysosomal surface (Fig 2C). The enrichment of mTOR on lysosomes was impaired once cells were starved, but was restored after amino acids re-feeding (Fig 2C, upper panel). Meanwhile, mTOR failed to localize to lysosomes in TDP-43-depleted cells regardless of the presence or absence of amino acids (Fig 2C, lower panel). Consistent with these data, biochemical assays showed that the level of phosphorylated p70S6K was relatively low after starvation. Re-treatment of amino acids failed to restore p70S6K phosphorylation in TDP-43-depleted cells, whereas it significantly induced p70S6K phosphorylation in control cells (Fig 2D). Figure 2. TDP-43 regulates mTORC1 activity and localization A. HeLa cells were transfected with the indicated siRNAs. After 72 h, the cells were subjected to immunofluorescence assay using antibody against mTOR (red). DAPI (blue) was used for nuclear staining. The stained cells were visualized using confocal microscopy. Scale bar, 5 μm. B. HeLa cells were transfected as in (A). After 72 h, cells lysates were subjected to immunoblot analysis using anti-TDP-43, -p70S6K, -phosphorylated p70S6K (T389), -mTOR, -RagB, -P18, -raptor, and -GAPDH antibodies. The relative densities of phosphorylated/total p70S6K are shown in the upper-right panel, and the relative densities of mTOR, RagB, P18, raptor, and TDP-43 to GAPDH are shown in the lower right panel. The data from three independent experiments are presented as means ± S.E.M.; ns, not significantly different; *, P < 0.05; **, P < 0.01; one-way ANOVA. C. HeLa cells were transfected with the indicated siRNAs. After 48 h, the cells were re-transfected with LAMP1-RFP (red) for 24 h. The cells were then incubated with Earle's balanced salt solution (Starvation) for 2 h or re-stimulated with amino acids (starvation + amino acids) for 30 min. The cells were stained with mTOR (green) and DAPI (blue) and then visualized using confocal microscopy. Regions within the dotted boxes are magnified in the insets. Scale bar, 5 μm. D. Similar transfection and treatment as in (C) were performed, and the lysates from starved and re-feeding cells were subjected to immunoblot analysis using anti-TDP-43, -p70S6K, -phosphorylated p70S6K, and -GAPDH antibodies. The relative densities are shown on the right side. The data from three independent experiments are presented as means ± S.E.M.; ns, not significantly different; **, P < 0.01; one-way ANOVA. E. Similar transfection as in (B) was performed, and transfected cells were processed for qRT–PCR analysis. The level of TDP-43, mTOR, raptor, RagB, and P18 mRNA was quantified and normalized relative to GAPDH. The data from three independent experiments are presented as means ± S.E.M.; ns, not significantly different; **, P < 0.01; one-way ANOVA. F, G. HeLa cells were transfected with the indicated siRNAs. After 48 h, the cells were transfected with LAMP1-RFP (red), with or without HA-raptor for 24 h. The cells were stained with anti-mTOR (green) antibody and DAPI (blue) and then visualized using confocal microscopy. Regions within the dotted boxes are magnified in the insets. Scale bar, 5 μm. The quantification of mTOR on lysosomes is shown in (G). Data from three independent experiments represented as means ± S.E.M.; **, P < 0.01; one-way ANOVA. Source data are available online for this figure. Source Data for Figure 2 [embj201591998-sup-0004-SDataFig2.pdf] Download figure Download PowerPoint Raptor, the adaptor of mTORC1, is involved in TDP-43-mediated mTOR lysosomal localization Previous studies have shown that the subcellular localization and activity of mTOR are highly controlled by mTORC1 adaptor protein raptor, Rag GTPases, and Ragulator complex which is comprised of p18, p14, and MP1 proteins that reside directly on the lysosomal membrane surface (Sancak et al, 2008, 2010; Zoncu et al, 2011a; Bar-Peled et al, 2012). In Ragulator–Rag–mTORC1 complex, Ragulator anchors the Rag GTPases to lysosomes, which is essential for Rag GTPases activation through an amino acids-dependent manner. Meanwhile, raptor interacts with active Rag GTPase heterodimers in which GTP-bound RagA or RagB (very similar to RagA) associates with GDP-bound RagC or RagD (similar to RagC), and raptor targets mTOR to lysosome surface where mTOR could be activated by its activator Rheb on the lysosomes. We examined the cellular localization and expression level of components of Ragulator–Rag–mTORC1 complex in TDP-43-deficient cells. TDP-43-depleted cells showed a decrease of raptor, but not mTOR, p18, and RagB, compared with control cells (Fig 2B). Moreover, knockdown of TDP-43 did not affect the cellular localization of Rag GTPases (Fig 3D). To further investigate the effects of TDP-43 on the expression of raptor, we performed quantitative RT–PCR assays. Downregulation of raptor mRNA was observed in TDP-43-depleted cells, whereas mRNA levels of other known members of mTOR-associated machinery on the lysosomes, such as mTOR itself, RagB, p18, MP1, p14, HBXIP, C7orf59, RagA, RagC, RagD, and FLCN, were not changed (Figs 2E and EV1C). The cells treated with siRNA against raptor had diminished mTOR localization to lysosomes, showing a similarity to the cells treated with siRNA against TDP-43 (Figs 2F and G, and EV1A and B). Similar effects were also observed in cells treated with siRNAs against both TDP-43 and raptor (Figs 2F and G, and EV1A and B). Given that raptor is the key adaptor of mTORC1 and directly regulates mTOR lysosomal localization, we wondered whether TDP-43 regulates mTOR lysosomal localization in a raptor-dependent manner. In TDP-43-depleted cells, mTOR was recruited to lysosomes, indicated by a co-localization of lysosomal marker LAMP1 and CD63 after raptor re-transfection (Figs 2F and G, and EV1D and E), suggesting a specific dependence of raptor in TDP-43-mediated mTOR lysosomal localization. Figure 3. TDP-43 regulates TFEB lysosomal localization A–C. HeLa cells were transfected with the indicated siRNAs. After 48 h, the cells were transfected with TFEB-EGFP (green) for 24 h. The cells were stained with anti-mTOR (red) antibody and DAPI (blue) and then visualized using confocal microscopy. Regions within the dotted boxes are magnified in the insets. Scale bar, 5 μm. The quantification data of TFEB puncta and nuclear localization are shown in (B and C), respectively. Data from three independent experiments represented as means ± S.E.M.; **, P < 0.01; one-way ANOVA. D. HEK 293 cells were transfected with the indicated siRNAs. After 48 h, the cells were re-transfected with LAMP1-RFP (red) and constitutively active HA-GST-tagged Rag GTPase mutants (RagA Q66L + RagC S75L = RagAGTP + RagCGDP) for 24 h. The cells were stained with anti-HA (green) antibody and DAPI (blue). Cells were visualized using microscope IX71. Regions within the dotted boxes are magnified in the insets. Scale bar, 5 μm. E. HEK 293 cells were transfected with siRNA targeting TDP-43. After 48 h, the cells were re-transfected with EGFP-tagged TFEB-WT, TFEB-S211A, TFEB-Q10A/L11A, or TFEB-Δ30, along with LAMP1-RFP for 24 h. Cells were visualized using microscope IX71. Regions within the dotted boxes are magnified in the insets. Scale bar, 5 μm. F. HEK 293 cells were transfected with the indicated siRNAs. After 48 h, the cells were re-transfected with EGFP-tagged TFEB-WT or TFEB-R245-247A, along with LAMP1-RFP for 24 h. Then, the cells were incubated with Torin-1 (250 nM) for 1 h, Earle's balanced salt solution (starvation) for 2 h or incubated with both. Cells were fixed and visualized using confocal microscopy. Regions within the dotted boxes are magnified in the insets. Scale bar, 5 μm. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. TDP-43 regulates mTOR lysosomal localization and mTORC1 activity in a raptor-dependent manner HeLa cells were transfected with the indicated siRNAs. After 72 h, cell lysates were subjected to immunoblot analysis using anti-TDP-43, -raptor, and -GAPDH antibodies. HeLa cells were transfected with the indicated siRNAs. After 72 h, the cells were stained with anti-mTOR (green) antibody and DAPI (blue). Cells were fixed and visualized using confocal microscopy. Scale bar, 5 μm. Similar transfection as in (A) was performed, and transfected cells were processed for qRT–PCR analysis. The level of MP1, p14, HBXIP, C7orf59, RagA, RagC, RagD, and FLCN mRNA was quantified and normalized relative to GAPDH. The data from three independent experiments are presented as means ± S.E.M.; ns, not significantly different; one-way ANOVA. HeLa cells were transfected with the indicated siRNAs. After 48 h, the cells were transfected with CD63-GFP (green), with or without HA-raptor for 24 h. The cells were stained with anti-mTOR (red) antibody and DAPI (blue). Cells were fixed and visualized using microscope IX71. Regions within the dotted boxes are magnified in the insets. Scale bar, 5 μm. Similar transfection as in (D) was performed, and transfected cells were subjected to immunoblot analysis using anti-TDP-43, -p70S6K, -p-p70S6K, -raptor, and -GAPDH antibodies. Download figure Download PowerPoint TDP-43 regulates the localization of TFEB by targeting raptor, but not Rag GTPases As we have shown that TDP-43 controls TFEB nuclear translocation and regulates the activity of mTORC1, we wonder how TDP-43 mediates TFEB localization. Recent studies showed that in fully fed cells, most TFEB appear to diffusely distributed in the cytoplasm and a little fraction of TFEB is recruited to activated Rag GTPases on the lysosomal surface, where TFEB is phosphorylated by mTORC1 on serine 211 (S211). Phosphorylated TFEB then interacts with cytosolic 14-3-3, resulting in the sequestration of TFEB in the cytosol. However, TFEB would transport into the nucleus once the interaction is abolished, resulting in an increased expression of TFEB targeting genes (Pena-Llopis et al, 2011; Martina et al, 2012; Roczniak-Ferguson et al, 2012; Settembre et al, 2012; Martina & Puertollano, 2013). In consistence with those findings, we observed that TFEB showed a diffuse cytoplasmic distribution in si-control cells, whereas mTOR was localized to lysosomes (Fig 3A–C). In contrast, in TDP-43 knockdown cells, TFEB and mTOR localization was dramatically changed, showing that TFEB was translocated from the cytosol to the nucleus and formed lysosomal puncta in cytoplasm, whereas mTOR had a diffusively cytoplasmic distribution (Figs 3A and EV2), suggesting an involvement of mTORC1 in TDP-43-mediated TFEB nuclear translocation. Moreover, similar results were obtained in raptor knockdown cells as well as in TDP-43 and raptor double-knockdown cells (Figs 3A and EV2). To further test whether TDP-43-induced redistribution of TFEB and mTOR depends on raptor, we performed raptor re-transfection assay in TDP-43-depleted cells and examined the subcellular localization of TFEB and mTOR. After a restoration of raptor, but not active Rag GTPases (RagAGTP/RagCGDP), TFEB shuttled back to the cytoplasm (Figs 3A, EV2, and EV3A and B), suggesting a critical role of raptor in TDP-43-mediated regulation of TFEB. Based on the studies that Rag GTPases functions in the recruitment of TFEB and mTOR to lysosomes (Martina & Puertollano, 2013), we next tested whether TDP-43 could influence the lysosomal localization of Rag GTPases. The enrichment of the active Rag GTPases (RagAGTP/RagCGDP) (Martina & Puertollano, 2013) on lysosomes was not disturbed after knockdown of TDP-43, but was strongly abolished after knockdown of p18, which anchors Rag GTPases to lysosomes (Fig 3D), indicating that los
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