Abstract
Aims/hypothesisDiabetic macular edema represents the main cause of visual loss in diabetic retinopathy. Besides inner blood retinal barrier breakdown, the role of the outer blood retinal barrier breakdown has been poorly analyzed. We characterized the structural and molecular alterations of the outer blood retinal barrier during the time course of diabetes, focusing on PKCζ, a critical protein for tight junction assembly, known to be overactivated by hyperglycemia.MethodsStudies were conducted on a type2 diabetes Goto-Kakizaki rat model. PKCζ level and subcellular localization were assessed by immunoblotting and immunohistochemistry. Cell death was detected by TUNEL assays. PKCζ level on specific layers was assessed by laser microdissection followed by Western blotting. The functional role of PKCζ was then evaluated in vivo, using intraocular administration of its specific inhibitor.ResultsPKCζ was localized in tight junction protein complexes of the retinal pigment epithelium and in photoreceptors inner segments. Strikingly, in outer segment PKCζ staining was restricted to cone photoreceptors. Short-term hyperglycemia induced activation and delocalization of PKCζ from both retinal pigment epithelium junctions and cone outer segment. Outer blood retinal barrier disruption and photoreceptor cone degeneration characterized long-term hyperglycemia. In vivo, reduction of PKCζ overactivation using a specific inhibitor, restored its tight-junction localization and not only improved the outer blood retinal barrier, but also reduced photoreceptor cell-death.ConclusionsIn the retina, hyperglycemia induced overactivation of PKCζ is associated with outer blood retinal barrier breakdown and photoreceptor degeneration. In vivo, short-term inhibition of PKCζ restores the outer barrier structure and reduces photoreceptor cell death, identifying PKCζ as a potential target for early and underestimated diabetes-induced retinal pathology.
Highlights
Diabetic retinopathy (DR) is one of the most severe complication of diabetes and the leading cause of visual loss among western working-age adults [1,2]
In retinal pigment epithelium (RPE) cells from 6-month-old diabetic rats, the PKCf staining was either barely or not detectable associated to the TJ but instead a diffuse staining of PKCf was observed in the cytoplasma (Fig 2b)
At 12 months of age, in diabetic RPE, PKCf and occludin could be observed in the nuclei of cells, while marked disruptions of the tight junctions with buttonhole formation could be observed (Fig 2c)
Summary
Diabetic retinopathy (DR) is one of the most severe complication of diabetes and the leading cause of visual loss among western working-age adults [1,2]. Visual impairment results mostly from macular edema (ME), defined as fluid accumulation in/or under the macula, which is the specialized retinal area responsible for visual acuity [3,4]. Fluid entry is controlled by the inner blood retinal barrier (BRB) made of TJ between the endothelial cells of the retinal vasculature together with a complex network of glial components. The retinal pigment epithelium (RPE) is a monolayer of pigmented cells with tight-junctions that control mostly fluid exit from the sub-retinal space through the choroid. We have recently evidenced that tight-like junctions containing occludin are present at the OLM, mainly between Muller glial cells and cones, the only photoreceptors of the macula [6]. Any protein loss of this complex leads to mislocalization of the others [14]. PKCf phosphorylates occludin and thereby promotes assembly of TJ [15] and it was shown that hyperglycemia modulates its activity.[16,17]
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