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Sense-encoded poly-GR dipeptide repeat proteins correlate to neurodegeneration and uniquely co-localize with TDP-43 in dendrites of repeat-expanded C9orf72 amyotrophic lateral sclerosis

Abstract

Hexanucleotide repeat expansions in C9orf72 are the most common genetic cause of amyotrophic lateral sclerosis (C9 ALS). The main hypothesized pathogenic mechanisms are C9orf72 haploinsufficiency and/or toxicity from one or more of bi-directionally transcribed repeat RNAs and their dipeptide repeat proteins (DPRs) poly-GP, poly-GA, poly-GR, poly-PR and poly-PA. Recently, nuclear import and/or export defects especially caused by arginine-containing poly-GR or poly-PR have been proposed as significant contributors to pathogenesis based on disease models. We quantitatively studied and compared DPRs, nuclear pore proteins and C9orf72 protein in clinically related and clinically unrelated regions of the central nervous system, and compared them to phosphorylated TDP-43 (pTDP-43), the hallmark protein of ALS. Of the five DPRs, only poly-GR was significantly abundant in clinically related areas compared to unrelated areas (p < 0.001), and formed dendritic-like aggregates in the motor cortex that co-localized with pTDP-43 (p < 0.0001). While most poly-GR dendritic inclusions were pTDP-43 positive, only 4% of pTDP-43 dendritic inclusions were poly-GR positive. Staining for arginine-containing poly-GR and poly-PR in nuclei of neurons produced signals that were not specific to C9 ALS. We could not detect significant differences of nuclear markers RanGap, Lamin B1, and Importin β1 in C9 ALS, although we observed subtle nuclear changes in ALS, both C9 and non-C9, compared to control. The C9orf72 protein itself was diffusely expressed in cytoplasm of large neurons and glia, and nearly 50% reduced, in both clinically related frontal cortex and unrelated occipital cortex, but not in cerebellum. In summary, sense-encoded poly-GR DPR was unique, and localized to dendrites and pTDP43 in motor regions of C9 ALS CNS. This is consistent with new emerging ideas about TDP-43 functions in dendrites.

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References

  1. Al-Sarraj S, King A, Troakes C, Smith B, Maekawa S, Bodi I, Rogelj B, Al-Chalabi A, Hortobagyi T, Shaw CE (2011) p62 positive, TDP-43 negative, neuronal cytoplasmic and intranuclear inclusions in the cerebellum and hippocampus define the pathology of C9orf72-linked FTLD and MND/ALS. Acta Neuropathol 122:691–702. https://doi.org/10.1007/s00401-011-0911-2

    CAS  Article  PubMed  Google Scholar 

  2. Alami NH, Smith RB, Carrasco MA, Williams LA, Winborn CS, Han SSW, Kiskinis E, Winborn B, Freibaum BD, Kanagaraj A et al (2014) Axonal transport of TDP-43 mRNA granules is impaired by ALS-causing mutations. Neuron 81:536–543. https://doi.org/10.1016/j.neuron.2013.12.018

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  3. Ash PE, Bieniek KF, Gendron TF, Caulfield T, Lin WL, Dejesus-Hernandez M, van Blitterswijk MM, Jansen-West K, Paul JW 3rd, Rademakers R et al (2013) Unconventional translation of C9ORF72 GGGGCC expansion generates insoluble polypeptides specific to c9FTD/ALS. Neuron 77:639–646. https://doi.org/10.1016/j.neuron.2013.02.004

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  4. Atanasio A, Decman V, White D, Ramos M, Ikiz B, Lee HC, Siao CJ, Brydges S, LaRosa E, Bai Y et al (2016) C9orf72 ablation causes immune dysregulation characterized by leukocyte expansion, autoantibody production, and glomerulonephropathy in mice. Sci Rep 6:23204. https://doi.org/10.1038/srep23204

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  5. Boxer AL, Mackenzie IR, Boeve BF, Baker M, Seeley WW, Crook R, Feldman H, Hsiung GY, Rutherford N, Laluz V et al (2011) Clinical, neuroimaging and neuropathological features of a new chromosome 9p-linked FTD-ALS family. J Neurol Neurosurg Psychiatry 82:196–203. https://doi.org/10.1136/jnnp.2009.204081

    Article  PubMed  Google Scholar 

  6. Brettschneider J, Del Tredici K, Toledo JB, Robinson JL, Irwin DJ, Grossman M, Suh E, Van Deerlin VM, Wood EM, Baek Y et al (2013) Stages of pTDP-43 pathology in amyotrophic lateral sclerosis. Ann Neurol 74:20–38. https://doi.org/10.1002/ana.23937

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  7. Burberry A, Suzuki N, Wang JY, Moccia R, Mordes DA, Stewart MH, Suzuki-Uematsu S, Ghosh S, Singh A, Merkle FT et al (2016) Loss-of-function mutations in the C9ORF72 mouse ortholog cause fatal autoimmune disease. Sci Transl Med 8:347ra93. https://doi.org/10.1126/scitranslmed.aaf6038

    Article  PubMed  PubMed Central  Google Scholar 

  8. Chew J, Gendron TF, Prudencio M, Sasaguri H, Zhang YJ, Castanedes-Casey M, Lee CW, Jansen-West K, Kurti A, Murray ME et al (2015) Neurodegeneration. C9ORF72 repeat expansions in mice cause TDP-43 pathology, neuronal loss, and behavioral deficits. Science (New York, NY) 348:1151–1154. https://doi.org/10.1126/science.aaa9344

    CAS  Article  Google Scholar 

  9. Davidson Y, Robinson AC, Liu X, Wu D, Troakes C, Rollinson S, Masuda-Suzukake M, Suzuki G, Nonaka T, Shi J et al (2016) Neurodegeneration in frontotemporal lobar degeneration and motor neurone disease associated with expansions in C9orf72 is linked to TDP-43 pathology and not associated with aggregated forms of dipeptide repeat proteins. Neuropathol Appl Neurobiol 42:242–254. https://doi.org/10.1111/nan.12292

    CAS  Article  PubMed  Google Scholar 

  10. Davidson YS, Robinson AC, Rollinson S, Pickering-Brown S, Xiao S, Robertson J, Mann DMA (2017) Immunohistochemical detection of C9orf72 protein in frontotemporal lobar degeneration and motor neurone disease: patterns of immunostaining and an evaluation of commercial antibodies. Amyotroph Lateral Scler Frontotemporal Degener 1–10. doi:https://doi.org/10.1080/21678421.2017.1359304

  11. DeJesus-Hernandez M, Mackenzie IR, Boeve BF, Boxer AL, Baker M, Rutherford NJ, Nicholson AM, Finch NA, Flynn H, Adamson J et al (2011) Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS. Neuron 72:245–256. https://doi.org/10.1016/j.neuron.2011.09.011

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  12. Devlin AC, Burr K, Borooah S, Foster JD, Cleary EM, Geti I, Vallier L, Shaw CE, Chandran S, Miles GB (2015) Human iPSC-derived motoneurons harbouring TARDBP or C9ORF72 ALS mutations are dysfunctional despite maintaining viability. Nature Commun 6:5999. https://doi.org/10.1038/ncomms6999

    CAS  Article  Google Scholar 

  13. Freibaum BD, Lu Y, Lopez-Gonzalez R, Kim NC, Almeida S, Lee KH, Badders N, Valentine M, Miller BL, Wong PC et al (2015) GGGGCC repeat expansion in C9orf72 compromises nucleocytoplasmic transport. Nature 525:129–133. https://doi.org/10.1038/nature14974

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  14. Gendron TF, Bieniek KF, Zhang YJ, Jansen-West K, Ash PE, Caulfield T, Daughrity L, Dunmore JH, Castanedes-Casey M, Chew J et al (2013) Antisense transcripts of the expanded C9ORF72 hexanucleotide repeat form nuclear RNA foci and undergo repeat-associated non-ATG translation in c9FTD/ALS. Acta Neuropathol 126:829–844. https://doi.org/10.1007/s00401-013-1192-8

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  15. Gijselinck I, Van Langenhove T, van der Zee J, Sleegers K, Philtjens S, Kleinberger G, Janssens J, Bettens K, Van Cauwenberghe C, Pereson S et al (2012) A C9orf72 promoter repeat expansion in a Flanders-Belgian cohort with disorders of the frontotemporal lobar degeneration-amyotrophic lateral sclerosis spectrum: a gene identification study. Lancet Neurol 11:54–65. https://doi.org/10.1016/s1474-4422(11)70261-7

    CAS  Article  PubMed  Google Scholar 

  16. Gomez-Deza J, Lee YB, Troakes C, Nolan M, Al-Sarraj S, Gallo JM, Shaw CE (2015) Dipeptide repeat protein inclusions are rare in the spinal cord and almost absent from motor neurons in C9ORF72 mutant amyotrophic lateral sclerosis and are unlikely to cause their degeneration. Acta Neuropathol Commun 3:38. https://doi.org/10.1186/s40478-015-0218-y

    Article  PubMed  PubMed Central  Google Scholar 

  17. Ishiguro A, Kimura N, Watanabe Y, Watanabe S, Ishihama A (2016) TDP-43 binds and transports G-quadruplex-containing mRNAs into neurites for local translation. Genes Cells 21:466–481. https://doi.org/10.1111/gtc.12352

    CAS  Article  PubMed  Google Scholar 

  18. Jiang J, Zhu Q, Gendron TF, Saberi S, McAlonis-Downes M, Seelman A, Stauffer JE, Jafar-Nejad P, Drenner K, Schulte D et al (2016) Gain of toxicity from ALS/FTD-Linked repeat expansions in C9ORF72 Is alleviated by antisense oligonucleotides targeting GGGGCC-containing RNAs. Neuron 90:535–550. https://doi.org/10.1016/j.neuron.2016.04.006

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  19. Jovicic A, Mertens J, Boeynaems S, Bogaert E, Chai N, Yamada SB, Paul JW 3rd, Sun S, Herdy JR, Bieri G et al (2015) Modifiers of C9orf72 dipeptide repeat toxicity connect nucleocytoplasmic transport defects to FTD/ALS. Nat Neurosci 18:1226–1229. https://doi.org/10.1038/nn.4085

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  20. King A, Maekawa S, Bodi I, Troakes C, Al-Sarraj S (2011) Ubiquitinated, p62 immunopositive cerebellar cortical neuronal inclusions are evident across the spectrum of TDP-43 proteinopathies but are only rarely additionally immunopositive for phosphorylation-dependent TDP-43. Neuropathology 31:239–249. https://doi.org/10.1111/j.1440-1789.2010.01171.x

    Article  PubMed  Google Scholar 

  21. Koppers M, Blokhuis AM, Westeneng HJ, Terpstra ML, Zundel CA, Vieira de Sa R, Schellevis RD, Waite AJ, Blake DJ, Veldink JH et al (2015) C9orf72 ablation in mice does not cause motor neuron degeneration or motor deficits. Ann Neurol 78:426–438. https://doi.org/10.1002/ana.24453

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  22. Kwon I, Xiang S, Kato M, Wu L, Theodoropoulos P, Wang T, Kim J, Yun J, Xie Y, McKnight SL (2014) Poly-dipeptides encoded by the C9orf72 repeats bind nucleoli, impede RNA biogenesis, and kill cells. Science (New York, NY) 345:1139–1145. https://doi.org/10.1126/science.1254917

    CAS  Article  Google Scholar 

  23. Lall D, Baloh RH (2017) Microglia and C9orf72 in neuroinflammation and ALS and frontotemporal dementia. J Clin Investig. https://doi.org/10.1172/jci90607

    PubMed  Google Scholar 

  24. Lee KH, Zhang P, Kim HJ, Mitrea DM, Sarkar M, Freibaum BD, Cika J, Coughlin M, Messing J, Molliex A et al (2016) C9orf72 dipeptide repeats impair the assembly, dynamics, and function of membrane-less organelles. Cell 167(774–788):e717. https://doi.org/10.1016/j.cell.2016.10.002

    Google Scholar 

  25. Lee YB, Chen HJ, Peres JN, Gomez-Deza J, Attig J, Stalekar M, Troakes C, Nishimura AL, Scotter EL, Vance C et al (2013) Hexanucleotide repeats in ALS/FTD form length-dependent RNA foci, sequester RNA binding proteins, and are neurotoxic. Cell Rep 5:1178–1186. https://doi.org/10.1016/j.celrep.2013.10.049

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  26. Lin Y, Mori E, Kato M, Xiang S, Wu L, Kwon I, McKnight SL (2016) Toxic PR poly-dipeptides encoded by the C9orf72 repeat expansion target LC domain polymers. Cell 167(789–802):e712. https://doi.org/10.1016/j.cell.2016.10.003

    Google Scholar 

  27. Liu-Yesucevitz L, Lin AY, Ebata A, Boon JY, Reid W, Xu YF, Kobrin K, Murphy GJ, Petrucelli L, Wolozin B (2014) ALS-linked mutations enlarge TDP-43-enriched neuronal RNA granules in the dendritic arbor. J Neurosci 34:4167–4174. https://doi.org/10.1523/jneurosci.2350-13.2014

    Article  PubMed  PubMed Central  Google Scholar 

  28. Liu Y, Pattamatta A, Zu T, Reid T, Bardhi O, Borchelt DR, Yachnis AT, Ranum LP (2016) C9orf72 BAC mouse model with motor deficits and neurodegenerative features of ALS/FTD. Neuron 90:521–534. https://doi.org/10.1016/j.neuron.2016.04.005

    CAS  Article  PubMed  Google Scholar 

  29. Lopez-Gonzalez R, Lu Y, Gendron TF, Karydas A, Tran H, Yang D, Petrucelli L, Miller BL, Almeida S, Gao FB (2016) Poly(GR) in C9ORF72-Related ALS/FTD compromises mitochondrial function and increases oxidative stress and DNA damage in iPSC-derived motor neurons. Neuron 92:383–391. https://doi.org/10.1016/j.neuron.2016.09.015

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  30. Mackenzie IR, Arzberger T, Kremmer E, Troost D, Lorenzl S, Mori K, Weng SM, Haass C, Kretzschmar HA, Edbauer D et al (2013) Dipeptide repeat protein pathology in C9ORF72 mutation cases: clinico-pathological correlations. Acta Neuropathol 126:859–879. https://doi.org/10.1007/s00401-013-1181-y

    CAS  Article  PubMed  Google Scholar 

  31. Mackenzie IR, Frick P, Grasser FA, Gendron TF, Petrucelli L, Cashman NR, Edbauer D, Kremmer E, Prudlo J, Troost D et al (2015) Quantitative analysis and clinico-pathological correlations of different dipeptide repeat protein pathologies in C9ORF72 mutation carriers. Acta Neuropathol 130:845–861. https://doi.org/10.1007/s00401-015-1476-2

    CAS  Article  PubMed  Google Scholar 

  32. Mann DM, Rollinson S, Robinson A, Bennion Callister J, Thompson JC, Snowden JS, Gendron T, Petrucelli L, Masuda-Suzukake M, Hasegawa M et al (2013) Dipeptide repeat proteins are present in the p62 positive inclusions in patients with frontotemporal lobar degeneration and motor neurone disease associated with expansions in C9ORF72. Acta Neuropathol Commun 1:68. https://doi.org/10.1186/2051-5960-1-68

    Article  PubMed  PubMed Central  Google Scholar 

  33. May S, Hornburg D, Schludi MH, Arzberger T, Rentzsch K, Schwenk BM, Grasser FA, Mori K, Kremmer E, Banzhaf-Strathmann J et al (2014) C9orf72 FTLD/ALS-associated Gly-Ala dipeptide repeat proteins cause neuronal toxicity and Unc119 sequestration. Acta Neuropathol 128:485–503. https://doi.org/10.1007/s00401-014-1329-4

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  34. Mizielinska S, Gronke S, Niccoli T, Ridler CE, Clayton EL, Devoy A, Moens T, Norona FE, Woollacott IO, Pietrzyk J et al (2014) C9orf72 repeat expansions cause neurodegeneration in Drosophila through arginine-rich proteins. Science (New York, NY) 345:1192–1194. https://doi.org/10.1126/science.1256800

    CAS  Article  Google Scholar 

  35. Mori K, Arzberger T, Grasser FA, Gijselinck I, May S, Rentzsch K, Weng SM, Schludi MH, van der Zee J, Cruts M et al (2013) Bidirectional transcripts of the expanded C9orf72 hexanucleotide repeat are translated into aggregating dipeptide repeat proteins. Acta Neuropathol 126:881–893. https://doi.org/10.1007/s00401-013-1189-3

    CAS  Article  PubMed  Google Scholar 

  36. Mori K, Weng SM, Arzberger T, May S, Rentzsch K, Kremmer E, Schmid B, Kretzschmar HA, Cruts M, Van Broeckhoven C et al (2013) The C9orf72 GGGGCC repeat is translated into aggregating dipeptide-repeat proteins in FTLD/ALS. Science (New York, NY) 339:1335–1338. https://doi.org/10.1126/science.1232927

    CAS  Article  Google Scholar 

  37. Nishihira Y, Tan CF, Onodera O, Toyoshima Y, Yamada M, Morita T, Nishizawa M, Kakita A, Takahashi H (2008) Sporadic amyotrophic lateral sclerosis: two pathological patterns shown by analysis of distribution of TDP-43-immunoreactive neuronal and glial cytoplasmic inclusions. Acta Neuropathol 116:169–182. https://doi.org/10.1007/s00401-008-0385-z

    CAS  Article  PubMed  Google Scholar 

  38. O’Rourke JG, Bogdanik L, Muhammad AK, Gendron TF, Kim KJ, Austin A, Cady J, Liu EY, Zarrow J, Grant S et al (2015) C9orf72 BAC transgenic mice display typical pathologic features of ALS/FTD. Neuron 88:892–901. https://doi.org/10.1016/j.neuron.2015.10.027

    Article  PubMed  PubMed Central  Google Scholar 

  39. O’Rourke JG, Bogdanik L, Yanez A, Lall D, Wolf AJ, Muhammad AK, Ho R, Carmona S, Vit JP, Zarrow J et al (2016) C9orf72 is required for proper macrophage and microglial function in mice. Science (New York, NY) 351:1324–1329. https://doi.org/10.1126/science.aaf1064

    Article  Google Scholar 

  40. Peters OM, Cabrera GT, Tran H, Gendron TF, McKeon JE, Metterville J, Weiss A, Wightman N, Salameh J, Kim J et al (2015) Human C9ORF72 hexanucleotide expansion reproduces RNA foci and dipeptide repeat proteins but not neurodegeneration in BAC transgenic mice. Neuron 88:902–909. https://doi.org/10.1016/j.neuron.2015.11.018

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  41. Renton AE, Majounie E, Waite A, Simon-Sanchez J, Rollinson S, Gibbs JR, Schymick JC, Laaksovirta H, van Swieten JC, Myllykangas L et al (2011) A hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD. Neuron 72:257–268. https://doi.org/10.1016/j.neuron.2011.09.010

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  42. Rossi S, Serrano A, Gerbino V, Giorgi A, Di Francesco L, Nencini M, Bozzo F, Schinina ME, Bagni C, Cestra G et al (2015) Nuclear accumulation of mRNAs underlies G4C2-repeat-induced translational repression in a cellular model of C9orf72 ALS. J Cell Sci 128:1787–1799. https://doi.org/10.1242/jcs.165332

    CAS  Article  PubMed  Google Scholar 

  43. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B et al (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676–682. https://doi.org/10.1038/nmeth.2019

    CAS  Article  PubMed  Google Scholar 

  44. Schludi MH, Becker L, Garrett L, Gendron TF, Zhou Q, Schreiber F, Popper B, Dimou L, Strom TM, Winkelmann J et al (2017) Spinal poly-GA inclusions in a C9orf72 mouse model trigger motor deficits and inflammation without neuron loss. Acta Neuropathol. https://doi.org/10.1007/s00401-017-1711-0

    PubMed  PubMed Central  Google Scholar 

  45. Schludi MH, May S, Grasser FA, Rentzsch K, Kremmer E, Kupper C, Klopstock T, Arzberger T, German Consortium for Frontotemporal Lobar D, Bavarian Brain Banking A et al (2015) Distribution of dipeptide repeat proteins in cellular models and C9orf72 mutation cases suggests link to transcriptional silencing. Acta Neuropathol 130:537–555. https://doi.org/10.1007/s00401-015-1450-z

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  46. Schwenk BM, Hartmann H, Serdaroglu A, Schludi MH, Hornburg D, Meissner F, Orozco D, Colombo A, Tahirovic S, Michaelsen M et al (2016) TDP-43 loss of function inhibits endosomal trafficking and alters trophic signaling in neurons. EMBO J 35:2350–2370. https://doi.org/10.15252/embj.201694221

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  47. Sudria-Lopez E, Koppers M, de Wit M, van der Meer C, Westeneng HJ, Zundel CA, Youssef SA, Harkema L, de Bruin A, Veldink JH et al (2016) Full ablation of C9orf72 in mice causes immune system-related pathology and neoplastic events but no motor neuron defects. Acta Neuropathol 132:145–147. https://doi.org/10.1007/s00401-016-1581-x

    Article  PubMed  PubMed Central  Google Scholar 

  48. Takeuchi R, Tada M, Shiga A, Toyoshima Y, Konno T, Sato T, Nozaki H, Kato T, Horie M, Shimizu H et al (2016) Heterogeneity of cerebral TDP-43 pathology in sporadic amyotrophic lateral sclerosis: evidence for clinico-pathologic subtypes. Acta Neuropathol Commun 4:61. https://doi.org/10.1186/s40478-016-0335-2

    Article  PubMed  PubMed Central  Google Scholar 

  49. Tran H, Almeida S, Moore J, Gendron TF, Chalasani U, Lu Y, Du X, Nickerson JA, Petrucelli L, Weng Z et al (2015) Differential toxicity of nuclear RNA foci versus dipeptide repeat proteins in a drosophila model of C9ORF72 FTD/ALS. Neuron 87:1207–1214. https://doi.org/10.1016/j.neuron.2015.09.015

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  50. van Blitterswijk M, Gendron TF, Baker MC, DeJesus-Hernandez M, Finch NA, Brown PH, Daughrity LM, Murray ME, Heckman MG, Jiang J et al (2015) Novel clinical associations with specific C9ORF72 transcripts in patients with repeat expansions in C9ORF72. Acta Neuropathol 130:863–876. https://doi.org/10.1007/s00401-015-1480-6

    Article  PubMed  PubMed Central  Google Scholar 

  51. Waite AJ, Baumer D, East S, Neal J, Morris HR, Ansorge O, Blake DJ (2014) Reduced C9orf72 protein levels in frontal cortex of amyotrophic lateral sclerosis and frontotemporal degeneration brain with the C9ORF72 hexanucleotide repeat expansion. Neurobiol Aging 35(7):1779.e5–1779.e13. https://doi.org/10.1016/j.neurobiolaging.2014.01.016

    CAS  Article  Google Scholar 

  52. Wen X, Tan W, Westergard T, Krishnamurthy K, Markandaiah SS, Shi Y, Lin S, Shneider NA, Monaghan J, Pandey UB et al (2014) Antisense proline-arginine RAN dipeptides linked to C9ORF72-ALS/FTD form toxic nuclear aggregates that initiate in vitro and in vivo neuronal death. Neuron 84:1213–1225. https://doi.org/10.1016/j.neuron.2014.12.010

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  53. Xiao S, MacNair L, McGoldrick P, McKeever PM, McLean JR, Zhang M, Keith J, Zinman L, Rogaeva E, Robertson J (2015) Isoform-specific antibodies reveal distinct subcellular localizations of C9orf72 in amyotrophic lateral sclerosis. Ann Neurol 78:568–583. https://doi.org/10.1002/ana.24469

    CAS  Article  PubMed  Google Scholar 

  54. Xu Z, Poidevin M, Li X, Li Y, Shu L, Nelson DL, Li H, Hales CM, Gearing M, Wingo TS et al (2013) Expanded GGGGCC repeat RNA associated with amyotrophic lateral sclerosis and frontotemporal dementia causes neurodegeneration. Proc Natl Acad Sci USA 110:7778–7783. https://doi.org/10.1073/pnas.1219643110

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  55. Yang D, Abdallah A, Li Z, Lu Y, Almeida S, Gao FB (2015) FTD/ALS-associated poly(GR) protein impairs the Notch pathway and is recruited by poly(GA) into cytoplasmic inclusions. Acta Neuropathol 130:525–535. https://doi.org/10.1007/s00401-015-1448-6

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  56. Yin S, Lopez-Gonzalez R, Kunz RC, Gangopadhyay J, Borufka C, Gygi SP, Gao FB, Reed R (2017) Evidence that C9ORF72 Dipeptide Repeat Proteins Associate with U2 snRNP to Cause Mis-splicing in ALS/FTD Patients. Cell Rep 19:2244–2256. https://doi.org/10.1016/j.celrep.2017.05.056

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  57. Zhang K, Donnelly CJ, Haeusler AR, Grima JC, Machamer JB, Steinwald P, Daley EL, Miller SJ, Cunningham KM, Vidensky S et al (2015) The C9orf72 repeat expansion disrupts nucleocytoplasmic transport. Nature 525:56–61. https://doi.org/10.1038/nature14973

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  58. Zhang YJ, Gendron TF, Grima JC, Sasaguri H, Jansen-West K, Xu YF, Katzman RB, Gass J, Murray ME, Shinohara M et al (2016) C9ORF72 poly(GA) aggregates sequester and impair HR23 and nucleocytoplasmic transport proteins. Nat Neurosci 19:668–677. https://doi.org/10.1038/nn.4272

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  59. Zhang YJ, Jansen-West K, Xu YF, Gendron TF, Bieniek KF, Lin WL, Sasaguri H, Caulfield T, Hubbard J, Daughrity L et al (2014) Aggregation-prone c9FTD/ALS poly(GA) RAN-translated proteins cause neurotoxicity by inducing ER stress. Acta Neuropathol 128:505–524. https://doi.org/10.1007/s00401-014-1336-5

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  60. Zu T, Liu Y, Banez-Coronel M, Reid T, Pletnikova O, Lewis J, Miller TM, Harms MB, Falchook AE, Subramony SH et al (2013) RAN proteins and RNA foci from antisense transcripts in C9ORF72 ALS and frontotemporal dementia. Proc Natl Acad Sci USA 110:E4968–4977. https://doi.org/10.1073/pnas.1315438110

    CAS  Article  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

This research was supported by Grants from ALS Association (5356S3), Target ALS (20134792), National Institute of Neurological Diseases and Stroke (NIH R01NS088578 and NS047101), and Pam Golden. JJ is a recipient of career development Grant from Muscular Dystrophy Association (479769) and was supported by postdoctoral training Grant (T32 AG00216) and postdoctoral fellowship (F32 NS087842) from the NIH. MB is supported by NIH NIGMS Award T32GM008666.

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Corresponding author

Correspondence to John Ravits.

Additional information

Shahram Saberi, Jennifer E. Stauffer and Jie Jiang contributed equally.

Electronic supplementary material

Below is the link to the electronic supplementary material.

401_2017_1793_MOESM1_ESM.pdf

Supplemental Figure 1 Dipeptide repeat protein distribution in different CNS regions. (a-h) Burden of all DPRs in different layers of cortex. Motor cortex (a) and frontal cortex (b) are disease-related parts of cortex, while occipital cortex (c) and parietal cortex (d) are disease-unrelated parts. Retrosplenial granular cortex, cerebellum, hippocampus and olfactory bulb are of uncertain relation to disease. All DPRs are more abundant in layers of brain with prominent neuronal population (layers 2-6). No DPRs could be seen in subcortical white matter with mostly glial and oligodendrocyte population. (i) Burden of DPRs in different parts of lumbar spinal cord. No DPRs could be seen in white matter (columns) of spinal cord with mostly glial and oligodendrocyte population. (j) Specificity of poly-GR immunoreactivity is demonstrated by quenching antibody with recombinant GST-(GR)10 proteins. (k-l) When analyzed by actual percentage as opposed to grading of neurons that contain DPRs, poly-GR is still uniquely significantly more abundant in disease-related areas of CNS (PDF 12142 kb)

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Supplemental Figure 2 RanGap, Lamin B1, Nup205 and Importin β1 immunohistochemical (IHC) staining in C9 ALS and controls. (a-d) IHC identifying RanGap in Betz cells (a-b) and spinal motor neurons (c-d) of C9 ALS (a and c) and controls (b and d). (e-h) IHC identifying Lamin B1 in Betz cells (e-f) and spinal motor neurons (g-h) of C9 ALS (e and g) and controls (f and h). Patterns of expression of Lamin B1 is very similar as RanGap. (i-l) IHC identifying Nup205 in Betz cells (i-j) and spinal motor neurons (k-l) of C9 ALS (i and k) and controls (j and l). Despite the prominent nuclear signal seen with RanGap and Lamin B1, the signal with Nup205 is mostly diffuse in the cytoplasm. (m-n) There is no significant differences between controls, SALS and C9-ALS cases in patterns of expression of Lamin B1 in spinal motor neurons. (o-p) IHC identifying Importin β1 in spinal motor neurons shows smooth, intermediate or irregular patterns (o) and there are no significant differences between controls, SALS and C9-ALS in patterns of expression (p) (PDF 9690 kb)

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Supplemental Figure 3 Diffuse nuclear pattern of RanGap is a three-dimensional effect. (a–d) Examples of diffuse nuclear RanGap signal in the motor neurons of spinal cord in C9 ALS cases when confocal images are stacked. On the left side and at the bottom of each panel are the transversal views of each sample. Below (red line) and to the left (yellow line) are the corresponding horizontal and transversal sections of each neuron. (a´–d´) show nuclear ring pattern seen by a three-dimensional plane in which image is cut through the cell (a´, b´, c´ and d´ are different co-focal planes through a, b, c and d, respectively) (PDF 9846 kb)

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Supplemental Figure 4 Aberrant nuclear shapes in C9 ALS and SALS patients. (a–i) Nuclear morphologies were evaluated based on RanGap IF staining. We classified them as normal (a–c), moderately aberrant (d–f), and severely aberrant (g–i). (j) Evaluating the shape of the spinal motor neuron nucleus with RanGap shows slightly more moderately and severely aberrant nucleus in ALS cases (both SALS and C9 ALS) in comparison to controls (PDF 16387 kb)

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Supplemental Figure 5 Short and long C9orf72 mRNA and proteins. (a–b) C9orf72 protein detection with immunohistochemical staining shows significant decrease after quenching with recombinant C9orf72 proteins in human tonsil epithelial lining. (c) Western blotting shows that C9orf72 protein expression is not decreased in cerebellum of C9 ALS compared to control and SALS cases. (d–e) Short (d) and long (e) mRNA foci (small black dot-like signals) could be seen in the epithelium of human tonsil, using CISH (chromogenic in situ hybridization), but not in the same tissue without applying probes (d and e-insert). (f) No predicted short isoform C9orf72 protein is detected using antibodies generated with amino acid 1-169, which should recognize both long and the predicted short isoform C9orf72 protein (PDF 24307 kb)

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Saberi, S., Stauffer, J.E., Jiang, J. et al. Sense-encoded poly-GR dipeptide repeat proteins correlate to neurodegeneration and uniquely co-localize with TDP-43 in dendrites of repeat-expanded C9orf72 amyotrophic lateral sclerosis. Acta Neuropathol 135, 459–474 (2018). https://doi.org/10.1007/s00401-017-1793-8

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  • DOI: https://doi.org/10.1007/s00401-017-1793-8

Keywords

  • C9orf72
  • Hexanucleotide repeat expansions
  • Amyotrophic lateral sclerosis
  • Dipeptide repeat proteins
  • Arginine-containing dipeptide repeat proteins
  • Poly-GR
  • Sense strand
  • Antisense strand
  • Nuclear pore complex and nucleocytoplasmic transport