FUS/TLS forms cytoplasmic aggregates, inhibits cell growth and interacts with TDP-43 in a yeast model of amyotrophic lateral sclerosis

Abstract

Amyotrophic lateral sclerosis (ALS) is a fatal disease characterized by the premature loss of motor neurons. While the underlying cellular mechanisms of neuron degeneration are unknown, the cytoplasmic aggregation of several proteins is associated with sporadic and familial forms of the disease. Both wild-type and mutant forms of the RNA-binding proteins FUS and TDP-43 accumulate in cytoplasmic inclusions in the neurons of ALS patients. It is not known if these so-called proteinopathies are due to a loss of function or a gain of toxicity resulting from the formation of cytoplasmic aggregates. Here we present a model of FUS toxicity using the yeast Saccharomyces cerevisiae in which toxicity is associated with greater expression and accumulation of FUS in cytoplasmic aggregates. We find that FUS and TDP-43 have a high propensity for co-aggregation, unlike the aggregation patterns of several other aggregation-prone proteins. Moreover, the biophysical properties of FUS aggregates in yeast are distinctly different from many amyloidogenic proteins, suggesting they are not composed of amyloid.

Keywords

amyotrophic lateral sclerosis (ALS) / fused in sarcoma (FUS) / TLS / proteinopathy / yeast

References

[1] Alberti, S., Halfmann, R., King, O., Kapila, A., and Lindquist, S. (2009). A systematic survey identifies prions and illuminates sequence features of prionogenic proteins. Cell 137, 146-158 .19345193
[2] Ayala, Y.M., Zago, P., D’Ambrogio, A., Xu, Y.F., Petrucelli, L., Buratti, E., and Baralle, F.E. (2008). Structural determinants of the cellular localization and shuttling of TDP-43. J Cell Sci 121, 3778-3785 .18957508
[3] Bharadwaj, P., Martins, R., and Macreadie, I. (2010). Yeast as a model for studying Alzheimer's disease. FEMS Yeast Res 10, 961-969 .
[4] Bosco, D.A., Lemay, N., Ko, H.K., Zhou, H., Burke, C., Kwiatkowski, T.J. Jr, Sapp, P., McKenna-Yasek, D., Brown, R.H. Jr, and Hayward, L.J. (2010). Mutant FUS proteins that cause amyotrophic lateral sclerosis incorporate into stress granules. Hum Mol Genet 19, 4160-4175 .20699327
[5] Braun, R.J., Büttner, S., Ring, J., Kroemer, G., and Madeo, F. (2010). Nervous yeast: modeling neurotoxic cell death. Trends Biochem Sci 35, 135-144 .19926288
[6] Buchan, J.R., Muhlrad, D., and Parker, R. (2008). P bodies promote stress granule assembly in Saccharomyces cerevisiae. J Cell Biol 183, 441-455 .18981231
[7] Buchan, J.R., and Parker, R. (2009). Eukaryotic stress granules: the ins and outs of translation. Mol Cell 36, 932-941 .20064460
[8] Chernoff, Y.O., Galkin, A.P., Lewitin, E., Chernova, T.A., Newnam, G.P., and Belenkiy, S.M. (2000). Evolutionary conservation of prion-forming abilities of the yeast Sup35 protein. Mol Microbiol 35, 865-876 .10692163
[9] Chernoff, Y.O., Lindquist, S.L., Ono, B., Inge-Vechtomov, S.G., and Liebman, S.W. (1995). Role of the chaperone protein Hsp104 in propagation of the yeast prion-like factor [psi+].Science 268, 880-884 .7754373
[10] Colombrita, C., Zennaro, E., Fallini, C., Weber, M., Sommacal, A., Buratti, E., Silani, V., and Ratti, A. (2009). TDP-43 is recruited to stress granules in conditions of oxidative insult. J Neurochem 111, 1051-1061 .19765185
[11] Cooper, A.A., Gitler, A.D., Cashikar, A., Haynes, C.M., Hill, K.J., Bhullar, B., Liu, K., Xu, K., Strathearn, K.E., Liu, F., (2006). Alpha-synuclein blocks ER-Golgi traffic and Rab1 rescues neuron loss in Parkinson’s models. Science 313, 324-328 .16794039
[12] Cushman, M., Johnson, B.S., King, O.D., Gitler, A.D., and Shorter, J. (2010). Prion-like disorders: blurring the divide between transmissibility and infectivity. J Cell Sci 123, 1191-1201 .20356930
[13] Deng, H.X., Zhai, H., Bigio, E.H., Yan, J., Fecto, F., Ajroud, K., Mishra, M., Ajroud-Driss, S., Heller, S., Sufit, R., (2010). FUS-immunoreactive inclusions are a common feature in sporadic and non-SOD1 familial amyotrophic lateral sclerosis. Ann Neurol 67, 739-748 .20517935
[14] Doi, H., Okamura, K., Bauer, P.O., Furukawa, Y., Shimizu, H., Kurosawa, M., Machida, Y., Miyazaki, H., Mitsui, K., Kuroiwa, Y., (2008). RNA-binding protein TLS is a major nuclear aggregate-interacting protein in huntingtin exon 1 with expanded polyglutamine-expressing cells. J Biol Chem 283, 6489-6500 .18167354
[15] Duennwald, M.L., and Lindquist, S. (2008). Impaired ERAD and ER stress are early and specific events in polyglutamine toxicity. Genes Dev 22, 3308-3319 .19015277
[16] Elden, A.C., Kim, H.J., Hart, M.P., Chen-Plotkin, A.S., Johnson, B.S., Fang, X., Armakola, M., Geser, F., Greene, R., Lu, M.M., (2010). Ataxin-2 intermediate-length polyglutamine expansions are associated with increased risk for ALS. Nature 466, 1069-1075 .20740007
[17] Fuentealba, R.A., Udan, M., Bell, S., Wegorzewska, I., Shao, J., Diamond, M.I., Weihl, C.C., and Baloh, R.H. (2010). Interaction with polyglutamine aggregates reveals a Q/N-rich domain in TDP-43. J Biol Chem 285, 26304-26314 .20554523
[18] Fushimi, K., Long, C., Jayaram, N., Chen, X., Li, L., and Wu, J.Y. (2011). Expression of human FUS/TLS in yeast leads to protein aggregation and cytotoxicity, recapitulating key features of FUS proteinopathy. Protein Cell 2, 141-149
[19] Gal, J., Zhang, J., Kwinter, D.M., Zhai, J., Jia, H., Jia, J., and Zhu, H. (2010). Nuclear localization sequence of FUS and induction of stress granules by ALS mutants. Neurobiol Aging . Jul29. [Epub ahead of print].10.1016/ j.neurobiolaging.2010.06.010.
[20] Iko, Y., Kodama, T.S., Kasai, N., Oyama, T., Morita, E.H., Muto, T., Okumura, M., Fujii, R., Takumi, T., Tate, S., (2004). Domain architectures and characterization of an RNA-binding protein, TLS. J Biol Chem 279, 44834-44840 .15299008
[21] Ito, D., Seki, M., Tsunoda, Y., Uchiyama, H., and Suzuki, N. (2011). Nuclear transport impairment of amyotrophic lateral sclerosis-linked mutations in FUS/TLS. Ann Neurol 69, 152-162 .21280085
[22] Johnson, B.S., McCaffery, J.M., Lindquist, S., and Gitler, A.D. (2008). A yeast TDP-43 proteinopathy model: Exploring the molecular determinants of TDP-43 aggregation and cellular toxicity. Proc Natl Acad Sci U S A 105, 6439-6444 .18434538
[23] Kaganovich, D., Kopito, R., and Frydman, J. (2008). Misfolded proteins partition between two distinct quality control compartments. Nature 454, 1088-1095 .18756251
[24] Khurana, V., and Lindquist, S. (2010). Modelling neurodegeneration in Saccharomyces cerevisiae: why cook with baker’s yeast? Nat Rev Neurosci 11, 436-449 .20424620
[25] Kim, S.H., Shanware, N.P., Bowler, M.J., and Tibbetts, R.S. (2010). Amyotrophic lateral sclerosis-associated proteins TDP-43 and FUS/TLS function in a common biochemical complex to co-regulate HDAC6 mRNA. J Biol Chem 285, 34097-34105 .20720006
[26] Krobitsch, S., and Lindquist, S. (2000). Aggregation of huntingtin in yeast varies with the length of the polyglutamine expansion and the expression of chaperone proteins. Proc Natl Acad Sci U S A 97, 1589-1594 .10677504
[27] Kryndushkin, D.S., Alexandrov, I.M., Ter-Avanesyan, M.D., and Kushnirov, V.V. (2003). Yeast [PSI+] prion aggregates are formed by small Sup35 polymers fragmented by Hsp104. J Biol Chem 278, 49636-49643 .14507919
[28] Kushnirov, V.V., Alexandrov, I.M., Mitkevich, O.V., Shkundina, I.S., and Ter-Avanesyan, M.D. (2006). Purification and analysis of prion and amyloid aggregates. Methods 39, 50-55 .16774835
[29] Kushnirov, V.V., Kochneva-Pervukhova, N.V., Chechenova, M.B., Frolova, N.S., and Ter-Avanesyan, M.D. (2000). Prion properties of the Sup35 protein of yeast Pichia methanolica. EMBO J 19, 324-331 .10654931
[30] Kwiatkowski, T.J. Jr, Bosco, D.A., Leclerc, A.L., Tamrazian, E., Vanderburg, C.R., Russ, C., Davis, A., Gilchrist, J., Kasarskis, E.J., Munsat, T., (2009). Mutations in the FUS/TLS gene on chromosome 16 cause familial amyotrophic lateral sclerosis. Science 323, 1205-1208 .19251627
[31] Lagier-Tourenne, C., Polymenidou, M., and Cleveland, D.W. (2010). TDP-43 and FUS/TLS: emerging roles in RNA processing and neurodegeneration. Hum Mol Genet 19, R46-R64 .20400460
[32] Ling, S.C., Albuquerque, C.P., Han, J.S., Lagier-Tourenne, C., Tokunaga, S., Zhou, H., and Cleveland, D.W. (2010). ALS-associated mutations in TDP-43 increase its stability and promote TDP-43 complexes with FUS/TLS. Proc Natl Acad Sci U S A 107, 13318-13323 .20624952
[33] McGlinchey, R., Kryndushkin, D., and Wickner, R.B. (2011). Suicidal [PSI+] is a lethal yeast prion. Proc Natl Acad Sci U S A. (In press)
[34] Meriin, A.B., Zhang, X., He, X., Newnam, G.P., Chernoff, Y.O., and Sherman, M.Y. (2002). Huntington toxicity in yeast model depends on polyglutamine aggregation mediated by a prion-like protein Rnq1. J Cell Biol 157, 997-1004 .12058016
[35] Meriin, A.B., Zhang, X., Miliaras, N.B., Kazantsev, A., Chernoff, Y.O., McCaffery, J.M., Wendland, B., and Sherman, M.Y. (2003). Aggregation of expanded polyglutamine domain in yeast leads to defects in endocytosis. Mol Cell Biol 23, 7554-7565 .14560003
[36] Muchowski, P.J., Schaffar, G., Sittler, A., Wanker, E.E., Hayer-Hartl, M.K., and Hartl, F.U. (2000). Hsp70 and hsp40 chaperones can inhibit self-assembly of polyglutamine proteins into amyloid-like fibrils. Proc Natl Acad Sci U S A 97, 7841-7846 .10859365
[37] Neumann, M., Roeber, S., Kretzschmar, H.A., Rademakers, R., Baker, M., and Mackenzie, I.R. (2009). Abundant FUS-immunoreactive pathology in neuronal intermediate filament inclusion disease. Acta Neuropathol 118, 605-616 .19669651
[38] Nonhoff, U., Ralser, M., Welzel, F., Piccini, I., Balzereit, D., Yaspo, M.L., Lehrach, H., and Krobitsch, S. (2007). Ataxin-2 interacts with the DEAD/H-box RNA helicase DDX6 and interferes with P-bodies and stress granules. Mol Biol Cell 18, 1385-1396 .17392519
[39] Outeiro, T.F., and Lindquist, S. (2003). Yeast cells provide insight into alpha-synuclein biology and pathobiology. Science 302, 1772-1775 .14657500
[40] Rosen, D.R., Siddique, T., Patterson, D., Figlewicz, D.A., Sapp, P., Hentati, A., Donaldson, D., Goto, J., O’Regan, J.P., Deng, H.X., (1993). Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis. Nature 362, 59-62 .8446170
[41] Salnikova, A.B., Kryndushkin, D.S., Smirnov, V.N., Kushnirov, V.V., and Ter-Avanesyan, M.D. (2005). Nonsense suppression in yeast cells overproducing Sup35 (eRF3) is caused by its non-heritable amyloids. J Biol Chem 280, 8808-8812 .15618222
[42] Santoso, A., Chien, P., Osherovich, L.Z., and Weissman, J.S. (2000). Molecular basis of a yeast prion species barrier. Cell 100, 277-288 .10660050
[43] Serio, T.R., Cashikar, A.G., Kowal, A.S., Sawicki, G.J., Moslehi, J.J., Serpell, L., Arnsdorf, M.F., and Lindquist, S.L. (2000). Nucleated conformational conversion and the replication of conformational information by a prion determinant. Science 289, 1317-1321 .10958771
[44] Sharma, N., Brandis, K.A., Herrera, S.K., Johnson, B.E., Vaidya, T., Shrestha, R., and Debburman, S.K. (2006). alpha-Synuclein budding yeast model: toxicity enhanced by impaired proteasome and oxidative stress. J Mol Neurosci 28, 161-178 .16679556
[45] Speransky, V.V., Taylor, K.L., Edskes, H.K., Wickner, R.B., and Steven, A.C. (2001). Prion filament networks in [URE3] cells of Saccharomyces cerevisiae. J Cell Biol 153, 1327-1336 .11402074
[46] Sreedharan, J., Blair, I.P., Tripathi, V.B., Hu, X., Vance, C., Rogelj, B., Ackerley, S., Durnall, J.C., Williams, K.L., Buratti, E., (2008). TDP-43 mutations in familial and sporadic amyotrophic lateral sclerosis. Science 319, 1668-1672 .18309045
[47] Toombs, J.A., McCarty, B.R., and Ross, E.D. (2010). Compositional determinants of prion formation in yeast. Mol Cell Biol 30, 319-332 .19884345
[48] Udan, M., and Baloh, R.H. (2011). Implications of the prion-related Q/N domains in TDP-43 and FUS. Prion 5, 1-5 .21135580
[49] Vance, C., Rogelj, B., Hortobágyi, T., De Vos, K.J., Nishimura, A.L., Sreedharan, J., Hu, X., Smith, B., Ruddy, D., Wright, P., (2009). Mutations in FUS, an RNA processing protein, cause familial amyotrophic lateral sclerosis type 6. Science 323, 1208-1211 .19251628
[50] Wickner, R.B., Edskes, H.K., and Shewmaker, F. (2006). How to find a prion: [URE3], [PSI+] and [beta]. [beta] Methods 39, 3-8 .16793280
[51] Wickner, R.B., Shewmaker, F., Edskes, H., Kryndushkin, D., Nemecek, J., McGlinchey, R., Bateman, D., and Winchester, C.L. (2010). Prion amyloid structure explains templating: how proteins can be genes. FEMS Yeast Res 10, 980-991 .20726897
[52] Woulfe, J., Gray, D.A., and Mackenzie, I.R. (2010). FUS-immunoreactive intranuclear inclusions in neurodegenerative disease. Brain Pathol 20, 589-597 .19832837
[53] Yeger-Lotem, E., Riva, L., Su, L.J., Gitler, A.D., Cashikar, A.G., King, O.D., Auluck, P.K., Geddie, M.L., Valastyan, J.S., Karger, D.R., (2009). Bridging high-throughput genetic and transcriptional data reveals cellular responses to alpha-synuclein toxicity. Nat Genet 41, 316-323 .19234470
[54] Zinszner, H., Sok, J., Immanuel, D., Yin, Y., and Ron, D. (1997). TLS (FUS) binds RNA in vivo and engages in nucleo-cytoplasmic shuttling. J Cell Sci 110, 1741-1750 .9264461