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A. van Rossum, J. Meulenman, W. Aartsen, A. Malysheva, I. Versteeg, J. Klooster, J. Wijnholds; Pals1 Is Required for Correct Localization of Crb1 at the Sub–Apical Region in Müller Glia Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):129.
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Mutations in the human Crumbs homologue–1 (CRB1) gene cause retinal diseases including Leber congenital amaurosis (LCA) and retinitis pigmentosa 12 (RP12). In the retina, the CRB1 transmembrane protein localizes at the sub–apical region (SAR) of the outer limiting membrane (OLM), just above the intercellular adherens junctions between photoreceptor and Müller glia (MG) cells. The Crb1–/– phenotype is characterized by OLM interruptions, upregulation of GFAP immunoreactivity in MG cells, formation of retinal folds, and pseudorosettes. Crb1 is essential for maintenance of the adherens junction complex to prevent retinal disorganization. In this study, we used primary retina cultures to study the mechanisms of CRB1–mediated retinal degeneration.
Wild type (WT) and Crb1–/– primary retinas from 1.5 days old mice were cultured for up to one month. An in vitro electroporation technique was used for both gain– and loss–of–function studies in primary retina cultures.
In primary retina cultures, the Crb1–/– phenotype is accelerated and intensified starting from 7 days in vitro. Reintroduction of CMV–driven CRB1 into Crb1–/– retinas resulted in human CRB1 localization at the SAR in MG cells. Electron microscopic immunohistochemistry showed strong Crb1 immunoreactivity at the SAR in MG cells but barely in photoreceptor cells. Down regulation of the Crb1 interacting protein Pals1 (Proteins Associated with Lin7, or Mpp5), in MG cells using RNAi, resulted in proportional down regulation of Crb1, Crb2 and Crb3 immunofluorescent reactivity.
Our results demonstrate that Pals1/Mpp5 is required for correct localization of Crb1, Crb2 and Crb3 at the SAR in Müller glia cells.
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