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Paulo A. Ferreira, Arjun Saha, Emdadul Haque, Yun-Zheng Le, Mason Webb; Conditional Knock-Out of Ran-binding protein 2 (RanBP2)/Nucleoporin 358 (NUP358) in the Retinal Pigment Epithelium Results in the Activation of Membrane to Nuclear Signaling Pathways and Hallmark Features of Age-Related Macular Degeneration (AMD). Invest. Ophthalmol. Vis. Sci. 2012;53(14):6508.
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The atrophy of the retinal pigment epithelium is a hallmark feature of geographic atrophy (GA). Yet, GA remains largely idiopathic. RanBP2/Nup358 is a highly pleiotropic protein with cell-context dependent roles. We have shown previously that haploinsufficiency of RanBP2 is neuroprotective against photo-oxidative stress. This study tests the hypothesis that genetic ablation of RanBP2 selectively in the RPE causes its degeneration.
We carried out the conditional ablation of RanBP2 in the RPE by crossing transgenic mice co-expressing the human vitelliform macular dystrophy-2 (VMD2) promoter (PVMD2)-directed reverse tetracycline-dependent transactivator (PVMD2-rtTA) and the tetracycline-responsive element (TRE)-directed cre (tetO-PhCMV-cre) with mice harboring the loxP-flanked RanBP2 allele in the presence or absence of a constitutively disrupted RanBP2 allele (RanBP2lacz-PLAP). These mice and RPE-specific Cre mice alone were compared for changes of the fundus, morphology and ultrastructure of retinas and RPE. The levels and subcellular localization of components of the RanBP2 interactome and RPE markers were examined by qRT-PCR, immuno-cytochemistry and immunoblot analyses.
Fundus photography showed degeneration of the RPE in RanBP2loxP/loxP or RanBP2loxP/lacz-PLAP mice with RPE-specific Cre, but not RPE-specific Cre mice alone. The latter present well tessellated RPE cells, whereas either conditional knockout RanBP2 mouse presents RPE cells with marked loss of shape, disruption of cell boundaries, widespread cell atrophy with interspersed large cells with multiple nuclei. Advanced atrophic RPE cells present nuclear atypia with nuclei pushed toward the edge of cell, sporadic detachment of atrophic RPE cells between the RPE and outer segments. RPE cells present impaired dispersion of melanin. Further, we found strong activation and subcellular delocalization of selective components of the RanBP2 interactome, such as of a subset of receptor tyrosine kinases and allied nuclear-cytoplasmic signaling regulators. These effects were accompanied by the down-regulation of RPE markers, such as RPE65, but not of other species, such as DICER, in conditional knockout RanBP2 mice, but not wild-type or RPE-specific Cre mice.
These studies support that RanBP2 is essential to RPE viability and provide insights into novel signaling mechanisms and molecular processes triggering RPE degeneration by deficits in RanBP2 and that contribute to our understanding of AMD pathogenesis.
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