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Ferulic acid attenuated cognitive deficits and increase in carbonyl proteins induced by buthionine-sulfoximine in mice
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Neuroscience Letters 430 (2008) 115–118
Ferulic acid attenuated cognitive deficits and increase in carbonyl proteins induced by buthionine-sulfoximine in mice Takayoshi Mamiya a,∗ , Mitsuo Kise b , Keiko Morikawa b a
Department of Chemical Pharmacology, Faculty of Pharmaceutical Sciences, Meijo University, 150 Yagotoyama, Tempaku-ku, Nagoya 468-8503, Japan b Applied Food Processing Group, FANCL Research Institute, FANCL Corporation, 12-13 Kamishinano, Totsuka-ku, Yokohama 244-0806, Japan Received 9 July 2007; received in revised form 8 October 2007; accepted 26 October 2007
Abstract -Amyloid peptide (A), the major constituent of the senile plaques observed in the brains of Alzheimer’s disease patients, is cytotoxic to neurons and plays a central role in the pathogenesis of this disease. Previous studies have suggested that oxidative stress is involved in the mechanisms of A-induced neurotoxicity in vivo. Here, we used a mouse model of brain dysfunction induced by dl-buthionine-(S,R)-sulfoximine (BSO: 3 mol/3 L/mouse, i.c.v.), an inhibitor of glutathione synthesis. In the novel object recognition test, we found impairments of exploratory preference in the retention trial but not the training trial 24 h after BSO treatment, suggesting that BSO produces cognitive dysfunction in mice. In the forebrain of this model, we observed increase in carbonyl protein levels, an index of biochemical oxidative damage of proteins, compared to vehicle-treated mice. Pretreatment with ferulic acid (5 mg/kg, s.c.) once a day for 6 days inhibited the induction of deficits in memory and increase in carbonyl protein levels by BSO. These findings suggest that pretreatment with FA may attenuate the memory deficits and increase the carbonyl protein levels induced by BSO in mice. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Buthionine sulfoximine; Ferulic acid; Novel object recognition test; Carbonyl protein; Short-term memory
Alzheimer’s disease (AD) is the most common cause of progressive cognitive impairment in the elderly. One of the principal pathophysiological features of this disease is the presence of extracellular senile plaques consisting essentially of -amyloid protein (A), a peptide that is thought to be a leading cause of neurotoxicity. There is accumulating evidence that oxidative stress (OS) contributes to A-induced neurotoxicity [see reviews: 3, 4, 17]. Glutathione, an endogenous antioxidant, plays a critical role in intracellular defense against OS in the normal physiological condition. In the brain, where it is present in millimolar concentrations [12], glutathione scavenges reactive oxygen species and is capable of neutralizing free radicals [16]. Depletion of glutathione in brain might thus result in free-radical-induced neuronal damage [1]. Buthionine sulfoximine (BSO) selectively inhibits ␥-glutamylcysteine synthetase, a key enzyme in glutathione biosynthesis, and thereby decreases glutathione concentration [6]. Treatment with BSO may thus
∗
Corresponding author. Tel.: +81 52 839 2737; fax: +81 52 834 8090. E-mail address: [email protected] (T. Mamiya).
0304-3940/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2007.10.029
decrease an organism’s antioxidant capacity, making it susceptible to neuronal damage by OS. In this study, we therefore examined whether BSO-induced impairment of cognitive behavior in the novel object recognition test and measured carbonyl protein levels to assess neuronal damage in the forebrain (because especially cortex and hippocampus play important roles in memory acquisition and storage) after the behavioral test. We recently reported that pre-germinated brown rice prevents A-induced learning and memory deficits in mice [8], although the mechanism by which it does so is unclear in detail. Ferulic acid (FA), one of the antioxidants in pre-germinated brown rice, is believed to have played an important role in the attenuation of A-induced learning and memory deficits in that study. We therefore also evaluated the effects of repeated administration of FA on this model of BSO-induced cognitive dysfunction. We used male ICR strain mice (25 weeks old, Nihon SLC Co., Shizuoka, Japan). The animals were housed in a controlled environment (23 ± 1 ◦ C, 50 ± 5% humidity) and given access to standard food pellet (AIN-93G, Oriental Yeast Co., Ltd., Japan) and tap water bottle ad libitum [8,9]. Room lights were on between 7:30 and 19:30 h. Ferulic acid (Sigma, MO, USA)
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and dl-buthionine-(S,R)-sulfoximine (BSO; Sigma, MO, USA, FW = 222.3) were dissolved in 0.9% physiological saline. ␣Tocopherol (vitamin E, Sigma, MO, USA) was dissolved in soybean oil. All experiments were performed in accordance with the Guidelines for Animal Experiments of Meijo University and the Guiding Principles for the Care and Use of Laboratory Animals approved by the Japanese Pharmacological Society (2006). BSO and the vehicle (3 L) were administered intracerebroventricularly (i.c.v.) for approximately 20 s under light ether anesthesia [10]. The site of administration was checked by injecting Indian ink in preliminary experiments. Neither insertion of the needle nor injection of the vehicle had a significant influence on survival, behavioral responses, or cognitive function. The experiment was conducted for 6 days. FA (0.5, 1, or 5 mg/kg) and vitamin E (300 mg/kg) were administered subcutaneously once a day for 6 days. BSO (3 mol/3 L/mouse) was administered intracerebroventricularly (i.c.v.) to mice 24 h before the training trial of the novel object recognition test. In the novel object recognition test, a mouse was habituated to a black plastic cage (30 cm × 30 cm floor × 50 cm height) for 15 min on days 4 and 5. On day 6, in the training trial, two same-sample objects (white film cases, Ø 3 cm × 6 cm) were placed in the cage (5 cm from the center each), and the mouse was allowed to explore them freely for 5 min. The time spent exploring each object was recorded manually. In the retention trial, which was conducted immediately after the training trial, the mouse was removed once and then placed back in the same cage, and one of the objects used in the training trial was replaced with a novel object (black dry battery cell, Ø 3 cm × 6 cm) for the mouse to explore freely for 5 min. Exploratory preference (%), which is
the ratio of time spent exploring any one of the two same-sample objects in the training trial or the novel object in the retention trial to the total time spent exploring both objects, was used as an index of recognition memory. After the behavioral test, some mice from each group were sacrificed by decapitation under light ether anesthesia and their forebrains were removed. Short-term memory was examined by monitoring spontaneous alternation behavior in the Y-maze. The maze was made of wood painted black and each arm was 40 cm long, 12 cm high, 3 cm wide at the bottom and 10 cm wide at the top. The arms converged at an equilateral triangular central area that was 4 cm at its longest axis. The apparatus was placed on the floor of the experimental room and illuminated with a 100 W bulb from 200 cm above. Each mouse was placed at the end of one arm and allowed to move freely through the maze during an 8 min session, and the series of arm entries was recorded visually. The alternation behavior was defined by the successive entry into the three arms, on overlapping triplet sets, and such behavior (%) was expressed as the ratio of actual alternations to possible alternations (defined as the total number of arm entries minus two), multiplied by 100 [10]. The forebrains were homogenized in 28% (w/v) trichloroacetic acid and left for 10 min on ice. The homogenate was centrifuged for 3 min at 10,000 rpm at 4 ◦ C. The extent of protein oxidation was assessed by measuring carbonyl proteins spectrophotometrically at 450 nm with the 2,4dinitrophenylhydrazine (DNP)-labeling procedure using a carbonyl protein enzyme immunoassay kit (Zentech PC Test, Zenith Technology Corp., Ltd., New Zealand). Protein content was determined according to the Lowry method utilizing bovine serum albumin as standard.
Fig. 1. Effects of ferulic acid on exploratory preference in training (A) and retention (B) trials of the novel object recognition test in mice. Columns indicate mean ± S.E.M. The number of mice included in each group is shown in each column. Sal, saline; VE, vitamin E 300 mg/kg; BSO, buthionine sulfoximine. **P < 0.01 vs. saline-treated group, ## P < 0.01 vs. BSO-treated group (Tukey’s test).
T. Mamiya et al. / Neuroscience Letters 430 (2008) 115–118
All results are expressed as means ± S.E.M. for each group. Results were examined by one-way ANOVA, followed by Tukey’s test. The level of significance was P < 0.05. Each group approaches one object for 13.8–24.3 s and as shown in Fig. 1, there were no differences in exploratory preference among the groups in the training trial (F5,69 = 0.61, P = 0.69). In the retention trial, the (Sal + Sal)-treated group exhibited greater preference for the novel object than the familiar object. The duration of exploration of the novel object was decreased by BSO (3 mol/mouse) without affecting ambulation. FA in dose-dependent manner attenuated the decrement in exploratory preference induced by BSO to a value comparable to that obtained with vitamin E administration (F5,69 = 6.27, P < 0.01, Tukey’s test; P < 0.05). We did not check the effects of FA alone on the carbonyl protein levels since FA alone failed to affect behavioral changes in our preliminary experiments. Table 1 shows the results of Y-maze test. When we administered BSO to mice, it impaired significantly the spontaneous alternation behavior in the control group (F5,65 = 4.88, P < 0.01, Tukey’s test; P < 0.01). FA (5 mg/kg) or vitamin E attenuated the impairments of spontaneous alternation behavior. On the other hand, there was no significant difference among the six groups on the number of arm entries (F5,65 = 0.92, P > 0.05). In Fig. 2, the extent of protein oxidation in forebrain was assessed by measuring carbonyl protein levels. Compared with the (Sal + Sal)-treated group, the BSO-treated group had markedly higher carbonyl protein levels (F5,29 = 7.63, P < 0.01, Tukey’s test; P < 0.05). FA (0.5–5 mg/kg) significantly reduced carbonyl protein levels (Tukey’s test; P < 0.05) to a value less than that when vitamin E was administered at 300 mg/kg. In the present study, we investigated the effects of FA on BSO-induced changes in exploratory preference and carbonyl protein levels in mice. Because brain glutathione depletion by BSO is marked and long-lasting, we administered i.c.v. BSO to induce OS [13,14]. Pileblad and Magnusson [13] have shown that rat striatum glutathione concentration is reduced by as much as 60% one day after treatment with BSO (14.4 mol, i.c.v.). In fact, we demonstrated that a low dose of BSO (3 mol, i.c.v.) increased carbonyl protein levels by 70%, with this level serving
Table 1 Effects of ferulic acid on spontaneous alternation behavior in the Y-maze test in mice Treatment (mg/kg)
N
Alternation behavior (%)
Sal + FA (0) BSO + FA (0) +FA (0.5) +FA (1) +FA (5) +VE (300)
15 13 10 10 10 8
71.7 61.8 61.6 60.8 71.7 68.7
± ± ± ± ± ±
1.7 2.1* 2.6 2.4 3.1# 3.2#
Number of arm entries 27.6 32.5 33.7 30.7 32.0 36.4
± ± ± ± ± ±
2.3 2.5 2.1 2.3 2.3 3.1
Values indicate mean ± S.E.M. Sal, saline; VE, vitamin E 300 mg/kg; BSO, buthionine sulfoximine. * P < 0.05 vs. Sal + FA (0)-treated group. # P < 0.05 vs. BSO + FA (0)-treated group (Tukey’s test).
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Fig. 2. Effects of ferulic acid on protein carbonyl levels in forebrains of mice. Columns indicate mean ± S.E.M. The number of mice included in each group is shown in each column. Sal, saline; VE, vitamin E 300 mg/kg; BSO, buthionine sulfoximine. *P < 0.05 vs. saline-treated group, ## P < 0.01, # P < 0.05 vs. BSOtreated group (Tukey’s test).
as an index of peroxidation in mouse forebrain. Statistical analysis did not show significant difference between (Sal + Sal)-group and (BSO + FA 5 mg/kg)-treated group. Because here we used 25-week-old mice based on the reports that brain glutathione may be decreased in aging [5,11], the value of (Sal + Sal)-group may be higher than that of younger mice and FA (5 mg/kg) alone may lower the oxidative stress to the level of young mouse. In the novel object recognition test, we observed no difference in exploratory time for same-sample objects among the groups in the training trial, indicating that mice in all groups had essentially the same levels of motivation to explore the two same-sample objects. Since we focused on the short-term memory, there is no time interval between the training and retention trials. In the retention trial, control (Sal + Sal-treated) mice required a longer time to approach the novel object than those in the training trial. In contrast, BSO-treated mice failed to explore the novel object for a long time, suggesting that BSO impaired performance of this task. Notably, this impairment of exploratory preference for the novel object was attenuated by FA (5 mg/kg). We observed similar effects of FA in the Y-maze test, which is also used to evaluate short-term memory. In the biochemical study, we found that FA inhibited the BSO-induced increase in protein carbonyl levels. It appears that doses higher than biochemical ones are needed to attenuate behavioral dysfunction. These findings suggest that FA may attenuate memory deficits and protein oxidation in the brain induced by BSO in mice, whereas we have to investigate effects of FA on long-term memory with 24 h interval. As observed in the brains of AD patients, exposure to A increases lipid peroxidation, protein oxidation, and the formation of H2 O2 in cultured cells [2]. The increase in oxidative stress by A contributes to A-induced neurotoxicity. This study focused on the oxidative stress and revealed that BSO elevated carbonyl protein and induced behavioral cognitive deficits, so
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that we believe that this model may reflect one of the biochemical and behavioral syndromes of AD. Antioxidants such as vitamin E, idebenone, and FA have been reported to be effective in an animal model of AD induced by A [7,18,19]. Yan et al. have shown that pretreatment with FA in drinking water for 28 days prevented A-induced impairment of learning and memory in mice [19]. In their study, A exhibited increased immunoreactivity for glial fibrillary acidic protein and interleukin-6, which are inflammatory markers, though direct involvement of A and FA in OS was not shown. Our study revealed that FA has potent efficacy in reversing the symptoms of OS, in addition to its anti-inflammatory effect. As noted above, we have reported that pre-germinated brown rice (PGBR) can reduce the impairments caused by A in mice [8]. PGBR includes not only FA but also ␥-oryzanol, which is mainly metabolized to FA in liver [15]. Taken together, these findings suggest that FA in PGBR may be a key compound in the attenuation of A-induced learning and memory deficits in mice. As a next step, we plan to clarify the details of this mechanism. In conclusion, FA may inhibit OS and thus be useful for treatment of AD. Acknowledgments This study was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology, Japan; a Sasakawa Scientific Research Grant from The Japan Science Society; the Mochida Memorial Foundation for Medical and Pharmaceutical Research; a Scientific Research Grant from Tokai Gakujutsu (Scientific) Bounty; a Research Grant from the Ichihara International Scholarship Foundation; and a Research Grant from the Japan Health and Research Institute. We are thankful to late Professor Makoto Ukai and Takamasa Asanuma for assistance with experiments. References [1] B.N. Ames, M.K. Shigenaga, T.M. Hagen, Oxidants, antioxidants, and the degenerative diseases of aging, Proc. Natl. Acad. Sci. U.S.A. 90 (1993) 7915–7922. [2] C. Behl, J.B. Davis, R. Lesley, D. Schubert, Hydrogen peroxide mediates amyloid beta protein toxicity, Cell 77 (1994) 817–827. [3] D.A. Butterfield, D. Boyd-Kimball, Amyloid beta-peptide(1–42) contributes to the oxidative stress and neurodegeneration found in Alzheimer disease brain, Brain Pathol. 14 (2004) 426–432.
[4] D.A. Butterfield, Amyloid beta-peptide (1–42)-induced oxidative stress and neurotoxicity: implications for neurodegeneration in Alzheimer’s disease brain. A review, Free Radic. Res. 36 (2002) 1307–1313. [5] T.S. Chen, J.P. Richie Jr., C.A. Lang, The effect of aging on glutathione and cysteine levels in different regions of the mouse brain, Proc. Soc. Exp. Biol. Med. 190 (1989) 399–402. [6] O.W. Griffith, Mechanism of action, metabolism, and toxicity of buthionine sulfoximine and its higher homologs, potent inhibitors of glutathione synthesis, J. Biol. Chem. 257 (1982) 13704–13712. [7] J.H. Jhoo, H.C. Kim, T. Nabeshima, K. Yamada, E.J. Shin, W.K. Jhoo, W. Kim, K.S. Kang, S.A. Jo, J.I. Woo, Beta-amyloid (1–42)-induced learning and memory deficits in mice: involvement of oxidative burdens in the hippocampus and cerebral cortex, Behav. Brain Res. 155 (2004) 185–196. [8] T. Mamiya, T. Asanuma, M. Kise, Y. Ito, A. Mizukuchi, H. Aoto, M. Ukai, Effects of pre-germinated brown rice on beta-amyloid proteininduced learning and memory deficits in mice, Biol. Pharm. Bull. 27 (2004) 1041–1045. [9] T. Mamiya, M. Kise, K. Morikawa, H. Aoto, M. Ukai, Y. Noda, Effects of pre-germinated brown rice on depression-like behavior in mice, Pharmacol. Biochem. Behav. 86 (2007) 62–67. [10] T. Mamiya, M. Ukai, [Gly14 ]-Humanin improved the learning and memory impairment induced by scopolamine in vivo, Br. J. Pharmacol. 134 (2001) 1597–1599. [11] J.Q. Mo, D.G. Hom, J.K. Andersen, Decreases in protective enzymes correlates with increased oxidative damage in the aging mouse brain, Mech. Ageing Dev. 81 (1995) 73–82. [12] E. Pileblad, T. Magnusson, Effective depletion of glutathione in rat striatum and substantia nigra by l-buthionine sulfoximine in combination with 2cyclohexene-1-one, Life Sci. 47 (1990) 2333–2342. [13] E. Pileblad, T. Magnusson, Intracerebroventricular administration of lbuthionine sulfoximine: a method for depleting brain glutathione, J. Neurochem. 53 (1989) 1878–1882. [14] B. Shukitt-Hale, S.A. Erat, J.A. Joseph, Spatial learning and memory deficits induced by dopamine administration with decreased glutathione, Free Radic. Biol. Med. 24 (1998) 1149–1158. [15] K. Umehara, Y. Shimokawa, G. Miyamoto, Effects of ␥-oryzanol on cytochrome P450 activities in human liver, Biol. Pharm. Bull. 27 (2004) 1151–1153. [16] A. Vanella, C. Di Giacomo, V. Sorrenti, A. Russo, C. Castorina, A. Campisi, M. Renis, J.R. Perez-Polo, Free radical scavenger depletion in post-ischemic reperfusion brain damage, Neurochem. Res. 18 (1993) 1337–1340. [17] K. Yamada, T. Nabeshima, Animal models of Alzheimer’s disease and evaluation of anti-dementia drugs, Pharmacol. Ther. 88 (2000) 93–113. [18] K. Yamada, T. Tanaka, D. Han, K. Senzaki, T. Kameyama, T. Nabeshima, Protective effects of idebenone and alpha-tocopherol on beta-amyloid(1–42)-induced learning and memory deficits in rats: implication of oxidative stress in beta-amyloid-induced neurotoxicity in vivo, Eur. J. Neurosci. 11 (1999) 83–90. [19] J.J. Yan, J.Y. Cho, H.S. Kim, K.L. Kim, J.S. Jung, S.O. Huh, H.W. Suh, Y.H. Kim, D.K. Song, Protection against beta-amyloid peptide toxicity in vivo with long-term administration of ferulic acid, Br. J. Pharmacol. 133 (2001) 89–96.
Neuroscience Letters 430 (2008) 115–118
Ferulic acid attenuated cognitive deficits and increase in carbonyl proteins induced by buthionine-sulfoximine in mice Takayoshi Mamiya a,∗ , Mitsuo Kise b , Keiko Morikawa b a
Department of Chemical Pharmacology, Faculty of Pharmaceutical Sciences, Meijo University, 150 Yagotoyama, Tempaku-ku, Nagoya 468-8503, Japan b Applied Food Processing Group, FANCL Research Institute, FANCL Corporation, 12-13 Kamishinano, Totsuka-ku, Yokohama 244-0806, Japan Received 9 July 2007; received in revised form 8 October 2007; accepted 26 October 2007
Abstract -Amyloid peptide (A), the major constituent of the senile plaques observed in the brains of Alzheimer’s disease patients, is cytotoxic to neurons and plays a central role in the pathogenesis of this disease. Previous studies have suggested that oxidative stress is involved in the mechanisms of A-induced neurotoxicity in vivo. Here, we used a mouse model of brain dysfunction induced by dl-buthionine-(S,R)-sulfoximine (BSO: 3 mol/3 L/mouse, i.c.v.), an inhibitor of glutathione synthesis. In the novel object recognition test, we found impairments of exploratory preference in the retention trial but not the training trial 24 h after BSO treatment, suggesting that BSO produces cognitive dysfunction in mice. In the forebrain of this model, we observed increase in carbonyl protein levels, an index of biochemical oxidative damage of proteins, compared to vehicle-treated mice. Pretreatment with ferulic acid (5 mg/kg, s.c.) once a day for 6 days inhibited the induction of deficits in memory and increase in carbonyl protein levels by BSO. These findings suggest that pretreatment with FA may attenuate the memory deficits and increase the carbonyl protein levels induced by BSO in mice. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Buthionine sulfoximine; Ferulic acid; Novel object recognition test; Carbonyl protein; Short-term memory
Alzheimer’s disease (AD) is the most common cause of progressive cognitive impairment in the elderly. One of the principal pathophysiological features of this disease is the presence of extracellular senile plaques consisting essentially of -amyloid protein (A), a peptide that is thought to be a leading cause of neurotoxicity. There is accumulating evidence that oxidative stress (OS) contributes to A-induced neurotoxicity [see reviews: 3, 4, 17]. Glutathione, an endogenous antioxidant, plays a critical role in intracellular defense against OS in the normal physiological condition. In the brain, where it is present in millimolar concentrations [12], glutathione scavenges reactive oxygen species and is capable of neutralizing free radicals [16]. Depletion of glutathione in brain might thus result in free-radical-induced neuronal damage [1]. Buthionine sulfoximine (BSO) selectively inhibits ␥-glutamylcysteine synthetase, a key enzyme in glutathione biosynthesis, and thereby decreases glutathione concentration [6]. Treatment with BSO may thus
∗
Corresponding author. Tel.: +81 52 839 2737; fax: +81 52 834 8090. E-mail address: [email protected] (T. Mamiya).
0304-3940/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2007.10.029
decrease an organism’s antioxidant capacity, making it susceptible to neuronal damage by OS. In this study, we therefore examined whether BSO-induced impairment of cognitive behavior in the novel object recognition test and measured carbonyl protein levels to assess neuronal damage in the forebrain (because especially cortex and hippocampus play important roles in memory acquisition and storage) after the behavioral test. We recently reported that pre-germinated brown rice prevents A-induced learning and memory deficits in mice [8], although the mechanism by which it does so is unclear in detail. Ferulic acid (FA), one of the antioxidants in pre-germinated brown rice, is believed to have played an important role in the attenuation of A-induced learning and memory deficits in that study. We therefore also evaluated the effects of repeated administration of FA on this model of BSO-induced cognitive dysfunction. We used male ICR strain mice (25 weeks old, Nihon SLC Co., Shizuoka, Japan). The animals were housed in a controlled environment (23 ± 1 ◦ C, 50 ± 5% humidity) and given access to standard food pellet (AIN-93G, Oriental Yeast Co., Ltd., Japan) and tap water bottle ad libitum [8,9]. Room lights were on between 7:30 and 19:30 h. Ferulic acid (Sigma, MO, USA)
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and dl-buthionine-(S,R)-sulfoximine (BSO; Sigma, MO, USA, FW = 222.3) were dissolved in 0.9% physiological saline. ␣Tocopherol (vitamin E, Sigma, MO, USA) was dissolved in soybean oil. All experiments were performed in accordance with the Guidelines for Animal Experiments of Meijo University and the Guiding Principles for the Care and Use of Laboratory Animals approved by the Japanese Pharmacological Society (2006). BSO and the vehicle (3 L) were administered intracerebroventricularly (i.c.v.) for approximately 20 s under light ether anesthesia [10]. The site of administration was checked by injecting Indian ink in preliminary experiments. Neither insertion of the needle nor injection of the vehicle had a significant influence on survival, behavioral responses, or cognitive function. The experiment was conducted for 6 days. FA (0.5, 1, or 5 mg/kg) and vitamin E (300 mg/kg) were administered subcutaneously once a day for 6 days. BSO (3 mol/3 L/mouse) was administered intracerebroventricularly (i.c.v.) to mice 24 h before the training trial of the novel object recognition test. In the novel object recognition test, a mouse was habituated to a black plastic cage (30 cm × 30 cm floor × 50 cm height) for 15 min on days 4 and 5. On day 6, in the training trial, two same-sample objects (white film cases, Ø 3 cm × 6 cm) were placed in the cage (5 cm from the center each), and the mouse was allowed to explore them freely for 5 min. The time spent exploring each object was recorded manually. In the retention trial, which was conducted immediately after the training trial, the mouse was removed once and then placed back in the same cage, and one of the objects used in the training trial was replaced with a novel object (black dry battery cell, Ø 3 cm × 6 cm) for the mouse to explore freely for 5 min. Exploratory preference (%), which is
the ratio of time spent exploring any one of the two same-sample objects in the training trial or the novel object in the retention trial to the total time spent exploring both objects, was used as an index of recognition memory. After the behavioral test, some mice from each group were sacrificed by decapitation under light ether anesthesia and their forebrains were removed. Short-term memory was examined by monitoring spontaneous alternation behavior in the Y-maze. The maze was made of wood painted black and each arm was 40 cm long, 12 cm high, 3 cm wide at the bottom and 10 cm wide at the top. The arms converged at an equilateral triangular central area that was 4 cm at its longest axis. The apparatus was placed on the floor of the experimental room and illuminated with a 100 W bulb from 200 cm above. Each mouse was placed at the end of one arm and allowed to move freely through the maze during an 8 min session, and the series of arm entries was recorded visually. The alternation behavior was defined by the successive entry into the three arms, on overlapping triplet sets, and such behavior (%) was expressed as the ratio of actual alternations to possible alternations (defined as the total number of arm entries minus two), multiplied by 100 [10]. The forebrains were homogenized in 28% (w/v) trichloroacetic acid and left for 10 min on ice. The homogenate was centrifuged for 3 min at 10,000 rpm at 4 ◦ C. The extent of protein oxidation was assessed by measuring carbonyl proteins spectrophotometrically at 450 nm with the 2,4dinitrophenylhydrazine (DNP)-labeling procedure using a carbonyl protein enzyme immunoassay kit (Zentech PC Test, Zenith Technology Corp., Ltd., New Zealand). Protein content was determined according to the Lowry method utilizing bovine serum albumin as standard.
Fig. 1. Effects of ferulic acid on exploratory preference in training (A) and retention (B) trials of the novel object recognition test in mice. Columns indicate mean ± S.E.M. The number of mice included in each group is shown in each column. Sal, saline; VE, vitamin E 300 mg/kg; BSO, buthionine sulfoximine. **P < 0.01 vs. saline-treated group, ## P < 0.01 vs. BSO-treated group (Tukey’s test).
T. Mamiya et al. / Neuroscience Letters 430 (2008) 115–118
All results are expressed as means ± S.E.M. for each group. Results were examined by one-way ANOVA, followed by Tukey’s test. The level of significance was P < 0.05. Each group approaches one object for 13.8–24.3 s and as shown in Fig. 1, there were no differences in exploratory preference among the groups in the training trial (F5,69 = 0.61, P = 0.69). In the retention trial, the (Sal + Sal)-treated group exhibited greater preference for the novel object than the familiar object. The duration of exploration of the novel object was decreased by BSO (3 mol/mouse) without affecting ambulation. FA in dose-dependent manner attenuated the decrement in exploratory preference induced by BSO to a value comparable to that obtained with vitamin E administration (F5,69 = 6.27, P < 0.01, Tukey’s test; P < 0.05). We did not check the effects of FA alone on the carbonyl protein levels since FA alone failed to affect behavioral changes in our preliminary experiments. Table 1 shows the results of Y-maze test. When we administered BSO to mice, it impaired significantly the spontaneous alternation behavior in the control group (F5,65 = 4.88, P < 0.01, Tukey’s test; P < 0.01). FA (5 mg/kg) or vitamin E attenuated the impairments of spontaneous alternation behavior. On the other hand, there was no significant difference among the six groups on the number of arm entries (F5,65 = 0.92, P > 0.05). In Fig. 2, the extent of protein oxidation in forebrain was assessed by measuring carbonyl protein levels. Compared with the (Sal + Sal)-treated group, the BSO-treated group had markedly higher carbonyl protein levels (F5,29 = 7.63, P < 0.01, Tukey’s test; P < 0.05). FA (0.5–5 mg/kg) significantly reduced carbonyl protein levels (Tukey’s test; P < 0.05) to a value less than that when vitamin E was administered at 300 mg/kg. In the present study, we investigated the effects of FA on BSO-induced changes in exploratory preference and carbonyl protein levels in mice. Because brain glutathione depletion by BSO is marked and long-lasting, we administered i.c.v. BSO to induce OS [13,14]. Pileblad and Magnusson [13] have shown that rat striatum glutathione concentration is reduced by as much as 60% one day after treatment with BSO (14.4 mol, i.c.v.). In fact, we demonstrated that a low dose of BSO (3 mol, i.c.v.) increased carbonyl protein levels by 70%, with this level serving
Table 1 Effects of ferulic acid on spontaneous alternation behavior in the Y-maze test in mice Treatment (mg/kg)
N
Alternation behavior (%)
Sal + FA (0) BSO + FA (0) +FA (0.5) +FA (1) +FA (5) +VE (300)
15 13 10 10 10 8
71.7 61.8 61.6 60.8 71.7 68.7
± ± ± ± ± ±
1.7 2.1* 2.6 2.4 3.1# 3.2#
Number of arm entries 27.6 32.5 33.7 30.7 32.0 36.4
± ± ± ± ± ±
2.3 2.5 2.1 2.3 2.3 3.1
Values indicate mean ± S.E.M. Sal, saline; VE, vitamin E 300 mg/kg; BSO, buthionine sulfoximine. * P < 0.05 vs. Sal + FA (0)-treated group. # P < 0.05 vs. BSO + FA (0)-treated group (Tukey’s test).
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Fig. 2. Effects of ferulic acid on protein carbonyl levels in forebrains of mice. Columns indicate mean ± S.E.M. The number of mice included in each group is shown in each column. Sal, saline; VE, vitamin E 300 mg/kg; BSO, buthionine sulfoximine. *P < 0.05 vs. saline-treated group, ## P < 0.01, # P < 0.05 vs. BSOtreated group (Tukey’s test).
as an index of peroxidation in mouse forebrain. Statistical analysis did not show significant difference between (Sal + Sal)-group and (BSO + FA 5 mg/kg)-treated group. Because here we used 25-week-old mice based on the reports that brain glutathione may be decreased in aging [5,11], the value of (Sal + Sal)-group may be higher than that of younger mice and FA (5 mg/kg) alone may lower the oxidative stress to the level of young mouse. In the novel object recognition test, we observed no difference in exploratory time for same-sample objects among the groups in the training trial, indicating that mice in all groups had essentially the same levels of motivation to explore the two same-sample objects. Since we focused on the short-term memory, there is no time interval between the training and retention trials. In the retention trial, control (Sal + Sal-treated) mice required a longer time to approach the novel object than those in the training trial. In contrast, BSO-treated mice failed to explore the novel object for a long time, suggesting that BSO impaired performance of this task. Notably, this impairment of exploratory preference for the novel object was attenuated by FA (5 mg/kg). We observed similar effects of FA in the Y-maze test, which is also used to evaluate short-term memory. In the biochemical study, we found that FA inhibited the BSO-induced increase in protein carbonyl levels. It appears that doses higher than biochemical ones are needed to attenuate behavioral dysfunction. These findings suggest that FA may attenuate memory deficits and protein oxidation in the brain induced by BSO in mice, whereas we have to investigate effects of FA on long-term memory with 24 h interval. As observed in the brains of AD patients, exposure to A increases lipid peroxidation, protein oxidation, and the formation of H2 O2 in cultured cells [2]. The increase in oxidative stress by A contributes to A-induced neurotoxicity. This study focused on the oxidative stress and revealed that BSO elevated carbonyl protein and induced behavioral cognitive deficits, so
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that we believe that this model may reflect one of the biochemical and behavioral syndromes of AD. Antioxidants such as vitamin E, idebenone, and FA have been reported to be effective in an animal model of AD induced by A [7,18,19]. Yan et al. have shown that pretreatment with FA in drinking water for 28 days prevented A-induced impairment of learning and memory in mice [19]. In their study, A exhibited increased immunoreactivity for glial fibrillary acidic protein and interleukin-6, which are inflammatory markers, though direct involvement of A and FA in OS was not shown. Our study revealed that FA has potent efficacy in reversing the symptoms of OS, in addition to its anti-inflammatory effect. As noted above, we have reported that pre-germinated brown rice (PGBR) can reduce the impairments caused by A in mice [8]. PGBR includes not only FA but also ␥-oryzanol, which is mainly metabolized to FA in liver [15]. Taken together, these findings suggest that FA in PGBR may be a key compound in the attenuation of A-induced learning and memory deficits in mice. As a next step, we plan to clarify the details of this mechanism. In conclusion, FA may inhibit OS and thus be useful for treatment of AD. Acknowledgments This study was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology, Japan; a Sasakawa Scientific Research Grant from The Japan Science Society; the Mochida Memorial Foundation for Medical and Pharmaceutical Research; a Scientific Research Grant from Tokai Gakujutsu (Scientific) Bounty; a Research Grant from the Ichihara International Scholarship Foundation; and a Research Grant from the Japan Health and Research Institute. We are thankful to late Professor Makoto Ukai and Takamasa Asanuma for assistance with experiments. References [1] B.N. Ames, M.K. Shigenaga, T.M. Hagen, Oxidants, antioxidants, and the degenerative diseases of aging, Proc. Natl. Acad. Sci. U.S.A. 90 (1993) 7915–7922. [2] C. Behl, J.B. Davis, R. Lesley, D. Schubert, Hydrogen peroxide mediates amyloid beta protein toxicity, Cell 77 (1994) 817–827. [3] D.A. Butterfield, D. Boyd-Kimball, Amyloid beta-peptide(1–42) contributes to the oxidative stress and neurodegeneration found in Alzheimer disease brain, Brain Pathol. 14 (2004) 426–432.
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