Increased mitochondrial DNA damage and down-regulation of DNA repair enzymes in aged rodent retinal pigment epithelium and choroid

Animal models

Six male C57BL/6 mice in each group (5 month and 22 month) and twelve male Brown-Norway rats in each group (5 month and 26 month) were provided by National Institute on Aging. Animal were housed conventionally, under ambient conditions (10 h dark, 14 h light) and were fed standard rodent chow and water ad libitum. Six frozen eyes of male C57BL/6 mice in each group (4, 12, 18, and 24 month) and ten frozen eyes of male F344xBN F1 rats in each group (4, 18, 24, and 32 month) were provided by the tissue bank of National Institute on Aging. All experiments were performed in accordance with the United States Public Health Service Policy on Humane Care and Use of Laboratory Animals, the National Institutes of Health Guide for the Care and Use of Laboratory Animals, the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and institutional, federal, and state guidelines regarding animal research.

Immunohistochemistry

Enucleated eyes were fixed in 2% wt/vol paraformaldehyde in 0.01 M phosphate buffered saline (PBS; pH 7.4) at 4 °C overnight. Six live animals were used for each group in all immunohistochemistry experiments. Immunohistochemistry was performed on paraffin sagittal sections of eyes after pigment bleaching for 1:200 dilution 8-hydroxy-2’-deoxy-guanosine (8-OHdG; Oxis, Foster City, CA), using the Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA) and diaminobenzidine as a substrate. As a negative control, sections were treated in the same manner, except that incubation with primary antibody was omitted. The sections were treated for equal time in DAB reagent and photographed at the same time.

8-hydroxy-2’-deoxy-guanosine ELISA

Genomic DNA was isolated using a DNeasy Blood & Tissue Kit (Qiagen, Valencia, CA) from six live rat RPE and choroid, following hydrolysis with a four enzyme mixture including DNase I, phosphodiesterases I and II and alkaline phosphatase [12]. Competitive ELISA assays for 8-OHdG were performed according to the manufacture’s protocol (Bioxytech 8-OHdG-EIA kit; Oxis). Briefly, the 8-OHdG antibody and the sample were added to ELISA plate which has been precoated with 8-OHdG. The 8-OHdG in the sample competes with the 8-OHdG bond on the plate for the 8-OHdG antibody bites binding sties. The antibodies that are bound to the 8-OHdG in the sample were washed out of the well, while those that have bound to the 8-OHdG coated on the plate will remain. Following secondary antibody and chromogen, the color reaction was terminated and the absorbance was measured. 
Line 100~102: Standard 8-OHdG was assayed over a concentration range of 0.125 to 20 ng/ml in duplicates for each experiment. The average concentration of 8-OHdG per microgram of DNA for each group was calculated for each sample. Sample DNA assays were performed in duplicate. Standard 8-OHdG was assayed over a concentration range of 0.125 to 20 ng/ml in duplicates for each experiment the average concentration of 8-OHdG per microgram of DNA for each group was calculated for each sample. Controls without added DNA and appropriate blanks were also incorporated into experiments.

Long extension-polymerase chain reaction

Long extension-polymerase chain reaction (LX-PCR) was performed as previously described [13]. Genomic DNA in RPE and choroid from five frozen rat eyes in each group was isolated with DNeasy Blood & Tissue Kit (Qiagen). The quantitation of the purified genomic DNA, as well as of PCR products, was performed fluorometrically using the PicoGreen dsDNA reagent (Invitrogen, Carlsbad, CA). LX-PCR was performed with the GeneAmp LX-PCR was performed with the GeneAmp XL PCR system (Applied Biosystems), using rTth DNA Polymerase XL enzyme which is designed to amplify target DNA sequences up to about 40 kb. The amounts of primers were 20 pmol, and the Mg²⁺ concentration was 1.1 mM. The four pairs of PCR primers employed in this study are given in Table 1. All the protocols were initiated by a hot start (75 °C, 2 min) before addition of rTth enzyme. For amplification of a long fragment of mtDNA, the standard thermocycler program included initial denaturation at 94 °C for 1 min, 18 cycles of 94 °C for 15 s, 65 °C for 12 min, with final extension at 72 °C for 10 min. Amplification of a short mtDNA fragment (235 bp) was done with the same program except the extension temperature was 60 °C. To amplify a long (12.5 kB) nDNA fragment, the thermocycler profile included initial denaturation at 94 °C for 1 min, 28 cycles of 94 °C 15 s, 65 °C 12 min, with final extension at 72 °C for 10 min. A short nDNA fragment (195 bp) was amplified with the same program except the extension temperature was 60 °C.

DNA damage was quantified by comparing the relative efficiency of amplification of large fragments of DNA (13.4 kB from mtDNA and 12.5 kB for nDNA) and normalizing this to the amplification of smaller (235 bp and 195 bp) fragments. The template DNA (5~15 ng) was varied so that PCR products were obtained during the long phase of the PCR amplification.

Amplification of total, non-deleted, and deleted mitochondrial DNA

Genomic DNA in RPE and choroid from five frozen rat eyes in each group was isolated. Three nonoverlapping regions of the mitochondrial genome were amplified by conventional PCR to evaluate the relative proportion of total, non-deleted, and deleted mtDNA in old rats, compared to those in young rats. The three pairs of 20-nucleotide PCR primers (Table 2) employed in this study were similar in GC content and melting temperatures. The total primer pair amplified a segment of mtDNA outside of the 4.8 kB common deletion; the non-deleted primer pair amplified a region within the common deletion; and the deletion primer pair amplified a product only in the presence of the common deletion, which would bring the primer pairs within 500 bp. Thermocycling included one cycle of 94 °C for 5 min, followed by 40 cycles of 94 °C denaturing for 45 s, 55 °C annealing for 45 s, and 72 °C extension for 60 s. A final 72 °C extension for 5 min completed the PCR. Negative controls were included that contained the PCR reagents without template DNA.

To determine the amounts of DNA products obtained in these reactions, we isolated total DNA and used the aforedescribed three primers for amplification of total, non-deleted, and deleted mtDNA in young and aged RPE and choroid. The amount of PCR product was determined fluorometrically using the PicoGreen dsDNA quantitation reagent (Invitrogen).

Real time reverse transcriptase–polymerase chain reaction for DNA repair enzymes

Total cellular RNA in RPE and choroid from 6 frozen mice eyes was isolated and purified (PicoPure^TM; Arcturus, Mountain View, CA). Samples of the total starting RNA were analyzed by capillary electrophoresis (Agilent Technologies, Palo Alto, CA) to assess the degree of purification. Real time quantitative RT–PCR (qRT–PCR) was done using the SYBR-Green dye binding method implemented on an Applied Biosystems 7900 genetic analyzer by the Functional Genomics Facility at the University of Chicago. Validated primers for each gene of interest were designed for each target mRNA (Table 3). Optimization of primers and determination of the input cDNA levels were done to ensure appropriate cycle time response. Relative expression was calculated from the differences in cycle time of an internal standard (18s RNA) compared to the target mRNA. All qRT–PCR reactions used 40 ng of cDNA produced as described above and were run with two sets of duplicates from six different animals in each group.

Immunoblot

RPE and choroid from six rats (4 month and 32 month) were dissected and pooled, lysed in buffer (20 mM HEPES, pH 7.0, 10 mM KCl, 2 mM MgCl₂, 0.5% Nonidet P-40, 1 mM Na₃VO₄, 1 mM PMSF, and 0.15 U ml^–1 aprotinin) and homogenized. Protein concentrations were determined using the Bradford colorimetric assay. Thirty micrograms of each protein lysate were loaded in each lane in sample buffer (2% SDS, 10% glycerol, 0.001% bromophenol blue, 1% DTT, and 0.05 M Tris-HCl, pH 6.8), separated on 10% SDS–PAGE (Invitrogen), and transferred to a PVDF membrane (Millipore, Temecula, CA). The blots were blocked with 5% nonfat milk in PBS and incubated with specific rabbit polyclonal antibody against 1:200 8-oxoguanine-DNA glycosylase 1 (OGG1), mutY homolog (MYH) (Novus) followed by peroxidase-conjugated goat antirabbit IgG_2a (Millipore) and the enhanced chemoluminescence detection system (Amersham Biosciences, Arlington Heights, IL). The differences in expression levels were determined by scanning gels and determining the integrated areas of the bands using Image-J software. Data are expressed as normalized ratios to actin.

Statistical analyses

Data are presented as mean±standard error of the mean (SEM) with statistical differences between groups analyzed by a standard Student two-tailed t-test using GraphPad Prism software. A p value of less than 0.05 was considered statistically significant.

8-hydroxy-2’-deoxy-guanosine immunohistochemistry

We compared young and aged RPE and choroid tissues for the presence of 8-OHdG as an indicator of oxidative DNA damage. The labeling intensity in mice and rats was higher in the old RPE and choroid than the young RPE and choroid. Figure 1 shows the immunolabeling of RPE and choroid from young mice (5 month, n=6) and young rats (5 month, n=6) compared to old mice (22 month, n=6) and old rats (26 month, n=6). Although relative intensity differed from cell to cell, RPE and choroid from old eyes has compact labeling, indicating increased oxidative damage to DNA. The specificity of the antibody that we used has been established [14], and we further confirmed the specificity for immunohistochemistry by observing that there was no reduction in labeling following pretreatment with DNAase-free RNAase (data not shown).

8-hydroxy-2’-deoxy-guanosine ELISA

We complemented the immunohistochemical results using ELISA. Because greater amounts of DNA were available from rat RPE and choroid tissues, which are substantially larger than mice, we performed ELISA determinations for 8-OHdG on young (5 month) and old (26 month) rat samples. ELISA also provided a quantitate estimate of oxidative DNA damage. In the young group, the level of 8-OHdG was 3.7±0.3 ng/ml/µg DNA. In the old group, the level of 8-OHdG was 5.8±0.5 ng/ml/µg DNA (Figure 1E). The amount of 8-OHdG in DNA was significantly increased in aged RPE and choroid (p=0.007, n=6).

Long extension quantitative polymerase chain reaction of mitochondrial DNA and nuclear DNA

Cellular DNA is composed of mtDNA and nDNA. In our study of the RPE and choroid, we used quantitative PCR-based measurements to evaluate oxidative damage in nDNA and mtDNA. These methods required only small amounts of genomic DNA and were used to directly compare damage to nDNA and mtDNA in the same sample [13,14]. In postmitotic cells, greater levels of oxidative DNA damage with age occur more commonly in mtDNA than in nDNA [15], which is consistent with our findings.

We used an independent method to determine global DNA damage to both nDNA and mtDNA. The quantitative PCR assay of DNA damage is based on the principle that many kinds of DNA lesions can slow down or block the progression of DNA polymerase [13]. Therefore, equal amounts of DNA in less damaged samples will amplify to a greater extent than samples with more damaged DNA. DNA products from the polymerase (rTth) mediated amplification of long fragments of mtDNA (13.4 kB) and nDNA (12.5 kB) from RPE and choroid of frozen eyes (4, 18, 24, and 32 month) were measured using the PicoGreen reagent that is selective for dsDNA products. These data were normalized by the measured level of the short fragment of mtDNA and nDNA obtained using the same DNA template.

As shown in Figure 2A, there was a significant decrease in mtDNA amplification beginning at 18 month (2.6±0.1; p=0.004, n=5), which progressively decreased at 24 month (1.9±0.1; p<0.0001, n=5) and at 32 month (1.7±0.1; p<0.0001, n=5), compared with the 4 month group (3.5±0.2; n=5). As shown in Figure 2B, there was no change in the relative nDNA amplification comparing the 4 month group (1.16±0.05, n=5) to 18 month group (1.18±0.13, p=0.54, n=5), or to 24 month group (1.09±0.12, p=0.06, n=5). However, the 32 month group (1.01±0.14, p=0.002, n=5) exhibited a significant decrease in nDNA amplification compared to the 4 month group (1.16±0.05, n=5).

Mitochondrial DNA polymerase chain reaction

Amplification of total, non-deleted (not damaged), and deleted (damaged) PCR products of mtDNA were also done using total genomic DNA from rat RPE and choroid of all groups as a template. The total input of genomic DNA was 10 ng for each PCR reaction. Amplification products representing the total, non-deleted, and deleted mtDNA were detected in young and old rat RPE and choroid (Figure 3). Non-deleted mtDNA remained stable through much of the lifespan at 18 month (0.9±0.02; p=0.02, n=5), 24 month (0.9 ±0.05; p=0.06, n=5), and 32 month (0.9±0.1; p=0.70, n=5), compared with 4 month (1.0 ±0.02, n=5). However, there was a significant increase in deleted mtDNA at 18 month (0.2±0.1) (p=0.003, n=5), at 24 month (0.4±0.05; p<0.0001, n=5) and at 32 month (0.3±0.1; p<0.0001, n=5), compared with the 4 month group (0.1±0.03). These data independently confirm the long extension PCR experiments that demonstrated an increase with age in the level of mtDNA containing deletion products in old RPE and choroid tissue.

Real time reverse transcriptase polymerase chain reaction for DNA repair enzymes

We investigated the changes in expression of a subset of enzymes associated with DNA repair functions in the mouse RPE and choroid. OGG1, MYH, thymine DNA glycosylase (TDG), and NTH1 remove modified bases in the first step of the DNA repair process [4,7]. Another commonly studied DNA repair enzyme is NTH1. Figure 4 illustrates the results of quantitative RT–PCR experiments for these enzymes comparing RPE and choroid at different ages. The expression level of OGG1 was decreased at 18 month (p=0.01, n=6), but showed no difference at 12 month (p=0.7, n=6) and 24 month (p=0.07, n=6), compared with the 4 month group (Figure 4A). The expression level of MYH was decreased at 18 month (p=0.001, n=6) and 24 month (p=0.007, n=6), but showed no difference at 12 month (p=0.87, n=6), compared with the 4 month group (Figure 4B). The expression levels of TDG was decreased at 18 month (p<0.0001, n=6) and at 24 month (p=0.03, n=6), but showed no difference at 12 month (p=0.35, n=6), compared with the 4 month group (Figure 4C). The expression level of NTH1 was not different at all ages tested (p>0.05, n=6; see Figure 4D).

Protein levels of DNA repair enzymes

Protein levels of the enzymes for DNA repair in RPE and choroid were determined using antibodies against OGG1 and MYH. As shown in Figure 5, there was significant decrease in OGG1 at 32 month (0.91±0.03) (p=0.02, n=3), compared with the 4 month group (1.04±0.01); in MYH at 32 month (0.78±0.01) (p=0.001, n=3), compared with the 4 month group (0.97±0.02).