Knockdown of transactive response DNA-binding protein (TDP-43) downregulates histone deacetylase 6
2009; Springer Nature; Volume: 29; Issue: 1 Linguagem: Inglês
10.1038/emboj.2009.324
ISSN1460-2075
AutoresFabienne C. Fiesel, Aaron Voigt, Stephanie Weber, Chris Van den Haute, Andrea Waldenmaier, Karin Görner, Michael Walter, Marlene L Anderson, Jeannine V. Kern, Tobias M. Rasse, Thorsten Schmidt, Wolfdieter Springer, Roland Kirchner, Michael Bonin, Manuela Neumann, Veerle Baekelandt, Marianna Alunni‐Fabbroni, Jörg B. Schulz, Philipp J. Kahle,
Tópico(s)Cholinesterase and Neurodegenerative Diseases
ResumoArticle12 November 2009free access Knockdown of transactive response DNA-binding protein (TDP-43) downregulates histone deacetylase 6 Fabienne C Fiesel Corresponding Author Fabienne C Fiesel Laboratory of Functional Neurogenetics, Department of Neurodegeneration, Hertie Institute for Clinical Brain Research, Tübingen, Germany Search for more papers by this author Aaron Voigt Aaron Voigt Department of Neurology, University Medical Centre, RWTH Aachen, Aachen, Germany Search for more papers by this author Stephanie S Weber Stephanie S Weber Laboratory of Functional Neurogenetics, Department of Neurodegeneration, Hertie Institute for Clinical Brain Research, Tübingen, Germany Search for more papers by this author Chris Van den Haute Chris Van den Haute Laboratory for Neurobiology and Gene Therapy, Division of Molecular Medicine, Department of Molecular and Cellular Medicine, Katholieke Universiteit Leuven, Leuven, Belgium Search for more papers by this author Andrea Waldenmaier Andrea Waldenmaier Beckman Coulter Biomedical GmbH, Munich, Germany Search for more papers by this author Karin Görner Karin Görner Beckman Coulter Biomedical GmbH, Munich, Germany Search for more papers by this author Michael Walter Michael Walter The Microarray Facility, University of Tübingen, Tübingen, Germany Search for more papers by this author Marlene L Anderson Marlene L Anderson Laboratory of Functional Neurogenetics, Department of Neurodegeneration, Hertie Institute for Clinical Brain Research, Tübingen, Germany Search for more papers by this author Jeannine V Kern Jeannine V Kern Synaptic Plasticity Group, Hertie Institute for Clinical Brain Research, Tübingen, Germany Search for more papers by this author Tobias M Rasse Tobias M Rasse Synaptic Plasticity Group, Hertie Institute for Clinical Brain Research, Tübingen, Germany Search for more papers by this author Thorsten Schmidt Thorsten Schmidt Department of Medical Genetics, University of Tübingen, Tübingen, Germany Search for more papers by this author Wolfdieter Springer Wolfdieter Springer Laboratory of Functional Neurogenetics, Department of Neurodegeneration, Hertie Institute for Clinical Brain Research, Tübingen, Germany Search for more papers by this author Roland Kirchner Roland Kirchner Beckman Coulter Biomedical GmbH, Munich, Germany Search for more papers by this author Michael Bonin Michael Bonin The Microarray Facility, University of Tübingen, Tübingen, Germany Search for more papers by this author Manuela Neumann Manuela Neumann Institute of Neuropathology, University of Zurich, Zurich, Switzerland Search for more papers by this author Veerle Baekelandt Veerle Baekelandt Laboratory for Neurobiology and Gene Therapy, Division of Molecular Medicine, Department of Molecular and Cellular Medicine, Katholieke Universiteit Leuven, Leuven, Belgium Search for more papers by this author Marianna Alunni-Fabbroni Marianna Alunni-Fabbroni Beckman Coulter Biomedical GmbH, Munich, Germany Search for more papers by this author Jörg B Schulz Jörg B Schulz Department of Neurology, University Medical Centre, RWTH Aachen, Aachen, Germany Search for more papers by this author Philipp J Kahle Corresponding Author Philipp J Kahle Laboratory of Functional Neurogenetics, Department of Neurodegeneration, Hertie Institute for Clinical Brain Research, Tübingen, Germany Search for more papers by this author Fabienne C Fiesel Corresponding Author Fabienne C Fiesel Laboratory of Functional Neurogenetics, Department of Neurodegeneration, Hertie Institute for Clinical Brain Research, Tübingen, Germany Search for more papers by this author Aaron Voigt Aaron Voigt Department of Neurology, University Medical Centre, RWTH Aachen, Aachen, Germany Search for more papers by this author Stephanie S Weber Stephanie S Weber Laboratory of Functional Neurogenetics, Department of Neurodegeneration, Hertie Institute for Clinical Brain Research, Tübingen, Germany Search for more papers by this author Chris Van den Haute Chris Van den Haute Laboratory for Neurobiology and Gene Therapy, Division of Molecular Medicine, Department of Molecular and Cellular Medicine, Katholieke Universiteit Leuven, Leuven, Belgium Search for more papers by this author Andrea Waldenmaier Andrea Waldenmaier Beckman Coulter Biomedical GmbH, Munich, Germany Search for more papers by this author Karin Görner Karin Görner Beckman Coulter Biomedical GmbH, Munich, Germany Search for more papers by this author Michael Walter Michael Walter The Microarray Facility, University of Tübingen, Tübingen, Germany Search for more papers by this author Marlene L Anderson Marlene L Anderson Laboratory of Functional Neurogenetics, Department of Neurodegeneration, Hertie Institute for Clinical Brain Research, Tübingen, Germany Search for more papers by this author Jeannine V Kern Jeannine V Kern Synaptic Plasticity Group, Hertie Institute for Clinical Brain Research, Tübingen, Germany Search for more papers by this author Tobias M Rasse Tobias M Rasse Synaptic Plasticity Group, Hertie Institute for Clinical Brain Research, Tübingen, Germany Search for more papers by this author Thorsten Schmidt Thorsten Schmidt Department of Medical Genetics, University of Tübingen, Tübingen, Germany Search for more papers by this author Wolfdieter Springer Wolfdieter Springer Laboratory of Functional Neurogenetics, Department of Neurodegeneration, Hertie Institute for Clinical Brain Research, Tübingen, Germany Search for more papers by this author Roland Kirchner Roland Kirchner Beckman Coulter Biomedical GmbH, Munich, Germany Search for more papers by this author Michael Bonin Michael Bonin The Microarray Facility, University of Tübingen, Tübingen, Germany Search for more papers by this author Manuela Neumann Manuela Neumann Institute of Neuropathology, University of Zurich, Zurich, Switzerland Search for more papers by this author Veerle Baekelandt Veerle Baekelandt Laboratory for Neurobiology and Gene Therapy, Division of Molecular Medicine, Department of Molecular and Cellular Medicine, Katholieke Universiteit Leuven, Leuven, Belgium Search for more papers by this author Marianna Alunni-Fabbroni Marianna Alunni-Fabbroni Beckman Coulter Biomedical GmbH, Munich, Germany Search for more papers by this author Jörg B Schulz Jörg B Schulz Department of Neurology, University Medical Centre, RWTH Aachen, Aachen, Germany Search for more papers by this author Philipp J Kahle Corresponding Author Philipp J Kahle Laboratory of Functional Neurogenetics, Department of Neurodegeneration, Hertie Institute for Clinical Brain Research, Tübingen, Germany Search for more papers by this author Author Information Fabienne C Fiesel 1, Aaron Voigt2, Stephanie S Weber1, Chris Van den Haute3, Andrea Waldenmaier4, Karin Görner4, Michael Walter5, Marlene L Anderson1, Jeannine V Kern6, Tobias M Rasse6, Thorsten Schmidt7, Wolfdieter Springer1, Roland Kirchner4, Michael Bonin5, Manuela Neumann8, Veerle Baekelandt3, Marianna Alunni-Fabbroni4, Jörg B Schulz2 and Philipp J Kahle 1 1Laboratory of Functional Neurogenetics, Department of Neurodegeneration, Hertie Institute for Clinical Brain Research, Tübingen, Germany 2Department of Neurology, University Medical Centre, RWTH Aachen, Aachen, Germany 3Laboratory for Neurobiology and Gene Therapy, Division of Molecular Medicine, Department of Molecular and Cellular Medicine, Katholieke Universiteit Leuven, Leuven, Belgium 4Beckman Coulter Biomedical GmbH, Munich, Germany 5The Microarray Facility, University of Tübingen, Tübingen, Germany 6Synaptic Plasticity Group, Hertie Institute for Clinical Brain Research, Tübingen, Germany 7Department of Medical Genetics, University of Tübingen, Tübingen, Germany 8Institute of Neuropathology, University of Zurich, Zurich, Switzerland *Corresponding authors. Laboratory of Functional Neurogenetics, Department of Neurodegeneration, Hertie Institute for Clinical Brain Research, University Clinics Tübingen, Otfried-Müller-Strasse 27, 72076 Tübingen, Germany. Tel.: +49 7071 29 81968; Fax: +49 7071 29 4620; E-mail: [email protected] or Tel.: +49 7071 29 81970; Fax: +49 7071 29 4620; E-mail: [email protected] The EMBO Journal (2010)29:209-221https://doi.org/10.1038/emboj.2009.324 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 TDP-43 is an RNA/DNA-binding protein implicated in transcriptional repression and mRNA processing. Inclusions of TDP-43 are hallmarks of frontotemporal dementia and amyotrophic lateral sclerosis. Besides aggregation of TDP-43, loss of nuclear localization is observed in disease. To identify relevant targets of TDP-43, we performed expression profiling. Thereby, histone deacetylase 6 (HDAC6) downregulation was discovered on TDP-43 silencing and confirmed at the mRNA and protein level in human embryonic kidney HEK293E and neuronal SH-SY5Y cells. This was accompanied by accumulation of the major HDAC6 substrate, acetyl-tubulin. HDAC6 levels were restored by re-expression of TDP-43, dependent on RNA binding and the C-terminal protein interaction domains. Moreover, TDP-43 bound specifically to HDAC6 mRNA arguing for a direct functional interaction. Importantly, in vivo validation in TDP-43 knockout Drosophila melanogaster confirmed the specific downregulation of HDAC6. HDAC6 is necessary for protein aggregate formation and degradation. Indeed, HDAC6-dependent reduction of cellular aggregate formation and increased cytotoxicity of polyQ-expanded ataxin-3 were found in TDP-43 silenced cells. In conclusion, loss of functional TDP-43 causes HDAC6 downregulation and might thereby contribute to pathogenesis. Introduction Transactive response DNA-binding protein (TDP-43) is a 43 kDa protein abundant in most tissues and well conserved among mammals and invertebrates (Wang et al, 2004; Ayala et al, 2005). TDP-43 contains two RNA-recognition motifs (RRMs) and a C-terminal glycine-rich domain (GRD) and was originally cloned as a novel protein binding to transactivator responsive DNA sequences within human immunodeficiency virus type 1 and acting as a strong transcriptional repressor (Ou et al, 1995). In the mouse, TDP-43 mediates epigenetic transcriptional repression of the spermatid-specific acrosomal protein SP-10 in somatic tissue (Abhyankar et al, 2007). In addition to its strong DNA binding and transcriptional shut-off activity, TDP-43 binds to RNA and mediates exon skipping of the cystic fibrosis transmembrane conductance regulator (CFTR) and apolipoprotein A2 (APOA2), in cooperation with heterogenous nuclear ribonucleoprotein (hnRNP) A/B (Buratti and Baralle, 2001; Buratti et al, 2001, 2005; Mercado et al, 2005). TDP-43 not only mediates exon skipping but also exon 7 inclusion of the survival motor neuron protein 2 (SMN2) (Bose et al, 2008). The reported functions of TDP-43 are mainly nuclear, where most of the protein resides. Within the nucleus, mouse TDP-43 isoforms were localized in a diffuse and a punctuated pattern (Wang et al, 2002). The punctuate structures were designated T-bodies and suggested to serve as a scaffold connecting nuclear bodies (NBs) (Wang et al, 2002). Consistently, we find co-localization of human TDP-43 with markers of RNA processing bodies suggesting an involvement in pre-mRNA splicing, and perhaps in other RNA processing events. The identification of TDP-43 as the major protein in the neuropathological hallmark lesions of patients with frontotemporal lobar degeneration with ubiquitinated inclusions (FTLD-U, meanwhile renamed to FTLD-TDP (Mackenzie et al, 2009)) and in amyotrophic lateral sclerosis (ALS) emphasizes the importance of TDP-43 (Neumann et al, 2006). Distinct neuropathological profiles distinguish particular subtypes of sporadic and familial FTLD-U and ALS, with TDP-43 immunoreactivity being present in dystrophic neurites and neuronal cytoplasmic inclusions as well as in neuronal intranuclear inclusions (Kwong et al, 2007). Moreover, TDP-43-positive cytosolic aggregates in muscle cells have been described in familiar or sporadic inclusion body myopathies (IBM) (Weihl et al, 2008; Olivé et al, 2009). Pathological modifications of TDP-43 include ubiquitination, phosphorylation, and protein fragmentation (Neumann et al, 2006). The association of TDP-43 with disease is further substantiated genetically. Meanwhile, 30 different TDP-43 mutations, which cluster in the C-terminal GRD, have been associated with motoneuron disease, ALS, and FTD (AD&FTD mutation database http://www.molgen.ua.ac.be/FTDmutations). Most remarkably, neurons (Neumann et al, 2006, 2007) and muscle fibres (Olivé et al, 2009; Salajegheh et al, 2009) with cytosolic inclusions showed dramatic loss of nuclear TDP-43 staining. It has been suggested that cytosolic redistribution of TDP-43 is an early event in sporadic ALS, preceding the formation of insoluble aggregates (Giordana et al, 2009). Accordingly, a common neuropathological finding in FTLD-TDP are ‘preinclusions’—defined as a cell body lacking nuclear TDP-43 but with diffuse/granular cytoplasmic TDP-43 staining (Brandmeir et al, 2008). Thus, a loss of nuclear TDP-43 protein and thereby loss of nuclear function could lead to RNA processing defects in pathologic conditions. Interestingly, only recently mutations in another DNA/RNA-binding protein, fused in sarcoma/translocation in liposarcoma, have been linked to familial ALS (Kwiatkowski et al, 2009; Vance et al, 2009), further supporting the hypothesis that defects in RNA processing may have a central function in FTLD/ALS. However, the currently known TDP-43 target mRNAs have not resolved mechanistic effects of TDP-43 misfunction in neurodegenerative diseases. To gain further insight into the (patho)physiologically relevant functions of TDP-43, we depleted cells of TDP-43 by RNA interference. To identify TDP-43 responsive genes, we performed differential microarray analysis of human embryonic kidney HEK293E cells treated with siRNATDP-43 versus scrambled siRNA. Among the consistently altered genes, we identified histone deacetylase 6 (HDAC6). Specific downregulation of HDAC6 on TDP-43 silencing was confirmed at the mRNA and protein level in transiently as well as stably silenced non-neuronal and neuronal cell lines. Concomitant with the downregulation of HDAC6, TDP-43 silencing led to the accumulation of a major HDAC6 substrate, acetyl-tubulin (Hubbert et al, 2002). HDAC6 downregulation could be reverted after transfection with wild-type (wt) TDP-43, but not mutants lacking the RRMs, the GRD, or the nuclear localization signal (NLS). In pull-down and immunoprecipitation experiments we found that TDP-43 specifically bound to HDAC6 mRNA. Specific reduction of HDAC6 mRNA and protein was confirmed in vivo in Drosophila melanogaster lacking the TDP-43 ortholog gene, TBPH. The pathological consequences of reduced HDAC6 expression on TDP-43 silencing were assessed using ataxin-3 (Atx3) as experimental proteotoxic protein. In an HDAC6-dependent manner, TDP-43 knockdown in HEK293E cells reduced the formation of aggregates and increased cell death on transfection with Atx3 containing an expanded polyQ tract. Therefore, HDAC6 reduction and thus lowered cytosolic deacetylase activity might contribute to pathogenesis due to TDP-43 impairment. Results Endogenous TDP-43 in human cell lines is localized mostly in the nucleus To characterize the distribution of endogenous TDP-43, we initially performed biochemical fractionation experiments in HEK293E cells. TDP-43 was detected mostly in the nuclear fraction and very little in the cytosol (Supplementary Figure S1A). Immunostaining further confirmed the predominant nuclear localization of TDP-43 in non-neuronal HEK293E cells (Supplementary Figure S1B) as well as in neuroblastoma SH-SY5Y cells (Supplementary Figure S1C). In addition, weak but specific granular staining of endogenous TDP-43 in the cytosol was observed. Thus, TDP-43 is a mostly nuclear protein with a small fraction in the cytosol (Wang et al, 2008). Within the nucleus, endogenous TDP-43 is distributed in a punctuate pattern throughout the chromatin but excluded from nucleoli. Most cells display a discrete number of NBs with enriched TDP-43 signal. To characterize these T-bodies further, we performed dual-label confocal fluorescence microscopy of endogenous TDP-43 and marker proteins for known NBs. Those NBs have functions in small nuclear ribonucleoprotein biogenesis as the Cajal bodies and the closely associated gemini of Cajal bodies (gems), act as sites of splicing factor storage like SC35 speckles or have a role in transcriptional regulation as the promyelocytic leukaemia (PML) NBs (reviewed in Bernardi and Pandolfi, 2007; reviewed in Collins and Penny, 2009). Most overlap of T-body signal was observed with gems and Cajal bodies (Supplementary Figure 2A–F). Note that TDP-43 was completely buried within Cajal bodies (Supplementary Movie S1). Furthermore, T-bodies were closely associated with SC35 speckles, yet much SC35 signal was found without TDP-43 immunoreactivity (Supplementary Figure 2G–I). Finally, there was little overlap with PML NBs (Supplementary Figure 2J–L). Thus, in general agreement with a previous description of transfected mouse TDP-43 (Wang et al, 2002), endogenous human TDP-43 is diffusely distributed within the nucleus and enriched in NBs showing considerable co-localization with gems and Cajal bodies. Such distribution of TDP-43 is consistent with its broad functions reported on transcription and RNA processing and possibly as nuclear body scaffolding protein. Identification of TDP-43 target genes To gain insight into the physiological functions of TDP-43, we performed RNA silencing experiments in HEK293E cells. TDP-43 knockdown of up to 90% was not overtly cytotoxic and did not alter the number or morphology of TDP-43 co-localizing NBs (results not shown). As knockdown experiments did not support a general structural role of TDP-43, we screened for specific mRNA targets by microarray expression profiling. Four independent cultures of HEK293E cells were treated with siRNA against TDP-43 or control scrambled siRNA. Total RNA was extracted and each hybridized on a Human Genome U133+ 2.0 array. Knockdown efficiency was simultaneously assessed by western blot (WB) analysis of the same samples used for RNA extraction and hybridization (Supplementary Figure S3). We first examined genes associated with the spectrum of FTDs (Kumar-Singh and Van Broeckhoven, 2007) and OMIM-annotated ALS genes (http://www.ncbi.nlm.nih.gov/sites/entrez?db=omim). As expected, TDP-43 mRNA levels were reduced more than three-fold after silencing (Table I). In contrast, expressions of the known FTD/ALS-associated genes were only minimally altered on TDP-43 knockdown. Thus, TDP-43 does not directly modulate the expression of known FTD and ALS genes in siRNATDP-43-treated HEK293E cells. Table 1. Influence of TDP-43 knockdown in HEK293E cells on FTD and ALS candidate genes Candidate genes Fold change FTD-associated genes MAPT +1.25 PGRN +1.16 VCP −1.15 CHMP2B +1.08 ALS-associated genes SOD1 +1.04 ALS2 +1.16 SETX −1.05 FUS/TLS −1.27 VAPB −1.31 ANG +1.05 TARDBP (TDP-43) −3.26 FIG4 +1.08 Novel TDP-43 target gene HDAC6 −2.04 Genes associated with FTD and ALS are listed together with their signal change of the GeneChip Human Genome U133+ 2.0 microarray. The known TDP-43 splice targets, CFTR and APOA2 (Buratti and Baralle, 2001; Mercado et al, 2005), were expressed below meaningful detection threshold levels in HEK293E cells, precluding the analysis of TDP-43 knockdown effects. Expression of the TDP-43 splice target SMN2 was only minimally changed after TDP-43 knockdown, consistent with the original report (Bose et al, 2008). Alterations of neurofilament light chain expression (Strong et al, 2007) were not confirmed in siRNATDP-43-treated HEK293E cells. Likewise, the reported targets of TDP-43 from another microarray study performed in HeLa cells (Ayala et al, 2008) could not be confirmed in our experimental system (data not shown). We identified a total of 402 differentially expressed genes in cells treated with siRNATDP-43 compared with scrambled siRNA controls (Supplementary Table SI). We further validated 15 altered transcripts by RT–PCR using four different TDP-43-specific siRNAs (Supplementary Figure S4). However, 60% of the tested transcripts did not pass the employed high stringency validation criteria and were thus considered as possible off-target effects. Other transcripts were regulated by each of the four TDP-43-directed siRNAs (see Supplementary Figure S4 for details). An appealing candidate among those was HDAC6. HDAC6 is an unusual deacetylase characterized by cytoplasmic localization, ubiquitin-binding capacity, and impact on tubulin and actin cytoskeleton (Boyault et al, 2007a). Furthermore, HDAC6 has been implicated in autophagic degradation of aggregated proteins and was shown to rescue neurodegeneration in a fly model of spinal muscular atrophy (Pandey et al, 2007). Together with valosin-containing protein (VCP), another gene associated with a particular form of FTD (Watts et al, 2004), HDAC6 decides the fate of polyubiquitinated proteins (Boyault et al, 2006) and therefore putatively has an important function in proteinopathies. Owing to its functions regulating the turnover of aggregation-prone proteins associated with neurodegenerative diseases, we selected HDAC6 for further validation. Validation of HDAC6 downregulation by TDP-43 knockdown To validate the HDAC6 downregulation by TDP-43 knockdown, we treated HEK293E cells with the same siRNATDP-43 and scrambled control as for the microarray hybridizations and additionally used two more siRNAs directed against TDP-43 (Figure 1A). All siRNAs reduced TDP-43 expression and significantly downregulated HDAC6 mRNA, as visualized by semi-quantitative qRT–PCR (Figure 1B; Supplementary Figure S4) and quantitated by real-time RT–PCR (Figure 1C). This effect was confirmed at the protein level: strong knockdown of TDP-43 protein was consistently accompanied by reduced HDAC6 protein on WBs (Figure 1D). Functional depletion of the HDAC6 enzyme was shown by the concomitant increase of acetyl-tubulin (Figure 1D), a major cellular HDAC6 substrate (Hubbert et al, 2002). In addition, downregulation of HDAC6 by TDP-43 silencing was confirmed at the single cell level by dual-label confocal microscopy. Effectively silenced cells expressing low levels of TDP-43 also showed low levels of HDAC6, in contrast to adjacent non-transfected cells showing robust expression of both TDP-43 and HDAC6 (Figure 1E–G). Figure 1.Validation of HDAC6 downregulation in TDP-43 silenced HEK293E cells. (A) Positions of siRNA sequences within the TDP-43 locus. Boxes represent exons (gold, coding; pink, non-coding). (B–D) HEK293E cells were mock transfected (m), treated with scrambled (scr) siRNA, or the indicated TDP-43-directed siRNAs. Total RNA was extracted and subjected to semi-quantitative RT–PCR (B) using primer pairs amplifying TDP-43, HDAC6 and the housekeeping gene porphobilinogen deaminase (PBGD), as indicated. Relative qRT–PCR quantification (C) of TDP-43 (black bars) and HDAC6 (grey bars) mRNA was normalized to PBGD. Values represent the mean of three independent experiments. Compared with mock transfection, TDP-43 knockdown significantly reduced TDP-43 and HDAC6 qRT–PCR signals (**P<0.002; ***P<0.0001). (D) Cell lysates were western blotted and probed with antibodies against TDP-43, HDAC6, total α-tubulin, and acetyl-tubulin, as indicated. (E–G) HEK293E cells were transfected with siRNATDP-43 B and stained with mouse anti-TDP-43 (E, red) and rabbit anti-HDAC6 (F, green). Arrows point to transfected cells with strong TDP-43 knockdown, asterisks label cells with little knockdown. Nuclei were counterstained with Hoechst 33342 (blue); merged image is shown to the right (G). Size bars=10 μm. Download figure Download PowerPoint Validation of HDAC6 decrease after stable lentiviral knockdown of TDP-43 in non-neuronal and neuronal cell lines To rule out artefacts because of acute cellular stress caused by transient transfection, we validated HDAC6 downregulation in HEK293E cells with stably integrated shRNATDP-43 after lentiviral transduction. In a dose-dependent manner TDP-43 knockdown was accompanied by HDAC6 protein downregulation (Figure 2A). Even partial transduction of HEK293E cells (vector dilution 1:1024) resulted in TDP-43 knockdown (Figure 2B), strong HDAC6 decrease, and consequent acetyl-tubulin increase on single cell level as seen by dual-label confocal microscopy (Figure 2C). Stable clones of shRNATDP-43-treated HEK293E cells showed robust TDP-43 knockdown as well as strong HDAC6 mRNA (Figure 2D) and protein reduction (Figure 2F). The concomitant accumulation of the HDAC6 substrate acetyl-tubulin was comparable to the effects achieved with the specific HDAC6 inhibitor tubacin (Haggarty et al, 2003) (Figure 2F). Figure 2.HDAC6 downregulation in stably TDP-43 silenced cells. (A) HEK293E cells were treated with serial dilutions of lenti-shRNATDP-43 (lane 1, 1:4; lane 2, 1:16; lane 3, 1:64; lane 4, 1:256; lane 5, 1:1024; lane 6, 1:4096) or left untreated (−). WBs were prepared from cell lysates and probed with antibodies against TDP-43, HDAC6, α-tubulin, and acetyl-tubulin, as indicated. (B, C) HEK293E cells transduced with the 5th dilution (1:1024 of vector particles) were stained with anti-TDP-43 (B) or with anti-HDAC6 and anti-acetyl-tubulin (C). Epifluorescence shows the expression of green fluorescent protein (GFP) by the viral vector cassette. Note that even after low-dose transduction, successfully transduced HEK293E cells (GFP positive) have considerably strong downregulation of TDP-43 (B) and show also decrease in HDAC6 and increase in acetyl-tubulin (arrows in C). Scale bars=10 μm. (D–G) Single cell clones of shRNATDP-43-treated HEK293E (D, F) or SH-SY5Y neuroblastoma cells (E, G) were analysed for RNA and protein levels. (D, E) RNA was extracted from parental cell lines (−) and single clones and subjected to semi-quantitative RT–PCR using primer pairs against TDP-43, HDAC6, and the housekeeping gene PBGD. (F, G) WBs were prepared from protein lysates and probed with antibodies against TDP-43, HDAC6, total α-tubulin, and acetyl-tubulin, as indicated. Some cultures were treated for 5 h with the indicated concentrations of tubacin. Download figure Download PowerPoint We furthermore used SH-SY5Y cells for validation in a neuronal cell type. Stable silencing of TDP-43 on lentiviral transduction of shRNATDP-43 in SH-SY5Y cell clones also consistently caused downregulation of HDAC6 mRNA (Figure 2E) and protein (Figure 2G). Thus, TDP-43 knockdown downregulates HDAC6 mRNA and protein expression, and reduces HDAC6 enzyme activity in non-neuronal as well as in neuronal cells. wt TDP-43 retransfection restores HDAC6 expression To show that HDAC6 decrease on TDP-43 silencing is a specific effect, we made use of one of the siRNAs, which targets the 3′UTR of TDP-43 (siRNATDP-43 B; see Figure 1A), and thus allows efficient retransfection with TDP-43 cDNA. HDAC6 mRNA (Figure 3A) and protein (Figure 3B) could be restored after retransfection with wt TDP-43, showing the specific dependence of HDAC6 levels on TDP-43. However, overexpression of TDP-43 wt alone did not cause further HDAC6 increase, suggesting that endogenous TDP-43 amounts are already saturating for HDAC6 expression. Figure 3.HDAC6 expression is specifically restored by TDP-43 retransfection. (A–D) HEK293E cells were mock transfected (m), treated with scrambled (scr) siRNA or siRNATDP-43 B. After 24 h cells were transfected with empty vector (−), or with mutant or wt Flag-TDP-43, as indicated. (A, C). Total RNA was extracted and subjected to semi-quantitative RT–PCR amplifying endogenous TDP-43, total TDP-43, HDAC6, and the housekeeping gene PBGD, as indicated. (B, D). WBs were prepared from cell lysates and probed with antibodies against TDP-43, HDAC6, total α-tubulin, or GAPDH, as indicated. (E, F). Stably silenced shRNATDP-43 HEK293E cells were retransfected with empty vector (−) or the indicated Flag-TDP-43 constructs. Extracted RNA was used for semi-quantitatve RT–PCR (E), protein lysates for WBs (F) as described above. (G, H) HEK293E cells were mock transfected (−) or transfected with siRNATDP-43 B. After 24 h cells were transfected with empty vector (−), or with wt or C-terminally truncated (ΔGRD) Flag-TDP-43. Extracted RNA was used for semi-quantitatve RT–PCR (G), protein lysates for WBs (H), as described above. (I) HEK293E cells were mock transfected (m), treated with scrambled (scr) or siRNATDP-43 B. After 24 h, TDP-43 silenced cells were transfected with empty vector (−), Flag-TDP-43 wt, or the indicated point mutants. Total RNA was extracted and subjected to semi-quantitative RT–PCR as described above. Download figure Download PowerPoint TDP-43 contains two RRM domains that are involved in specific RNA processing functions. We have generated deletion constructs lacking RRM1 (ΔRRM1), RRM2 (ΔRRM2), and both RRMs (ΔRRM1/2). These deletion constructs failed to restore HDAC6 mRNA and protein levels on TDP-43 knockdown in transiently and stably silenced HEK293E cells (Figure 3C–F). Moreover, TDP-43 mutant variants with either impaired nuclear localization (NLSmut) (Winton et al, 2008) (Figure 3D) or lacking the C-terminal GRD (ΔGRD) (Figure 3G
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