Mice with endogenous TDP ‐43 mutations exhibit gain of splicing function and characteristics of amyotrophic lateral sclerosis
2018; Springer Nature; Volume: 37; Issue: 11 Linguagem: Inglês
10.15252/embj.201798684
ISSN1460-2075
AutoresPietro Fratta, Prasanth Sivakumar, Jack Humphrey, Kitty Lo, Thomas C. Ricketts, Hugo M. De Oliveira, J Armas, Bernadett Kalmár, Agnieszka M Ule, Yichao Yu, Nicol Birsa, Cristian Bodo, Toby Collins, Alexander E. Conicella, Alan Mejia Maza, Alessandro Marrero‐Gagliardi, Michelle Stewart, Joffrey Mianné, Silvia Corrochano, Warren Emmett, Gemma Codner, Michael J. Groves, Ryutaro Fukumura, Yoichi Gondo, Mark F. Lythgoe, Erwin Pauws, Emma Peskett, Philip Stanier, Lydia Teboul, Martina Hallegger, Andrea Calvo, Adriano Chiò, Adrian M. Isaacs, Nicolas L. Fawzi, Eric T. Wang, David E. Housman, Francisco E. Baralle, Linda Greensmith, Emanuele Buratti, Vincent Plagnol, Elizabeth Fisher, Abraham Acevedo‐Arozena,
Tópico(s)Prion Diseases and Protein Misfolding
ResumoArticle15 May 2018Open Access Source DataTransparent process Mice with endogenous TDP-43 mutations exhibit gain of splicing function and characteristics of amyotrophic lateral sclerosis Pietro Fratta Corresponding Author Pietro Fratta [email protected] orcid.org/0000-0002-8762-8188 UCL Institute of Neurology, and MRC Centre for Neuromuscular Disease, London, UK Search for more papers by this author Prasanth Sivakumar Prasanth Sivakumar UCL Institute of Neurology, and MRC Centre for Neuromuscular Disease, London, UK Search for more papers by this author Jack Humphrey Jack Humphrey UCL Institute of Neurology, and MRC Centre for Neuromuscular Disease, London, UK UCL Genetics Institute, London, UK Search for more papers by this author Kitty Lo Kitty Lo UCL Genetics Institute, London, UK Search for more papers by this author Thomas Ricketts Thomas Ricketts MRC Mammalian Genetics Unit, Harwell, UK Search for more papers by this author Hugo Oliveira Hugo Oliveira MRC Mammalian Genetics Unit, Harwell, UK Search for more papers by this author Jose M Brito-Armas Jose M Brito-Armas Unidad de Investigación, Hospital Universitario de Canarias, Fundación Canaria de Investigación Sanitaria and Instituto de Tecnologías Biomédicas (CIBICAN), La Laguna, Spain Search for more papers by this author Bernadett Kalmar Bernadett Kalmar UCL Institute of Neurology, and MRC Centre for Neuromuscular Disease, London, UK Search for more papers by this author Agnieszka Ule Agnieszka Ule UCL Institute of Neurology, and MRC Centre for Neuromuscular Disease, London, UK Search for more papers by this author Yichao Yu Yichao Yu UCL Centre for Advanced Biomedical Imaging, University College London, London, UK Search for more papers by this author Nicol Birsa Nicol Birsa UCL Institute of Neurology, and MRC Centre for Neuromuscular Disease, London, UK Search for more papers by this author Cristian Bodo Cristian Bodo UCL Institute of Neurology, and MRC Centre for Neuromuscular Disease, London, UK Search for more papers by this author Toby Collins Toby Collins UCL Institute of Neurology, and MRC Centre for Neuromuscular Disease, London, UK Search for more papers by this author Alexander E Conicella Alexander E Conicella Graduate Program in Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, RI, USA Search for more papers by this author Alan Mejia Maza Alan Mejia Maza UCL Institute of Neurology, and MRC Centre for Neuromuscular Disease, London, UK Search for more papers by this author Alessandro Marrero-Gagliardi Alessandro Marrero-Gagliardi Unidad de Investigación, Hospital Universitario de Canarias, Fundación Canaria de Investigación Sanitaria and Instituto de Tecnologías Biomédicas (CIBICAN), La Laguna, Spain Search for more papers by this author Michelle Stewart Michelle Stewart MRC Mary Lyon Centre, Harwell, UK Search for more papers by this author Joffrey Mianne Joffrey Mianne orcid.org/0000-0003-0893-9947 MRC Mary Lyon Centre, Harwell, UK Search for more papers by this author Silvia Corrochano Silvia Corrochano MRC Mammalian Genetics Unit, Harwell, UK Search for more papers by this author Warren Emmett Warren Emmett UCL Genetics Institute, London, UK Search for more papers by this author Gemma Codner Gemma Codner MRC Mary Lyon Centre, Harwell, UK Search for more papers by this author Michael Groves Michael Groves UCL Institute of Neurology, and MRC Centre for Neuromuscular Disease, London, UK Search for more papers by this author Ryutaro Fukumura Ryutaro Fukumura Mutagenesis and Genomics Team, RIKEN BioResource Center, Tsukuba, Ibaraki, Japan Search for more papers by this author Yoichi Gondo Yoichi Gondo Mutagenesis and Genomics Team, RIKEN BioResource Center, Tsukuba, Ibaraki, Japan Search for more papers by this author Mark Lythgoe Mark Lythgoe UCL Centre for Advanced Biomedical Imaging, University College London, London, UK Search for more papers by this author Erwin Pauws Erwin Pauws UCL Institute of Child Health, London, UK Search for more papers by this author Emma Peskett Emma Peskett UCL Institute of Child Health, London, UK Search for more papers by this author Philip Stanier Philip Stanier orcid.org/0000-0001-9340-8117 UCL Institute of Child Health, London, UK Search for more papers by this author Lydia Teboul Lydia Teboul MRC Mary Lyon Centre, Harwell, UK Search for more papers by this author Martina Hallegger Martina Hallegger UCL Institute of Neurology and Francis Crick Institute, London, UK Search for more papers by this author Andrea Calvo Andrea Calvo Rita Levi Montalcini Department of Neuroscience, University of Turin, Turin, Italy Search for more papers by this author Adriano Chiò Adriano Chiò Rita Levi Montalcini Department of Neuroscience, University of Turin, Turin, Italy Search for more papers by this author Adrian M Isaacs Adrian M Isaacs orcid.org/0000-0002-6820-5534 UCL Institute of Neurology, and MRC Centre for Neuromuscular Disease, London, UK UK Dementia Research Institute at UCL, UCL Institute of Neurology, London, UK Search for more papers by this author Nicolas L Fawzi Nicolas L Fawzi orcid.org/0000-0001-5483-0577 Department of Molecular Pharmacology, Physiology & Biotechnology, Brown University, Providence, RI, USA Search for more papers by this author Eric Wang Eric Wang Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA Search for more papers by this author David E Housman David E Housman Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA Search for more papers by this author Francisco Baralle Francisco Baralle International Center for Genomic Engineering and Biotechnology (ICGEB), Trieste, Italy Search for more papers by this author Linda Greensmith Linda Greensmith UCL Institute of Neurology, and MRC Centre for Neuromuscular Disease, London, UK Search for more papers by this author Emanuele Buratti Emanuele Buratti orcid.org/0000-0002-1356-9074 International Center for Genomic Engineering and Biotechnology (ICGEB), Trieste, Italy Search for more papers by this author Vincent Plagnol Vincent Plagnol UCL Genetics Institute, London, UK Search for more papers by this author Elizabeth MC Fisher Elizabeth MC Fisher UCL Institute of Neurology, and MRC Centre for Neuromuscular Disease, London, UK Search for more papers by this author Abraham Acevedo-Arozena Corresponding Author Abraham Acevedo-Arozena [email protected] orcid.org/0000-0001-6127-7116 MRC Mammalian Genetics Unit, Harwell, UK Unidad de Investigación, Hospital Universitario de Canarias, Fundación Canaria de Investigación Sanitaria and Instituto de Tecnologías Biomédicas (CIBICAN), La Laguna, Spain Search for more papers by this author Pietro Fratta Corresponding Author Pietro Fratta [email protected] orcid.org/0000-0002-8762-8188 UCL Institute of Neurology, and MRC Centre for Neuromuscular Disease, London, UK Search for more papers by this author Prasanth Sivakumar Prasanth Sivakumar UCL Institute of Neurology, and MRC Centre for Neuromuscular Disease, London, UK Search for more papers by this author Jack Humphrey Jack Humphrey UCL Institute of Neurology, and MRC Centre for Neuromuscular Disease, London, UK UCL Genetics Institute, London, UK Search for more papers by this author Kitty Lo Kitty Lo UCL Genetics Institute, London, UK Search for more papers by this author Thomas Ricketts Thomas Ricketts MRC Mammalian Genetics Unit, Harwell, UK Search for more papers by this author Hugo Oliveira Hugo Oliveira MRC Mammalian Genetics Unit, Harwell, UK Search for more papers by this author Jose M Brito-Armas Jose M Brito-Armas Unidad de Investigación, Hospital Universitario de Canarias, Fundación Canaria de Investigación Sanitaria and Instituto de Tecnologías Biomédicas (CIBICAN), La Laguna, Spain Search for more papers by this author Bernadett Kalmar Bernadett Kalmar UCL Institute of Neurology, and MRC Centre for Neuromuscular Disease, London, UK Search for more papers by this author Agnieszka Ule Agnieszka Ule UCL Institute of Neurology, and MRC Centre for Neuromuscular Disease, London, UK Search for more papers by this author Yichao Yu Yichao Yu UCL Centre for Advanced Biomedical Imaging, University College London, London, UK Search for more papers by this author Nicol Birsa Nicol Birsa UCL Institute of Neurology, and MRC Centre for Neuromuscular Disease, London, UK Search for more papers by this author Cristian Bodo Cristian Bodo UCL Institute of Neurology, and MRC Centre for Neuromuscular Disease, London, UK Search for more papers by this author Toby Collins Toby Collins UCL Institute of Neurology, and MRC Centre for Neuromuscular Disease, London, UK Search for more papers by this author Alexander E Conicella Alexander E Conicella Graduate Program in Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, RI, USA Search for more papers by this author Alan Mejia Maza Alan Mejia Maza UCL Institute of Neurology, and MRC Centre for Neuromuscular Disease, London, UK Search for more papers by this author Alessandro Marrero-Gagliardi Alessandro Marrero-Gagliardi Unidad de Investigación, Hospital Universitario de Canarias, Fundación Canaria de Investigación Sanitaria and Instituto de Tecnologías Biomédicas (CIBICAN), La Laguna, Spain Search for more papers by this author Michelle Stewart Michelle Stewart MRC Mary Lyon Centre, Harwell, UK Search for more papers by this author Joffrey Mianne Joffrey Mianne orcid.org/0000-0003-0893-9947 MRC Mary Lyon Centre, Harwell, UK Search for more papers by this author Silvia Corrochano Silvia Corrochano MRC Mammalian Genetics Unit, Harwell, UK Search for more papers by this author Warren Emmett Warren Emmett UCL Genetics Institute, London, UK Search for more papers by this author Gemma Codner Gemma Codner MRC Mary Lyon Centre, Harwell, UK Search for more papers by this author Michael Groves Michael Groves UCL Institute of Neurology, and MRC Centre for Neuromuscular Disease, London, UK Search for more papers by this author Ryutaro Fukumura Ryutaro Fukumura Mutagenesis and Genomics Team, RIKEN BioResource Center, Tsukuba, Ibaraki, Japan Search for more papers by this author Yoichi Gondo Yoichi Gondo Mutagenesis and Genomics Team, RIKEN BioResource Center, Tsukuba, Ibaraki, Japan Search for more papers by this author Mark Lythgoe Mark Lythgoe UCL Centre for Advanced Biomedical Imaging, University College London, London, UK Search for more papers by this author Erwin Pauws Erwin Pauws UCL Institute of Child Health, London, UK Search for more papers by this author Emma Peskett Emma Peskett UCL Institute of Child Health, London, UK Search for more papers by this author Philip Stanier Philip Stanier orcid.org/0000-0001-9340-8117 UCL Institute of Child Health, London, UK Search for more papers by this author Lydia Teboul Lydia Teboul MRC Mary Lyon Centre, Harwell, UK Search for more papers by this author Martina Hallegger Martina Hallegger UCL Institute of Neurology and Francis Crick Institute, London, UK Search for more papers by this author Andrea Calvo Andrea Calvo Rita Levi Montalcini Department of Neuroscience, University of Turin, Turin, Italy Search for more papers by this author Adriano Chiò Adriano Chiò Rita Levi Montalcini Department of Neuroscience, University of Turin, Turin, Italy Search for more papers by this author Adrian M Isaacs Adrian M Isaacs orcid.org/0000-0002-6820-5534 UCL Institute of Neurology, and MRC Centre for Neuromuscular Disease, London, UK UK Dementia Research Institute at UCL, UCL Institute of Neurology, London, UK Search for more papers by this author Nicolas L Fawzi Nicolas L Fawzi orcid.org/0000-0001-5483-0577 Department of Molecular Pharmacology, Physiology & Biotechnology, Brown University, Providence, RI, USA Search for more papers by this author Eric Wang Eric Wang Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA Search for more papers by this author David E Housman David E Housman Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA Search for more papers by this author Francisco Baralle Francisco Baralle International Center for Genomic Engineering and Biotechnology (ICGEB), Trieste, Italy Search for more papers by this author Linda Greensmith Linda Greensmith UCL Institute of Neurology, and MRC Centre for Neuromuscular Disease, London, UK Search for more papers by this author Emanuele Buratti Emanuele Buratti orcid.org/0000-0002-1356-9074 International Center for Genomic Engineering and Biotechnology (ICGEB), Trieste, Italy Search for more papers by this author Vincent Plagnol Vincent Plagnol UCL Genetics Institute, London, UK Search for more papers by this author Elizabeth MC Fisher Elizabeth MC Fisher UCL Institute of Neurology, and MRC Centre for Neuromuscular Disease, London, UK Search for more papers by this author Abraham Acevedo-Arozena Corresponding Author Abraham Acevedo-Arozena [email protected] orcid.org/0000-0001-6127-7116 MRC Mammalian Genetics Unit, Harwell, UK Unidad de Investigación, Hospital Universitario de Canarias, Fundación Canaria de Investigación Sanitaria and Instituto de Tecnologías Biomédicas (CIBICAN), La Laguna, Spain Search for more papers by this author Author Information Pietro Fratta *,1,‡,‡, Prasanth Sivakumar1,‡, Jack Humphrey1,2,‡, Kitty Lo2,‡, Thomas Ricketts3,‡, Hugo Oliveira3,‡, Jose M Brito-Armas4, Bernadett Kalmar1, Agnieszka Ule1, Yichao Yu5, Nicol Birsa1, Cristian Bodo1, Toby Collins1, Alexander E Conicella6, Alan Mejia Maza1, Alessandro Marrero-Gagliardi4, Michelle Stewart7, Joffrey Mianne7, Silvia Corrochano3, Warren Emmett2, Gemma Codner7, Michael Groves1, Ryutaro Fukumura8, Yoichi Gondo8, Mark Lythgoe5, Erwin Pauws9, Emma Peskett9, Philip Stanier9, Lydia Teboul7, Martina Hallegger10, Andrea Calvo11, Adriano Chiò11, Adrian M Isaacs1,12, Nicolas L Fawzi13, Eric Wang14, David E Housman14, Francisco Baralle15, Linda Greensmith1, Emanuele Buratti15, Vincent Plagnol2, Elizabeth MC Fisher1,‡ and Abraham Acevedo-Arozena *,3,4,‡ 1UCL Institute of Neurology, and MRC Centre for Neuromuscular Disease, London, UK 2UCL Genetics Institute, London, UK 3MRC Mammalian Genetics Unit, Harwell, UK 4Unidad de Investigación, Hospital Universitario de Canarias, Fundación Canaria de Investigación Sanitaria and Instituto de Tecnologías Biomédicas (CIBICAN), La Laguna, Spain 5UCL Centre for Advanced Biomedical Imaging, University College London, London, UK 6Graduate Program in Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, RI, USA 7MRC Mary Lyon Centre, Harwell, UK 8Mutagenesis and Genomics Team, RIKEN BioResource Center, Tsukuba, Ibaraki, Japan 9UCL Institute of Child Health, London, UK 10UCL Institute of Neurology and Francis Crick Institute, London, UK 11Rita Levi Montalcini Department of Neuroscience, University of Turin, Turin, Italy 12UK Dementia Research Institute at UCL, UCL Institute of Neurology, London, UK 13Department of Molecular Pharmacology, Physiology & Biotechnology, Brown University, Providence, RI, USA 14Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA 15International Center for Genomic Engineering and Biotechnology (ICGEB), Trieste, Italy ‡These authors contributed equally to this work ‡These authors contributed equally to this work *Corresponding author. Tel: +44 2034 484112; E-mail: [email protected] *Corresponding author. Tel: +34 9226 78108; E-mail: [email protected] The EMBO Journal (2018)37:e98684https://doi.org/10.15252/embj.201798684 See also: C Rouaux et al (June 2018) 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 TDP-43 (encoded by the gene TARDBP) is an RNA binding protein central to the pathogenesis of amyotrophic lateral sclerosis (ALS). However, how TARDBP mutations trigger pathogenesis remains unknown. Here, we use novel mouse mutants carrying point mutations in endogenous Tardbp to dissect TDP-43 function at physiological levels both in vitro and in vivo. Interestingly, we find that mutations within the C-terminal domain of TDP-43 lead to a gain of splicing function. Using two different strains, we are able to separate TDP-43 loss- and gain-of-function effects. TDP-43 gain-of-function effects in these mice reveal a novel category of splicing events controlled by TDP-43, referred to as "skiptic" exons, in which skipping of constitutive exons causes changes in gene expression. In vivo, this gain-of-function mutation in endogenous Tardbp causes an adult-onset neuromuscular phenotype accompanied by motor neuron loss and neurodegenerative changes. Furthermore, we have validated the splicing gain-of-function and skiptic exons in ALS patient-derived cells. Our findings provide a novel pathogenic mechanism and highlight how TDP-43 gain of function and loss of function affect RNA processing differently, suggesting they may act at different disease stages. Synopsis Point mutations within the low complexity C-terminal domain of TDP-43 induce phenotypes resembling amyotrophic lateral sclerosis and motor neuron disease in mice. Molecularly, these mutations induce a novel gain of splicing activity leading to skipping of certain constitutive exons, a phenomenon designated as "skiptic" exons here. Two new mouse lines express endogenous TDP-43 with mutations in either the RNA-recognition motif or in the C-terminal low complexity domain. Mice carrying a C-terminal TDP-43 mutation develop a progressive neuromuscular phenotype accompanied by loss of motor neurons. Motor neuron degeneration occurs in the absence of TDP-43 splicing loss-of-function. Endogenous TDP-43 C-terminal mutations induce gain-of-function splicing activity. TDP-43 gain-of-function activity leads to the skipping of constitutive exons, referred to as "skiptic" exons. Introduction TDP-43 is a ubiquitously expressed, predominantly nuclear, RNA binding protein, which is involved in multiple steps of RNA processing and maturation, including transcription and splicing (Ratti & Buratti, 2016; Ederle & Dormann, 2017). TDP-43 has two RNA binding domains (RRM1 and RRM2) and a C-terminal low complexity glycine-rich domain (LCD), in which mutations causative for amyotrophic lateral sclerosis (ALS) are clustered (Ratti & Buratti, 2016; Ederle & Dormann, 2017). Mutations in TARDBP account for a small percentage (< 5%) of ALS cases; however, cytoplasmic TDP-43 inclusions, accompanied by nuclear depletion of the protein, are the key pathological hallmark of > 98% of ALS indicating the profound importance of TDP-43 in pathogenesis (Mackenzie et al, 2010; Sreedharan et al, 2008); furthermore, TDP-43 deposition has been consistently described in a range of other neurodegenerative diseases most prominently frontotemporal dementia (Neumann et al, 2006). Despite this, the mechanism(s) by which TDP-43 mutations lead to disease remains unknown, and both nuclear loss of function (LOF) and cytoplasmic gain of function (GOF) have been proposed to play a role (Ling et al, 2013). TDP-43 deposition and nuclear depletion in post-mortem tissue support a potential role for LOF in end-stage ALS. However, little is known about the early-stage effects of TARDBP mutations, including the impact on RNA metabolism (Koyama et al, 2016). TDP-43 is extremely dosage sensitive and is tightly autoregulated by a mechanism that involves TDP-43 protein binding to its own 3′UTR; these properties have proven challenging for studying the effects of TARDBP mutations in vivo (Ayala et al, 2011; Polymenidou et al, 2011; Avendaño-Vázquez et al, 2012). Thus, the main approach to studying LOF, by using null mice, has been hampered because TDP-43 knockout (KO) causes very early embryonic lethality, and heterozygous mice have normal levels of TDP-43 due to autoregulation (Kraemer et al, 2010; Sephton et al, 2010; Wu et al, 2010; Ricketts et al, 2014). However, conditional KO strategies and downregulation of TDP-43 via siRNA suggest that acute TDP-43 loss of function could lead to neurodegeneration (Chiang et al, 2010; Wu et al, 2012; Iguchi et al, 2013; Yang et al, 2014). Similarly, in transgenic mice, even low levels of overexpression of wild-type (WT) or mutant TDP-43 cause multiple RNA changes, making it difficult to identify the pathogenic RNA profile that occurs physiologically in disease (Arnold et al, 2013). Here, we have addressed these issues by working with animals from an allelic series carrying point mutations within mouse endogenous Tardbp to dissect the molecular effects of TDP-43 LOF and GOF in vivo in a physiological setting and in the absence of the confounding effects of ectopically expressed transgenes. One of our novel mouse mutants has decreased TDP-43 RNA binding capacity, which allowed us to characterise TDP-43 LOF. A second strain carries a mutation within the C-terminal low complexity glycine-rich domain (LCD) of TDP-43, which induces a splicing GOF. The analysis of this GOF uncovered entirely novel splicing events in genes not known to be controlled by TDP-43, such that a set of constitutive exons—here named "skiptic exons" (SE)—are skipped due to TDP-43 GOF. Remarkably, the skipping of constitutive exons induced by GOF is in contrast to the previously identified TDP-43 LOF-induced cryptic exons, which are normally repressed but in LOF are aberrantly included in mRNAs (Ling et al, 2015; Humphrey et al, 2017). Furthermore, and importantly for modelling of TDP-43 disease processes, our LCD mutant mouse strain with a GOF develops a progressive neuromuscular phenotype, in the absence of LOF changes, thus showing that GOF is sufficient to initiate pathogenesis. Lastly, we show that splicing GOF and SEs occur also in another C-terminal ALS-causative Tardbp mutation and in fibroblasts derived from ALS patients carrying TARDBP mutations. Our results shed light on novel aspects of TDP-43 biology and provide powerful new tools to gain insight into the early stages of TDP-43 neurodegeneration in vivo. Results Novel endogenous missense mutations in TDP-43 RRM2 and LCD To study the effects of mutations in different domains of TDP-43 in vivo, at physiological levels, we selected two endogenous Tardbp mutations from a mouse allelic series found by screening mutagenised DNA archives from two large N-ethyl-N-nitrosourea (ENU) mouse mutagenesis programmes (Acevedo-Arozena et al, 2008; Gondo et al, 2010). The first mutation (F210I) is located in the important RNA recognition motif 2 (RRM2) of TDP-43; hence, we refer to this mutation as RRM2mut hereafter (Fig 1A). Figure 1. Novel endogenous mutations RRM2mut and LCDmut have a loss- and gain-of-function effect on splicing Diagram of TDP-43 illustrates the location of non-synonymous mutations used in this study. Agarose gel and quantification of CFTR minigene splicing assay performed on MEFs from RRM2mut and LCDmut homozygous (HOM), heterozygous (HET) and littermate (WT) controls show an increase in exon inclusion in RRM2mut and a decrease in LCDmut. RRM2mut ANOVA P < 0.0001; LCDmut ANOVA P = 0.0166; Bonferroni multiple comparison tests are plotted as: *P < 0.05; **P < 0.01; ***P < 0.001; mean and SEM plotted; N = 3 per genotype. Quantification of Sortilin 1 RT–PCR products from RRM2mut and LCDmut MEFs shows exon 18 inclusion increases in RRM2mut and decreases in LCDmut. RRM2mut and LCDmut ANOVA P < 0.0001; Bonferroni multiple comparison tests are plotted as: **P < 0.01; ***P < 0.001; mean and SEM plotted; N = 3 per genotype. EMSA assay performed with increasing quantities of recombinant WT and RRM2mut TDP-43 with radiolabelled (UG)6 RNA and TDPBR RNA shows reduced RNA binding of recombinant RRM2mut. Scatter plot of pentamer enrichment in TDP-43 iCLIP shows a canonical TDP-43 pattern of enrichment in TG/GT-containing pentamers in RRM2mut (left) and LCDmut (right) along with littermate WT controls. Mapping of TDP-43 binding sites in RRM2mut (left) and LCDmut (right) along with littermate WT controls iCLIP datasets shows a similar distribution genome-wide. Source data are available online for this figure. Source Data for Figure 1 [embj201798684-sup-0003-SDataFig1.pdf] Download figure Download PowerPoint The second mutation results in an M323K change in TDP-43 and falls within the C-terminal LCD where almost all known ALS-causing mutations are clustered—specifically within a 20 amino acid stretch that influences TDP-43 alpha-helix structure which is important for liquid phase separation, aggregation and protein self-interaction (Conicella et al, 2016). Importantly, we found in an in vitro turbidity assay that the M323K mutation containing C-terminal domain decreased the propensity for phase separation compared to wild type—similarly to the human ALS-causing mutation TDP-43 Q331K (Appendix Fig S1). Hereafter, we refer the M323K mutation strain as LCDmut (Fig 1A). To generate cohorts of animals for in vivo analysis, we produced founder mice from the ENU programmes' sperm archives and generated litters by in vitro fertilisation; F1 mice were then backcrossed for at least five generations to eliminate non-linked ENU mutations and to place the mutations on congenic inbred backgrounds. We found both mutations, RRM2mut and LCDmut, were embryonic lethal on a congenic C57BL/6J background, supporting a significant impact of both amino acid changes on TDP-43 function (Appendix Table S1). Genetic background effects can profoundly modify phenotype, and there are several reports in the literature of different backgrounds, or combinations of backgrounds, rescuing embryonic lethality (LeCouter et al, 1998). To determine whether we could produce homozygous animals by changing genetic background, we undertook a standard mouse genetics approach and backcrossed both RRM2mut and LCDmut lines onto C57BL/6J and onto DBA/2J for at least five generations and then worked with F1 C57BL/6J-DBA/2J intercrosses to produce homozygotes. We found the RRM2mut allele remained homozygous embryonic lethal and no live mice were detected. However, LCDmut homozygous mice were viable in this mixed genetic background (Appendix Table S1). RRM2mut and LCDmut cause splicing LOF and GOF, respectively To investigate the effects of these TDP-43 mutations on splicing in an endogenous physiological context, we performed the CFTR minigene assay in mouse embryonic fibroblasts (MEFs). RRM2mut caused a highly significant shift towards exon inclusion, similar to the well-documented effect of TDP-43 knockdown (Appendix Fig S2), showing a dose-dependent LOF (Fig 1B). Surprisingly, the LCDmut showed an opposite effect, leading to an increase in exon exclusion, suggesting GOF (Fig 1B). We then investigated by RT–PCR whether a similar effect was present in a well-characterised endogenous TDP-43 splicing target, exon 18 of Sortilin 1. Exon 18 inclusion was increased in RRM2mut, as previously described in TDP-43 knockdown (Polymenidou et al, 2011), but had an opposite change in LCDmut, further supporting the splicing GOF (Fig 1C). Since TDP-43 protein levels appeared to be unchanged in both lines in MEFs (Appendix Fig S3), we performed electromobility shift assays (EMSA) to evaluate the RNA binding capacity of WT, RRM2mut and LCDmut recombinant TDP-43 (Buratti & Baralle, 2001). RNA binding was reduced in RRM2mut TDP-43, whereas it was unchanged with the LCDmut mutation (Fig 1D and Appendix Fig S4). To address whether the reduced RNA binding capacity in the RRM2mut was associated with changes in RNA binding specificity, we performed TDP-43 iCLIP (Tollervey et al, 2011) on CNS tissue. As RRM2mut homozygotes are not viable, we performed iCLIP in embryonic brains for that strain. However, as homozygous LCDmut are viable on a hybrid background, we were able to perform iCLIP on adult brain—the most relevant age for the effects of the TDP-43 mutation. The iCLIP data cannot be used to draw quantitative binding conclusions, but in each case, the binding motifs and transcriptome-wide distribution of RRM2mut and LCDmut TDP-43 binding sites within exons, introns, UTRs and intergenic regions showed no changes from wild-type TDP-43 (Fig 1E and F). Thus, RRM2mut is a hypomorphic allele, as it shows reduced RNA binding affinity whilst maintaining the typical TDP-43 sequence recognition and leads to a splicing loss of function. LCDmut instead induces a splicing gain of function whilst maintaining normal RNA binding capacity and specificity. RRM2mut and LCDmut show opposing effects transcriptome-wide and counteracting biological effects We next sought to directly compare our two mutants to a bona fide TDP-43 LOF. We could not use TDP-43 null mice as they die before E6.5, so we performed RNA-seq on MEFs homozygous for the RRM2mut and the LCDmut alleles comparing them each to their wild-type controls, in parallel with wild-type MEFs in which we induced TDP-43 knockdown with shRNA (Tardbp-shRNA) comparing them to scramble sequence shRNA (Appendix Fig S5). Two-way comparisons showed that RRM2mut aligns with Tardbp-shRNA, with all significantly differentially expressed exons in both datasets up- or downregulated in agreement. Conversely and strikingly, LCDmut showed all of the significant differentially expressed exons changing in the opposing direction to RRM2mut and to Tardbp-shRNA (Fig 2A, Appendix Table S2). Figure 2. TDP-43 RRM2mut and LCDmut show opposing effects transcriptome-wide and counteracting biological effects Two-way comparisons of MEF single exon differential expression changes between RRM2mut, LCDmut and TDP-43
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