Limits...
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.

Show MeSH

Related in: MedlinePlus

Ogg1 messenger in mouse retina, forebrain and cerebellum with associated densitometric analysis of the PCR bands. A shows relative amount of 8-oxoguanine glycosylase (Ogg1) mRNA in the mouse retina, forebrain (brain) and cerebellum (Cereb). To determine the expression of Ogg1 mRNA in adult C57BL/6J mouse neuroretinal cells (n=3), forebrain (n=3), and cereb (n=3), we performed semiquantitative RT–PCR using specific primers for mouse Ogg1 mRNA and cyclophilin A mRNA (Cyclo). Cyclo mRNA was used as an internal control. The 400 bp band corresponds to Ogg1 PCR products and the 311 bp band corresponds to cyclophilin A PCR products. B represents comparison of the Ogg1 mRNA expression in neuroretina, liver, and testis. Quantitative RT–PCR was performed to determine the relative levels of Ogg1 mRNA in adult C57BL/6 mouse neuroretina (n=5), liver (n=5), and testis (n=5), Cyclo mRNA was used as an internal standard for normalization. Significant, higher levels of Ogg1 mRNA expression were observed in testis as compared to those obtained in the liver and the neuroretina. Values are means±SEM.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2690988&req=5

f2: Ogg1 messenger in mouse retina, forebrain and cerebellum with associated densitometric analysis of the PCR bands. A shows relative amount of 8-oxoguanine glycosylase (Ogg1) mRNA in the mouse retina, forebrain (brain) and cerebellum (Cereb). To determine the expression of Ogg1 mRNA in adult C57BL/6J mouse neuroretinal cells (n=3), forebrain (n=3), and cereb (n=3), we performed semiquantitative RT–PCR using specific primers for mouse Ogg1 mRNA and cyclophilin A mRNA (Cyclo). Cyclo mRNA was used as an internal control. The 400 bp band corresponds to Ogg1 PCR products and the 311 bp band corresponds to cyclophilin A PCR products. B represents comparison of the Ogg1 mRNA expression in neuroretina, liver, and testis. Quantitative RT–PCR was performed to determine the relative levels of Ogg1 mRNA in adult C57BL/6 mouse neuroretina (n=5), liver (n=5), and testis (n=5), Cyclo mRNA was used as an internal standard for normalization. Significant, higher levels of Ogg1 mRNA expression were observed in testis as compared to those obtained in the liver and the neuroretina. Values are means±SEM.

Mentions: Using semiquantitative RT–PCR analyses, we determined the expression of Ogg1 mRNA in the adult mouse neuroretina. (Figure 2A). The housekeeping gene, cyclophilin A, was used as a common internal standard (311 bp product). RT–PCR was also performed on mRNA extracted from mouse cerebellum and forebrain neuronal tissues in which Ogg1 gene expression has previously been described [25]. RT–PCR with mouse neuroretina RNA yielded an expected 400 bp product similar to that amplified from cerebellum and forebrain, and it seemed that there was no difference in Ogg1 mRNA levels between the three postmitotic tissues studied. Using quantitative RT–PCR analyses, we detected significantly higher level of Ogg1 mRNA expression in testis than in the liver, as previously described [26,27], and the neuroretina (Figure 2B). In addition, we showed that the amounts of Ogg1 mRNA were similar in neuroretina and liver (p=0.557).


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)

Ogg1 messenger in mouse retina, forebrain and cerebellum with associated densitometric analysis of the PCR bands. A shows relative amount of 8-oxoguanine glycosylase (Ogg1) mRNA in the mouse retina, forebrain (brain) and cerebellum (Cereb). To determine the expression of Ogg1 mRNA in adult C57BL/6J mouse neuroretinal cells (n=3), forebrain (n=3), and cereb (n=3), we performed semiquantitative RT–PCR using specific primers for mouse Ogg1 mRNA and cyclophilin A mRNA (Cyclo). Cyclo mRNA was used as an internal control. The 400 bp band corresponds to Ogg1 PCR products and the 311 bp band corresponds to cyclophilin A PCR products. B represents comparison of the Ogg1 mRNA expression in neuroretina, liver, and testis. Quantitative RT–PCR was performed to determine the relative levels of Ogg1 mRNA in adult C57BL/6 mouse neuroretina (n=5), liver (n=5), and testis (n=5), Cyclo mRNA was used as an internal standard for normalization. Significant, higher levels of Ogg1 mRNA expression were observed in testis as compared to those obtained in the liver and the neuroretina. Values are means±SEM.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Ogg1 messenger in mouse retina, forebrain and cerebellum with associated densitometric analysis of the PCR bands. A shows relative amount of 8-oxoguanine glycosylase (Ogg1) mRNA in the mouse retina, forebrain (brain) and cerebellum (Cereb). To determine the expression of Ogg1 mRNA in adult C57BL/6J mouse neuroretinal cells (n=3), forebrain (n=3), and cereb (n=3), we performed semiquantitative RT–PCR using specific primers for mouse Ogg1 mRNA and cyclophilin A mRNA (Cyclo). Cyclo mRNA was used as an internal control. The 400 bp band corresponds to Ogg1 PCR products and the 311 bp band corresponds to cyclophilin A PCR products. B represents comparison of the Ogg1 mRNA expression in neuroretina, liver, and testis. Quantitative RT–PCR was performed to determine the relative levels of Ogg1 mRNA in adult C57BL/6 mouse neuroretina (n=5), liver (n=5), and testis (n=5), Cyclo mRNA was used as an internal standard for normalization. Significant, higher levels of Ogg1 mRNA expression were observed in testis as compared to those obtained in the liver and the neuroretina. Values are means±SEM.
Mentions: Using semiquantitative RT–PCR analyses, we determined the expression of Ogg1 mRNA in the adult mouse neuroretina. (Figure 2A). The housekeeping gene, cyclophilin A, was used as a common internal standard (311 bp product). RT–PCR was also performed on mRNA extracted from mouse cerebellum and forebrain neuronal tissues in which Ogg1 gene expression has previously been described [25]. RT–PCR with mouse neuroretina RNA yielded an expected 400 bp product similar to that amplified from cerebellum and forebrain, and it seemed that there was no difference in Ogg1 mRNA levels between the three postmitotic tissues studied. Using quantitative RT–PCR analyses, we detected significantly higher level of Ogg1 mRNA expression in testis than in the liver, as previously described [26,27], and the neuroretina (Figure 2B). In addition, we showed that the amounts of Ogg1 mRNA were similar in neuroretina and liver (p=0.557).

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