Histochemistry and Cell Biology

, Volume 122, Issue 1, pp 51–59 | Cite as

Immunolocalization of tight junction proteins in the adult and developing human brain

  • Daniela VirgintinoEmail author
  • Mariella Errede
  • David Robertson
  • Carmen Capobianco
  • Francesco Girolamo
  • Antonella Vimercati
  • Mirella Bertossi
  • Luisa Roncali
Original Paper


The formation of endothelial tight junctions (TJs) is crucial in blood-brain barrier (BBB) differentiation, and the expression and targeting of TJ-associated proteins mark the beginning of BBB functions. Using confocal microscopy, this study analyzed endothelial TJs in adult human cerebral cortex and the fetal telencephalon and leptomeninges in order to compare the localization of two TJ-associated transmembrane proteins, occludin and claudin-5. In the arterioles and microvessels of adult brain, occludin and claudin-5 form continuous bands of endothelial immunoreactivity. During fetal development, occludin and claudin-5 immunoreactivity is first detected as a diffuse labeling of endothelial cytoplasm. Later, at 14 weeks, the immunosignal for both proteins shifts from the cytoplasm to the interface of adjacent endothelial cells, forming a linear, widely discontinuous pattern of immunoreactivity that achieves an adult-like appearance within a few weeks. These results demonstrate that occludin and claudin-5 expression is an early event in human brain development, followed shortly by assembly of both proteins at the junctional areas. This incremental process suggests more rapid establishment of the human BBB, consistent with its specific function of creating a suitable environment for neuron differentiation and neurite outgrowth during neocortical histogenesis.


Occludin Claudin-5 Tight junctions Blood-brain barrier Human brain development 



The authors are grateful to Prof. S. Nag for critical reading of the manuscript. The authors also thank Ms M.V.C. Pragnell, BA, for linguistic help and Ms M. Ambrosi for excellent technical assistance. This work was supported by grants from the Ministero dell’Istruzione, dell’Università e della Ricerca (M.I.U.R.) (to L.R.).

Supplementary material

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  1. Abbott NJ (2002) Astrocyte-endothelial interactions and blood-brain barrier permeability. J Anat 200:629–638CrossRefPubMedGoogle Scholar
  2. Balabanov R, Dore-Duffy P (1998) Role of the CNS microvascular pericyte in the blood-brain barrier. J Neurosci Res 53:637–644CrossRefPubMedGoogle Scholar
  3. Bär T (1980) The vascular system of the cerebral cortex. Adv Anat Embryol Cell Biol 59:1–62Google Scholar
  4. Bauer H-C, Bauer H (2000) Neural induction of the blood-brain barrier: still an enigma. Cell Mol Neurobiol 20:13–28CrossRefPubMedGoogle Scholar
  5. Bauer H-C, Bauer H, Lametschwandtner A, Amberger A, Ruiz PL, Steiner M (1993) Neovascularization and the appearance of morphological characteristics of the blood-brain barrier in the embryonic mouse central nervous system. Dev Brain Res 75:269–278CrossRefGoogle Scholar
  6. Bauer H, Sonnleitner U, Lametschwandtner A, Steiner M, Adam H, Bauer H-C (1995) Ontogenic expression of the erythroid-type glucose transporter (GLUT 1) in the telencephalon of the mouse: correlation to the tightening of the blood-brain barrier. Dev Brain Res 86:317–325CrossRefGoogle Scholar
  7. Bertossi M, Virgintino D, Errede M, Roncali L (1999) Immunohistochemical and ultrastructural characterization of cortical plate microvasculature in the human fetus telencephalon. Microvasc Res 58:49–61CrossRefPubMedGoogle Scholar
  8. Bertossi M, Girolamo F, Errede M, Benagiano V, Virgintino D, Roncali L (2002) Developmental changes of HT7 expression in the microvessels of the chick embryo brain. Anat Embryol 205:229–233CrossRefPubMedGoogle Scholar
  9. Cancilla PA, Bready J, Berliner J (1993) Brain endothelial-astrocyte interactions. In: Pardridge WM (ed) The blood-brain barrier. Cellular and molecular biology. Raven, New York, pp 25–46Google Scholar
  10. Cassella JP, Lawrenson JG, Allt G, Firth JA (1996) Ontogeny of four blood-brain barrier markers: an immunocytochemical comparison of pial and cerebral microvessels. J Anat 189:407–415PubMedGoogle Scholar
  11. Dobrogowska DH, Vorbrodt AW (1999) Quantitative immunocytochemical study of blood-brain barrier glucose transporter (GLUT-1) in four regions of mouse brain. J Histochem Cytochem 47:1021–1029PubMedGoogle Scholar
  12. Dziegielewska KM, Evans CAN, Malinowska DH, Møllgård K, Reynolds JM, Reynolds ML, Sauders NR (1979) Studies of the development of brain barrier systems to lipid insoluble molecules in fetal sheep. J Physiol 292:207–231PubMedGoogle Scholar
  13. Fanning AS, Jameson BJ, Jesaitis LA, Anderson JM (1998) The tight junction protein ZO-1 establishes a link between the transmembrane protein occludin and the actin cytoskeleton. J Biol Chem 273:29745–29753PubMedGoogle Scholar
  14. Furuse M, Tetsuaki H, Itoh M, Nagafuchi A, Yonemura S, Tsukita S, Tsukita S (1993) Occludin: a novel integral membrane protein localizing at tight junctions. J Cell Biol 123:1777–1788PubMedGoogle Scholar
  15. Furuse M, Fujita K, Hiiragi T, Fujimoto K, Tsukita S (1998) Claudin-1 and -2: novel integral membrane proteins localizing at tight junctions with no sequence similarity to occludin. J Cell Biol 141:1539–1550PubMedGoogle Scholar
  16. Furuse M, Sasaki H, Tsukita S (1999) Manner of interaction of heterogeneous claudin species within and between tight junction strands. J Cell Biol 147:891–903PubMedGoogle Scholar
  17. Gerhart DZ, Le Vasseur RJ, Broderius MA, Drewes LR (1989) Glucose transporter localization in brain using light and electron immunocytochemistry. J Neurosci Res 22:464–472Google Scholar
  18. González-Mariscal L, Betanzos A, Nava P, Jaramillo BE (2003) Tight junction proteins. Prog Biophys Mol Biol 81:1–44CrossRefPubMedGoogle Scholar
  19. Goodenough DA (1999) Plugging the leaks. Proc Natl Acad Sci U S A 96:319–321CrossRefPubMedGoogle Scholar
  20. Hirase T, Staddon JM, Saitou M, Ando-Akatsuka Y, Itoh M, Furuse M, Fujimoto K, Tsukita S, Rubin LL (1997) Occludin as a possible determinant of tight junction permeability in endothelial cells. J Cell Sci 110:1603–1613PubMedGoogle Scholar
  21. Itoh M, Sasaki H, Furuse M, Ozaki H, Kita T, Tsukita S (2001) Junctional adhesion molecule (JAM) binds to PAR-3: a possible mechanism for the recruitment of PAR-3 to tight junctions. J Cell Biol 154:491–497CrossRefPubMedGoogle Scholar
  22. Ivanov AI, Nusrat A, Parkos CA (2004) Endocytosis of epithelial apical junctional proteins by a clathrin-mediated pathway into a unique storage compartment. Mol Biol Cell 15:176–188CrossRefPubMedGoogle Scholar
  23. Janzer RC, Raff MC (1987) Astrocytes induce blood-brain barrier properties in endothelial cells. Nature 325:253–257PubMedGoogle Scholar
  24. Kacem K, LaCombe P, Seylaz J, Bonvento G (1998) Structural organization of the perivascular astrocyte endfeet and their relationship with the endothelial glucose transporter: a confocal microscopy study. Glia 23:1–10CrossRefPubMedGoogle Scholar
  25. Kniesel U, Wolburg H (2000) Tight junctions of the blood-brain barrier. Cell Mol Neurobiol 20:57–76CrossRefPubMedGoogle Scholar
  26. Kniesel U, Risau W, Wolburg H (1996) Development of blood-brain barrier tight junctions in the rat cortex. Dev Brain Res 96:229–240CrossRefGoogle Scholar
  27. Lacaz-Vieira F, Jaeger MM, Farshori P, Kachar B (1999) Small synthetic peptides homologous to segments of the first external loop of occludin impair tight junction resealing. J Membr Biol 168:289–297CrossRefPubMedGoogle Scholar
  28. Laterra J, Guerin C, Goldstein GW (1990) Astrocyte induce neural microvascular endothelial cells to form capillary-like structure in vitro. J Cell Physiol 144:204–215PubMedGoogle Scholar
  29. Liebner S, Kniesel U, Kalbacher H, Wolburg H (2000) Correlation of tight junction morphology with the expression of tight junction proteins in blood-brain barrier endothelial cells. Eur J Cell Biol 79:707–717PubMedGoogle Scholar
  30. Lippoldt A, Liebner S, Andbjer B, Kalbacher H, Wolburg H, Haller H, Fuxe K (2000a) Organization of choroid plexus epithelial and endothelial cell tight junctions and regulation of claudin-1, -2 and -5 expression by protein kinase C. Neuroreport 11:1427–1431PubMedGoogle Scholar
  31. Lippoldt A, Kniesel U, Liebner S, Kalbacher H, Kirsch T, Wolburg H, Haller H (2000b) Structural alterations of tight junctions are associated with loss of polarity in stroke-prone spontaneously hypertensive rat blood-brain barrier endothelial cells. Brain Res 885:251–261CrossRefPubMedGoogle Scholar
  32. Marín-Padilla M (1987) Embryogenesis of the early vascularization of the central nervous system. In: Yasargil MG (ed) Microneurosurgery, vol 3. Thieme, Stuttgart, pp 23–44Google Scholar
  33. Marín-Padilla M (1995) Prenatal development of fibrous (white matter), protoplasmic (gray matter), and layer I astrocytes in the human cerebral cortex: a Golgi study. J Comp Neurol 357:554–572PubMedGoogle Scholar
  34. Matter K, Balda S (2003) Holey barrier: claudins and the regulation of brain endothelial permeability. J Cell Biol 161:459–460CrossRefPubMedGoogle Scholar
  35. Meyer G, Schaaps JP, Moreau L, Goffinet AM (2000) Embryonic and early fetal development of the human neocortex. J Neurosci 20:1858–1868PubMedGoogle Scholar
  36. Møllgård K, Saunders NR (1986) The development of the human blood-brain and blood-CSF barriers. Neuropathol Appl Neurobiol 12:337–358PubMedGoogle Scholar
  37. Morcos Y, Hosie MJ, Bauer H-C, Chan-Ling T (2001) Immunolocalization of occludin and claudin-1 to tight junctions in intact CNS vessels of mammalian retina. J Neurocytol 30:107–123CrossRefPubMedGoogle Scholar
  38. Morita K, Furuse M, Fujimoto K, Tsukita S (1999a) Claudin multigene family encoding four-transmembrane domain protein components of tight junction strands. Proc Natl Acad Sci U S A 96:511–516PubMedGoogle Scholar
  39. Morita K, Sasaki H, Furuse M, Tsukita S (1999b) Endothelial claudin: claudin-5/TMVCF constitutes tight junction strands in endothelial cells. J Cell Biol 147:185–194CrossRefPubMedGoogle Scholar
  40. Nag S (2003) Morphology and molecular properties of cellular components of normal cerebral vessels. In: Nag S (ed) The blood-brain barrier. Biology and research protocols. Humana, Totowa, NJ, pp 3–36Google Scholar
  41. Nagy Z, Peters H, Huttner I (1984) Fracture faces of cell junctions in cerebral endothelium during normal and hyperosmotic conditions. Lab Invest 50:313–322PubMedGoogle Scholar
  42. Nitta T, Hata M, Gotoh S, Seo Y, Sasaki H, Hashimoto N, Furuse M, Tsukita S (2003) Size-selective loosening of the blood-brain barrier in claudin-5-deficient mice. J Cell Biol 161:653–660Google Scholar
  43. Norman MG, O’Kusky JR (1986) The growth and development of microvasculature in human cerebral cortex. J Neuropathol Exp Neurol 45:222–232Google Scholar
  44. Nusrat A, Turner JR, Madara JL (2000) Molecular physiology and pathophysiology of tight junctions. IV. Regulation of tight junctions by extracellular stimuli: nutrients, cytokines, and immune cells. Am J Physiol Gastrointest Liver Physiol 279:G851–G857PubMedGoogle Scholar
  45. Papadopoulos MC, Saadoun S, Woodrow CJ, Davies DC, Costa-Martins P, Moss RF, Krishna S, Bell BA (2001) Occludin expression in microvessels of neoplastic and non-neoplastic human brain. Neuropathol Appl Neurobiol 27:384–395CrossRefPubMedGoogle Scholar
  46. Pekny M, Stanness KA, Eliasson C, Betsholtz C, Janigro D (1998) Impaired induction of blood-brain barrier properties in aortic endothelial cells by astrocytes from GFAP-deficient mice. Glia 22:390–400CrossRefPubMedGoogle Scholar
  47. Plate KH (1999) Mechanisms of angiogenesis in the brain. J Neuropathol Exp Neurol 58:313–320PubMedGoogle Scholar
  48. Plumb J, McQuaid S, Mirakhur M, Kirk J (2002) Abnormal endothelial tight junctions in active lesions and normal-appearing white matter in multiple sclerosis. Brain Pathol 12:154–169PubMedGoogle Scholar
  49. Risau W (1993) Development of the vascular system of organs and tissues. In: Schaper W, Schaper J (eds) Collateral circulation. Kluwer Academic, Norwell, pp 17–28Google Scholar
  50. Risau W (1997) Mechanisms of angiogenesis. Nature 386:671–674PubMedGoogle Scholar
  51. Roncali L, Nico B, Ribatti D, Bertossi M, Mancini L (1986) Microscopical and ultrastructural investigation on the development of the blood-brain barrier in the chick embryo optic tectum. Acta Neuropathol 70:193–201PubMedGoogle Scholar
  52. Rubin LL, Staddon JM (1999) The cell biology of the blood-brain barrier. Annu Rev Neurosci 22:11–28PubMedGoogle Scholar
  53. Saitou M, Furuse M, Sasaki H, Schulzke J-D, Fromm M, Takano H, Noda T, Tsukita S (2000) Complex phenotype of mice lacking occludin, a component of tight junction strands. Am Soc Cell Biol 11:4131–4142Google Scholar
  54. Sasaki H, Matsui C, Furuse K, Mimori-Kiyosue Y, Furuse M, Tsukita S (2003) Dynamic behavior of paired claudin strands within apposing plasma membranes. Proc Natl Acad Sci U S A 100:3971–3976CrossRefPubMedGoogle Scholar
  55. Saunders NR (1977) Ontogeny of the blood-brain barrier. Exp Eye Res S25:523–550Google Scholar
  56. Savettieri G, Di Liegro I, Catania C, Licata L, Pitarresi GL, D’Agostino S, Schiera G, De Caro V, Giandalia G, Giannola LI, Cestelli A (2000) Neurons and ECM regulate occludin localization in brain endothelial cells. Neuroreport 11:1081–1084PubMedGoogle Scholar
  57. Smart IHM, Dehay C, Giroud P, Berland M, Kennedy H (2002) Unique morphological features of the proliferative zones and postmitotic compartments of the neural epithelium giving rise to striate and extrastriate cortex in the monkey. Cereb Cortex 12:37–53CrossRefPubMedGoogle Scholar
  58. Sobue K, Yamamoto N, Yoneda K, Hodgson ME, Yamashiro K, Tsuruoka N, Tsuda T, Katsuya H, Miura Y, Asai K, Kato T (1999) Induction of blood-brain barrier properties in immortalized bovine brain endothelial cells by astrocytic factors. Neurosci Res 35:155–164CrossRefPubMedGoogle Scholar
  59. Tagami M, Yamagata K, Fujino H, Kubota A, Nara Y, Yamori Y (1992) Morphological differentiation of endothelial cells co-cultured with astrocytes on type-I or type-IV collagen. Cell Tissue Res 268:225–232PubMedGoogle Scholar
  60. Tilling T, Korte D, Hoheisel D, Galla HJ (1998) Basement membrane proteins influence brain capillary endothelial barrier function in vitro. J Neurochem 71:1151–1157PubMedGoogle Scholar
  61. Tsukita S, Furuse M (1999) Occludin and claudins in tight-junction strands: leading or supporting players? Trends Cell Biol 9:268–273PubMedGoogle Scholar
  62. Tsukita S, Furuse M, Itoh M (2001) Multifunctional strands in tight junctions. Nat Rev Mol Cell Biol 2:285–293CrossRefPubMedGoogle Scholar
  63. Virgintino D, Robertson D, Monaghan P, Errede M, Ambrosi G, Roncali L, Bertossi M (1998a) Glucose transporter GLUT1 localization in human foetus telencephalon. Neurosci Lett 256:147–150CrossRefPubMedGoogle Scholar
  64. Virgintino D, Maiorano E, Errede M, Vimercati A, Greco P, Selvaggi L, Roncali L, Bertossi M (1998b) Astroglia-microvessel relationship in the developing human telencephalon. Int J Dev Biol 42:1165–1168PubMedGoogle Scholar
  65. Virgintino D, Robertson D, Benagiano V, Errede M, Bertossi M, Ambrosi G, Roncali L (2000) Immunogold cytochemistry of the blood-brain barrier glucose transporter GLUT1 and endogenous albumin in the developing human brain. Dev Brain Res 123:95–101CrossRefGoogle Scholar
  66. Vorbrodt A, Dobrogowska DH (1994) Immunocytochemical evaluation of blood-brain barrier to endogenous albumin in adult, newborn and aged mice. Folia Histochem Cytobiol 32:63–70PubMedGoogle Scholar
  67. Vorbrodt A, Dobrogowska DH (2003) Molecular anatomy of intercellular junctions in brain endothelial and epithelial barriers: electron microscopist’s view. Brain Res Rev 42:221–242CrossRefPubMedGoogle Scholar
  68. Vorbrodt A, Dobrogowska DH, Tarnawski M (2001) Immunogold study of interendothelial junction-associated and glucose transporter proteins during postnatal maturation of the mouse blood-brain barrier. J Neurocytol 30:705–716CrossRefPubMedGoogle Scholar
  69. Willis CL, Nolan CC, Reith SN, Lister T, Prior MJW, Guerin CJ, Mavroudis G, Ray DE (2004) Focal astrocyte loss is followed by microvascular damage, with subsequent repair of the blood-brain barrier in the apparent absence of direct astrocytic contact. Glia 45:325–337CrossRefPubMedGoogle Scholar
  70. Wolburg H, Lippoldt A (2002) Tight junctions of the blood-brain barrier: development, composition and regulation. Vascul Pharmacol 38:323–337CrossRefPubMedGoogle Scholar
  71. Wolburg H, Wolburg-Buchholz K, Kraus J, Rascher-Eggstein G, Liebner S, Hamm S, Duffner F, Grote E-H, Risau W, Engelhardt B (2003) Localization of claudin-3 in tight junctions of the blood-brain barrier is selectively lost during experimental autoimmune encephalomyelitis and human glioblastoma multiforme. Acta Neuropathol 105:586–592PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  • Daniela Virgintino
    • 1
    Email author
  • Mariella Errede
    • 1
  • David Robertson
    • 2
  • Carmen Capobianco
    • 3
  • Francesco Girolamo
    • 1
  • Antonella Vimercati
    • 4
  • Mirella Bertossi
    • 5
  • Luisa Roncali
    • 1
  1. 1.Department of Human Anatomy and HistologyUniversity of Bari School of MedicineBariItaly
  2. 2.Breakthrough CentreInstitute of Cancer ResearchLondonUK
  3. 3.Department of Emergency and Organ TransplantationUniversity of Bari School of MedicineBariItaly
  4. 4.Institute of Obstetric and Gynecology IIUniversity of Bari School of MedicineBariItaly
  5. 5.Department of Biomedical SciencesUniversity of Foggia School of MedicineFoggiaItaly

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