Developmental lead exposure alters gene expression of metabotropic glutamate receptors in rat hippocampal neurons

Neuroscience Letters 413 (2007) 222–226

Developmental lead exposure alters gene expression of metabotropic glutamate receptors in rat hippocampal neurons Jian Xu, Chong-Huai Yan ∗ , Sheng-Hu Wu, Xiao-Dan Yu, Xiao-Gang Yu, Xiao-Ming Shen ∗ Shanghai XinHua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai Institute for Pediatric Research, Shanghai Key Laboratory of Children’s Environmental Health, Shanghai 200092, China Received 31 August 2006; received in revised form 24 October 2006; accepted 31 October 2006

Abstract Exposure to lead in utero and in infancy is associated with a risk of impaired cognitive development. Increasing evidence suggests that the family of metabotropic glutamate receptors (mGluRs) plays an important role in synaptic plasticity and memory formation. We determined whether mGluRs subtypes 1, 3, and 7 (mGluR1, mGluR3, and mGluR7) were involved in developmental neurotoxicity due to lead. Embryonic rat hippocampal neurons were cultured for 21 days and exposed to lead chloride beginning on the fourth day of incubation. We investigated levels of mGluR1, mGluR3, and mGluR7 mRNA expression by using quantitative real-time reverse-transcription polymerase chain reaction (RT-PCR) with lead exposure at 10 nM, 1 ␮M, and 100 ␮M. Lead exposure in vitro downregulated the expression of mGluR1 mRNA and upregulated the expression of mGluR3 and mGluR7 mRNA in a dose-dependent manner. We speculate that mGluRs may be involved in lead neurotoxicity. Pathways that likely contribute to lead neurotoxicity by means of mGluRs are impairment of long-term potentiation, effects on N-methyl-d-aspartate (NMDA) receptor functions, and depotentiation. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Lead; Gene expression; Metabotropic glutamate receptors; Hippocampus; Development

Lead is one of the oldest known poisons. Although various actions have been taken to decrease the use and distribution of lead in the environment, it remains a considerable health hazard. The developing central nervous system is particularly susceptible to lead. Membrane ionic channels and signaling molecules seem to be among the most relevant molecular targets contributing to lead neurotoxicity. Numerous studies have shown that hippocampal N-methyl-d-aspartate (NMDA) receptors play an important role in synaptic plasticity and brain development, which are a principal target in lead-induced neurotoxicity [8,14,25]. However, the exact neurotoxic mechanism of lead poisoning remains unclear. Metabotropic glutamate receptors (mGluRs) have been extensively studied in recent times. Eight different mGluRs subunits have been identified and are divided into three groups based on sequence homology, ligand selectivity, and signaling

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Corresponding author. Tel.: +86 21 6502 8710; fax: +86 21 6579 5173. E-mail addresses: [email protected], [email protected] (C.-H. Yan), [email protected], [email protected] (X.-M. Shen). 0304-3940/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2006.10.070

properties. Group I receptors (mGluR1 and mGluR5) neighbor NMDA receptors and actively interact with them to regulate various cellular and synaptic activities, stimulating phospholipase C (PLC) and targeting NMDA receptors [4]. Group II (mGluR2 and mGluR3) and group III (mGluR4, mGluR6, and mGluR7) receptors are negatively coupled to adenylate cyclase by means of Go and possibly Gi protein [13,33]. Previous studies showed that antagonists and agonists of mGluRs could modulate the induction, formation, and maintenance of long-term potentiation (LTP) [13,23,32,33], a form of neuronal plasticity that is involved in memory and learning. Given these findings, whether mGluRs participate in lead neurotoxicity is an open issue. We used hippocampal neuronal cultures and real-time reverse-transcription polymerase chain reaction (RT-PCR) assay to evaluate the expression of mGluR1, mGluR3, and mGluR7 mRNA after exposure to different levels of lead in vitro. The aim of this study was to determine the effect of lead on mGluR gene expression during late gestation and lactation. Pregnant Sprague–Dawley rats at 18 ± 0.5 days of gestation (200–250 g) were purchased from Animal House Center

J. Xu et al. / Neuroscience Letters 413 (2007) 222–226

at Chinese Academy of Sciences (Shanghai, China). Dissociated hippocampal cultures were prepared from fetal rats, as previously described [21,20,24]. All procedures complied with institutional guidelines regarding the ethical care and use of animals. On the fourth day of incubation, lead chloride (Sigma, St. Louis, MO, USA) was randomly added to the medium to produce final concentrations of lead (Pb2+ ) of 1 ␮M, 100 ␮M, or 10 nM. The concentrations were maintained by replacing the lead when the medium was changed every 4 days. Quality control for the measurement of lead was satisfactory throughout the course of the current study. Overall, three groups of neurons were studied. Embryonic neurons at 18 days of gestation were chosen because hippocampi during this stage are easy to separate and because the neurons cultured survive better than those of newborn rats [9]. Hippocampal neurons cultured from 18-day-old fetal Sprague–Dawley rats were exposed to 100 nM of lead for 10–30 days induced numerous changes in synaptic events, such as modulation of glutamatergic transmission [7]. In addition, concentrations of lead ranging from 25 to 100 nM have been found in the cerebrospinal fluid of humans not known to be occupationally exposed to lead [5,6]. Therefore, we selected three doses of Pb2+ of 10 nM, 1 ␮M and 100 ␮M to cover a range of effects of daily exposure or non-occupational lead exposure level, medium and high levels of lead exposure. Hippocampal neurons and glial cells present in the cultures were identified by using immunocytochemistry with antibodies against monoclonal anti-microtubule-associated protein 2 (MAP2) and polyclonal anti-glial fibrillary acidic protein (GFAP), respectively [20]. MAP2 (1:200, Dako, Carpinteria, CA, USA) and GFAP (1:100, Dako) immunostaining were conducted according to the manufacturer’s instructions after 4 days of culture. Two negative controls were performed in each experiment. Cells were observed by using a compound microscope (Zeiss, Oberkochen, Germany) at 400× magnification with phase contrast. Neurons and glial cells were counted to quantify the proportion of neurons and glial cells. RNA was isolated by using a Trizol kit (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Extracted RNA concentrations and purity were evaluated by measuring the A260 nm-to-A280 nm absorbance ratio with an ultraviolet spectrophotometer (Perkin Elmer, Wellesley, MA). Its integrity was assessed by means of agarose gel electrophoresis. Highly purified oligonucleotide primers were commercially generated (Shanghai Sangon, China). Primer design and optimization were done with Oligo4.1 software (National Biosciences Inc., Plymouth, MN) [10]. Primers used were: mGluR1 (GenBank accession number X57569), sense 5 -GCG GAA TGG TAC GAT CTG TGT GC-3 , antisense 5 -GGC AAG AAA AGG CGA TGG CTA TG-3 , 255 bp; mGluR3 (GenBank accession number M92076), sense 5 -GAC GTG GTC CTG GTG ATC CTA T-3 , antisense 5 -CTA ACG GAG ATG CAC ATT G-3 , 197 bp; mGluR7 (GenBank accession number D16817), sense 5 -CCA GAC AAC AAA CAC AAC CAACC-3 , antisense 5 -GCG TTC CCT TCT GTG TCT TCT TC-3 , 173 bp; ␤-actin, sense 5 -AGA CCT CTA TGC CAA CAC AGT GCT

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G-3 , antisense 5 -TCA TCG TAC TCC TGC TTG CTG A-3 , 218 bp. One-step, real-time quantitative RT-PCR was carried out with a LightCycler instrument (Roche, Mannheim, Germany) by using the LightCycler SYBR Green I RNA Master Kit (Roche). All reactions were conducted in duplicate. Negative control was performed with sterile purified deionized water. Each cycle of PCR included denaturation at 95 ◦ C for 5 s, primers annealing at 62 ◦ C (69 ◦ C for mGluR1) for 5 s, and a final extension at 72 ◦ C for 12 s. The fluorescence of each sample was measured at 5 ◦ C below the product’s melting temperature (Tm ) to eliminate background fluorescence due to primer-dimer [34]. Results were analyzed with LightCycler Software Version 3.5 by using the second-derivative maximum method to set the threshold cycle (CT ). PCR efficiency (E) was calculated using the equation E = 10(−1/slope) [28–30]. Relative quantification was carried out with the Relative Expression Software Tool (REST, Roche). Relative and normalized expression ratios were calculated on the basis of the median of the performed duplicates and computed according to the following equation: R = Etarget exp(CTtarget )/Eref exp(CTref ) [10,29,30]. Agarose gel electrophoresis analyses were also performed to verify whether the amplified product corresponded to the size predicted for gene-specific product. Because of expression level of ␤-actin gene was constant regardless of lead exposure [36], relative quantification was presented to calculate the relative expression ratio by means of normalization with ␤-actin gene. Lead, particularly at high concentration, caused neuronal cell death. Therefore, it was necessary to apply a relative quantification method to normalize the numbers of neurons alive in the culture media by using the house-keeping gene and correct data analysis. In real-time PCR experiments, small sample sizes were used realistically. Therefore, a distribution-free Wilcoxon test will be a more robust and appropriate alternative in this case to compare effects of different Pb exposure on expression of targeted genes, as previously described [35]. Variations in gene expression were compared by using SAS 6.12 software (SAS Institute, Cary, NC) with coefficients of variability (CV) and Wilcoxon two group test (significant level p = 0.05) [35] and by using Relative Expression Software Tool with relative quantification. Hippocampal neurons were confirmed by means of positive MAP2 immunoreactivity (Fig. 1A) and astrocytes stained positive for GFAP (Fig. 1B). Estimated cell counts indicated that the yield of hippocampal neurons was 95–97%. Compared with controls, lead-exposed neurons grew poorly, often with abnormal nuclei or soma and inhibited neurite initiation. These effects showed a dose-dependent relationship with the concentration of lead in the culture media. In high exposure group (100 ␮M), many neuronal deaths or cell fragments were observed in the culture media. Optical-density ratios at 260–280 nm for total RNA were all between 1.8 and 2.0. Agarose gel electrophoresis showed that the 28S and 18S ribosomal RNA bands were clearly visible at a staining intensity of about 2:1 (28S:18S).

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Fig. 2. Gene expression of mGluR1, mGluR3, and mGluR7 mRNA in hippocampal neurons after lead exposure in vitro. Relative and normalized expression ratios and its confidence intervals are expressed in the chart. A ratio of 1 indicates no change in gene expression; <1 indicates reduced expression; >1 indicates increased expression. (*) Significantly different from the 1 ␮M group (p < 0.05); () significantly different from the 10 nM group (p < 0.05). Lead exposure of 10 nM did not substantially change expression in any of the targeted genes compared with controls (p > 0.05).

Fig. 1. Representative photomicrographs showing immunocytochemical results for hippocampal neurons and glial cells: (A) embryonic neurons positively stain for MAP2 after 4 days in culture and (B) glial cells positively stain for GFAP. The background blue nuclei of neurons are seen indistinctly. Scale bar = 35 ␮m. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

By drawing standard curves for ␤-actin and other targeted genes, we found a linear relationship between the CT value and the logarithm of the starting concentration of the cDNA standard. Melting-curve analysis showed that all PCR amplifications led to a single and specific product. Products were identified on high-resolution agarose gel electrophoresis. PCR efficiency of ␤-actin, mGluR1, mGluR3, and mGluR7 were 1.96, 1.88, 1.94, and 1.76, respectively, CV of PCR efficiency were 0, 2.7‰, 2‰, and 3‰. Tm of ␤-actin, mGluR1, mGluR3, and mGluR7 were 84.6 ◦ C, 86.97 ◦ C, 81.13 ◦ C, and 82.54 ◦ C, respectively, CV of Tm were 2.1‰, 3.1‰, 2.3‰, and 3.7‰. As Fig. 2 shows, gene expression of mGluR3 and mGluR7 mRNA was stimulated and gene expression of mGluR1 mRNA decreased in a dose-dependent manner after exposure to lead (Wilcoxon two group test showed p < 0.05 between doses). Highest lead exposure of 100 ␮M resulted in maximum expression of mGluR3 and mGluR7 mRNA and minimal expression of mGluR1 mRNA. Medium exposure with 1 ␮M result in expression between those observed with 100 ␮M and 10 nM lead

exposure. Lead exposure of 10 nM did not substantially change expression in any of the targeted genes compared with controls (p > 0.05). We evaluated potential roles of mGluRs in developmental neurotoxicity of lead. Our results suggested that lead exposure in vitro interfered with the expression of mGluRs mRNA in rat hippocampal cultures. As studied previously, antagonists and agonists of mGluRs had a significant impact on the formation of LTP [13,23,32,33]. LTP is characterized by two steps: an early one mediated by NMDA receptors that leads to a partial and short-term potentiation, and a late one mediated by mGluRs, which allows for the enhancement and consolidation of potentiation that transforms it into a full LTP [12]. The mechanism by which activation of mGluR1 facilitates LTP is mainly associated with a presynaptic increase of glutamate release [1,18]. mGluR1 is necessary to facilitate synaptic plasticity in medial vestibular nuclei and mGluR1a-dependent LTP in hippocampal interneurons [23]. Group II and III mGluRs inhibit and reverse vestibular LTP during the early inductive phase, which prevents LTP from achieving its full expression and consolidation [14]. Our results showed that lead exposure downregulated expression of mGluR1 mRNA and upregulated expression of mGluR3 and mGluR7 mRNA in a dose-dependent manner, these two effects might act synergistically to block LTP, this may be a potential mechanism of lead neurotoxicity. Group I mGluRs are positively coupled to the NMDA receptor NR1 subunit for protein phosphorylation. Selective activation of group I mGluRs stimulates tyrosine phosphorylation of NR2A/B subunits in cortical neurons, which is mediated specifically by mGluR1 and an intracellular cascade involving PLC, calmodulin, Pyk2, and the Src family of kinases (Src/Fyn). Tyro-

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sine phosphorylation of NMDA receptors mediated by mGluR1 has functional consequences, such as increasing the NMDA receptor-mediated current [15]. This altered phosphorylation of NR1 and NR2A/B may underlie group I mGluRs regulation of NMDA receptor functions. Lead decreased expression of mGluR1 in the hippocampus, a change that may in turn reduce expression of the NR1 and NR2A/B subunits of NMDA receptors, reduce postsynaptic Ca2+ influx, and finally affect synaptic transmission. Huang and Hsu [16] and Huang et al. [17] found out that afferent low-frequency stimulation or pharmacologic treatments that interrupted cell–cell or cell–matrix interactions, when given shortly after LTP induction, could reverse hippocampal CA1 LTP. They named this reversal of synaptic strength from potentiated state to pre-LTP levels depotentiation. The activation of group II mGluRs and subsequent Gi/Go protein-coupled signaling cascade are responsible for the depotentiation. In the present study, upregulation of mGluR3 mRNA was verified. Depotentiation is reported to impair memory in animals [16]. Therefore, a plausible explanation is that depotentiation may be involved in lead neurotoxicity by activating group II mGluRs. Numerous reports mention that pharmacologic activation of group II or group III mGluR agonists or mGluR1 antagonists are neuroprotective in a variety of models of neuronal degeneration, including neuronal cultures, brain slices, and in vivo models of excitotoxicity [3,11,19,22,27,31]. The prominent role of mGluR4 and mGluR7 in mediating group III agonist-induced neuroprotection is also consistent with the high expression levels and broad distribution of mGluR4 and mGluR7 in many regions of the mammalian brain. Because of its neuroprotective role, dose-dependent upregulation of mGluR3 and mGluR7 mRNA and downregulation of mGluR1 mRNA might be a protective and stress-induced reaction to lead-induced neuronal impairment or injuries in vitro. Hence, we speculated that a dual effect, including primary neurotoxicity and accessory neuroprotection induced by lead, might simultaneously occur when neurons were exposed to lead. mGluR1 activation may reduce nerve cell susceptibility to excitotoxic injury and facilitate neurogenesis in a PLCdependent manner [2]. mGluR1 stimulates phosphoinositidespecific PLC activity and causes the release of Ca2+ from cytoplasmic stores, which in turn activates protein kinase C (PKC). Previous studies showed that acute administration of picomolar concentrations of lead in vitro activated PKC [6]. However, long-term lead administration in vivo reduces hippocampal PKC expression [26]. Group II and group III mGluRs negatively couple to adenylyl cyclase. Downregulation of mGluR1 mRNA and upregulation of mGluR3 or mGluR7 mRNA inhibit neurogenesis and disturb the expression of PKC or cyclic adenosine monophosphate, which may impair synaptic transmission and induce lead neurotoxicity. Lead exposure in vitro altered expression levels of mGluR1, mGluR3, and mGluR7 mRNA in developmental hippocampal neurons, and the involvement of mGluRs in lead neurotoxicity was inferred. Further studies are required to reveal the outcomes of five spliced variants of mGluR2, mGluR4, mGluR5, mGluR6, and mGluR8 and protein-expression levels of mGluRs

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