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ALS-derived fibroblasts exhibit reduced proliferation rate, cytoplasmic TDP-43 aggregation and a higher susceptibility to DNA damage

Abstract

Background

Dermic fibroblasts have been proposed as a potential genetic-ALS cellular model. This study aimed to explore whether dermic fibroblasts from patients with sporadic-ALS (sALS) recapitulate alterations typical of ALS motor neurons and exhibit abnormal DNA-damage response.

Methods

Dermic fibroblasts were obtained from eight sALS patients and four control subjects. Cellular characterization included proliferation rate analysis, cytoarchitecture studies and confocal immunofluorescence assessment for TDP-43. Additionally, basal and irradiation-induced DNA damage was evaluated by confocal immunofluorescence and biochemical techniques.

Results

sALS-fibroblasts showed decreased proliferation rates compared to controls. Additionally, whereas control fibroblasts exhibited the expected normal spindle-shaped morphology, ALS fibroblasts were thinner, with reduced cell size and enlarged nucleoli, with frequent cytoplasmic TDP-43aggregates. Also, baseline signs of DNA damage were evidenced more frequently in ALS-derived fibroblasts (11 versus 4% in control-fibroblasts). Assays for evaluating the irradiation-induced DNA damage demonstrated that DNA repair was defective in ALS-fibroblasts, accumulating more than double of γH2AX-positive DNA damage foci than controls. Very intriguingly, the proportion of fibroblasts particularly vulnerable to irradiation (with more than 15 DNA damage foci per nucleus) was seven times higher in ALS-derived fibroblasts than in controls.

Conclusions

Dermic-derived ALS fibroblasts recapitulate relevant cellular features of sALS and show a higher susceptibility to DNA damage and defective DNA repair responses. Altogether, these results support that dermic fibroblasts may represent a convenient and accessible ALS cellular model to study pathogenetic mechanisms, particularly those related to DNA damage response, as well as the eventual response to disease-modifying therapies.

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References

  1. 1.

    Logroscino G, Traynor BJ, Hardiman O, Chio A, Mitchell D, Swingler RJ et al (2010) Incidence of amyotrophic lateral sclerosis in Europe. J Neurol Neurosurg Psychiatry 81(4):385–390

  2. 2.

    Riancho J, Lozano-Cuesta P, Santurtun A, Sanchez-Juan P, Lopez-Vega JM, Berciano J et al (2016) Amyotrophic lateral sclerosis in Northern Spain 40 years later: what has changed? Neurodegener Dis 16(5–6):337–341

  3. 3.

    Brown RH, Al CA (2017) Amyotrophic Lateral Sclerosis. N Engl J Med 377(2):162–172

  4. 4.

    Zufiria M, Gil-Bea FJ, Fernandez-Torron R, Poza JJ, Munoz-Blanco JL, Rojas-Garcia R et al (2016) ALS: a bucket of genes, environment, metabolism and unknown ingredients. Prog Neurobiol 142:104–129

  5. 5.

    Riancho J, Bosque-Varela P, Perez-Pereda S, Povedano M, de Munain AL, Santurtun A (2018) The increasing importance of environmental conditions in amyotrophic lateral sclerosis. Int J Biometeorol 62:1361–1374

  6. 6.

    Ittner LM, Halliday GM, Kril JJ, Gotz J, Hodges JR, Kiernan MC (2015) FTD and ALS–translating mouse studies into clinical trials. Nat Rev Neurol 11(6):360–366

  7. 7.

    Lutz C (2018) Mouse models of ALS: Past, present and future. Brain Res 1693(Pt A):1–10

  8. 8.

    Picher-Martel V, Valdmanis PN, Gould PV, Julien JP, Dupre N (2016) From animal models to human disease: a genetic approach for personalized medicine in ALS. Acta Neuropathol Commun 4(1):70

  9. 9.

    Auburger G, Klinkenberg M, Drost J, Marcus K, Morales-Gordo B, Kunz WS et al (2012) Primary skin fibroblasts as a model of Parkinson's disease. Mol Neurobiol 46(1):20–27

  10. 10.

    Van Linthout S, Miteva K, Tschope C (2014) Crosstalk between fibroblasts and inflammatory cells. Cardiovasc Res 102(2):258–269

  11. 11.

    Sabatelli M, Zollino M, Conte A, Del Grande A, Marangi G, Lucchini M et al (2015) Primary fibroblasts cultures reveal TDP-43 abnormalities in amyotrophic lateral sclerosis patients with and without SOD1 mutations. Neurobiol Aging 36(5):2005

  12. 12.

    Imamura K, Izumi Y, Watanabe A, Tsukita K, Woltjen K, Yamamoto T et al (2017) The Src/c-Abl pathway is a potential therapeutic target in amyotrophic lateral sclerosis. Sci Transl Med 9:3962

  13. 13.

    Mata-Garrido J, Tapia O, Casafont I, Berciano MT, Cuadrado A, Lafarga M (2018) Persistent accumulation of unrepaired DNA damage in rat cortical neurons: nuclear organization and ChIP-seq analysis of damaged DNA. Acta Neuropathol Commun 6(1):68

  14. 14.

    Chow HM, Herrup K (2015) Genomic integrity and the ageing brain. Nat Rev Neurosci 16(11):672–684

  15. 15.

    Madabhushi R, Pan L, Tsai LH (2014) DNA damage and its links to neurodegeneration. Neuron 83(2):266–282

  16. 16.

    Sanders LH, Laganiere J, Cooper O, Mak SK, Vu BJ, Huang YA et al (2014) LRRK2 mutations cause mitochondrial DNA damage in iPSC-derived neural cells from Parkinson's disease patients: reversal by gene correction. Neurobiol Dis 62:381–386

  17. 17.

    Lovell MA, Markesbery WR (2007) Oxidative DNA damage in mild cognitive impairment and late-stage Alzheimer's disease. Nucleic Acids Res 35(22):7497–7504

  18. 18.

    Farg MA, Konopka A, Soo KY, Ito D, Atkin JD (2017) The DNA damage response (DDR) is induced by the C9orf72 repeat expansion in amyotrophic lateral sclerosis. Hum Mol Genet 26(15):2882–2896

  19. 19.

    Wang H, Hegde ML (2019) New mechanisms of DNA repair defects in fused in sarcoma-associated neurodegeneration: stage set for DNA repair-based therapeutics? J Exp Neurosci 13:1179069519856358

  20. 20.

    Mitra J, Guerrero EN, Hegde PM, Liachko NF, Wang H, Vasquez V et al (2019) Motor neuron disease-associated loss of nuclear TDP-43 is linked to DNA double-strand break repair defects. Proc Natl Acad Sci USA 116(10):4696–4705

  21. 21.

    Naumann M, Pal A, Goswami A, Lojewski X, Japtok J, Vehlow A et al (2018) Impaired DNA damage response signaling by FUS-NLS mutations leads to neurodegeneration and FUS aggregate formation. Nat Commun 9(1):335

  22. 22.

    Brooks BR, Miller RG, Swash M, Munsat TL (2000) El Escorial revisited: revised criteria for the diagnosis of amyotrophic lateral sclerosis. Amyotroph Lateral Scler Other Motor Neuron Disord 1(5):293–299

  23. 23.

    Vangipuram M, Ting D, Kim S, Diaz R, Schule B (2013) Skin punch biopsy explant culture for derivation of primary human fibroblasts. J Vis Exp 77:e3779

  24. 24.

    Mata-Garrido J, Casafont I, Tapia O, Berciano MT, Lafarga M (2016) Neuronal accumulation of unrepaired DNA in a novel specific chromatin domain: structural, molecular and transcriptional characterization. Acta Neuropathol Commun 4:41

  25. 25.

    Fernandez-Capetillo O, Lee A, Nussenzweig M, Nussenzweig A (2004) H2AX: the histone guardian of the genome. DNA Repair 3(8–9):959–967

  26. 26.

    Delgado-Calle J, Arozamena J, Garcia-Renedo R, Garcia-Ibarbia C, Pascual-Carra MA, Gonzalez-Macias J et al (2011) Osteocyte deficiency in hip fractures. Calcif Tissue Int 89(4):327–334

  27. 27.

    Taylor JP, Brown RH Jr, Cleveland DW (2016) Decoding ALS: from genes to mechanism. Nature 539(7628):197–206

  28. 28.

    Casafont I, Bengoechea R, Tapia O, Berciano MT, Lafarga M (2009) TDP-43 localizes in mRNA transcription and processing sites in mammalian neurons. J Struct Biol 167(3):235–241

  29. 29.

    Palanca A, Casafont I, Berciano MT, Lafarga M (2014) Reactive nucleolar and Cajal body responses to proteasome inhibition in sensory ganglion neurons. Biochim Biophys Acta 1842(6):848–859

  30. 30.

    Panier S, Boulton SJ (2014) Double-strand break repair: 53BP1 comes into focus. Nat Rev Mol Cell Biol 15(1):7–18

  31. 31.

    Konrad C, Kawamata H, Bredvik KG, Arreguin AJ, Cajamarca SA, Hupf JC et al (2017) Fibroblast bioenergetics to classify amyotrophic lateral sclerosis patients. Mol Neurodegener 12(1):76

  32. 32.

    Liu WC, Liu T, Liu ZH, Deng M (2016) Detection the mutated protein aggregation and mitochondrial function in fibroblasts from amyotrophic lateral sclerosis patients with SOD1 gene mutations. Zhonghua Yi Xue Za Zhi 96(25):1982–1986

  33. 33.

    Orru S, Coni P, Floris A, Littera R, Carcassi C, Sogos V et al (2016) Reduced stress granule formation and cell death in fibroblasts with the A382T mutation of TARDBP gene: evidence for loss of TDP-43 nuclear function. Hum Mol Genet 25(20):4473–4483

  34. 34.

    Lo BM, Di Fini F, Notaro A, Spataro R, Conforti FL, La BV (2017) ALS-related mutant FUS protein is mislocalized to cytoplasm and is recruited into stress granules of fibroblasts from asymptomatic FUS P525L mutation carriers. Neurodegener Dis 17(6):292–303

  35. 35.

    Alberts, Johnson, Lewis, Raf, Roberts, Walter (2015) Biología molecular de la célula. 6ª Edición ed. Omega, Sabadell. ISBN: 9788428216388

  36. 36.

    Riancho J, Ruiz-Soto M, Villagra NT, Berciano J, Berciano MT, Lafarga M (2014) Compensatory motor neuron response to chromatolysis in the murine hSOD1(G93A) model of amyotrophic lateral sclerosis. Front Cell Neurosci 8:346

  37. 37.

    Lee SM, Asress S, Hales CM, Gearing M, Vizcarra JC, Fournier CN et al. (2019) TDP-43 cytoplasmic inclusion formation is disrupted in C9orf72-associated amyotrophic lateral sclerosis/frontotemporal lobar degeneration. Brain Commun 1(1):fcz014.

  38. 38.

    Codron P, Cassereau J, Vourch P, Veyrat-Durebex C, Blasco H, Kane S et al (2018) Primary fibroblasts derived from sporadic amyotrophic lateral sclerosis patients do not show ALS cytological lesions. Amyotroph Lateral Scler Frontotemporal Degener 19:446–456

  39. 39.

    De Marco G, Lupino E, Calvo A, Moglia C, Buccinna B, Grifoni S et al (2011) Cytoplasmic accumulation of TDP-43 in circulating lymphomonocytes of ALS patients with and without TARDBP mutations. Acta Neuropathol 121(5):611–622

  40. 40.

    De Marco G, Lomartire A, Calvo A, Risso A, De Luca E, Mostert M et al (2017) Monocytes of patients with amyotrophic lateral sclerosis linked to gene mutations display altered TDP-43 subcellular distribution. Neuropathol Appl Neurobiol 43(2):133–153

  41. 41.

    Casafont I, Palanca A, Lafarga V, Berciano MT, Lafarga M (2011) Effect of ionizing radiation in sensory ganglion neurons: organization and dynamics of nuclear compartments of DNA damage/repair and their relationship with transcription and cell cycle. Acta Neuropathol 122(4):481–493

  42. 42.

    Riancho J, Gil-Bea FJ, Castanedo-Vazquez D, Sedano MJ, Zufiria M, de Eulate GFG et al (2018) Clinical evidences supporting the Src/c-Abl pathway as potential therapeutic target in amyotrophic lateral sclerosis. J Neurol Sci 393:80–82

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Acknowledgements

This work was supported by grants from the Carlos III Health Institute (ISCII, FEDER, PI18/01066), CIBERNED (2016-04), Bioef (BIO17/ND/023-BD) and the Basque Government (2017222027 and 2015111122) and the Institute of Research Valdecilla (IDIVAL) (INNVAL 16/11).

Author information

JR: conception, experimental design, writing; DCV, OT, JA, CDV, MJS: participated in the acquisition of data, and experiment performance; FGB, MTB, ALM: manuscript revision and interpretation of the results; ML: final revision of the manuscript.

Correspondence to Javier Riancho.

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All authors declare that they have no conflict of interests.

Ethical standards

This study was approved by the IRB. Informed consent was obtained from all the participants in the study.

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Cite this article

Riancho, J., Castanedo-Vázquez, D., Gil-Bea, F. et al. ALS-derived fibroblasts exhibit reduced proliferation rate, cytoplasmic TDP-43 aggregation and a higher susceptibility to DNA damage. J Neurol (2020) doi:10.1007/s00415-020-09704-8

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Keywords

  • Amyotrophic lateral sclerosis
  • ALS
  • DNA-damage
  • Fibroblasts
  • TDP-43