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A constitutively active G?i3 protein corrects the abnormal retinal pigment epithelium phenotype of Oa1-/- mice.

ABSTRACT: Ocular Albinism type 1 (OA1) is a disease caused by mutations in the OA1 gene and characterized by the presence of macromelanosomes in the retinal pigment epithelium (RPE) as well as abnormal crossing of the optic axons at the optic chiasm. We showed in our previous studies in mice that Oa1 activates specifically G?i3 in its signaling pathway and thus, hypothesized that a constitutively active G?i3 in the RPE of Oa1-/- mice might keep on the Oa1 signaling cascade and prevent the formation of macromelanosomes. To test this hypothesis, we have generated transgenic mice that carry the constitutively active G?i3 (Q204L) protein in the RPE of Oa1-/- mice and are now reporting the effects that the transgene produced on the Oa1-/- RPE phenotype.Transgenic mice carrying RPE-specific expression of the constitutively active G?i3 (Q204L) were generated by injecting fertilized eggs of Oa1-/- females with a lentivirus containing the G?i3 (Q204L) cDNA. PCR, Southern blots, Western blots and confocal microscopy were used to confirm the presence of the transgene in the RPE of positive transgenic mice. Morphometrical analyses were performed using electron microscopy to compare the size and number of melanosomes per RPE area in putative Oa1-/-, G?i3 (Q204L) transgenic mice with those of wild-type NCrl and Oa1-/- mice.We found a correlation between the presence of the constitutively active G?i3 (Q204L) transgene and the rescue of the normal phenotype of RPE melanosomes in Oa1-/-, G?i3 (Q204L) mice. These mice have higher density of melanosomes per RPE area and a larger number of small melanosomes than Oa1-/- mice, and their RPE phenotype is similar to that of wild-type mice.Our results show that a constitutively active G?i3 protein can by-pass the lack of Oa1 protein in Oa1-/- mice and consequently rescue the RPE melanosomal phenotype.

SUBMITTER: Young A

PROVIDER: S-EPMC3787026 | biostudies-other | 2013

REPOSITORIES: biostudies-other

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A constitutively active Gαi3 protein corrects the abnormal retinal pigment epithelium phenotype of Oa1-/- mice.

Young Alejandra A Wang Ying Y Ahmedli Novruz B NB Jiang Meisheng M Farber Debora B DB

PloS one 20130930 9

<h4>Purpose</h4>Ocular Albinism type 1 (OA1) is a disease caused by mutations in the OA1 gene and characterized by the presence of macromelanosomes in the retinal pigment epithelium (RPE) as well as abnormal crossing of the optic axons at the optic chiasm. We showed in our previous studies in mice that Oa1 activates specifically Gαi3 in its signaling pathway and thus, hypothesized that a constitutively active Gαi3 in the RPE of Oa1-/- mice might keep on the Oa1 signaling cascade and prevent the ...[more]

PMID: 24098784

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Project description:Lipofuscin accumulates with age in the retinal pigment epithelium (RPE) in discrete granular organelles and may contribute to age-related macular degeneration. Because previous studies suggest that lipofuscin contains protein that may impact pathogenic mechanisms, we pursued proteomics analysis of lipofuscin. The composition of RPE lipofuscin and its mechanisms of pathogenesis are poorly understood in part because of the heterogeneity of isolated preparations. We purified RPE lipofuscin granules by treatment with proteinase K or SDS and showed by light, confocal, and transmission electron microscopy that the purified granules are free of extragranular material and associated membranes. Crude and purified lipofuscin preparations were quantitatively compared by (i) LC MS/MS proteomics analyses, (ii) immunoanalyses of oxidative protein modifications, (iii) amino acid analysis, (iv) HPLC of bisretinoids, and (v) assaying phototoxicity to RPE cells. From crude lipofuscin preparations 186 proteins were identified, many of which appeared to be modified. In contrast, very little protein ( approximately 2% (w/w) by amino acid analysis) and no identifiable protein were found in the purified granules, which retained full phototoxicity to cultured RPE cells. Our analyses showed that granules in purified and crude lipofuscin preparations exhibit no statistically significant differences in diameter or circularity or in the content of the bisretinoids A2E, isoA2E, and all-trans-retinal dimer-phosphatidylethanolamine. The finding that the purified granules contain minimal protein yet retain phototoxic activity suggests that RPE lipofuscin pathogenesis is largely independent of associated protein. The purified granules also exhibited oxidative protein modifications, including nitrotyrosine generated from reactive nitrogen oxide species and carboxyethylpyrrole and iso[4]levuglandin E(2) adducts generated from reactive lipid fragments. This finding is consistent with previous studies demonstrating RPE lipofuscin to be a potent generator of reactive oxygen species and supports the hypothesis that such species, including reactive fragments from lipids and retinoids, contribute to the mechanisms of RPE lipofuscin pathogenesis.

Dataset Information

A constitutively active G?i3 protein corrects the abnormal retinal pigment epithelium phenotype of Oa1-/- mice.

Publications

A constitutively active Gαi3 protein corrects the abnormal retinal pigment epithelium phenotype of Oa1-/- mice.

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