The long range goal of this proposal is to understand the role that tight junctions play in regulating the outer blood-retinal barrier. Malfunction of the blood-retinal barrier is implicated in many retinopathies. This proposal examines how immature, leaky, tight junctions are converted into a low permeability form, and how this process is regulated by the neural retina. During development, tight junctions of the retinal pigment epithelium (RPE) undergoes this conversion to form the outer blood-retinal barrier. Tight junctions are semi-selective, and that selectivity can be regulated. Consequently, epithelial tight junctions vary in their permeability to solutes of different size and different charge. This laboratory devised an innovative culture model that shows permeability is partially regulated by secretions of the neural retina. Those experiments led to the novel finding that junction selectivity involves multiple mechanisms. It is now imperative to define these mechanisms to understand the different regulatory pathways that affect tight junctions. This proposal addresses three hypotheses to define these mechanisms and to begin dissecting the regulatory pathways: 1) Tight junctions use size and charge discrimination to regulate diffusion across them, and these mechanisms are regulated independently by distinct pathways. This will be tested electrophysiologically and by the permeation of charged and uncharged monosaccharides. With a clear understanding of these mechanisms, it becomes possible to examine how they are regulated. 2) The phosphorylation of the tight junction proteins, occluding and 7H6, is part of regulatory mechanism that modulates specific functions of tight junctions. This will be tested by using 32/P orthophosphate to metabolically label RPE that is cultured in presence or absence of stimulatory retinal factors. These experiments will dissect the role of phosphorylation in different regulatory pathways. This will be tested with dominant negative mutants that are designed to compete with the native protein for regulatory factors. The mutations will be expressed by a retroviral expression system in primary cell culture. The regulation of the tight junction has proved to be enigmatic. Recent advances in the development of reagents coupled with this novel culture model will advance our understanding of this essential structure of the outer blood-retinal barrier.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
2R01EY008694-07
Application #
2766113
Study Section
Visual Sciences C Study Section (VISC)
Project Start
1993-07-01
Project End
2001-11-30
Budget Start
1998-12-01
Budget End
1999-11-30
Support Year
7
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Yale University
Department
Surgery
Type
Schools of Medicine
DUNS #
082359691
City
New Haven
State
CT
Country
United States
Zip Code
06520
Sun, Ru; Peng, Shaomin; Chen, Xiang et al. (2008) Diffusible retinal secretions regulate the expression of tight junctions and other diverse functions of the retinal pigment epithelium. Mol Vis 14:2237-62
Cong, Lidan; Sun, Dawei; Zhang, Zhongyu et al. (2008) A novel rabbit model for studying RPE transplantation. Invest Ophthalmol Vis Sci 49:4115-25
Rizzolo, Lawrence J (2007) Development and role of tight junctions in the retinal pigment epithelium. Int Rev Cytol 258:195-234
Rizzolo, Lawrence J; Chen, Xiang; Weitzman, Matthew et al. (2007) Analysis of the RPE transcriptome reveals dynamic changes during the development of the outer blood-retinal barrier. Mol Vis 13:1259-73
Luo, Yan; Fukuhara, Masayuki; Weitzman, Matthew et al. (2006) Expression of JAM-A, AF-6, PAR-3 and PAR-6 during the assembly and remodeling of RPE tight junctions. Brain Res 1110:55-63
Luo, Yan; Zhuo, Yehong; Fukuhara, Masayuki et al. (2006) Effects of culture conditions on heterogeneity and the apical junctional complex of the ARPE-19 cell line. Invest Ophthalmol Vis Sci 47:3644-55
Rahner, Christoph; Fukuhara, Masayuki; Peng, Shaomin et al. (2004) The apical and basal environments of the retinal pigment epithelium regulate the maturation of tight junctions during development. J Cell Sci 117:3307-18
Peng, Shaomin; Rahner, Christoph; Rizzolo, Lawrence J (2003) Apical and basal regulation of the permeability of the retinal pigment epithelium. Invest Ophthalmol Vis Sci 44:808-17
Kojima, S; Rahner, C; Peng, S et al. (2002) Claudin 5 is transiently expressed during the development of the retinal pigment epithelium. J Membr Biol 186:81-8
Bumsted, K M; Rizzolo, L J; Barnstable, C J (2001) Defects in the MITF(mi/mi) apical surface are associated with a failure of outer segment elongation. Exp Eye Res 73:383-92

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