Skip to main content

Nuclear ribonucleoprotein-containing foci increase in size in non-dividing cells from patients with myotonic dystrophy type 2

Histochemistry and Cell Biology volume 138, pages 699–707 (2012)Cite this article

Abstract

Myotonic dystrophies (DM) are genetically based neuromuscular disorders characterized by the accumulation of mutant transcripts into peculiar intranuclear foci, where different splicing factors (among which the alternative splicing regulator muscleblind-like 1 protein, MBNL1) are ectopically sequestered. The aim of the present investigation was to describe the dynamics of the DM-specific intranuclear foci in interphase nuclei and during mitosis, as well as after the exit from the cell cycle. Primary cultures of skin fibroblasts from DM2 patients were used, as a model system to reproduce in vitro, as accurately as possible, the in vivo conditions. Cycling and resting fibroblasts were investigated by immunocytochemical and morphometric techniques, and the relative amounts of MBNL1 were also estimated by western blotting. MBNL1-containing foci were exclusively found in the nucleus during most of the interphase, while being observed in the cytoplasm during mitosis when they never associate with the chromosomes; the foci remained in the cytoplasm at cytodieresis, and underwent disassembly in early G1 to be reformed in the nucleus at each cell cycle. After fibroblasts had stopped dividing in late-passage cultures, the nuclear foci were observed to progressively increase in size. Interestingly, measurements on muscle biopsies taken from the same DM2 patients at different ages demonstrated that, in the nuclei of myofibers, the MBNL1-containing foci become larger with increasing patient’s age. As a whole, these results suggest that in non-dividing cells of DM2 patients the sequestration in the nuclear foci of factors needed for RNA processing would be continuous and progressive, eventually leading to the onset (and the worsening with time) of the pathological traits. This is consistent with the evidence that in DM patients the most affected organs or tissues are those where non-renewing cells are mainly present, i.e., the central nervous system, heart and skeletal muscle.

This is a preview of subscription content, access via your institution.

Access options

Buy single article

Instant access to the full article PDF.

43,34 €

Price includes VAT (Finland)
Tax calculation will be finalised during checkout.

Fig. 1
Fig. 2
Fig. 3
Fig. 4

References

  • Andrade LE, Tan EM, Chan EK (1993) Immunocytochemical analysis of the coiled body in the cell cycle and during cell proliferation. Proc Natl Acad Sci USA 90:1947–1951

    PubMed  Article  CAS  Google Scholar 

  • Bachinski LL, Udd B, Meola G, Sansone V, Bassez G, Eymard B, Thornton CA, Moxley RT, Harper PS, Rogers MT, Jurkat-Rott K, Lehmann-Horn F, Wieser T, Gamez J, Navarro C, Bottani A, Kohler A, Shriver MD, Sallinen R, Wessman M, Zhang S, Wright FA, Krahe R (2003) Confirmation of the type 2 myotonic dystrophy (CCTG)n expansion mutation in patients with proximal myotonic myopathy/proximal myotonic dystrophy of different European origins: a single shared haplotype indicates an ancestral founder effect. Am J Hum Gen 73:835–848

    Article  CAS  Google Scholar 

  • Biggiogera M, Pellicciari C (2000) Heterogeneous ectopic RNP-derived structures (HERDS) are markers of transcriptional arrest. FASEB J 14:828–834

    PubMed  CAS  Google Scholar 

  • Biggiogera M, Bottone MG, Scovassi AI, Soldani C, Vecchio L, Pellicciari C (2004) Rearrangement of nuclear ribonucleoprotein (RNP)-containing structures during apoptosis and transcriptional arrest. Biol Cell 96:603–615

    PubMed  Article  CAS  Google Scholar 

  • Biggiogera M, Cisterna B, Bottone MG, Soldani C, Pellicciari C (2007) Nuclear RNP and nucleolar-associated proteins during apoptosis: a politically correct form of segregation? Dynamic Cell Biol 1:65–71

    Google Scholar 

  • Brook JD, McCurrach ME, Harley HG, Buckler AJ, Church D, Aburatani H, Hunter K, Stanton VP, Thirion JP, Hudson T, Sohn R, Zemelman B, Snell RG, Rundle SA, Crow S, Davies J, Shelbourne P, Buxton J, Jones C, Juvonen V, Johnson K, Harper PS, Shaw DJ, Housman DE (1992) Molecular basis of myotonic dystrophy: expansion of a trinucleotide (CTG) repeat at the 3′ end of a transcript encoding a protein kinase family member. Cell 69:385–387

    PubMed  CAS  Google Scholar 

  • Cardani R, Mancinelli E, Sansone V, Rotondo G, Meola G (2004) Biomolecular identification of (CCTG)n mutation in myotonic dystrophy type 2 (DM2) by FISH on muscle biopsy. Eur J Histochem 48:437–442

    PubMed  CAS  Google Scholar 

  • Cardani R, Mancinelli E, Rotondo G, Sansone V, Meola G (2006) Muscleblind-like protein 1 nuclear sequestration is a molecular pathology marker of DM1 and DM2. Eur J Histochem 50:177–182

    PubMed  CAS  Google Scholar 

  • Cardani R, Mancinelli E, Giagnacovo M, Sansone V, Meola G (2009) Ribonuclear inclusions as biomarker of myotonic dystrophy type 2, even in improperly frozen or defrozen skeletal muscle biopsies. Eur J Histochem 53:107–112

    PubMed  CAS  Google Scholar 

  • Chen YC, Kappel C, Beaudouin J, Eils R, Spector DL (2008) Live cell dynamics of promyelocytic leukemia nuclear bodies upon entry into and exit from mitosis. Mol Biol Cell 19:3147–3162

    PubMed  Article  CAS  Google Scholar 

  • Day JW, Roelofs R, Leroy B, Pech I, Benzow K, Ranum LP (1999) Clinical and genetic characteristics of a five-generation family with a novel form of myotonic dystrophy (DM2). Neuromuscul Disord 9:19–27

    PubMed  Article  CAS  Google Scholar 

  • Day JW, Ricker K, Jacobsen JF, Rasmussen LJ, Dick KA, Kress W, Schneider C, Koch MC, Beilman GJ, Harrison AR, Dalton JC, Ranum LP (2003) Myotonic dystrophy type 2: molecular, diagnostic and clinical spectrum. Neurology 60:657–664

    PubMed  Article  CAS  Google Scholar 

  • Dimri GP, Lee X, Basile G, Acosta M, Scott G, Roskelley C, Medrano EE, Linskens M, Rubelj I, Pereira Smith O, Peacocke M, Campisi J (1995) A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci USA 92:9363–9367

    PubMed  Article  CAS  Google Scholar 

  • Dundr M (2011) Seed and grow: a two-step model for nuclear body biogenesis. J Cell Biol 193:605–606

    PubMed  Article  CAS  Google Scholar 

  • Fakan S (2004) The functional architecture of the nucleus as analysed by ultrastructural cytochemistry. Histochem Cell Biol 122:83–93

    PubMed  Article  CAS  Google Scholar 

  • Fardaei M, Rogers MT, Thorpe HM, Larkin K, Hamshere MG, Harper PS, Brook JD (2002) Three proteins, MBNL, MBLL and MBXL, co-localize in vivo with nuclear foci of expanded-repeat transcripts in DM1 and DM2 cells. Hum Mol Genet 11:805–814

    PubMed  Article  CAS  Google Scholar 

  • Fu YH, Pizzuti A, Fenwick RG Jr, King J, Rajnarayan S, Dunne PW, Dubel J, Nasser GA, Ashizawa T, de Jong P, Wieringa B, Korneluk R, Perryman MB, Epstein HF, Caskey CT (1992) An unstable triplet repeat in a gene related to myotonic muscular dystrophy. Science 255:1256–1258

    PubMed  Article  CAS  Google Scholar 

  • Hayflick L, Moorhead PS (1961) The serial cultivation of human diploid cell strains. Exp Cell Res 25:585–621

    Article  Google Scholar 

  • Holt I, Jacquemin V, Fardaei M, Sewry CA, Butler-Browne GS, Furling D, Brook JD, Morris GE (2009) Muscleblind-like proteins: similarities and differences in normal and myotonic dystrophy muscle. Am J Pathol 174:216–227

    PubMed  Article  CAS  Google Scholar 

  • Jones K, Jin B, Iakova P, Huichalaf C, Sarkar P, Schneider-Gold C, Schoser B, Meola G, Shyu AB, Timchenko N, Timchenko L (2011) RNA Foci, CUGBP1, and ZNF9 are the primary targets of the mutant CUG and CCUG repeats expanded in myotonic dystrophies type 1 and type 2. Am J Pathol. doi:10.1016/j.ajpath.2011.07.013 (in press)

    Google Scholar 

  • Ladd AN, Charlet N, Cooper TA (2001) The CELF family of RNA binding proteins is implicated in cell-specific and developmentally regulated alternative splicing. Mol Cell Biol 21:1285–1296

    PubMed  Article  CAS  Google Scholar 

  • Liquori CL, Ricker K, Moseley ML, Jacobsen JF, Kress W, Naylor SL, Day JW, Ranum LP (2001) Myotonic dystrophy type 2 caused by a CCTG expansion in intron 1 of ZNF9. Science 293:864–867

    PubMed  Article  CAS  Google Scholar 

  • Llorian M, Smith CW (2011) Decoding muscle alternative splicing. Curr Opin Genet Dev 21:380–387

    PubMed  Article  CAS  Google Scholar 

  • Mahadevan M, Tsilfidis C, Sabourin L, Shutler G, Amemiya C, Jansen G, Neville C, Narang M, Barceló J, O’Hoy K, Leblond S, Earle-MacDonald J, de Jong PJ, Wieringa B, Korneluk RG (1992) Myotonic dystrophy mutation: an unstable CTG repeat in the 3′ untranslated region of the gene. Science 255:1253–1255

    PubMed  Article  CAS  Google Scholar 

  • Malatesta M, Zancanaro C, Martin TE, Chan EKL, Amalric F, Lührmann R, Vogel P, Fakan S (1994) Cytochemical and immunocytochemical characterization of nuclear bodies during hibernation. Eur J Cell Biol 65:82–93

    PubMed  CAS  Google Scholar 

  • Malatesta M, Cardinali A, Battistelli S, Zancanaro C, Martin TE, Fakan S, Gazzanelli G (1999) Nuclear bodies are usual constituents in tissues of hibernating dormice. Anat Rec 254:389–395

    PubMed  Article  CAS  Google Scholar 

  • Malatesta M, Giagnacovo M, Cardani R, Meola G, Pellicciari C (2011) RNA processing is altered in skeletal muscle nuclei of patients affected by myotonic dystrophy. Histochem Cell Biol 135:419–425

    PubMed  Article  CAS  Google Scholar 

  • Mankodi A, Teng-Umnuay P, Krym M, Henderson D, Swanson M, Thornton CA (2003) Ribonuclear inclusions in skeletal muscle in myotonic dystrophy types 1 and 2. Ann Neurol 54:760–768

    PubMed  Article  CAS  Google Scholar 

  • Mehta IS, Figgitt M, Clements CS, Kill IR, Bridger JM (2007) Alterations to nuclear architecture and genome behavior in senescent cells. Ann NY Acad Sci 1100:250–263

    PubMed  Article  CAS  Google Scholar 

  • Meola G, Moxley RT 3rd (2004) Myotonic dystrophy type 2 and related myotonic disorders. J Neurol 251:1173–1182

    PubMed  Article  Google Scholar 

  • Miller JW, Urbinati CR, Teng-Umnuay P, Stenberg MG, Byrne BJ, Thornton CA, Swanson MS (2000) Recruitment of human muscleblind proteins to (CUG)(n) expansions associated with myotonic dystrophy. EMBO J 19:4439–4448

    PubMed  Article  CAS  Google Scholar 

  • Moxley RT, Meola G (2008) The myotonic dystrophies. Chapter 47. In: Rosenberg RN, Di Mauro S, Paulson HL, Ptacek L, Nestler EJ (eds) The molecular and genetic basis of neurologic and psychiatric disease, 4th edn. Lippincot Williams, Philadelphia

    Google Scholar 

  • Mulders SAM, van den Broek WJAA, Wheeler TM, Croes HJE, van Kuik-Romeijn P, de Kimpe SJ, Furling D, Platenburg GJ, Gourdon G, Thornton CA, Wieringa B, Wansink DG (2009) Triplet-repeat oligonucleotide-mediated reversal of RNA toxicity in myotonic dystrophy. Proc Natl Acad Sci USA 106:13915–13920

    PubMed  Article  CAS  Google Scholar 

  • Perdoni F, Malatesta M, Cardani R, Giagnacovo M, Mancinelli E, Meola G, Pellicciari C (2009) RNA/MBNL1-containing foci in myoblast nuclei from patients affected by myotonic dystrophy type 2: an immunocytochemical study. Eur J Histochem 53:151–158

    PubMed  CAS  Google Scholar 

  • Querido E, Gallardo F, Beaudoin M, Ménard C, Chartrand P (2011) Stochastic and reversible aggregation of mRNA with expanded CUG-triplet repeats. J Cell Sci 124:1703–1714

    PubMed  Article  CAS  Google Scholar 

  • Ranum LP, Rasmussen PF, Benzow KA, Koob MD, Day JW (1998) Genetic mapping of a second myotonic dystrophy locus. Nat Genet 19:196–198

    PubMed  Article  CAS  Google Scholar 

  • Rubin H (1997) Cell aging in vivo and in vitro. Mech Ageing Dev 98:1–35

    PubMed  Article  CAS  Google Scholar 

  • Savkur RS, Philips AV, Cooper TA (2001) Aberrant regulation of insulin receptor alternative splicing is associated with insulin resistance in myotonic dystrophy. Nat Genet 29:40–47

    PubMed  Article  CAS  Google Scholar 

  • Savkur RS, Philips AV, Cooper TA, Dalton JC, Moseley ML, Ranum LP, Day JW (2004) Insulin receptor splicing alteration in myotonic dystrophy type 2. Am J Hum Genet 74:1309–1313

    PubMed  Article  CAS  Google Scholar 

  • Schoser B, Timchenko L (2010) Myotonic dystrophies 1 and 2: complex diseases with complex mechanisms. Curr Genomics 11:77–90

    PubMed  Article  CAS  Google Scholar 

  • Sikora E, Arendt T, Bennett M, Narita M (2011) Impact of cellular senescence signature on ageing research. Ageing Res Rev 10:146–152

    PubMed  Article  CAS  Google Scholar 

  • Smolinski DJ, Wrobel B, Noble A, Zienkiewicz A, Gorska-Brylass A (2011) Periodic expression of Sm proteins parallels formation of nuclear Cajal bodies and cytoplasmic snRNP-rich bodies. Histochem Cell Biol 136:527–541

    PubMed  Article  CAS  Google Scholar 

  • Suominen T, Bachinski LL, Auvinen S, Hackman P, Baggerly KA, Angelini C, Peltonen L, Krahe R, Udd B (2011) Population frequency of myotonic dystrophy: higher than expected frequency of myotonic dystrophy type 2 (DM2) mutation in Finland. Eur J Hum Gen 19:776–782

    Article  CAS  Google Scholar 

  • Udd B, Meola G, Krahe R, Wansink DG, Bassez G, Kress W, Schoser B, Moxley R (2011) Myotonic dystrophy type 2 (DM2) and related disorders. Neuromusc Disord 21:443–450

    PubMed  Article  CAS  Google Scholar 

  • Vihola A, Bassez G, Meola G, Zhang S, Haapasalo H, Paetau A, Mancinelli E, Rouche A, Hogrel JY, Laforêt P, Maisonobe T, Pellissier JF, Krahe R, Eymard B, Udd B (2003) Histopathological differences of myotonic dystrophy type 1 (DM1) and PROMM/DM2. Neurology 60:1854–1857

    PubMed  Article  CAS  Google Scholar 

  • Wang G-S, Kearney DL, De Biasi M, Taffet G, Cooper TA (2007) Elevation of RNA-binding protein CUGBP1 is an early event in an inducible heart-specific mouse model of myotonic dystrophy. J Clin Invest 117:2802–2811

    PubMed  Article  CAS  Google Scholar 

  • Warf MB, Nakamori M, Matthys CM, Thornton CA, Berglund JA (2009) Pentamidine reverses the splicing defects associated with myotonic dystrophy. Proc Natl Acad Sci USA 106:18551–18556

    PubMed  Article  CAS  Google Scholar 

  • White AE, Leslie ME, Calvi BR, Marzluff WF, Duronio RJ (2007) Developmental and cell cycle regulation of the drosophila histone locus body. Mol Biol Cell 18:2491–2502

    PubMed  Article  CAS  Google Scholar 

Download references

Acknowledgments

We express our gratitude to Prof. C. A. Thornton for kindly providing us with the anti-MBNL1 antibody. Marzia Giagnacovo is a Ph.D. student in receipt of a fellowship from the Dottorato di Ricerca in Biologia Cellulare (University of Pavia).

Author information

Affiliations

  1. Dipartimento di Biologia e Biotecnologie “Lazzaro Spallanzani”, Laboratorio di Biologia cellulare e Neurobiologia, Università di Pavia, via A. Ferrata 9, 27100, Pavia, Italy

    M. Giagnacovo & C. Pellicciari

  2. Dipartimento di Scienze Neurologiche, Neuropsicologiche, Morfologiche e Motorie, Sezione di Anatomia e Istologia, Università di Verona, Verona, Italy

    M. Malatesta

  3. Dipartimento di Biologia Molecolare e Biotecnologie, Università degli Studi di Milano, Milano, Italy

    R. Cardani

  4. Centro per lo Studio delle Malattie Neuromuscolari, CMN, Milano, Italy

    R. Cardani

  5. Dipartimento di Neurologia, IRCCS Policlinico San Donato, Università degli Studi di Milano, Piazza E. Malan 1, San Donato Milanese, MI, Italy

    G. Meola

Authors
  1. M. Giagnacovo
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. M. Malatesta
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. R. Cardani
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. G. Meola
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. C. Pellicciari
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to C. Pellicciari.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Giagnacovo, M., Malatesta, M., Cardani, R. et al. Nuclear ribonucleoprotein-containing foci increase in size in non-dividing cells from patients with myotonic dystrophy type 2. Histochem Cell Biol 138, 699–707 (2012). https://doi.org/10.1007/s00418-012-0984-6

Download citation

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00418-012-0984-6

Keywords

  • Myotonic dystrophy
  • Fibroblasts
  • Skeletal muscle fibers
  • Nuclear foci
  • Cell cycle
  • Fluorescence microscopy
  • Immunocytochemistry