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Inhibition of RNA lariat debranching enzyme suppresses TDP-43 toxicity in ALS disease models

Abstract

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease primarily affecting motor neurons. Mutations in the gene encoding TDP-43 cause some forms of the disease, and cytoplasmic TDP-43 aggregates accumulate in degenerating neurons of most individuals with ALS. Thus, strategies aimed at targeting the toxicity of cytoplasmic TDP-43 aggregates may be effective. Here, we report results from two genome-wide loss-of-function TDP-43 toxicity suppressor screens in yeast. The strongest suppressor of TDP-43 toxicity was deletion of DBR1, which encodes an RNA lariat debranching enzyme. We show that, in the absence of Dbr1 enzymatic activity, intronic lariats accumulate in the cytoplasm and likely act as decoys to sequester TDP-43, preventing it from interfering with essential cellular RNAs and RNA-binding proteins. Knockdown of Dbr1 in a human neuronal cell line or in primary rat neurons is also sufficient to rescue TDP-43 toxicity. Our findings provide insight into TDP-43–mediated cytotoxicity and suggest that decreasing Dbr1 activity could be a potential therapeutic approach for ALS.

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Figure 1: DBR1 deletion suppresses TDP-43 toxicity in yeast.
Figure 2: DBR1 knockdown reduces TDP-43 toxicity in a human neuronal cell line.
Figure 3: Dbr1 knockdown reduces TDP-43 toxicity in primary rat neurons.
Figure 4: Dbr1 lariat debranching enzymatic activity is required for TDP-43 toxicity, and TDP-43 toxicity suppression is mediated by lariat intron accumulation.
Figure 5: Intronic lariats colocalize with TDP-43 cytoplasmic foci in yeast.

References

  1. Boillée, S., Vande Velde, C. & Cleveland, D.W. ALS: a disease of motor neurons and their nonneuronal neighbors. Neuron 52, 39–59 (2006).

    PubMed  Google Scholar 

  2. Rosen, D.R. et al. Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis. Nature 362, 59–62 (1993).

    CAS  PubMed  Google Scholar 

  3. Bruijn, L.I. et al. Aggregation and motor neuron toxicity of an ALS-linked SOD1 mutant independent from wild-type SOD1. Science 281, 1851–1854 (1998).

    CAS  PubMed  Google Scholar 

  4. Smith, R.A. et al. Antisense oligonucleotide therapy for neurodegenerative disease. J. Clin. Invest. 116, 2290–2296 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Lagier-Tourenne, C., Polymenidou, M. & Cleveland, D.W. TDP-43 and FUS/TLS: emerging roles in RNA processing and neurodegeneration. Hum. Mol. Genet. 19, R46–R64 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Gitler, A.D. & Shorter, J. RNA-binding proteins with prion-like domains in ALS and FTLD-U. Prion 5, 179–187 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Mackenzie, I.R. et al. Pathological TDP-43 distinguishes sporadic amyotrophic lateral sclerosis from amyotrophic lateral sclerosis with SOD1 mutations. Ann. Neurol. 61, 427–434 (2007).

    CAS  PubMed  Google Scholar 

  8. Neumann, M. et al. Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Science 314, 130–133 (2006).

    CAS  PubMed  Google Scholar 

  9. Pesiridis, G.S., Lee, V.M. & Trojanowski, J.Q. Mutations in TDP-43 link glycine-rich domain functions to amyotrophic lateral sclerosis. Hum. Mol. Genet. 18, R156–R162 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Rutherford, N.J. et al. Novel mutations in TARDBP (TDP-43) in patients with familial amyotrophic lateral sclerosis. PLoS Genet. 4, e1000193 (2008).

    PubMed  PubMed Central  Google Scholar 

  11. Sreedharan, J. et al. TDP-43 mutations in familial and sporadic amyotrophic lateral sclerosis. Science 319, 1668–1672 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Van Deerlin, V.M. et al. TARDBP mutations in amyotrophic lateral sclerosis with TDP-43 neuropathology: a genetic and histopathological analysis. Lancet Neurol. 7, 409–416 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Yokoseki, A. et al. TDP-43 mutation in familial amyotrophic lateral sclerosis. Ann. Neurol. 63, 538–542 (2008).

    CAS  PubMed  Google Scholar 

  14. Belzil, V.V. et al. Mutations in FUS cause FALS and SALS in French and French Canadian populations. Neurology 73, 1176–1179 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Corrado, L. et al. Mutations of FUS gene in sporadic amyotrophic lateral sclerosis. J. Med. Genet. 47, 190–194 (2010).

    CAS  PubMed  Google Scholar 

  16. Hewitt, C. et al. Novel FUS/TLS mutations and pathology in familial and sporadic amyotrophic lateral sclerosis. Arch. Neurol. 67, 455–461 (2010).

    PubMed  Google Scholar 

  17. Kwiatkowski, T.J. Jr. et al. Mutations in the FUS/TLS gene on chromosome 16 cause familial amyotrophic lateral sclerosis. Science 323, 1205–1208 (2009).

    CAS  PubMed  Google Scholar 

  18. Vance, C. et al. Mutations in FUS, an RNA processing protein, cause familial amyotrophic lateral sclerosis type 6. Science 323, 1208–1211 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Johnson, B.S., McCaffery, J.M., Lindquist, S. & Gitler, A.D. A yeast TDP-43 proteinopathy model: exploring the molecular determinants of TDP-43 aggregation and cellular toxicity. Proc. Natl. Acad. Sci. USA 105, 6439–6444 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Johnson, B.S. et al. TDP-43 is intrinsically aggregation-prone, and amyotrophic lateral sclerosis–linked mutations accelerate aggregation and increase toxicity. J. Biol. Chem. 284, 20329–20339 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Sun, Z. et al. Molecular determinants and genetic modifiers of aggregation and toxicity for the ALS disease protein FUS/TLS. PLoS Biol. 9, e1000614 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Elden, A.C. et al. Ataxin-2 intermediate-length polyglutamine expansions are associated with increased risk for ALS. Nature 466, 1069–1075 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Lagier-Tourenne, C. & Cleveland, D.W. Neurodegeneration: an expansion in ALS genetics. Nature 466, 1052–1053 (2010).

    CAS  PubMed  Google Scholar 

  24. Willingham, S., Outeiro, T.F., DeVit, M.J., Lindquist, S.L. & Muchowski, P.J. Yeast genes that enhance the toxicity of a mutant huntingtin fragment or α-synuclein. Science 302, 1769–1772 (2003).

    CAS  PubMed  Google Scholar 

  25. Giorgini, F., Guidetti, P., Nguyen, Q., Bennett, S.C. & Muchowski, P.J. A genomic screen in yeast implicates kynurenine 3-monooxygenase as a therapeutic target for Huntington disease. Nat. Genet. 37, 526–531 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Tong, A.H. et al. Systematic genetic analysis with ordered arrays of yeast deletion mutants. Science 294, 2364–2368 (2001).

    CAS  PubMed  Google Scholar 

  27. Schuldiner, M. et al. Exploration of the function and organization of the yeast early secretory pathway through an epistatic miniarray profile. Cell 123, 507–519 (2005).

    CAS  PubMed  Google Scholar 

  28. Barmada, S.J. et al. Cytoplasmic mislocalization of TDP-43 is toxic to neurons and enhanced by a mutation associated with familial amyotrophic lateral sclerosis. J. Neurosci. 30, 639–649 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Arrasate, M., Mitra, S., Schweitzer, E.S., Segal, M.R. & Finkbeiner, S. Inclusion body formation reduces levels of mutant huntingtin and the risk of neuronal death. Nature 431, 805–810 (2004).

    CAS  PubMed  Google Scholar 

  30. Arenas, J. & Hurwitz, J. Purification of a RNA debranching activity from HeLa cells. J. Biol. Chem. 262, 4274–4279 (1987).

    CAS  PubMed  Google Scholar 

  31. Chapman, K.B. & Boeke, J.D. Isolation and characterization of the gene encoding yeast debranching enzyme. Cell 65, 483–492 (1991).

    CAS  PubMed  Google Scholar 

  32. Domdey, H. et al. Lariat structures are in vivo intermediates in yeast pre-mRNA splicing. Cell 39, 611–621 (1984).

    CAS  PubMed  Google Scholar 

  33. Khalid, M.F., Damha, M.J., Shuman, S. & Schwer, B. Structure-function analysis of yeast RNA debranching enzyme (Dbr1), a manganese-dependent phosphodiesterase. Nucleic Acids Res. 33, 6349–6360 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Franks, T.M., Singh, G. & Lykke-Andersen, J. Upf1 ATPase-dependent mRNP disassembly is required for completion of nonsense-mediated mRNA decay. Cell 143, 938–950 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Gatfield, D. & Izaurralde, E. Nonsense-mediated messenger RNA decay is initiated by endonucleolytic cleavage in Drosophila. Nature 429, 575–578 (2004).

    CAS  PubMed  Google Scholar 

  36. Lejeune, F., Li, X. & Maquat, L.E. Nonsense-mediated mRNA decay in mammalian cells involves decapping, deadenylating, and exonucleolytic activities. Mol. Cell 12, 675–687 (2003).

    CAS  PubMed  Google Scholar 

  37. Larimer, F.W., Hsu, C.L., Maupin, M.K. & Stevens, A. Characterization of the XRN1 gene encoding a 5′→3′ exoribonuclease: sequence data and analysis of disparate protein and mRNA levels of gene-disrupted yeast cells. Gene 120, 51–57 (1992).

    CAS  PubMed  Google Scholar 

  38. van Dijk, E.L. et al. XUTs are a class of Xrn1-sensitive antisense regulatory non-coding RNA in yeast. Nature 475, 114–117 (2011).

    CAS  PubMed  Google Scholar 

  39. Igaz, L.M. et al. Dysregulation of the ALS-associated gene TDP-43 leads to neuronal death and degeneration in mice. J. Clin. Invest. 121, 726–738 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Zhang, Y.J. et al. Aberrant cleavage of TDP-43 enhances aggregation and cellular toxicity. Proc. Natl. Acad. Sci. USA 106, 7607–7612 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Winton, M.J. et al. Disturbance of nuclear and cytoplasmic TAR DNA-binding protein (TDP-43) induces disease-like redistribution, sequestration, and aggregate formation. J. Biol. Chem. 283, 13302–13309 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Haim, L., Zipor, G., Aronov, S. & Gerst, J.E. A genomic integration method to visualize localization of endogenous mRNAs in living yeast. Nat. Methods 4, 409–412 (2007).

    CAS  PubMed  Google Scholar 

  43. Haim-Vilmovsky, L. & Gerst, J.E. m-TAG: a PCR-based genomic integration method to visualize the localization of specific endogenous mRNAs in vivo in yeast. Nat. Protoc. 4, 1274–1284 (2009).

    CAS  PubMed  Google Scholar 

  44. Haim-Vilmovsky, L. & Gerst, J.E. Visualizing endogenous mRNAs in living yeast using m-TAG, a PCR-based RNA aptamer integration method, and fluorescence microscopy. Methods Mol. Biol. 714, 237–247 (2011).

    CAS  PubMed  Google Scholar 

  45. Mayas, R.M., Maita, H., Semlow, D.R. & Staley, J.P. Spliceosome discards intermediates via the DEAH box ATPase Prp43p. Proc. Natl. Acad. Sci. USA 107, 10020–10025 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Buratti, E. & Baralle, F.E. Characterization and functional implications of the RNA binding properties of nuclear factor TDP-43, a novel splicing regulator of CFTR exon 9. J. Biol. Chem. 276, 36337–36343 (2001).

    CAS  PubMed  Google Scholar 

  47. Polymenidou, M. et al. Long pre-mRNA depletion and RNA missplicing contribute to neuronal vulnerability from loss of TDP-43. Nat. Neurosci. 14, 459–468 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Suzuki, H. et al. Characterization of RNase R–digested cellular RNA source that consists of lariat and circular RNAs from pre-mRNA splicing. Nucleic Acids Res. 34, e63 (2006).

    PubMed  PubMed Central  Google Scholar 

  49. Voigt, A. et al. TDP-43–mediated neuron loss in vivo requires RNA-binding activity. PLoS ONE 5, e12247 (2010).

    PubMed  PubMed Central  Google Scholar 

  50. Li, Y. et al. A Drosophila model for TDP-43 proteinopathy. Proc. Natl. Acad. Sci. USA 107, 3169–3174 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Yang, C. et al. The C-terminal TDP-43 fragments have a high aggregation propensity and harm neurons by a dominant-negative mechanism. PLoS ONE 5, e15878 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Estes, P.S. et al. Wild-type and A315T mutant TDP-43 exert differential neurotoxicity in a Drosophila model of ALS. Hum. Mol. Genet. 20, 2308–2321 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Sephton, C.F. et al. Identification of neuronal RNA targets of TDP-43–containing ribonucleoprotein complexes. J. Biol. Chem. 286, 1204–1215 (2011).

    CAS  PubMed  Google Scholar 

  54. Tollervey, J.R. et al. Characterizing the RNA targets and position-dependent splicing regulation by TDP-43. Nat. Neurosci. 14, 452–458 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Xiao, S. et al. RNA targets of TDP-43 identified by UV-CLIP are deregulated in ALS. Mol. Cell Neurosci. 47, 167–180 (2011).

    CAS  PubMed  Google Scholar 

  56. Ye, Y., De Leon, J., Yokoyama, N., Naidu, Y. & Camerini, D. DBR1 siRNA inhibition of HIV-1 replication. Retrovirology 2, 63 (2005).

    PubMed  PubMed Central  Google Scholar 

  57. Xia, H., Mao, Q., Paulson, H.L. & Davidson, B.L. siRNA-mediated gene silencing in vitro and in vivo. Nat. Biotechnol. 20, 1006–1010 (2002).

    CAS  PubMed  Google Scholar 

  58. Xia, H. et al. RNAi suppresses polyglutamine-induced neurodegeneration in a model of spinocerebellar ataxia. Nat. Med. 10, 816–820 (2004).

    CAS  PubMed  Google Scholar 

  59. Singer, O. et al. Targeting BACE1 with siRNAs ameliorates Alzheimer disease neuropathology in a transgenic model. Nat. Neurosci. 8, 1343–1349 (2005).

    CAS  PubMed  Google Scholar 

  60. Kordasiewicz, H.B. et al. Sustained therapeutic reversal of Huntington′s disease by transient repression of Huntingtin synthesis. Neuron 74, 1031–1044 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  61. Da Cruz, S. & Cleveland, D.W. Understanding the role of TDP-43 and FUS/TLS in ALS and beyond. Curr. Opin. Neurobiol. 21, 904–919 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  62. Cooper, A.A. et al. Alpha-synuclein blocks ER-Golgi traffic and Rab1 rescues neuron loss in Parkinson′s models. Science 313, 324–328 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  63. Duennwald, M.L. & Lindquist, S. Impaired ERAD and ER stress are early and specific events in polyglutamine toxicity. Genes Dev. 22, 3308–3319 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  64. Tong, A.H. et al. Global mapping of the yeast genetic interaction network. Science 303, 808–813 (2004).

    CAS  PubMed  Google Scholar 

  65. Tong, A.H. & Boone, C. Synthetic genetic array analysis in Saccharomyces cerevisiae. Methods Mol. Biol. 313, 171–192 (2006).

    CAS  PubMed  Google Scholar 

  66. Armakola, M., Hart, M.P. & Gitler, A.D. TDP-43 toxicity in yeast. Methods 53, 238–245 (2011).

    CAS  PubMed  Google Scholar 

  67. Collins, S.R., Schuldiner, M., Krogan, N.J. & Weissman, J.S. A strategy for extracting and analyzing large-scale quantitative epistatic interaction data. Genome Biol. 7, R63 (2006).

    PubMed  PubMed Central  Google Scholar 

  68. Sharma, P., Ando, D.M., Daub, A., Kaye, J.A. & Finkbeiner, S. High-throughput screening in primary neurons. Methods Enzymol. 506, 331–360 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  69. Arrasate, M. & Finkbeiner, S. Automated microscope system for determining factors that predict neuronal fate. Proc. Natl. Acad. Sci. USA 102, 3840–3845 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  70. Shinbo, Y. et al. Proper SUMO-1 conjugation is essential to DJ-1 to exert its full activities. Cell Death Differ. 13, 96–108 (2006).

    CAS  PubMed  Google Scholar 

  71. Rothrock, C.R., House, A.E. & Lynch, K.W. HnRNP L represses exon splicing via a regulated exonic splicing silencer. EMBO J. 24, 2792–2802 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank K. Lynch and S. Smith for helpful suggestions and discussions about RNA and splicing; T. Nakaya for advice and assistance with lentivirus transduction experiments and analysis; C. Kurischko for advice and assistance with visualizing P bodies and stress granules; Q. Mitrovich and A. Plocik for advice with running the two-dimensional nucleic acid gels; S. Collins and D. Cameron for useful advice and assistance in performing the data analysis for screen 2; B. Hodges, D. Hosangadi, P. Patel, P. Nathanson and C. Mrejen for assistance with yeast experiments; J. Epstein and A. Raphael for critical comments on the manuscript and helpful suggestions; and G. Howard for editorial assistance. A. Elden helped with initial stages of this project. We are grateful to J. Gerst (Weizmann Institute) for providing the yeast m-TAG plasmids, R. Parker (University of Arizona) for sharing the P-body and stress granule marker plasmids and J. Weibezahn (University of California, San Francisco) for providing temperature-sensitive CDC48 (cdc48-3, SM 4783) and wild-type CDC48 isogenic (SM 5124) yeast strains. We thank B. Schwer (Weill Cornell Medical College) for providing the yeast mutant Dbr1 expression plasmids. We thank C. Boone (University of Toronto) for the MATα strain Y7092. This work was supported by US National Institutes of Health (NIH) Director's New Innovator Awards 1DP2OD004417 (to A.D.G.) and 1DP2OD002177 (to J.S.), NIH grants NS065317 and NS065317 (to A.D.G.), NS067354 (to J.S.), GM084448, GM084279, GM081879 and GM098101 (to N.J.K.), NS39074 and NS045491 (to S.F.) and NS072233 (to S.J.B.), a New Scholar in Aging Award from the Ellison Medical Foundation (to J.S.), a grant from the Packard Center for ALS Research at Johns Hopkins (A.D.G. and J.S.), a grant from the Consortium for Frontotemporal Research (to R.V.F.), NIH grant 2P01AG02074 (to S.F.), a grant from the ALS Association (to S.F.) and the Taube-Koret Center and Hellman Family Foundation (to S.F.). A.D.G. is a Pew Scholar in the Biomedical Sciences, supported by The Pew Charitable Trusts, and a Rita Allen Foundation Scholar. N.J.K. is a Searle Scholar and a Keck Young Investigator. R.V.F. is an Investigator of the Gladstone Institutes. The J. David Gladstone Institutes received support from National Center for Research Resources Grant RR18928.

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M.A., M.J.H., M.D.F., S.J.B., J.S., N.J.K., S.F., R.V.F. and A.D.G. designed the experiments. M.A., M.J.H., M.D.F., S.J.B., J.S., E.A.S., Z.D., X.F. and A.D.G. performed the research. M.A., M.J.H., M.D.F., S.J.B., J.S., N.J.K., S.F., R.V.F. and A.D.G. analyzed and interpreted data. M.A., M.J.H., M.D.F., S.J.B., S.F., R.V.F. and A.D.G. wrote the manuscript with contributions from all authors.

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Correspondence to Robert V Farese Jr or Aaron D Gitler.

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A.D.G. is an inventor on patents and patent applications that have been licensed to FoldRx.

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Supplementary Text and Figures

Supplementary Figures 1–6, Supplementary Table 1 and Supplementary Note (PDF 7108 kb)

Supplementary Data

Results of TDP-43 toxicity modifier screen (supplied as separate Excel file) (XLSX 542 kb)

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Armakola, M., Higgins, M., Figley, M. et al. Inhibition of RNA lariat debranching enzyme suppresses TDP-43 toxicity in ALS disease models. Nat Genet 44, 1302–1309 (2012). https://doi.org/10.1038/ng.2434

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