- Email: [email protected]
The tight-junction-specific protein ZO-l is a component of the human and rat blood-brain barriers
Neuroscience Letters, 129 (1991 ) 6-10 (c~ 1991 Elsevier Scientific Publishers Ireland Ltd. 0304-3940/91/$ 03.50 A D ON1S 030439409100383 Y NSL 07849
The tight-junction-specific protein ZO-1 is a component of the human and rat blood-brain barriers Patricia M. W a t s o n 1, James M. A n d e r s o n 2, Christina M. Vanltallie 2 a n d Susan R. D o c t r o w 1 IAIkermes, Inc., Cambridge, MA 02139 (U.S.A.) and 2Department of Medicine, Yale University School of Medieine, New Haven, CT06510 (U.S.A.) (Received 22 January 1991; Revised version received 2 April 1991; Accepted 3 April 1991)
Key words." Blood brain barrier; Tight junction; ZO-1; Endothelium Continuous tight junctions between vascular endothelial ceils, the principal anatomical basis for the blood-brain barrier, have been investigated functionally and morphologically but their molecular components have not been defined. This communication reports that the protein ZO-I, a specific constituent of epithelial tight junctions, is found in human and rat brain vasculature. ZO-l-positive immunocytochemical staining forms a tightly banded pattern outlining individual endothelial cells in blood vessels of the human cerebral cortex. Rat brain exhibits a similar staining of blood vessels as well as ZO-l-positive staining around individual epithelial cells of the choroid plexus. The antiserum used for immunocytochemistry recognizes a protein of about 200 kDa in rat brain microvessels by Western blot. These findings indicate that ZO-I is located at the interendothelial junctions of brain vasculature, implicating its importance as a component of the blood-brain barrier.
The brain, unlike other organs, requires stringent protection from fluctuations in the chemical composition of its tissue since such changes might have profound neurological effects. The 'blood-brain barrier' ensures a controlled neurochemical environment by preventing bloodborne agents from crossing into the brain parenchyma [9, 14]. Specialized vascular endothelial cells linked by continuous highly restrictive tight junctions are generally regarded as the primary anatomical basis for the bloodbrain barrier [15]. These tight junctions, in combination with specific transport systems located on the endothelial cell surface [13], endow the brain vasculature with its highly selective transport properties. That is, while molecules required for brain metabolic processes are delivered via specific transcellular mechanisms, the tight junctions prevent agents from entering indiscriminately through paracellular pathways. It is clear, therefore, that the interendothelial tight junction plays an extremely important role in preserving neurological function. While the brain endothelial tight junction has been investigated both functionally [15] and morphologically [4], its molecular components have not been defined. ZO-1 was the first protein to be identified as a specific component of the mammalian epithelial tight junction, or zonula occludens [1, 19]. In this communication, we report that ZO-I is localized at the interendothelial borCorrespondence: S.R. Doctrow, Alkermes, Inc., 26 Landsdowne Street, Cambridge, MA 02139, U.S.A., Fax: ( 1) 617-494-9263.
ders of brain vasculature, suggesting that it is a component of the highly specialized brain endothelial tight junction. Rabbit polyclonal antiserum to human ZO-1 against a recombinant glutathione S-transferase fusion protein was produced, affinity purified and characterized as described previously [2]. Concentrations of rabbit IgG in the affinity purified anti-human ZO-1 and pre-immune serum samples were estimated using a competitive enzyme-linked immunoassay with purified rabbit IgG (Sigma Chemical Co., St. Louis, MO) as a standard. Frozen sections (7-10/~m) were prepared from human cerebral cortex tissue, obtained I--2 h postmortem, in the laboratory of Dr. Miklos Palkovits (Semmelweis University, Budapest). Sections mounted on glass slides were fixed in cold acetone and shipped to our laboratory on dry ice. Brain samples from nine male subjects, with ages at death ranging from 35 to 72, were examined and exhibited no apparent differences in staining for ZO-I. Sections from normal adult female Sprague Dawley rat brain were prepared immediately postmortem. Immunocytochemistry utilizing the avidin-biotin interaction [10] was conducted using the Vectastain Elite ABC Kit (Vector Labs., Burlingame CA) essentially per the manufacturer's protocol. Dulbecco's phosphate-buffered saline (DPBS) with 1% (w/v) bovine serum albumin (BSA, Sigma Chemical Co.) and 0.2% methyl ct-Dmannopyranoside was used as a blocking solution and antibody dilutions were prepared in DPBS with 1%
Fig. 1. Localization of ZO-I in blood vessels of the human cerebral cortex. Immunoeytochemistry was conducted on sections from human cerebral cortex as described in the text. ZO-l-stained blood vessels, photosraphed under (A,B) 200 x (arrows denote endothelial cell nuclei) and (C) 400 x magnification. D: control staining with pre-immune serum, under 200 x magnification.
BSA. The final concentrations of primary antibody corresponded to about 1/tg rabbit IgG per ml for the antiZO-1 affinity purified preparation and 4/tg rabbit IgG per ml for pre-immune serum. The secondary antibody, biotinylated goat anti rabbit IgG (Vector Labs., Burlingame, CA), was used at a concentration of 7.5/tg/ml. After immunocytochemistry, sections were counterstained with 0.1% Nuclear fast red (AJP Scientific, Clifton, N J) and 1% Methyl green (Aldrich Chemical Co., Milwaukee, WI), coverslipped, and photographed under a Nikon Optiphot microscope. Immunocytochemistry revealed the presence of ZO-1 in the blood vessels of the human cerebral cortex (Fig. 1A--C). ZO-l-positive staining formed a continuous, tightly banded pattern in these vessels, consistent with the localization of the protein at the tight junctions between endothelial cells. Control sections stained with pre-immune serum did not exhibit this characteristic banded pattern (Fig. 1D), even at a 10-fold higher concentration of pre-immune serum (not shown). The staining pattern for ZO-1 in rat brain vasculature (Fig. 2A) was similar to that exhibited in human tissue. In the rat sections, ZO-1 staining was also found outlining the individual epithelial cells of the choroid plexus (Fig. 2B). Identical staining patterns in rat brain were observed using a monoclonal antibody to mouse ZO-1 [19] (not shown). To confirm the identity of the antigen detected by
immunocytochemistry, Western blot analysis was performed on samples from fresh rat brain. Rat cerebral cortical tissue was fractionated into microvesselenriched and microvessel-depleted samples as described previously [3, 20] except that, to minimize proteolysis, digestion with collagenase [3] was omitted and all procedures were conducted at 4°C in a preparation buffer of DPBS with 5 mM EDTA and 1 mM phenylmethylsulfonylfluoride (Sigma Chemical Co.), pH 7.4. Thus, the tissue was minced, homogenized, suspended in preparation buffer with 15% (w/v) Dextran and centrifuged. This yielded a pellet containing most of the microvessels [3, 20], as confirmed by its appearance under phase contrast microscopy. The bulk of the material, consisting of microvessel-depleted neural tissue [3, 20], floated in a dense pale-colored layer on top of the Dextran cushion. The two fractions were separately resuspended in preparation buffer and their protein concentrations were estimated with the BioRad protein assay. For Western blot analysis, an aliquot of each was dissolved in SDS-PAGE sample buffer [8] at 0.6 mg protein/ml. The samples (36 pg protein each), along with pre-stained molecular weight markers, were subjected to SDS-PAGE [8] on a 6% acrylamide gel. Electrophoresed proteins were then transferred from the gel to a Millipore Immobilon transfer membrane using a Millipore Milliblot-SDE apparatus. Immunoblotting was performed using Vectastain Elite ABC reagents essentially per the manufac-
Fig. 2. Localizationof ZO-1 in rat brain sections.Stainingwasconductedas for Fig. 1, exceptthat sectionsfromrat brainwereused.A: ZO-l-stained blood vessels,photographedunder200 x magnification.B: ZO-I stainingof the choroidplexusepithelium,under200 x magnification.
turer's protocol. The primary antibodies were used at the same dilutions as for immunocytochemistry. Western blot analysis (Fig. 3) showed that the microvessel-enriched sample contained a protein band of about 200 kDa molecular weight that was heavily stained by the anti-human ZO-1 serum. This molecular weight is in good agreement with that reported for ZO-1 [1]. A much fainter band of the same molecular weight, likely due to contaminating microvessels, was also visible in the microvessel-depleted sample blotted with the anti-human ZO-1 serum. The 200 kDa band was not visible in either sample blotted with pre-immune serum. The microvessel-enriched sample also exhibited two fainter protein bands at about 90 and 60 kDa that appeared to be stained by the anti-human ZO-1 serum. Although their identity is not yet known, these may be proteolysis products of ZO- 1. In summary, this study demonstrates that the tight junction protein ZO-1 is found in blood vessels of the human cerebral cortex and is specifically localized to the
1
2
3
4
-200 dmt
-92.5 -69 -46 -BPB
Fig. 3. Western blot analysis of samples from rat brain. Rat brain tissue was fractionated into microvessel-enricbed and -depleted samples and analyzed as described in the text to produce these Western blots: microvessel-depleted (lanes 1 and 2) and microvessel-enriched (3 and 4) samples blotted with either pre-immune serum (1 and 3) or antiserum raised against the human ZO-1 fusion protein (lanes 2 and 4). The positions of molecular weight markers are given in kDa. BPB denotes the position of the Bromophenol blue dye front.
junctional regions between endothelial cells. The immunocytochemistry results obtained with human brain tissue have been confirmed using brain sections from a second species, the adult rat. Results with the rat brain sections further indicate that ZO-1 is also localized between the epithelial cells of the choroid plexus. Two types of evidence help to confirm that ZO-1 is the antigen being detected in our immunocytochemical staining. First, an identical staining pattern is observed in rat brain stained with a monoclonal antibody that recognizes authentic ZO-1 from mouse liver [19] as that seen with the antibody to human ZO-1 fusion protein. Second, the major immunoreactive protein detected in rat microvessel-enriched samples by Western blot analysis appears to be ZO-1. These results implicate the tight junctional protein ZO1 as a component of the junctions comprising the human and rat blood-brain barriers. Because the blood-brain barrier is crucial to neurological function, it is important to begin to define its molecular composition and these findings represent a step toward that definition. The presence of ZO-1 at the borders of the choroid plexus epithelium in the rat suggests that ZO-1 may contribute to the blood-cerebrospinal barrier [16] as well. In considering these findings, it should be noted that observations made at the light microscopic level cannot unambiguously prove that a protein is localized at the endothelial zonula occludens. However, ultrastructural evidence supports a specific association of ZO-1 with the epithelial zonula occludens [19]. These data, as well as its distinct localization at the borders of endothelial cells, justify our interpretation that ZO-1 appears to be a component of the brain endothelial tight junction. While ZO-1 has been investigated most thoroughly in epithelial tissues and cell lines [1, 2, 18, 19], its presence has also been noted in endothelium of the mouse kidney and testis [19] and it appears to be expressed by cultured endothelial cells from several sources [2, 11]. This is consistent with the fact that even 'non-barrier' endothelial cells in other tissues have the capacity to form tight junctions, allowing them to exhibit the limited permeability barrier necessary for normal vascular function. In the brain, however, this vascular permeability barrier is dramatically enhanced due largely to the increased length, width and complexity of the inter-endothelial tight junctions [4, 6, 15]. This results in a vasculature that sustains a high electrical resistance across the vessel wall [7] and displays the unusually selective transport properties that are hallmarks of the blood-brain barrier [13]. Since, apparently, the presence of ZO- 1 is not alone predictive of the very low permeability of brain endothelial tight junctions, additional factors that might account for this property will need to be identified. For example, brain
10 vasculature m a y differ f r o m n o n - b r a i n v a s c u l a t u r e in having m o r e ZO-1 molecules p e r m i c r o n j u n c t i o n [18]. A n o t h e r factor t h a t m a y c o n t r i b u t e to the b r a i n j u n c t i o nal p e r m e a b i l i t y p r o p e r t i e s is c o v a l e n t m o d i f i c a t i o n o f ZO-1, since it is r e p o r t e d to be a p h o s p h o p r o t e i n [18]. In a d d i t i o n , the n a t u r e o f a s s o c i a t i o n s between ZO-1 a n d other, as yet unidentified, tight j u n c t i o n c o m p o n e n t s is likely to influence j u n c t i o n a l i m p e r m e a b i l i t y [17]. To date, one o t h e r tight junction-specific c o m p o n e n t , n a m e d cingulin [5], has been identified a n d its structural relationship to ZO-1 at the tight j u n c t i o n is as yet undefined. F o r the m o m e n t , o u r d e m o n s t r a t i o n t h a t ZO-1 is present at the b l o o d - b r a i n b a r r i e r implies that future research on its expression, regulation, a n d m o l e c u l a r associations should c o n t r i b u t e significantly to o u r u n d e r s t a n d i n g o f this crucial n e u r o p r o t e c t i v e entity. This research was s u p p o r t e d in p a r t by U.S. Public H e a l t h Service G r a n t R43 NS28256-01 (S.R.D.). J . M . A . is a Lucille P. M a r k e y Scholar a n d this w o r k was supp o r t e d in p a r t by a g r a n t from the Lucille P. M a r k e y C h a r i t a b l e Trust. W e t h a n k Dr. M i k l o s P a l k o v i t s o f Semmelweis University, B u d a p e s t for h u m a n b r a i n tissue sections.
1 Anderson, J.M., Stevenson, B.R., Jesaitis, L.A., Goodenough, D.A. and Mooseker, M.S., Characterization of ZO-I, a protein component of the tight junction from mouse liver and MadinDarby cainine kidney cells, J. Cell Biol. 106 (1988) 1141-1149. 2 Anderson, J.M., Vanltallie, C.M., Peterson, M.D., Stevenson, B.R., Carew, E.A. and Mooseker, M.S., ZO-1 mRNA and protein expression during tight junction assembly in Caco-2 cells, J. Cell Biol., 109 (1989) 1047-1056. 3 Bowman, P.D., Ennis, S.R., Rarey, K.E., Betz, A.L. and Goldstein, G.W., Brain microvessel endothelial cells in tissue culture: a model for study of blood-brain barrier permeability, Ann. Neurol., 14 (1983) 396-402. 4 Brightman, M.W. and Reese, T.S., Junctions between intimately apposed cell membranes in the vertebrate brain, J. Cell Biol., 40 (1969) 648~677.
5 Citi, S., Sabanay, H., Jakes, R., Geiger, B. and Kendrick-Jones, J. Cingulin, a new peripheral component of tight junctions, Nature, 333 (1988) 272-276. 6 Connel, C.J. and Mercer, K.L., Freeze-fracture appearance of the capillary endothelium in the cerebral cortex of mouse brain, Am. J. Anat., 140 (1974) 595-599. 7 Crone, C. and Olesen, S.P., Electrical resistance of brain microvascular endothelium, Brain Res., 241 (1982)49-55. 8 Doctrow, S.R. and Lowenstein, J.M., Adenosine and 5'-chloro-5'deoxyadenosine inhibit the phosphorylation of phosphatidylinositol and myosin light chain in calf aorta smooth muscle, J. Biol. Chem., 260 (1985) 3469-3476. 9 Goldstein, G.W. and Betz, A.L., The blood-brain barrier, Sci. Amer., (1986) 74-83. 10 Hsu, S.M., Raine, L. and Fanger, H., Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures, J. Histochem. Cytochem., 29 (1981) 577-580. 11 Merwin, J.R., Anderson, J.M., Kocher, O., Vanltallie, C.M. and Madri, J.A., Transforming growth factor beta 1 modulates extracellular matrix organization and cell-celljunctional complex formation during in vitro angiogenesis, J. Cell Physiol., 142 (1990) 117 128. 13 Pardridge, W.M., Recent advances in blood-brain barrier transport, Annu. Rev. Pharmacol. Toxicol., 28 (1988) 25 39. 14 Rapoport, S., Blood-Brain Barrier in Physiology and Medicine, Raven, New York, 1976, 316 pp. 15 Reese, T.S. and Karnovsky, M.J., Fine structural localization of a blood-brain barrier to exogenous peroxidase, J. Cell Biol., 34 (1967) 207 217. 16 Spector, R. and Johanson, C.E., The mammalian choroid plexus, Sci. Am., (1989) 68 74. 17 Stevenson, B.R., Anderson, J.M. and Bullivant, S., The epithelial tight junction: structure, function, and preliminary biochemical characterization, Mol. Cell Biochem., 83 (1988) 129-145. 18 Stevenson, B.R., Anderson, J.M., Goodenough, D.A. and Mooseker, M.S., Tight junction structure and ZO-1 content are identical in two strains of Madin-Darby canine kidney cells which differ in transepithelial resistance, J. Cell Biol., 107 (1988) 2401-2408. 19 Stevenson, B.R., Siliciano, J.D., Mooseker, M.S. and Goodenough, D.A., Identification of ZO-1: a high molecular weight polypeptide associated with the tight junction (zonula occludens) in a variety of epithelia, J. Cell Biol., 103 (1986) 755-766. 20 Triguero, D., Buciak, J. and Pardridge, W.M., Capillary depletion method for quantification of blood-brain barrier transport of circulating peptides and plasma proteins, J. Neurochem., 54 (1990) 188~1888.
The tight-junction-specific protein ZO-1 is a component of the human and rat blood-brain barriers Patricia M. W a t s o n 1, James M. A n d e r s o n 2, Christina M. Vanltallie 2 a n d Susan R. D o c t r o w 1 IAIkermes, Inc., Cambridge, MA 02139 (U.S.A.) and 2Department of Medicine, Yale University School of Medieine, New Haven, CT06510 (U.S.A.) (Received 22 January 1991; Revised version received 2 April 1991; Accepted 3 April 1991)
Key words." Blood brain barrier; Tight junction; ZO-1; Endothelium Continuous tight junctions between vascular endothelial ceils, the principal anatomical basis for the blood-brain barrier, have been investigated functionally and morphologically but their molecular components have not been defined. This communication reports that the protein ZO-I, a specific constituent of epithelial tight junctions, is found in human and rat brain vasculature. ZO-l-positive immunocytochemical staining forms a tightly banded pattern outlining individual endothelial cells in blood vessels of the human cerebral cortex. Rat brain exhibits a similar staining of blood vessels as well as ZO-l-positive staining around individual epithelial cells of the choroid plexus. The antiserum used for immunocytochemistry recognizes a protein of about 200 kDa in rat brain microvessels by Western blot. These findings indicate that ZO-I is located at the interendothelial junctions of brain vasculature, implicating its importance as a component of the blood-brain barrier.
The brain, unlike other organs, requires stringent protection from fluctuations in the chemical composition of its tissue since such changes might have profound neurological effects. The 'blood-brain barrier' ensures a controlled neurochemical environment by preventing bloodborne agents from crossing into the brain parenchyma [9, 14]. Specialized vascular endothelial cells linked by continuous highly restrictive tight junctions are generally regarded as the primary anatomical basis for the bloodbrain barrier [15]. These tight junctions, in combination with specific transport systems located on the endothelial cell surface [13], endow the brain vasculature with its highly selective transport properties. That is, while molecules required for brain metabolic processes are delivered via specific transcellular mechanisms, the tight junctions prevent agents from entering indiscriminately through paracellular pathways. It is clear, therefore, that the interendothelial tight junction plays an extremely important role in preserving neurological function. While the brain endothelial tight junction has been investigated both functionally [15] and morphologically [4], its molecular components have not been defined. ZO-1 was the first protein to be identified as a specific component of the mammalian epithelial tight junction, or zonula occludens [1, 19]. In this communication, we report that ZO-I is localized at the interendothelial borCorrespondence: S.R. Doctrow, Alkermes, Inc., 26 Landsdowne Street, Cambridge, MA 02139, U.S.A., Fax: ( 1) 617-494-9263.
ders of brain vasculature, suggesting that it is a component of the highly specialized brain endothelial tight junction. Rabbit polyclonal antiserum to human ZO-1 against a recombinant glutathione S-transferase fusion protein was produced, affinity purified and characterized as described previously [2]. Concentrations of rabbit IgG in the affinity purified anti-human ZO-1 and pre-immune serum samples were estimated using a competitive enzyme-linked immunoassay with purified rabbit IgG (Sigma Chemical Co., St. Louis, MO) as a standard. Frozen sections (7-10/~m) were prepared from human cerebral cortex tissue, obtained I--2 h postmortem, in the laboratory of Dr. Miklos Palkovits (Semmelweis University, Budapest). Sections mounted on glass slides were fixed in cold acetone and shipped to our laboratory on dry ice. Brain samples from nine male subjects, with ages at death ranging from 35 to 72, were examined and exhibited no apparent differences in staining for ZO-I. Sections from normal adult female Sprague Dawley rat brain were prepared immediately postmortem. Immunocytochemistry utilizing the avidin-biotin interaction [10] was conducted using the Vectastain Elite ABC Kit (Vector Labs., Burlingame CA) essentially per the manufacturer's protocol. Dulbecco's phosphate-buffered saline (DPBS) with 1% (w/v) bovine serum albumin (BSA, Sigma Chemical Co.) and 0.2% methyl ct-Dmannopyranoside was used as a blocking solution and antibody dilutions were prepared in DPBS with 1%
Fig. 1. Localization of ZO-I in blood vessels of the human cerebral cortex. Immunoeytochemistry was conducted on sections from human cerebral cortex as described in the text. ZO-l-stained blood vessels, photosraphed under (A,B) 200 x (arrows denote endothelial cell nuclei) and (C) 400 x magnification. D: control staining with pre-immune serum, under 200 x magnification.
BSA. The final concentrations of primary antibody corresponded to about 1/tg rabbit IgG per ml for the antiZO-1 affinity purified preparation and 4/tg rabbit IgG per ml for pre-immune serum. The secondary antibody, biotinylated goat anti rabbit IgG (Vector Labs., Burlingame, CA), was used at a concentration of 7.5/tg/ml. After immunocytochemistry, sections were counterstained with 0.1% Nuclear fast red (AJP Scientific, Clifton, N J) and 1% Methyl green (Aldrich Chemical Co., Milwaukee, WI), coverslipped, and photographed under a Nikon Optiphot microscope. Immunocytochemistry revealed the presence of ZO-1 in the blood vessels of the human cerebral cortex (Fig. 1A--C). ZO-l-positive staining formed a continuous, tightly banded pattern in these vessels, consistent with the localization of the protein at the tight junctions between endothelial cells. Control sections stained with pre-immune serum did not exhibit this characteristic banded pattern (Fig. 1D), even at a 10-fold higher concentration of pre-immune serum (not shown). The staining pattern for ZO-1 in rat brain vasculature (Fig. 2A) was similar to that exhibited in human tissue. In the rat sections, ZO-1 staining was also found outlining the individual epithelial cells of the choroid plexus (Fig. 2B). Identical staining patterns in rat brain were observed using a monoclonal antibody to mouse ZO-1 [19] (not shown). To confirm the identity of the antigen detected by
immunocytochemistry, Western blot analysis was performed on samples from fresh rat brain. Rat cerebral cortical tissue was fractionated into microvesselenriched and microvessel-depleted samples as described previously [3, 20] except that, to minimize proteolysis, digestion with collagenase [3] was omitted and all procedures were conducted at 4°C in a preparation buffer of DPBS with 5 mM EDTA and 1 mM phenylmethylsulfonylfluoride (Sigma Chemical Co.), pH 7.4. Thus, the tissue was minced, homogenized, suspended in preparation buffer with 15% (w/v) Dextran and centrifuged. This yielded a pellet containing most of the microvessels [3, 20], as confirmed by its appearance under phase contrast microscopy. The bulk of the material, consisting of microvessel-depleted neural tissue [3, 20], floated in a dense pale-colored layer on top of the Dextran cushion. The two fractions were separately resuspended in preparation buffer and their protein concentrations were estimated with the BioRad protein assay. For Western blot analysis, an aliquot of each was dissolved in SDS-PAGE sample buffer [8] at 0.6 mg protein/ml. The samples (36 pg protein each), along with pre-stained molecular weight markers, were subjected to SDS-PAGE [8] on a 6% acrylamide gel. Electrophoresed proteins were then transferred from the gel to a Millipore Immobilon transfer membrane using a Millipore Milliblot-SDE apparatus. Immunoblotting was performed using Vectastain Elite ABC reagents essentially per the manufac-
Fig. 2. Localizationof ZO-1 in rat brain sections.Stainingwasconductedas for Fig. 1, exceptthat sectionsfromrat brainwereused.A: ZO-l-stained blood vessels,photographedunder200 x magnification.B: ZO-I stainingof the choroidplexusepithelium,under200 x magnification.
turer's protocol. The primary antibodies were used at the same dilutions as for immunocytochemistry. Western blot analysis (Fig. 3) showed that the microvessel-enriched sample contained a protein band of about 200 kDa molecular weight that was heavily stained by the anti-human ZO-1 serum. This molecular weight is in good agreement with that reported for ZO-1 [1]. A much fainter band of the same molecular weight, likely due to contaminating microvessels, was also visible in the microvessel-depleted sample blotted with the anti-human ZO-1 serum. The 200 kDa band was not visible in either sample blotted with pre-immune serum. The microvessel-enriched sample also exhibited two fainter protein bands at about 90 and 60 kDa that appeared to be stained by the anti-human ZO-1 serum. Although their identity is not yet known, these may be proteolysis products of ZO- 1. In summary, this study demonstrates that the tight junction protein ZO-1 is found in blood vessels of the human cerebral cortex and is specifically localized to the
1
2
3
4
-200 dmt
-92.5 -69 -46 -BPB
Fig. 3. Western blot analysis of samples from rat brain. Rat brain tissue was fractionated into microvessel-enricbed and -depleted samples and analyzed as described in the text to produce these Western blots: microvessel-depleted (lanes 1 and 2) and microvessel-enriched (3 and 4) samples blotted with either pre-immune serum (1 and 3) or antiserum raised against the human ZO-1 fusion protein (lanes 2 and 4). The positions of molecular weight markers are given in kDa. BPB denotes the position of the Bromophenol blue dye front.
junctional regions between endothelial cells. The immunocytochemistry results obtained with human brain tissue have been confirmed using brain sections from a second species, the adult rat. Results with the rat brain sections further indicate that ZO-1 is also localized between the epithelial cells of the choroid plexus. Two types of evidence help to confirm that ZO-1 is the antigen being detected in our immunocytochemical staining. First, an identical staining pattern is observed in rat brain stained with a monoclonal antibody that recognizes authentic ZO-1 from mouse liver [19] as that seen with the antibody to human ZO-1 fusion protein. Second, the major immunoreactive protein detected in rat microvessel-enriched samples by Western blot analysis appears to be ZO-1. These results implicate the tight junctional protein ZO1 as a component of the junctions comprising the human and rat blood-brain barriers. Because the blood-brain barrier is crucial to neurological function, it is important to begin to define its molecular composition and these findings represent a step toward that definition. The presence of ZO-1 at the borders of the choroid plexus epithelium in the rat suggests that ZO-1 may contribute to the blood-cerebrospinal barrier [16] as well. In considering these findings, it should be noted that observations made at the light microscopic level cannot unambiguously prove that a protein is localized at the endothelial zonula occludens. However, ultrastructural evidence supports a specific association of ZO-1 with the epithelial zonula occludens [19]. These data, as well as its distinct localization at the borders of endothelial cells, justify our interpretation that ZO-1 appears to be a component of the brain endothelial tight junction. While ZO-1 has been investigated most thoroughly in epithelial tissues and cell lines [1, 2, 18, 19], its presence has also been noted in endothelium of the mouse kidney and testis [19] and it appears to be expressed by cultured endothelial cells from several sources [2, 11]. This is consistent with the fact that even 'non-barrier' endothelial cells in other tissues have the capacity to form tight junctions, allowing them to exhibit the limited permeability barrier necessary for normal vascular function. In the brain, however, this vascular permeability barrier is dramatically enhanced due largely to the increased length, width and complexity of the inter-endothelial tight junctions [4, 6, 15]. This results in a vasculature that sustains a high electrical resistance across the vessel wall [7] and displays the unusually selective transport properties that are hallmarks of the blood-brain barrier [13]. Since, apparently, the presence of ZO- 1 is not alone predictive of the very low permeability of brain endothelial tight junctions, additional factors that might account for this property will need to be identified. For example, brain
10 vasculature m a y differ f r o m n o n - b r a i n v a s c u l a t u r e in having m o r e ZO-1 molecules p e r m i c r o n j u n c t i o n [18]. A n o t h e r factor t h a t m a y c o n t r i b u t e to the b r a i n j u n c t i o nal p e r m e a b i l i t y p r o p e r t i e s is c o v a l e n t m o d i f i c a t i o n o f ZO-1, since it is r e p o r t e d to be a p h o s p h o p r o t e i n [18]. In a d d i t i o n , the n a t u r e o f a s s o c i a t i o n s between ZO-1 a n d other, as yet unidentified, tight j u n c t i o n c o m p o n e n t s is likely to influence j u n c t i o n a l i m p e r m e a b i l i t y [17]. To date, one o t h e r tight junction-specific c o m p o n e n t , n a m e d cingulin [5], has been identified a n d its structural relationship to ZO-1 at the tight j u n c t i o n is as yet undefined. F o r the m o m e n t , o u r d e m o n s t r a t i o n t h a t ZO-1 is present at the b l o o d - b r a i n b a r r i e r implies that future research on its expression, regulation, a n d m o l e c u l a r associations should c o n t r i b u t e significantly to o u r u n d e r s t a n d i n g o f this crucial n e u r o p r o t e c t i v e entity. This research was s u p p o r t e d in p a r t by U.S. Public H e a l t h Service G r a n t R43 NS28256-01 (S.R.D.). J . M . A . is a Lucille P. M a r k e y Scholar a n d this w o r k was supp o r t e d in p a r t by a g r a n t from the Lucille P. M a r k e y C h a r i t a b l e Trust. W e t h a n k Dr. M i k l o s P a l k o v i t s o f Semmelweis University, B u d a p e s t for h u m a n b r a i n tissue sections.
1 Anderson, J.M., Stevenson, B.R., Jesaitis, L.A., Goodenough, D.A. and Mooseker, M.S., Characterization of ZO-I, a protein component of the tight junction from mouse liver and MadinDarby cainine kidney cells, J. Cell Biol. 106 (1988) 1141-1149. 2 Anderson, J.M., Vanltallie, C.M., Peterson, M.D., Stevenson, B.R., Carew, E.A. and Mooseker, M.S., ZO-1 mRNA and protein expression during tight junction assembly in Caco-2 cells, J. Cell Biol., 109 (1989) 1047-1056. 3 Bowman, P.D., Ennis, S.R., Rarey, K.E., Betz, A.L. and Goldstein, G.W., Brain microvessel endothelial cells in tissue culture: a model for study of blood-brain barrier permeability, Ann. Neurol., 14 (1983) 396-402. 4 Brightman, M.W. and Reese, T.S., Junctions between intimately apposed cell membranes in the vertebrate brain, J. Cell Biol., 40 (1969) 648~677.
5 Citi, S., Sabanay, H., Jakes, R., Geiger, B. and Kendrick-Jones, J. Cingulin, a new peripheral component of tight junctions, Nature, 333 (1988) 272-276. 6 Connel, C.J. and Mercer, K.L., Freeze-fracture appearance of the capillary endothelium in the cerebral cortex of mouse brain, Am. J. Anat., 140 (1974) 595-599. 7 Crone, C. and Olesen, S.P., Electrical resistance of brain microvascular endothelium, Brain Res., 241 (1982)49-55. 8 Doctrow, S.R. and Lowenstein, J.M., Adenosine and 5'-chloro-5'deoxyadenosine inhibit the phosphorylation of phosphatidylinositol and myosin light chain in calf aorta smooth muscle, J. Biol. Chem., 260 (1985) 3469-3476. 9 Goldstein, G.W. and Betz, A.L., The blood-brain barrier, Sci. Amer., (1986) 74-83. 10 Hsu, S.M., Raine, L. and Fanger, H., Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures, J. Histochem. Cytochem., 29 (1981) 577-580. 11 Merwin, J.R., Anderson, J.M., Kocher, O., Vanltallie, C.M. and Madri, J.A., Transforming growth factor beta 1 modulates extracellular matrix organization and cell-celljunctional complex formation during in vitro angiogenesis, J. Cell Physiol., 142 (1990) 117 128. 13 Pardridge, W.M., Recent advances in blood-brain barrier transport, Annu. Rev. Pharmacol. Toxicol., 28 (1988) 25 39. 14 Rapoport, S., Blood-Brain Barrier in Physiology and Medicine, Raven, New York, 1976, 316 pp. 15 Reese, T.S. and Karnovsky, M.J., Fine structural localization of a blood-brain barrier to exogenous peroxidase, J. Cell Biol., 34 (1967) 207 217. 16 Spector, R. and Johanson, C.E., The mammalian choroid plexus, Sci. Am., (1989) 68 74. 17 Stevenson, B.R., Anderson, J.M. and Bullivant, S., The epithelial tight junction: structure, function, and preliminary biochemical characterization, Mol. Cell Biochem., 83 (1988) 129-145. 18 Stevenson, B.R., Anderson, J.M., Goodenough, D.A. and Mooseker, M.S., Tight junction structure and ZO-1 content are identical in two strains of Madin-Darby canine kidney cells which differ in transepithelial resistance, J. Cell Biol., 107 (1988) 2401-2408. 19 Stevenson, B.R., Siliciano, J.D., Mooseker, M.S. and Goodenough, D.A., Identification of ZO-1: a high molecular weight polypeptide associated with the tight junction (zonula occludens) in a variety of epithelia, J. Cell Biol., 103 (1986) 755-766. 20 Triguero, D., Buciak, J. and Pardridge, W.M., Capillary depletion method for quantification of blood-brain barrier transport of circulating peptides and plasma proteins, J. Neurochem., 54 (1990) 188~1888.