Molecular Neurobiology

, Volume 51, Issue 3, pp 1168–1183 | Cite as

MicroRNA-34a Modulates Neural Stem Cell Differentiation by Regulating Expression of Synaptic and Autophagic Proteins

  • Ana L. Morgado
  • Joana M. Xavier
  • Pedro A. Dionísio
  • Maria F. C. Ribeiro
  • Raquel B. Dias
  • Ana M. Sebastião
  • Susana SoláEmail author
  • Cecília M. P. Rodrigues


We have previously demonstrated the involvement of specific apoptosis-associated microRNAs (miRNAs), including miR-34a, in mouse neural stem cell (NSC) differentiation. In addition, a growing body of evidence points to a critical role for autophagy during neuronal differentiation, as a response-survival mechanism to limit oxidative stress and regulate synaptogenesis associated with this process. The aim of this study was to further investigate the precise role of miR-34a during NSC differentiation. Our results showed that miR-34a expression was markedly downregulated during neurogenesis. Neuronal differentiation and cell morphology, synapse function, and electrophysiological maturation were significantly impaired in miR-34a-overexpressing NSCs. In addition, synaptotagmin 1 (Syt1) and autophagy-related 9a (Atg9a) significantly increased during neurogenesis. Pharmacological inhibition of autophagy impaired both neuronal differentiation and cell morphology. Notably, we showed that Syt1 and Atg9a are miR-34a targets in neural differentiation context, markedly decreasing after miR-34a overexpression. Syt1 overexpression and rapamycin-induced autophagy partially rescued the impairment of neuronal differentiation by miR-34a. In conclusion, our results demonstrate a novel role for miR-34a regulation of NSC differentiation, where miR-34a downregulation and subsequent increase of Syt1 and Atg9a appear to be crucial for neurogenesis progression.


Autophagy MicroRNA Neural stem cell Neurogenesis Synaptogenesis 


3′ UTR

3′ Untranslated region


Action potential


Autophagy-related 9a


Basic fibroblast growth factor


Fetal bovine serum


Microtubule-associated protein light chain 3


Microtubule-associated protein 2




Neuronal nuclei


Neural stem cell


Phosphate-buffered saline


Quantitative reverse transcription polymerase chain reaction


Reverse transcription


Synaptotagmin 1



We thank Dr. Massimiliano Agostini (Medical Research Council, Leicester University, Leicester, UK) for kindly providing Syt1 overexpression and Luc-Syt1 3′ UTR vectors. We also thank all members of the laboratory for the insightful discussions.

Conflict of Interest

The authors declare that they have no conflict of interest. This work was supported by grants PTDC/SAU-NMC/117877/2010 and PTDC/BIM-MED/0251/2012 and by fellowships SFRH/BD/80060/2011 (ALM) and SFRH/BD/68368/2010 (JMX) from Fundação para a Ciência e a Tecnologia, Lisbon, Portugal.

Supplementary material

12035_2014_8794_Fig8_ESM.gif (8 kb)
Figure S1

Cell viability after miR-34a overexpression.Mouse NSCs were induced to differentiate and collected for qRT-PCR analysis and flow cytometry quantification of cell viability by measuring annexin V- and PI-negative cells, as described in Materials and Methods. (A) MiR-34a expression after 48 hours, 12 and 14 days of transfection with either pre-miR control or pre-miR-34a. (B) Quantification of cell viability after 48 hours of differentiation and transfection with pre-miR control or pre-miR-34a. Results are expressed as mean ± SEM for at least three independent experiments.§ p < 0.05 and *p < 0.01 compared to pre-miR control. (GIF 8 kb)

12035_2014_8794_MOESM1_ESM.tif (195 kb)
ESM 1 (TIFF 195 kb)
12035_2014_8794_Fig9_ESM.gif (11 kb)
Figure S2

Modulation of Syt1 during neuronal differentiation. NSCs were induced to differentiate, co-transfected with pre-miR-34a and Syt1 overexpression plasmid, and collected for immunoblotting analysis, as described in Materials and Methods. Representative immunoblots (top) of Syt1 expression, and respective densitometry analysis (bottom).Results are expressed as mean ± SEM for at least three independent experiments.*p < 0.01 compared to pre-miR control plus mock; p < 0.01 compared to pre-miR-34a plus mock. (GIF 11 kb)

12035_2014_8794_MOESM2_ESM.tif (216 kb)
ESM 2 (TIFF 215 kb)
12035_2014_8794_Fig10_ESM.gif (9 kb)
Figure S3

Modulation of Atg9a after rapamycin treatment. NSCs were induced to differentiate, transfected with pre-miR-34a, and treated with rapamycin after 4 hours, and collected for immunoblotting analysis, as described in Materials and Methods.Representative immunoblots (top) of Atg9a expression, and respective densitometry (bottom).Results are expressed as mean ± SEM for at least three independent experiments.*p < 0.01 compared to pre-miR control plus DMSO; p < 0.01 compared to pre-miR-34a plus DMSO. (GIF 8 kb)

12035_2014_8794_MOESM3_ESM.tif (205 kb)
ESM 3 (TIFF 205 kb)


  1. 1.
    Conti L, Cattaneo E (2010) Neural stem cell systems: physiological players or in vitro entities? Nat Rev Neurosci 11:176–187CrossRefPubMedGoogle Scholar
  2. 2.
    Lindvall O, Kokaia Z (2010) Stem cells in human neurodegenerative disorders—time for clinical translation? J Clin Invest 120:29–40CrossRefPubMedCentralPubMedGoogle Scholar
  3. 3.
    Kim H, Cooke MJ, Shoichet MS (2012) Creating permissive microenvironments for stem cell transplantation into the central nervous system. Trends Biotechnol 30:55–63CrossRefPubMedGoogle Scholar
  4. 4.
    Sola S, Aranha MM, Rodrigues CM (2012) Driving apoptosis-relevant proteins toward neural differentiation. Mol Neurobiol 46:316–331CrossRefPubMedGoogle Scholar
  5. 5.
    Galluzzi L, Kepp O, Trojel-Hansen C, Kroemer G (2012) Non-apoptotic functions of apoptosis-regulatory proteins. EMBO Rep 13:322–330CrossRefPubMedCentralPubMedGoogle Scholar
  6. 6.
    Aranha MM, Sola S, Low WC, Steer CJ, Rodrigues CM (2009) Caspases and p53 modulate FOXO3A/Id1 signaling during mouse neural stem cell differentiation. J Cell Biochem 107:748–758CrossRefPubMedGoogle Scholar
  7. 7.
    Santos DM, Xavier JM, Morgado AL, Solá S, Rodrigues CM (2012) Distinct regulatory functions of calpain 1 and 2 during neural stem cell self-renewal and differentiation. PLoS One 7:e33468CrossRefPubMedCentralPubMedGoogle Scholar
  8. 8.
    Solá S, Xavier JM, Santos DM, Aranha MM, Morgado AL, Jepsen K, Rodrigues CM (2011) p53 interaction with JMJD3 results in its nuclear distribution during mouse neural stem cell differentiation. PLoS One 6:e18421CrossRefPubMedCentralPubMedGoogle Scholar
  9. 9.
    Ivey KN, Srivastava D (2010) MicroRNAs as regulators of differentiation and cell fate decisions. Cell Stem Cell 7:36–41CrossRefPubMedGoogle Scholar
  10. 10.
    Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116:281–297CrossRefPubMedGoogle Scholar
  11. 11.
    Ambros V (2004) The functions of animal microRNAs. Nature 431:350–355CrossRefPubMedGoogle Scholar
  12. 12.
    He L, He X, Lim LP, de Stanchina E, Xuan Z, Liang Y, Xue W, Zender L, Magnus J, Ridzon D, Jackson AL, Linsley PS, Chen C, Lowe SW, Cleary MA, Hannon GJ (2007) A microRNA component of the p53 tumour suppressor network. Nature 447:1130–1134CrossRefPubMedGoogle Scholar
  13. 13.
    Bommer GT, Gerin I, Feng Y, Kaczorowski AJ, Kuick R, Love RE, Zhai Y, Giordano TJ, Qin ZS, Moore BB, MacDougald OA, Cho KR, Fearon ER (2007) p53-mediated activation of miRNA34 candidate tumor-suppressor genes. Curr Biol 17:1298–1307CrossRefPubMedGoogle Scholar
  14. 14.
    Raver-Shapira N, Marciano E, Meiri E, Spector Y, Rosenfeld N, Moskovits N, Bentwich Z, Oren M (2007) Transcriptional activation of miR-34a contributes to p53-mediated apoptosis. Mol Cell 26:731–743CrossRefPubMedGoogle Scholar
  15. 15.
    Chang TC, Wentzel EA, Kent OA, Ramachandran K, Mullendore M, Lee KH, Feldmann G, Yamakuchi M, Ferlito M, Lowenstein CJ, Arking DE, Beer MA, Maitra A, Mendell JT (2007) Transactivation of miR-34a by p53 broadly influences gene expression and promotes apoptosis. Mol Cell 26:745–752CrossRefPubMedCentralPubMedGoogle Scholar
  16. 16.
    Tarasov V, Jung P, Verdoodt B, Lodygin D, Epanchintsev A, Menssen A, Meister G, Hermeking H (2007) Differential regulation of microRNAs by p53 revealed by massively parallel sequencing: miR-34a is a p53 target that induces apoptosis and G1-arrest. Cell Cycle 6:1586–1593CrossRefPubMedGoogle Scholar
  17. 17.
    Corney DC, Flesken-Nikitin A, Godwin AK, Wang W, Nikitin AY (2007) MicroRNA-34b and MicroRNA-34c are targets of p53 and cooperate in control of cell proliferation and adhesion-independent growth. Cancer Res 67:8433–8438CrossRefPubMedGoogle Scholar
  18. 18.
    Aranha MM, Santos DM, Sola S, Steer CJ, Rodrigues CM (2011) miR-34a regulates mouse neural stem cell differentiation. PLoS One 6:e21396CrossRefPubMedCentralPubMedGoogle Scholar
  19. 19.
    Aranha MM, Santos DM, Xavier JM, Low WC, Steer CJ, Sola S, Rodrigues CM (2010) Apoptosis-associated microRNAs are modulated in mouse, rat and human neural differentiation. BMC Genomics 11:514CrossRefPubMedCentralPubMedGoogle Scholar
  20. 20.
    Agostini M, Tucci P, Steinert JR, Shalom-Feuerstein R, Rouleau M, Aberdam D, Forsythe ID, Young KW, Ventura A, Concepcion CP, Han YC, Candi E, Knight RA, Mak TW, Melino G (2011) microRNA-34a regulates neurite outgrowth, spinal morphology, and function. Proc Natl Acad Sci U S A 108:21099–21104CrossRefPubMedCentralPubMedGoogle Scholar
  21. 21.
    Agostini M, Tucci P, Killick R, Candi E, Sayan BS, Rivetti Val di Cervo P, Nicotera P, McKeon F, Knight RA, Mak TW, Melino G (2011) Neuronal differentiation by TAp73 is mediated by microRNA-34a regulation of synaptic protein targets. Proc Natl Acad Sci U S A 108:21093–21098CrossRefPubMedCentralPubMedGoogle Scholar
  22. 22.
    Yang J, Chen D, He Y, Melendez A, Feng Z, Hong Q, Bai X, Li Q, Cai G, Wang J, Chen X (2013) MiR-34 modulates Caenorhabditis elegans lifespan via repressing the autophagy gene atg9. Age (Dordr) 35:11–22CrossRefGoogle Scholar
  23. 23.
    Orsi A, Razi M, Dooley HC, Robinson D, Weston AE, Collinson LM, Tooze SA (2012) Dynamic and transient interactions of Atg9 with autophagosomes, but not membrane integration, are required for autophagy. Mol Biol Cell 23:1860–1873CrossRefPubMedCentralPubMedGoogle Scholar
  24. 24.
    Yamamoto H, Kakuta S, Watanabe TM, Kitamura A, Sekito T, Kondo-Kakuta C, Ichikawa R, Kinjo M, Ohsumi Y (2012) Atg9 vesicles are an important membrane source during early steps of autophagosome formation. J Cell Biol 198:219–233CrossRefPubMedCentralPubMedGoogle Scholar
  25. 25.
    Mizushima N, Levine B (2010) Autophagy in mammalian development and differentiation. Nat Cell Biol 12:823–830CrossRefPubMedCentralPubMedGoogle Scholar
  26. 26.
    Pollard SM, Conti L, Sun Y, Goffredo D, Smith A (2006) Adherent neural stem (NS) cells from fetal and adult forebrain. Cereb Cortex 16(Suppl 1):i112–i120CrossRefPubMedGoogle Scholar
  27. 27.
    Conti L, Pollard SM, Gorba T, Reitano E, Toselli M, Biella G, Sun Y, Sanzone S, Ying QL, Cattaneo E, Smith A (2005) Niche-independent symmetrical self-renewal of a mammalian tissue stem cell. PLoS Biol 3:e283CrossRefPubMedCentralPubMedGoogle Scholar
  28. 28.
    Glaser T, Pollard SM, Smith A, Brustle O (2007) Tripotential differentiation of adherently expandable neural stem (NS) cells. PLoS One 2:e298CrossRefPubMedCentralPubMedGoogle Scholar
  29. 29.
    Spiliotopoulos D, Goffredo D, Conti L, Di Febo F, Biella G, Toselli M, Cattaneo E (2009) An optimized experimental strategy for efficient conversion of embryonic stem (ES)-derived mouse neural stem (NS) cells into a nearly homogeneous mature neuronal population. Neurobiol Dis 34:320–331CrossRefPubMedGoogle Scholar
  30. 30.
    Xavier JM, Morgado AL, Sola S, Rodrigues CM (2014) Mitochondrial translocation of p53 modulates neuronal fate by preventing differentiation-induced mitochondrial stress. Antioxid Redox Signal [Epub ahead of print]Google Scholar
  31. 31.
    Meijering E, Jacob M, Sarria JC, Steiner P, Hirling H, Unser M (2004) Design and validation of a tool for neurite tracing and analysis in fluorescence microscopy images. Cytometry A 58:167–176CrossRefPubMedGoogle Scholar
  32. 32.
    Dias RB, Rombo DM, Ribeiro JA, Sebastiao AM (2013) Ischemia-induced synaptic plasticity drives sustained expression of calcium-permeable AMPA receptors in the hippocampus. Neuropharmacology 65:114–122CrossRefPubMedGoogle Scholar
  33. 33.
    Naundorf B, Wolf F, Volgushev M (2006) Unique features of action potential initiation in cortical neurons. Nature 440:1060–1063CrossRefPubMedGoogle Scholar
  34. 34.
    Pratt T, Sharp L, Nichols J, Price DJ, Mason JO (2000) Embryonic stem cells and transgenic mice ubiquitously expressing a tau-tagged green fluorescent protein. Dev Biol 228:19–28CrossRefPubMedGoogle Scholar
  35. 35.
    Silva J, Chambers I, Pollard S, Smith A (2006) Nanog promotes transfer of pluripotency after cell fusion. Nature 441:997–1001CrossRefPubMedGoogle Scholar
  36. 36.
    Fonseca MB, Sola S, Xavier JM, Dionisio PA, Rodrigues CM (2013) Amyloid beta peptides promote autophagy-dependent differentiation of mouse neural stem cells: Abeta-mediated neural differentiation. Mol Neurobiol 48:829–840CrossRefPubMedGoogle Scholar
  37. 37.
    Sundelacruz S, Levin M, Kaplan DL (2009) Role of membrane potential in the regulation of cell proliferation and differentiation. Stem Cell Rev 5:231–246CrossRefPubMedGoogle Scholar
  38. 38.
    Johnson MA, Weick JP, Pearce RA, Zhang SC (2007) Functional neural development from human embryonic stem cells: accelerated synaptic activity via astrocyte coculture. J Neurosci 27:3069–3077CrossRefPubMedCentralPubMedGoogle Scholar
  39. 39.
    Mizushima N, Yoshimori T, Levine B (2010) Methods in mammalian autophagy research. Cell 140:313–326CrossRefPubMedCentralPubMedGoogle Scholar
  40. 40.
    Schratt G (2009) Fine-tuning neural gene expression with microRNAs. Curr Opin Neurobiol 19:213–219CrossRefPubMedGoogle Scholar
  41. 41.
    Guessous F, Zhang Y, Kofman A, Catania A, Li Y, Schiff D, Purow B, Abounader R (2010) microRNA-34a is tumor suppressive in brain tumors and glioma stem cells. Cell Cycle 9:1031–1036CrossRefPubMedCentralPubMedGoogle Scholar
  42. 42.
    Politis PK, Thomaidou D, Matsas R (2008) Coordination of cell cycle exit and differentiation of neuronal progenitors. Cell Cycle 7:691–697CrossRefPubMedGoogle Scholar
  43. 43.
    Wei JS, Song YK, Durinck S, Chen QR, Cheuk AT, Tsang P, Zhang Q, Thiele CJ, Slack A, Shohet J, Khan J (2008) The MYCN oncogene is a direct target of miR-34a. Oncogene 27:5204–5213CrossRefPubMedCentralPubMedGoogle Scholar
  44. 44.
    Fox MA, Sanes JR (2007) Synaptotagmin I and II are present in distinct subsets of central synapses. J Comp Neurol 503:280–296CrossRefPubMedGoogle Scholar
  45. 45.
    Xu J, Pang ZP, Shin OH, Sudhof TC (2009) Synaptotagmin-1 functions as a Ca2+ sensor for spontaneous release. Nat Neurosci 12:759–766CrossRefPubMedCentralPubMedGoogle Scholar
  46. 46.
    Greif KF, Asabere N, Lutz GJ, Gallo G (2013) Synaptotagmin-1 promotes the formation of axonal filopodia and branches along the developing axons of forebrain neurons. Dev Neurobiol 73:27–44CrossRefPubMedGoogle Scholar
  47. 47.
    Frankel LB, Lund AH (2012) MicroRNA regulation of autophagy. Carcinogenesis 33:2018–2025CrossRefPubMedGoogle Scholar
  48. 48.
    Torres CA, Sulzer D (2012) Macroautophagy can press a brake on presynaptic neurotransmission. Autophagy 8:1540–1541CrossRefPubMedCentralPubMedGoogle Scholar
  49. 49.
    Hernandez D, Torres CA, Setlik W, Cebrian C, Mosharov EV, Tang G, Cheng HC, Kholodilov N, Yarygina O, Burke RE, Gershon M, Sulzer D (2012) Regulation of presynaptic neurotransmission by macroautophagy. Neuron 74:277–284CrossRefPubMedCentralPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Ana L. Morgado
    • 1
  • Joana M. Xavier
    • 1
  • Pedro A. Dionísio
    • 1
  • Maria F. C. Ribeiro
    • 1
  • Raquel B. Dias
    • 3
    • 4
  • Ana M. Sebastião
    • 3
    • 4
  • Susana Solá
    • 1
    • 2
    Email author
  • Cecília M. P. Rodrigues
    • 1
    • 2
  1. 1.Research Institute for Medicines (iMed.ULisboa), Faculty of PharmacyUniversidade de LisboaLisbonPortugal
  2. 2.Department of Biochemistry and Human Biology, Faculty of PharmacyUniversidade de LisboaLisbonPortugal
  3. 3.Faculty of Medicine, Institute of Pharmacology and NeurosciencesUniversidade de LisboaLisbonPortugal
  4. 4.Unit of Neurosciences, Instituto de Medicina MolecularUniversidade de LisboaLisbonPortugal

Personalised recommendations