Skip to main content
SpringerLink
Log in

Evaluation of Skin Fibroblasts from Amyotrophic Lateral Sclerosis Patients for the Rapid Study of Pathological Features

  • Original Article
  • Published:
Neurotoxicity Research Aims and scope Submit manuscript

Abstract

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterised by the progressive degeneration of brain and spinal cord motor neurons. Ubiquitin–proteasome system (UPS) dysfunction and oxidative stress have been implicated in ALS pathogenesis. However, it is unknown whether the defects in these pathways extend to non-neuronal tissues such as fibroblasts. Fibroblasts, unlike neuronal tissue, are readily available and may hold potential for short-term, rapid diagnostic and prognostic purposes. We investigated whether primary skin fibroblasts from ALS patients share, or can be manipulated to develop, functional and pathological abnormalities seen in affected neuronal cells. We inhibited UPS function and induced oxidative stress in the fibroblasts and found that ALS-related cellular changes, such as aggregate formation and ubiquitination of ALS-associated proteins (TDP-43 and ubiquilin 2), can be reproduced in these cells. Higher levels of TDP-43 ubiquitination, as evident by colocalization between TDP-43 and ubiquitin, were found in all six ALS cases compared to controls following extracellular insults. In contrast, colocalization between ubiquilin 2 and ubiquitin was not markedly different between ALS cases and control. A UPS reporter assay revealed UPS abnormalities in patient fibroblasts. Despite the presence of ALS-related cellular changes in the patient fibroblasts, no elevated toxicity was observed. This suggests that aggregate formation and colocalization of ALS-associated proteins may be insufficient alone to confer toxicity in fibroblasts used in the present study. Chronic exposure to ALS-linked stresses and the ALS-linked cellular pathologies may be necessary to breach an unknown threshold that triggers cell death.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Abe K, Pan LH, Watanabe M, Konno H, Kato T, Itoyama Y (1997) Upregulation of protein-tyrosine nitration in the anterior horn cells of amyotrophic lateral sclerosis. Neurol Res 19:124–128

    CAS  PubMed  Google Scholar 

  • Bence NF, Bennett EJ, Kopito RR (2005) Application and analysis of the GFPu family of ubiquitin-proteasome system reporters. Methods Enzymol 399:481–490

    Article  CAS  PubMed  Google Scholar 

  • Blair IP, Williams KL, Warraich ST, Durnall JC, Thoeng AD, Manavis J, Blumbergs PC, Vucic S, Kiernan MC, Nicholson GA (2010) FUS mutations in amyotrophic lateral sclerosis: clinical, pathological, neurophysiological and genetic analysis. J Neurol Neurosur Psychiatry 81:639–645

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Cohen TJ, Hwang AW, Unger T, Trojanowski JQ, Lee VMY (2012) Redox signalling directly regulates TDP-43 via cysteine oxidation and disulphide cross-linking. EMBO J 31:1241–1252

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Coyle CH, Kader KN (2007) Mechanisms of H2O2-induced oxidative stress in endothelial cells exposed to physiologic shear stress. ASAIO J 53(1):17–22

    Article  CAS  PubMed  Google Scholar 

  • Daoud H, Rouleau GA (2011) Motor neuron disease: a role for ubiquilin 2 mutations in neurodegeneration. Nat Rev Neurol 7:599–600

    Article  CAS  PubMed  Google Scholar 

  • DeJesus-Hernandez M, Mackenzie IR, Boeve BF, Boxer AL, Baker M, Rutherford NJ, Nicholson AM, Finch NA, Flynn H, Adamson J, Kouri N, Wojtas A, Sengdy P, Hsiung G, Yuek R, Karydas A, Seeley WW, Keith JA, Coppola G, Geschwind DH, Wszolek ZK, Feldman H, Knopman DS, Petersen RC, Miller BL, Dickson DW, Boylan KB, Graff-Radford NR, Rademakers R (2011) Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS. Neuron 72:245–256

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Del Razo LM, Quintanilla-Vega B, Brambila-Colombres E, Calderón-Aranda ES, Manno M, Albores A (2001) Stress proteins induced by arsenic. Toxicol Appl Pharm 177:132–148

    Article  Google Scholar 

  • Deng HX, Chen W, Hong ST, Boycott KM, Gorrie GH, Siddique N, Yang Y, Fecto F, Shi Y, Zhai H, Jiang H, Hirano M, Rampersaud E, Jansen GH, Donkervoort S, Bigio EH, Brooks BR, Ajroud K, Sufit RL, Haines JL, Mugnaini E, Pericak-Vance MA, Siddique T (2011) Mutations in UBQLN2 cause dominant X-linked juvenile and adult-onset ALS and ALS/dementia. Nature 477:211–215

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Dimos JT, Rodolfa KT, Niakan KK, Weisenthal LM, Mitsumoto H, Chung W, Croft GF, Saphier G, Leibel R, Goland R, Wichterle H, Henderson CE, Eggan K (2008) Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons. Science 321:1218–1221

    Article  CAS  PubMed  Google Scholar 

  • Hardiman O (2010) Amyotrophic lateral sclerosis. In: Encyclopedia of life sciences (eLS). Wiley, Chichester

  • Iguchi Y, Katsuno M, Takagi S, Ishigaki S, J-i Niwa, Hasegawa M, Tanaka F, Sobue G (2012) Oxidative stress induced by glutathione depletion reproduces pathological modifications of TDP-43 linked to TDP-43 proteinopathies. Neurobiol Dis 45:862–870

    Article  CAS  PubMed  Google Scholar 

  • Ling S-C, Polymenidou M, Cleveland Don W (2013) Converging mechanisms in ALS and FTD: disrupted RNA and protein homeostasis. Neuron 79(3):416–438

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Liu-Yesucevitz L, Bilgutay A, Zhang YJ, Vanderwyde T, Citro A, Mehta T, Zaarur N, McKee A, Bowser R, Sherman M, Petrucelli L, Wolozin B (2010) Tar DNA binding protein-43 (TDP-43) associates with stress granules: analysis of cultured cells and pathological brain tissue. PLoS One 5:e13250

    Article  PubMed Central  PubMed  Google Scholar 

  • Mackenzie IRA, Rademakers R, Neumann M (2010) TDP-43 and FUS in amyotrophic lateral sclerosis and frontotemporal dementia. Lancet Neurol 9:995–1007

    Article  CAS  PubMed  Google Scholar 

  • Neumann M, Sampathu DM, Kwong LK, Truax AC, Micsenyi MC, Chou TT, Bruce J, Schuck T, Grossman M, Clark CM, McCluskey LF, Miller BL, Masliah E, Mackenzie IR, Feldman H, Feiden W, Kretzschmar HA, Trojanowski JQ, Lee VM (2006) Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Science 314:130–133

    Article  CAS  PubMed  Google Scholar 

  • Nriagu LBJ (2000) Molecular aspects of arsenic stress. J Toxicol Environ Health Part B 3(4):293–322

    Article  Google Scholar 

  • Oketa Y, Higashida K, Fukasawa H, Tsukie T, Ono S (2013) Abundant FUS-immunoreactive pathology in the skin of sporadic amyotrophic lateral sclerosis. Acta Neurol Scand 128:257–264

    Article  CAS  PubMed  Google Scholar 

  • Pampliega O, Orhon I, Patel B, Sridhar S, Diaz-Carretero A, Beau I, Codogno P, Satir BH, Satir P, Cuervo AM (2013) Functional interaction between autophagy and ciliogenesis. Nature 502:194–200

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Redler RL, Dokholyan NV (2012) The complex molecular biology of amyotrophic lateral sclerosis (ALS). Prog Mol Biol Transl Sci 107:215–262

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Renton AE, Majounie E, Waite A et al (2011) A hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD. Neuron 72:257–268

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Rosen DR, Siddique T, Patterson D, Figlewicz DA, Sapp P, Hentati A, Donaldson D, Goto J, O’Regan JP, Deng HX et al (1993) Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis. Nature 362:59–62

    Article  CAS  PubMed  Google Scholar 

  • Rothenberg C, Srinivasan D, Mah L, Kaushik S, Peterhoff CM, Ugolino J, Fang S, Cuervo AM, Nixon RA, Monteiro MJ (2010) Ubiquilin functions in autophagy and is degraded by chaperone-mediated autophagy. Hum Mol Genet 19(16):3219–3232

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Sabatelli M, Zollino M, Conte A, Del Grande A, Marangi G, Lucchini M, Mirabella M, Romano A, Piacentini R, Bisogni G, Lattante S, Luigetti M, Rossini PM, Moncada A (2015) Primary fibroblasts cultures reveal TDP-43 abnormalities in amyotrophic lateral sclerosis patients with and without SOD1 mutations. Neurobiol Aging 36(5):2005.e5–2005.e13

    Article  Google Scholar 

  • Shibata N, Nagai R, Uchida K, Horiuchi S, Yamada S, Hirano A, Kawaguchi M, Yamamoto T, Sasaki S, Kobayashi M (2001) Morphological evidence for lipid peroxidation and protein glycoxidation in spinal cords from sporadic amyotrophic lateral sclerosis patients. Brain Res 917:97–104

    Article  CAS  PubMed  Google Scholar 

  • Solski JA, Yang S, Nicholson GA, Luquin N, Williams KL, Fernando R, Pamphlett R, Blair IP (2012) A novel TARDBP insertion/deletion mutation in the flail arm variant of amyotrophic lateral sclerosis. Amyotroph Later Scler 13:465–470

    Article  CAS  Google Scholar 

  • Sreedharan J, Blair IP, Tripathi VB, Hu X, Vance C, Rogelj B, Ackerley S, Durnall JC, Williams KL, Buratti E, Baralle F, de Belleroche J, Mitchell JD, Leigh PN, Al-Chalabi A, Miller CC, Nicholson G, Shaw CE (2008) TDP-43 mutations in familial and sporadic amyotrophic lateral sclerosis. Science 319:1668–1672

    Article  CAS  PubMed  Google Scholar 

  • Suzuki M, Mikami H, Watanabe T, Yamano T, Yamazaki T, Nomura M, Yasui K, Ishikawa H, Ono S (2010) Increased expression of TDP-43 in the skin of amyotrophic lateral sclerosis. Acta Neurol Scand 122:367–372

    Article  CAS  PubMed  Google Scholar 

  • Tu PH, Gurney ME, Julien JP, Lee VM, Trojanowski JQ (1997) Oxidative stress, mutant SOD1, and neurofilament pathology in transgenic mouse models of human motor neuron disease. Lab Invest 76(4):441–456

    CAS  PubMed  Google Scholar 

  • Urushitani M, Kurisu J, Tsukita K, Takahashi R (2002) Proteasomal inhibition by misfolded mutant superoxide dismutase 1 induces selective motor neuron death in familial amyotrophic lateral sclerosis. J Neurochem 83:1030–1042

    Article  CAS  PubMed  Google Scholar 

  • Vance C, Rogelj B, Hortobagyi T, De Vos KJ, Nishimura AL, Sreedharan J, Hu X, Smith B, Ruddy D, Wright P, Ganesalingam J, Williams KL, Tripathi V, Al-Saraj S, Al-Chalabi A, Leigh PN, Blair IP, Nicholson G, de Belleroche J, Gallo J-M, Miller CC, Shaw CE (2009) Mutations in FUS, an RNA processing protein, cause familial amyotrophic lateral sclerosis type 6. Science 323:1208–1211

    Article  CAS  PubMed  Google Scholar 

  • Wang X, Fan H, Ying Z, Li B, Wang H, Wang G (2010) Degradation of TDP-43 and its pathogenic form by autophagy and the ubiquitin-proteasome system. Neurosci Lett 469:112–116

    Article  CAS  PubMed  Google Scholar 

  • Warraich ST, Yang S, Nicholson GA, Blair IP (2010) TDP-43: a DNA and RNA binding protein with roles in neurodegenerative diseases. Int J Biochem Cell B 42:1606–1609

    Article  CAS  Google Scholar 

  • Watanabe T, Okeda Y, Yamano T, Ono S (2010) An immunohistochemical study of ubiquitin in the skin of sporadic amyotrophic lateral sclerosis. J Neurol Sci 298:52–56

    Article  CAS  PubMed  Google Scholar 

  • Williams KL, Warraich ST, Yang S, Solski JA, Fernando R, Rouleau GA, Nicholson GA, Blair IP (2012) UBQLN2/ubiquilin 2 mutation and pathology in familial amyotrophic lateral sclerosis. Neurobiol aging 33:2527.e3–2527.e10

    Article  CAS  Google Scholar 

  • Gellera C, Tiloca C, Del Bo R, Corrado L, Pensato V, Agostini J, Cereda C, Ratti A, Castellotti B, Corti S, Bagarotti A, Cagnin A, Milani P, Gabelli C, Riboldi G, Mazzini L, Sorarù G, D’Alfonso S, Taroni F, Comi GP, Ticozzi N, Silani V, Consortium TS (2013) Ubiquilin 2 mutations in Italian patients with amyotrophic lateral sclerosis and frontotemporal dementia. J Neurol Neurosurg Psychiatry 84:183–187

    Article  PubMed  Google Scholar 

  • Zanotto-Filho A, Braganhol E, Battastini A, Moreira J (2012) Proteasome inhibitor MG132 induces selective apoptosis in glioblastoma cells through inhibition of PI3 K/Akt and NFkappaB pathways, mitochondrial dysfunction, and activation of p38-JNK1/2 signaling. Invest New Drugs 30:2252–2262

    Article  CAS  PubMed  Google Scholar 

  • Zhang KY, Yang S, Warraich ST, Blair IP (2014) Ubiquilin 2: a component of the ubiquitin-proteasome system with an emerging role in neurodegeneration. Int J Biochem Cell Biol 50:123–126

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

We thank all patients and family members for their participation and support, C. Cecere and M. Edwards for assistance with patient recruitment. This work was supported by the NHMRC of Australia (1004670), MND Australia (Leadership grant to IPB), as well as a Bill Gole fellowship (to SY) and a grant-in-aid (to LO and JY) from the MND Research Institute of Australia. MB is supported by the Rotary Club of Cronulla Motor Neurone Disease Research Ph.D. scholarship, Australian Rotary Health and the University of Wollongong. We also thank G. Guillemin for critical review of the manuscript. We acknowledge Ruben K. Dagda (University of Nevada School of Medicine, Pharmacology Department), Daniel Shiwarski (Carneggie Mellon University) and Charleen T Chu (University of Pittsburgh) for developing the Red and Green Puncta Colocalization Macro for Image J.

Conflict of interest

No conflicts of interest are disclosed by all authors.

Author information

Authors and Affiliations

  1. Faculty of Medicine and Health Sciences, Macquarie University, Sydney, NSW, 2109, Australia

    Shu Yang, Katharine Y. Zhang, Jennifer A. Fifita, Garth A. Nicholson & Ian P. Blair

  2. Northcott Neuroscience Laboratory, ANZAC Research Institute, Sydney, NSW, 2139, Australia

    Shu Yang, Ruvini Kariawasam, Jennifer A. Fifita, Garth A. Nicholson & Ian P. Blair

  3. School of Biological Sciences, Illawarra Health and Medical Research Institute, University of Wollongong, Wollongong, NSW, 2522, Australia

    Monique Bax, Lezanne Ooi & Justin J. Yerbury

  4. Sydney Medical School, University of Sydney, Sydney, NSW, 2006, Australia

    Garth A. Nicholson & Ian P. Blair

  5. Molecular Medicine Laboratory, Concord Hospital, Concord, NSW, 2139, Australia

    Garth A. Nicholson

Authors
  1. Shu Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Katharine Y. Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ruvini Kariawasam
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Monique Bax
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jennifer A. Fifita
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Lezanne Ooi
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Justin J. Yerbury
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Garth A. Nicholson
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Ian P. Blair
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ian P. Blair.

Electronic Supplementary Material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 4577 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Yang, S., Zhang, K.Y., Kariawasam, R. et al. Evaluation of Skin Fibroblasts from Amyotrophic Lateral Sclerosis Patients for the Rapid Study of Pathological Features. Neurotox Res 28, 138–146 (2015). https://doi.org/10.1007/s12640-015-9532-1

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12640-015-9532-1

Keywords

Search

Navigation