Mol Neurobiol DOI 10.1007/s12035-015-9169-3

Identification of a Novel Rat NR2B Subunit Gene Promoter Region Variant and Its Association with Microwave-Induced Neuron Impairment Li-Feng Wang 1 & Da-Wei Tian 2,3 & Hai-Juan Li 1 & Ya-Bing Gao 1 & Chang-Zhen Wang 1 & Li Zhao 1 & Hong-Yan Zuo 1 & Ji Dong 1 & Si-Mo Qiao 1 & Yong Zou 1 & Lu Xiong 1 & Hong-Mei Zhou 4 & Yue-Feng Yang 5 & Rui-Yun Peng 1 & Xiang-Jun Hu 1

Received: 4 November 2014 / Accepted: 2 April 2015 # Springer Science+Business Media New York 2015

Abstract Microwave radiation has been implicated in cognitive dysfunction and neuronal injury in animal models and in human investigations; however, the mechanism of these effects is unclear. In this study, single nucleotide polymorphism (SNP) sites in the rat GRIN2B promoter region were screened. The associations of these SNPs with microwave-induced rat brain dysfunction and with rat pheochromocytoma-12 (PC12) cell function were investigated. Wistar rats (n=160) were exposed to microwave radiation (30 mW/cm2 for 5 min/day, 5 days/week, over a period of 2 months). Screening of the GRIN2B promoter region revealed a stable C-to-T variant at nucleotide position −217 that was not induced by microwave exposure. The learning and memory ability, amino acid contents in the hippocampus and cerebrospinal fluid, and NR2B expression were then investigated in the different genotypes. Following microwave exposure, NR2B protein expression Li-Feng Wang and Da-Wei Tian are co-first authors. * Rui-Yun Peng [email protected] * Xiang-Jun Hu [email protected] 1

Laboratory of Experimental Pathology, Beijing Institute of Radiation Medicine, 27 Taiping Road, Beijing 100850, China

2

Vestibular Laboratory, Institute of Aviation Medicine, 28 Fucheng Road, Beijing 100142, China

3

Department of Aerospace Medicine Aerospace Biodynamics, The Fourth Military Medical University, 169 Changlexi Road, Xian 100032, China

4

Radiation Protection, Beijing Institute of Radiation Medicine, 27 Taiping Road, Beijing 100850, China

5

Experimental Hematology, Beijing Institute of Radiation Medicine, 27 Taiping Road, Beijing 100850, China

decreased, while the Glu contents in the hippocampus and CSF increased, and memory impairment was observed in the TT genotype but not the CC and CT genotypes. In PC12 cells, the effects of the T allele were more pronounced than those of the C allele on transcription factor binding ability, transcriptional activity, NR2B mRNA, and protein expression. These effects may be related to the detrimental role of the T allele and the protective role of the C allele in rat brain function and PC12 cells exposed to microwave radiation. Keywords NR2B . Microwave . Promoter . Polymorphism . Neurons . Association Electromagnetic fields at microwave frequencies are used in communication and countermeasure as well as in clinical therapy and detection applications and, as such, constitute an environmental factor for personnel in the relevant fields [1–3]. The potential of these fields to cause cognitive dysfunction and neuronal injury has been shown in animals exposed to microwaves [4–10]. Although some of the findings can be attributed to elevated tissue temperature, heating by absorption of microwave energy does not fully explain all findings. Furthermore, some studies showed differential changes in cognitive function and structure between individuals. In populations, the prevalence of self-reported electromagnetic hypersensitivity has usually been attributed to somatization disorders [11, 12]. A possible nonpsychological basis for electromagnetic hypersensitivity was evidenced by the occurrence of field-onset and field-offset brain potentials [13], and the induction of steadystate changes in brain electrical activity that persisted in the presence of the field [14], but the mechanism is still not clear. N-methyl-d-aspartate receptors (NMDARs) represent a unique family of central synaptic receptors, with members that differ widely in their biophysical and pharmacological

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properties [15, 16]. Experimental scrutiny of NMDARs provides insights into their involvement in excitatory synaptic transmission and related processes, such as synaptic plasticity and neural development [17, 18]. NMDARs are involved in several forms of synaptic plasticity, which have been linked to cognitive processes related to learning and memory; in particular, NR2B receptor subunits have been associated with cognitive functions [19, 20]. Polymorphisms in the NMDA receptor subunit 2B gene (GRIN2B) are associated with cognitive disorders such as schizophrenia, Alzheimer’s disease, Parkinson’s disease, obsessive-compulsive disorder, and bipolar disorder [21–28]. Furthermore, some studies have shown that exposure to electromagnetic fields is associated with increased risk for childhood acute leukemia, glioma, and acoustic neuroma [29, 30], although little is known about the relationship between GRIN2B and microwave exposure. Our previous study indicated that microwave radiation induces changes in the expression and activity of NR2B, which is involved in cognitive dysfunction [31, 32]. In this study, single nucleotide polymorphism (SNP) sites of the rat GRIN2B promoter region were screened, and the association of these SNPs with microwave-induced neurons dysfunction and PC12 cell function was investigated.

Materials and Methods Ethics Statement All animal work was conducted according to relevant national and international Guidelines. The study was approved by Beijing Institute of Radiation Medicine Animal Care and Use Committee. Experimental Groups and Exposure Male Wistar rats (n = 160) from different animal centers weighing 160–200 g were housed at a constant temperature (22±1 °C) and relative humidity (60 %) under a regular darklight schedule (light on 7 a.m. to 7 p.m.). Food and water were freely available. Animals in the exposed group (n=100 rats) were placed in individual polypropylene cages under microwave exposure (mean power density, 30 mW/cm2) for 5 min per day, 5 days per week over a period of 2 months. Shamtreated controls (n=60 rats) were placed in individual polypropylene cages without microwave exposure. The temperature of rats was measured by infrared thermodetector before and after microwave exposure. The NR2B promoter sequences (711 bp) including −217C/ T polymorphic sites of both genotypes were cloned into the pGL3-Basic plasmid (Promega; Madison, WI, USA). Rat pheochromocytoma-12 (PC12) cells were cultured at 37 °C and 5 % CO2 in DMEM supplemented with 5 % fetal bovine

serum and 10 % horse serum, 100 U/ml penicillin, and streptomycin. Cells were plated in 6-well tissue culture plates (2– 5×104 cells/well) coated with poly-D-lysine. Exposed and control groups of PC12 cells were then transfected with pGL3-Basic-NR2B-C, pGL3-Basic-NR2B-T, or pGL3-Basic using Lipofectamine2000 (Invitrogen; Rockford, IL, USA). The exposed group was exposed to microwave radiation (mean power density, 30 mW/cm2) for 5 min. The control group was treated under the same conditions as the exposed group, with the exception that the microwave generators were not energized. The supernatant temperature was measured using a fiber optic thermometer m3300 (Luxtron Corp.; Santa Clara, CA, USA) before and after microwave exposure. DNA Sequencing and Genotyping Genomic DNA was extracted from whole blood samples of rats using a QuickGene DNA Isolation kit (Kurabo; Tokyo, Japan). Pairs of polymerase chain reaction (PCR) primers were designed to amplify 705-bp fragments (262 to −443 bp). GRIN2B promoter region variants were initially screened by DNA sequencing. The variants were genotyped by PCR-restriction fragment length polymorphism (PCRRFLP) using a mismatched forward primer to produce an MspI recognition site. The sequences of the primer pair were as follows: forward, 5′-CTGCCTTCCTCCTTGCTTCCCA CTTC-3′; reverse, 5′-CTCTTCTCGCTTGCATATCCACAT AA-3′. Fragments were PCR-amplified according to the following parameters: 30 cycles of denaturation at 94 °C for 5 min, annealing at 60 °C for 20 s, and extension at 72 °C for 20 s, with a final extension at 72 °C for 15 min. The predicted 705-bp products were digested with MspI, and then resolved by 1 % agarose gel electrophoresis. The sizes of the digested fragments were 705 bp for the T allele and 497 and 208 bp for the C allele. Four SNPs of GRIN1 and GRIN2B reported in the SNP database were examined: rs8155766, rs8155765, rs8168565, and rs8169392. No variation in these sites was detected in our samples. Morris Water Maze The Morris water maze (MWM) task was performed in a circular pool (150 cm in diameter) filled with water maintained at 23±0.5 °C in a suitably equipped room at constant temperature, humidity, and brightness. The surface of a clear movable escape platform (12×15 cm) was submerged 1.5 cm below the water surface at a specific location for the entire session. The pool was surrounded by thick curtains to hide extra-maze visual cues from the rats. Rats were trained to find a submerged escape platform, located in a fixed position relative to the extra-maze visual cues, during four consecutive daily sessions. Each session consisted of four trials. Four different starting positions, equally spaced around the perimeter

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of the pool, were used in a fixed order. At 1–4, 14, and 28 days after microwave exposure, spatial memory was assessed. The memory test had an identical format to the training session, consisting of four consecutive trials. Rat behavior in the MWM experiments during the training and memory test procedures was digitally recorded using a SLY-MW system (Beijing Sunny Instrument Co., China), and the average escape latency (AEL) was analyzed. Determination of Amino Acid Content At 28 days after microwave exposure, the animals were anesthetized with sodium pentobarbital (50 mg/kg ip), and the cerebrospinal fluid (CSF) was collected; then, the brains of rats were quickly removed, and the CA3 region of the hippocampus was isolated. The hippocampal tissues were homogenized (1:100 or 1:1) in ice-cold 10 % sulfonic salicylate and then centrifuged at 15,000 rpm for 20 min at 4 °C. The pellets were discarded, and the supernatants were stored at −20 °C for later analysis. Subsequently, the amino acid content of the hippocampal tissue homogenate supernatants and CSF samples was analyzed by high-performance liquid chromatography (HPLC) (Hp1050 pump, Hp1046A fluorometric detector, Hewlett Packard; Palo Alto, CA, USA). RNA Extraction and qRT-PCR Total RNA was extracted from hippocampus tissues at 28 days or from PC12 cells at 6 h following microwave exposure using TRIZOL reagent (Invitrogen) according to the manufacturer’s protocol. Synthesis of cDNA and qRT-PCR reactions was performed using the PrimeScript TMRT reagent kit (Takara; Otsu, Japan). Primers were designed using Primer 5.0 software. The forward and reverse primer sequences were as follows: NR2B F, 5′-TGGCTATCCTGCAGCTGTTTG-3′, NR2B R, 5′-TGGCTGCTCATCACCTCATTC-3′ (103 bp); GAPDH F, 5′-GTGCTGAGTATGTCGTGGAG-3′, GAPDH R, 5′-CGGAGATGATGACCCTTTT-3′ (166 bp). The GAPD H gene was used as an internal standard. The ΔΔCt method was used to transform Ct values into relative quantities (mean ±standard deviation [SD]). Changes were expressed as a percentage of the controls. Immunoblotting Hippocampus tissue at 28 days or PC12 cells at 6 h following microwave exposure was transferred to ice-cold buffer containing 20 mM EDTA, 0.1 mM sodium orthovanadate, 20 μg/ml aprotinin, 10 μg/ml leupeptin, and 0.1 mg/ml phenylmethylsulfonyl fluoride. Protein concentrations were determined using the BCA protein assay (Pierce; Rockford, IL, USA).

Homogenates were mixed in the ratio of 3:2 with sample buffer containing 4 % SDS, 250 mM Tris, 3 mM EDTA, 20 % glycerol, 5 % β-mercaptoethanol, and 0.05 % bromophenol blue (pH 8.0) at 95 °C for 10 min. For each condition, 25 μg of protein was loaded into each well on a 10 % SDSpolyacrylamide gel. After separation by electrophoresis, protein samples were electrotransferred onto a nitrocellulose membrane in transfer buffer (25 mM Tris/192 mM glycine/ 0.02 % SDS/20 % methanol) for 180 min at a constant current (26 mA). Additional protein binding sites on the nitrocellulose membrane were saturated by incubation in 10 mM PBS with 0.1 % Tween-20 (PBST, pH 7.4) containing 10 % dried milk powder for 1 h at room temperature. After a short wash with PBST, blots were incubated with anti-NR2B antibody (1:500, polyclonal rabbit IgG, Santa Cruz, Dallas, Texas, USA) overnight at 4 °C. After rinsing three times with PBST, membranes were incubated with an anti-rabbit and HRP-conjugated monoclonal mouse IgG specific for GAPDH (1:10,000, Nanjingjiancheng, Nanjing, China) for 1 h at room temperature. Bands were developed on autoradiographic film by using an enhanced chemiluminescence kit (Pierce), and the integrated density value (IDV) of the scanned images was measured. Electrophoretic Mobility Shift Assay At 6 h after microwave exposure, PC12 cells were homogenized in 10 mM sodium HEPES (pH 7.9) containing 0.75 mM spermidine, 1.5 mM spermine, 10 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.1 mM EGTA, 5 mM dithiothreitol, the phosphatase inhibitors (10 mM sodium β-glycerophosphate and 1 mM sodium orthovanadate), and 1 μg/ml each of the protease inhibitors phenylmethane sulfonyl fluoride (PMSF), benzamidine, leupeptin, and aprotinin. After the addition of Nonidet P-40 at a final concentration of 0.5 %, the homogenates were centrifuged at 15,000 rpm for 5 min to pellet the nuclei-containing fraction. The pellets were suspended in 50 mM Tris–HCl (pH 7.5) containing 20 % glycerol, 10 % sucrose, 420 mM KCl, 5 mM MgCl2, 0.1 mM EDTA, 0.2 mM EGTA, and the phosphatase and protease inhibitors (previously described) and kept on ice for 30 min. The suspensions were then centrifuged at 15,000 rpm for 5 min, and the supernatants containing the nuclear extracts were stored at −80 °C for later analysis. The probes used in the electrophoretic mobility shift assays (EMSA) contained the following: the NR2B-C binding sequence (F: 5′-AGATAAAAAAAACCGGCATGCA-3′ (biotin labeled or no-biotin labeled), R: 5′-TGCATGCCGGAAAAAAAATATCT-3′), the NR2B-T binding sequence (F: 5′-AGATAAAAAAAATCGGCATGCA-3′ (biotin labeled or no-biotin labeled), R: 5′-TGCATGCCGAAAAAAAAATA TCT-3′), and the CREB positive control (F: 5′-AGAGATTG CCTGACGTCAGAGAGCTAG-3′ (biotin labeled or nobiotin labeled), R: 5′-CTAGCTCTCTGACGTCAGGCAA

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TCTCT-3′). Aliquots (containing 5 μg of protein) of nuclear extracts were incubated with 50 fmol probe (0.5–5×106 cpm/ pmol) in 10 mM Tris–HCl buffer (pH 7.5) containing 250 mM NaCl, 5 mM MgCl2, 2.5 mM EDTA, 20 % glycerol, and 2.5 mM DTT, 5 mM each of the aforementioned phosphatase inhibitors, and 1 μg/mL each of the protease inhibitors for 30 min at 2 °C. Bound and free probes were separated by electrophoresis on a 5 % (w/v) polyacrylamide gel in buffer (pH 8.5) containing 50 mM Tris, 0.38 M glycine, and 2 mM EDTA at a constant voltage of 11 V/cm for 1.5 h in an ice bath. Gels were fixed, dried, and exposed to X-ray films for different periods to obtain autoradiograms suitable for subsequent quantitative densitometry.

Dual-Luciferase Reporter Assay The luciferase reporter plasmids, constructed with both −217C/T alleles, were transiently transfected into PC12 cells using Lipofectamine2000 (Invitrogen). The Renilla luciferase expression plasmid, pRL-TK, was cotransfected as an internal standard. After transfection and at 6 h after microwave exposure, the Dual-Luciferase® Reporter Assay (Promega) was used to detect the transfection activity according to the manufacturer’s protocols. All experiments were repeated at least three times.

Analysis of Cell Cycle, Apoptosis, and Necrosis After transfection and at 6 h after microwave exposure, PC12 cells were collected and centrifuged at 1000 rpm for 5 min. For cell cycle analysis, 300-μl 5 % calf serum and 700-μl absolute ethanol were added to the pellets, mixed, and stored at −20 °C. Propidium iodide (PI, Molecular Probes; Rockford, IL, USA) was used to label cell nuclei. After removal of ethanol by centrifugation at 3000 rpm for 1 min, 100 μl RNase A (1 mg/ml) and 300 μl PI (100 μg/ml) were added, and cells numbers in the G0-G1, S, and G2-M phases were detected by flow cytometry (FCM). For analysis of the rates of apoptosis and necrosis, the pellets were suspended in the 200-μl binding buffer prior to the incubation with10-μl Annexin V-FITC (20 μg/ml, Molecular Probes) and 5 μl PI (50 μg/ml) for 15 min. Subsequently, 300-μl binding buffer was added, and the rates of apoptosis and necrosis were detected by FCM.

Statistical Analysis The results were expressed as means±SEM, and differences between them groups were analyzed by ANOVA using SPSS 11.1 software. Differences at P < 0.05 were considered significant.

Results Detection and Characterization GRIN2B Promoter Region Variants DNA sequence analysis revealed a C-to-T variant at nucleotide position −217 (Fig. 1a). PCR-RFLP analysis of samples from all animals revealed three genotypes: one homozygous for the C allele, one heterozygous for the C and T alleles, and one homozygous for the T allele (Fig. 1b). Comparison of DNA sequences obtained from animals before and after microwave exposure using DNAMAN software (Lynnon Biosoft, USA) revealed that the variant existed stably and did not arise as a result of microwave exposure (Fig. 1c). The C allele was identified as the wild type, because it appeared more frequently than the T allele in all animals (Table 1). The TFSEARCH program (http://www.cbrc.jp/ research/db/TFSEARCH.html) revealed several putative transcription factor binding sites within the 705-bp promoter region of the transcription initiation site. The sequences containing the C allele had three Hb binding sites, an HSF site, and a C-ETs binding site. The sequences containing the T allele had four Hb binding sites and a single HSF site (Table 2). Analysis of the Association of the C-217T Variant with Microwave-Induced Brain Function Abnormalities The association of the C-217T variant with microwaveinduced brain function abnormalities was investigated in Wistar rats (CT, CC, and TT genotypes) through analysis of the following factors: expression of NR2B mRNA and protein, amino acid contents of the hippocampus and CSF, learning and memory ability. Higher expression of NR2B mRNA and protein was detected in the CT and TT genotypes compared with that in the CC genotype, and shorter AELs were measured in MWM tests in the TT genotype compared with those in the CC genotype (P