Tight Junction Dysfunction: Altered ZO‐1 and Occludin in the Diabetic Cornea

Abstract

The cornea is the outermost part of the eye, and as a result, can be damaged by debris and other irritants. While the cornea can repair itself from minor injuries, improper wound repair can further complicate the issue. Type II Diabetes is known to hinder the body's wound healing ability, with complications in epithelial wound repair being detected in the corneas of diabetic rats. Tight junctions are multiprotein complexes involved in a variety of processes required for proper wound healing. Occludin, a transmembrane protein, and ZO‐1, a scaffolding protein, are found in the tight junction complex. Prior studies documented that occludin plays an important role in corneal wound healing and that downregulation of occludin impairs tight junction integrity. Our lab also has evidence that the localization of Crumbs3 (Crb3), a polarity protein associated with ZO‐1, is altered in the diabetic cornea. We hypothesized that occludin and ZO‐1 localization would be altered in the diabetic tissue. Our lab uses the diabetic induced obesity (DIO) mice as a model for Pre‐Type II diabetes. Previously we demonstrated a peripheral neuropathy and impaired wound healing in the DIO tissue. Corneal epithelium cells from DIO mice were examined 7.5 and 15 weeks (wk) after onset of a high fat diet and compared to WT C57BL6 mice. Corneas were wounded and allowed to heal for 18 hours after injury. Wounded (W) and unwounded (UW) cells were fixed in 4% paraformaldehyde, stained with antibodies to occludin and ZO‐1, and then imaged. Fixed corneas were imaged on a Zeiss LSM 700 Confocal Microscope using a 40x‐ and 63x‐oil objectives and analyzed using FIJI/NIHImageJ. We demonstrated that the localization patterns of occludin and ZO‐1 were altered in the UW and W DIO tissue at both the apical and basal cell layers of the corneal epithelium. ZO‐1 is apically located in the UW control tissue, while occludin is present throughout the tissue. Upon wounding, staining patterns of occludin are maintained, while ZO‐1 staining is observed at the apical and basal cells. In the UW 7.5 wk DIO tissue, staining of occludin appears punctate at the apical cell layers, while staining at the basal cell layers there is a loss of occludin staining at the cell membrane. Upon wounding, staining at the apical cells is similar to the UW control tissue and staining at the basal cells is similar to the W control tissue, however occludin and ZO‐1 staining is also localized along the wound edge. In the UW 15 wk DIO tissue, staining is similar to that of the UW control tissue. Upon wounding, staining is similar to the W control tissue; however there is a loss of ZO‐1 staining at the apical cell membranes. The change in continuous ZO‐1 occurs prior to changes in occludin. Our results support the results demonstrating that Crb3 is altered. Since Crb3 mediates trafficking of ZO‐1, our data suggest that there is an irregularity in tight junction stability and membrane polarity in the diabetic tissue.Support or Funding InformationSummer Training as Research Scholars Program, The Trinkaus‐Randall Lab, NIH EY06000 to VTR, and Funding from NIH MARC GM008422This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.