Arsenite exposure altered the expression of NMDA receptor and postsynaptic signaling proteins in rat hippocampus

Toxicology Letters 211 (2012) 39–44

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Arsenite exposure altered the expression of NMDA receptor and postsynaptic signaling proteins in rat hippocampus Jiao-hua Luo, Zhi-qun Qiu, Liang Zhang, Wei-qun Shu ∗ Department of Environmental Hygiene, College of Preventive Medicine, Third Military Medical University, Chongqing 400038, PR China

a r t i c l e

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Article history: Received 3 January 2012 Received in revised form 24 February 2012 Accepted 27 February 2012 Available online 8 March 2012 Keywords: Arsenite Neurotoxicity NMDA receptor PSD-95 SynGAP CaMKII ERK

a b s t r a c t Chronic arsenic exposure has an adverse effect on neurobehavioral function. Our previous study demonstrated an elevated arsenic level, ultra-structure changes and reduced NR2A gene expression in hippocampus, and impaired spatial learning in arsenite-exposed rats. The NMDA receptor and the postsynaptic signaling proteins CaMKII, postsynaptic density protein 95 (PSD-95), synaptic Ras GTPaseactivating protein (SynGAP) and nuclear activated extracellular-signal regulated kinase (ERK1/2) play important roles in synaptic plasticity, learning and memory. We hypothesized that the above molecular expression changes may contribute to arsenic neurotoxicity. In present study, the expression of NMDA receptor and postsynaptic signaling proteins in hippocampus were evaluated in rats exposed to 0, 2.72, 13.6 and 68 mg/L sodium arsenite for 3 months. Decreased protein expression of NR2A, PSD-95 and pCaMKII ␣ in the hippocampus of arsenite-exposed rats was observed, while the expression of SynGAP, a negative regulator of Ras-MAPK activity, was increased when compared with the controls. Additionally, decreased p-ERK1/2 activity was found in the hippocampus of arsenite-exposed rats. These data suggest that altered expression of NMDA receptor complex and postsynaptic signaling proteins may explain arsenic-induced neurotoxicity. © 2012 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Arsenic (As) is one of the most toxic naturally existed environmental contaminants. Arsenic-contaminated drinking water is a serious public health problem that affects many countries and millions of exposed people worldwide (Smith et al., 2011). Arsenic toxicity can cause diseases of gastrointestinal, respiratory, cardiovascular, neurological, genitourinary, endocrine, hematopoietic and cutaneous systems (Ratnaike, 2003; Duker et al., 2005). Recent studies on children have revealed an association between neurobehavioral function and arsenic exposure from drinking water or industrial sources (Calderon et al., 2001; Rosado et al., 2007; Tsai et al., 2003; von Ehrenstein et al., 2007; Wang et al., 2007; Wasserman et al., 2004, 2007). Data from animal studies demonstrate that arsenic exposure via drinking water has adverse effects

Abbreviations: AMPAR, alpha-amino-3-hydroxy-5-methyl-4isoxazolepropionic acid receptor; CaMKII, calcium/calmodulin-dependent protein kinase II; ERK, extracellular-signal regulated kinase; LTD, long-term depression; LTP, long-term potentiation; MAPK, mitogen-activated protein kinase; NMDAR, Nmethyl-d-aspartate receptor; NOS, nitric oxide synthase; PSD, postsynaptic density; PSD-95, postsynaptic density protein 95; SynGAP, synaptic Ras GTPase-activating protein. ∗ Corresponding author. Tel.: +86 2368752294; fax: +86 2368752294. E-mail addresses: [email protected] (J.-h. Luo), [email protected] (W.-q. Shu). 0378-4274/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.toxlet.2012.02.021

on neural behavior and learning tasks (Rodriguez et al., 2001, 2002; Martinez-Finley et al., 2008, 2009; Luo et al., 2009). Although evidence for neurodevelopmental neurotoxicity of arsenic is accumulating, the causative mechanism remains unclear (Wang et al., 2010). N-methyl-d-aspartate receptors (NMDARs), which are glutamate-gated ion channel receptors, are widely expressed in the central nervous system and play key roles in excitatory synaptic transmission, synaptic plasticity regulation, learning and memory (Papadia and Hardingham, 2007). NMDARs are heteromeric complexes that incorporate different subunits within a repertoire of three subtypes: NR1, NR2 (NR2A-D) and NR3 (NR3A and NR3B) (Paoletti and Neyton, 2007). The most widely expressed NMDARs contain the obligate subunit NR1 and either NR2B or NR2A or a mixture of the two (Papadia and Hardingham, 2007). Many of the important NMDA receptor properties are influenced by the subunit composition of the receptor assembly (Cull-Candy and Brickley, 2001). In excitatory synapses of the brain, specific receptors in the postsynaptic membrane lie ready to respond to the release of the neurotransmitter glutamate from the presynaptic terminal. Upon stimulation, these glutamate receptors activate multiple biochemical pathways that transduce signals into the postsynaptic neuron (Sheng and Kim, 2002). Different patterns of NMDAR activation can lead to either long-term potentiation (LTP) or long-term depression (LTD) of synaptic strength. These long-lasting forms of synaptic

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plasticity may represent ways of encoding “memories” in the brain (Sheng and Kim, 2002). NMDARs are embedded in the postsynaptic density (PSD), a microscopic structure associated with the postsynaptic membrane that contains a variety of scaffolding and signaling proteins. Many of the prominent proteins in the PSD fraction bind directly or indirectly to the NMDA receptor. Thus, the PSD fraction contains a core NMDA receptor-signaling complex (Kennedy, 2000). Calcium/calmodulin-dependent protein kinase II (CaMKII) is a Ca2+ -activated enzyme that is highly abundant in brain, in which it constitutes 1–2% of the total protein. CaMKII is persistently activated after NMDAR stimulation and is essential for NMDARdependent LTP (Lisman et al., 2002). At many excitatory synapses, LTP is triggered by Ca2+ entry into the postsynaptic cell. Evidence indicates that CaMKII senses the elevated Ca2+ and initiates the biochemical cascade that potentiates synaptic transmission (Lisman et al., 2002). CaMKII activation stimulates its binding to the cytoplasmic domain of the NMDAR subunit NR2B. By interfering with the autoinhibitory interactions within CaMKII, binding to NR2B locks CaMKII in an activated state that cannot be reversed by phosphatases (Bayer et al., 2001). Activated CaMKII can phosphorylate a number of synaptic targets, including alpha-amino-3-hydroxy-5methyl-4-isoxazolepropionic acid receptor (AMPAR), nitric oxide synthase (NOS) and synaptic Ras GTPase-activating protein (SynGAP), thereby facilitating an increase in synaptic efficacy that results in LTP (Rongo, 2002). Scaffold protein PSD-95 is another central component of the NMDA receptor-signaling complex. PSD-95 serves as the signaling scaffold to bridge NMDARs to the intracellular signaling complexes (Xu, 2011) and is required to sustain the molecular organization of the postsynaptic density (Chen et al., 2011). The PSD-95 family is comprised of four closely related proteins, each of which contains five protein-binding domains. Three PDZ domains are followed by an SH3 domain and a GK domain (Kennedy, 2000). The COOHterminal cytoplasmic tails of the NMDAR subunits bind to the first and the second PDZ domains of PSD-95. PSD-95, in turn, interacts with a host of cytoplasmic signaling molecules, including neuronal NOS and SynGAP, thereby connecting NMDARs to divergent signal transduction pathways (Sheng and Kim, 2002). PSD-95 is an important regulator of synaptic strength and plasticity. In PSD-95 mutant mice, LTP was greatly enhanced, whereas LTD was absent in these mice. Consistent with these results, the acute knockdown of PSD95 blocked or decreased LTD (Xu et al., 2008; Ehrlich et al., 2007), whereas overexpression of PSD-95 occluded LTP and decreased the threshold for LTD induction (Stein et al., 2003; Béïque and Andrade, 2003). SynGAP is a synaptic Ras GTPase-activating protein (RasGAP) that interacts with the PDZ domains of PSD-95 and SAP-102 in vitro and in vivo. SynGAP is specifically expressed in brain and is highly enriched at excitatory synapses in hippocampus, in which it is present in a large macromolecular complex with PSD-95 and NMDA receptor (Kim et al., 1998). SynGAP stimulates GTPase activity of Ras, which suggests that it negatively regulates Ras activity at excitatory synapses (Kim et al., 1998). SynGAP is deactivated by CaMKII phosphorylation. SynGAP inhibition by CaMKII will stop GTP-bound Ras inactivation and therefore could result in mitogen-activated protein kinase (MAPK) pathway activation in hippocampal neurons upon NMDA receptor activation (Chen et al., 1998). SynGAP plays a critical role in the regulation of neuronal MAPK signaling, AMPAR membrane trafficking and excitatory synaptic transmission (Rumbaugh et al., 2006). Neural SynGAP overexpression results in a significant reduction of ERK1/2 activation, a reduction of AMPAR expression on synaptic surface, and depression of AMPAR-mediated miniature excitatory postsynaptic currents (Rumbaugh et al., 2006). ERKs are members of the MAPK superfamily and form a major signal transduction pathway,

mediating extracellular stimuli to the nucleus (Schaeffer and Weber, 1999). The MAPK/ERK pathway plays a crucial role in learning and memory (Sweatt, 2004). Our previous study found that arsenite exposure could inhibit NR2A gene expression (Luo et al., 2009). We hypothesize that arsenite exposure could interfere with the expression of NMDA receptor and postsynaptic signaling proteins. In this study, Western blot analyses and enzyme activity assays were performed to determine whether there were alterations in the NMDA receptor complexassociated molecules following arsenite exposure. 2. Materials and methods 2.1. Chemical Sodium arsenite (analytical grade) was obtained from Merck Limited and dissolved in ddH2 O. Primary antibodies of rabbit anti-NR1 (06-311, 1:500), rabbit anti-NR2A (07-632, 1:1000), and rabbit anti-NR2B (06-600, 1:1000) were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Primary antibodies of mouse antiCaMKII␣ (6G9), rabbit anti-p-CaMKII␣ (Thr 286), rabbit anti-ERK2 (C-14), mouse anti-p-ERK (E-4), mouse anti-PSD-95 (2Q128), goat anti-SynGAP (R-19), mouse anti-GAPDH (D-6) and rabbit anti-␤-actin (I-19) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The dilution of antibodies was 1:200 unless indicated above. 2.2. Animals All animal procedures were performed according to a protocol approved by the Institutional Animal Use and Care Committee of the Third Military Medical University (TMMU) and were carried out by individuals with appropriate licenses. A total of 80 weaned and specific-pathogen-free (SPF) Sprague–Dawley male rats (55–80 g) were obtained from the Experimental Animal Center of TMMU (license number: SCXK-2002-008). Rats were kept on a 12-h light/dark cycle in a temperature-controlled room maintained at 25 ± 1 ◦ C with a relative humidity of 50 ± 5% and were habituated to the conditions for 1 week prior to arsenite exposure. Rats were randomly assigned into four groups (n = 20 for each group) and given water containing 0 mg/L (control), 2.72 mg/L (group A), 13.6 mg/L (group B) and 68 mg/L (group C) sodium arsenite for 3 months. The LD50 of sodium arsenite for rat is 41 mg/kg (orally). To avoid acute toxicity caused by sodium arsenite, the highest dose we selected was 68 mg/L sodium arsenite in drinking water. The concentrations achieved in the rats are higher than the concentrations human exposed to. The As-containing water was freshly prepared every 2 days. Rats were given food and water ad libitum. Rodriguez et al. (2002) reported that a 300 g rat consumes 24–34 ml water daily, and our results were consistent with theirs. Based on these estimates, a 300 g rat in our experiment would expectedly consume 0 ␮g As/day, 65.28–92.48 ␮g As/day, 326.4–462.4 ␮g As/day or 1632.0–2312 ␮g As/day, which is equivalent to 0, 0.22–0.31, 1.09–1.54 and 5.44–7.71 mg As/kg/day, respectively. The weights of the rats were measured every 7 days, and the water consumption volumes were measured every 2 days. After a 3-month arsenite exposure, hippocampus samples from 2 rats from each group were collected for examination by transmission electron microscopy, and 10 rats from each group were tested in a Morris water maze according to a modified procedure by Morris (1984). At the end of the spatial learning tasks, these rats were anesthetized with sodium pentobarbital (i.p.). Blood was collected by heart puncture. hippocampus were isolated, immediately transferred into liquid nitrogen and stored at −80 ◦ C for later use. 2.3. Preparation of the rat hippocampus extract for enzyme assays and Western blots Frozen hippocampi were homogenized using a Dounce homogenizer (1:10, w/v) in an RIPA Lysis Buffer (Beyotime, JiangShu, China) solution containing Complete Protease Inhibitor Tablets (Roche Biochemicals), and Phosphatase Inhibitor Cocktails I and II (Calbiochem). Homogenates were centrifuged for 5 min at 350 g at 4 ◦ C, and the supernatant protein concentration was determined using a BCA protein assay kit (Beyotime, JiangShu, China). For the CaMKII enzyme activity assay, aliquots of the supernatant were frozen at −80 ◦ C. For Western blotting, samples were diluted 1:1 with a 2× Laemmli buffer, boiled at 100 ◦ C, and stored at −80 ◦ C for later use. 2.4. Western blotting Western blot analyses were performed to validate the expression patterns of postsynaptic signaling proteins. A minimum of three independent sets of experiments was performed for all treatment groups using three animals per group in each set. The expression levels of ␤-actin and GAPDH (housekeeping protein), and the proteins of interest were assessed using Western blots. In brief, these proteins were transferred onto a PVDF membrane (Millipore, USA) after SDS-PAGE gel separation. The membranes were blocked with 5% nonfat milk in a Tris-buffered saline

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Table 1 CaMKII enzyme activity. Group

Activity (pmol/min/␮g protein)

Control A B C

0.422 0.291 0.226 0.205

± ± ± ±

0.05 0.04* 0.02* 0.03*

Values were mean ± SD (n = 6) as pmol/min/␮g protein. A: Group A (2.72 mg/L); B: Group B (13.6 mg/L); C: Group C (68 mg/L). * Different from control group, P < 0.05 as determined by one-way-ANOVA with LSD test.

solution containing 0.1% Tween-20 (TBST buffer) and were incubated overnight at 4 ◦ C with specific primary antibodies. Subsequently, the blots were washed 3 times with TBS containing 0.2% Tween 20 to remove the unbound antibodies and incubated for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibodies. The blots were extensively washed and then the proteins were detected via an enhanced chemiluminescence method (Amersham). The immunoblot densities were calculated using Quantity One software (Bio-Rad Laboratories, USA). The band densities were normalized to ␤-actin. 2.5. CaMKII enzyme activity assay ®

enzyme activity was analyzed using the SignaTECT CaMKII Calcium/Calmodulin-Dependent Protein Kinase Assay System (Promega, Madison, WI) as described previously (Toscano et al., 2005) with minor modifications. The SignaTECT® System utilizes a specific biotinylated substrate for CaMKII to assess CaMKII-dependent phosphorylation. Briefly, hippocampal homogenates were diluted to an approximate protein concentration in 50 mM Tris–HCl (pH 7.5), 10 mM MgCl2 , 0.5 mM DTT and 0.1 mg/ml BSA. Diluted hippocampal homogenates (5 ␮l) were added to a 30 ◦ C preheated reaction mix composed of (final concentrations): 50 mM Tris–HCl (pH 7.5); 10 mM MgCl2 ; 0.5 mM DTT; 100 ␮M ATP (0.5 ␮Ci [␥-32 P-ATP]; Furui Biotech, Beijing); 0.05 mM biotinylated peptide substrate; and with (activated reactions) or without (basal reactions) 1 mM CaCl2 and 1 ␮M calmodulin in a final reaction volume of 25 ␮l. The reactions proceeded at 30 ◦ C for 2 min and were terminated by the addition of guanidine hydrochloride to a final concentration of 2.5 M. The terminated reactions were spotted onto a streptavidin-impregnated membrane and washed 4 times with 2 M NaCl, 4 times with 2 M NaCl in 1% phosphoric acid, and 2 times in distilled water. The membranes were dried and then assessed for retained radioactivity by liquid scintillation counting (Beckman). Radioactivity counts were converted into specific activity, and the enzyme activity, which was expressed as pmol/min per ␮g protein, was determined using a specific formula. All reactions were run in duplicate, and background reactions (without the peptide addition) were run for each sample. 2.6. Statistical analysis Statistical analysis was performed using SPSS 10.0 software. Data are presented as the mean ± SD, and statistical significance was determined using one-way ANOVA, followed by a least significant difference (LSD) test. A P-value <0.05 was considered statistically significant.

3. Results The general appearance and physical condition of the arseniteexposed and control rats were closely observed, and no obvious differences were noticed. There were no differences in food intake between groups during the study. The arsenic levels in the blood and brain increased significantly following the 3-month arsenite exposure. Morris water maze performance was impaired in the rats that drank water with 68 mg/L arsenite for 3 months. Additionally, ultra-structure changes and reduced expression of NR2A mRNA were demonstrated in the hippocampus. The abovementioned results were previously published (Luo et al., 2009). In the hippocampus of arsenite-exposed rats, decreased NR2A protein expression was found (Fig. 1). Compared with the control, NR2A protein levels were reduced by 23%, 21% and 50% in groups A, B and C, respectively. There was no obvious change in the expression of NR2B (Fig. 1) and NR1 (Fig. 1) proteins in all groups. The results of CaMKII enzyme activity in hippocampal homogenates are shown in Table 1. Significant intergroup differences were observed after arsenite exposure for 3 months (P < 0.05). Compared with the control, CaMKII enzyme activity was reduced by

Fig. 1. Protein expression of NR1, NR2A and NR2B, subunits of NMDAR, in rat hippocampus after arsenite exposure. (A) Analyzed by Western blot, the expression of NR2A, but not NR1 and NR2B, was decreased compared with the control group. (B) The quantification of the Western blot results. The expression levels of NR1, NR2A and NR2B were normalized against ␤-actin. The values shown represent the mean ± SD of three separate experiments. **P < 0.01 versus control.

31%, 46% and 51% in groups A, B and C, respectively. The expression levels of CaMKII ␣ and ␣-CaMKII phosphorylation at Thr286 were detected using Western blot analysis. CaMKII ␣ protein expression did not show significant changes in the hippocampus among all groups (Fig. 2). Compared with the control, arsenite exposure significantly reduced p-CaMKII ␣ level (P < 0.05), declined by 34%, 63% and 61% in the hippocampus of rats in groups A, B and C, respectively (Fig. 2). In arsenite-exposed rats, decreased PSD-95 protein expression in the hippocampus was observed (Fig. 3). Compared with the control, PSD-95 protein levels were reduced by 49%, 42% and 63% in the hippocampus of rats in groups A, B and C, respectively. In contrast, an increased SynGAP protein expression was observed in arsenite-exposed rats with 8%, 26% and 79% upregulation in groups A, B and C, respectively (Fig. 3). Because SynGAP negatively regulates the Ras-MAPK pathway, and MAPK/ERK activation is necessary for both consolidation and reconsolidation of memory, the effect of arsenite exposure on ERK expression was further analyzed. As shown in Fig. 4, arsenite exposure did not evoke significant change of ERK1/2 expression in all groups. However, the phosporylation of ERK1/2 was significantly declined in arsenite-exposed rats (P < 0.05) (Fig. 4). 4. Discussion In this study, the effects of arsenite exposure on the expression of NMDA receptor and postsynaptic signaling proteins in rat hippocampus were evaluated. The arsenic overload was confirmed by the significantly increased arsenic level in blood and brain. We found brain arsenic levels in exposed rats were significantly higher than that in control rats, indicating the model was convincible for studying the toxical effect of arsenic on neurobehavior. In humans and most rodents, inorganic arsenic (iAs) taken up is metabolized in liver to monomethylarsonic acid (MAs) and dimethylarsinic acid (DMAs) by consecutive reduction and oxidative methylations (Jin et al., 2006). The toxic methylated trivalent metabolites of iAs, MAs(III) and DMAs(III) play a key role in the etiology of adverse health effects caused by iAs exposure. The MAs(III) and DMAs(III) in blood and brain used for arsenic level measurement are stable under this condition (Currier et al., 2011).

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Fig. 2. Protein expression of PSD-95 and SynGAP detected by Western blot in rat hippocampus after arsenite exposure. (A) Compared with the control, the PSD-95 expression was decreased after arsenite exposure significantly. In contrast, SynGAP expression increased in a dose-dependent manner. (B) Quantitative analysis on the Western blot. The expression levels of PSD-95 and SynGAP were normalized against ␤-actin. The values shown represent the mean ± SD of three separate experiments. **P < 0.01 versus control.

The findings herein of decreased NR2A in the hippocampus of arsenite-exposed rats are consistent with our previously reported results (Luo et al., 2009). This finding supports our assumed alteration of NMDA receptor in arsenite-exposed rats. Neurotoxicant Pb2+ exposure in the developing and matured rat brain resulted in a reduction of NR2A subunit expression (Guilarte and McGlothan, 1998; Nihei and Guilarte, 1999; Nihei et al., 2000; Zhang et al., 2002). Both Pb2+ and arsenite implement their toxic effects via bonding to sulphydryl groups of proteins and depletion of glutathione, which elicits oxidative stress, and subsequently induces DNA damage, lipid peroxidation, protein modification, etc., all symptomatic for numerous diseases (Jomova and Valko, 2011). Many of the important NMDA receptor properties are influenced by the subunits composing the receptor assembly (Cull-Candy and Brickley, 2001). It was reported that LTP in the hippocampus is specifically related to NR2A-containing NMDARs (Liu et al., 2004). One study found that acute arsenite exposure significantly inhibited the induction and establishment of LTP in adult rats (Kruger et al., 2006). Altered NMDA receptors would influence postsynaptic protein activation. CaMKII is persistently activated after NMDA receptor stimulation and is essential for NMDAR-dependent LTP (Lisman et al., 2002). CaMKII expression and threonine-286 residue phosphorylation have been proven crucial for learning, memory, and synaptic plasticity (Hardingham et al., 2003). Alterations in CaMKII activity and expression were observed in Pb2+ -exposed rats (Toscano et al., 2005) and 3,4-methylenedioxymethamphetamine (MDMA)-treated rats (Moyano et al., 2004). In this study, reduced NR2A expression and CaMKII enzyme activity were found in arsenite-exposed rats. This finding demonstrates that arsenite can disrupt the normal NMDA receptor assembly and the function of CaMKII. To further investigate potential mechanisms by which arsenite exposure decreases CaMKII activity, the

expression of CaMKII ␣ and ␣-CaMKII phosphorylation at Thr286 was measured. Arsenite exposure significantly decreased the phosphorylation of ␣-CaMKII at Thr286, which in turn decreased CaMKII activity. In mammals, CaMKII is encoded by four genes, ␣, ␤, ␥ and ␦; ␣ and ␤ isozymes are the predominant forms in the brain (Griffith, 2004). Thus, any expression changes of other

Fig. 3. The expression and phosphorylation of CaMKII ␣ protein in the hippocampus detected by Western blot. (A) Compared with the control, the expression of CaMKII ␣ protein did not obviously changed, but the phosphorylation of the protein was significantly decreased after arsenite exposures. (B) Quantitative analysis on the Western blot. The expressions of CaMKII ␣ and p-CaMKII ␣ were normalized against ␤-actin. The values shown represent the mean ± SD of three separate experiments. **P < 0.01 versus control.

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Fig. 4. The levels of ERK1/2 and p-ERK1/2 in the hippocampus detected by Western blot. (A) Compared with the control, the p-ERK1/2 level was significantly decreased in rat hippocampus after arsenite exposure, whereas ERK1/2 expression did not show a difference among all groups. (B) Quantitative analysis on the Western blot. The expressions of ERK1/2 and p-ERK1/2 were normalized against GAPDH. The values shown represent the mean ± SD of three separate experiments. **P < 0.01 versus control.

two isozymes may also contribute to the reduced CaMKII activity. As a signaling scaffold, PSD-95 brings intracellular signaling complexes close to NMDAR channels. PSD-95 bridges the calcium influx to the specific downstream signaling events (Xu, 2011). A decreased PSD-95 expression was found in the hippocampus of arsenite-exposed rats. As we did not evaluated the mRNA level of PSD-95, it is uncertain that the decreased PSD95 is caused by alteration at transcription level, translation level, or protein degradation, as arsenic has previously been found to promote the proteasomal degradation of some proteins (Yan et al., 2011). This reduction would impair the molecular organization of the postsynaptic density (Chen et al., 2011), synaptic strength and plasticity. A reduction in post-synaptic scaffolding PSD-95 protein levels is correlated with disease pathology in the inferior temporal cortex in Alzheimer’s disease (Proctor et al., 2010). SynGAP is a negative regulator of Ras at excitatory synapses (Kim et al., 1998). SynGAP is inhibited by CaMKII phosphorylation (Chen et al., 1998). In this study, decreased CaMKII activity and increased expression of SynGAP were found, resulting in the inhibition of MAPK/ERK activation as indicated by the decreased p-ERK1/2 protein in the hippocampus of arsenite-treated rats. ERK activation is necessary for both the consolidation and reconsolidation of recognition memory (Davis and Laroche, 2006). A study (Martinez-Finley et al., 2011) found reduced ERK2 phosphorylation in prenatal arsenic-exposed animals. Inhibition of this pathway has been shown to produce behavioral learning and memory deficits in arsenic-exposed animals and in other models (Martinez-Finley et al., 2009; Blum et al., 1999; Selcher et al., 1999). In summary, arsenic exposure was found to reduce the expression of NR2A and PSD-95, and CaMKII ␣ phosphorylation and enzymatic activity; increase SynGAP expression (a negative regulator of Ras-MAPK activity); and inhibit p-ERK1/2 level in the hippocampus of arsenite-exposed rats (Fig. 5). The disrupted expression of NR2A and postsynaptic proteins in the hippocampus may explain the neurobehavioral dysfunction induced by arsenite

exposure. At present, we could not address how arsenite exposure induces these alterations. Further investigations are needed to understand the mechanism by which arsenite cause neurobehavioral dysfunction.

Fig. 5. A schematic diagram of how arsenite affects NMDA receptor and postsynaptic signaling proteins in the hippocampus of arsenite-exposed rats. Arsenite exposure reduced the expression of NR2A subunit of NMDA receptor, which would influence the function of NMDA receptor, for example the NMDA receptor-mediated Ca2+ influx. A decreased PSD-95 expression could impair the molecular organization of the postsynaptic density. Normally, SynGAP, a negative regulator of Ras at excitatory synapses, is inhibited by CaMKII phosphorylation. After arsenite exposure, the decreased CaMKII activity and increased expression of SynGAP led to the inhibition of MAPK/ERK activation in terms of the decreased ERK1/2 phosphorylation. MAPK/ERK plays important role in synaptic plasticity and memory formation, and the declined p-ERK1/2 level may be involved in the impairment of neurobehavioral function in arsenite exposed rats.

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