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Age-related alterations in the expression of MTH2 in the hippocampus of the SAMP8 mouse with learning and memory deterioration
Journal of the Neurological Sciences 287 (2009) 188–196
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Journal of the Neurological Sciences j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j n s
Age-related alterations in the expression of MTH2 in the hippocampus of the SAMP8 mouse with learning and memory deterioration Jun-De Zheng a,1,2, Ai-Lian Hei a,2, Ping-Ping Zuo b, Yi-Long Dong b, Xiao-Ning Song a, Yasumitsu Takagi c, Mutsuo Sekiguchi c, Jian-Ping Cai a,⁎ a b c
The Key Laboratory of Geriatrics, Beijing Hospital & Beijing Institute of Geriatrics, Ministry of Health, No. 1, DaHua Road, Dong Dan, Beijing 100730, PR China Peking Union Medical College & Chinese Academy of Medical Sciences, No. 9, Dong Dan Santiao, DongCheng District, Beijing 100730, PR China Frontier Research Center, Fukuoka Dental College, Fukuoka 814-0193, Japan
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Article history: Received 17 June 2009 Accepted 31 July 2009 Available online 6 September 2009 Keywords: Mouse model Oxidative stress 8-oxoguanine MTH2 Aging Alzheimer's disease Mutation 8-oxo-dGTPase
a b s t r a c t MutT-related proteins degrade 8-oxo-7,8-dihydrodeoxyguanosine triphosphate (8-oxo-dGTP), a mutagenic substrate for DNA synthesis in the nucleotide pool, thereby preventing DNA replication errors. MTH2 (Mut T homolog 2), which belongs to this family of proteins, possesses 8-oxo-7,8-dihydro-2′-deoxyguanosine triphosphatase (8-oxo-dGTPase) activity and appears to function in the protection of the genetic material from the untoward effects of endogenous oxygen radicals. To examine the roles of MTH2 in the aging process, we used the senescence-accelerated prone mouse 8 (SAMP8), which exhibits early aging syndromes and declining abilities of learning and memory. Immunohistochemical and western blot analysis revealed that the level of MTH2 protein in the hippocampus of the SAMP8 mouse progressively decreases beginning from four months after birth, whereas no such change was observed in the control senescence-accelerated resistant mouse 1 (SAMR1). Under these conditions, 8-oxoguanine accumulates in the nuclear DNA in the CA1 and CA3 subregions of the hippocampus of SAMP8 in an age-dependent manner. In SAMR1 mice, accumulation of 8-oxoguanine in the DNA was not observed. These results suggest that the MTH2 deficiency might be one of the causative factors for accelerated aging. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Reactive oxygen species (ROS), such as superoxide and hydroxyl radicals, are produced through normal cellular metabolism, and the formation of such radicals is further enhanced by the exposure to certain physical and chemical agents [1,2]. Nucleic acids exposed to ROS generate various modified bases, among which 8-oxo-7,8-dihydroguanine (8-oxoguanine) is the most important. Unlike other types of oxidatively altered bases, 8-oxoguanine does not block nucleic acid synthesis but rather induces base mispairing, since it has the potential to pair with adenine as well as cytosine. This mispairing is thought to significantly contribute to the levels of spontaneous mutation frequency [3–6], and, when 8-oxoguanine is present in messenger RNA, it possibly causes alterations in gene expression [7,8]. Organisms are equipped with elaborate mechanisms for counteracting such deleterious effects of 8-oxoguanine. In mammalian cells, two types of glycosylases, MYH and OGG1, appear to function in the prevention of mutations caused by 8-oxoguanine in DNA. The MYH
⁎ Corresponding author. E-mail address: [email protected] (J.-P. Cai). 1 Present address: The First Affiliated Hospital of Guangzhou Medical College, Guangzhou 510120, PR China. 2 These authors contributed equally to this work. 0022-510X/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jns.2009.07.027
protein excises adenine paired with 8-oxoguanine, while the OGG1 protein removes 8-oxoguanine paired with cytosine from oxidatively damaged DNA [9–12]. Mammalian cells also possess MutT-related enzymes, which are capable of eliminating 8-oxoguanine-containing nucleotides from the DNA precursor pool [13–15]. These include MTH1 (NUDT1) and MTH2 (NUDT15), which hydrolyze an oxidized form of dGTP, 8-oxo-dGTP, to 8-oxo-dGMP, a non-usable form for DNA synthesis [16,17]. The central nervous system (CNS) is thought to be particularly susceptible to oxidative stress due to the high rate of oxygen consumption in the brain and the low level of antioxidant enzymes compared with other somatic tissues [18,19]. Therefore, ROS have been proposed as the pathogenic factor in various neurological disorders, including Alzheimer's disease (AD) [20–23]. Recent studies have focused on the relationship between AD and enzymes acting on oxidatively damaged DNA and its precursors. The activity of human OGG1 has been shown to be significantly decreased in the nuclear protein samples from brains of AD patients in comparison to the age-matched control subjects [24]. The expression of the mitochondrial form of human OGG1 protein, hOGG1-2a, is also reduced in the orbitofrontal gyrus and ethorhinal cortex in AD, and the reduction is associated with neurofibrillary tangles [25]. The expression of MTH1 has been reported to be decreased in the CA3 subregion of brains in AD subjects [26]. In a search for proteins homologous to the Escherichia coli MutT in mammalian cells, we found
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MTH2, which possesses the same sequence module as MTH1 [16]. Mouse MTH2 protein can hydrolyze 8-oxo-dGTP to 8-oxo-dGMP, and expression of the MTH2 cDNA reduced the elevated level of spontaneous mutation frequency of the E. coli mutT− mutant. Since MTH2 could act as an MTH1 redundancy factor, it is of interest to know how this protein behaves during aging. In this study, SAMP8, with its characteristics of accelerated aging and early abnormalities in learning and memory [27–29], was used to investigate MTH2 expression and its possible role in protecting neurons from the deleterious effects of oxidative stress. 2. Materials and methods 2.1. Antibodies Rabbit anti-APP (amyloid precursor protein) polyclonal antibodies were purchased from Upstate (Charlottesville, VA). Rabbit anti-β-amyloid polyclonal antibodies were obtained from Chemicon (Temecula, CA). Anti-α-tubulin, anti-β-action and IgG-HRP conjugate were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse N45.1 mAb was obtained from jalCA (Fukuoka, Japan). All the Histostain SP kits were purchased from Zymed Laboratories Inc. (South San Francisco, CA). Rabbit polyclonal antibodies against mouse MTH2 protein were prepared in rabbits using a His-tagged mouse MTH2 fusion protein. The fusion protein was produced in E. coli M15 cells carrying pQE30: mMTH2 and separated by SDS-polyacrylamide gel electrophoresis [16]. The region of the gels containing the fusion protein (approximately 200 μg) was excised to inoculate into the dorsum of a rabbit together with adjuvant. Four weeks later, the first booster injection (100 μg) was given, followed by three booster injections at two-week intervals. The immunoglobulin was purified by ammonium sulfate precipitation, followed by affinity chromatography. Specific antibodies were obtained by elution from the column with a buffer at pH 2.3–2.5 and then dialyzed against 10 mM Tris–HCl (pH 7.4) containing 0.9% NaCl. This preparation was designated as anti-mMTH2. 2.2. Subjects The specific pathogen-free male mice of the SAMP8 and SAMR1 strains at 1, 4, 8, and 12 months of age were purchased from the Laboratory Animal Center of Peking University and were maintained under clean conventional conditions (24 ± 2 °C, 12 h light/dark cycle with light on at 7:00 am). The mice were allowed free access to water and food ad libitum. 2.3. Grading score of senescence A grading score system established by Hosokawa et al. [27] was adopted for evaluating the degrees of senescence of mice. Briefly, the items to be examined in this system include 11 categories selected from the clinical signs and gross lesions considered to be associated with the aging process. The degree of the senescence in each category was graded with scores from 0 to 4 according to the detailed criteria. The degrees of senescence of the SAMP8 and SAMR1 mice were evaluated using this grading system at 1, 4, 8, and 12 months of age. 2.4. Water maze The Morris water maze task was used to evaluate spatial learning and memory. The apparatus was a black circular tank with a diameter of 100 cm and a height of 50 cm. The tank was filled with water (21 ± 1 °C, height 35 cm). The water was made opaque with black nontoxic paint. A black escape platform was submerged 1 cm below the surface of the water and remained in the same location for all hidden platform trials. First, the mice were pretrained to find the hidden platform for 2 days. Four trials per day were conducted. Each mouse was placed by the tail into the water immediately, facing the perimeter, in a fixed
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position opposite the hidden platform quadrant and given 120 s to find the hidden platform in each trial. If the mouse did not find the platform within this time, then the experimenter led the mouse to the platform where it sat for 10 s. Train testing on the hidden platform water maze task began 24 h after the last pretraining trial. It consisted of 3 consecutive days of testing, with four trials per day. For each trial, the latency to reach the platform, distance covered, and mean swim speed were recorded via a video camera that was mounted directly above the water maze to record the mouse's swimming paths, and a tracking analysis system was measured up to a maximum of 120 s. After the hidden platform trial, four visible platform trials were performed with the platform on the side of the pool opposite its location during hidden platform training to check the vision of all mice. The maze was located in a sound-isolated room. Many cues (e. g., the ceiling light, the experimenter, a rack, an animal cage trolley, instruments) that were visible from inside the pool external to the maze were maintained constant throughout testing to avoid any possible interference with the results.
2.5. Tissue processing After the water maze testing, five SAMP8 mice and five SAMR1 mice were deeply anesthetized with pentobarbital (30 mg/kg, i.p.) and then transaortically perfused with cold 0.1 M phosphate buffer (PB, pH 7.4), followed by chilled 4% paraformaldehyde (PFA). The brains were dissected, post-fixed for 12 h, cryoprotected with 30% sucrose-0.1 M PB for 5 h at 4 °C. Consequently, the brains were frozen at −80 °C until use. Serial coronal free-floating sections (40 μm) were produced on a cryostat (Leica, Germany) for immunohistochemistry (IHC) analysis.
2.6. Immunohistochemical detection of MTH2, APP, and 8-oxoguanine For detection of MTH2 and APP, the free-floating sections were first pretreated with 0.3% hydrogen peroxide for 30 min at room temperature and then incubated in blocking buffer for 1 h prior to the addition of the primary polyclonal antibodies (anti-mMTH2 1:200 or anti-β amyloid 1:400). Sections were incubated with primary antibodies overnight at 4 °C with continuous agitation. For the detection of 8-oxoguanine in nuclear DNA (nDNA), the sections were first pretreated in 10 mM Tris–HCl (pH 7.4) containing 15 mM NaCl and 5 mg/ml DNase-free RNase (Sigma, St. Louis, MO) for 60 min at 37 °C to eliminate cellular RNA. Then, the RNase-treated sections were incubated in 3 N HCl for 30 min at room temperature to denature the nDNA before they were subjected to IHC with N45.1 mAb. Pretreated sections were treated with 3% hydrogen peroxide for 15 min and then incubated with blocking buffer, followed by the primary monoclonal antibody, N45.1(1:25), overnight at 4 °C. On day 2, the sections were incubated with a goat anti-rat biotin-conjugated secondary antibody for 1 h, followed by peroxidase conjugated streptavidin (Santa Cruz Biotech. Inc, USA) for 1 h. Immunoreactions were developed with 3′3′-diaminobenzidinetetrahydrochloride (DAB) (Zymed, South San Francisco). The final immunoreaction product of DAB was brown–yellow in color. After being washed three times with PBS (5 min each), the sections were mounted onto slides and air-dried. The slides were dehydrated in a graded series of ethanol, cleared in xylene, and cover-slipped. Controls for the immunohistochemical procedure were obtained by running some slides through the entire procedure with the omission of the primary antibodies as a safeguard against nonspecific staining by the primary antibody. For the 8-oxoguanine detection, controls also included some slides which were treated with RNase-free DNase I (1000 U/ml, TakaRa Biotechnology Co., Ltd) for 60 min at 37 °C to eliminate nDNA as blank controls.
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2.7. Quantitative morphometric analysis All acquired digital images were processed uniformly at a threshold in a gray scale mode to subtract any background corresponding to the area without tissue using a Leica microscope (Leica, Germany). The optical density (OD) in an area comprising the cytoplasm and nucleus was determined using the Leica Q550CW Image Analysis System linked to a Leica microscope. Six adjacent fields of the CA1 and CA3 subregions of the hippocampi from three mice were selected. The OD value was corrected for background by subtracting the OD of the white matter on the same section and averaged. All measurements were done under the same optical and light conditions, and electronic shading correction was used to compensate for any unevenness that might be present in the illumination. 2.8. Western blotting After the water maze testing, six mice from each group were decapitated under anesthesia. The brains were removed, and then the hippocampi were rapidly dissected on ice and then immediately immersed into liquid nitrogen for storage until use. The hippocampi were homogenized with glass homogenizer on ice in ice-cold 50 mM Tris–HCl (pH 7.4) containing 150 mM NaCl, 5 mM EDTA, 1% NP-40, 0.1 mg/ml PMSF, and 1 μg/ml aprotinin. The protein content was measured with the BCA protein assay (Pierce, USA). For detection of MTH2, aliquots of protein (200 μg) were boiled in 5× loading buffer (Huatesheng, China) for 5 min before loading the samples. Then the denatured protein was separated on a 12% SDS-polyacrylamide gel. For APP, 30 μg of protein in 2× loading buffer was applied to a 6% SDSpolyacrylamide gel. Electrophoresis was performed at 80 V for concentration and 150 V for separation. Next, the gels were semidried, and proteins were transferred onto a nitrocellulose membrane. The membranes were blocked in blocking buffer (20 mM Tris–HCl, pH 7.4, containing 150 mM NaCl and 0.05% Tween-20 with 5% non-fat dry milk) for 2 h and then incubated with primary antibodies antimMTH2 (1:800) or anti-APP (1:1000), anti-α-tubulin (1:500), or anti-β-actin (1:1000) overnight at 4 °C. After being washed with TBST five times for 50 min (10 min each), the membranes were incubated with HRP-conjugated anti-rabbit IgG antibodies (1:2000) in blocking buffer for 2 h at room temperature. The blots were washed five times with TBST, and signals were detected by enhanced chemiluminescence (Pierce, USA) followed by exposure to Kodak film (Kodak, China). The bands were quantified by densitometry using a Scion Image software program, and the values were normalized to either α-tubulin or β-actin.
significantly higher scores were observed for 12-month-old SAMP8 than for 12-month-old SAMR1 mice (F(1,18) = 25.632, p b 0.05). Therefore, the SAMP8 mice exhibited significantly accelerated aging characteristics in comparison to SAMR1. The two types of mice were then subjected to the Morris water maze test to find the hidden platform in water. Fig. 2 shows the average latencies of SAMP8 and SAMR1 mice at the ages of 1, 4, 8, and 12 months. No significant difference was observed in 1-month-old and 4-month-old SAMP8 mice in comparison to the age-matched SAMR1 control groups. Significantly longer latencies were observed in the 8month-old SAMP8 (F(1,98) = 102.98, p b 0.05) and 12-month-old SAMP8 (F(1,98) = 28.722, p b 0.05) than in the age-matched SAMR1 mice. These results suggested that younger SAMP8 mice were better able to learn this task and that the learning function of older SAMP8 mice had declined. All mice learned to swim to the visible platform during the four visible trials, indicating adequate visual acuity to perform this task using the spatial cues. Meanwhile, no significant difference was observed in swimming speed for the two types of SAM mice during the performance of hidden platform trials (data not shown). Therefore, the effects of motivational (swimming speed) or sensorimotor (visible platform trial) factors on an animal's learning performance could be excluded.
3.2. APP expression in the hippocampi of SAMP8 and SAMR1 mice APP is mostly present in the cytoplasm of neurons throughout the hippocampus, as seen in both strains of mice (Fig. 3A: a and b). Of interest is the observation that there is a significant elevation in the level of APP in the CA1 and CA3 subregions of SAMP8 at the age of 8 and 12 months in comparison to the 1-month-old mice (p b 0.05) (Fig. 3A: d and f). In the SAMR1 hippocampi, its expression in the CA1 (F(3,12) = 1.303) and CA3 (F(3,12) = 0.409) subregions did not significantly change with increasing age, and there was no significant difference among the four different age groups (p N 0.05) (Fig. 3A: c and e). The difference between the two strains reached the highest level at the age of 8 months in the CA1 region (t(8) = 5.680, p b 0.05) and at 12 months in the CA3 region (t(7) = −3.844, p b 0.05) (Fig. 3B). These results were further verified by a western blot analysis (Fig. 3C). When the intensities for the band corresponding to a 110kDa protein, which can be stained with anti-APP antibodies, were compared with those for actin, a considerable increase was observed in the 12-month-old SAMP8 as compared with younger groups of SAMP8 and also with age-matched SAMR1 (p b 0.05) (Fig. 3D).
2.9. Statistical analysis All statistical analyses in this study were performed using the SPSS 11.5 version software. All data are presented as the mean ± S.D. The differences among different age groups were statistically tested using the ANOVA method, then post-hoc comparisons were performed using Student–Newman–Keuls or Dunnett's t-test method, and the alpha level was corrected. The difference between SAMP8 and SAMR1 strains at the same age were tested using Student's t-test. The level of p b 0.05 was accepted as significant. 3. Results 3.1. Age-related responses of two types of mice The grading of senescence was examined for the SAMP8 and the normal counterpart SAMR1. The values tended to increase with age, but the rate of increase in SAMP8 was far greater than that in SAMR1 (Fig. 1). The scores of 8-month-old SAMP8 were significantly higher than in the 8-month-old SAMR1 control group (F(1,18) = 32.817, p b 0.05). Also,
Fig. 1. The senescence grading scores of SAMP8 and SAMR1 mice. The black and red lines indicate the values for SAMR1 and SAMP8, respectively. ⁎p b 0.05, in comparison to 1-month-old SAMP8. #p b 0.05, in comparison to age-matched SAMR1. The data are presented as the mean ± SD (n = 10 for each group).
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Fig. 2. The latencies of SAMP8 and SAMR1 mice to find the submerged platform in the Morris water maze. (A to D): Escape latencies of the 1-, 4-, 8-, and 12-month-old mice. The black and red lines indicate values for SAMR1 and SAMP8, respectively. *p b 0.05, in comparison to age-matched SAMR1. The data are presented as the mean ± SD (n = 10 for each group).
3.3. Accumulation of 8-oxoguanine in the nDNA of SAMP8 hippocampus
3.4. Expression of MTH2 in SAMP8 and SAMR1 mice
To detect 8-oxoguanine in the nuclear DNA of the hippocampi, immunostaining using antibodies against 8-oxo-dG (8-oxo-7,8-dihydrodeoxyguanosine) was performed (Fig. 4A). The Con 1 (control 1) panel results are the controls with the omission of primary antibody during the incubation, and the Con 2 (control 2) panel results are the control sections which were treated with DNase I to eliminate nDNA prior to immunohistochemical analysis. No immunoreactivity was observed in the nuclei of the controls. When antibodies against 8-oxo-dG were applied to the samples (1 M to 12 M, Fig. 4A), specific staining of the nuclei was found in the CA1 and CA3 regions of the hippocampi of mice. In all the samples, the nuclei were exclusively stained, and more intensive staining was observed with samples derived from SAMP8 mice in comparison to SAMR1. The averaged optical density was measured for the subregions of the hippocampi of the two types of mice, and the digitized data are shown in Fig. 4B. It was observed that a large amount of 8-oxoguanine is significantly present in the DNA of the CA1 of the hippocampi of 8- and 12-month-old SAMP8 mice in comparison to those in the age-matched SAMR1 control mice, with t(8) = − 7.860 and t(8) = −7.192, respectively (p b 0.05). Similar results were seen in the CA3 subregion of the two age groups, with t(7) = −6.899 for 8month-old mice and t(8) = − 7.069 for 12-month-old mice, respectively, (p b 0.05).
Immunostaining with polyclonal antibodies against mouse MTH2 was performed for the hippocampi of the two types of mice (Fig. 5A). The MTH2 protein was detected mostly in the cytoplasm of neurons throughout the whole hippocampi in both SAMR1 and SAMP8 examined at all ages, whereas no immunoreactivity was observed in controls with the omission of primary antibodies (Con). In the SAMP8 mice, a significant decrease in the MTH2 signal was observed in the CA1 (F(3,16) = 52.410, p b 0.05) and CA3 (F(3,14) = 19.498, p b 0.05) regions of the hippocampus; meanwhile, no significant change in MTH2 was detected in the CA1 and CA3 regions of the control SAMR1 group (F(3,16) = 0.129, p N 0.05 for CA1 and F(3,15) = 0.408, p N 0.05 for CA3). The optical density analysis showed that an age-related decline of the MTH2 content in the CA1 and CA3 regions of SAMP8 mice began at 8 months after birth (Fig. 5B). The levels of decline are statistically significant, with respect to both age and strain. The results of immunostaining were verified by western blot analysis, in which a 20-kDa protein was identified as MTH2 by the specific antibodies. As shown in Fig. 5C, there was an age-dependent decrease in the amount of MTH2 protein in SAMP8 mice. On the other hand, no significant change in the MTH2 level was observed in SAMR1 mice. Fig. 5D shows the relative expression levels of MTH2 against internal control tubulin at the different ages of the two types of mice. In SAMP8, the decline in MTH2 expression was significant (F(3,8) =
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Fig. 3. The age-related increase of APP expression in SAMP8 mice. (A) The immunohistochemical analyses of free-floating sections (40 μm in thickness). The two panels on the left (a: SAMR1, b: SAMP8) show a lower magnification (25×), whereas the magnified views (400×) of the CA1 (c: SAMR1, d: SAMP8) and CA3 (e: SAMR1, f: SAMP8) subregions are shown in the panels in the middle and the right, respectively. The sections shown in the top panel (Con) are blank controls without antibodies. Scale bars: a and b = 200 μm; c to f = 10 μm. (B) The averaged optical density for the APP index. The left panel shows the CA1 subregion and the right panel the CA3. (C) The western blot analysis of APP from the hippocampi of SAMP8 and SAMR1 mice. A total of 30 μg of protein isolated from hippocampus homogenates was subjected to SDS-PAGE (6% acrylamide) for western blot analysis of APP. The membranes were immunostained with anti-APP (upper panel) and anti-β-actin (lower panel as a loading control). Lanes 1 to 4: 1-, 4-, 8-, 12-month-old SAMR1; lanes 5 to 8: 1-, 4-, 8-, 12-month-old SAMP8 mice. (D) Quantification of the bands by densitometry using Scion image. The data are given as the mean ± SD. The statistical analysis was performed using ANOVA followed by Dunnett's test. * p b 0.05, in comparison to 1-month-old SAMP8. #p b 0.05, in comparison to age-matched SAMR1.
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Fig. 4. Immunohistochemical detection of 8-oxoguanine in the nuclear DNA of hippocampus. Free-floating sections were pretreated with RNase and HCl before incubation with N45.1 mAb. (A) The two panels on the left (a: SAMR1, b: SAMP8) at a lower magnification (25×), and magnified views (400×) of CA1 (c: SAMR1, d: SAMP8) and CA3 (e: SAMR1, f: SAMP8) subregions in the middle and right panels. The top two panels are blank controls (Con 1, without the primary antibodies; Con 2, sections pretreated with RNase-free DNase I before incubation with N45.1). Scale bars: a and b = 200 μm; c to f = 10 μm. (B) The averaged optical density for 8-oxoguanine in the CA1 and CA3 regions of the hippocampi of SAMR1 and SAMP8 mice. The data are presented as the means ± SD. * p b 0.05, in comparison to 1-month-old SAMP8, #p b 0.05, in comparison to age-matched SAMR1.
57.892, p b 0.05) as early as 4 months after birth, and the extent of the reduction increases with the progression of age. The levels of MTH2 expression in 8-month-old SAMP8 mice were about 43% of the level attained with 1-month-old mice, and the level for 12-month-old mice was only 4% of that for 1-month-old mice. This decrease in the MTH2 content is thus considered to be a defining characteristic in SAMP8 mice.
4. Discussion To examine the relationship between the expression of MTH2 and the progression of aging, we therefore chose the SAMP8 animal model, which has been characterized by a facilitated impairment of learning and memory [30]. In this study, our Morris water maze results suggested that 1- and 4-month-old SAMP8 mice were better
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Fig. 5. Age-related decrease in the level of MTH2 expression in the hippocampus of SAMP8. (A) Immunohistochemistry performed with free-floating sections. The two panels on the left (a: SAMR1, b: SAMP8) show a lower magnification (25×), whereas magnified views (400×) of CA1 (c: SAMR1, d: SAMP8) and CA3 (e: SAMR1, f: SAMP8) subregions are shown in the middle and the right. Sections in the top panel are blank control (Con) without anti-MTH2. Scales bars: a and b = 200 μm; c to f = 10 μm. (B) Optical density measured on the CA1 and CA3 regions. * p b 0.05, in comparison to 1-month-old SAMP8, #p b 0.05, in comparison to age-matched SAMR1. The data are presented as the mean ± SD. (C) Western blot analysis. Hippocampus homogenates (200 μg) were subjected to SDS-PAGE (12% acrylamide), and the membranes were immunostained with anti-MTH2 or anti-α-tubulin. Lanes 1 to 4: 1-, 4-, 8-, 12-month-old SAMR1; lanes 5 to 8: 1-, 4-, 8-, 12-month-old SAMP8. (D) Quantification of the western blot analysis. The relative intensities of the MTH2 bands against the tubulin bands are shown. The data are given as the means± SD of six animals. The statistical analysis was performed using ANOVA followed by Dunnett's test. *p b 0.05, in comparison to 1-month-old SAMP8. # p b 0.05, in comparison to the age-matched control.
able to learn the water maze task; however, older SAMP8 mice (8 and 12 months old) exhibited a declined learning ability. These results are consistent with the findings of others [31–33]. Meanwhile, the
grading scores of senescence were significantly higher in the 8- and 12-month-old SAMP8 mice in comparison to the levels found in agematched SAMR1 mice. Studies on the expression of APP in the brain
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also proved the usefulness of this strain of mouse as a murine model for aging. Both IHC and the western blotting analysis showed a significant increase in APP expression from 8 to 12 months after birth in SAMP8 mice, while there was only a slight change in the SAMR1 control mice over the same time period. These results are in agreement with the findings of Morley et al. [34]. The elevated levels of the grading score of senescence and impairment of special cognitive performance occurred at almost the same time when the amount of 8-oxoguanine in the DNA increased in the CA1 and CA3 subregions of the hippocampi of the SAMP8 mice. There was no significant change in the 8-oxoguanine content in the corresponding regions of the SAMR1 mice. Morley et al. [34] reported that the visible deposition of senile plaques appeared 16 months after birth in SAMP8 mice. Since the accumulation of 8-oxoguanine in DNA becomes evident as early as 8 months after birth, the increase of 8-oxoguanine may be the initiating factor rather than the consequence of APP deposition, although the latter would play an important role in the vicious cycle. These results are reminiscent of the findings of Wang et al. [35] and Nunomura et al. [36], who showed that there is a significant increase in 8-oxoguanine content in DNA in the cases of AD in comparison to normal subjects. An over-expression of APP is also considered as an important factor in AD, and there have been numerous studies that have indicated that APP is over-expressed in AD patients [37,38]. The immunohistochemical results revealed that the MTH2 protein is present in the CA1 and CA3 regions of mouse hippocampus. In SAMP8 mice, the amount of this protein in these regions decreased progressively, beginning from 8 months after birth. No such change in MTH2 content was observed in the control SAMR1 mice. This result suggests that the decrease in the level of MTH2 might be related to the deterioration of learning and memory, which emerges at the same age. The result of the western blot analysis revealed that the decline in MTH2 level actually started as early as 4 months after birth in the SAMP8 mice, which is inferred as the causative effect of MTH2 decrease in the onset of the disease. How this could happen is unknown. We know that MTH2 possesses the specific enzymatic activity to convert the deleterious 8-oxo-dGTP to 8-oxo-dGMP. Considering the significant increase of 8-oxoguanine in DNA, we hypothesized that the decline of MTH2 expression in the hippocampus might lead to the insufficient sanitization of 8-oxo-dGTP, which might consequently result in the increase of 8-oxoguanine in DNA. The overload of 8-oxo-dGTP may be involved in the onset and progression of APP over-expression and the final formation of Aβ deposition. Further studies are in progress to address these possibilities. Overall, our findings show that MTH2 deficiency is associated with the progression of aging, yet the exact mechanism still needs to be studied further. It has been suggested that the decline of hippocampal synapses may be a cause of the cognition impairment in AD [39]. In a western blot analysis of various tissues, we found that all organs except the liver contain substantial amounts of MTH2 protein and that the highest levels of the protein are present in the brain and testis of both types of mouse strains (data not shown). If we assume that high oxygen consumption causes the high level of oxidative damage to DNA [40], the level of the MTH2 proteins might be concerned with this outcome. To better understand the role of MTH2 in this process, we need to develop a mouse line defective in the Mth2 gene, and studies regarding this aspect are now in progress. Acknowledgement This work was supported by the Key International Science and Technology Cooperation Projects (2006DFB31410). We thank the members of the Institute of Geriatrics of the Ministry of Health for advice and assistance. We thank B. Quinn for language assistance. Disclosure statement There is no conflict of interest.
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Journal of the Neurological Sciences j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j n s
Age-related alterations in the expression of MTH2 in the hippocampus of the SAMP8 mouse with learning and memory deterioration Jun-De Zheng a,1,2, Ai-Lian Hei a,2, Ping-Ping Zuo b, Yi-Long Dong b, Xiao-Ning Song a, Yasumitsu Takagi c, Mutsuo Sekiguchi c, Jian-Ping Cai a,⁎ a b c
The Key Laboratory of Geriatrics, Beijing Hospital & Beijing Institute of Geriatrics, Ministry of Health, No. 1, DaHua Road, Dong Dan, Beijing 100730, PR China Peking Union Medical College & Chinese Academy of Medical Sciences, No. 9, Dong Dan Santiao, DongCheng District, Beijing 100730, PR China Frontier Research Center, Fukuoka Dental College, Fukuoka 814-0193, Japan
a r t i c l e
i n f o
Article history: Received 17 June 2009 Accepted 31 July 2009 Available online 6 September 2009 Keywords: Mouse model Oxidative stress 8-oxoguanine MTH2 Aging Alzheimer's disease Mutation 8-oxo-dGTPase
a b s t r a c t MutT-related proteins degrade 8-oxo-7,8-dihydrodeoxyguanosine triphosphate (8-oxo-dGTP), a mutagenic substrate for DNA synthesis in the nucleotide pool, thereby preventing DNA replication errors. MTH2 (Mut T homolog 2), which belongs to this family of proteins, possesses 8-oxo-7,8-dihydro-2′-deoxyguanosine triphosphatase (8-oxo-dGTPase) activity and appears to function in the protection of the genetic material from the untoward effects of endogenous oxygen radicals. To examine the roles of MTH2 in the aging process, we used the senescence-accelerated prone mouse 8 (SAMP8), which exhibits early aging syndromes and declining abilities of learning and memory. Immunohistochemical and western blot analysis revealed that the level of MTH2 protein in the hippocampus of the SAMP8 mouse progressively decreases beginning from four months after birth, whereas no such change was observed in the control senescence-accelerated resistant mouse 1 (SAMR1). Under these conditions, 8-oxoguanine accumulates in the nuclear DNA in the CA1 and CA3 subregions of the hippocampus of SAMP8 in an age-dependent manner. In SAMR1 mice, accumulation of 8-oxoguanine in the DNA was not observed. These results suggest that the MTH2 deficiency might be one of the causative factors for accelerated aging. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Reactive oxygen species (ROS), such as superoxide and hydroxyl radicals, are produced through normal cellular metabolism, and the formation of such radicals is further enhanced by the exposure to certain physical and chemical agents [1,2]. Nucleic acids exposed to ROS generate various modified bases, among which 8-oxo-7,8-dihydroguanine (8-oxoguanine) is the most important. Unlike other types of oxidatively altered bases, 8-oxoguanine does not block nucleic acid synthesis but rather induces base mispairing, since it has the potential to pair with adenine as well as cytosine. This mispairing is thought to significantly contribute to the levels of spontaneous mutation frequency [3–6], and, when 8-oxoguanine is present in messenger RNA, it possibly causes alterations in gene expression [7,8]. Organisms are equipped with elaborate mechanisms for counteracting such deleterious effects of 8-oxoguanine. In mammalian cells, two types of glycosylases, MYH and OGG1, appear to function in the prevention of mutations caused by 8-oxoguanine in DNA. The MYH
⁎ Corresponding author. E-mail address: [email protected] (J.-P. Cai). 1 Present address: The First Affiliated Hospital of Guangzhou Medical College, Guangzhou 510120, PR China. 2 These authors contributed equally to this work. 0022-510X/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jns.2009.07.027
protein excises adenine paired with 8-oxoguanine, while the OGG1 protein removes 8-oxoguanine paired with cytosine from oxidatively damaged DNA [9–12]. Mammalian cells also possess MutT-related enzymes, which are capable of eliminating 8-oxoguanine-containing nucleotides from the DNA precursor pool [13–15]. These include MTH1 (NUDT1) and MTH2 (NUDT15), which hydrolyze an oxidized form of dGTP, 8-oxo-dGTP, to 8-oxo-dGMP, a non-usable form for DNA synthesis [16,17]. The central nervous system (CNS) is thought to be particularly susceptible to oxidative stress due to the high rate of oxygen consumption in the brain and the low level of antioxidant enzymes compared with other somatic tissues [18,19]. Therefore, ROS have been proposed as the pathogenic factor in various neurological disorders, including Alzheimer's disease (AD) [20–23]. Recent studies have focused on the relationship between AD and enzymes acting on oxidatively damaged DNA and its precursors. The activity of human OGG1 has been shown to be significantly decreased in the nuclear protein samples from brains of AD patients in comparison to the age-matched control subjects [24]. The expression of the mitochondrial form of human OGG1 protein, hOGG1-2a, is also reduced in the orbitofrontal gyrus and ethorhinal cortex in AD, and the reduction is associated with neurofibrillary tangles [25]. The expression of MTH1 has been reported to be decreased in the CA3 subregion of brains in AD subjects [26]. In a search for proteins homologous to the Escherichia coli MutT in mammalian cells, we found
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MTH2, which possesses the same sequence module as MTH1 [16]. Mouse MTH2 protein can hydrolyze 8-oxo-dGTP to 8-oxo-dGMP, and expression of the MTH2 cDNA reduced the elevated level of spontaneous mutation frequency of the E. coli mutT− mutant. Since MTH2 could act as an MTH1 redundancy factor, it is of interest to know how this protein behaves during aging. In this study, SAMP8, with its characteristics of accelerated aging and early abnormalities in learning and memory [27–29], was used to investigate MTH2 expression and its possible role in protecting neurons from the deleterious effects of oxidative stress. 2. Materials and methods 2.1. Antibodies Rabbit anti-APP (amyloid precursor protein) polyclonal antibodies were purchased from Upstate (Charlottesville, VA). Rabbit anti-β-amyloid polyclonal antibodies were obtained from Chemicon (Temecula, CA). Anti-α-tubulin, anti-β-action and IgG-HRP conjugate were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse N45.1 mAb was obtained from jalCA (Fukuoka, Japan). All the Histostain SP kits were purchased from Zymed Laboratories Inc. (South San Francisco, CA). Rabbit polyclonal antibodies against mouse MTH2 protein were prepared in rabbits using a His-tagged mouse MTH2 fusion protein. The fusion protein was produced in E. coli M15 cells carrying pQE30: mMTH2 and separated by SDS-polyacrylamide gel electrophoresis [16]. The region of the gels containing the fusion protein (approximately 200 μg) was excised to inoculate into the dorsum of a rabbit together with adjuvant. Four weeks later, the first booster injection (100 μg) was given, followed by three booster injections at two-week intervals. The immunoglobulin was purified by ammonium sulfate precipitation, followed by affinity chromatography. Specific antibodies were obtained by elution from the column with a buffer at pH 2.3–2.5 and then dialyzed against 10 mM Tris–HCl (pH 7.4) containing 0.9% NaCl. This preparation was designated as anti-mMTH2. 2.2. Subjects The specific pathogen-free male mice of the SAMP8 and SAMR1 strains at 1, 4, 8, and 12 months of age were purchased from the Laboratory Animal Center of Peking University and were maintained under clean conventional conditions (24 ± 2 °C, 12 h light/dark cycle with light on at 7:00 am). The mice were allowed free access to water and food ad libitum. 2.3. Grading score of senescence A grading score system established by Hosokawa et al. [27] was adopted for evaluating the degrees of senescence of mice. Briefly, the items to be examined in this system include 11 categories selected from the clinical signs and gross lesions considered to be associated with the aging process. The degree of the senescence in each category was graded with scores from 0 to 4 according to the detailed criteria. The degrees of senescence of the SAMP8 and SAMR1 mice were evaluated using this grading system at 1, 4, 8, and 12 months of age. 2.4. Water maze The Morris water maze task was used to evaluate spatial learning and memory. The apparatus was a black circular tank with a diameter of 100 cm and a height of 50 cm. The tank was filled with water (21 ± 1 °C, height 35 cm). The water was made opaque with black nontoxic paint. A black escape platform was submerged 1 cm below the surface of the water and remained in the same location for all hidden platform trials. First, the mice were pretrained to find the hidden platform for 2 days. Four trials per day were conducted. Each mouse was placed by the tail into the water immediately, facing the perimeter, in a fixed
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position opposite the hidden platform quadrant and given 120 s to find the hidden platform in each trial. If the mouse did not find the platform within this time, then the experimenter led the mouse to the platform where it sat for 10 s. Train testing on the hidden platform water maze task began 24 h after the last pretraining trial. It consisted of 3 consecutive days of testing, with four trials per day. For each trial, the latency to reach the platform, distance covered, and mean swim speed were recorded via a video camera that was mounted directly above the water maze to record the mouse's swimming paths, and a tracking analysis system was measured up to a maximum of 120 s. After the hidden platform trial, four visible platform trials were performed with the platform on the side of the pool opposite its location during hidden platform training to check the vision of all mice. The maze was located in a sound-isolated room. Many cues (e. g., the ceiling light, the experimenter, a rack, an animal cage trolley, instruments) that were visible from inside the pool external to the maze were maintained constant throughout testing to avoid any possible interference with the results.
2.5. Tissue processing After the water maze testing, five SAMP8 mice and five SAMR1 mice were deeply anesthetized with pentobarbital (30 mg/kg, i.p.) and then transaortically perfused with cold 0.1 M phosphate buffer (PB, pH 7.4), followed by chilled 4% paraformaldehyde (PFA). The brains were dissected, post-fixed for 12 h, cryoprotected with 30% sucrose-0.1 M PB for 5 h at 4 °C. Consequently, the brains were frozen at −80 °C until use. Serial coronal free-floating sections (40 μm) were produced on a cryostat (Leica, Germany) for immunohistochemistry (IHC) analysis.
2.6. Immunohistochemical detection of MTH2, APP, and 8-oxoguanine For detection of MTH2 and APP, the free-floating sections were first pretreated with 0.3% hydrogen peroxide for 30 min at room temperature and then incubated in blocking buffer for 1 h prior to the addition of the primary polyclonal antibodies (anti-mMTH2 1:200 or anti-β amyloid 1:400). Sections were incubated with primary antibodies overnight at 4 °C with continuous agitation. For the detection of 8-oxoguanine in nuclear DNA (nDNA), the sections were first pretreated in 10 mM Tris–HCl (pH 7.4) containing 15 mM NaCl and 5 mg/ml DNase-free RNase (Sigma, St. Louis, MO) for 60 min at 37 °C to eliminate cellular RNA. Then, the RNase-treated sections were incubated in 3 N HCl for 30 min at room temperature to denature the nDNA before they were subjected to IHC with N45.1 mAb. Pretreated sections were treated with 3% hydrogen peroxide for 15 min and then incubated with blocking buffer, followed by the primary monoclonal antibody, N45.1(1:25), overnight at 4 °C. On day 2, the sections were incubated with a goat anti-rat biotin-conjugated secondary antibody for 1 h, followed by peroxidase conjugated streptavidin (Santa Cruz Biotech. Inc, USA) for 1 h. Immunoreactions were developed with 3′3′-diaminobenzidinetetrahydrochloride (DAB) (Zymed, South San Francisco). The final immunoreaction product of DAB was brown–yellow in color. After being washed three times with PBS (5 min each), the sections were mounted onto slides and air-dried. The slides were dehydrated in a graded series of ethanol, cleared in xylene, and cover-slipped. Controls for the immunohistochemical procedure were obtained by running some slides through the entire procedure with the omission of the primary antibodies as a safeguard against nonspecific staining by the primary antibody. For the 8-oxoguanine detection, controls also included some slides which were treated with RNase-free DNase I (1000 U/ml, TakaRa Biotechnology Co., Ltd) for 60 min at 37 °C to eliminate nDNA as blank controls.
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2.7. Quantitative morphometric analysis All acquired digital images were processed uniformly at a threshold in a gray scale mode to subtract any background corresponding to the area without tissue using a Leica microscope (Leica, Germany). The optical density (OD) in an area comprising the cytoplasm and nucleus was determined using the Leica Q550CW Image Analysis System linked to a Leica microscope. Six adjacent fields of the CA1 and CA3 subregions of the hippocampi from three mice were selected. The OD value was corrected for background by subtracting the OD of the white matter on the same section and averaged. All measurements were done under the same optical and light conditions, and electronic shading correction was used to compensate for any unevenness that might be present in the illumination. 2.8. Western blotting After the water maze testing, six mice from each group were decapitated under anesthesia. The brains were removed, and then the hippocampi were rapidly dissected on ice and then immediately immersed into liquid nitrogen for storage until use. The hippocampi were homogenized with glass homogenizer on ice in ice-cold 50 mM Tris–HCl (pH 7.4) containing 150 mM NaCl, 5 mM EDTA, 1% NP-40, 0.1 mg/ml PMSF, and 1 μg/ml aprotinin. The protein content was measured with the BCA protein assay (Pierce, USA). For detection of MTH2, aliquots of protein (200 μg) were boiled in 5× loading buffer (Huatesheng, China) for 5 min before loading the samples. Then the denatured protein was separated on a 12% SDS-polyacrylamide gel. For APP, 30 μg of protein in 2× loading buffer was applied to a 6% SDSpolyacrylamide gel. Electrophoresis was performed at 80 V for concentration and 150 V for separation. Next, the gels were semidried, and proteins were transferred onto a nitrocellulose membrane. The membranes were blocked in blocking buffer (20 mM Tris–HCl, pH 7.4, containing 150 mM NaCl and 0.05% Tween-20 with 5% non-fat dry milk) for 2 h and then incubated with primary antibodies antimMTH2 (1:800) or anti-APP (1:1000), anti-α-tubulin (1:500), or anti-β-actin (1:1000) overnight at 4 °C. After being washed with TBST five times for 50 min (10 min each), the membranes were incubated with HRP-conjugated anti-rabbit IgG antibodies (1:2000) in blocking buffer for 2 h at room temperature. The blots were washed five times with TBST, and signals were detected by enhanced chemiluminescence (Pierce, USA) followed by exposure to Kodak film (Kodak, China). The bands were quantified by densitometry using a Scion Image software program, and the values were normalized to either α-tubulin or β-actin.
significantly higher scores were observed for 12-month-old SAMP8 than for 12-month-old SAMR1 mice (F(1,18) = 25.632, p b 0.05). Therefore, the SAMP8 mice exhibited significantly accelerated aging characteristics in comparison to SAMR1. The two types of mice were then subjected to the Morris water maze test to find the hidden platform in water. Fig. 2 shows the average latencies of SAMP8 and SAMR1 mice at the ages of 1, 4, 8, and 12 months. No significant difference was observed in 1-month-old and 4-month-old SAMP8 mice in comparison to the age-matched SAMR1 control groups. Significantly longer latencies were observed in the 8month-old SAMP8 (F(1,98) = 102.98, p b 0.05) and 12-month-old SAMP8 (F(1,98) = 28.722, p b 0.05) than in the age-matched SAMR1 mice. These results suggested that younger SAMP8 mice were better able to learn this task and that the learning function of older SAMP8 mice had declined. All mice learned to swim to the visible platform during the four visible trials, indicating adequate visual acuity to perform this task using the spatial cues. Meanwhile, no significant difference was observed in swimming speed for the two types of SAM mice during the performance of hidden platform trials (data not shown). Therefore, the effects of motivational (swimming speed) or sensorimotor (visible platform trial) factors on an animal's learning performance could be excluded.
3.2. APP expression in the hippocampi of SAMP8 and SAMR1 mice APP is mostly present in the cytoplasm of neurons throughout the hippocampus, as seen in both strains of mice (Fig. 3A: a and b). Of interest is the observation that there is a significant elevation in the level of APP in the CA1 and CA3 subregions of SAMP8 at the age of 8 and 12 months in comparison to the 1-month-old mice (p b 0.05) (Fig. 3A: d and f). In the SAMR1 hippocampi, its expression in the CA1 (F(3,12) = 1.303) and CA3 (F(3,12) = 0.409) subregions did not significantly change with increasing age, and there was no significant difference among the four different age groups (p N 0.05) (Fig. 3A: c and e). The difference between the two strains reached the highest level at the age of 8 months in the CA1 region (t(8) = 5.680, p b 0.05) and at 12 months in the CA3 region (t(7) = −3.844, p b 0.05) (Fig. 3B). These results were further verified by a western blot analysis (Fig. 3C). When the intensities for the band corresponding to a 110kDa protein, which can be stained with anti-APP antibodies, were compared with those for actin, a considerable increase was observed in the 12-month-old SAMP8 as compared with younger groups of SAMP8 and also with age-matched SAMR1 (p b 0.05) (Fig. 3D).
2.9. Statistical analysis All statistical analyses in this study were performed using the SPSS 11.5 version software. All data are presented as the mean ± S.D. The differences among different age groups were statistically tested using the ANOVA method, then post-hoc comparisons were performed using Student–Newman–Keuls or Dunnett's t-test method, and the alpha level was corrected. The difference between SAMP8 and SAMR1 strains at the same age were tested using Student's t-test. The level of p b 0.05 was accepted as significant. 3. Results 3.1. Age-related responses of two types of mice The grading of senescence was examined for the SAMP8 and the normal counterpart SAMR1. The values tended to increase with age, but the rate of increase in SAMP8 was far greater than that in SAMR1 (Fig. 1). The scores of 8-month-old SAMP8 were significantly higher than in the 8-month-old SAMR1 control group (F(1,18) = 32.817, p b 0.05). Also,
Fig. 1. The senescence grading scores of SAMP8 and SAMR1 mice. The black and red lines indicate the values for SAMR1 and SAMP8, respectively. ⁎p b 0.05, in comparison to 1-month-old SAMP8. #p b 0.05, in comparison to age-matched SAMR1. The data are presented as the mean ± SD (n = 10 for each group).
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Fig. 2. The latencies of SAMP8 and SAMR1 mice to find the submerged platform in the Morris water maze. (A to D): Escape latencies of the 1-, 4-, 8-, and 12-month-old mice. The black and red lines indicate values for SAMR1 and SAMP8, respectively. *p b 0.05, in comparison to age-matched SAMR1. The data are presented as the mean ± SD (n = 10 for each group).
3.3. Accumulation of 8-oxoguanine in the nDNA of SAMP8 hippocampus
3.4. Expression of MTH2 in SAMP8 and SAMR1 mice
To detect 8-oxoguanine in the nuclear DNA of the hippocampi, immunostaining using antibodies against 8-oxo-dG (8-oxo-7,8-dihydrodeoxyguanosine) was performed (Fig. 4A). The Con 1 (control 1) panel results are the controls with the omission of primary antibody during the incubation, and the Con 2 (control 2) panel results are the control sections which were treated with DNase I to eliminate nDNA prior to immunohistochemical analysis. No immunoreactivity was observed in the nuclei of the controls. When antibodies against 8-oxo-dG were applied to the samples (1 M to 12 M, Fig. 4A), specific staining of the nuclei was found in the CA1 and CA3 regions of the hippocampi of mice. In all the samples, the nuclei were exclusively stained, and more intensive staining was observed with samples derived from SAMP8 mice in comparison to SAMR1. The averaged optical density was measured for the subregions of the hippocampi of the two types of mice, and the digitized data are shown in Fig. 4B. It was observed that a large amount of 8-oxoguanine is significantly present in the DNA of the CA1 of the hippocampi of 8- and 12-month-old SAMP8 mice in comparison to those in the age-matched SAMR1 control mice, with t(8) = − 7.860 and t(8) = −7.192, respectively (p b 0.05). Similar results were seen in the CA3 subregion of the two age groups, with t(7) = −6.899 for 8month-old mice and t(8) = − 7.069 for 12-month-old mice, respectively, (p b 0.05).
Immunostaining with polyclonal antibodies against mouse MTH2 was performed for the hippocampi of the two types of mice (Fig. 5A). The MTH2 protein was detected mostly in the cytoplasm of neurons throughout the whole hippocampi in both SAMR1 and SAMP8 examined at all ages, whereas no immunoreactivity was observed in controls with the omission of primary antibodies (Con). In the SAMP8 mice, a significant decrease in the MTH2 signal was observed in the CA1 (F(3,16) = 52.410, p b 0.05) and CA3 (F(3,14) = 19.498, p b 0.05) regions of the hippocampus; meanwhile, no significant change in MTH2 was detected in the CA1 and CA3 regions of the control SAMR1 group (F(3,16) = 0.129, p N 0.05 for CA1 and F(3,15) = 0.408, p N 0.05 for CA3). The optical density analysis showed that an age-related decline of the MTH2 content in the CA1 and CA3 regions of SAMP8 mice began at 8 months after birth (Fig. 5B). The levels of decline are statistically significant, with respect to both age and strain. The results of immunostaining were verified by western blot analysis, in which a 20-kDa protein was identified as MTH2 by the specific antibodies. As shown in Fig. 5C, there was an age-dependent decrease in the amount of MTH2 protein in SAMP8 mice. On the other hand, no significant change in the MTH2 level was observed in SAMR1 mice. Fig. 5D shows the relative expression levels of MTH2 against internal control tubulin at the different ages of the two types of mice. In SAMP8, the decline in MTH2 expression was significant (F(3,8) =
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Fig. 3. The age-related increase of APP expression in SAMP8 mice. (A) The immunohistochemical analyses of free-floating sections (40 μm in thickness). The two panels on the left (a: SAMR1, b: SAMP8) show a lower magnification (25×), whereas the magnified views (400×) of the CA1 (c: SAMR1, d: SAMP8) and CA3 (e: SAMR1, f: SAMP8) subregions are shown in the panels in the middle and the right, respectively. The sections shown in the top panel (Con) are blank controls without antibodies. Scale bars: a and b = 200 μm; c to f = 10 μm. (B) The averaged optical density for the APP index. The left panel shows the CA1 subregion and the right panel the CA3. (C) The western blot analysis of APP from the hippocampi of SAMP8 and SAMR1 mice. A total of 30 μg of protein isolated from hippocampus homogenates was subjected to SDS-PAGE (6% acrylamide) for western blot analysis of APP. The membranes were immunostained with anti-APP (upper panel) and anti-β-actin (lower panel as a loading control). Lanes 1 to 4: 1-, 4-, 8-, 12-month-old SAMR1; lanes 5 to 8: 1-, 4-, 8-, 12-month-old SAMP8 mice. (D) Quantification of the bands by densitometry using Scion image. The data are given as the mean ± SD. The statistical analysis was performed using ANOVA followed by Dunnett's test. * p b 0.05, in comparison to 1-month-old SAMP8. #p b 0.05, in comparison to age-matched SAMR1.
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Fig. 4. Immunohistochemical detection of 8-oxoguanine in the nuclear DNA of hippocampus. Free-floating sections were pretreated with RNase and HCl before incubation with N45.1 mAb. (A) The two panels on the left (a: SAMR1, b: SAMP8) at a lower magnification (25×), and magnified views (400×) of CA1 (c: SAMR1, d: SAMP8) and CA3 (e: SAMR1, f: SAMP8) subregions in the middle and right panels. The top two panels are blank controls (Con 1, without the primary antibodies; Con 2, sections pretreated with RNase-free DNase I before incubation with N45.1). Scale bars: a and b = 200 μm; c to f = 10 μm. (B) The averaged optical density for 8-oxoguanine in the CA1 and CA3 regions of the hippocampi of SAMR1 and SAMP8 mice. The data are presented as the means ± SD. * p b 0.05, in comparison to 1-month-old SAMP8, #p b 0.05, in comparison to age-matched SAMR1.
57.892, p b 0.05) as early as 4 months after birth, and the extent of the reduction increases with the progression of age. The levels of MTH2 expression in 8-month-old SAMP8 mice were about 43% of the level attained with 1-month-old mice, and the level for 12-month-old mice was only 4% of that for 1-month-old mice. This decrease in the MTH2 content is thus considered to be a defining characteristic in SAMP8 mice.
4. Discussion To examine the relationship between the expression of MTH2 and the progression of aging, we therefore chose the SAMP8 animal model, which has been characterized by a facilitated impairment of learning and memory [30]. In this study, our Morris water maze results suggested that 1- and 4-month-old SAMP8 mice were better
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Fig. 5. Age-related decrease in the level of MTH2 expression in the hippocampus of SAMP8. (A) Immunohistochemistry performed with free-floating sections. The two panels on the left (a: SAMR1, b: SAMP8) show a lower magnification (25×), whereas magnified views (400×) of CA1 (c: SAMR1, d: SAMP8) and CA3 (e: SAMR1, f: SAMP8) subregions are shown in the middle and the right. Sections in the top panel are blank control (Con) without anti-MTH2. Scales bars: a and b = 200 μm; c to f = 10 μm. (B) Optical density measured on the CA1 and CA3 regions. * p b 0.05, in comparison to 1-month-old SAMP8, #p b 0.05, in comparison to age-matched SAMR1. The data are presented as the mean ± SD. (C) Western blot analysis. Hippocampus homogenates (200 μg) were subjected to SDS-PAGE (12% acrylamide), and the membranes were immunostained with anti-MTH2 or anti-α-tubulin. Lanes 1 to 4: 1-, 4-, 8-, 12-month-old SAMR1; lanes 5 to 8: 1-, 4-, 8-, 12-month-old SAMP8. (D) Quantification of the western blot analysis. The relative intensities of the MTH2 bands against the tubulin bands are shown. The data are given as the means± SD of six animals. The statistical analysis was performed using ANOVA followed by Dunnett's test. *p b 0.05, in comparison to 1-month-old SAMP8. # p b 0.05, in comparison to the age-matched control.
able to learn the water maze task; however, older SAMP8 mice (8 and 12 months old) exhibited a declined learning ability. These results are consistent with the findings of others [31–33]. Meanwhile, the
grading scores of senescence were significantly higher in the 8- and 12-month-old SAMP8 mice in comparison to the levels found in agematched SAMR1 mice. Studies on the expression of APP in the brain
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also proved the usefulness of this strain of mouse as a murine model for aging. Both IHC and the western blotting analysis showed a significant increase in APP expression from 8 to 12 months after birth in SAMP8 mice, while there was only a slight change in the SAMR1 control mice over the same time period. These results are in agreement with the findings of Morley et al. [34]. The elevated levels of the grading score of senescence and impairment of special cognitive performance occurred at almost the same time when the amount of 8-oxoguanine in the DNA increased in the CA1 and CA3 subregions of the hippocampi of the SAMP8 mice. There was no significant change in the 8-oxoguanine content in the corresponding regions of the SAMR1 mice. Morley et al. [34] reported that the visible deposition of senile plaques appeared 16 months after birth in SAMP8 mice. Since the accumulation of 8-oxoguanine in DNA becomes evident as early as 8 months after birth, the increase of 8-oxoguanine may be the initiating factor rather than the consequence of APP deposition, although the latter would play an important role in the vicious cycle. These results are reminiscent of the findings of Wang et al. [35] and Nunomura et al. [36], who showed that there is a significant increase in 8-oxoguanine content in DNA in the cases of AD in comparison to normal subjects. An over-expression of APP is also considered as an important factor in AD, and there have been numerous studies that have indicated that APP is over-expressed in AD patients [37,38]. The immunohistochemical results revealed that the MTH2 protein is present in the CA1 and CA3 regions of mouse hippocampus. In SAMP8 mice, the amount of this protein in these regions decreased progressively, beginning from 8 months after birth. No such change in MTH2 content was observed in the control SAMR1 mice. This result suggests that the decrease in the level of MTH2 might be related to the deterioration of learning and memory, which emerges at the same age. The result of the western blot analysis revealed that the decline in MTH2 level actually started as early as 4 months after birth in the SAMP8 mice, which is inferred as the causative effect of MTH2 decrease in the onset of the disease. How this could happen is unknown. We know that MTH2 possesses the specific enzymatic activity to convert the deleterious 8-oxo-dGTP to 8-oxo-dGMP. Considering the significant increase of 8-oxoguanine in DNA, we hypothesized that the decline of MTH2 expression in the hippocampus might lead to the insufficient sanitization of 8-oxo-dGTP, which might consequently result in the increase of 8-oxoguanine in DNA. The overload of 8-oxo-dGTP may be involved in the onset and progression of APP over-expression and the final formation of Aβ deposition. Further studies are in progress to address these possibilities. Overall, our findings show that MTH2 deficiency is associated with the progression of aging, yet the exact mechanism still needs to be studied further. It has been suggested that the decline of hippocampal synapses may be a cause of the cognition impairment in AD [39]. In a western blot analysis of various tissues, we found that all organs except the liver contain substantial amounts of MTH2 protein and that the highest levels of the protein are present in the brain and testis of both types of mouse strains (data not shown). If we assume that high oxygen consumption causes the high level of oxidative damage to DNA [40], the level of the MTH2 proteins might be concerned with this outcome. To better understand the role of MTH2 in this process, we need to develop a mouse line defective in the Mth2 gene, and studies regarding this aspect are now in progress. Acknowledgement This work was supported by the Key International Science and Technology Cooperation Projects (2006DFB31410). We thank the members of the Institute of Geriatrics of the Ministry of Health for advice and assistance. We thank B. Quinn for language assistance. Disclosure statement There is no conflict of interest.
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