Inhibition of JNK phosphorylation reverses memory deficit induced by β-amyloid (1–42) associated with decrease of apoptotic factors

Behavioural Brain Research 217 (2011) 424–431

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Inhibition of JNK phosphorylation reverses memory deficit induced by ␤-amyloid (1–42) associated with decrease of apoptotic factors Mahmoudreza Ramin, Pegah Azizi, Fereshteh Motamedi, Abbas Haghparast, Fariba Khodagholi ∗ Neuroscience Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran

a r t i c l e

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Article history: Received 7 September 2010 Received in revised form 25 October 2010 Accepted 5 November 2010 Available online 11 November 2010 Keywords: ␤-amyloid Alzheimer Apoptosis JNK SP600125 Morris water maze Rat

a b s t r a c t Alzheimer’s disease (AD) is the most common form of dementia that is degenerative and terminal disease. The main reason of the disease is still unknown. ␤-amyloid (A␤) plaques are the important hallmarks of memory impairment in patients suffering from AD. Aggregation of these plaques in the hippocampus appears during the development of the disease. One of the prominent factors having crucial impact in this process is MAPK. JNK, as a member of MAPK family has a pivotal role, especially in cell survival. We hypothesized that JNK may have beneficial effect on the process of memory improvement. Hence, we performed Morris water maze to investigate the possible impact of JNK inhibitor on spatial memory in A␤injected rats. Our data indicated that intracerebroventricular administration of SP600125, a JNK inhibitor, could significantly decrease escape latency and increase time spent in target quadrant, in treatment group. Furthermore, we evaluated some of the apoptotic factors in the hippocampus of the treated rats. Based on our data, the inhibitor led to the significant decrease in the amount of caspase-3, TUNEL positive cells, cyclooxygenase-2 and increase in Bcl-2/Bax ratio. Given the possible neuroprotective effects of SP600125 on A␤-induced memory impairment and apoptosis, our results may open a new avenue for the treatment of AD. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Alzheimer’s disease (AD) is a multi-factorial neurodegenerative disease characterized by progressive synaptic loss and neuronal death with gradual cognitive decline. The illness starts insidiously with early signs of patchy memory loss and gradually progresses to impaired language comprehend dementia and ultimately death. The pathological characteristics of AD include accumulation and deposition of ␤-amyloid (A␤) peptides in brain parenchyma (senile plaques) and cerebral vessels and the formation of neurofibrillary tangles [45]. One of the main hypotheses about the pathogenesis of AD, the A␤ hypothesis, is supported by a number of epidemiological, genetic and experimental studies [53,55]. Deposition of A␤ peptides in the brain and cerebral vessels results in neuroinflammation and neurovascular inflammation [9,26,49]. Loss of the normal physiological functions of A␤ is also thought to contribute to neuronal dysfunction [1]. A␤42 , a major component of amyloid plaques in the AD brain, is an A␤ peptide with 42 amino acids that is produced by the amyloidogenic pathway [6]. Mitogen-activated protein kinases (MAPKs) are serine– threonine kinases that mediate intracellular signaling associated

with a variety of cellular activities including cell proliferation, survival, death, and transformation [12,25,51]. JNK is a major cellular stress response protein induced by oxidative stress and plays an important role in AD, and its activation is considered as an early event in AD [67]. Activated JNK is found in the hippocampal and cortical regions of individuals with severe AD and localized with neurofibrillar alterations [66,67]. A␤ peptides induce JNK signaling which mediates A␤ toxicity and adverse effects on long-term potentiation in the hippocampus [3,30,52,60]. Application of A␤ peptides triggers the JNK signaling pathway resulting in phosphorylation of c-Jun [1,41,65]. A very recent study has demonstrated the possibility of JNK activity inhibition on providing therapeutic benefit in the context of AD [5]. Thus, due to the above-stated reasons, in this study we tried to investigate the effects of JNK inhibitor, SP600125, on behavioral response of rats in order to demonstrate the practical effect of JNK inhibitor on spatial memory. Besides, we studied some of the apoptotic factors such as caspase-3, Bax, and Bcl-2 that we expected to get impact from JNK. 2. Materials and methods 2.1. Animals

∗ Corresponding author. Tel.: +98 21 22429768, fax: +98 21 22432047. E-mail address: [email protected] (F. Khodagholi). 0166-4328/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2010.11.017

Thirty-five adult male albino Wistar rats (Pasteur Institute, Tehran, Iran) weighing 210–280 g were used in these experiments. Animals were housed in groups of three per cage in a 12/12 h light/dark cycle (light on between 7:00 a.m. and 7:00 p.m.)

M. Ramin et al. / Behavioural Brain Research 217 (2011) 424–431 with free access to chow and tap water. The animals were randomly allocated to different experimental groups. Each animal was used only once. Rats were habituated to their new environment and handled for 1 week before the experimental procedure was started. All experiments were executed in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication No. 80-23, revised 1996) and were approved by the Research and Ethics Committee of Shahid Beheshti University of Medical Sciences. 2.2. Drugs Antibodies directed against Caspase-3, Bax, Bcl-2, Cyclooxygenase-2 (COX-2), p-JNK, and ␤-actin were obtained from Cell Signaling Technology. Electrochemiluminescence (ECL) kit was provided from Amersham Bioscience, USA. A␤ (1–42) and SP600125 were obtained from Sigma–Aldrich (St. Louis, MO). TUNEL (Apoptag plus peroxidase in situ Apoptosis detection) kit was gotten from Chemicon. 2.3. Preparation of beta-amyloid peptide 1–42 (Aˇ1–42) and fibrilization The A␤1–42 was dissolved, and aliquots were stored at −20 ◦ C until use. Aliquots of A␤1–42 at a concentration of 200 ng/␮l prepared in Phosphate Buffer Saline (PBS 0.1 M) were incubated for 5 days at 37 ◦ C. On the test day, PBS was added to the solution to reach the final concentration of 10 ng/␮l. 2.4. Stereotaxic surgery Rats were anesthetized by intraperitoneal injection of xylazine (10 mg/kg) and ketamine (100 mg/kg), and placed into stereotaxic device (Stoelting, USA). An incision was made along the midline, the scalp was retracted, and the area surrounding bregma was cleaned and dried. In addition, lidocaine with epinephrine solution (0.2 ml) was injected in several locations around the incision. Microinjections were performed by 30-gauge injector cannula. Polyethylene tube (PE-10) was used to attach injector cannula to the 5 ␮l Hamilton syringe. For intracerebroventricular (ICV) administration of JNK inhibitor solution, 30-gauge injector cannula was aimed at the lateral ventricle (stereotaxic coordinates: incisor bar -3.3 mm, 0.5 mm posterior to the bregma, 1.5 mm lateral to the sagittal suture and 4 mm down from top of the skull) [39]. For intra-hippocampal administration, stainless steel guide cannulae (23-gauge), 6 mm in length, were aimed at the CA1 area of hippocampus (stereotaxic coordinates: incisor bar −3.3 mm, 3.8 mm posterior to the bregma, ±3.2 mm lateral to the sagittal suture and 2.7 mm down from top of the skull) bilaterally [39]. Cannulae were secured with jewelers’ screws and dental acrylic cement. After the cement was completely dried and hardened, two stainless steel stylets were used to occlude the guide cannulae. Penicillin-G 200,000 IU/ml (0.2–0.3 ml/rat, single dose, intramuscular) was administered immediately after surgery. Animals received a total volume of 5 ␮l JNK inhibitor into the left or right ventricle, and 3 ␮l/side A␤ microinjection into the CA1. All microinjections were performed slowly over a period of 60 s, and injection needles were left in place for an additional 60 s to facilitate diffusion of the drugs. 2.5. Behavioral test: Morris water maze (MWM) 2.5.1. Apparatus The water maze that was used has been described extensively [27,42,46]. Briefly, it consisted of a dark circular pool (140 cm in diameter and 55 cm high) filled with water (20 ± 1 ◦ C) to a depth of 25 cm. A transparent Plexiglas platform (11 cm diameter) was located 1 cm below the water surface in the center of one of the arbitrarily designed north-east (NE), south-east (SE), south-west (SW) or north-west (NW) orthogonal quadrants. The platform provided the only escape from the water. Many extra-maze cues such as racks, a window, a door, bookshelves and pictures on the walls surrounded the room where the water maze was performed. These were kept in fixed positions with respect to the swimming pool to allow the rat to locate the escape platform hidden below the water surface. The position of the animal was monitored by a camera that was mounted above the center of the pool. Animal displacement was recorded using a 3CCD camera (Panasonic Inc., Japan) placed 2 m above the MWM apparatus and locomotion tracking was measured by ethovision software (version XT7), a video tracking system for automation of behavioral experiments (Noldus Information Technology, the Netherlands). In these series of experiments, escape latency and swimming speed as well as time spent in the target quadrant were recorded during 60 s, in both probe and training trials. 2.5.2. Habituation Twenty-four hours prior to the start of training, rats were habituated to the pool by allowing them to perform a 60 s swimming without the platform. 2.5.3. Procedure 19 days after surgery, the behavioral tests were started. The single training session consisted of eight trials with four different starting positions that were equally distributed around the perimeter of the maze [11]. Each rat was placed in the water facing the wall of the tank at one of the four designated starting points (north, east, south and west) and was allowed to swim

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and find the hidden platform located in the SW quadrant (target quadrant) of the maze. Each of four starting positions was used twice in eight training sessions; their order was randomized. During each trial, each rat was given 60 s to find the hidden platform. After mounting the platform, the animals were allowed to remain there for 20 s, and were then placed in a holding cage for 30 s until the start of next trial. After completion of training, the animals were returned to their home cages until the probe trial 24 h later (on the test day). In the probe trial the hidden platform was removed and the animals were released from the north location and allowed to swim freely for 60 s. After the probe trial, the platform was elevated above the water surface and placed in the different position (SE quadrant) and rats were allowed to swim freely for 120 s in order to test their visual ability. All of experiments were conducted between 9:00 and 13:00. 2.6. Western blot analysis Western blot analysis was carried out using protein extract 7 days after A␤ injection. For this purpose, the hippocampi were homogenized in lysis buffer containing complete protease inhibitor cocktail. Then, the total proteins were electrophoresed in 12% SDS-PAGE gels, transferred to polyvinylidene fluoride membranes and probed with specific antibodies. Immunoreactive polypeptides were detected by chemiluminescence using enhanced ECL reagents and subsequent autoradiography. Quantification of the results was performed by densitometric scan of films. Data analysis was done by Image. J., measuring integrated density of bands after background subtraction. Protein concentrations were determined according to Bradford’s method [4]. Standard plot was generated using bovine serum albumin. 2.7. Immunostaining To detect cells undergoing apoptosis, we used the technique of TerminalTransferase dUTP Nick End labeling (TUNEL). After killing, brains were removed, two hemispheres separated and rapidly fixed in formalin 10% for 24 h. The tissues were processed and paraffin embedded. The blocks were cronally sectioned by microtome. Sections (10 ␮m) were mounted on slides and a proteinase K digestion (20 ␮g/ml) was carried out for 15 min. Endogenous hydrogen peroxidase activity was quenched in 3% hydrogen peroxide. After a series of rinsing, nucleotides labeled with digoxigenin were enzymatically added to DNA by terminal deoxy nucleotidyl transferase enzyme (TdT). The incubation was carried out for 60 min and the labeled DNA was detected using anti-digoxigenin-peroxidase for 30 min. Addition of the chromogen diaminobenzidine tetra hydrochloride (DAB) resulted in a brown reaction product that was evaluated by light microscopy. Positive and negative controls were carried out on slides from the same block. Incubation without TdT served as the negative control. For TUNEL staining, 10 fields were chosen from each groups (4 groups) and the percent of TUNEL-positive cells were calculated according to this relation: %TUNEL-positive neurons = (TUNEL-positive neurons/TUNEL-positive neurons (brown) + normal neurons(green)) × 100. 2.8. Experimental design In the present study, animals were divided into four groups: (i) A␤-injected group, which received unilateral ICV administration of DMSO (5 ␮l/rat) 4 h before the bilateral intra-CA1 injection of A␤ (30 ng/3 ␮l PBS per side), without receiving any treatment; (ii) Vehicle group, that only received carriers [DMSO (5 ␮l/rat) in lateral ventricle and PBS (3 ␮l/side) in both CA1 regions]; (iii) JNK inhibitor group, which received ICV infusion of SP600125 (30 ␮g/5 ␮l 1% DMSO in PBS) with PBS injection (3 ␮l/side) in CA1; and (iv) treatment group which received ICV administration of SP600125 (30 ␮g/5 ␮l 1% DMSO in PBS) 4 h prior to intra-hippocampal A␤ (30 ng/3 ␮l PBS per side) injection. The aforementioned groups entered two experimental protocols: behavioral experiments and molecular studies. 2.9. Statistics Data were expressed as mean ± SEM (standard error of mean) and processed by commercially available software GraphPad Prism® 5.0. One-way analysis of variance (ANOVA) and randomized block model followed by post-hoc analysis (Newman–Keuls test) were used. On the other hand, the mean value of training data for the first and second four trials was compared by paired student t-test. P-value less than 0.05 (P < 0.05) was considered to be statistically significant.

3. Results 3.1. Behavioral results 3.1.1. SP600125, a JNK specific inhibitor, had influence on spatial learning in MWM Data obtained in training session showed that there is a significant difference between the first and second four trials in escape latency in all experimental groups, except in A␤-injected rats which was not significant [t(4) = 1.0570, P = 0.3502; Fig. 1A]. The swimming speed did not show any significant alteration between the first and

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Fig. 1. Effects of SP600125, a JNK specific inhibitor, on spatial learning in MWM. Animals received intra-CA1 injection of A␤ (30 ng/3 ␮l PBS per side), or PBS (3 ␮l/side) as a vehicle, prior to SP600125. Difference of (A) escape latency and (B) swimming speed between the first and second four trials were evaluated in training session. Each point shows the mean ± SEM for 5–8 rats. * P < 0.05; ** P < 0.01 different from the first four trials

second four training trials indicating no motor disturbance in the treated animals (Fig. 1B). Neither DMSO nor PBS administrations had effect on escape latency, time spent in target quadrant, and swimming speed.

PBS had no effect on escape latency, time spent in target quadrant, and swimming speed.

3.1.2. SP600125 affected the Aˇ-induced spatial memory impairment in MWM In these series of experiments, one-way ANOVA revealed that escape latency reduced significantly [F(3,21) = 12.42; P < 0.0001; Fig. 2A] in treatment group of animals compared to that in respective A␤-injected group. Also, there was no significant difference between treatment and vehicle groups. As shown in Fig. 2B, time spent in target quadrant increased significantly in treatment group of animals compared to the A␤-injected group in probe trial [F(3,21) = 7.689; P = 0.0016]. Furthermore, the time spent in other quadrants did not show any significant difference between all experimental and control groups (supplementary Fig.). The swimming speed did not show any significant alteration during testing period, indicating no motor disturbance in the treated animals [F(3,21) = 0.3863; P = 0.7642; Fig. 2C]. Microinjection of DMSO and

3.2.1. Inhibition of JNK phosphorylation by SP600125 in Aˇ-injected rats SP600125 is a reversible inhibitor that competes with ATP for binding to kinases and has the highest selectivity toward JNK [2]. Using this compound, we found that it inhibited A␤-induced JNK phosphorylation in rats, as indicated by western blot analysis. As shown in Fig. 3, densitometric analysis revealed about 1.61 fold increase in JNK phosphorylation level in A␤-injected group, compared to the vehicle group (P < 0.001). This increase was inhibited in the treatment group that was pretreated with SP600125 (P < 0.001).

3.2. Molecular results

3.2.2. Increase of Bcl-2/Bax ratio by SP600125 in Aˇ-injected rats As shown in Fig. 4, we found that the increase of apoptosis in A␤-injected rats went along with a significant increase in Bax expression and a decrease in Bcl-2 expression. The ratio of Bcl-

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Fig. 3. Decrease of JNK phosphorylation level by ICV administration of SP600125 (A) p-JNK level in A␤-injected rats with or without SP600125 pretreatment (One representative western blot is shown; n = 6). (B) The densities of corresponding bands were measured and the ratio of p-JNK to JNK was calculated. Each point shows the mean ± SEM. *** P < 0.001 different from the vehicle group. ††† P < 0.001 different from the A␤-injected group.

caspase-3, we measured the level of cleaved caspase-3 by western blot analysis in the presence of this compound. As shown in Fig. 5, A␤ induced the appearance of cleaved active caspase-3, 7 days after its injection, arguing for involvement of caspase-3 in A␤induced cell death in in vivo model. In treatment group pretreated with SP600125, the level of caspase-3 decreased by about 2.44 fold compared to A␤-injected group (P < 0.001).

Fig. 2. Effects of ICV administration of SP600125 (30 ␮g/5 ␮l 1% DMSO in PBS) on (A) escape latency, (B) time spent in target quadrant and (C) swimming speed in probe trial. Animals received intra-CA1 injection of A␤ (30 ng/3 ␮l PBS per side) or PBS (3 ␮l/side) as a vehicle, prior to SP600125. Each point shows the mean ± SEM for 5–8 rats. * P < 0.05; *** P < 0.001 different from the DMSO + PBS (vehicle) group. † P < 0.05; ††† P < 0.001 different from the DMSO + A␤ (A␤-injected) group.

2/Bax decreased by about 4.62 fold in A␤-injected group, compared to vehicle group. However, in treatment group, ICV administration of JNK inhibitor (30 ␮g/5 ␮l 1% DMSO in PBS) increased Bcl-2/Bax ratio by about 2.90 fold compared to A␤-injected group (Fig. 4; P < 0.01). 3.2.3. Inhibition of caspase-3 expression by SP600125 in Aˇ-injected rats Another data, confirming the protective effect of SP600125 was obtained from western blot analysis of caspase-3. The progress of apoptosis is regulated by a series of signal cascades. Caspases are cysteine-dependent enzymes that play pivotal role in the induction, transduction and amplification of intracellular apoptotic signals, and a recent study has demonstrated the efficacy of caspase inhibitor in preventing A␤-induced apoptosis in rats [62]. To verify whether SP600125 interferes with apoptosis via suppressing

Fig. 4. Bcl-2 and Bax levels in A␤-injected rats pretreated with SP600125. (A) The hippocampus samples were homogenized in lysis buffer. Then 60 ␮g proteins were separated on SDS-PAGE, western blotted, probed with anti-Bax and/or anti-Bcl-2 antibodies and reprobed with anti-␤-actin antibody (One representative western blot is shown; n = 6). (B) The densities of corresponding bands were measured and the ratio of Bcl-2 to Bax was evaluated. Each point shows the mean ± SEM. ** P < 0.01; *** P < 0.001 different from the vehicle group. †† P < 0.01; ††† P < 0.001 different from the A␤-injected group.

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Fig. 5. Decrease of caspase-3 level in the hippocampus of A␤-injected rats pretreated with SP600125 (A) 60 ␮g proteins were separated on SDS-PAGE, western blotted, probed with anti-caspase antibody, and reprobed with anti-␤-actin antibody (One representative western blot is shown; n = 6). (B) The densities of corresponding bands were measured and their ratio to actin was calculated. Each point shows the mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001 different from the vehicle group. †† P < 0.01; ††† P < 0.001 different from the A␤-injected group.

3.2.4. SP600125 pretreatment suppressed Aˇ-induced apoptosis in Aˇ-injection rat model In the next step, to confirm the protective effect of SP600125 on A␤-induced apoptosis we did TUNEL assay and morphology study. As shown in Fig. 6 in hippocampus slide of A␤-injected rats, there were several TUNEL-positive (apoptotic) cells versus control slide. However, SP600125 pre-administration did not induce apoptosis.

Fig. 7. COX-2 levels in A␤-injected rats pretreated with SP600125 (A) 60 ␮g proteins were separated on SDS-PAGE, western blotted, probed with anti-COX-2 antibody, and reprobed with anti-␤-actin antibody (One representative western blot is shown; n = 6). (B) The densities of corresponding bands were measured and their ratio to actin was calculated. Each point shows the mean ± SEM. ** P < 0.01; *** P < 0.001 different from the vehicle group. †† P < 0.01; ††† P < 0.001 different from the A␤-injected group.

3.2.5. Suppression of Aˇ-induced COX-2 expression by SP600125 in Aˇ-injected rats Accumulating evidence strongly supports the idea that neuronal COX-2, but not COX-1 levels elevated in AD brain, showing that COX-2 is involved in the mechanism of neuronal death/survival [38]. As shown in Fig. 7, we found that in A␤-injected group, the level of COX-2 increased by about 2.26 fold, compared to vehicle group, as determined by western blot analysis. Microinjection of SP600125 in treatment group decreased COX-2 level by about 1.35 fold, compared to A␤-injected group. In these conditions, neither A␤-injected, nor treatment group showed any signifi-

Fig. 6. Hippocampal TUNEL-positive neurons 7 days after stereotaxic injection of A␤. (A) First panel from left shows staining in vehicle group. Second panel shows A␤-injected group. Several TUNEL-positive neurons (arrows) were observed. Third panel shows JNK inhibitor group. Fourth panel shows the A␤-injected group pretreated by SP600125. In this group, number of TUNEL-positive neurons has been reduced. Normal cells (green), TUNEL-positive cells (brown); light microscope (100×) was utilized for taking picture. (B) Represents the quantitative analysis of the data. *** P < 0.001 different from the JNK inhibitor group. ††† P < 0.001 different from the A␤-injected group.

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cant change in COX-1 level, compared to vehicle group (data not shown).

4. Discussion AD is the most common neurodegenerative disease characterized by the progressive deterioration of cognition and memory in association with the presence of senile plaques of A␤ fibrils, neurofibrillary tangles, and massive loss of neurons, mainly in the cerebral cortex and hippocampus [47]. One of the crucial signaling pathways which could be involved in all of the pathological hallmarks of AD is JNK cascade. Among the MAPKs, JNK is one of essential mediators of relevant proinflammatory functions in microglia [44,56]. JNK is a component of signaling pathways that result in inflammation, and it can control the synthesis and releases of proinflammatory substances by LPS activated microglia [20]. Furthermore, Vukic et al. demonstrated that the JNK-AP1 signaling pathway is responsible for increased expression of inflammatory genes induced by A␤ peptides in human brain endothelial cells and in Alzheimer’s brain [55]. The JNK pathway has been demonstrated to cause phosphorylation of tau proteins, therefore JNKs may lie at an intersection between both of the major pathological hallmarks of AD [54]. A recent study proved that JNK plays a fundamental role in phosphorylation of amyloid precursor protein (APP) since its specific inhibition, with the JNK inhibitor peptide (D-JNKI1), induced APP degradation and prevented APP phosphorylation [8]. Recently, it has been shown that inhibition of JNK provides neuroprotection in brain slice model of APP-induced neurodegeneration [5]. In the present study, to further explore the beneficial effect of JNK inhibition in AD, we evaluated the role of JNK in A␤-induced learning and memory deficits, along with apoptotic factors. Our data demonstrate that inhibition of JNK phosphorylation by using a small molecule inhibitor, SP600125, has neuroprotective function in A␤-injected rats, as it has potent memory enhancing effects and blocks learning deficits induced by A␤. SP600125 is a selective inhibitor of JNK whose inhibitory action on JNK phosphorylation has been studied [2]. Some investigations study the effects of peripheral administration of SP600125 on the caerulein-induced JNK phosphorylation. For example, Namkung et al. showed that the peripheral administration of SP600125 inhibited the caeruleininduced JNK phosphorylation [32] and Liu et al. investigated the role of JNK in memory storage in the hippocampus [24]; while Mitsoyama et al. studies using a specific JNK inhibitor demonstrated a decrease in gastric damage in animals treated with SP600125 [29]. However, in support of our results, another study confirms the investigation of central effects of SP600125 [59]. Moreover, biochemical analysis showed the possible ability of this inhibitor in attenuation of A␤-induced apoptosis. To date, there is no animal model available which could mimic all the cognitive, behavioral, biochemical, and histopathological abnormalities observed in patients with AD. However, partial reproductions of AD neuropathology and cognitive deficits have been achieved by pharmacological and genetic approaches [14,23]. Since it has been well established that no significant neurodegeneration can be found in transgenic models of amyloidosis [5], we used the injection model. Among several animal models used to induce memory impairment, we used A␤ protein (1–42)-infused rats which showed delayed, progressive memory impairment, as was done by Oka et al. [35]. It is well known that the hippocampal formation is involved in learning and memory, and it was reported that the hippocampus plays an important role in processing, and remembering spatial and contextual information [18]. Furthermore, subregional analysis of the hippocampus indicates that the CA1 area is involved in spatial learning [21]. In agreement with Yamada et al., our study confirmed the memory impairment induced by intra-CA1 injec-

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tion of A␤ [63]. Several studies have found that intra-hippocampal injections of A␤ produced deficits under a number of behavioral paradigms [13,19,48], and that such effects became significant after 30 days following A␤ injection [34]. In this study, we showed that even 20 days after A␤ infusion, these effects could be significant. Since various lines of investigations revealed that for the observation of spatial learning and memory of rats MWM is more useful than other apparatuses (T-maze, radial arm maze) due to its reasonable design [10,31,61], we chose MWM test to monitor spatial memory. This study indicated that JNK inhibitor could reduce the escape latency in SP600125 treated animals. It could also decrease the escape latency and increase duration in target quadrant, in treatment group. Moreover, there was no change in swimming speed which was used as a criterion for locomotor activity. The molecular mechanisms underlying A␤-mediated neurotoxicity still remain to be elucidated, but mounting evidence suggests the involvement of caspases in the disease process with AD. The activation of caspases may be responsible for the neurodegeneration associated with AD, and several recent studies have suggested that caspases may also play a role in promoting the mechanisms associated with this disease. Our data showed that caspase-3 and Bax protein levels were remarkably up-regulated 7 days after A␤ injection and then significantly decreased in the group pretreated with SP600125. Concomitantly, a significant decrease in Bcl-2 protein was observed in A␤-injected rats that was increased by SP600125 pretreatment. One mechanism in the programmed cell death is up-regulation of the pro-apoptotic Bax protein. The increase of Bax expression affects the permeability of mitochondrial membrane leading to the activation of caspases which finally proteolyse cellular components. In contrast, Bcl-2 is one of the key genes known to down regulate Bax, and therefore, has the potency to inhibit the mitochondrial pathway of apoptosis [22]. This possibility is consistent with other’s findings. Previous studies show that A␤-induced apoptosis is characterized by decreased expression of the antiapoptotic Bcl-2 and Bcl-xL [15,36,50,58]. Besides, Yao et al. showed that Bcl-w expression is downregulated in A␤induced neuronal apoptosis. They found that SP600125 effectively prevents A␤-induced Bcl-w downregulation, indicating that this critical step in the A␤ cell-death pathway is dependent on JNK activation [64]. From the other side, it has been shown that over expression of Bcl-2 attenuates JNK activation, as well as caspase activity [7], opening a possible cross-talk between these factors. In addition, there is a convincing evidence for the involvement of inflammation caused by increased expression of COX-2 [37]. Consistent with this notion, neurons in the AD brain display increased levels of COX-2 [17,38]. In this study, we showed that A␤ injection resulted in elevated expression COX-2 protein 7 days later. There are multiple lines of compelling evidence supporting the notion that COX-2 plays a role in apoptosis. Neurons derived from transgenic mice over expressing COX-2 were more vulnerable to A␤-mediated neurotoxicity [16]. Besides, Pasinetti and Aisen have demonstrated that up-regulation of COX-2 overlapped the cellular and morphological features of apoptosis in frontal cortex of AD brain [38]. In the present work, pretreatment with SP600125 resulted in inhibition of COX-2 protein level, along with inhibition of apoptotic factors. Collectively based on the obtained results, JNK inhibitor can inhibit apoptotic factors, which confirms the role of JNK in apoptosis. Intra-peritoneal administration of SP600125 in the studies about pancreatitis [28] and ischemic renal injury [57] give us an idea of using this JNK inhibitor to investigate whether it can be used as an impressive drug in AD patients. Although in a very recent study, intraperitoneal administration of a JNK-inhibitor, TAT-JBD, immediately after perinatal hypoxic-ischemic prevented AP-1 activation [33], we need further studies to show whether or not SP600125 can be used in the clinical aspects of memory recov-

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