NSL 07849 Neuroscience Letters, 129 (1991 ) 6-10 (c~ 1991 Elsevier Scientific Publishers Ireland Ltd. 0304-3940/91/$ 03.50 A D ON1S 030439409100383 Y The tight-junction-specific protein ZO-1 is a component of the human and rat blood-brain barriers Patricia M. Watson 1, James M. Anderson 2, Christina M. Vanltallie 2 and Susan R. Doctrow 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 immunocyto- chemistry recognizes a protein of about 200 kDa in rat brain microvessels by Western blot. These findings indicate that ZO-I is located at the inter- endothelial junctions of brain vasculature, implicating its importance as a component of the blood-brain barrier. The brain, unlike other organs, requires stringent pro- tection from fluctuations in the chemical composition of its tissue since such changes might have profound neuro- logical effects. The 'blood-brain barrier' ensures a con- trolled neurochemical environment by preventing blood- borne 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 blood- brain 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 mole- cules required for brain metabolic processes are deliv- ered 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 im- portant 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 bor- Correspondence: 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 compo- nent 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 de- scribed 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 in- teraction [10] was conducted using the Vectastain Elite ABC Kit (Vector Labs., Burlingame CA) essentially per the manufacturer's protocol. Dulbecco's phosphate-buf- fered saline (DPBS) with 1% (w/v) bovine serum albu- min (BSA, Sigma Chemical Co.) and 0.2% methyl ct-D- mannopyranoside was used as a blocking solution and antibody dilutions were prepared in DPBS with 1%