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Linjing Li, Nageswara Rao Kollu, Hemant Khanna; Evaluation of Cone Photoreceptor Pathogenesis in mouse models of X-linked retinitis pigmentosa due to mutations in RPGR and RP2. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4652. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Cone photoreceptors mediate the bulk of our vision. However, paucity of cones has hampered our understanding of effect of mutations in genes causing retinal degenerative diseases specifically on cone photoreceptors. The objective of our study is to understand the effect of mutations in Retinitis pigmentosa GTPase Regulator (RPGR) and RP2, which account for >80% of X-linked RP cases, on cone photoreceptors with an aim to identify cone-autonomous pathways altered during retinal degeneration.
We generated Rpgr-ko and Rp2null mice in Nrl-/- (neural retinal leucine zipper knock out) background. The Nrl-/- mice develop S-cone enriched cone-only retina and express cone-specific genes. Age-matched male Nrl-/-:Rpgr-ko (RPGR-DKO) or Nrl-/-:Rp2null (RP2-DKO) mice were used for further studies. All mice are on C57/BL6J background. Immunoblotting was performed to assess the expression of RPGR or RP2 proteins in the cognate mutant mice. Electroretinography (ERG) was used to analyze cone function. Cone distribution was observed on whole-mount retinas. Electron microscopy (EM) and immunofluorescence staining were used to investigate morphology and protein trafficking in cones.
RPGR-DKO and RP2-DKO showed undetectable expression of RPGR and RP2, respectively. ERG analysis revealed improvement in photopic b-wave amplitude in RPGR-DKO (~ 3-fold increase) and RP2-DKO (~2-fold increase) mice as compared to Nrl-/- mice, up to 6 months of age. Immunoblot analysis revealed comparable amount of S-cone opsin and cone phosphodiesterase 6. Whereas ablation of Rpgr does not affect cone photoreceptor structure or organization, RP2-DKO mice showed loss of rosettes, which are a hallmark of Nrl-/- mice and are predicted to be due to defective outer limiting membrane (OLM) structure. Further analyses revealed abnormally elongated but disorganized cone outer segment (COS) with vesicle accumulation in RP2-DKO retina.
Increased cone function in the RPGR-DKO and RP2-DKO albeit at different levels indicates an effect of loss of RPGR and RP2 on the trafficking or function of cone phototransduction-associated proteins. Almost complete absence of rosettes and abnormally elongated COS in RP2-DKO mice suggests that RP2 regulates the morphology of the OLM and process of OS elongation. Further studies are underway to investigate cone-autonomous pathways regulated by RP2 and RPGR.
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