Clostridium perfringens epsilon toxin induces blood brain barrier permeability via caveolae-dependent transcytosis and requires expression of MAL

English version

Autoři: Jennifer R. Linden ^aff001; Claudia Flores ^aff001; Eric F. Schmidt ^aff002; Francisco A. Uzal ^aff003; Adam O. Michel ^aff004; Marissa Valenzuela ^aff001; Sebastian Dobrow ^aff001; Timothy K. Vartanian ^aff001
Působiště autorů: The Brain and Mind Research Institute and the Department of Neurology, Weill Cornell Medical College, New York, New York, United States of America ^aff001; Laboratory of Molecular Biology, The Rockefeller University, New York, New York, United States of America ^aff002; California Animal Health & Food Safety Laboratory System, San Bernardino Branch, University of California, Davis, San Bernardino, California, United States of America ^aff003; Laboratory of Comparative Pathology, Center of Comparative Medicine and Pathology, Memorial Sloan Kettering Cancer Center, The Rockefeller University, Weill Cornell Medicine, New York, New York, United States of America ^aff004
Vyšlo v časopise: Clostridium perfringens epsilon toxin induces blood brain barrier permeability via caveolae-dependent transcytosis and requires expression of MAL. PLoS Pathog 15(11): e32767. doi:10.1371/journal.ppat.1008014
Kategorie: Research Article
doi: https://doi.org/10.1371/journal.ppat.1008014

Souhrn

Clostridium perfringens epsilon toxin (ETX) is responsible for causing the economically devastating disease, enterotoxaemia, in livestock. It is well accepted that ETX causes blood brain barrier (BBB) permeability, however the mechanisms involved in this process are not well understood. Using in vivo and in vitro methods, we determined that ETX causes BBB permeability in mice by increasing caveolae-dependent transcytosis in brain endothelial cells. When mice are intravenously injected with ETX, robust ETX binding is observed in the microvasculature of the central nervous system (CNS) with limited to no binding observed in the vasculature of peripheral organs, indicating that ETX specifically targets CNS endothelial cells. ETX binding to CNS microvasculature is dependent on MAL expression, as ETX binding to CNS microvasculature of MAL-deficient mice was not detected. ETX treatment also induces extravasation of molecular tracers including 376Da fluorescein salt, 60kDA serum albumin, 70kDa dextran, and 155kDA IgG. Importantly, ETX-induced BBB permeability requires expression of both MAL and caveolin-1, as mice deficient in MAL or caveolin-1 did not exhibit ETX-induced BBB permeability. Examination of primary murine brain endothelial cells revealed an increase in caveolae in ETX-treated cells, resulting in dynamin and lipid raft-dependent vacuolation without cell death. ETX-treatment also results in a rapid loss of EEA1 positive early endosomes and accumulation of large, RAB7-positive late endosomes and multivesicular bodies. Based on these results, we hypothesize that ETX binds to MAL on the apical surface of brain endothelial cells, causing recruitment of caveolin-1, triggering caveolae formation and internalization. Internalized caveolae fuse with early endosomes which traffic to late endosomes and multivesicular bodies. We believe that these multivesicular bodies fuse basally, releasing their contents into the brain parenchyma.

Klíčová slova:

Cell death – Central nervous system – Coated pits – Endothelial cells – Fluorescence imaging – Toxins – Blood-brain barrier permeability assay – Endosomes

Zdroje

1. Zlokovic BV. The blood-brain barrier in health and chronic neurodegenerative disorders. Neuron. 2008;57(2):178–201. doi: 10.1016/j.neuron.2008.01.003 18215617.

2. Persidsky Y, Ramirez SH, Haorah J, Kanmogne GD. Blood-brain barrier: structural components and function under physiologic and pathologic conditions. J Neuroimmune Pharmacol. 2006;1(3):223–36. doi: 10.1007/s11481-006-9025-3 18040800.

3. Hawkins BT, Davis TP. The blood-brain barrier/neurovascular unit in health and disease. Pharmacol Rev. 2005;57(2):173–85. doi: 10.1124/pr.57.2.4 15914466.

4. Engelhardt B. Development of the blood-brain barrier. Cell Tissue Res. 2003;314(1):119–29. doi: 10.1007/s00441-003-0751-z 12955493.

5. Balabanov R, Dore-Duffy P. Role of the CNS microvascular pericyte in the blood-brain barrier. J Neurosci Res. 1998;53(6):637–44. doi: 10.1002/(SICI)1097-4547(19980915)53:6<637::AID-JNR1>3.0.CO;2-6 9753191.

6. Alvarez JI, Katayama T, Prat A. Glial influence on the blood brain barrier. Glia. 2013;61(12):1939–58. doi: 10.1002/glia.22575 24123158.

7. Abbott NJ, Friedman A. Overview and introduction: the blood-brain barrier in health and disease. Epilepsia. 2012;53 Suppl 6:1–6. doi: 10.1111/j.1528-1167.2012.03696.x 23134489.

8. Abbott NJ, Ronnback L, Hansson E. Astrocyte-endothelial interactions at the blood-brain barrier. Nat Rev Neurosci. 2006;7(1):41–53. doi: 10.1038/nrn1824 16371949.

9. Gloor SM, Wachtel M, Bolliger MF, Ishihara H, Landmann R, Frei K. Molecular and cellular permeability control at the blood-brain barrier. Brain Res Brain Res Rev. 2001;36(2–3):258–64. doi: 10.1016/s0165-0173(01)00102-3 11690623.

10. Tietz S, Engelhardt B. Brain barriers: Crosstalk between complex tight junctions and adherens junctions. J Cell Biol. 2015;209(4):493–506. doi: 10.1083/jcb.201412147 26008742.

11. Wolburg H, Lippoldt A. Tight junctions of the blood-brain barrier: development, composition and regulation. Vascul Pharmacol. 2002;38(6):323–37. 12529927.

12. De Bock M, Van Haver V, Vandenbroucke RE, Decrock E, Wang N, Leybaert L. Into rather unexplored terrain-transcellular transport across the blood-brain barrier. Glia. 2016;64(7):1097–123. doi: 10.1002/glia.22960 26852907.

13. Preston JE, Joan Abbott N, Begley DJ. Transcytosis of macromolecules at the blood-brain barrier. Adv Pharmacol. 2014;71:147–63. doi: 10.1016/bs.apha.2014.06.001 25307216.

14. Herve F, Ghinea N, Scherrmann JM. CNS delivery via adsorptive transcytosis. AAPS J. 2008;10(3):455–72. doi: 10.1208/s12248-008-9055-2 18726697.

15. Wioland L, Dupont JL, Bossu JL, Popoff MR, Poulain B. Attack of the nervous system by Clostridium perfringens Epsilon toxin: from disease to mode of action on neural cells. Toxicon. 2013;75:122–35. doi: 10.1016/j.toxicon.2013.04.003 23632158.

16. Popoff MR. Epsilon toxin: a fascinating pore-forming toxin. FEBS J. 2011;278(23):4602–15. doi: 10.1111/j.1742-4658.2011.08145.x 21535407.

17. Alves GG, Machado de Avila RA, Chavez-Olortegui CD, Lobato FC. Clostridium perfringens epsilon toxin: the third most potent bacterial toxin known. Anaerobe. 2014;30:102–7. doi: 10.1016/j.anaerobe.2014.08.016 25234332.

18. Stiles BG, Barth G, Barth H, Popoff MR. Clostridium perfringens epsilon toxin: a malevolent molecule for animals and man? Toxins (Basel). 2013;5(11):2138–60. doi: 10.3390/toxins5112138 24284826.

19. Sayeed S, Fernandez-Miyakawa ME, Fisher DJ, Adams V, Poon R, Rood JI, et al. Epsilon-toxin is required for most Clostridium perfringens type D vegetative culture supernatants to cause lethality in the mouse intravenous injection model. Infect Immun. 2005;73(11):7413–21. doi: 10.1128/IAI.73.11.7413-7421.2005 16239541.

20. Garcia JP, Adams V, Beingesser J, Hughes ML, Poon R, Lyras D, et al. Epsilon toxin is essential for the virulence of Clostridium perfringens type D infection in sheep, goats, and mice. Infect Immun. 2013;81(7):2405–14. doi: 10.1128/IAI.00238-13 23630957.

21. Hatheway CL. Toxigenic clostridia. Clin Microbiol Rev. 1990;3(1):66–98. doi: 10.1128/cmr.3.1.66 2404569.

22. Rood JI. Virulence genes of Clostridium perfringens. Annu Rev Microbiol. 1998;52:333–60. doi: 10.1146/annurev.micro.52.1.333 9891801.

23. Hassan KA, Elbourne LD, Tetu SG, Melville SB, Rood JI, Paulsen IT. Genomic analyses of Clostridium perfringens isolates from five toxinotypes. Res Microbiol. 2015;166(4):255–63. doi: 10.1016/j.resmic.2014.10.003 25445567.

24. Petit L, Gibert M, Popoff MR. Clostridium perfringens: toxinotype and genotype. Trends Microbiol. 1999;7(3):104–10. doi: 10.1016/s0966-842x(98)01430-9 10203838.

25. Bokori-Brown M, Savva CG, Fernandes da Costa SP, Naylor CE, Basak AK, Titball RW. Molecular basis of toxicity of Clostridium perfringens epsilon toxin. FEBS J. 2011;278(23):4589–601. doi: 10.1111/j.1742-4658.2011.08140.x 21518257.

26. Cole AR, Gibert M, Popoff M, Moss DS, Titball RW, Basak AK. Clostridium perfringens epsilon-toxin shows structural similarity to the pore-forming toxin aerolysin. Nat Struct Mol Biol. 2004;11(8):797–8. doi: 10.1038/nsmb804 15258571.

27. Rood JI, Adams V, Lacey J, Lyras D, McClane BA, Melville SB, et al. Expansion of the Clostridium perfringens toxin-based typing scheme. Anaerobe. 2018. doi: 10.1016/j.anaerobe.2018.04.011 29866424.

28. Navarro MA, McClane BA, Uzal FA. Mechanisms of Action and Cell Death Associated with Clostridium perfringens Toxins. Toxins (Basel). 2018;10(5). doi: 10.3390/toxins10050212 29786671.

29. Rumah KR, Linden J, Fischetti VA, Vartanian T. Isolation of Clostridium perfringens type B in an individual at first clinical presentation of multiple sclerosis provides clues for environmental triggers of the disease. PLoS One. 2013;8(10):e76359. doi: 10.1371/journal.pone.0076359 24146858.

30. Wagley S, Bokori-Brown M, Morcrette H, Malaspina A, D’Arcy C, Gnanapavan S, et al. Evidence of Clostridium perfringens epsilon toxin associated with multiple sclerosis. Mult Scler. 2018:1352458518767327. doi: 10.1177/1352458518767327 29681209.

31. Freedman JC, Li J, Uzal FA, McClane BA. Proteolytic processing and activation of Clostridium perfringens epsilon toxin by caprine small intestinal contents. MBio. 2014;5(5):e01994–14. doi: 10.1128/mBio.01994-14 25336460.

32. Goldstein J, Morris WE, Loidl CF, Tironi-Farinati C, McClane BA, Uzal FA, et al. Clostridium perfringens epsilon toxin increases the small intestinal permeability in mice and rats. PLoS One. 2009;4(9):e7065. doi: 10.1371/journal.pone.0007065 19763257.

33. Fernandez Miyakawa ME, Uzal FA. The early effects of Clostridium perfringens type D epsilon toxin in ligated intestinal loops of goats and sheep. Vet Res Commun. 2003;27(3):231–41. doi: 10.1023/a:1023348708599 12777097.

34. Losada-Eaton DM, Uzal FA, Fernandez Miyakawa ME. Clostridium perfringens epsilon toxin is absorbed from different intestinal segments of mice. Toxicon. 2008;51(7):1207–13. doi: 10.1016/j.toxicon.2008.02.008 18457853.

35. Tamai E, Ishida T, Miyata S, Matsushita O, Suda H, Kobayashi S, et al. Accumulation of Clostridium perfringens epsilon-toxin in the mouse kidney and its possible biological significance. Infect Immun. 2003;71(9):5371–5. doi: 10.1128/IAI.71.9.5371-5375.2003 12933886.

36. Nagahama M, Sakurai J. Distribution of labeled Clostridium perfringens epsilon toxin in mice. Toxicon. 1991;29(2):211–7. doi: 10.1016/0041-0101(91)90105-z 2048139.

37. Soler-Jover A, Blasi J, Gomez de Aranda I, Navarro P, Gibert M, Popoff MR, et al. Effect of epsilon toxin-GFP on MDCK cells and renal tubules in vivo. J Histochem Cytochem. 2004;52(7):931–42. doi: 10.1369/jhc.4A6254.2004 15208360.

38. Soler-Jover A, Dorca J, Popoff MR, Gibert M, Saura J, Tusell JM, et al. Distribution of Clostridium perfringens epsilon toxin in the brains of acutely intoxicated mice and its effect upon glial cells. Toxicon. 2007;50(4):530–40. doi: 10.1016/j.toxicon.2007.04.025 17572464.

39. Buxton D. The use of an immunoperoxidase technique to investigate by light and electron microscopy the sites of binding of Clostridium welchii type-D epsilon toxin in mice. J Med Microbiol. 1978;11(3):289–92. doi: 10.1099/00222615-11-3-289 210278.

40. Fernandez Miyakawa ME, Ibarra CA, Uzal FA. In vitro effects of Clostridium perfringens type D epsilon toxin on water and ion transport in ovine and caprine intestine. Anaerobe. 2003;9(3):145–9. doi: 10.1016/S1075-9964(03)00069-6 16887702.

41. Losada-Eaton DM, Fernandez-Miyakawa ME. Clostridium perfringens epsilon toxin inhibits the gastrointestinal transit in mice. Res Vet Sci. 2010;89(3):404–8. doi: 10.1016/j.rvsc.2010.04.006 20434186.

42. Uzal FA, Kelly WR, Morris WE, Bermudez J, Baison M. The pathology of peracute experimental Clostridium perfringens type D enterotoxemia in sheep. J Vet Diagn Invest. 2004;16(5):403–11. doi: 10.1177/104063870401600506 15460322.

43. Uzal FA, Kelly WR, Morris WE, Assis RA. Effects of intravenous injection of Clostridium perfringens type D epsilon toxin in calves. J Comp Pathol. 2002;126(1):71–5. doi: 10.1053/jcpa.2001.0514 11814324.

44. Garcia JP, Giannitti F, Finnie JW, Manavis J, Beingesser J, Adams V, et al. Comparative neuropathology of ovine enterotoxemia produced by Clostridium perfringens type D wild-type strain CN1020 and its genetically modified derivatives. Vet Pathol. 2015;52(3):465–75. doi: 10.1177/0300985814540543 24964921.

45. Fernandez-Miyakawa ME, Sayeed S, Fisher DJ, Poon R, Adams V, Rood JI, et al. Development and application of an oral challenge mouse model for studying Clostridium perfringens type D infection. Infect Immun. 2007;75(9):4282–8. doi: 10.1128/IAI.00562-07 17562765.

46. Worthington RW, Mulders MS. Effect of Clostridium perfringens epsilon toxin on the blood brain barrier of mice. Onderstepoort J Vet Res. 1975;42(1):25–7. 171606.

47. Finnie JW. Histopathological changes in the brain of mice given Clostridium perfringens type D epsilon toxin. J Comp Pathol. 1984;94(3):363–70. doi: 10.1016/0021-9975(84)90024-0 6088597.

48. Finnie JW. Ultrastructural changes in the brain of mice given Clostridium perfringens type D epsilon toxin. J Comp Pathol. 1984;94(3):445–52. doi: 10.1016/0021-9975(84)90031-8 6088599.

49. Finnie JW, Hajduk P. An immunohistochemical study of plasma albumin extravasation in the brain of mice after the administration of Clostridium perfringens type D epsilon toxin. Aust Vet J. 1992;69(10):261–2. doi: 10.1111/j.1751-0813.1992.tb09878.x 1445075.

50. Ghabriel MN, Zhu C, Reilly PL, Blumbergs PC, Manavis J, Finnie JW. Toxin-induced vasogenic cerebral oedema in a rat model. Acta Neurochir Suppl. 2000;76:231–6. doi: 10.1007/978-3-7091-6346-7_47 11450014.

51. Zhu C, Ghabriel MN, Blumbergs PC, Reilly PL, Manavis J, Youssef J, et al. Clostridium perfringens prototoxin-induced alteration of endothelial barrier antigen (EBA) immunoreactivity at the blood-brain barrier (BBB). Exp Neurol. 2001;169(1):72–82. doi: 10.1006/exnr.2001.7652 11312560.

52. Dorca-Arevalo J, Soler-Jover A, Gibert M, Popoff MR, Martin-Satue M, Blasi J. Binding of epsilon-toxin from Clostridium perfringens in the nervous system. Vet Microbiol. 2008;131(1–2):14–25. doi: 10.1016/j.vetmic.2008.02.015 18406080.

53. Finnie JW, Manavis J, Chidlow G. Loss of endothelial barrier antigen immunoreactivity as a marker of Clostridium perfringens type D epsilon toxin-induced microvascular damage in rat brain. J Comp Pathol. 2014;151(2–3):153–6. doi: 10.1016/j.jcpa.2014.04.010 24973988.

54. Finnie JW, Manavis J, Casson RJ, Chidlow G. Retinal microvascular damage and vasogenic edema produced by Clostridium perfringens type D epsilon toxin in rats. J Vet Diagn Invest. 2014;26(3):470–2. doi: 10.1177/1040638714530127 24741023.

55. Dorca-Arevalo J, Pauillac S, Diaz-Hidalgo L, Martin-Satue M, Popoff MR, Blasi J. Correlation between in vitro cytotoxicity and in vivo lethal activity in mice of epsilon toxin mutants from Clostridium perfringens. PLoS One. 2014;9(7):e102417. doi: 10.1371/journal.pone.0102417 25013927.

56. Finnie JW, Blumbergs PC, Manavis J. Neuronal damage produced in rat brains by Clostridium perfringens type D epsilon toxin. J Comp Pathol. 1999;120(4):415–20. doi: 10.1053/jcpa.1998.0289 10208737.

57. Miyamoto O, Minami J, Toyoshima T, Nakamura T, Masada T, Nagao S, et al. Neurotoxicity of Clostridium perfringens epsilon-toxin for the rat hippocampus via the glutamatergic system. Infect Immun. 1998;66(6):2501–8. 9596708.

58. Lonchamp E, Dupont JL, Wioland L, Courjaret R, Mbebi-Liegeois C, Jover E, et al. Clostridium perfringens epsilon toxin targets granule cells in the mouse cerebellum and stimulates glutamate release. PLoS One. 2010;5(9). doi: 10.1371/journal.pone.0013046 20941361.

59. Cases M, Llobet A, Terni B, Gomez de Aranda I, Blanch M, Doohan B, et al. Acute Effect of Pore-Forming Clostridium perfringens epsilon-Toxin on Compound Action Potentials of Optic Nerve of Mouse. eNeuro. 2017;4(4). doi: 10.1523/ENEURO.0051-17.2017 28798954.

60. Morris WE, Goldstein J, Redondo LM, Cangelosi A, Geoghegan P, Brocco M, et al. Clostridium perfringens epsilon toxin induces permanent neuronal degeneration and behavioral changes. Toxicon. 2017;130:19–28. doi: 10.1016/j.toxicon.2017.02.019 28237716.

61. Wioland L, Dupont JL, Doussau F, Gaillard S, Heid F, Isope P, et al. Epsilon toxin from Clostridium perfringens acts on oligodendrocytes without forming pores, and causes demyelination. Cell Microbiol. 2015;17(3):369–88. doi: 10.1111/cmi.12373 25287162.

62. Linden JR, Ma Y, Zhao B, Harris JM, Rumah KR, Schaeren-Wiemers N, et al. Clostridium perfringens Epsilon Toxin Causes Selective Death of Mature Oligodendrocytes and Central Nervous System Demyelination. MBio. 2015;6(3):e02513. doi: 10.1128/mBio.02513-14 26081637.

63. Freedman JC, McClane BA, Uzal FA. New insights into Clostridium perfringens epsilon toxin activation and action on the brain during enterotoxemia. Anaerobe. 2016;41:27–31. doi: 10.1016/j.anaerobe.2016.06.006 27321761.

64. Uzal FA, Kelly WR. Effects of the intravenous administration of Clostridium perfringens type D epsilon toxin on young goats and lambs. J Comp Pathol. 1997;116(1):63–71. doi: 10.1016/s0021-9975(97)80044-8 9076601.

65. Uzal FA, Pasini MI, Olaechea FV, Robles CA, Elizondo A. An outbreak of enterotoxaemia caused by Clostridium perfringens type D in goats in Patagonia. Vet Rec. 1994;135(12):279–80. doi: 10.1136/vr.135.12.279 7817506.

66. Finnie JW. Neurological disorders produced by Clostridium perfringens type D epsilon toxin. Anaerobe. 2004;10(2):145–50. doi: 10.1016/j.anaerobe.2003.08.003 16701511.

67. Finnie JW, Manavis J, Blumbergs PC. Aquaporin-4 in acute cerebral edema produced by Clostridium perfringens type D epsilon toxin. Vet Pathol. 2008;45(3):307–9. doi: 10.1354/vp.45-3-307 18487486.

68. Robertson SL, Li J, Uzal FA, McClane BA. Evidence for a prepore stage in the action of Clostridium perfringens epsilon toxin. PLoS One. 2011;6(7):e22053. doi: 10.1371/journal.pone.0022053 21814565.

69. Petit L, Gibert M, Gourch A, Bens M, Vandewalle A, Popoff MR. Clostridium perfringens epsilon toxin rapidly decreases membrane barrier permeability of polarized MDCK cells. Cell Microbiol. 2003;5(3):155–64. 12614459.

70. Kang J, Gao J, Yao W, Kang L, Gao S, Yang H, et al. F199E substitution reduced toxicity of Clostridium perfringens epsilon toxin by depriving the receptor binding capability. Hum Vaccin Immunother. 2017;13(7):1598–608. doi: 10.1080/21645515.2017.1303022 28304231.

71. Chassin C, Bens M, de Barry J, Courjaret R, Bossu JL, Cluzeaud F, et al. Pore-forming epsilon toxin causes membrane permeabilization and rapid ATP depletion-mediated cell death in renal collecting duct cells. Am J Physiol Renal Physiol. 2007;293(3):F927–37. doi: 10.1152/ajprenal.00199.2007 17567938.

72. Ivie SE, Fennessey CM, Sheng J, Rubin DH, McClain MS. Gene-trap mutagenesis identifies mammalian genes contributing to intoxication by Clostridium perfringens epsilon-toxin. PLoS One. 2011;6(3):e17787. doi: 10.1371/journal.pone.0017787 21412435.

73. Fennessey CM, Sheng J, Rubin DH, McClain MS. Oligomerization of Clostridium perfringens epsilon toxin is dependent upon caveolins 1 and 2. PLoS One. 2012;7(10):e46866. doi: 10.1371/journal.pone.0046866 23056496.

74. Rumah KR, Ma Y, Linden JR, Oo ML, Anrather J, Schaeren-Wiemers N, et al. The Myelin and Lymphocyte Protein MAL Is Required for Binding and Activity of Clostridium perfringens epsilon-Toxin. PLoS Pathog. 2015;11(5):e1004896. doi: 10.1371/journal.ppat.1004896 25993478.

75. Blanch M, Dorca-Arevalo J, Not A, Cases M, Gomez de Aranda I, Martinez Yelamos A, et al. The cytotoxicity of Epsilon toxin from Clostridium perfringens on lymphocytes is mediated by MAL protein expression. Mol Cell Biol. 2018. doi: 10.1128/MCB.00086-18 29987189.

76. Daneman R, Zhou L, Agalliu D, Cahoy JD, Kaushal A, Barres BA. The mouse blood-brain barrier transcriptome: a new resource for understanding the development and function of brain endothelial cells. PLoS One. 2010;5(10):e13741. doi: 10.1371/journal.pone.0013741 21060791.

77. Zhang Y, Chen K, Sloan SA, Bennett ML, Scholze AR, O’Keeffe S, et al. An RNA-sequencing transcriptome and splicing database of glia, neurons, and vascular cells of the cerebral cortex. J Neurosci. 2014;34(36):11929–47. doi: 10.1523/JNEUROSCI.1860-14.2014 25186741.

78. Doyle JP, Dougherty JD, Heiman M, Schmidt EF, Stevens TR, Ma G, et al. Application of a translational profiling approach for the comparative analysis of CNS cell types. Cell. 2008;135(4):749–62. doi: 10.1016/j.cell.2008.10.029 19013282.

79. Nectow AR, Moya MV, Ekstrand MI, Mousa A, McGuire KL, Sferrazza CE, et al. Rapid Molecular Profiling of Defined Cell Types Using Viral TRAP. Cell Rep. 2017;19(3):655–67. doi: 10.1016/j.celrep.2017.03.048 28423326.

80. Nakajima M, Gorlich A, Heintz N. Oxytocin modulates female sociosexual behavior through a specific class of prefrontal cortical interneurons. Cell. 2014;159(2):295–305. doi: 10.1016/j.cell.2014.09.020 25303526.

81. Dorca-Arevalo J, Martin-Satue M, Blasi J. Characterization of the high affinity binding of epsilon toxin from Clostridium perfringens to the renal system. Vet Microbiol. 2012;157(1–2):179–89. doi: 10.1016/j.vetmic.2011.12.020 22264388.

82. Schaeren-Wiemers N, Bonnet A, Erb M, Erne B, Bartsch U, Kern F, et al. The raft-associated protein MAL is required for maintenance of proper axon—glia interactions in the central nervous system. J Cell Biol. 2004;166(5):731–42. doi: 10.1083/jcb.200406092 15337780.

83. Nitta T, Hata M, Gotoh S, Seo Y, Sasaki H, Hashimoto N, et al. Size-selective loosening of the blood-brain barrier in claudin-5-deficient mice. J Cell Biol. 2003;161(3):653–60. doi: 10.1083/jcb.200302070 12743111.

84. Liu J, Jin X, Liu KJ, Liu W. Matrix metalloproteinase-2-mediated occludin degradation and caveolin-1-mediated claudin-5 redistribution contribute to blood-brain barrier damage in early ischemic stroke stage. J Neurosci. 2012;32(9):3044–57. doi: 10.1523/JNEUROSCI.6409-11.2012 22378877.

85. Liu J, Weaver J, Jin X, Zhang Y, Xu J, Liu KJ, et al. Nitric Oxide Interacts with Caveolin-1 to Facilitate Autophagy-Lysosome-Mediated Claudin-5 Degradation in Oxygen-Glucose Deprivation-Treated Endothelial Cells. Mol Neurobiol. 2016;53(9):5935–47. doi: 10.1007/s12035-015-9504-8 26515186.

86. Nag S, Venugopalan R, Stewart DJ. Increased caveolin-1 expression precedes decreased expression of occludin and claudin-5 during blood-brain barrier breakdown. Acta Neuropathol. 2007;114(5):459–69. doi: 10.1007/s00401-007-0274-x 17687559.

87. Stamatovic SM, Keep RF, Wang MM, Jankovic I, Andjelkovic AV. Caveolae-mediated internalization of occludin and claudin-5 during CCL2-induced tight junction remodeling in brain endothelial cells. J Biol Chem. 2009;284(28):19053–66. doi: 10.1074/jbc.M109.000521 19423710.

88. Parton RG, Simons K. The multiple faces of caveolae. Nat Rev Mol Cell Biol. 2007;8(3):185–94. doi: 10.1038/nrm2122 17318224.

89. Williams TM, Lisanti MP. The caveolin proteins. Genome Biol. 2004;5(3):214. doi: 10.1186/gb-2004-5-3-214 15003112.

90. Schnitzer JE, Oh P, Pinney E, Allard J. Filipin-sensitive caveolae-mediated transport in endothelium: reduced transcytosis, scavenger endocytosis, and capillary permeability of select macromolecules. J Cell Biol. 1994;127(5):1217–32. doi: 10.1083/jcb.127.5.1217 7525606.

91. Schubert W, Frank PG, Razani B, Park DS, Chow CW, Lisanti MP. Caveolae-deficient endothelial cells show defects in the uptake and transport of albumin in vivo. J Biol Chem. 2001;276(52):48619–22. doi: 10.1074/jbc.C100613200 11689550.

92. Frank PG, Woodman SE, Park DS, Lisanti MP. Caveolin, caveolae, and endothelial cell function. Arterioscler Thromb Vasc Biol. 2003;23(7):1161–8. doi: 10.1161/01.ATV.0000070546.16946.3A 12689915.

93. Lajoie P, Nabi IR. Lipid rafts, caveolae, and their endocytosis. Int Rev Cell Mol Biol. 2010;282:135–63. doi: 10.1016/S1937-6448(10)82003-9 20630468.

94. Liu P, Rudick M, Anderson RG. Multiple functions of caveolin-1. J Biol Chem. 2002;277(44):41295–8. doi: 10.1074/jbc.R200020200 12189159.

95. Nabi IR, Le PU. Caveolae/raft-dependent endocytosis. J Cell Biol. 2003;161(4):673–7. doi: 10.1083/jcb.200302028 12771123.

96. Sverdlov M, Shajahan AN, Minshall RD. Tyrosine phosphorylation-dependence of caveolae-mediated endocytosis. J Cell Mol Med. 2007;11(6):1239–50. doi: 10.1111/j.1582-4934.2007.00127.x 18205698.

97. Linden JR, Telesford K, Shetty S, Winokour P, Haigh S, E. C-M, et al. A Novel Panel of Rabbit Monoclonal Antibodies and Their Diverse Applications Including Inhibition of Clostridium perfringens Epsilon Toxin Oligomerization. Antibodies. 2018;7(4)(37). Epub October 25, 2018.

98. Ferrarezi MC, Curci VC, Cardoso TC. Cellular vacuolation and mitochondrial-associated factors induced by Clostridium perfringens epsilon toxin detected using acoustic flow cytometry. Anaerobe. 2013;24:55–9. doi: 10.1016/j.anaerobe.2013.09.009 24076036.

99. Oh P, McIntosh DP, Schnitzer JE. Dynamin at the neck of caveolae mediates their budding to form transport vesicles by GTP-driven fission from the plasma membrane of endothelium. J Cell Biol. 1998;141(1):101–14. doi: 10.1083/jcb.141.1.101 9531551.

100. Nagahama M, Itohayashi Y, Hara H, Higashihara M, Fukatani Y, Takagishi T, et al. Cellular vacuolation induced by Clostridium perfringens epsilon-toxin. FEBS J. 2011;278(18):3395–407. doi: 10.1111/j.1742-4658.2011.08263.x 21781280.

101. Anton O, Batista A, Millan J, Andres-Delgado L, Puertollano R, Correas I, et al. An essential role for the MAL protein in targeting Lck to the plasma membrane of human T lymphocytes. J Exp Med. 2008;205(13):3201–13. doi: 10.1084/jem.20080552 19064697.

102. Caduff J, Sansano S, Bonnet A, Suter U, Schaeren-Wiemers N. Characterization of GFP-MAL expression and incorporation in rafts. Microsc Res Tech. 2001;52(6):645–55. doi: 10.1002/jemt.1049 11276117.

103. Magal LG, Yaffe Y, Shepshelovich J, Aranda JF, de Marco Mdel C, Gaus K, et al. Clustering and lateral concentration of raft lipids by the MAL protein. Mol Biol Cell. 2009;20(16):3751–62. doi: 10.1091/mbc.E09-02-0142 19553470.

104. Marazuela M, Acevedo A, Adrados M, Garcia-Lopez MA, Alonso MA. Expression of MAL, an integral protein component of the machinery for raft-mediated pical transport, in human epithelia. J Histochem Cytochem. 2003;51(5):665–74. doi: 10.1177/002215540305100512 12704214.

105. Martin-Belmonte F, Arvan P, Alonso MA. MAL mediates apical transport of secretory proteins in polarized epithelial Madin-Darby canine kidney cells. J Biol Chem. 2001;276(52):49337–42. doi: 10.1074/jbc.M106882200 11673461.

106. Martin-Belmonte F, Puertollano R, Millan J, Alonso MA. The MAL proteolipid is necessary for the overall apical delivery of membrane proteins in the polarized epithelial Madin-Darby canine kidney and fischer rat thyroid cell lines. Mol Biol Cell. 2000;11(6):2033–45. doi: 10.1091/mbc.11.6.2033 10848627.

107. Puertollano R, Martin-Belmonte F, Millan J, de Marco MC, Albar JP, Kremer L, et al. The MAL proteolipid is necessary for normal apical transport and accurate sorting of the influenza virus hemagglutinin in Madin-Darby canine kidney cells. J Cell Biol. 1999;145(1):141–51. doi: 10.1083/jcb.145.1.141 10189374.

108. Zacchetti D, Peranen J, Murata M, Fiedler K, Simons K. VIP17/MAL, a proteolipid in apical transport vesicles. FEBS Lett. 1995;377(3):465–9. doi: 10.1016/0014-5793(95)01396-2 8549777.

109. Dorca-Arevalo J, Blanch M, Pradas M, Blasi J. Epsilon toxin from Clostridium perfringens induces cytotoxicity in FRT thyroid epithelial cells. Anaerobe. 2018. doi: 10.1016/j.anaerobe.2018.05.011 29895394.

110. Etxaniz A, Gonzalez-Bullon D, Martin C, Ostolaza H. Membrane Repair Mechanisms against Permeabilization by Pore-Forming Toxins. Toxins (Basel). 2018;10(6). doi: 10.3390/toxins10060234 29890730.

111. Gonzalez MR, Bischofberger M, Freche B, Ho S, Parton RG, van der Goot FG. Pore-forming toxins induce multiple cellular responses promoting survival. Cell Microbiol. 2011;13(7):1026–43. doi: 10.1111/j.1462-5822.2011.01600.x 21518219.

112. Husmann M, Beckmann E, Boller K, Kloft N, Tenzer S, Bobkiewicz W, et al. Elimination of a bacterial pore-forming toxin by sequential endocytosis and exocytosis. FEBS Lett. 2009;583(2):337–44. doi: 10.1016/j.febslet.2008.12.028 19101547.

113. Romero M, Keyel M, Shi G, Bhattacharjee P, Roth R, Heuser JE, et al. Intrinsic repair protects cells from pore-forming toxins by microvesicle shedding. Cell Death Differ. 2017;24(5):798–808. doi: 10.1038/cdd.2017.11 28186501.

114. Lisanti MP, Scherer PE, Tang Z, Sargiacomo M. Caveolae, caveolin and caveolin-rich membrane domains: a signalling hypothesis. Trends Cell Biol. 1994;4(7):231–5. doi: 10.1016/0962-8924(94)90114-7 14731661.

115. Zimnicka AM, Husain YS, Shajahan AN, Sverdlov M, Chaga O, Chen Z, et al. Src-dependent phosphorylation of caveolin-1 Tyr-14 promotes swelling and release of caveolae. Mol Biol Cell. 2016;27(13):2090–106. doi: 10.1091/mbc.E15-11-0756 27170175.

116. Heiman M, Schaefer A, Gong S, Peterson JD, Day M, Ramsey KE, et al. A translational profiling approach for the molecular characterization of CNS cell types. Cell. 2008;135(4):738–48. doi: 10.1016/j.cell.2008.10.028 19013281.

117. Saunders NR, Dziegielewska KM, Mollgard K, Habgood MD. Markers for blood-brain barrier integrity: how appropriate is Evans blue in the twenty-first century and what are the alternatives? Front Neurosci. 2015;9:385. doi: 10.3389/fnins.2015.00385 26578854.

118. Stewart PA, Farrell CR, Farrell CL, Hayakawa E. Horseradish peroxidase retention and washout in blood-brain barrier lesions. J Neurosci Methods. 1992;41(1):75–84. doi: 10.1016/0165-0270(92)90125-w 1578903.

119. Hoffmann A, Bredno J, Wendland M, Derugin N, Ohara P, Wintermark M. High and Low Molecular Weight Fluorescein Isothiocyanate (FITC)-Dextrans to Assess Blood-Brain Barrier Disruption: Technical Considerations. Transl Stroke Res. 2011;2(1):106–11. doi: 10.1007/s12975-010-0049-x 21423333.

120. Welser-Alves JV, Boroujerdi A, Milner R. Isolation and culture of primary mouse brain endothelial cells. Methods Mol Biol. 2014;1135:345–56. doi: 10.1007/978-1-4939-0320-7_28 24510877.