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Molecular Neurobiology

, Volume 54, Issue 4, pp 3062–3077 | Cite as

C9ORF72 Regulates Stress Granule Formation and Its Deficiency Impairs Stress Granule Assembly, Hypersensitizing Cells to Stress

  • Niran Maharjan
  • Christina Künzli
  • Kilian Buthey
  • Smita SaxenaEmail author
Article

Abstract

Hexanucleotide repeat expansions in the C9ORF72 gene are causally associated with frontotemporal lobar dementia (FTLD) and/or amyotrophic lateral sclerosis (ALS). The physiological function of the normal C9ORF72 protein remains unclear. In this study, we characterized the subcellular localization of C9ORF72 to processing bodies (P-bodies) and its recruitment to stress granules (SGs) upon stress-related stimuli. Gain of function and loss of function experiments revealed that the long isoform of C9ORF72 protein regulates SG assembly. CRISPR/Cas9-mediated knockdown of C9ORF72 completely abolished SG formation, negatively impacted the expression of SG-associated proteins such as TIA-1 and HuR, and accelerated cell death. Loss of C9ORF72 expression further compromised cellular recovery responses after the removal of stress. Additionally, mimicking the pathogenic condition via the expression of hexanucleotide expansion upstream of C9ORF72 impaired the expression of the C9ORF72 protein, caused an abnormal accumulation of RNA foci, and led to the spontaneous formation of SGs. Our study identifies a novel function for normal C9ORF72 in SG assembly and sheds light into how the mutant expansions might impair SG formation and cellular-stress-related adaptive responses.

Keywords

C9ORF72 ALS Motor neuron degeneration Stress granules Cell recovery 

Notes

Acknowledgments

This work was supported by the Synapsis Foundation, Frick foundation for ALS research, and Swiss National Science Foundation Professorship grant (150756), to S.S. We thank Gabor Morotz, Kings College London, UK, for help with cortical neuron cultures.

Supplementary material

12035_2016_9850_MOESM1_ESM.pdf (894 kb)
Fig. S1 a) Examination of the phosphorylation status of eIF2a (peIF2a) revealed that C9-CRISPR cells with or without DTT treatment displayed significantly lower amounts of phosphorylated eIF2a as compared to control condition. b) Quantification of peIF2a intensity in C9-CRISPR positive and negative cells in the presence or absence of DTT treatment. Each value represents the mean of ± SEM of 15 cells each from 3 independent experiments, ***p=<0.001. c) and d) Immunostaining of n2a cells overexpressing C9(LF) myc construct with TDP-43 and FUS/TLS, displayed normal nuclear localization and expression levels as in GFP transfected cells. (Scale bar = 4 μm for all image panels) (PDF 893 kb)
12035_2016_9850_MOESM2_ESM.pdf (682 kb)
Fig. S2 Transfection of cortical neurons with GFP construct having long repeat expansions led to the formation of TIA-1 positive SGs (arrow), (Scale bar = 4 μm) (PDF 682 kb)
12035_2016_9850_MOESM3_ESM.pdf (434 kb)
Fig. S3 a) Representative image of FISH against hexanucleotide repeat showing formation of RNA foci with increment in repeat length, (Scale bar = 4 μm). b) Western blot analysis of n2a cells transfected with GFP constructs with different hexanucleotide repeat expansion shows formation of dipeptide-repeat protein with higher repeat expansion (shown by arrow). c) Cell viability assay using Propidium Iodide (PI) after transfection of cortical neurons with GFP construct with different hexanucleotide repeat expansion length shows increase in dead cells with increment in repeat length. Each value represents the mean of ± SEM of 15 cells each from 3 independent experiments (PDF 433 kb)

References

  1. 1.
    Rowland L, Shneider N (2001) Amyotrophic lateral sclerosis. N Engl J Med 344:1688–1700. doi: 10.1056/NEJM200105313442207 CrossRefPubMedGoogle Scholar
  2. 2.
    Ratnavalli E, Brayne C, Dawson K, Hodges JR (2002) The prevalence of frontotemporal dementia. Neurology 58:1615–1621CrossRefPubMedGoogle Scholar
  3. 3.
    Liscic RM, Storandt M, Cairns NJ, Morris JC (2007) Clinical and psychometric distinction of frontotemporal and Alzheimer dementias. Arch Neurol 64:535–540CrossRefPubMedGoogle Scholar
  4. 4.
    Phukan J, Pender NP, Hardiman O (2007) Cognitive impairment in amyotrophic lateral sclerosis. Lancet Neurol 6(11):994–1003CrossRefPubMedGoogle Scholar
  5. 5.
    Giordana MT, Ferrero P, Grifoni S, Pellerino A, Naldi A, Montuschi A (2011) Dementia and cognitive impairment in amyotrophic lateral sclerosis: a review. Neurol Sci 32:9–16. doi: 10.1007/s10072-010-0439-6 CrossRefPubMedGoogle Scholar
  6. 6.
    Lomen-Hoerth C, Anderson T, Miller B. The overlap of amyotrophic lateral sclerosis and frontotemporal dementia. Neurology. 2002. pp. 1077–1079. doi: 10.1212/WNL.59.7.1077
  7. 7.
    Neumann M, Roeber S, Kretzschmar H a, Rademakers R, Baker M, Mackenzie IR a (2009) Abundant FUS-immunoreactive pathology in neuronal intermediate filament inclusion disease. Acta Neuropathol 118:605–616. doi: 10.1007/s00401-009-0581-5 CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Neumann M, Sampathu DM, Kwong LK, Truax AC, Micsenyi MC, Chou TT, Bruce J, Schuck T et al (2006) Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Science 314:130–3. doi: 10.1126/science.1134108
  9. 9.
    Renton AE, Majounie E, Waite A, Simón-Sánchez J, Rollinson S, Gibbs JR, Schymick JC, Laaksovirta H et al (2011) A hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD. Neuron 72:257–68. doi: 10.1016/j.neuron.2011.09.010
  10. 10.
    DeJesus-Hernandez M, Mackenzie IR, Boeve BF, Boxer AL, Baker M, Rutherford NJ, Nicholson AM, Finch NA et al (2011) Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS. Neuron 72:245–256. doi: 10.1016/j.neuron.2011.09.011
  11. 11.
    Mori K, Weng S-M, Arzberger T, May S, Rentzsch K, Kremmer E, Schmid B, Kretzschmar HA et al (2013) The C9orf72 GGGGCC repeat is translated into aggregating dipeptide-repeat proteins in FTLD/ALS. Science 339:1335–8. doi: 10.1126/science.1232927
  12. 12.
    Gijselinck I, Van Langenhove T, van der Zee J, Sleegers K, Philtjens S, Kleinberger G, Janssens J, Bettens K 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 11:54–65Google Scholar
  13. 13.
    Ash PE a, Bieniek KF, Gendron TF, Caulfield T, Lin W-L, Dejesus-Hernandez M, van Blitterswijk MM, Jansen-West K et al (2013) Unconventional translation of C9ORF72 GGGGCC expansion generates insoluble polypeptides specific to c9FTD/ALS. Neuron 77:639–646. doi: 10.1016/j.neuron.2013.02.004
  14. 14.
    Mackenzie IR, Arzberger T, Kremmer E, Troost D, Lorenzl S, Mori K, Weng SM, Haass C et al (2013) Dipeptide repeat protein pathology in C9ORF72 mutation cases: clinico-pathological correlations. Acta Neuropathol 126:859–79. doi: 10.1007/s00401-013-1181-y
  15. 15.
    Farg MA, Sundaramoorthy V, Sultana JM, Yang S, Atkinson RAK, Levina V, Halloran MA, Gleeson PA et al (2014) C9ORF72, implicated in amytrophic lateral sclerosis and frontotemporal dementia, regulates endosomal trafficking. Hum Mol Genet 23:3579–3595. doi: 10.1093/hmg/ddu068
  16. 16.
    Xu Z, Poidevin M, Li X, Li Y, Shu L, Nelson DL, Li H, Hales CM et al (2013) Expanded GGGGCC repeat RNA associated with amyotrophic lateral sclerosis and frontotemporal dementia causes neurodegeneration. Proc Natl Acad Sci U S A 110:7778–83. doi: 10.1073/pnas.1219643110
  17. 17.
    Strober W. Trypan blue exclusion test of cell viability. Curr Protoc Immunol. 2001;Appendix 3: Appendix 3B. doi: 10.1002/0471142735.ima03bs21
  18. 18.
    Waite AJ, Bäumer 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 35:1779.e5–1779.e13. doi: 10.1016/j.neurobiolaging.2014.01.016
  19. 19.
    Wilczynska A, Aigueperse C, Kress M, Dautry F, Weil D (2005) The translational regulator CPEB1 provides a link between dcp1 bodies and stress granules. J Cell Sci 118:981–992CrossRefPubMedGoogle Scholar
  20. 20.
    Kedersha N, Stoecklin G, Ayodele M, Yacono P, Lykke-Andersen J, Fitzler MJ, Scheuner D, Kaufman RJ et al (2005) Stress granules and processing bodies are dynamically linked sites of mRNP remodeling. J Cell Biol 169:871–884Google Scholar
  21. 21.
    Anderson P, Kedersha N (2006) RNA granules. J Cell Biol 172:803–808CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Kedersha N, Anderson P (2002) Stress granules: sites of mRNA triage that regulate mRNA stability and translatability. Biochem Soc Trans 30:963–969CrossRefPubMedGoogle Scholar
  23. 23.
    Zhang D, Iyer LM, He F, Aravind L. Discovery of Novel DENN Proteins: Implications for the Evolution of Eukaryotic Intracellular Membrane Structures and Human Disease. Front Genet. 2012;3. doi: 10.3389/fgene.2012.00283
  24. 24.
    Levine TP, Daniels RD, Gatta AT, Wong LH, Hayes MJ (2013) The product of C9orf72, a gene strongly implicated in neurodegeneration, is structurally related to DENN Rab-GEFs. Bioinformatics 29:499–503. doi: 10.1093/bioinformatics/bts725 CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Behrends C, Sowa ME, Gygi SP, Harper JW (2010) Network organization of the human autophagy system. Nature 466:68–76CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Nonhoff U, Ralser M, Welzel F, Piccini I, Balzereit D, Yaspo M, Lehrach H, 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 18:1385–96. doi: 10.1091/mbc.E06-12-1120
  27. 27.
    Figley MD, Bieri G, Kolaitis R-M, Taylor JP, Gitler AD (2014) Profilin 1 associates with stress granules and ALS-linked mutations alter stress granule dynamics. J Neurosci 34:8083–97. doi: 10.1523/JNEUROSCI.0543-14.2014 CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Zou T, Yang X, Pan D, Huang J, Sahin M, Zhou J (2011) SMN deficiency reduces cellular ability to form stress granules, sensitizing cells to stress. Cell Mol Neurobiol 31:541–50. doi: 10.1007/s10571-011-9647-8 CrossRefPubMedGoogle Scholar
  29. 29.
    Seguin SJ, Morelli FF, Vinet J, Amore D, De Biasi S, Poletti A, Rubinsztein DC, Carra S (2014) Inhibition of autophagy, lysosome and VCP function impairs stress granule assembly. Cell Death Differ. Nat Publ Group 21:1838–1851. doi: 10.1038/cdd.2014.103
  30. 30.
    Cooper-Knock J, Walsh MJ, Higginbottom A, Highley JR, Dickman MJ, Edbauer D, Ince PG, Wharton SB et al (2014) Sequestration of multiple RNA recognition motif-containing proteins by C9orf72 repeat expansions. Brain 137:2040–2051. doi: 10.1093/brain/awu120
  31. 31.
    McGurk L, Lee VM, Trojanowksi JQ, Van Deerlin VM, Lee EB, Bonini NM (2014) Poly-a binding protein-1 localization to a subset of TDP-43 inclusions in amyotrophic lateral sclerosis occurs more frequently in patients harboring an expansion in C9orf72. J Neuropathol Exp Neurol 73(9):837–845. doi: 10.1097/NEN.0000000000000102
  32. 32.
    Jamison JT, Kayali F, Rudolph J, Marshall M, Kimball SR, DeGracia DJ (2008) Persistent redistribution of poly-adenylated mRNAs correlates with translation arrest and cell death following global brain ischemia and reperfusion. Neuroscience 154:504–520CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Therrien M, Rouleau G a, Dion P a, Parker JA (2013) Deletion of C9ORF72 results in motor neuron degeneration and stress sensitivity in C. elegans. PLoS One 8, e83450. doi: 10.1371/journal.pone.0083450 CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Almeida S, Gascon E, Tran H, Chou HJ, Gendron TF, Degroot S, Tapper AR, Sellier C et al (2013) Modeling key pathological features of frontotemporal dementia with C9ORF72 repeat expansion in iPSC-derived human neurons. Acta Neuropathol 126:385–399Google Scholar
  35. 35.
    Ciura S, Lattante S, Le Ber I, Latouche M, Tostivint H, Brice A, Kabashi E (2013) Loss of function of C9orf72 causes motor deficits in a zebrafish model of amyotrophic lateral sclerosis. Ann Neurol 74:180–187Google Scholar
  36. 36.
    Lagier-Tourenne C, Baughn M, Rigo F, Sun S, Liu P, Li H-R, Jiang J, Watt AT et al (2013) Targeted degradation of sense and antisense C9orf72 RNA foci as therapy for ALS and frontotemporal degeneration. Proc Natl Acad Sci U S A 110:E4530–9. doi: 10.1073/pnas.1318835110
  37. 37.
    Mizielinska S, Grönke S, Niccoli T, Ridler CE, Clayton EL, Devoy A, Moens T, Norona FE et al (2014) C9orf72 repeat expansions cause neurodegeneration in Drosophila through arginine-rich proteins. Science 345:1192–1194. doi: 10.1126/science.1256800
  38. 38.
    Zhang Y-J, Jansen-West K, Xu Y-F, Gendron TF, Bieniek KF, Lin W-L, Sasaguri H, Caulfield T et al (2014) Aggregation-prone c9FTD/ALS poly(GA) RAN-translated proteins cause neurotoxicity by inducing ER stress. Acta Neuropathol 128:505–524. doi: 10.1007/s00401-014-1336-5
  39. 39.
    Kwon I, Xiang S, Kato M, Wu L, Theodoropoulos P, Wang T, Kim J, Yun J et al (2014) Poly-dipeptides encoded by the C9ORF72 repeats bind nucleoli, impede RNA biogenesis, and kill cells. Science 345:1254917. doi: 10.1126/science.1254917
  40. 40.
    Wen X, Tan W, Westergard T, Krishnamurthy K, Markandaiah SS, Shi Y, Lin S, Shneider NA 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. doi: 10.1016/j.neuron.2014.12.010
  41. 41.
    Tao Z, Wang H, Xia Q, Li K, Li K, Jiang X, Xu G, Wang G et al (2015) Nucleolar stress and impaired stress granule formation contribute to C9orf72 RAN translation-induced cytotoxicity. Hum Mol Genet 24:2426–41. doi: 10.1093/hmg/ddv005
  42. 42.
    Peters OM, Cabrera GT, Tran H, Gendron TF, McKeon JE, Metterville J, Weiss A, Wightman N et al (2015) Human C9ORF72 hexanucleotide expansion reproduces RNA foci and dipeptide repeat proteins but not neurodegeneration in BAC transgenic mice. Neuron Elsevier Ltd 88:902–909. doi: 10.1016/j.neuron.2015.11.018
  43. 43.
    O’Rourke JG, Bogdanik L, Muhammad AKMG, Gendron TF, Kim KJ, Austin A, Cady J, Liu EY et al (2015) C9orf72 BAC transgenic mice display typical pathologic features of ALS/FTD. Neuron 88:892–901. doi: 10.1016/j.neuron.2015.10.027

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Institute of Cell BiologyUniversity of BernBernSwitzerland
  2. 2.Graduate School for Cellular and Biomedical SciencesUniversity of BernBernSwitzerland

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