NeuroMolecular Medicine

, Volume 18, Issue 4, pp 551–560 | Cite as

Identification of miRNAs as Potential Biomarkers in Cerebrospinal Fluid from Amyotrophic Lateral Sclerosis Patients

  • Michele Benigni
  • Claudia RicciEmail author
  • Ashley R. Jones
  • Fabio Giannini
  • Ammar Al-Chalabi
  • Stefania Battistini
Original Paper


Amyotrophic lateral sclerosis (ALS) is a progressive, fatal neurodegenerative disorder. Since no diagnostic laboratory test exists, the identification of specific biomarkers could be fundamental in clinical practice. microRNAs (miRNAs) are considered promising biomarkers for neurodegenerative diseases. The aim of the study was to identify a CSF miRNA set that could differentiate ALS from non-ALS condition. miRNA profiling in CSF from ALS patients (n = 24; eight with C9orf72 expansion) and unaffected control subjects (n = 24) by quantitative reverse transcription PCR identified fourteen deregulated miRNAs. Validation experiments confirmed eight miRNAs as significantly deregulated in ALS. No significant differences were observed between ALS patients with or without C9orf72 expansion. The receiver operator characteristic (ROC) curve analyses revealed the highest diagnostic accuracy for the upregulated miR181a-5p and the downregulated miR21-5p and miR15b-5p. The miR181a-5p/miR21-5p and miR181a-5p/miR15b-5p ratios detected ALS with 90 and 85 % sensitivity and 87 and 91 % specificity, respectively, confirming the application potential as disease biomarkers. These deregulated miRNAs are implicated in apoptotic way and provide insight into processes responsible for motor neuron degeneration.


Amyotrophic lateral sclerosis Cerebrospinal fluid microRNA C9orf72 expansion Biomarkers 



The authors thank Dr. Donatella Moschettini for providing CSF samples from control subjects and Dr. Marisa Carone for help in collecting clinical data. AAC and AJ thank the Motor Neurone Disease Association and the ALS Association for support.


The work leading up to this publication was funded by the European Community’s Health Seventh Framework Programme (FP7/2007–2013; Grant Agreement Number 259867) (AAC) and by the Associazione Polisportiva Torrenieri a.s.d.- Ice Bucket Challenge (2268-2014-BS-CONTRLIB_001) (SB). MB was supported by a doctoral Fellowship award from the Dottorato Toscano in Neuroscienze.

Compliance with Ethical Standards

Conflict of interest

This is an EU Joint Programme—Neurodegenerative Disease Research (JPND) project. The project is supported through the following funding organizations under the aegis of JPND— (United Kingdom, Medical Research Council and Economic and Social Research Council). AAC receives salary support from the National Institute for Health Research (NIHR) Dementia Biomedical Research Unit and Biomedical Research Centre in Mental Health at South London and Maudsley NHS Foundation Trust and King’s College Hospital. All the other authors do not have any conflict of interest.


  1. Akers, J. C., Ramakrishnan, V., Kim, R., Phillips, S., Kaimal, V., Mao, Y., et al. (2015). miRNA contents of cerebrospinal fluid extracellular vesicles in glioblastoma patients. Journal of Neuro-Oncology, 123(2), 205–216.CrossRefPubMedPubMedCentralGoogle Scholar
  2. Akers, J. C., Ramakrishnan, V., Kim, R., Skog, J., Nakano, I., Pingle, S., et al. (2013). MiR-21 in the extracellular vesicles (EVs) of cerebrospinal fluid (CSF): a platform for glioblastoma biomarker development. PLoS ONE, 8(10), e78115.CrossRefPubMedPubMedCentralGoogle Scholar
  3. Andersen, P. M., & Al-Chalabi, A. (2011). Clinical genetics of amyotrophic lateral sclerosis: what do we really know? Nature Reviews Neurology, 7(11), 603–615.CrossRefPubMedGoogle Scholar
  4. Baraniskin, A., Kuhnhenn, J., Schlegel, U., Maghnouj, A., Zöllner, H., Schmiegel, W., et al. (2012). Identification of microRNAs in the cerebrospinal fluid as biomarker for the diagnosis of glioma. Neuro-Oncology, 14(1), 29–33.CrossRefPubMedGoogle Scholar
  5. Bartel, D. P. (2009). MicroRNAs: Target recognition and regulatory functions. Cell, 136(2), 215–233.CrossRefPubMedPubMedCentralGoogle Scholar
  6. Brooks, B. R., Miller, R. G., Swash, M., & Munsat, T. L. (2000). World Federation of Neurology Research Group on Motor Neuron Diseases. El Escorial revisited: revised criteria for the diagnosis of amyotrophic lateral sclerosis. Amyotroph Lateral Sclerosis and Other Motor Neuron Disorders, 1(5), 293–299.CrossRefGoogle Scholar
  7. Burgos, K. L., Javaherian, A., Bomprezzi, R., Ghaffari, L., Rhodes, S., Courtright, A., et al. (2013). Identification of extracellular miRNA in human cerebrospinal fluid by next-generation sequencing. RNA, 19(5), 712–722.CrossRefPubMedPubMedCentralGoogle Scholar
  8. Burgos, K., Malenica, I., Metpally, R., Courtright, A., Rakela, B., Beach, T., et al. (2014). Profiles of extracellular miRNA in cerebrospinal fluid and serum from patients with Alzheimer’s and Parkinson’s diseases correlate with disease status and features of pathology. PLoS ONE, 9(5), e94839.CrossRefPubMedPubMedCentralGoogle Scholar
  9. Cogswell, J. P., Ward, J., Taylor, I. A., Waters, M., Shi, Y., et al. (2008). Identification of miRNA changes in Alzheimer’s disease brain and CSF yields putative biomarkers and insights into disease pathways. Journal of Alzheimer’s Disease, 14(1), 27–41.PubMedGoogle Scholar
  10. Conti, A., Aguennouz, M., La Torre, D., Tomasello, C., Cardali, S., Angileri, F. F., et al. (2009). miR-21 and 221 upregulation and miR-181b downregulation in human grade II-IV astrocytic tumors. Journal of Neuro-Oncology, 93(3), 325–332.CrossRefPubMedGoogle Scholar
  11. De Felice, B., Annunziata, A., Fiorentino, G., Borra, M., Biffali, E., Coppola, C., et al. (2014). miR-338-3p is over-expressed in blood, CFS, serum and spinal cord from sporadic amyotrophic lateral sclerosis patients. Neurogenetics, 15(4), 243–253.CrossRefPubMedGoogle Scholar
  12. De Felice, B., Guida, M., Guida, M., Coppola, C., De Mieri, G., & Cotrufo, R. (2012). A miRNA signature in leukocytes from sporadic amyotrophic lateral sclerosis. Gene, 508(1), 35–40.CrossRefPubMedGoogle Scholar
  13. Denk, J., Boelmans, K., Siegismund, C., Lassner, D., Arlt, S., & Jahn, H. (2015). MicroRNA profiling of CSF reveals potential biomarkers to detect alzheimer’s disease. PLoSOne, 10(5), e0126423.CrossRefGoogle Scholar
  14. Droppelmann, C. A., Campos-Melo, D., Ishtiaq, M., Volkening, K., & Strong, M. J. (2014). RNA metabolism in ALS: When normal processes become pathological. Amyotroph Lateral Sclerosis and Other Motor Neuron Disorders, 15(5–6), 321–336.Google Scholar
  15. Eitan, C., & Hornstein, E. (2016). Vulnerability of microRNA biogenesis in FTD-ALS. Brain Research. doi: 10.1016/j.brainres.2015.12.063.
  16. Freischmidt, A., Müller, K., Ludolph, A. C., & Weishaupt, J. H. (2013). Systemic dysregulation of TDP-43 binding microRNAs in amyotrophic lateral sclerosis. Acta Neuropathologica Communications, 1, 42.CrossRefPubMedPubMedCentralGoogle Scholar
  17. Freischmidt, A., Müller, K., Zondler, L., Weydt, P., Mayer, B., von Arnim, C. A., et al. (2015). Serum microRNAs in sporadic amyotrophic lateral sclerosis. Neurobiology of Aging, 36(9), 2660.e15-20.CrossRefPubMedGoogle Scholar
  18. Freischmidt, A., Müller, K., Zondler, L., Weydt, P., Volk, A. E., Božič, A. L., et al. (2014). Serum microRNAs in patients with genetic amyotrophic lateral sclerosis and pre-manifest mutation carriers. Brain, 137(Pt11), 2938–2950.CrossRefPubMedGoogle Scholar
  19. Gallego, J. A., Gordon, M. L., Claycomb, K., Bhatt, M., Lencz, T., & Malhotra, A. K. (2012). In vivo microRNA detection and quantitation in cerebrospinal fluid. Journal of Molecular Neuroscience, 47(2), 243–248.CrossRefPubMedPubMedCentralGoogle Scholar
  20. Garg, N., Vijayakumar, T., Bakhshinyan, D., Venugopal, C., & Singh, S. K. (2015). MicroRNA regulation of brain tumour initiating cells in central nervous system tumours. Stem Cells International, 141793.Google Scholar
  21. Gibert, B., Delloye-Bourgeois, C., Gattolliat, C. H., Meurette, O., Le Guernevel, S., Fombonne, J., et al. (2014). Regulation by miR181 family of the dependence receptor CDON tumor suppressive activity in neuroblastoma. Journal of the National Cancer Institute, 106(11), 318.CrossRefGoogle Scholar
  22. Goodall, E. F., Heath, P. R., Bandmann, O., Kirby, J., & Shaw, P. J. (2013). Neuronal dark matter: The emerging role of microRNAs in neurodegeneration. Frontiers In Cellular Neuroscience, 7, 178.CrossRefPubMedPubMedCentralGoogle Scholar
  23. Haghikia, A., Haghikia, A., Hellwig, K., Baraniskin, A., Holzmann, A., Décard, B. F., et al. (2012). Regulated microRNAs in the CSF of patients with multiple sclerosis: A case-control study. Neurology, 79(22), 2166–2170.CrossRefPubMedGoogle Scholar
  24. Maciotta, S., Meregalli, M., & Torrente, Y. (2013). The involvement of microRNAs in neurodegenerative diseases. Front Cell Neurosci, 7, 265.CrossRefPubMedPubMedCentralGoogle Scholar
  25. Marangi, G., & Traynor, B. J. (2015). Genetic causes of amyotrophic lateral sclerosis: New genetic analysis methodologies entailing new opportunities and challenges. Brain Research, 1607, 75–93.CrossRefPubMedGoogle Scholar
  26. Millecamps, S., Boillée, S., Le Ber, I., Seilhean, D., Teyssou, E., Giraudeau, M., et al. (2012). Phenotype difference between ALS patients with expanded repeats in C9ORF72 and patients with mutations in other ALS-related genes. Journal of Medical Genetics, 49(4), 258–263.CrossRefPubMedGoogle Scholar
  27. Mitchell, P. S., Parkin, R. K., Kroh, E. M., Fritz, B. R., Wyman, S. K., Pogosova-Agadjanyan, E. L., et al. (2008). Circulating microRNAs as stable blood-based markers for cancer detection. Proceedings of the National Academy of Sciences of the United States of America, 105(30), 10513–10518.CrossRefPubMedPubMedCentralGoogle Scholar
  28. Müller, M., Kuiperij, H. B., Claassen, J. A., Küsters, B., & Verbeek, M. M. (2014). MicroRNAs in Alzheimer’s disease: Differential expression in hippocampus and cell-free cerebrospinal fluid. Neurobiology of Aging, 35(1), 152–158.CrossRefPubMedGoogle Scholar
  29. Pacifici, M., Delbue, S., Kadri, F., & Peruzzi, F. (2014). Cerebrospinal fluid MicroRNA profiling using quantitative real time PCR. Journal of Visualized Experiments, 83, e51172.Google Scholar
  30. Paez-Colasante, X., Figueroa-Romero, C., Sakowski, S. A., Goutman, S. A., & Feldman, E. L. (2015). Amyotrophic lateral sclerosis: mechanisms and therapeutics in the epigenomic era. Nature Reviews Neurology, 11(5), 266–279.CrossRefPubMedGoogle Scholar
  31. Pang, C., Guan, Y., Zhao, K., Chen, L., Bao, Y., Cui, R., et al. (2015). Up-regulation of microRNA-15b correlates with unfavorable prognosis and malignant progression of human glioma. International Journal of Clinical and Experimental Pathology, 8(5), 4943–4952.PubMedPubMedCentralGoogle Scholar
  32. Papagiannakopoulos, T., Shapiro, A., & Kosik, K. S. (2008). MicroRNA-21 targets a network of key tumor-suppressive pathways in glioblastoma cells. Cancer Research, 68(19), 8164–8172.CrossRefPubMedGoogle Scholar
  33. Pritchard, C. C., Cheng, H. H., & Tewari, M. (2012). MicroRNA profiling: Approaches and considerations. Nature Reviews Genetics, 13(5), 358–369.CrossRefPubMedPubMedCentralGoogle Scholar
  34. Rao, P., Benito, E., & Fischer, A. (2013). MicroRNAs as biomarkers for CNS disease. Frontiers in Molecular Neuroscience, 6, 39.CrossRefPubMedPubMedCentralGoogle Scholar
  35. Renton, A. E., Chiò, A., & Traynor, B. J. (2014). State of play in amyotrophic lateral sclerosis genetics. Nature Neuroscience, 17(1), 17–23.CrossRefPubMedGoogle Scholar
  36. Robberecht, W., & Philips, T. (2013). The changing scene of amyotrophic lateral sclerosis. Nature Reviews Neuroscience, 14(4), 248–264.CrossRefPubMedGoogle Scholar
  37. Roberts, R. A., Aschner, M., Calligaro, D., Guilarte, T. R., Hanig, J. P., Herr, D. W., et al. (2015). Translational biomarkers of neurotoxicity: A health and environmental sciences institute perspective on the way forward. Toxicological Sciences, 148(2), 332–340.CrossRefPubMedPubMedCentralGoogle Scholar
  38. Sheinerman, K. S., Tsivinsky, V. G., Crawford, F., Mullan, M. J., Abdullah, L., & Umansky, S. R. (2012). Plasma microRNA biomarkers for detection of mild cognitive impairment. Aging, 4(9), 590–605.CrossRefPubMedPubMedCentralGoogle Scholar
  39. Shi, L., Cheng, Z., Zhang, J., Li, R., Zhao, P., Fu, Z., et al. (2008). hsa-mir-181a and hsa-mir-181b function as tumor suppressors in human glioma cells. Brain Research, 1236, 185–193.CrossRefPubMedGoogle Scholar
  40. Teplyuk, N. M., Mollenhauer, B., Gabriely, G., Giese, A., Kim, E., Smolsky, M., et al. (2012). MicroRNAs in cerebrospinal fluid identify glioblastoma and metastatic brain cancers and reflect disease activity. Neuro-Oncology, 14(6), 689–700.CrossRefPubMedPubMedCentralGoogle Scholar
  41. Teunissen, C. E., Tumani, H., Engelborghs, S., & Mollenhauer, B. (2014). Biobanking of CSF: International standardization to optimize biomarker development. Clinical Biochemistry, 47(4–5), 288–292.CrossRefPubMedGoogle Scholar
  42. Wahid, F., Shehzad, A., Khan, T., & Kim, Y. Y. (2010). MicroRNAs: synthesis, mechanism, function, and recent clinical trials. Biochimica et Biophysica Acta, 1803(11), 1231–1243.CrossRefPubMedGoogle Scholar
  43. Xie, F., Xiao, P., Chen, D., Xu, L., & Zhang, B. (2012). miRDeepFinder: a miRNA analysis tool for deep sequencing of plant small RNAs. Plant Molecular Biology (Epub ahead of print).Google Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Michele Benigni
    • 1
  • Claudia Ricci
    • 1
    Email author
  • Ashley R. Jones
    • 2
  • Fabio Giannini
    • 1
  • Ammar Al-Chalabi
    • 2
  • Stefania Battistini
    • 1
  1. 1.Department of Medical, Surgical and Neurological SciencesUniversity of SienaSienaItaly
  2. 2.Department of Basic and Clinical Neuroscience, Institute of Psychiatry, Psychology and NeuroscienceKing’s College LondonLondonUK

Personalised recommendations