MutT Homolog-1 Attenuates Oxidative DNA Damage and Delays Photoreceptor Cell Death in Inherited Retinal Degeneration
2012; Elsevier BV; Volume: 181; Issue: 4 Linguagem: Inglês
10.1016/j.ajpath.2012.06.026
ISSN1525-2191
AutoresYusuke Murakami, Yasuhiro Ikeda, Noriko Yoshida, Shoji Notomi, Toshio Hisatomi, Sugako Oka, Gabriele De Luca, Yoshikazu Yonemitsu, Margherita Bignami, Yusaku Nakabeppu, Tatsuro Ishibashi,
Tópico(s)Cytomegalovirus and herpesvirus research
ResumoRetinitis pigmentosa (RP) is a genetically heterogenous group of inherited retinal degenerative diseases resulting from photoreceptor cell death and affecting >1 million persons globally. Although oxidative stress has been implicated in the pathogenesis of RP, the mechanisms by which oxidative stress mediates photoreceptor cell death are largely unknown. Here, we show that oxidation of nucleic acids is a key component in the initiation of death-signaling pathways in rd10 mice, a model of RP. Accumulation of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dG) increased in photoreceptor cells, and especially within their nuclei, in rd10 mice as well as in Royal College of Surgeons rats, another model of RP caused by different genetic mutations. Vitreous samples from humans with RP contained higher levels of 8-oxo-dG excreted than samples from nondegenerative controls. Transgenic overexpression of human MutT homolog-1, which hydrolyzes oxidized purine nucleoside triphosphates in the nucleotide pool, significantly attenuated 8-oxo-dG accumulation in nuclear DNA and photoreceptor cell death in rd10 mice, in addition to suppressing DNA single-strand break formation, poly(ADP-ribose) polymerase activation, and nuclear translocation of apoptosis-inducing factor. These findings indicate that oxidative DNA damage is an important process for the triggering of photoreceptor cell death in rd10 mice and suggest that stimulation of DNA repair enzymes may be a novel therapeutic approach to attenuate photoreceptor cell loss in RP. Retinitis pigmentosa (RP) is a genetically heterogenous group of inherited retinal degenerative diseases resulting from photoreceptor cell death and affecting >1 million persons globally. Although oxidative stress has been implicated in the pathogenesis of RP, the mechanisms by which oxidative stress mediates photoreceptor cell death are largely unknown. Here, we show that oxidation of nucleic acids is a key component in the initiation of death-signaling pathways in rd10 mice, a model of RP. Accumulation of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dG) increased in photoreceptor cells, and especially within their nuclei, in rd10 mice as well as in Royal College of Surgeons rats, another model of RP caused by different genetic mutations. Vitreous samples from humans with RP contained higher levels of 8-oxo-dG excreted than samples from nondegenerative controls. Transgenic overexpression of human MutT homolog-1, which hydrolyzes oxidized purine nucleoside triphosphates in the nucleotide pool, significantly attenuated 8-oxo-dG accumulation in nuclear DNA and photoreceptor cell death in rd10 mice, in addition to suppressing DNA single-strand break formation, poly(ADP-ribose) polymerase activation, and nuclear translocation of apoptosis-inducing factor. These findings indicate that oxidative DNA damage is an important process for the triggering of photoreceptor cell death in rd10 mice and suggest that stimulation of DNA repair enzymes may be a novel therapeutic approach to attenuate photoreceptor cell loss in RP. Retinitis pigmentosa (RP) is a group of inherited retinal degenerative diseases resulting from rod and cone photoreceptor cell death and affecting 1.5 million people globally. Molecular genetic analyses have identified >45 genetic mutations in patients with RP1Hartong D.T. Berson E.L. Dryja T.P. Retinitis pigmentosa.Lancet. 2006; 368: 1795-1809Abstract Full Text Full Text PDF PubMed Scopus (2291) Google Scholar; however, the mechanisms by which these mutations cause photoreceptor cell death are largely unknown and these diseases remain intractable. Oxidative stress has been implicated in the pathogenesis of neurodegenerative disorders, including retinal degeneration. Retinal oxidative damage increases in animal models of RP and antioxidant treatment suppresses rod and cone photoreceptor cell death in these animals,2Komeima K. Rogers B.S. Lu L. Campochiaro P.A. Antioxidants reduce cone cell death in a model of retinitis pigmentosa.Proc Natl Acad Sci U S A. 2006; 103: 11300-11305Crossref PubMed Scopus (350) Google Scholar, 3Komeima K. Rogers B.S. Campochiaro P.A. Antioxidants slow photoreceptor cell death in mouse models of retinitis pigmentosa.J Cell Physiol. 2007; 213: 809-815Crossref PubMed Scopus (216) Google Scholar indicating the deleterious effects of oxidative damage to photoreceptor cells. By contrast, oxidative stress is also involved in cell survival signaling.4Chong Z.Z. Li F. Maiese K. Oxidative stress in the brain: novel cellular targets that govern survival during neurodegenerative disease.Prog Neurobiol. 2005; 75: 207-246Crossref PubMed Scopus (459) Google Scholar For example, hydrogen peroxide generation in response to cellular stress activates the Akt pathway and mediates the survival of retina-derived cells.5Mackey A.M. Sanvicens N. Groeger G. Doonan F. Wallace D. Cotter T.G. Redox survival signalling in retina-derived 661W cells.Cell Death Differ. 2008; 15: 1291-1303Crossref PubMed Scopus (60) Google Scholar Given the diverse functions of oxidative stress, identification of the processes by which oxidative stress induces photoreceptor cell death is critical to developing new treatments for retinal and neurodegenerative diseases. Reactive oxygen species affect cellular functions by oxidizing lipids, proteins, and nucleic acids. Excessive oxidation of DNA leads to the formation of abasic (apurinic/apyrimidinic) sites and single-strand breaks (SSBs) and initiates death-signaling pathways.6Chen J. Jin K. Chen M. Pei W. Kawaguchi K. Greenberg D.A. Simon R.P. Early detection of DNA strand breaks in the brain after transient focal ischemia: implications for the role of DNA damage in apoptosis and neuronal cell death.J Neurochem. 1997; 69: 232-245Crossref PubMed Scopus (293) Google Scholar, 7Guillet M. Boiteux S. Endogenous DNA abasic sites cause cell death in the absence of Apn1. Apn2 and Rad1/Rad10 in Saccharomyces cerevisiae.EMBO J. 2002; 21: 2833-2841Crossref PubMed Scopus (109) Google Scholar Accumulation of oxidative DNA damage is associated with various neurodegenerative diseases, such as Parkinson's disease and Alzheimer's disease.8Zhang J. Perry G. Smith M.A. Robertson D. Olson S.J. Graham D.G. Montine T.J. Parkinson's disease is associated with oxidative damage to cytoplasmic DNA and RNA in substantia nigra neurons.Am J Pathol. 1999; 154: 1423-1429Abstract Full Text Full Text PDF PubMed Scopus (528) Google Scholar, 9Nunomura A. Perry G. Aliev G. Hirai K. Takeda A. Balraj E.K. Jones P.K. Ghanbari H. Wataya T. Shimohama S. Chiba S. Atwood C.S. Petersen R.B. Smith M.A. Oxidative damage is the earliest event in Alzheimer disease.J Neuropathol Exp Neurol. 2001; 60: 759-767Crossref PubMed Scopus (1584) Google Scholar Mammalian cells have multiple repair mechanisms against oxidative damage to nucleic acids. 8-Oxo-7,8-dihydroguanine (8-oxoG) DNA glycosylase-1 excises oxidized guanine from DNA.10Boiteux S. Radicella J.P. The human OGG1 gene: structure, functions, and its implication in the process of carcinogenesis.Arch Biochem Biophys. 2000; 377: 1-8Crossref PubMed Scopus (416) Google Scholar Alternatively, the MutT homolog-1 (MTH1) hydrolyzes oxidized purine nucleoside triphosphates to the monophosphate form, thereby avoiding incorporation of oxidized nucleotides into DNA.11Maki H. Sekiguchi M. MutT protein specifically hydrolyses a potent mutagenic substrate for DNA synthesis.Nature. 1992; 355: 273-275Crossref PubMed Scopus (789) Google Scholar, 12Sakumi K. Furuichi M. Tsuzuki T. Kakuma T. Kawabata S. Maki H. Sekiguchi M. Cloning and expression of cDNA for a human enzyme that hydrolyzes 8-oxo-dGTP, a mutagenic substrate for DNA synthesis.J Biol Chem. 1993; 268: 23524-23530Abstract Full Text PDF PubMed Google Scholar We previously reported that Mth1 deficiency increases mitochondrial oxidative DNA damage and cell death in dopamine neurons after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine administration.13Yamaguchi H. Kajitani K. Dan Y. Furuichi M. Ohno M. Sakumi K. Kang D. Nakabeppu Y. MTH1, an oxidized purine nucleoside triphosphatase, protects the dopamine neurons from oxidative damage in nucleic acids caused by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.Cell Death Differ. 2006; 13: 551-563Crossref PubMed Scopus (74) Google Scholar Conversely, transgenic overexpression of human MTH1 (hMTH1) protects against the striatal degeneration induced by 3-nitropropionic acid.14De Luca G. Russo M.T. Degan P. Tiveron C. Zijno A. Meccia E. Ventura I. Mattei E. Nakabeppu Y. Crescenzi M. Pepponi R. Pezzola A. Popoli P. Bignami M. A role for oxidized DNA precursors in Huntington's disease-like striatal neurodegeneration.PLoS Genet. 2008; 4: e1000266Crossref PubMed Scopus (60) Google Scholar These findings suggest that oxidative DNA damage may play a role in the pathogenesis of neurodegeneration. However, little is known about the contribution of DNA oxidation to the photoreceptor cell death caused by genetic factors. Furthermore, it remains unclear which kind of oxidative DNA damage—mitochondrial or nuclear DNA damage or both—accumulates and how it causes photoreceptor cell death in inherited retinal degeneration. In the present study, we show that DNA oxidation was increased in an early phase of retinal degeneration in two different models of RP and in the eyes of patients with RP. Transgenic overexpression of hMTH1 in a mouse RP model attenuated accumulation of oxidative DNA damage, prevented the subsequent poly(ADP-ribose) polymerase (PARP) activation, and delayed photoreceptor cell death. These findings implicate oxidative DNA damage as an important component in the pathogenesis of RP. All animal experiments adhered to the statement of the Association for Research in Vision and Ophthalmology, and protocols were approved by the Committee for Animals, Recombinant DNA, and Infectious Pathogens Experiments at Kyushu University. B6.CXB1-Pde6βrd10/J (rd10) and age-matched wild-type (WT) C57BL/6 mice were purchased from The Jackson Laboratories (Bar Harbor, ME). Adult Royal College of Surgeons (RCS) rats and age-matched SD rats were maintained humanely. hMTH1 transgenic (hMTH1-Tg) mice were generated as described previously14De Luca G. Russo M.T. Degan P. Tiveron C. Zijno A. Meccia E. Ventura I. Mattei E. Nakabeppu Y. Crescenzi M. Pepponi R. Pezzola A. Popoli P. Bignami M. A role for oxidized DNA precursors in Huntington's disease-like striatal neurodegeneration.PLoS Genet. 2008; 4: e1000266Crossref PubMed Scopus (60) Google Scholar and backcrossed to the C57BL/6 background. Rd10 mice were crossed with hMTH1-Tg mice, and the littermates from rd10;hMTH1-Tg+/− males and rd10;hMTH1-Tg+/− females were used for the experiments. Rd10 mutation in exon 13 of the Pde6β gene [CGC (Arg) → TGC (Cys)] was identified by PCR with the use of the forward primer 5′-TCTCAGAACCCACATGTACT-3′ and reverse primer 5′-TGATTCATCTAGCCCATCC-3′ and subsequent direct sequencing. The hMTH1 transgene was analyzed by PCR with the use of previously described primers.14De Luca G. Russo M.T. Degan P. Tiveron C. Zijno A. Meccia E. Ventura I. Mattei E. Nakabeppu Y. Crescenzi M. Pepponi R. Pezzola A. Popoli P. Bignami M. A role for oxidized DNA precursors in Huntington's disease-like striatal neurodegeneration.PLoS Genet. 2008; 4: e1000266Crossref PubMed Scopus (60) Google Scholar Heterozygous and homozygous hMTH1-Tg mice were distinguished by real-time PCR with the use of primers for hMTH1 (forward primer, 5′-TTGAGTTCGTGGGCGAGC-3′; reverse primer, 5′-GCATGGGCGCATTTCG-3′; and MGB probe, 5′-FAM-CACAGACAGCATCCAG-MGB-3′) and for RNase P (forward primer, 5′-GCCGGAGCTTGGAACAGA-3′; reverse primer, 5′-GGTGCCTCACCTCAGCCAT-3′; TaqMan probe, 5′-VIC-CACGGCCAGCAGTGCGAGTTCA-TAMRA-3′; Applied Biosystems, Foster City, CA). Immunostaining for 8-oxo-dG in nuclear and mitochondrial DNA was performed as previously described.13Yamaguchi H. Kajitani K. Dan Y. Furuichi M. Ohno M. Sakumi K. Kang D. Nakabeppu Y. MTH1, an oxidized purine nucleoside triphosphatase, protects the dopamine neurons from oxidative damage in nucleic acids caused by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.Cell Death Differ. 2006; 13: 551-563Crossref PubMed Scopus (74) Google Scholar Deparaffinized sections were pretreated with 5 mg/mL RNase (Sigma-Aldrich, St. Louis, MO) at 37°C for 1 hour to eliminate cellular RNA. To increase the sensitivity of 8-oxo-dG in nuclear DNA, the sections were further treated with 2N HCl at room temperature for 10 minutes, and then washed with Tris-HCl, pH 7.5, for 10 minutes. Samples pretreated with RNase or RNase + HCl were incubated with N45.1 anti-8-oxo-dG antibody [1:100; Japan International Cooperation Agency (JAICA), Fukuroi, Japan] at 4°C overnight. Then signals were developed with the avidin-biotinylated peroxidase complex method (Dako, Carpinteria, CA). A nonimmune serum was used as a negative control. A total of 20 eyes with epiretinal membrane (ERM; idiopathic: 13 eyes of 12 patients; secondary to RP: 7 eyes of 6 patients, respectively) underwent a pars plana vitrectomy at Kyushu University Hospital (Fukuoka, Japan), and the vitreous fluid was collected. The investigation was performed with approval from the institutional review board and was conducted in accordance with the tenets of the Declaration of Helsinki on biomedical research involving human subjects. The details on patients with RP are given (see Supplemental Table S1 at http://ajp.amjpathol.org). All six patients with RP developed rod-cone dystrophy bilaterally, with a variable extent of visual restriction. The visual fields were evaluated with Goldmann kinetic perimetry, and the vertical diameter of the visual field for the I/4 target was measured as previously described.15Murakami T. Akimoto M. Ooto S. Suzuki T. Ikeda H. Kawagoe N. Takahashi M. Yoshimura N. Association between abnormal autofluorescence and photoreceptor disorganization in retinitis pigmentosa.Am J Ophthalmol. 2008; 145: 687-694Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar No patient had any associated hearing loss or other systemic diseases. The mode of inheritance was autosomal dominant in one patient, autosomal recessive in two patients, and spontaneous (an individual patient with no family history of RP) in three patients. The frequency of the type of inheritance was consistent with a previous study of Japanese patients with RP (16.9% dominant, 25.2% recessive, 1.6% X-linked, and 56.3% spontaneous).16Hayakawa M. Fujiki K. Kanai A. et al.Multicenter genetic study of retinitis pigmentosa in Japan, I: genetic heterogeneity in typical retinitis pigmentosa.Jpn J Ophthalmol. 1997; 41: 1-6Crossref PubMed Scopus (23) Google Scholar Genetic tests were not performed. Total DNA in the mouse and rat retinas was extracted with a DNeasy Blood and Tissue Kit (QIAGEN, Valencia, CA). At all of the stages of manipulation 10 mmol/L 2,2,6,6-tetramethylpiperidine-N-oxyl (Wako, Osaka, Japan) was added, as previously described.17Tsuruya K. Furuichi M. Tominaga Y. Shinozaki M. Tokumoto M. Yoshimitsu T. Fukuda K. Kanai H. Hirakata H. Iida M. Nakabeppu Y. Accumulation of 8-oxoguanine in the cellular DNA and the alteration of the OGG1 expression during ischemia-reperfusion injury in the rat kidney.DNA Repair (Amst). 2003; 2: 211-229Crossref PubMed Scopus (85) Google Scholar The DNA samples were digested with 5 units of Nuclease P1 (Sigma-Aldrich) at 37°C for 1 hour in 20 mmol/L sodium acetate, pH 5.2, followed by treatment with 10 units of alkaline phosphatase (Sigma-Aldrich) at 37°C for 1 hour in 100 mmol/L Tris, pH 7.5. The reaction mixture was centrifuged for 5 minutes at 10,000 × g, and the supernatant fluid was used for 8-oxo-dG enzyme-linked immunosorbent assay (ELISA) (Cell Biolabs, San Diego, CA) according to the instructions of the manufacturer. Briefly, 1 μg of digested DNA samples or 8-oxo-dG standards was incubated with anti-8-oxo-dG antibody in wells precoated with 8-oxo-dG. The antibodies that bound to 8-oxo-dG in the digested DNA samples were washed away, and the antibodies that bound to 8-oxo-dG on the plate were quantified with enzyme-labeled secondary antibody. The 8-oxo-dG concentration obtained from a standard curve was converted to the 8-oxo-dG amount in 1 μg total DNA with the following calculation: [8-oxo-dG concentration (pg/μL) × 50 μL]/1 μg total DNA. Vitreous samples were centrifuged at 10,000 × g for 5 minutes, and the supernatant fluids were subjected to 8-oxo-dG ELISA assay as described earlier. The mouse eyes were enucleated, fixed with 4% paraformaldehyde in PBS for 24 hours, and then mounted in paraffin. Sections 5 μm thick were prepared along the horizontal meridian. The sections were subsequently stained with H&E. Five sections were randomly selected from each eye. The number of cells in the outer nuclear layer (ONL) was counted in a 200 μm × 200 μm square area in the central (400 μm from the optic nerve), mid-peripheral (800 μm from the optic nerve), and peripheral (1200 μm from the optic nerve) regions of the retina in the nasal hemisphere. The tissue samples were assigned numbers and letters, and the conditions were masked from the observers. The TUNEL procedure and quantification of TUNEL-positive cells were performed with an ApopTag Fluorescein Direct In Situ Apoptosis Detection Kit (Chemicon International, Temecula, CA) according to the instructions of the manufacturer. The mid-peripheral retina was photographed, and the number of TUNEL-positive cells in the ONL was counted by masked observers. The retinal area was measured by Image J software version 1.43u (National Institutes of Health, Bethesda, MD). The data are expressed as TUNEL-positive cells/mm2 of retinal area. Immunofluorescence was performed as previously reported.18Murakami Y. Ikeda Y. Yonemitsu Y. Onimaru M. Nakagawa K. Kohno R. Miyazaki M. Hisatomi T. Nakamura M. Yabe T. Hasegawa M. Ishibashi T. Sueishi K. Inhibition of nuclear translocation of apoptosis-inducing factor is an essential mechanism of the neuroprotective activity of pigment epithelium-derived factor in a rat model of retinal degeneration.Am J Pathol. 2008; 173: 1326-1338Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar Paraffinized sections were used for single-strand DNA and poly(ADP-ribose) (PAR) staining, and cryosections were used for apoptosis-inducing factor (AIF) and voltage-dependent anion-selective channel (VDAC) staining. Samples were incubated with anti-single strand DNA (1:200; Dako), anti-PAR (1:200; Trevigen, Gaithersburg, MD), anti-AIF (1:100; Cell Signaling, Danvers, MA), or anti-VDAC (1:100; Cell Signaling) at 4°C overnight. A nonimmune serum was used as a negative control. Alexa Fluor 488- or Alexa Fluor 594-conjugated antibodies (Invitrogen, Carlsbad, CA) was used as secondary antibody. Immunofluorescent images were acquired with an Olympus BX51 microscope (Olympus, Tokyo, Japan) with a fluorescence attachment or Leica confocal microscope (Leica, Wetzlar, Germany). Double-staining pictures were taken with Leica confocal microscope. The neural retina was collected at the indicated time points. Protein (30 μg) per sample was separated on SDS-PAGE and transferred onto a polyvinylidene difluoride membrane. After blocking with 3% nonfat dried milk, the membrane was reacted with anti-PAR (1:3000; Trevigen). Immunoreactivity for PAR was visualized with the ECL Plus detection reagents (Amersham Biosciences, Buckinghamshire, United Kingdom). Lane-loading differences were normalized by β-actin (1:1000; Cell Signaling). Calpain activity was measured with a Calpain Activity Assay kit (Biovision, Mountain View, CA) according to the instructions of the manufacturer. The calpain substrate (Ac-LLY-AFC) was incubated with retinal lysates (200 μg of protein) for 60 minutes at 37°C in the dark. 3,4-Dihydro-5-[4-(1-piperidinyl)butoxy]−1(2H)-isoquinolinone (DPQ; PARP inhibitor) and MDL28170 (calpain inhibitor) were purchased from Enzo Lifesciences (Plymouth Meeting, PA). Rd10 mice received once daily intraperitoneal injection of DPQ (20 μg/g), MDL28170 (25 μg/g), or vehicle (1% dimethyl sulfoxide in PBS) from p19 to p26. All values were expressed as the mean ± SEM. Statistical differences between two groups were analyzed by Mann–Whitney U-test. Multiple group comparison was performed by analysis of variance followed by Tukey–Kramer adjustments. Differences were considered significant at P < 0.05. Rd10 mice are an animal model of RP harboring homozygous mutation in the β-subunit of the Pde6β gene that develop progressive retinal degeneration with onset around p18.19Chang B. Hawes N.L. Hurd R.E. Davisson M.T. Nusinowitz S. Heckenlively J.R. Retinal degeneration mutants in the mouse.Vision Res. 2002; 42: 517-525Crossref PubMed Scopus (636) Google Scholar The course of retinal degeneration in rd10 mice is slower than that of rd1 mice, another Pde6β-mutant mouse model, and it has been suggested that rd10 mice might be more analogous to later onset forms of RP.20Pang J.J. Boye S.L. Kumar A. Dinculescu A. Deng W. Li J. Li Q. Rani A. Foster T.C. Chang B. Hawes N.L. Boatright J.H. Hauswirth W.W. AAV-mediated gene therapy for retinal degeneration in the rd10 mouse containing a recessive PDEbeta mutation.Invest Ophthalmol Vis Sci. 2008; 49: 4278-4283Crossref PubMed Scopus (111) Google Scholar To assess the levels and distribution of oxidative DNA damage in retinal degeneration, we performed immunohistochemistry for 8-oxo-dG, a major oxidative lesion in DNA or nucleotides.21Kasai H. Nishimura S. Hydroxylation of deoxyguanosine at the C-8 position by ascorbic acid and other reducing agents.Nucleic Acids Res. 1984; 12: 2137-2145Crossref PubMed Scopus (883) Google Scholar We previously described that the N45.1 monoclonal antibody preferentially detects 8-oxo-dG in mitochondrial DNA in samples pretreated with RNase, whereas it reacts with 8-oxo-dG in nuclear DNA in samples pretreated with RNase and HCl.13Yamaguchi H. Kajitani K. Dan Y. Furuichi M. Ohno M. Sakumi K. Kang D. Nakabeppu Y. MTH1, an oxidized purine nucleoside triphosphatase, protects the dopamine neurons from oxidative damage in nucleic acids caused by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.Cell Death Differ. 2006; 13: 551-563Crossref PubMed Scopus (74) Google Scholar Thus, we applied these two different pretreatments for the retinal sections. Among samples pretreated with RNase alone, the p21 rd10 retina sections showed stronger immunoreactivity for 8-oxo-dG in the photoreceptor cell layer compared with the WT retina sections. Unexpectedly, however, the signal was mainly localized in the ONL but not in the inner segment (IS), a mitochondria-rich portion of photoreceptor cells (Figure 1A). Double immunostaining with 8-oxo-dG and a mitochondrial marker, VDAC, showed that most of the 8-oxo-dG staining was not colocalized with VDAC (see Supplemental Figure S1 at http://ajp.amjpathol.org), suggesting that the photoreceptor nuclear DNA in rd10 mice may be strongly oxidized and that the N45.1 antibody may detect the strong 8-oxo-dG accumulation in the photoreceptor nuclei even without HCl pretreatment. In accordance with this idea, rd10 retina samples pretreated with RNase and HCl exhibited intense 8-oxo-dG immunoreactivity in photoreceptor nuclei (Figure 1A). In contrast, the WT retina showed minimal immunoreactivity even with HCl pretreatment (Figure 1A). No positive staining was found by nonimmune mouse IgG in samples with either pretreatment (data not shown). We next assessed oxidative DNA damage in RCS rats, another clinically relevant model of RP caused by a Mertk mutation.22D'Cruz P.M. Yasumura D. Weir J. Matthes M.T. Abderrahim H. LaVail M.M. Vollrath D. Mutation of the receptor tyrosine kinase gene Mertk in the retinal dystrophic RCS rat.Hum Mol Genet. 2000; 9: 645-651Crossref PubMed Scopus (727) Google Scholar, 23Gal A. Li Y. Thompson D.A. Weir J. Orth U. Jacobson S.G. Apfelstedt-Sylla E. Vollrath D. Mutations in MERTK, the human orthologue of the RCS rat retinal dystrophy gene, cause retinitis pigmentosa.Nat Genet. 2000; 26: 270-271Crossref PubMed Scopus (550) Google Scholar The retina of p28 RCS rats showed strong immunoreactivity for 8-oxo-dG in the ONL, which was further enhanced by HCl pretreatment (Figure 1B). In contrast, the normal rat retina sections used as controls showed minimal staining for 8-oxo-dG with either pretreatment (Figure 1B). Quantitative measurement of 8-oxo-dG by ELISA confirmed that the amounts of 8-oxo-dG in total DNA prepared from the retina of rd10 mice and RCS rats were significantly higher than those in the controls (Figure 1, C and D). 8-Oxo-dG content excreted in the urine, serum, or cerebrospinal fluid has been shown to serve as a biomarker of oxidative damage24Shigenaga M.K. Gimeno C.J. Ames B.N. Urinary 8-hydroxy-2′-deoxyguanosine as a biological marker of in vivo oxidative DNA damage.Proc Natl Acad Sci U S A. 1989; 86: 9697-9701Crossref PubMed Scopus (699) Google Scholar and to be increased in patients with Parkinson's disease or acute encephalopathy.25Sato S. Mizuno Y. Hattori N. Urinary 8-hydroxydeoxyguanosine levels as a biomarker for progression of Parkinson disease.Neurology. 2005; 64: 1081-1083Crossref PubMed Scopus (100) Google Scholar, 26Shiihara T. Kato M. Ichiyama T. Takahashi Y. Tanuma N. Miyata R. Hayasaka K. Acute encephalopathy with refractory status epilepticus: bilateral mesial temporal and claustral lesions, associated with a peripheral marker of oxidative DNA damage.J Neurol Sci. 2006; 250: 159-161Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar Therefore, we next examined the levels of 8-oxo-dG excretion in the vitreous samples from human patients with RP and nondegenerative `controls (idiopathic ERM). The vitreous levels of 8-oxo-dG in patients with ERM secondary to RP (0.75 ± 0.06 ng/mL) were significantly higher than levels in patients with ERM alone (Figure 1E). To assess the role of oxidative DNA damage in inherited retinal degeneration, we crossed rd10 mice with transgenic mice expressing hMTH1 cDNA.14De Luca G. Russo M.T. Degan P. Tiveron C. Zijno A. Meccia E. Ventura I. Mattei E. Nakabeppu Y. Crescenzi M. Pepponi R. Pezzola A. Popoli P. Bignami M. A role for oxidized DNA precursors in Huntington's disease-like striatal neurodegeneration.PLoS Genet. 2008; 4: e1000266Crossref PubMed Scopus (60) Google Scholar The number of photoreceptor cells was counted in the peripheral, mid-peripheral, and central regions of the retina at p26. The results showed that there was a 1.5-fold increase in the number of photoreceptor cells at the mid-peripheral retina of rd10 mice with a hemizygous hMTH1 transgene (rd10;hMTH1-Tg+/−) compared with control rd10 mice (rd10;hMTH1-Tg−/−) (Figure 2, A and B). Rd10 mice with a homozygous hMTH1 transgene (rd10;hMTH1-Tg+/+) exhibited significant protection of photoreceptor cells at the mid-peripheral and peripheral retina (Figure 2, A and B) and retained 1.8-fold higher number of photoreceptor cells than control rd10 mice at the mid-peripheral retina. In contrast, the retinal structure in WT mice was not affected by the homozygous hMTH1 transgene (see Supplemental Figure S2 at http://ajp.amjpathol.org). Because the mid-peripheral region showed the greatest prevention of photoreceptor cell loss by hMTH1 overexpression in rd10 mice, we assessed this region of the retina in the following experiments. TUNEL staining confirmed that TUNEL-positive cells in the ONL were substantially reduced in rd10;hMTH1-Tg+/− and rd10;hMTH1-Tg+/+ mice compared with TUNEL-positive cells in rd10;hMTH1-Tg−/− mice (Figure 2, C and D). Furthermore, immunohistochemistry for 8-oxo-dG showed that the hemizygous hMTH1 transgene decreased 8-oxo-dG accumulation in photoreceptor nuclei, and this decrease was augmented by the homozygous hMTH1 transgene (Figure 2E). These data indicate that oxidative DNA damage in photoreceptor nuclei contributes to the photoreceptor cell death in rd10 mice. We recently described that accumulation of oxidative DNA damage results in the formation of SSBs in nuclear and mitochondrial DNA in mouse embryonic fibroblasts.27Oka S. Ohno M. Tsuchimoto D. Sakumi K. Furuichi M. Nakabeppu Y. Two distinct pathways of cell death triggered by oxidative damage to nuclear and mitochondrial DNAs.EMBO J. 2008; 27: 421-432Crossref PubMed Scopus (179) Google Scholar In addition, we reported that DNA oxidation in the nucleus and mitochondria independently triggers two distinct cell death pathways: nuclear DNA oxidation mediates PARP activation and mitochondrial DNA oxidation activates calpain. Thus, we next examined the involvement of these pathways during retinal degeneration. Immunofluorescence for single-strand DNA showed intense staining in photoreceptor nuclei of rd10 mice but not of WT mice at p21 (Figure 3A). We also performed immunofluorescence for PAR polymer, because this polymer is known to be produced on PARP activation. The results showed intense staining for PAR in the ONL of rd10 mice (Figure 3B). In contrast, the WT retina showed weak staining for PAR in the ONL (Figure 3B). Western blot analysis for PAR found that the rd10 retina contained poly(ADP)-ribosylated proteins of ∼110 kDa and ∼170 kDa, whereas the WT retina contained mainly the ∼110-kDa protein (Figure 3C). This higher band of PAR-protein is reported to be produced during PARP-dependent cell death.28Yu S.W. Wang H. Poitras M.F. Coombs C. Bowers W.J. Federoff H.J. Poirier G.G. Dawson T.M. Dawson V.L. Mediation of poly(ADP-ribose) polymerase-1-dependent cell death by apoptosis-inducing factor.Science. 2002; 297: 259-263Crossref PubMed Scopus (1569) Google Scholar Densitometric analysis showed that the amount of the ∼170-kDa PAR-protein was increased 28-fold in the rd10 retina compared with the WT retina (Figure 3D), suggesting that the PARP pathway is activated in rd10 mice. However, calpain activity in retinal extracts incre
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