Exosome microRNAs in Amyotrophic Lateral Sclerosis: A Pilot Study
Abstract
:1. Introduction
2. Materials and Methods
2.1. Participants and Samples
2.2. Exosome Isolation and Characterization
2.3. RNA Extraction
2.4. Real-Time PCR Panel Analysis of miRNAs
2.5. Data Analysis and Statistics
3. Results
3.1. Exosome Characterization
3.2. Overview of miRNA Profile
3.3. Enrichment Analysis of Relevant miRNA
4. Discussion
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
- Tay, Y.; Zhang, J.; Thomson, A.M.; Lim, B.; Rigoutsos, I. MicroRNAs to Nanog, Oct4 and Sox2 coding regions modulate embryonic stem cell differentiation. Nature 2008, 455, 1124–1128. [Google Scholar] [CrossRef] [PubMed]
- O’Brien, J.; Hayder, H.; Zayed, Y.; Peng, C. Overview of microRNA biogenesis, mechanisms of actions, and circulation. Front. Endocrinol. 2018, 9, 402. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Stoicea, N.; Du, A.; Lakis, C.D.; Tipton, C.; Arias-Morales, C.E.; Bergese, S.D. The miRNA journey from theory to practice as a CNS biomarker. Front. Genet. 2016, 7, 11. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Kumar, S.; Reddy, P.H. Are circulating microRNAs peripheral biomarkers for Alzheimer’s disease? Biochim. Biophys. Acta-Mol. Basis Dis. 2016, 1862, 1617–1627. [Google Scholar] [CrossRef]
- Xu, J.; Zhao, J.; Evan, G.; Xiao, C.; Cheng, Y.; Xiao, J. Circulating microRNAs: Novel biomarkers for cardiovascular diseases. J. Mol. Med. 2012, 90, 865–875. [Google Scholar] [CrossRef]
- Bryzgunova, O.; Konoshenko, M.; Zaporozhchenko, I.; Yakovlev, A.; Laktionov, P. Isolation of cell-free mirna from biological fluids: Influencing factors and methods. Diagnostics 2021, 11, 865. [Google Scholar] [CrossRef]
- Heijnen, H.F.G.; Schiel, A.E.; Fijnheer, R.; Geuze, H.J.; Sixma, J.J. Activated platelets release two types of membrane vesicles: Microvesicles by surface shedding and exosomes derived from exocytosis of multivesicular bodies and α-granules. Blood 1999. [Google Scholar] [CrossRef]
- Théry, C.; Zitvogel, L.; Amigorena, S. Exosomes: Composition, biogenesis and function. Nat. Rev. Immunol. 2002, 2, 569–579. [Google Scholar] [CrossRef]
- Chung, I.M.; Rajakumar, G.; Venkidasamy, B.; Subramanian, U.; Thiruvengadam, M. Exosomes: Current use and future applications. Clin. Chim. Acta 2020, 500, 226–232. [Google Scholar] [CrossRef]
- Gallo, A.; Tandon, M.; Alevizos, I.; Illei, G.G. The majority of microRNAs detectable in serum and saliva is concentrated in exosomes. PLoS ONE 2012, 7, e30679. [Google Scholar] [CrossRef] [Green Version]
- Valadi, H.; Ekström, K.; Bossios, A.; Sjöstrand, M.; Lee, J.J.; Lötvall, J.O. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat. Cell Biol. 2007, 9, 654–659. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Mori, M.A.; Ludwig, R.G.; Garcia-Martin, R.; Brandão, B.B.; Kahn, C.R. Extracellular miRNAs: From Biomarkers to Mediators of Physiology and Disease. Cell Metab. 2019, 30, 656–673. [Google Scholar] [CrossRef]
- Fauré, J.; Lachenal, G.; Court, M.; Hirrlinger, J.; Chatellard-Causse, C.; Blot, B.; Grange, J.; Schoehn, G.; Goldberg, Y.; Boyer, V.; et al. Exosomes are released by cultured cortical neurones. Mol. Cell. Neurosci. 2006, 31, 642–648. [Google Scholar] [CrossRef] [PubMed]
- Chen, C.C.; Liu, L.; Ma, F.; Wong, C.W.; Guo, X.E.; Chacko, J.V.; Farhoodi, H.P.; Zhang, S.X.; Zimak, J.; Ségaliny, A.; et al. Elucidation of Exosome Migration Across the Blood–Brain Barrier Model In Vitro. Cell. Mol. Bioeng. 2016, 9, 509–529. [Google Scholar] [CrossRef]
- Rajendran, L.; Honsho, M.; Zahn, T.R.; Keller, P.; Geiger, K.D.; Verkade, P.; Simons, K. Alzheimer’s disease β-amyloid peptides are released in association with exosomes. Proc. Natl. Acad. Sci. USA 2006, 103, 11172–11177. [Google Scholar] [CrossRef] [Green Version]
- Silverman, J.M.; Fernando, S.M.; Grad, L.I.; Hill, A.F.; Turner, B.J.; Yerbury, J.J.; Cashman, N.R. Disease Mechanisms in ALS: Misfolded SOD1 Transferred Through Exosome-Dependent and Exosome-Independent Pathways. Cell. Mol. Neurobiol. 2016, 36, 377–381. [Google Scholar] [CrossRef] [PubMed]
- Kawabe, Y.; Mori, K.; Yamashita, T.; Gotoh, S.; Ikeda, M. The RNA exosome complex degrades expanded hexanucleotide repeat RNA in C9orf72 FTLD/ALS. EMBO J. 2020, 39, e102700. [Google Scholar] [CrossRef] [PubMed]
- Brown, R.H.; Al-Chalabi, A. Amyotrophic lateral sclerosis. N. Engl. J. Med. 2017, 377, 162–172. [Google Scholar] [CrossRef] [Green Version]
- Neumann, M.; Sampathu, D.M.; Kwong, L.K.; Truax, A.C.; Micsenyi, M.C.; Chou, T.T.; Bruce, J.; Schuck, T.; Grossman, M.; Clark, C.M.; et al. Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Science 2006, 314, 130–133. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Hosaka, T.; Yamashita, T.; Tamaoka, A.; Kwak, S. Extracellular RNAs as biomarkers of sporadic amyotrophic lateral sclerosis and other neurodegenerative diseases. Int. J. Mol. Sci. 2019, 20, 3148. [Google Scholar] [CrossRef] [Green Version]
- De Andrade, H.M.T.; de Albuquerque, M.; Avansini, S.H.; Rocha, C.D.S.; Dogini, D.B.; Nucci, A.; Carvalho, B.; Lopes-Cendes, I.; França, M.C. MicroRNAs-424 and 206 are potential prognostic markers in spinal onset amyotrophic lateral sclerosis. J. Neurol. Sci. 2016, 368, 19–24. [Google Scholar] [CrossRef]
- Sheinerman, K.S.; Toledo, J.B.; Tsivinsky, V.G.; Irwin, D.; Grossman, M.; Weintraub, D.; Hurtig, H.I.; Chen-Plotkin, A.; Wolk, D.A.; McCluskey, L.F.; et al. Circulating brain-enriched microRNAs as novel biomarkers for detection and differentiation of neurodegenerative diseases. Alzheimer’s Res. Ther. 2017, 9, 89. [Google Scholar] [CrossRef]
- Vrabec, K.; Boštjančič, E.; Koritnik, B.; Leonardis, L.; Dolenc Grošelj, L.; Zidar, J.; Rogelj, B.; Glavač, D.; Ravnik-Glavač, M. Differential expression of several miRNAs and the host genes AATK and DNM2 in leukocytes of sporadic als patients. Front. Mol. Neurosci. 2018, 11, 106. [Google Scholar] [CrossRef] [PubMed]
- Waller, R.; Wyles, M.; Heath, P.R.; Kazoka, M.; Wollff, H.; Shaw, P.J.; Kirby, J. Small RNA sequencing of sporadic amyotrophic lateral sclerosis cerebrospinal fluid reveals differentially expressed miRNAs related to neural and glial activity. Front. Neurosci. 2018, 11, 731. [Google Scholar] [CrossRef] [PubMed]
- Raheja, R.; Regev, K.; Healy, B.C.; Mazzola, M.A.; Beynon, V.; Von Glehn, F.; Paul, A.; Diaz-Cruz, C.; Gholipour, T.; Glanz, B.I.; et al. Correlating serum micrornas and clinical parameters in amyotrophic lateral sclerosis. Muscle Nerve 2018, 58, 261–269. [Google Scholar] [CrossRef] [PubMed]
- Yelick, J.; Men, Y.; Jin, S.; Seo, S.; Espejo-Porras, F.; Yang, Y. Elevated exosomal secretion of miR-124-3p from spinal neurons positively associates with disease severity in ALS. Exp. Neurol. 2020, 333, 113414. [Google Scholar] [CrossRef]
- Katsu, M.; Hama, Y.; Utsumi, J.; Takashina, K.; Yasumatsu, H.; Mori, F.; Wakabayashi, K.; Shoji, M.; Sasaki, H. MicroRNA expression profiles of neuron-derived extracellular vesicles in plasma from patients with amyotrophic lateral sclerosis. Neurosci. Lett. 2019, 708, 134176. [Google Scholar] [CrossRef]
- Banack, S.A.; Dunlop, R.A.; Cox, P.A. An miRNA fingerprint using neural-enriched extracellular vesicles from blood plasma: Towards a biomarker for amyotrophic lateral sclerosis/motor neuron disease. Open Biol. 2020, 10, 200116. [Google Scholar] [CrossRef] [PubMed]
- Lee, E.C.; Whitehead, A.L.; Jacques, R.M.; Julious, S.A. The statistical interpretation of pilot trials: Should significance thresholds be reconsidered? BMC Med. Res. Methodol. 2014, 14, 41. [Google Scholar] [CrossRef] [PubMed]
- Vandesompele, J.; De Preter, K.; Pattyn, F.; Poppe, B.; Van Roy, N.; De Paepe, A.; Speleman, F. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 2002, 3, research0034.1. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Benjamini, Y.; Hochberg, Y. Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple Testing. J. R. Stat. Soc. Ser. B 1995, 57, 289–300. [Google Scholar] [CrossRef]
- Vlachos, I.S.; Zagganas, K.; Paraskevopoulou, M.D.; Georgakilas, G.; Karagkouni, D.; Vergoulis, T.; Dalamagas, T.; Hatzigeorgiou, A.G. DIANA-miRPath v3.0: Deciphering microRNA function with experimental support. Nucleic Acids Res. 2015, 43, W460–W466. [Google Scholar] [CrossRef]
- Joilin, G.; Leigh, P.N.; Newbury, S.F.; Hafezparast, M. An Overview of MicroRNAs as Biomarkers of ALS. Front. Neurol. 2019, 10, 186. [Google Scholar] [CrossRef] [Green Version]
- Saucier, D.; Wajnberg, G.; Roy, J.; Beauregard, A.P.; Chacko, S.; Crapoulet, N.; Fournier, S.; Ghosh, A.; Lewis, S.M.; Marrero, A.; et al. Identification of a circulating miRNA signature in extracellular vesicles collected from amyotrophic lateral sclerosis patients. Brain Res. 2019, 1708, 100–108. [Google Scholar] [CrossRef] [PubMed]
- Takahashi, I.; Hama, Y.; Matsushima, M.; Hirotani, M.; Kano, T.; Hohzen, H.; Yabe, I.; Utsumi, J.; Sasaki, H. Identification of plasma microRNAs as a biomarker of sporadic Amyotrophic Lateral Sclerosis. Mol. Brain 2015, 8, 1–9. [Google Scholar] [CrossRef] [PubMed]
- Robichaud, P.P.; Arseneault, M.; O’Connell, C.; Ouellette, R.J.; Morin, P.J. Circulating cell-free DNA as potential diagnostic tools for amyotrophic lateral sclerosis. Neurosci. Lett. 2021, 750, 135813. [Google Scholar] [CrossRef]
- Liguori, M.; Nuzziello, N.; Introna, A.; Consiglio, A.; Licciulli, F.; D’Errico, E.; Scarafino, A.; Distaso, E.; Simone, I.L. Dysregulation of MicroRNAs and Target Genes Networks in Peripheral Blood of Patients With Sporadic Amyotrophic Lateral Sclerosis. Front. Mol. Neurosci. 2018, 11, 288. [Google Scholar] [CrossRef] [PubMed]
- Taguchi, Y.H.; Wang, H. Exploring microRNA biomarker for amyotrophic lateral sclerosis. Int. J. Mol. Sci. 2018, 19, 1318. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Benigni, M.; Ricci, C.; Jones, A.R.; Giannini, F.; Al-Chalabi, A.; Battistini, S. Identification of miRNAs as Potential Biomarkers in Cerebrospinal Fluid from Amyotrophic Lateral Sclerosis Patients. Neuromol. Med. 2016, 18, 551–560. [Google Scholar] [CrossRef]
- Matamala, J.M.; Arias-Carrasco, R.; Sanchez, C.; Uhrig, M.; Bargsted, L.; Matus, S.; Maracaja-Coutinho, V.; Abarzua, S.; van Zundert, B.; Verdugo, R.; et al. Genome-wide circulating microRNA expression profiling reveals potential biomarkers for amyotrophic lateral sclerosis. Neurobiol. Aging 2018, 64, 123–138. [Google Scholar] [CrossRef]
- Huang, E.J.; Reichardt, L.F. Neurotrophins: Roles in neuronal development and function. Annu. Rev. Neurosci. 2001, 24, 677–736. [Google Scholar] [CrossRef] [Green Version]
- Alcántara, S.; Frisén, J.; Del Río, J.A.; Soriano, E.; Barbacid, M.; Silos-Santiago, I. TrkB signaling is required for postnatal survival of CNS neurons and protects hippocampal and motor neurons from axotomy-induced cell death. J. Neurosci. 1997, 17, 3623–3633. [Google Scholar] [CrossRef] [Green Version]
- Pradhan, J.; Noakes, P.G.; Bellingham, M.C. The Role of Altered BDNF/TrkB Signaling in Amyotrophic Lateral Sclerosis. Front. Cell. Neurosci. 2019, 13, 368. [Google Scholar] [CrossRef] [PubMed]
- Tsai, T.; Klausmeyer, A.; Conrad, R.; Gottschling, C.; Leo, M.; Faissner, A.; Wiese, S. 7,8-Dihydroxyflavone leads to survival of cultured embryonic motoneurons by activating intracellular signaling pathways. Mol. Cell. Neurosci. 2013, 56, 18–28. [Google Scholar] [CrossRef]
- Hu, P.; Kalb, R.G. BDNF heightens the sensitivity of motor neurons to excitotoxic insults through activation of TrkB. J. Neurochem. 2003, 84, 1421–1430. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Mojsilovic-Petrovic, J.; Jeong, G.B.; Crocker, A.; Arneja, A.; David, S.; Russell, D.; Kalb, R.G. Protecting motor neurons from toxic insult by antagonism of adenosine A2a and Trk receptors. J. Neurosci. 2006, 26, 9250–9263. [Google Scholar] [CrossRef]
- Aulas, A.; Vande Velde, C. Alterations in stress granule dynamics driven by TDP-43 and FUS: A link to pathological inclusions in ALS? Front. Cell. Neurosci. 2015, 9, 423. [Google Scholar] [CrossRef] [Green Version]
- Ilieva, E.V.; Ayala, V.; Jové, M.; Dalfó, E.; Cacabelos, D.; Povedano, M.; Bellmunt, M.J.; Ferrer, I.; Pamplona, R.; Portero-Otín, M. Oxidative and endoplasmic reticulum stress interplay in sporadic amyotrophic lateral sclerosis. Brain 2007, 130, 3111–3123. [Google Scholar] [CrossRef]
- Hetz, C.; Saxena, S. ER stress and the unfolded protein response in neurodegeneration. Nat. Rev. Neurol. 2017, 13, 477–491. [Google Scholar] [CrossRef]
- Sambataro, F.; Pennuto, M. Post-translational modifications and protein quality control in motor neuron and polyglutamine diseases. Front. Mol. Neurosci. 2017, 10, 82. [Google Scholar] [CrossRef] [Green Version]
- Xue, Y.C.; Feuer, R.; Cashman, N.; Luo, H. Enteroviral infection: The forgotten link to amyotrophic lateral sclerosis? Front. Mol. Neurosci. 2018, 11, 63. [Google Scholar] [CrossRef] [PubMed]
- Grad, L.I.; Fernando, S.M.; Cashman, N.R. From molecule to molecule and cell to cell: Prion-like mechanisms in amyotrophic lateral sclerosis. Neurobiol. Dis. 2015, 77, 257–265. [Google Scholar] [CrossRef]
- Bird, S.W.; Maynard, N.D.; Covert, M.W.; Kirkegaard, K. Nonlytic viral spread enhanced by autophagy components. Proc. Natl. Acad. Sci. USA. 2014, 111, 13081–13086. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Robinson, S.M.; Tsueng, G.; Sin, J.; Mangale, V.; Rahawi, S.; McIntyre, L.L.; Williams, W.; Kha, N.; Cruz, C.; Hancock, B.M.; et al. Coxsackievirus B Exits the Host Cell in Shed Microvesicles Displaying Autophagosomal Markers. PLoS Pathog. 2014, 10, e1004045. [Google Scholar] [CrossRef]
- Chen, Y.H.; Du, W.; Hagemeijer, M.C.; Takvorian, P.M.; Pau, C.; Cali, A.; Brantner, C.A.; Stempinski, E.S.; Connelly, P.S.; Ma, H.C.; et al. Phosphatidylserine vesicles enable efficient en bloc transmission of enteroviruses. Cell 2015, 160, 619–630. [Google Scholar] [CrossRef] [Green Version]
- Too, I.H.K.; Yeo, H.; Sessions, O.M.; Yan, B.; Libau, E.A.; Howe, J.L.C.; Lim, Z.Q.; Suku-Maran, S.; Ong, W.Y.; Chua, K.B.; et al. Enterovirus 71 infection of motor neuron-like NSC-34 cells undergoes a non-lytic exit pathway. Sci. Rep. 2016, 6, 36983. [Google Scholar] [CrossRef] [PubMed]
Sporadic (n = 4) | TDP-43 Mutated (n = 3) | Healthy Donors (n = 3) | |
---|---|---|---|
Age at inclusion, mean (SD) | 53 (17.8) | 51.7 (22.9) | 52 (30) |
Gender, % M | 100 | 100 | 100 |
Duration of the disease (months), median [IQR] | 16 [19.5] | 14 [19.5] | NA |
Riluzole | Yes | Yes | NA |
Onset % bulbar/spinal | 25/75 | 0/100 | NA |
Fast/Slow | N | N | NA |
miRname | SD ALS pts | SD Controls | Average dCq ALS pts | Average dCq Controls | ddCq | RQ |
---|---|---|---|---|---|---|
hsa-miR-15a-5p | 1.67 | 0.88 | 0.64 | 0.24 | 0.40 | 0.76 |
hsa-miR-451a | 1.27 | 0.41 | 3.27 | 3.96 | −0.69 | 1.61 |
hsa-miR-21-5p | 1.11 | 0.99 | 0.51 | −0.23 | 0.74 | 0.60 |
hsa-miR-16-5p | 0.73 | 0.25 | 2.91 | 3.84 | −0.92 | 1.90 |
hsa-miR-223-3p | 1.35 | 0.70 | −0.01 | −0.15 | 0.14 | 0.91 |
hsa-miR-142-3p | 1.03 | 0.20 | 1.43 | −0.61 | 2.03 | 0.25 |
hsa-miR-126-3p | 0.78 | 3.30 | −0.87 | −3.12 | 2.26 | 0.21 |
hsa-miR-19a-3p | 0.69 | 0.58 | −1.18 | −0.54 | −0.65 | 1.57 |
hsa-miR-20a-5p | 0.53 | 0.19 | −1.76 | −0.51 | −1.26 | 2.39 |
hsa-miR-27b-3p | 1.00 | 0.99 | −2.88 | −4.44 | 1.56 | 0.34 |
miRname | Reference | ALS Type | Biofluid | Variation Reported | qPCR Normalization | Validated Target Genes |
---|---|---|---|---|---|---|
miR-15a-5p | Liguori et al., 2018 [37] | Sporadic | whole blood | decrease | miR-484 | hmga1, ucp2 |
miR-451a | Liguori et al., 2018; [37] | Sporadic | whole blood | decrease | miR-484 | cdkn2d |
Taguchi [38] and Wang, 2018 | Sporadic, familial and mutated carriers | Serum | increase | RMA + DBAG algorithm (Affymetrix) | ||
miR-21-5p | Benigni et al., 2016 [39] | Sporadic | CSF | decrease | spiked in cel-miR39-3p, miR608, miR328-3p | |
miR-16-5p | Liguori et al., 2018 [37] | Sporadic | whole blood | decrease | miR-484 | arhgdia, hdgf, hmga1, zyx |
miR-223-3p | Liguori et al., 2018 [37] | Sporadic | whole blood | decrease | miR-484 | cdkn2d |
miR-142-3p | Matamala et al., 2018 [40] | Sporadic | Serum | increase | Spiked in cel-miR39-3p | |
miR-27b-3p | Liguori et al., 2018 [37] | Sporadic | whole blood | decrease | miR-484 |
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. |
© 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Pregnolato, F.; Cova, L.; Doretti, A.; Bardelli, D.; Silani, V.; Bossolasco, P. Exosome microRNAs in Amyotrophic Lateral Sclerosis: A Pilot Study. Biomolecules 2021, 11, 1220. https://doi.org/10.3390/biom11081220
Pregnolato F, Cova L, Doretti A, Bardelli D, Silani V, Bossolasco P. Exosome microRNAs in Amyotrophic Lateral Sclerosis: A Pilot Study. Biomolecules. 2021; 11(8):1220. https://doi.org/10.3390/biom11081220
Chicago/Turabian StylePregnolato, Francesca, Lidia Cova, Alberto Doretti, Donatella Bardelli, Vincenzo Silani, and Patrizia Bossolasco. 2021. "Exosome microRNAs in Amyotrophic Lateral Sclerosis: A Pilot Study" Biomolecules 11, no. 8: 1220. https://doi.org/10.3390/biom11081220