Molecular Neurobiology

, Volume 56, Issue 10, pp 6928–6940 | Cite as

Zinc Uptake and Storage During the Formation of the Cerebral Cortex in Mice

  • Jessy Hasna
  • Sylvain Bohic
  • Sophie Lemoine
  • Corinne Blugeon
  • Alexandre BouronEmail author


The cerebral cortex (or neocortex) is a brain structure formed during embryogenesis. The present study seeks to provide a detailed characterization of the Zn homeostatic mechanisms during cerebral cortex formation and development. To reach that goal, we have combined high-throughput RNA-sequencing analysis of the whole murine genome, X-ray fluorescence nanoimaging (XRF), inductively coupled plasma-atomic emission spectrometry (ICP-AES), and live-cell imaging of dissociated cortical neurons loaded with the Zn fluorescent probe FluoZin-3. The transcriptomic analysis was conducted from mRNAs isolated from cortices collected at embryonic (E) days 11 (E11), E13, and E17 and on postnatal day 1 (PN1) pups. This permitted to characterize the temporal pattern of expression of the main genes participating in the cellular transport, storage, and release of Zn during corticogenesis. It appears that cells of the immature cortex express a wide diversity of actors involved in Zn homeostasis with Zip7, SOD1, and metallothioneins being the most abundant transcripts throughout corticogenesis. The quantification of total Zn with XRF and ICP-AES reveals a reduction of Zn levels. Moreover, this is accompanied by a diminution of the size of the internal pools of mobilizable Zn. This study illustrates the tight temporal and spatial regulation of Zn homeostasis during cerebral brain development.


Brain development Zinc Corticogenesis Transcriptome RNA-seq 



We wish to thank Dr. J. Pérard for his assistance with the ICP-AES experiments and L. Macari for her help with the cell cultures.

Funding Information

The study was supported by a grant from l’Agence Nationale de la Recherche (ANR-16-CE29-0024 to AB). We also wish to acknowledge the support from the Centre National de la Recherche Scientifique (CNRS), the Commissariat à l’Energie Atomique et aux Energies Alternatives (CEA), the Université de Grenoble Alpes (UGA), and the France Génomique infrastructure, funded as part of the “Investissements d’Avenir” program managed by the Agence Nationale de la Recherche (contract ANR-10-INBS-09).

Compliance with Ethical Standards

The experimental protocol was approved by the ethical committee of the CEA’s Life Sciences Division (CETEA, # A14-006).

Conflict of Interest

The authors declare that they have no conflict of interest.

Supplementary material

12035_2019_1581_MOESM1_ESM.docx (5.4 mb)
ESM 1 (DOCX 5578 kb)


  1. 1.
    Finlay BL, Uchiyama R (2015) Developmental mechanisms channeling cortical evolution. Trends Neurosci 38(2):69–76. CrossRefGoogle Scholar
  2. 2.
    Gotz M, Huttner WB (2005) The cell biology of neurogenesis. Nat Rev Mol Cell Biol 6(10):777–788. CrossRefGoogle Scholar
  3. 3.
    Takahashi T, Nowakowski RS, Caviness VS, Jr. (1996) The leaving or Q fraction of the murine cerebral proliferative epithelium: a general model of neocortical neuronogenesis. J Neurosci 16 (19):6183–6196Google Scholar
  4. 4.
    Sensi SL, Paoletti P, Bush AI, Sekler I (2009) Zinc in the physiology and pathology of the CNS. Nat Rev Neurosci 10(11):780–791CrossRefGoogle Scholar
  5. 5.
    Frederickson CJ, Suh SW, Silva D, Thompson RB (2000) Importance of zinc in the central nervous system: the zinc-containing neuron. J Nutr 130(5S Suppl):1471S–1483SCrossRefGoogle Scholar
  6. 6.
    Paoletti P, Vergnano AM, Barbour B, Casado M (2009) Zinc at glutamatergic synapses. Neuroscience 158(1):126–136CrossRefGoogle Scholar
  7. 7.
    Barr CA, Burdette SC (2017) The zinc paradigm for metalloneurochemistry. Essays Biochem 61(2):225–235. CrossRefGoogle Scholar
  8. 8.
    Takeda A, Tamano H (2017) The impact of synaptic Zn(2+) dynamics on cognition and its decline. Int J Mol Sci 18(11):2411. CrossRefGoogle Scholar
  9. 9.
    Vergnano AM, Rebola N, Savtchenko LP, Pinheiro PS, Casado M, Kieffer BL, Rusakov DA, Mulle C et al (2014) Zinc dynamics and action at excitatory synapses. Neuron 82(5):1101–1114. CrossRefGoogle Scholar
  10. 10.
    Aizenman E, Stout AK, Hartnett KA, Dineley KE, McLaughlin B, Reynolds IJ (2000) Induction of neuronal apoptosis by thiol oxidation: putative role of intracellular zinc release. J Neurochem 75(5):1878–1888CrossRefGoogle Scholar
  11. 11.
    Sensi SL, Ton-That D, Weiss JH (2002) Mitochondrial sequestration and Ca(2+)-dependent release of cytosolic Zn(2+) loads in cortical neurons. Neurobiol Dis 10(2):100–108CrossRefGoogle Scholar
  12. 12.
    Sun T, Hevner RF (2014) Growth and folding of the mammalian cerebral cortex: from molecules to malformations. Nat Rev Neurosci 15(4):217–232. CrossRefGoogle Scholar
  13. 13.
    Jourdren L, Bernard M, Dillies MA, Le Crom S (2012) Eoulsan: a cloud computing-based framework facilitating high throughput sequencing analyses. Bioinformatics 28(11):1542–1543. CrossRefGoogle Scholar
  14. 14.
    Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, Batut P, Chaisson M et al (2013) STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29(1):15–21. CrossRefGoogle Scholar
  15. 15.
    Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G et al (2009) The sequence alignment/map format and SAMtools. Bioinformatics 25(16):2078–2079. CrossRefGoogle Scholar
  16. 16.
    Anders S, Pyl PT, Huber W (2015) HTSeq--a Python framework to work with high-throughput sequencing data. Bioinformatics 31(2):166–169. CrossRefGoogle Scholar
  17. 17.
    Love MI, Huber W, Anders S (2014) Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15(12):550. CrossRefGoogle Scholar
  18. 18.
    Gibon J, Deloulme J-C, Chevallier T, Ladevèze E, Abrous DN, Bouron A (2013) The antidepressant hyperforin increases the phosphorylation of CREB and the expression of TrkB in a tissue-specific manner. Int J Neuropsychopharmacol 16(1):189–198. CrossRefGoogle Scholar
  19. 19.
    Gee KR, Zhou ZL, Ton-That D, Sensi SL, Weiss JH (2002) Measuring zinc in living cells. A new generation of sensitive and selective fluorescent probes. Cell Calcium 31(5):245–251CrossRefGoogle Scholar
  20. 20.
    Tu P, Gibon J, Bouron A (2010) The TRPC6 channel activator hyperforin induces the release of zinc and calcium from mitochondria. J Neurochem 112:204–213CrossRefGoogle Scholar
  21. 21.
    Gibon J, Tu P, Bohic S, Richaud P, Arnaud J, Zhu M, Boulay G, Bouron A (2011) The over-expression of TRPC6 channels in HEK-293 cells favours the intracellular accumulation of zinc. Biochim Biophys Acta 1808(12):2807–2818CrossRefGoogle Scholar
  22. 22.
    Kiedrowski L (2011) Cytosolic zinc release and clearance in hippocampal neurons exposed to glutamate--the role of pH and sodium. J Neurochem 117(2):231–243. CrossRefGoogle Scholar
  23. 23.
    De Samber B, Meul E, Laforce B, De Paepe B, Smet J, De Bruyne M, De Rycke R, Bohic S et al (2018) Nanoscopic X-ray fluorescence imaging and quantification of intracellular key-elements in cryofrozen Friedreich’s ataxia fibroblasts. PLoS One 13(1):e0190495. CrossRefGoogle Scholar
  24. 24.
    Carmona A, Zogzas CE, Roudeau S, Porcaro F, Garrevoet J, Spiers KM, Salome M, Cloetens P et al (2018) SLC30A10 mutation involved in parkinsonism results in manganese accumulation within nanovesicles of the Golgi apparatus. ACS Chem Neurosci 10:599–609. CrossRefGoogle Scholar
  25. 25.
    Colvin RA, Jin Q, Lai B, Kiedrowski L (2016) Visualizing metal content and intracellular distribution in primary hippocampal neurons with synchrotron X-ray fluorescence. PLoS One 11(7):e0159582. CrossRefGoogle Scholar
  26. 26.
    Colvin RA, Lai B, Holmes WR, Lee D (2015) Understanding metal homeostasis in primary cultured neurons. Studies using single neuron subcellular and quantitative metallomics. Metallomics 7(7):1111–1123. CrossRefGoogle Scholar
  27. 27.
    Pearce LL, Gandley RE, Han W, Wasserloos K, Stitt M, Kanai AJ, McLaughlin MK, Pitt BR et al (2000) Role of metallothionein in nitric oxide signaling as revealed by a green fluorescent fusion protein. Proc Natl Acad Sci U S A 97(1):477–482CrossRefGoogle Scholar
  28. 28.
    Sensi SL, Ton-That D, Sullivan PG, Jonas EA, Gee KR, Kaczmarek LK, Weiss JH (2003) Modulation of mitochondrial function by endogenous Zn2+ pools. Proc Natl Acad Sci U S A 100(10):6157–6162CrossRefGoogle Scholar
  29. 29.
    Wagner GP, Kin K, Lynch VJ (2012) Measurement of mRNA abundance using RNA-seq data: RPKM measure is inconsistent among samples. Theory Biosci 131(4):281–285. CrossRefGoogle Scholar
  30. 30.
    Wagner GP, Kin K, Lynch VJ (2013) A model based criterion for gene expression calls using RNA-seq data. Theory Biosci 132(3):159–164. CrossRefGoogle Scholar
  31. 31.
    Cousins RJ, Liuzzi JP, Lichten LA (2006) Mammalian zinc transport, trafficking, and signals. J Biol Chem 281(34):24085–24089CrossRefGoogle Scholar
  32. 32.
    Kambe T, Matsunaga M, Takeda TA (2017) Understanding the contribution of zinc transporters in the function of the early secretory pathway. Int J Mol Sci 18(10).
  33. 33.
    Kambe T, Tsuji T, Hashimoto A, Itsumura N (2015) The physiological, biochemical, and molecular roles of zinc transporters in zinc homeostasis and metabolism. Physiol Rev 95(3):749–784. CrossRefGoogle Scholar
  34. 34.
    Juarez-Rebollar D, Rios C, Nava-Ruiz C, Mendez-Armenta M (2017) Metallothionein in brain disorders. Oxidative Med Cell Longev 2017:5828056. CrossRefGoogle Scholar
  35. 35.
    Homma K, Fujisawa T, Tsuburaya N, Yamaguchi N, Kadowaki H, Takeda K, Nishitoh H, Matsuzawa A et al (2013) SOD1 as a molecular switch for initiating the homeostatic ER stress response under zinc deficiency. Mol Cell 52(1):75–86. CrossRefGoogle Scholar
  36. 36.
    Hershfinkel M (2018) The zinc sensing receptor, ZnR/GPR39, in health and disease. Int J Mol Sci 19(2).
  37. 37.
    Bosomworth HJ, Thornton JK, Coneyworth LJ, Ford D, Valentine RA (2012) Efflux function, tissue-specific expression and intracellular trafficking of the Zn transporter ZnT10 indicate roles in adult Zn homeostasis. Metallomics 4(8):771–779. CrossRefGoogle Scholar
  38. 38.
    Nitzan YB, Sekler I, Hershfinkel M, Moran A, Silverman WF (2002) Postnatal regulation of ZnT-1 expression in the mouse brain. Brain Res Dev Brain Res 137(2):149–157CrossRefGoogle Scholar
  39. 39.
    Hartl D, Irmler M, Romer I, Mader MT, Mao L, Zabel C, de Angelis MH, Beckers J et al (2008) Transcriptome and proteome analysis of early embryonic mouse brain development. Proteomics 8(6):1257–1265. CrossRefGoogle Scholar
  40. 40.
    Perez Y, Shorer Z, Liani-Leibson K, Chabosseau P, Kadir R, Volodarsky M, Halperin D, Barber-Zucker S et al (2017) SLC30A9 mutation affecting intracellular zinc homeostasis causes a novel cerebro-renal syndrome. Brain J Neurol 140:928–939. CrossRefGoogle Scholar
  41. 41.
    Palmiter RD, Cole TB, Quaife CJ, Findley SD (1996) ZnT-3, a putative transporter of zinc into synaptic vesicles. Proc Natl Acad Sci U S A 93(25):14934–14939CrossRefGoogle Scholar
  42. 42.
    Nitzan YB, Sekler I, Silverman WF (2004) Histochemical and histofluorescence tracing of chelatable zinc in the developing mouse. J Histochem Cytochem : Off J Histochem Soc 52(4):529–539. CrossRefGoogle Scholar
  43. 43.
    Czupryn A, Skangiel-Kramska J (1997) Distribution of synaptic zinc in the developing mouse somatosensory barrel cortex. J Comp Neurol 386(4):652–660CrossRefGoogle Scholar
  44. 44.
    Dufner-Beattie J, Huang ZL, Geiser J, Xu W, Andrews GK (2005) Generation and characterization of mice lacking the zinc uptake transporter ZIP3. Mol Cell Biol 25(13):5607–5615. CrossRefGoogle Scholar
  45. 45.
    Nishikawa M, Mori H, Hara M (2017) Analysis of ZIP (Zrt-, Irt-related protein) transporter gene expression in murine neural stem/progenitor cells. Environ Toxicol Pharmacol 53:81–88. CrossRefGoogle Scholar
  46. 46.
    Hogstrand C, Kille P, Nicholson RI, Taylor KM (2009) Zinc transporters and cancer: a potential role for ZIP7 as a hub for tyrosine kinase activation. Trends Mol Med 15(3):101–111. CrossRefGoogle Scholar
  47. 47.
    Grubman A, Lidgerwood GE, Duncan C, Bica L, Tan JL, Parker SJ, Caragounis A, Meyerowitz J et al (2014) Deregulation of subcellular biometal homeostasis through loss of the metal transporter, Zip7, in a childhood neurodegenerative disorder. Acta Neuropathol Commun 2:25. CrossRefGoogle Scholar
  48. 48.
    Chowanadisai W, Graham DM, Keen CL, Rucker RB, Messerli MA (2013) Neurulation and neurite extension require the zinc transporter ZIP12 (slc39a12). Proc Natl Acad Sci U S A 110(24):9903–9908. CrossRefGoogle Scholar
  49. 49.
    Lee SJ, Koh JY (2010) Roles of zinc and metallothionein-3 in oxidative stress-induced lysosomal dysfunction, cell death, and autophagy in neurons and astrocytes. Mol Brain 3(1):30. CrossRefGoogle Scholar
  50. 50.
    Gunes C, Heuchel R, Georgiev O, Muller KH, Lichtlen P, Bluthmann H, Marino S, Aguzzi A et al (1998) Embryonic lethality and liver degeneration in mice lacking the metal-responsive transcriptional activator MTF-1. EMBO J 17(10):2846–2854. CrossRefGoogle Scholar
  51. 51.
    Pardo CA, Xu Z, Borchelt DR, Price DL, Sisodia SS, Cleveland DW (1995) Superoxide dismutase is an abundant component in cell bodies, dendrites, and axons of motor neurons and in a subset of other neurons. Proc Natl Acad Sci U S A 92(4):954–958CrossRefGoogle Scholar
  52. 52.
    Bouron A, Oberwinkler J (2014) Contribution of calcium-conducting channels to the transport of zinc ions. Pflugers Arch 466(3):381–387. CrossRefGoogle Scholar
  53. 53.
    Bouron A, Altafaj X, Boisseau S, De Waard M (2005) A store-operated Ca2+ influx activated in response to the depletion of thapsigargin-sensitive Ca2+ stores is developmentally regulated in embryonic cortical neurons from mice. Brain Res Dev Brain Res 159(1):64–71CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Université Grenoble Alpes, CNRS, CEA, BIG-LCBMGrenobleFrance
  2. 2.ESRFGrenobleFrance
  3. 3.Institut de biologie de l’Ecole normale supérieure (IBENS), Ecole Normale Supérieure, CNRS, INSERMPSL Université ParisParisFrance
  4. 4.Laboratoire de Chimie et Biologie des MétauxUMR CNRS 5249, CEAGrenobleFrance

Personalised recommendations