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Lucie Pellissier, Jan Klooster, Jan Wijnholds; CRB proteins expression and localization in human retina. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2686.
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© ARVO (1962-2015); The Authors (2016-present)
Mutations in the Crumbs homologue 1 (Crb1) gene are associated with Leber congenital amaurosis (LCA) and retinitis pigmentosa (RP). At the subapical region in murine retinas, CRB1 is restricted to Müller glial microvilli and CRB2 is present in both photoreceptor and Müller glial cells. CRB proteins are present at the subapical region of human retina but the cellular localization is still unknown.
Using quantitative PCR, immuno-electron microscopy and immunohistochemistry, we studied the cellular localization of CRB1 and its variants, CRB2 and CRB3 transcripts and proteins in human donor retinas.
Using quantitative PCR, we showed that full length CRB1, CRB1 variant lacking the C-terminal cytosolic and transmembrane domains, CRB1 variant carrying an in-frame deletion of exons 3 and 4 leading to the deletion of the EGF domains 4-6, CRB2 and CRB3 transcripts were expressed in the human retina and in similar levels in the macula and the peripheral retinas and were not detectable in RPE/choroid tissues. Immuno-electron microscopy on human donor retinas revealed strong CRB1 staining at the subapical region above the adherens junctions in the microvilli of Müller glia cells and in the inner segments of photoreceptor cells. CRB2 staining was obvious in the microvilli of Müller glia cells, but barely detectable in photoreceptor cells. Immunohistochemistry of human retinas showed strong and similar intensity at the subapical region of the macula and in the peripheral retina for CRB1, CRB2 and CRB3. CRB1 antibody raised against the extracellular domain, which might recognize all the protein variants, and CRB1 antibody against the intracellular domain, showed the same expression pattern at the subapical region and in the cone inner segment of the macula. CRB1 and CRB2 co-localized at the subapical region, but CRB1 co-localized partially with glutamine synthetase (a marker for Müller glia cells) whereas CRB2 co-localized completely. CRB3 localized at the subapical region but also at the synaptic outer plexiform layer.
In contrast to mice, in the human retina CRB1 protein was expressed at the subapical region in photoreceptors and Müller glia cells, and CRB2 only in Müller glia cells. This inverted pattern of expression may explain the differences between the phenotype in mice and in CRB1-related patients.
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