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      Proteomic and cellular localisation studies suggest non‐tight junction cytoplasmic and nuclear roles for occludin in astrocytes

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          Abstract

          Occludin is a component of tight junctions, which are essential structural components of the blood–brain barrier. However, occludin is expressed in cells without tight junctions, implying additional functions. We determined the expression and localisation of occludin in astrocytes in cell culture and in human brain tissue, and sought novel binding partners using a proteomic approach. Expression was investigated by immunocytochemistry and immunoblotting in the 1321N1 astrocytoma cell line and ScienCell human primary astrocytes, and by immunohistochemistry in human autopsy brain tissue. Recombinant N‐ and C‐terminal occludin was used to pull‐down proteins from 1321N1 cell lysates and protein‐binding partners identified by mass spectrometry analysis. Occludin was expressed in both the cytoplasm and nucleus of astrocytes in vitro and in vivo. Mass spectrometry identified binding to nuclear and cytoplasmic proteins, particularly those related to RNA metabolism and nuclear function. Occludin is expressed in several subcellular compartments of brain cell‐types that do not form tight junctions and the expression patterns in cell culture reflect those in human brain tissue, indicating they are suitable model systems. Proteomic analysis suggests that occludin has novel functions in neuroepithelial cells that are unrelated to tight junction formation. Further research will establish the roles of these functions in both cellular physiology and in disease states.

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          Most cited references44

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          Claudin-based tight junctions are crucial for the mammalian epidermal barrier

          The tight junction (TJ) and its adhesion molecules, claudins, are responsible for the barrier function of simple epithelia, but TJs have not been thought to play an important role in the barrier function of mammalian stratified epithelia, including the epidermis. Here we generated claudin-1–deficient mice and found that the animals died within 1 d of birth with wrinkled skin. Dehydration assay and transepidermal water loss measurements revealed that in these mice the epidermal barrier was severely affected, although the layered organization of keratinocytes appeared to be normal. These unexpected findings prompted us to reexamine TJs in the epidermis of wild-type mice. Close inspection by immunofluorescence microscopy with an antioccludin monoclonal antibody, a TJ-specific marker, identified continuous TJs in the stratum granulosum, where claudin-1 and -4 were concentrated. The occurrence of TJs was also confirmed by ultrathin section EM. In claudin-1–deficient mice, claudin-1 appeared to have simply been removed from these TJs, leaving occludin-positive (and also claudin-4–positive) TJs. Interestingly, in the wild-type epidermis these occludin-positive TJs efficiently prevented the diffusion of subcutaneously injected tracer (∼600 D) toward the skin surface, whereas in the claudin-1–deficient epidermis the tracer appeared to pass through these TJs. These findings provide the first evidence that continuous claudin-based TJs occur in the epidermis and that these TJs are crucial for the barrier function of the mammalian skin.
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            Complex phenotype of mice lacking occludin, a component of tight junction strands.

            Occludin is an integral membrane protein with four transmembrane domains that is exclusively localized at tight junction (TJ) strands. Here, we describe the generation and analysis of mice carrying a null mutation in the occludin gene. Occludin -/- mice were born with no gross phenotype in the expected Mendelian ratios, but they showed significant postnatal growth retardation. Occludin -/- males produced no litters with wild-type females, whereas occludin -/- females produced litters normally when mated with wild-type males but did not suckle them. In occludin -/- mice, TJs themselves did not appear to be affected morphologically, and the barrier function of intestinal epithelium was normal as far as examined electrophysiologically. However, histological abnormalities were found in several tissues, i.e., chronic inflammation and hyperplasia of the gastric epithelium, calcification in the brain, testicular atrophy, loss of cytoplasmic granules in striated duct cells of the salivary gland, and thinning of the compact bone. These phenotypes suggested that the functions of TJs as well as occludin are more complex than previously supposed.
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              The molecular constituents of the blood-brain barrier.

              The blood-brain barrier (BBB) maintains the optimal microenvironment in the central nervous system (CNS) for proper brain function. The BBB comprises specialized CNS endothelial cells with fundamental molecular properties essential for the function and integrity of the BBB. The restrictive nature of the BBB hinders the delivery of therapeutics for many neurological disorders. In addition, recent evidence shows that BBB dysfunction can precede or hasten the progression of several neurological diseases. Despite the physiological significance of the BBB in health and disease, major discoveries of the molecular regulators of BBB formation and function have occurred only recently. This review highlights recent findings describing the molecular determinants and core cellular pathways that confer BBB properties on CNS endothelial cells.
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                Author and article information

                Contributors
                c.garwood@sheffield.ac.uk
                Journal
                Eur J Neurosci
                Eur. J. Neurosci
                10.1111/(ISSN)1460-9568
                EJN
                The European Journal of Neuroscience
                John Wiley and Sons Inc. (Hoboken )
                0953-816X
                1460-9568
                30 May 2018
                June 2018
                : 47
                : 12 ( doiID: 10.1111/ejn.2018.47.issue-12 )
                : 1444-1456
                Affiliations
                [ 1 ] Sheffield Institute for Translational Neuroscience University of Sheffield Sheffield UK
                [ 2 ] Department of Chemical and Biological Engineering University of Sheffield Sheffield UK
                Author notes
                [*] [* ] Correspondence

                Claire J. Garwood, Sheffield Institute for Translational Neuroscience, University of Sheffield, Sheffield, UK.

                Email: c.garwood@ 123456sheffield.ac.uk

                [†]

                Joint first author.

                Author information
                http://orcid.org/0000-0001-8611-177X
                Article
                EJN13933
                10.1111/ejn.13933
                6079634
                29738614
                8dd8cf11-8823-44a3-a7e9-1fed5bf9025d
                © 2018 The Authors. European Journal of Neuroscience published by Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

                This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

                History
                : 26 June 2017
                : 16 April 2018
                Page count
                Figures: 3, Tables: 4, Pages: 13, Words: 8447
                Funding
                Funded by: Alzheimer's Research UK
                Award ID: ARUK‐PhD2012‐7
                Award ID: PG2010‐5
                Funded by: Alzheimer's Society
                Award ID: AS#248
                Funded by: Consejo Nacional de Ciencia y Tecnologia (CONACYT)
                Funded by: Biotechnology and Biological Sciences Research Council UK (BBSRC)
                Award ID: BB/M012166/1
                Categories
                Research Report
                Neurosystems
                Custom metadata
                2.0
                ejn13933
                June 2018
                Converter:WILEY_ML3GV2_TO_NLMPMC version:version=5.4.4 mode:remove_FC converted:07.08.2018

                Neurosciences
                astrocytes,rna metabolism,tight junction proteins
                Neurosciences
                astrocytes, rna metabolism, tight junction proteins

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