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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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Expression of BER mRNA and proteins in the adult mouse retina. A: Semiquantitative RT–PCR experiments were performed to determine the APE1, DNA polymerase β (DNA pol β), and XRCC1 mRNA levels of expression in C57BL/6 mouse neuroretinal cells. Cyclophilin A (Cyclo) was used as an internal control. Specific primers for amplifying mouse APE1, DNA pol β, XRCC1, and Cyclophilin A cDNAs were used. The expected size for each specific amplified product was obtained: 446 bp for APE1, 693 bp for DNA pol β, 418 bp for XRCC1, 311 bp for Cyclophilin A. Immunohistological localization of APE1, DNA polymerase β, and XRCC1 in the adult mouse retina was performed using an antibody raised against APE1 (B), DNA pol β (C) or XRCC1 (D). The staining appears in brown. Sections were counterstained with a methyl green solution. No signal was detected when the specific anti-APE1 antibody was omitted (E). APE1 and DNA polymerase β were detected in the ganglion cell layer (GCL), the inner nuclear layer (INL), and the photoreceptor inner segments (IS). Labeling was also observed in the INL and outer plexiform layers (ONL). Surprisingly, XRCC1 was not detected in the IS. Scale bar equals 50 μm in B, C, and E, and 10 µm in D.
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f6: Expression of BER mRNA and proteins in the adult mouse retina. A: Semiquantitative RT–PCR experiments were performed to determine the APE1, DNA polymerase β (DNA pol β), and XRCC1 mRNA levels of expression in C57BL/6 mouse neuroretinal cells. Cyclophilin A (Cyclo) was used as an internal control. Specific primers for amplifying mouse APE1, DNA pol β, XRCC1, and Cyclophilin A cDNAs were used. The expected size for each specific amplified product was obtained: 446 bp for APE1, 693 bp for DNA pol β, 418 bp for XRCC1, 311 bp for Cyclophilin A. Immunohistological localization of APE1, DNA polymerase β, and XRCC1 in the adult mouse retina was performed using an antibody raised against APE1 (B), DNA pol β (C) or XRCC1 (D). The staining appears in brown. Sections were counterstained with a methyl green solution. No signal was detected when the specific anti-APE1 antibody was omitted (E). APE1 and DNA polymerase β were detected in the ganglion cell layer (GCL), the inner nuclear layer (INL), and the photoreceptor inner segments (IS). Labeling was also observed in the INL and outer plexiform layers (ONL). Surprisingly, XRCC1 was not detected in the IS. Scale bar equals 50 μm in B, C, and E, and 10 µm in D.

Mentions: When initiated by Ogg1, the base excision repair of 8-oxoG involves a highly coordinated process mediated by several proteins [20]. Using both RT–PCR and immunohistochemistry, we determined whether APE1, DNA polymerase β, and XRCC1, all of which are involved in BER, were present in mouse neuroretina. Amplification of APE1 (446 bp), DNA polymerase β (693 bp), and XRCC1 (418 bp), through the use of specific primers, confirmed the expression of these genes in the mouse retina (Figure 6A).


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)

Expression of BER mRNA and proteins in the adult mouse retina. A: Semiquantitative RT–PCR experiments were performed to determine the APE1, DNA polymerase β (DNA pol β), and XRCC1 mRNA levels of expression in C57BL/6 mouse neuroretinal cells. Cyclophilin A (Cyclo) was used as an internal control. Specific primers for amplifying mouse APE1, DNA pol β, XRCC1, and Cyclophilin A cDNAs were used. The expected size for each specific amplified product was obtained: 446 bp for APE1, 693 bp for DNA pol β, 418 bp for XRCC1, 311 bp for Cyclophilin A. Immunohistological localization of APE1, DNA polymerase β, and XRCC1 in the adult mouse retina was performed using an antibody raised against APE1 (B), DNA pol β (C) or XRCC1 (D). The staining appears in brown. Sections were counterstained with a methyl green solution. No signal was detected when the specific anti-APE1 antibody was omitted (E). APE1 and DNA polymerase β were detected in the ganglion cell layer (GCL), the inner nuclear layer (INL), and the photoreceptor inner segments (IS). Labeling was also observed in the INL and outer plexiform layers (ONL). Surprisingly, XRCC1 was not detected in the IS. Scale bar equals 50 μm in B, C, and E, and 10 µm in D.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f6: Expression of BER mRNA and proteins in the adult mouse retina. A: Semiquantitative RT–PCR experiments were performed to determine the APE1, DNA polymerase β (DNA pol β), and XRCC1 mRNA levels of expression in C57BL/6 mouse neuroretinal cells. Cyclophilin A (Cyclo) was used as an internal control. Specific primers for amplifying mouse APE1, DNA pol β, XRCC1, and Cyclophilin A cDNAs were used. The expected size for each specific amplified product was obtained: 446 bp for APE1, 693 bp for DNA pol β, 418 bp for XRCC1, 311 bp for Cyclophilin A. Immunohistological localization of APE1, DNA polymerase β, and XRCC1 in the adult mouse retina was performed using an antibody raised against APE1 (B), DNA pol β (C) or XRCC1 (D). The staining appears in brown. Sections were counterstained with a methyl green solution. No signal was detected when the specific anti-APE1 antibody was omitted (E). APE1 and DNA polymerase β were detected in the ganglion cell layer (GCL), the inner nuclear layer (INL), and the photoreceptor inner segments (IS). Labeling was also observed in the INL and outer plexiform layers (ONL). Surprisingly, XRCC1 was not detected in the IS. Scale bar equals 50 μm in B, C, and E, and 10 µm in D.
Mentions: When initiated by Ogg1, the base excision repair of 8-oxoG involves a highly coordinated process mediated by several proteins [20]. Using both RT–PCR and immunohistochemistry, we determined whether APE1, DNA polymerase β, and XRCC1, all of which are involved in BER, were present in mouse neuroretina. Amplification of APE1 (446 bp), DNA polymerase β (693 bp), and XRCC1 (418 bp), through the use of specific primers, confirmed the expression of these genes in the mouse retina (Figure 6A).

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