Regulation of the Glycine Transporter GLYT1 by microRNAs


The glycine transporter GLYT1 participates in inhibitory and excitatory neurotransmission by controlling the reuptake of this neuroactive substance from synapses. Over the past few years, microRNAs have emerged as potent negative regulators of gene expression. In this report, we investigate the possible regulation of GLYT1 by microRNAs. TargetScan software predicted the existence of multiple targets for microRNAs within the 3′ UTR of the human GLYT1 (miR-7, miR-30, miR-96, miR-137 and miR-141), and as they are all conserved among mammalian orthologues, their effects on GLYT1 expression were determined experimentally. Dual reporter bioluminescent assays showed that only miR-96 and miR-137 down-regulated expression of the Renilla reporter fused to the 3′ UTR of GLYT1. Mutations introduced into the target sequences blocked this inhibitory effect. Consistently, these two microRNAs downregulated the uptake of [3H]glycine into glial C6 cells, a cell line where GLYT1 is the main carrier for glycine. Moreover, the expression of endogenous GLYT1 in primary mixed cultures from rat spinal cord was decreased upon lentiviral expression of miR-96 and miR-137. Although the bulk of GLYT1 is glial, it is abundantly expressed in glycinergic neurons of the retina and in smaller amounts in glutamatergic neurons though the brain. Since miR-96 in the retina is strongly downregulated by light exposure, when rats were maintained in darkness for a few hours we observed a concomitant increase of GLYT1 expression, suggesting that at least miR-96 might be an important negative regulator of GLYT1 under physiological conditions.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4


  1. 1.

    Gomeza J, Hülsmann S, Ohno K et al (2003) Inactivation of the glycine transporter 1 gene discloses vital role of glial glycine uptake in glycinergic inhibition. Neuron 40:785–796.

    CAS  Article  PubMed  Google Scholar 

  2. 2.

    Zafra F, Aragón C, Olivares L et al (1995) Glycine transporters are differentially expressed among CNS cells. J Neurosci 15:3952–3969.

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  3. 3.

    Cubelos B, Giménez C, Zafra F (2005) Localization of the GLYT1 glycine transporter at glutamatergic synapses in the rat brain. Cereb Cortex 15:448–459.

    Article  PubMed  Google Scholar 

  4. 4.

    Zafra F, Ibáñez I, Bartolomé-Martín D et al (2017) Glycine transporters and its coupling with NMDA receptors. Adv Neurobiol 16:55–83.

    Article  PubMed  Google Scholar 

  5. 5.

    Le Bail M, Martineau M, Sacchi S et al (2015) Identity of the NMDA receptor coagonist is synapse specific and developmentally regulated in the hippocampus. Proc Natl Acad Sci U S A 112:E204–E213.

    CAS  Article  PubMed  Google Scholar 

  6. 6.

    Wässle H, Heinze L, Ivanova E et al (2009) Glycinergic transmission in the mammalian retina. Front Mol Neurosci 2:6.

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  7. 7.

    Eulenburg V, Knop G, Sedmak T et al (2018) GlyT1 determines the glycinergic phenotype of amacrine cells in the mouse retina. Brain Struct Funct 223:3251–3266.

    CAS  Article  PubMed  Google Scholar 

  8. 8.

    Borowsky B, Hoffman BJ (1998) Analysis of a gene encoding two glycine transporter variants reveals alternative promoter usage and a novel gene structure. J Biol Chem 273:29077–29085.

    CAS  Article  PubMed  Google Scholar 

  9. 9.

    Adams RH, Sato K, Shimada S et al (1995) Gene structure and glial expression of the glycine transporter GlyT1 in embryonic and adult rodents. J Neurosci 15:2524–2532.

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  10. 10.

    Fernández-Sánchez E, Martínez-Villarreal J, Giménez C, Zafra F (2009) Constitutive and regulated endocytosis of the glycine transporter GLYT1b is controlled by ubiquitination. J Biol Chem 284:19482–19492.

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  11. 11.

    Vargas-Medrano J, Castrejon-Tellez V, Plenge F et al (2011) PKCβ-dependent phosphorylation of the glycine transporter 1. Neurochem Int 59:1123–1132.

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  12. 12.

    Cubelos B, González-González IM, Giménez C, Zafra F (2005) The scaffolding protein PSD-95 interacts with the glycine transporter GLYT1 and impairs its internalization. J Neurochem 95:1047–1058.

    CAS  Article  PubMed  Google Scholar 

  13. 13.

    Flynt AS, Lai EC (2008) Biological principles of microRNA-mediated regulation: shared themes amid diversity. Nat Rev Genet 9:831–842.

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  14. 14.

    Ha M, Kim VN (2014) Regulation of microRNA biogenesis. Nat Rev Mol Cell Biol 15:509–524.

    CAS  Article  PubMed  Google Scholar 

  15. 15.

    Lai X, Wolkenhauer O, Vera J (2016) Understanding microRNA-mediated gene regulatory networks through mathematical modelling. Nucleic Acids Res 44:6019–6035.

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  16. 16.

    Bhalala OG, Srikanth M, Kessler JA (2013) The emerging roles of microRNAs in CNS injuries. Nat Rev Neurol 9:328–339.

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  17. 17.

    Higa GSV, de Sousa E, Walter LT et al (2014) MicroRNAs in neuronal communication. Mol Neurobiol 49:1309–1326.

    CAS  Article  PubMed  Google Scholar 

  18. 18.

    Baudry A, Mouillet-Richard S, Schneider B et al (2010) MiR-16 targets the serotonin transporter: a new facet for adaptive responses to antidepressants. Science 329:1537–1541.

    CAS  Article  PubMed  Google Scholar 

  19. 19.

    Launay JM, Mouillet-Richard S, Baudry A et al (2011) Raphe-mediated signals control the hippocampal response to SRI antidepressants via miR-16. Transl Psychiatry 1:e56–e56.

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  20. 20.

    Moya PR, Wendland JR, Salemme J et al (2013) miR-15a and miR-16 regulate serotonin transporter expression in human placental and rat brain raphe cells. Int J Neuropsychopharmacol 16:621–629.

    CAS  Article  Google Scholar 

  21. 21.

    Liao X-J, Mao W-M, Wang Q et al (2016) MicroRNA-24 inhibits serotonin reuptake transporter expression and aggravates irritable bowel syndrome. Biochem Biophys Res Commun 469:288–293.

    CAS  Article  Google Scholar 

  22. 22.

    Gu J, Zhang H, Ji B et al (2017) Vesicle miR-195 derived from endothelial cells inhibits expression of serotonin transporter in vessel smooth muscle cells. Sci Rep 7:43546.

    Article  PubMed Central  PubMed  Google Scholar 

  23. 23.

    Issler O, Haramati S, Paul ED et al (2014) MicroRNA 135 is essential for chronic stress resiliency, antidepressant efficacy, and intact serotonergic activity. Neuron 83:344–360.

    CAS  Article  Google Scholar 

  24. 24.

    Hommers LG, Richter J, Yang Y et al (2018) A functional genetic variation of SLC6A2 repressor hsa-miR-579-3p upregulates sympathetic noradrenergic processes of fear and anxiety. Transl Psychiatry 8:226.

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  25. 25.

    Jia X, Wang F, Han Y et al (2016) miR-137 and miR-491 negatively regulate dopamine transporter expression and function in neural cells. Neurosci Bull 32:512–522.

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  26. 26.

    Morel L, Regan M, Higashimori H et al (2013) Neuronal exosomal miRNA-dependent translational regulation of astroglial glutamate transporter GLT1. J Biol Chem 288:7105–7116.

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  27. 27.

    Piniella D, Martínez-Blanco E, Ibáñez I et al (2018) Identification of novel regulatory partners of the glutamate transporter GLT-1. Glia 66:2737–2755.

    Article  Google Scholar 

  28. 28.

    Wu J, Bonsra AN, Du G (2009) pSM155 and pSM30 vectors for miRNA and shRNA expression. Methods Mol Biol 487:205–219.

    CAS  Article  Google Scholar 

  29. 29.

    Villarejo-López L, Jiménez E, Bartolomé-Martín D et al (2017) P2X receptors up-regulate the cell-surface expression of the neuronal glycine transporter GlyT2. Neuropharmacology 125:99–116.

    CAS  Article  Google Scholar 

  30. 30.

    Zafra F, Giménez C (1989) Characteristics and adaptive regulation of glycine transport in cultured glial cells. Biochem J 258:403–408.

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  31. 31.

    Agarwal V, Bell GW, Nam J-W, Bartel DP (2015) Predicting effective microRNA target sites in mammalian mRNAs. Elife 4.

  32. 32.

    Gomeza J, Zafra F, Olivares L et al (1995) Regulation by phorbol esters of the glycine transporter (GLYT1) in glioblastoma cells. Biochim Biophys Acta 1233:41–46.

    Article  PubMed  Google Scholar 

  33. 33.

    Pearlman RJ, Aubrey KR, Vandenberg RJ (2003) Arachidonic acid and anandamide have opposite modulatory actions at the glycine transporter, GLYT1a. J Neurochem 84:592–601.

    CAS  Article  PubMed  Google Scholar 

  34. 34.

    Krol J, Busskamp V, Markiewicz I et al (2010) Characterizing light-regulated retinal microRNAs reveals rapid turnover as a common property of neuronal microRNAs. Cell 141:618–631.

    CAS  Article  PubMed  Google Scholar 

  35. 35.

    Li H, Kloosterman W, Fekete DM (2010) MicroRNA-183 family members regulate sensorineural fates in the inner ear. J Neurosci 30:3254–3263.

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  36. 36.

    Li H, Fekete DM (2010) MicroRNAs in hair cell development and deafness. Curr Opin Otolaryngol Head Neck Surg 18:459–465.

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  37. 37.

    Mencía A, Modamio-Høybjør S, Redshaw N et al (2009) Mutations in the seed region of human miR-96 are responsible for nonsyndromic progressive hearing loss. Nat Genet 41:609–613.

    CAS  Article  PubMed  Google Scholar 

  38. 38.

    Lewis MA, Quint E, Glazier AM et al (2009) An ENU-induced mutation of miR-96 associated with progressive hearing loss in mice. Nat Genet 41:614–618.

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  39. 39.

    Weston MD, Pierce ML, Jensen-Smith HC et al (2011) MicroRNA-183 family expression in hair cell development and requirement of microRNAs for hair cell maintenance and survival. Dev Dyn 240:808–819.

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  40. 40.

    Sacheli R, Nguyen L, Borgs L et al (2009) Expression patterns of miR-96, miR-182 and miR-183 in the development inner ear. Gene Expr Patterns 9:364–370.

    CAS  Article  PubMed  Google Scholar 

  41. 41.

    Schlüter T, Berger C, Rosengauer E et al (2018) miR-96 is required for normal development of the auditory hindbrain. Hum Mol Genet 27:860–874.

    CAS  Article  PubMed  Google Scholar 

  42. 42.

    Xu S, Witmer PD, Lumayag S et al (2007) MicroRNA (miRNA) transcriptome of mouse retina and identification of a sensory organ-specific miRNA cluster. J Biol Chem 282:25053–25066.

    CAS  Article  PubMed  Google Scholar 

  43. 43.

    Xiang L, Chen X-J, Wu K-C et al (2017) miR-183/96 plays a pivotal regulatory role in mouse photoreceptor maturation and maintenance. Proc Natl Acad Sci U S A 114:6376–6381.

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  44. 44.

    Li H, Gong Y, Qian H et al (2015) Brain-derived neurotrophic factor is a novel target gene of the has-miR-183/96/182 cluster in retinal pigment epithelial cells following visible light exposure. Mol Med Rep 12:2793–2799.

    CAS  Article  PubMed  Google Scholar 

  45. 45.

    Müller F, Wässle H, Voigt T (1988) Pharmacological modulation of the rod pathway in the cat retina. J Neurophysiol 59:1657–1672.

    Article  PubMed  Google Scholar 

  46. 46.

    Yang XL, Wu SM (1989) Effects of prolonged light exposure, GABA, and glycine on horizontal cell responses in tiger salamander retina. J Neurophysiol 61:1025–1035.

    CAS  Article  PubMed  Google Scholar 

  47. 47.

    Guella I, Sequeira A, Rollins B et al (2013) Analysis of miR-137 expression and rs1625579 in dorsolateral prefrontal cortex. J Psychiatr Res 47:1215–1221.

    Article  PubMed Central  PubMed  Google Scholar 

  48. 48.

    Mahmoudi E, Cairns MJ (2017) MiR-137: an important player in neural development and neoplastic transformation. Mol Psychiatry 22:44–55.

    CAS  Article  PubMed  Google Scholar 

  49. 49.

    Schizophrenia Psychiatric Genome-Wide Association Study (GWAS) Consortium (2011) Genome-wide association study identifies five new schizophrenia loci. Nat Genet 43:969–976.

    CAS  Article  Google Scholar 

  50. 50.

    Schizophrenia Working Group of the Psychiatric Genomics Consortium (2014) Biological insights from 108 schizophrenia-associated genetic loci. Nature 511:421–427.

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  51. 51.

    Warburton A, Breen G, Bubb VJ, Quinn JP (2016) A GWAS SNP for schizophrenia is linked to the internal MIR137 promoter and supports differential allele-specific expression. Schizophr Bull 42:1003–1008.

    Article  Google Scholar 

  52. 52.

    Wu S, Zhang R, Nie F et al (2016) MicroRNA-137 inhibits EFNB2 expression affected by a genetic variant and is expressed aberrantly in peripheral blood of schizophrenia patients. EBioMedicine 12:133–142.

    Article  PubMed Central  PubMed  Google Scholar 

  53. 53.

    Olde Loohuis NFM, Ba W, Stoerchel PH et al (2015) MicroRNA-137 controls AMPA-receptor-mediated transmission and mGluR-dependent LTD. Cell Rep 11:1876–1884.

    CAS  Article  Google Scholar 

  54. 54.

    de Sena CA, Berkel S, Cristian F-B et al (2018) A direct regulatory link between microRNA-137 and SHANK2: implications for neuropsychiatric disorders. J Neurodev Disord 10:15.

    Article  Google Scholar 

  55. 55.

    Siegert S, Seo J, Kwon EJ et al (2015) The schizophrenia risk gene product miR-137 alters presynaptic plasticity. Nat Neurosci 18:1008–1016.

    CAS  Article  PubMed Central  PubMed  Google Scholar 

Download references


We would like to thank Enrique Núñez for his expert technical contribution. The pSM30 plasmid was a generous donation of Dr. G. Du (StonyBrook University). The professional editing service NB Revisions was used for technical preparation of the text prior to submission.


This work was supported by grants from the Spanish Ministerio de Ciencia e Innovación (RTI2018-098712-B-I00) and Fundación Ramón Areces, the later providing an institutional grant to CBMSO.

Author information



Corresponding author

Correspondence to Francisco Zafra.

Ethics declarations

Conflict of interest

The authors have declared no conflict of interest.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Special issue in honor of Prof Baruch Kanner

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Jiménez, E., Piniella, D., Giménez, C. et al. Regulation of the Glycine Transporter GLYT1 by microRNAs. Neurochem Res (2021).

Download citation


  • Glycine transporters
  • microRNAs
  • Neurotransmission
  • Retina