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Expression of 8-oxoguanine DNA glycosylase (Ogg1) in mouse retina.

Bigot K, Leemput J, Vacher M, Campalans A, Radicella JP, Lacassagne E, Provost A, Masson C, Menasche M, Abitbol M - Mol. Vis. (2009)

Bottom Line: Ogg1, the enzyme responsible for the recognition and excision of the oxidized base, is present in its active form and found mainly in ganglion cells and photoreceptor inner segments.The cellular distribution of these proteins was similar to that of Ogg1.This result suggests that the elimination of 8-oxoG is coordinated through two pathways, which differ slightly according to the cellular localization of the abnormally oxidized guanine.

View Article: PubMed Central - PubMed

Affiliation: Université Paris-Descartes, CERTO, Centre de Recherche Thérapeutique en Ophtalmologie, Paris, France.

ABSTRACT

Purpose: The retina is highly exposed to oxidative stress due to the high level of oxygen consumption in this tissue and its exposure to light. The main DNA base lesion generated by oxygen free radicals is 8-oxoguanine (8-oxoG). However, its presence in retinal cells and the mechanisms underlying its repair remain undetermined.

Methods: 8-oxoguanine DNA glycosylase (Ogg1) gene expression and messenger localization in adult mouse ocular tissues was analyzed by RT-PCR and in situ hybridization. Using immunohistochemistry, we determined the localization of Ogg1 protein and three base excision repair (BER) enzymes: apurinic/apyrimidic endonuclease (APE1), DNA polymerase beta, and X-ray repair cross-complementation group 1 (XRCC1). Ogg1 and AP-lyase activities in the neuroretina were obtained using double-stranded oligonucleotides harboring either an 8-oxoG residue or a tetrahydrofuran.

Results: We report here that 8-oxoG is abundant in the retina. Ogg1, the enzyme responsible for the recognition and excision of the oxidized base, is present in its active form and found mainly in ganglion cells and photoreceptor inner segments. We show that APE1 and DNA polymerase beta, two BER proteins involved in 8-oxoG repair, are also present in these cells. The cellular distribution of these proteins was similar to that of Ogg1. XRRC1 is present in both inner nuclear and ganglion cells layers; however, this protein is absent from photoreceptor inner segments.

Conclusions: This is the first study to demonstrate the presence of a functional 8-oxoG BER pathway in retinal neurons. The study of three BER proteins involved in 8-oxoG elimination demonstrates that XRCC1 localization differs from those of Ogg1, APE1, and DNA polymerase beta. This result suggests that the elimination of 8-oxoG is coordinated through two pathways, which differ slightly according to the cellular localization of the abnormally oxidized guanine.

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Distribution of Ogg1 transcript in the mouse adult eye. In situ hybridization signals of 8-oxoguanine glycosylase (Ogg1) mRNA were detected in neuroretina (A, C, and D), ciliary body (E), and cornea (F) using a specific antisense digoxigenin-labeled riboprobe. Labeled Ogg1 mRNA appears as purple precipitates. No signal was detected with a control sense probe (B). Ogg1 mRNA was present in all retinal nuclear layers, in photoreceptor inner segments (IS), and in retinal pigment epithelium (RPE). Ogg1 mRNA was also detected in non-neuronal cells: corneal epithelial (Cep) and endothelial cells (Cep), keratocytes of the corneal stroma, pigmentary epithelium (PCE), and nonpigmentary ciliary epithelium (NPCE). C and D represent a high magnification of the inner portion of the neuroretina, the retinal pigment epithelium (RPE), and the choroid (Ch), respectively. Scale bar equals 70 μm in A, B, E, and F, and 25 µm in C and D. Abbreviations: ganglion cell layer (GCL); inner nuclear layer (INL); outer nuclear layer (ONL).
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f3: Distribution of Ogg1 transcript in the mouse adult eye. In situ hybridization signals of 8-oxoguanine glycosylase (Ogg1) mRNA were detected in neuroretina (A, C, and D), ciliary body (E), and cornea (F) using a specific antisense digoxigenin-labeled riboprobe. Labeled Ogg1 mRNA appears as purple precipitates. No signal was detected with a control sense probe (B). Ogg1 mRNA was present in all retinal nuclear layers, in photoreceptor inner segments (IS), and in retinal pigment epithelium (RPE). Ogg1 mRNA was also detected in non-neuronal cells: corneal epithelial (Cep) and endothelial cells (Cep), keratocytes of the corneal stroma, pigmentary epithelium (PCE), and nonpigmentary ciliary epithelium (NPCE). C and D represent a high magnification of the inner portion of the neuroretina, the retinal pigment epithelium (RPE), and the choroid (Ch), respectively. Scale bar equals 70 μm in A, B, E, and F, and 25 µm in C and D. Abbreviations: ganglion cell layer (GCL); inner nuclear layer (INL); outer nuclear layer (ONL).

Mentions: We performed in situ hybridization on paraffin-embedded sections of adult C57BL/6 mouse eyes to determine the tissue distribution of Ogg1 transcripts. No signal was observed in the retina using the Ogg1 sense probe (Figure 3B). A strong Ogg1 mRNA signal was detected in several cell layers of the neuroretina: GCL, INL, ONL, and IS (Figure 3A,C,D). No labeling was observed in the inner and outer plexiform layers. Ogg1 mRNA was also present in RPE cells. These cells, like the neuroretina, are derived from the prosencephalon. Ogg1 transcripts were also detected in other ocular cells derived from the nervous system such as choroidal cells (melanocytes derived from the neural crest, unlike RPE cells derived from the CNS), and ciliary body epithelial cells (pigmented and nonpigmented, Figure 3E). In non-neuronal cells, Ogg1 transcripts were present in corneal epithelial (Cep) and endothelial cells (Cen) as well as in keratocytes of the corneal stroma (Figure 3F).


Expression of 8-oxoguanine DNA glycosylase (Ogg1) in mouse retina.

Bigot K, Leemput J, Vacher M, Campalans A, Radicella JP, Lacassagne E, Provost A, Masson C, Menasche M, Abitbol M - Mol. Vis. (2009)

Distribution of Ogg1 transcript in the mouse adult eye. In situ hybridization signals of 8-oxoguanine glycosylase (Ogg1) mRNA were detected in neuroretina (A, C, and D), ciliary body (E), and cornea (F) using a specific antisense digoxigenin-labeled riboprobe. Labeled Ogg1 mRNA appears as purple precipitates. No signal was detected with a control sense probe (B). Ogg1 mRNA was present in all retinal nuclear layers, in photoreceptor inner segments (IS), and in retinal pigment epithelium (RPE). Ogg1 mRNA was also detected in non-neuronal cells: corneal epithelial (Cep) and endothelial cells (Cep), keratocytes of the corneal stroma, pigmentary epithelium (PCE), and nonpigmentary ciliary epithelium (NPCE). C and D represent a high magnification of the inner portion of the neuroretina, the retinal pigment epithelium (RPE), and the choroid (Ch), respectively. Scale bar equals 70 μm in A, B, E, and F, and 25 µm in C and D. Abbreviations: ganglion cell layer (GCL); inner nuclear layer (INL); outer nuclear layer (ONL).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2690988&req=5

f3: Distribution of Ogg1 transcript in the mouse adult eye. In situ hybridization signals of 8-oxoguanine glycosylase (Ogg1) mRNA were detected in neuroretina (A, C, and D), ciliary body (E), and cornea (F) using a specific antisense digoxigenin-labeled riboprobe. Labeled Ogg1 mRNA appears as purple precipitates. No signal was detected with a control sense probe (B). Ogg1 mRNA was present in all retinal nuclear layers, in photoreceptor inner segments (IS), and in retinal pigment epithelium (RPE). Ogg1 mRNA was also detected in non-neuronal cells: corneal epithelial (Cep) and endothelial cells (Cep), keratocytes of the corneal stroma, pigmentary epithelium (PCE), and nonpigmentary ciliary epithelium (NPCE). C and D represent a high magnification of the inner portion of the neuroretina, the retinal pigment epithelium (RPE), and the choroid (Ch), respectively. Scale bar equals 70 μm in A, B, E, and F, and 25 µm in C and D. Abbreviations: ganglion cell layer (GCL); inner nuclear layer (INL); outer nuclear layer (ONL).
Mentions: We performed in situ hybridization on paraffin-embedded sections of adult C57BL/6 mouse eyes to determine the tissue distribution of Ogg1 transcripts. No signal was observed in the retina using the Ogg1 sense probe (Figure 3B). A strong Ogg1 mRNA signal was detected in several cell layers of the neuroretina: GCL, INL, ONL, and IS (Figure 3A,C,D). No labeling was observed in the inner and outer plexiform layers. Ogg1 mRNA was also present in RPE cells. These cells, like the neuroretina, are derived from the prosencephalon. Ogg1 transcripts were also detected in other ocular cells derived from the nervous system such as choroidal cells (melanocytes derived from the neural crest, unlike RPE cells derived from the CNS), and ciliary body epithelial cells (pigmented and nonpigmented, Figure 3E). In non-neuronal cells, Ogg1 transcripts were present in corneal epithelial (Cep) and endothelial cells (Cen) as well as in keratocytes of the corneal stroma (Figure 3F).

Bottom Line: Ogg1, the enzyme responsible for the recognition and excision of the oxidized base, is present in its active form and found mainly in ganglion cells and photoreceptor inner segments.The cellular distribution of these proteins was similar to that of Ogg1.This result suggests that the elimination of 8-oxoG is coordinated through two pathways, which differ slightly according to the cellular localization of the abnormally oxidized guanine.

View Article: PubMed Central - PubMed

Affiliation: Université Paris-Descartes, CERTO, Centre de Recherche Thérapeutique en Ophtalmologie, Paris, France.

ABSTRACT

Purpose: The retina is highly exposed to oxidative stress due to the high level of oxygen consumption in this tissue and its exposure to light. The main DNA base lesion generated by oxygen free radicals is 8-oxoguanine (8-oxoG). However, its presence in retinal cells and the mechanisms underlying its repair remain undetermined.

Methods: 8-oxoguanine DNA glycosylase (Ogg1) gene expression and messenger localization in adult mouse ocular tissues was analyzed by RT-PCR and in situ hybridization. Using immunohistochemistry, we determined the localization of Ogg1 protein and three base excision repair (BER) enzymes: apurinic/apyrimidic endonuclease (APE1), DNA polymerase beta, and X-ray repair cross-complementation group 1 (XRCC1). Ogg1 and AP-lyase activities in the neuroretina were obtained using double-stranded oligonucleotides harboring either an 8-oxoG residue or a tetrahydrofuran.

Results: We report here that 8-oxoG is abundant in the retina. Ogg1, the enzyme responsible for the recognition and excision of the oxidized base, is present in its active form and found mainly in ganglion cells and photoreceptor inner segments. We show that APE1 and DNA polymerase beta, two BER proteins involved in 8-oxoG repair, are also present in these cells. The cellular distribution of these proteins was similar to that of Ogg1. XRRC1 is present in both inner nuclear and ganglion cells layers; however, this protein is absent from photoreceptor inner segments.

Conclusions: This is the first study to demonstrate the presence of a functional 8-oxoG BER pathway in retinal neurons. The study of three BER proteins involved in 8-oxoG elimination demonstrates that XRCC1 localization differs from those of Ogg1, APE1, and DNA polymerase beta. This result suggests that the elimination of 8-oxoG is coordinated through two pathways, which differ slightly according to the cellular localization of the abnormally oxidized guanine.

Show MeSH
Related in: MedlinePlus