Differential Effects Between One Week and Four Weeks Exposure to Same Mass of SO2 on Synaptic Plasticity in Rat Hippocampus Gaoyi Yao, Yang Yun, Nan Sang College of Environment and Resource, Research Center of Environment and Health, Institute of Environmental Science, Shanxi University, Taiyuan, Shanxi 030006, People’s Republic of China
Received 2 October 2014; revised 2 December 2014; accepted 7 December 2014 ABSTRACT: Sulfur dioxide (SO2) is a ubiquitous air pollutant. The previous studies have documented the adverse effects of SO2 on nervous system health, suggesting that acutely SO2 inhalation at high concentration may be associated with neurotoxicity and increase risk of hospitalization and mortality of many brain disorders. However, the remarkable features of air pollution exposure are lifelong duration and at low concentration; and it is rarely reported that whether there are different responses on synapse when rats inhaled same mass of SO2 at low concentration with a longer term. In this study, we evaluated the synaptic plasticity in rat hippocampus after exposure to same mass of SO2 at various concentrations and durations (3.5 and 7 mg/m3, 6 h/day, for 4 weeks; and 14 and 28 mg/m3, 6 h/day, for 1 week). The results showed that the mRNA level of synaptic plasticity marker Arc, glutamate receptors (GRIA1, GRIA2, GRIN1, GRIN2A, and GRIN2B) and the protein expression of memory related kinase p-CaMKga were consistently inhibited by SO2 both in 1 week and 4 weeks exposure cases; the protein expression of presynaptic marker synaptophysin, postsynaptic density protein 95 (PSD-95), protein kinase A (PKA), and protein kinase C (PKC) were increased in 1 week exposure case, and decreased in 4 weeks exposure case. Our results indicated that SO2 inhalation caused differential synaptic injury in 1 week and 4 weeks exposure cases, and implied the differential effects might result from different PKA- and/or PKCC 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 820–829, 2016. mediated signal pathway. V Keywords: sulfur dioxide; synaptic injury; synaptic plasticity; glutamate receptors; hippocampus
INTRODUCTION Correspondence to: N. Sang; e-mail: [email protected] Contract grant sponsor: National Science Foundation of People’s Republic of China. Contract grant numbers: 21377076, 21307079, 21222701 Contract grant sponsor: Specialized Research Fund for the Doctoral Program of Higher Education (SRFDP). Contract grant number: 20121401110003, 20131401110005 Contract grant sponsor: Program for the Top Young and Middle-aged Innovative Talents of Higher Learning Institutions of Shanxi (TYMIT). Contract grant number: 20120201 Published online 23 December 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/tox.22093
Sulfur dioxide (SO2) is a ubiquitous air pollutant, and the negative effects of SO2 on public health have attracted more attention of public due to the severe air pollution of SO2 caused primarily by combustion of fossil and biomass fuels for industrial and domestic energy. An increasing body of epidemiologic literatures has indicated that SO2 exposure not only induced many respiratory responses (Herbarth et al., 2001; Iwasawa et al., 2009), but linked with many neurological disorders such as migraine headache, stroke and brain cancer (Hong et al., 2002; Kampaa and Castanas, C 2014 Wiley Periodicals, Inc. V
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2008; Szyszkowicz et al., 2009). Paralleling experimental studies indicated that acutely SO2 exposure at high concentration enhanced oxidative damage and release of proinflammatory cytokines, contributed to the development and progression of ischemic stroke, and even were responsible for DNA breaks in brains of rats (Meng et al., 2004; Sang et al., 2008; Migliore and Coppede, 2009; Sang et al., 2010). These results suggested the neurological dysfunction and neurotoxicity of acutely SO2 inhalation at high concentration. However, when individuals are exposed to air pollution, the remarkable features are lifelong duration and at low concentration; and it is rarely reported that whether there are different responses on synapse when rats inhaled same mass of SO2 at low concentration with a longer term. The Environmental Protection Agency (EPA) set the 3 h average secondary standard of SO2 at 0.5 parts per million (ppm). Given the concentrations in previous studies and the interval of exposure under the experimental conditions, the mean concentration of SO2 in four weeks exposure case was about 3.5 or 7 mg/m3 (about 1.2 or 2.4 ppm). As for the one week exposure case, the mean concentration of SO2 was four-fold higher than what in four weeks exposure case, by which way rats were kept exposing to same mass of SO2. Synaptic plasticity is crucial to the development of the nervous system and thereafter to the ability of an individual to learn and remember new information and to adjust its behavior accordingly (Martin et al., 2000; Neves et al., 2008; Mayford et al., 2012), so it is believed to be important in neurological disorders, such as epilepsy, neurodegeneration, and in recovery from neuronal injury (Kim and Diamond, 2002; Battaglia et al., 2007; Neves et al., 2008). The AMPA and NMDA receptors, as important structural components of excitatory postsynaptic membrane, are critical for synaptic transmission and plasticity, even in cognitive processes (Collingridge and Singer, 1990; Liu et al., 2004; Prybylowski and Wenthold, 2004; Sprengel, 2006; Kessels and Malinow, 2009). The AMPA receptors are the principal molecular units for fast excitatory synaptic transmission in the central nervous system (Malinow and Malenka, 2002; Kessels and Malinow, 2009). It was also shown that the transient activation of NMDARs are the trigger for the induction of long-term potentiation (LTP) at synapses in the hippocampus (Collingridge et al., 1983; Liu, et al., 2004), and are also required for formation of hippocampus dependent learning and memory (Morris et al., 1986; Tsien et al., 1996; Nakazawa et al., 2004). Thus, in order to investigate the differential effects of long-term (4 weeks) and short-term (1 week) exposure to same mass of SO2 on synaptic plasticity, we treated adult rats with SO2 at different concentrations and durations to ensure the rats inhaled same mass of SO2 (3.5 and 7 mg/m3, 6 h/day, for 4 weeks; and 14 and 28 mg/m3, 6 h/day, for 1 week), and then analyzed the expression of the genes and proteins in hippocampus for synaptic structure and signal transduction involved in synaptic plasticity. The results of
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this study conducted to disclose previously undetected differential effects of long-term and short-term SO2 inhalation.
MATERIALS AND METHODS Experimental Animals and Exposure Treatments Male Wistar rats, weighing 180–200 g, were purchased from Center of Experimental Animal of Hebei Province and used according to the guidelines approved by the Institutional Animal Care and Use Committee of Shanxi University. The rats were divided randomly into 6 equal groups of six animals each for two scenarios, in one of which, two groups were placed in 1 m3 exposure chambers containing continuous concentrations of 14.00 6 0.89, and 28.00 6 4.12 mg/m3 SO2 for 6 h/day for one week, respectively; and one control group was placed in another identical chamber, which was continually pumped with filtered air for the same period of time. Following the schedule, in another scenario, with one control group using the same condition for four weeks, the other two groups were exposed to 3.5 6 0.39 and 7.1 6 1.13 mg/m3 SO2 in the same chambers for 6 h/day for four weeks, respectively. The atmosphere of SO2 in the exposure chamber was obtained by mixing continuously at a constant flow rate with the filtered ambient air pumped to the chamber at a flow rate of 30 L/min. The SO2 was diluted with the filtered air at the intake port of the chamber to yield the desired SO2 concentrations. The SO2 within the chambers was measured every 30 min by pararosaniline hydrochloride spectrophotometry (Goyal, 2001). When not being treated, the rats had free access to food and water. Rats were killed 18 h after the last exposure. After the brain was removed, entire hippocampus was excised, half for gene results and half for protein assays, frozen quickly in liquid nitrogen and stored at 280 C.
Protein Isolation and Immunoblot Analysis Proteins were extracted from hippocampal tissues in ice-cold lysis buffer (1% Nonidet P-40, 1 mmol/L EDTA, 125 mmol/ L sodium fluoride, 0.5 mmol/L sodium vanadate, 2.5 lg/mL of aprotinin, 5 lg/mL of pepstatin, 50 lg/mL of leupeptin, 25 lmol/L PMSF, and 25 lg/mL of trypsin inhibitor). The lysates were centrifuged at 13,000 rpm for 15 min and supernatants were collected. Protein concentration was determined according to the method of Bradford using BSA as the standard protein (Bradford, 1976) to ensure equal loading for assessment by Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). Briefly, 50 mg total proteins was separated by SDS–PAGE, then transferred to a nitrocellulose membrane, and blocked with 5% nonfat milk. After blocking, proteins were incubated overnight at 4 C with antibodies to targeted proteins (anti-b-actin antibody, CST; anti-PKA antibody, Bioss; anti-PKC antibody, Bioss;
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TABLE I. Primers sequences used for real-time RT-PCR Gene
Accession No.
Sequence
b-actin Arc GRIA1 GRIA2 GRIN1 GRIN2A GRIN2B
NM_031144 forward reverse NM_019361.1 forward reverse NM_031608.1 forward reverse NM_001083811.1 forward reverse NM_017010.1 forward reverse NM_012573.3 forward reverse NM_012574.1 forward reverse
50 -GCCCTAGACTTCGAGCAAGAG-30 50 -AGCAAGATCAGAGTTACAGTGT-30 50 -CCCTGCAGCCCAAGTTCAAG-30 50 -GAAGGCTCAGCTGCCTGCTC-30 50 -AACTCAAGCGTCCAGAATA-30 50 -ACAGTAGCCCTCATAGCG-30 50 -GGCAAAGAATCCACAGAA-30 50 -ATCACTCAAGGTCATCCC-30 50 -GCTGCACGCCTTTATCTG-30 50 -CTCATGGGACTTGAGTATGGA-30 50 -CGCAGCCGTCTTGAACTA-30 50 -ACTGGAGCAGAGCGAGGT-30 50 -ATGGGATAATGACTCTGGAT-30 50 -TAAGAAAGACGGAGGATAAA-30
anti-p-CaMKga antibody, Bioss; anti-SYP antibody, Bioss, and anti-PSD-95 antibody, Bioss). Exposure to fluorescently labeled secondary antibody (1:2000) (IRDye 800CW goat antirabbit IgG (H 1 L), LI-COR) was followed by scanning and detecting with LI-COR Odyssey Infrared Fluorescent System.
uranyl acetate for 1 h at room temperature in dark, dehydrated by graded ethanol and embedded in beam capsules. Sections, 70–80 nm-thick, were cut from the embedded tissue and collected onto grids to air dry overnight. Stained grids with uranyl acetate for 15–30 min and lead citrate for 3–15 min, and then observe under transmission electron microscope (JEOL, JEM 1400).
RNA Isolation and Real Time PCR Total RNA was isolated from hippocampus tissues using TRIzol Reagent (TaKaRa, China) according to the manufacturer’s instructions. RNA quality was insured by 1% gel electrophoresis (28S/18S RNA). The OD260/OD280 ratio was in the range of 1.9–2.1. Total RNA was then treated with DNase I (TaKaRa, China), and cDNA was synthesized using the First Strand cDNA Synthesis kit (Fermentas, Glen Burnie, MD). The cDNA product was stored at 280 C until used. Primer Designer software was used to design all of the PCR primers. The sequences of primers were as Table I. The real-time quantitative PCR (qPCR) was performed using a Rotor-Gene 3000 Real-Time Cycler (Corbett Research, Sydney, Australia) and Maxima SYBR Green qPCR Master Mix kit (Takara, Dalian, China). A melting curve was established for each sample. Cycling conditions were as follows: 3 min at 95 C, 45 cycles of 20 s at 94 C, 20 s at 55–60 C, and 20 s at 72 C. Fluorescence data were acquired at the 72 C step. The threshold cycle (Ct) value for each dilution was then plotted against the log of its concentration, and Ct values for the experimental samples were plotted onto this dilution series standard curve. Target quantities were calculated from separate standard curves generated for each experiment. Relative expression values (REVs) were then determined by dividing the quantities of the target sequence of interest with the quantity obtained for b-actin as an internal reference gene. The qPCR was repeated three times for each gene.
Transmission Electron Microscope (TEM) Observation About 1 3 1 3 1 mm3 pieces were rapidly cut from hippocampus in brain, fixed in 4% formaldehyde and 1% glutaraldehyde in 0.1M Phosphate Buffer (PB) (pH 7.4) for 2 h at room temperature and then postfixed in 2.5% osmium tetroxide in 0.1M PB. After that, En bloc stain with 2% aqueous
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Statistical Analysis Results were expressed as mean 6 SE. Unless stated otherwise, one-way ANOVA followed by the Fisher’s least significant difference (LSD) test was used for statistical comparison when appropriate, using SPSS software. Differences were considered significant when P < 0.05.
RESULTS Differential Effects of SO2 on Expression of ARC mRNA in Short-Term and Long-Term Cases The activity-regulated cytoskeletal associated gene Arc is necessary for the stabilization of the synaptic plasticity. As shown in Figure 1, in short-term exposure case, the expression of Arc mRNA in the hippocampus was significantly reduced in SO2 rats as compared with control (0.58- and 0.47-fold of control for 14 and 28 mg/m3). Similar significant results were shown in long-term exposure case (0.61and 0.50-fold of control for 3.5 and 7 mg/m3), whereas the severity of the decrement was alleviated.
Differential Effects of SO2 on Expression of Glutamate Receptors mRNA in Short-Term and Long-Term Cases AMPA and NMDA receptors in excitatory synapses are critical for synaptic transmission and plasticity. As shown in Figure 2, in short-term exposure case, the expression of GRIA1, GRIA2, GRIN1, GRIN2A,and GRIN2B mRNA in the hippocampus was significantly reduced in SO2 rats as compared with control rats. The similar results were obtained in long-term exposure case, SO2 rats showed significantly down-regulated expression of GRIA1 mRNA, on the
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Fig. 1. Differential effects on ARC mRNA in hippocampus of rats exposure to SO2 in short-term (a) and long-term (b) cases. (a) Male Wistar rats were exposed to 14 and 28 mg/m3 SO2 for 6 h/day for 1 week. (b) Male Wistar rats were exposed to 3.5 and 7 mg/m3 SO2 for 6 h/day for 4 weeks. Control group rats were exposed to filtered air using the same schedule, respectively. Value in each treated group was expressed as a fold increase as compared to mean value in control group, which has been ascribed as an arbitrary value of 1. Data were expressed as means 6 SE (n 5 6); *P < 0.05, **P < 0.01 versus negative control; Fisher’s least significant difference (LSD) test.
level of GRIA2 and GRIN2B, significant difference appeared only at relatively high concentration, and no significant change was observed on the level of GRIN1 and GRIN2A.
Differential Effects of SO2 on Expression of Protein Kinases in Short-Term and LongTerm Cases Evidence is emerging that protein kinases, such as protein kinase A (PKA) and protein kinase C (PKC), are
critically involved in synaptic plasticity. As shown in Figure 3, in 1 week exposure case, the expression of PKA and PKC in the hippocampus was significantly increased in SO2 rats as compared with control (1.43and 1.37-fold in PKA and 1.32- and 1.43-fold in PKC of control for 14 and 28 mg/m3, respectively). Opposite significant results were shown in 4 weeks exposure case (0.87- and 0.60-fold in PKA and 0.82- and 0.77fold in PKC of control for 3.5 and 7 mg/m3, respectively).
Fig. 2. Differential effects on glutamate receptors subunits mRNA in hippocampus of rats exposure to SO2 in short-term (a) and long-term (b) cases. (a) Male Wistar rats were exposed to 14 and 28 mg/m3 SO2 for 6 h/day for 1 week. (b) Male Wistar rats were exposed to 3.5 and 7 mg/m3 SO2 for 6 h/day for 4 weeks. Control group rats were exposed to filtered air using the same schedule, respectively. Value in each treated group was expressed as a fold increase as compared to mean value in control group, which has been ascribed as an arbitrary value of 1. Data were expressed as means 6 SE (n 5 6); *P < 0.05, **P < 0.01 versus negative control; Fisher’s least significant difference (LSD) test.
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Fig. 3. Differential effects on PKA, PKC, and p-CaMKga in hippocampus of rats exposure to SO2 in short-term (a and b) and long-term (a and c) cases. (b) Male Wistar rats were exposed to 14 and 28 mg/m3 SO2 for 6 h/day for 1 week. (c) Male Wistar rats were exposed to 3.5 and 7 mg/m3 SO2 for 6 h/day for 4 weeks. Control group rats were exposed to filtered air using the same schedule, respectively. Value in each treated group was expressed as a fold increase as compared to mean value in control group, which has been ascribed as an arbitrary value of 1. Data were expressed as means 6 SE (n 5 6); *P < 0.05, **P < 0.01 versus negative control; Fisher’s least significant difference (LSD) test.
As shown in Figure 3, in short-term exposure case, the expression of p-CaMKga in the hippocampus was significantly decreased in SO2 rats when compared with control. Also, the similar result was obtained in long-term exposure case, SO2 rats exhibited a significant decrease in pCaMKga, but significant difference appeared only at relatively higher concentration.
Differential Effects of SO2 on Expression of Postsynaptic Marker Protein PSD-95 in Short-Term and Long-Term Cases It is reported that postsynaptic density protein 95 (PSD95) is an important postsynaptic scaffolding protein that plays a critical role in anchoring PKA and PKC at postsynaptic density. As shown in Figure 4, in 1 week exposure case, the expression of PDS-95 in the hippocampus was significantly increased in SO2 rats as compared with control (1.19- and 1.17-fold of control for 14 and 28 mg/m3). Opposite significant result was shown in 4 weeks exposure case (0.83- and 0.70-fold of control for 3.5 and 7 mg/m3).
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Differential Effects of SO2 on Expression of Presynaptic Marker SYP in Short-Term and Long-Term Cases It is also reported that the protein kinase A plays a critical role for synaptic plasticity in presynaptically by regulating the readily releasable pool of synaptic vesicles. Further to investigate the differential effects of 4 weeks and 1 week exposure of SO2 on presynaptic terminals, we analyzed the expression of Synaptophysin (SYP). As shown in Figure 5, in 1 week exposure case, the expression of SYP in the hippocampus was significantly increased in SO2 rats as compared with control (1.22- and 1.29-fold of control for 14 and 28 mg/m3). Opposite significant results were shown in 4 weeks exposure case (0.76- and 0.60-fold of control for 3.5 and 7 mg/m3).
Synapse Ultrastructural Alterations in Rats Exposure to SO2 in Short-Term and LongTerm Cases To further validate the alterations of presynaptic and postsynaptic marker proteins, the ultrastructural changes of synapses
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Fig. 4. Differential effects on PSD-95 in hippocampus of rats exposure to SO2 in short-term (a and b) and long-term (a and c) cases. (b) Male Wistar rats were exposed to 14 and 28 mg/m3 SO2 for 6 h/day for 1 week. (c) Male Wistar rats were exposed to 3.5 and 7 mg/m3 SO2 for 6 h/day for 4 weeks. Control group rats were exposed to filtered air using the same schedule, respectively. Value in each treated group was expressed as a fold increase compared to mean value in control group, which has been ascribed as an arbitrary value of 1. Data were expressed as means 6 SE (n 5 6); *P < 0.05, **P < 0.01 versus negative control; Fisher’s least significant difference (LSD) test.
in hippocampus of rats after SO2 exposure were studied by TEM (Fig. 6). As shown in Figure 6(a), in 1 week exposure case, SO2 rats showed increased presynaptic vesicles density and thickened/expanded postsynaptic density (PSD). Opposite results, reduced vesicles density and thinned PSD [Fig. 6(b)], were shown in 4 weeks exposure case.
DISCUSSION SO2 is a ubiquitous air pollutant, and the negative effects of SO2 on public health have attracted more attention of public. It is well known that the toxicological effects of chemicals are differential during the two kinds of exposure modes, uniform concentration of lifespan exposure and higher concentration of short-term exposure. The present study reported the differential toxicological effects on synaptic plasticity of long-term and short-term SO2 inhalation. Additionally, in this study the animals were subjected to regular periods of extended exposure, with relief periods between protocols (i.e., 6 h/day, with 18 h between exposures). This may provide a corollary to individuals exposed to the gas in an occupational setting. The OSHA standard for SO2 is 5 ppm (about 13 mg/m3) averaged over an 8 h work shift. The 1
week exposure concentration in this study is 14 or 28 mg/ m3, which is just above or 2 times higher than the OSHA standard. And NIOSH has recommended that the permissible exposure limit be reduced to 2 ppm (about 5 mg/m3) as a time-weighted average concentration. The 4 weeks exposure concentration in this study is 3.5 or 7 mg/m3, which is around the NIOSH standard. Therefore, the exposure model in this study is more in line with the exposure case of occupational groups, and our results provide a series of basic experimental evidences on synaptic damage of SO2 air pollution. Synaptic plasticity is crucial to the development of the nervous system and thereafter to the ability of learning and memory (Martin et al., 2000; Battaglia et al., 2007; Neves et al., 2008). A lot of works have been done in previous studies on SO2 exposure acutely at high concentrations, therefore in order to avoid duplication of these works in this study, the typical indicators of synaptic plasticity and synaptic transmission were select to investigate the differential responses on synaptic plasticity between long-term and short-term exposure cases. The immediate early gene Arc is necessary for the stabilization of the synaptic plasticity and for the formation of long-term memory (Guzowski et al., 2000; Plath et al.,
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Fig. 5. Differential effects on SYP in hippocampus of rats exposure to SO2 in short-term (a and b) and long-term (a and c) cases. (b) Male Wistar rats were exposed to 14 and 28 mg/m3 SO2 for 6 h/day for 1 week. (c) Male Wistar rats were exposed to 3.5 and 7 mg/m3 SO2 for 6 h/day for 4 weeks. Control group rats were exposed to filtered air using the same schedule, respectively. Value in each treated group was expressed as a fold increase compared to mean value in control group, which has been ascribed as an arbitrary value of 1. Data were expressed as means 6 SE (n 5 6); *P < 0.05, **P < 0.01 versus negative control; Fisher’s least significant difference (LSD) test.
Fig. 6. Morphological alterations of hippocampal synapses in rats exposure to SO2 in short-term (a) and long-term (b) cases. Synapses were marked with black arrows. (a) Male Wistar rats were exposed to 14 and 28 mg/m3 SO2 for 6 h/day for 1 week. (b) Male Wistar rats were exposed to 3.5 and 7 mg/m3 SO2 for 6 h/day for 4 weeks. Control group rats were exposed to filtered air using the same schedule, respectively. Scale bars are 1 lm.
Environmental Toxicology DOI 10.1002/tox
SULFUR DIOXIDE AND SYNAPTIC INJURY IN RAT HIPPOCAMPUS
2006). In this study, we demonstrated that SO2 reduced the expression of Arc at mRNA level, suggesting that SO2 disrupted the expression of Arc to change the synaptic plasticity. As glutamate receptors in excitatory synapses play a critical role in synaptic transmission, we examined the mRNA level of glutamate receptors subunits. In present study, SO2 rats showed reduced expression of glutamate receptors at mRNA level, and the influence was serious when rats exposed to SO2 at higher concentration. These results suggested that SO2 inhalation might cause changes of the synaptic structure by impeding the expression of glutamate receptors mRNA. Besides, protein kinases play a crucial role in the process of synaptic transmission involved in synaptic plasticity. Previous studies have shown that activated CaMKga can potentiate and prolong synaptic transmission by its autophosphorylation at Thr-286 when activated (Miller and Kennedy, 1986). In this study, SO2 rats exhibited a significant decrease expression of CaMKga, and this influence was serious when rats exposed to SO2 at higher concentration. These suggest that SO2 suppresses the expression of p-CaMKga, which could repress the phosphorylation of postsynaptic density proteins to induce the interruption of synaptic transmission and result in synaptic dysfunction. Interestingly, in present study, we observed that SO2 rats in 1 week exposure case showed increased expression of PKA, while decreased in 4 weeks exposure case. This may be due to that the protein kinase A is critical for synaptic plasticity not only in postsynaptic mechanisms to coordinate glutamate receptor trafficking or b-adrenergic receptor signaling to mediated synaptic plasticity and memory (Skeberdis et al., 2006; Robertson, et al., 2009; Havekes et al., 2012; Zhang et al., 2013), but also presynaptically regulates the readily releasable pool of synaptic vesicles (Trudeau et al., 1996; Moulder et al., 2008; Park et al., 2014). Further to test the presynaptic role of PKA in the process of synaptic injury induced by SO2 inhalation, we examined the expression of Synaptophysin (SYP), a synaptic vesicle membrane protein involved in synaptic vesicle trafficking and initiation of neurotransmitter release (Valtorta et al., 2004). Also, we obtained similar result in the expression of SYP, SO2 rats showed increased expression of SYP in 1week exposure case, but decreased in 4 weeks exposure case, which were consistent with the results in electron microscopy, increased vesicles density in 1 week exposure case and decreased in 4 weeks exposure case. These data suggested that SO2 may disrupt the expression of SYP to change the neurotransmitter release in synaptic transmission, and indicated that the role of PKA-mediated signal pathway in presynaptic mechanisms is involved in SO2-induced injury in 1 week and 4 weeks exposure cases. In addition, evidences are emerging that the elevated actived PKC, translocating from the cytosolic to membrane compartments (Olds et al., 1989), could enhance synaptic remodeling, synaptogenesis and protein synthesis (Alkon et al., 2005; Sun et al., 2010). Similarly, it is noteworthy that
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SO2 inhalation rats in 1 week exposure case show increased expression of PKC, while the effects were opposite in 4 weeks exposure case. These data suggest that the role of PKCmediated signal pathway might be involved in SO2-induced synaptic injury in 1 week and 4 weeks exposure cases. Numerous studies have shown that the signaling enzymes PKA and PKC at postsynaptic density are binding to Akinase-anchoring protein (AKAP)79/150 (Klauck et al., 1996; Faux, 1999; Dell’Acqua et al., 2006), which linked to glutamate receptors through the postsynaptic marker protein PSD95 (Colledge, 2000). PSD-95 is an important postsynaptic membrane-associated guanylate kinase scaffolding protein that plays a critical role in protein assembly, synaptic development and neural plasticity (Kim and Sheng, 2004; Sheng and Hoogenraad, 2007). In present study, we observed that SO2 rats showed increased expression of PSD-95 in 1 week exposure case, and decreased in 4 weeks exposure case, which was consistent with the results of PKA and PKC. Also, the electron microscopy showed that SO2 inhalation thickened/ expanded postsynaptic density in 1 week exposure case and thinned postsynaptic density in 4 weeks exposure case. These data indicated that the role of PKA- and PKC-mediated signal pathway in postsynaptic mechanisms is also involved in SO2induced injury in 1 week and 4 weeks exposure cases. Our results in present study indicate SO2 inhalationinduced synaptic injury could result not only from postsynaptically inhibiting expression of glutamate receptors and interfering related kinases to mediate synaptic signaling and plasticity, but also from presynaptically regulating the initiation of neurotransmitter release by PKA-mediated signal pathway. It is well known that PKC has high affinity for diacylglycerol (DAG) and it can be activated by DAG (Sun and Alkon, 2009), and in the CNS, most 2-AG is postsynaptically synthesized from DAG via activation of DAG lipase and then across the synaptic cleft to presynaptic side (Murataeva et al., 2014). Both on the presynaptic and postsynaptic side, 2-AG can be broken down into glycerol and arachidonic acid (AA). Our previous studies have confirmed that acutely SO2 exposure at high concentration induced the neurotoxicity via the mechanisms that postsynaptic inducible enzyme cyclooxygenase-2 (COX-2)mediated AA and the metabolite bind to its presynaptic receptors to activate PKA-mediated signal pathway (Sang et al., 2011), these data may imply that DAG-PKC-mediated signal pathway, especially in crosstalk of phospholipid metabolism and endocannabinoids synthesis, might be involved in SO2induced synaptic transmission and synaptic dysfunction, via which mechanism cause differential synaptic responses between high concentration and low concentration exposure cases. However further details are needed in order to work towards preventing CNS damage provoked by polluted air.
CONCLUSIONS In this study, we reported that exposure to same mass of SO2 in both short-term and long-term cases could lead to
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disruption of immediate early gene Arc, alteration of synaptic glutamate receptors (GRIA1, GRIA2, GRIN1, GRIN2A, and GRIN2B) and suppression of CaMKga phosphorylation in rats hippocampal synapses, whereas the expression of presynaptic marker synaptophysin (SYP), postsynaptic density protein 95(PSD-95), Protein kinase A (PKA) and Protein kinase C (PKC) were increased in 1week exposure case, and decreased in 4 weeks exposure case. Furthermore, these adverse effects were more pronounced in rats exposed to SO2 in short-term case with high concentration. These results implied that SO2 inhalation could cause differential synaptic injury participated in synaptic transmission and even might in cognitive impairment.
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Plath N, Ohana O, Dammermann B, Errington ML, Schmitz D, Gross C, Mao X, Engelsberg A, Mahlke C, Welzl H, Kobalz U, Stawrakakis A, Fernandez E, Waltereit R, Bick-Sander A, Therstappen E, Cooke SF, Blanquet V, Wurst W, Salmen B, B€osl MR, Lipp HP, Grant SG, Bliss TV, Wolfer DP, Kuhl D. 2006. Arc/Arg3.1 is essential for the consolidation of synaptic plasticity and memories. Neuron 52:437–444. Prybylowski K, Wenthold RJ. 2004. N-Methyl-D-aspartate receptors: subunit assembly and trafficking to the synapse. J Biol Chem 279:9673–9676. Robertson HR, Gibson ES, Benke TA, Dell’Acqua ML. 2009. Regulation of postsynaptic structure and function by an A-kinase anchoring protein-membrane-associated guanylate kinase scaffolding complex. J Neurosci 29:7929–7943. Sang N, Yun Y, Hou L, Li GK. 2008. SO2 inhalation-induced effects on cytokine, intracellular calcium and immediately early genes in rat hippocampus. Acta Sci Circumst 28: 2315–2320. Sang N, Yun Y, Li HY, Hou L, Han M, Li GK. 2010. SO2 inhalation contributes to the development and progression of ischemic stroke in the brain. Toxicol Sci 114:226–236. Sang N, Yun Y, Yao GY, Li HY, Guo L, Li GK. 2011. SO2-induced neurotoxicity is mediated by cyclooxygenases-2-derived prostaglandin E2 and its downstream signaling pathway in rat hippocampal neurons. Toxicol Sci 124:400–413.
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Environmental Toxicology DOI 10.1002/tox
Received 2 October 2014; revised 2 December 2014; accepted 7 December 2014 ABSTRACT: Sulfur dioxide (SO2) is a ubiquitous air pollutant. The previous studies have documented the adverse effects of SO2 on nervous system health, suggesting that acutely SO2 inhalation at high concentration may be associated with neurotoxicity and increase risk of hospitalization and mortality of many brain disorders. However, the remarkable features of air pollution exposure are lifelong duration and at low concentration; and it is rarely reported that whether there are different responses on synapse when rats inhaled same mass of SO2 at low concentration with a longer term. In this study, we evaluated the synaptic plasticity in rat hippocampus after exposure to same mass of SO2 at various concentrations and durations (3.5 and 7 mg/m3, 6 h/day, for 4 weeks; and 14 and 28 mg/m3, 6 h/day, for 1 week). The results showed that the mRNA level of synaptic plasticity marker Arc, glutamate receptors (GRIA1, GRIA2, GRIN1, GRIN2A, and GRIN2B) and the protein expression of memory related kinase p-CaMKga were consistently inhibited by SO2 both in 1 week and 4 weeks exposure cases; the protein expression of presynaptic marker synaptophysin, postsynaptic density protein 95 (PSD-95), protein kinase A (PKA), and protein kinase C (PKC) were increased in 1 week exposure case, and decreased in 4 weeks exposure case. Our results indicated that SO2 inhalation caused differential synaptic injury in 1 week and 4 weeks exposure cases, and implied the differential effects might result from different PKA- and/or PKCC 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 820–829, 2016. mediated signal pathway. V Keywords: sulfur dioxide; synaptic injury; synaptic plasticity; glutamate receptors; hippocampus
INTRODUCTION Correspondence to: N. Sang; e-mail: [email protected] Contract grant sponsor: National Science Foundation of People’s Republic of China. Contract grant numbers: 21377076, 21307079, 21222701 Contract grant sponsor: Specialized Research Fund for the Doctoral Program of Higher Education (SRFDP). Contract grant number: 20121401110003, 20131401110005 Contract grant sponsor: Program for the Top Young and Middle-aged Innovative Talents of Higher Learning Institutions of Shanxi (TYMIT). Contract grant number: 20120201 Published online 23 December 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/tox.22093
Sulfur dioxide (SO2) is a ubiquitous air pollutant, and the negative effects of SO2 on public health have attracted more attention of public due to the severe air pollution of SO2 caused primarily by combustion of fossil and biomass fuels for industrial and domestic energy. An increasing body of epidemiologic literatures has indicated that SO2 exposure not only induced many respiratory responses (Herbarth et al., 2001; Iwasawa et al., 2009), but linked with many neurological disorders such as migraine headache, stroke and brain cancer (Hong et al., 2002; Kampaa and Castanas, C 2014 Wiley Periodicals, Inc. V
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2008; Szyszkowicz et al., 2009). Paralleling experimental studies indicated that acutely SO2 exposure at high concentration enhanced oxidative damage and release of proinflammatory cytokines, contributed to the development and progression of ischemic stroke, and even were responsible for DNA breaks in brains of rats (Meng et al., 2004; Sang et al., 2008; Migliore and Coppede, 2009; Sang et al., 2010). These results suggested the neurological dysfunction and neurotoxicity of acutely SO2 inhalation at high concentration. However, when individuals are exposed to air pollution, the remarkable features are lifelong duration and at low concentration; and it is rarely reported that whether there are different responses on synapse when rats inhaled same mass of SO2 at low concentration with a longer term. The Environmental Protection Agency (EPA) set the 3 h average secondary standard of SO2 at 0.5 parts per million (ppm). Given the concentrations in previous studies and the interval of exposure under the experimental conditions, the mean concentration of SO2 in four weeks exposure case was about 3.5 or 7 mg/m3 (about 1.2 or 2.4 ppm). As for the one week exposure case, the mean concentration of SO2 was four-fold higher than what in four weeks exposure case, by which way rats were kept exposing to same mass of SO2. Synaptic plasticity is crucial to the development of the nervous system and thereafter to the ability of an individual to learn and remember new information and to adjust its behavior accordingly (Martin et al., 2000; Neves et al., 2008; Mayford et al., 2012), so it is believed to be important in neurological disorders, such as epilepsy, neurodegeneration, and in recovery from neuronal injury (Kim and Diamond, 2002; Battaglia et al., 2007; Neves et al., 2008). The AMPA and NMDA receptors, as important structural components of excitatory postsynaptic membrane, are critical for synaptic transmission and plasticity, even in cognitive processes (Collingridge and Singer, 1990; Liu et al., 2004; Prybylowski and Wenthold, 2004; Sprengel, 2006; Kessels and Malinow, 2009). The AMPA receptors are the principal molecular units for fast excitatory synaptic transmission in the central nervous system (Malinow and Malenka, 2002; Kessels and Malinow, 2009). It was also shown that the transient activation of NMDARs are the trigger for the induction of long-term potentiation (LTP) at synapses in the hippocampus (Collingridge et al., 1983; Liu, et al., 2004), and are also required for formation of hippocampus dependent learning and memory (Morris et al., 1986; Tsien et al., 1996; Nakazawa et al., 2004). Thus, in order to investigate the differential effects of long-term (4 weeks) and short-term (1 week) exposure to same mass of SO2 on synaptic plasticity, we treated adult rats with SO2 at different concentrations and durations to ensure the rats inhaled same mass of SO2 (3.5 and 7 mg/m3, 6 h/day, for 4 weeks; and 14 and 28 mg/m3, 6 h/day, for 1 week), and then analyzed the expression of the genes and proteins in hippocampus for synaptic structure and signal transduction involved in synaptic plasticity. The results of
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this study conducted to disclose previously undetected differential effects of long-term and short-term SO2 inhalation.
MATERIALS AND METHODS Experimental Animals and Exposure Treatments Male Wistar rats, weighing 180–200 g, were purchased from Center of Experimental Animal of Hebei Province and used according to the guidelines approved by the Institutional Animal Care and Use Committee of Shanxi University. The rats were divided randomly into 6 equal groups of six animals each for two scenarios, in one of which, two groups were placed in 1 m3 exposure chambers containing continuous concentrations of 14.00 6 0.89, and 28.00 6 4.12 mg/m3 SO2 for 6 h/day for one week, respectively; and one control group was placed in another identical chamber, which was continually pumped with filtered air for the same period of time. Following the schedule, in another scenario, with one control group using the same condition for four weeks, the other two groups were exposed to 3.5 6 0.39 and 7.1 6 1.13 mg/m3 SO2 in the same chambers for 6 h/day for four weeks, respectively. The atmosphere of SO2 in the exposure chamber was obtained by mixing continuously at a constant flow rate with the filtered ambient air pumped to the chamber at a flow rate of 30 L/min. The SO2 was diluted with the filtered air at the intake port of the chamber to yield the desired SO2 concentrations. The SO2 within the chambers was measured every 30 min by pararosaniline hydrochloride spectrophotometry (Goyal, 2001). When not being treated, the rats had free access to food and water. Rats were killed 18 h after the last exposure. After the brain was removed, entire hippocampus was excised, half for gene results and half for protein assays, frozen quickly in liquid nitrogen and stored at 280 C.
Protein Isolation and Immunoblot Analysis Proteins were extracted from hippocampal tissues in ice-cold lysis buffer (1% Nonidet P-40, 1 mmol/L EDTA, 125 mmol/ L sodium fluoride, 0.5 mmol/L sodium vanadate, 2.5 lg/mL of aprotinin, 5 lg/mL of pepstatin, 50 lg/mL of leupeptin, 25 lmol/L PMSF, and 25 lg/mL of trypsin inhibitor). The lysates were centrifuged at 13,000 rpm for 15 min and supernatants were collected. Protein concentration was determined according to the method of Bradford using BSA as the standard protein (Bradford, 1976) to ensure equal loading for assessment by Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). Briefly, 50 mg total proteins was separated by SDS–PAGE, then transferred to a nitrocellulose membrane, and blocked with 5% nonfat milk. After blocking, proteins were incubated overnight at 4 C with antibodies to targeted proteins (anti-b-actin antibody, CST; anti-PKA antibody, Bioss; anti-PKC antibody, Bioss;
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TABLE I. Primers sequences used for real-time RT-PCR Gene
Accession No.
Sequence
b-actin Arc GRIA1 GRIA2 GRIN1 GRIN2A GRIN2B
NM_031144 forward reverse NM_019361.1 forward reverse NM_031608.1 forward reverse NM_001083811.1 forward reverse NM_017010.1 forward reverse NM_012573.3 forward reverse NM_012574.1 forward reverse
50 -GCCCTAGACTTCGAGCAAGAG-30 50 -AGCAAGATCAGAGTTACAGTGT-30 50 -CCCTGCAGCCCAAGTTCAAG-30 50 -GAAGGCTCAGCTGCCTGCTC-30 50 -AACTCAAGCGTCCAGAATA-30 50 -ACAGTAGCCCTCATAGCG-30 50 -GGCAAAGAATCCACAGAA-30 50 -ATCACTCAAGGTCATCCC-30 50 -GCTGCACGCCTTTATCTG-30 50 -CTCATGGGACTTGAGTATGGA-30 50 -CGCAGCCGTCTTGAACTA-30 50 -ACTGGAGCAGAGCGAGGT-30 50 -ATGGGATAATGACTCTGGAT-30 50 -TAAGAAAGACGGAGGATAAA-30
anti-p-CaMKga antibody, Bioss; anti-SYP antibody, Bioss, and anti-PSD-95 antibody, Bioss). Exposure to fluorescently labeled secondary antibody (1:2000) (IRDye 800CW goat antirabbit IgG (H 1 L), LI-COR) was followed by scanning and detecting with LI-COR Odyssey Infrared Fluorescent System.
uranyl acetate for 1 h at room temperature in dark, dehydrated by graded ethanol and embedded in beam capsules. Sections, 70–80 nm-thick, were cut from the embedded tissue and collected onto grids to air dry overnight. Stained grids with uranyl acetate for 15–30 min and lead citrate for 3–15 min, and then observe under transmission electron microscope (JEOL, JEM 1400).
RNA Isolation and Real Time PCR Total RNA was isolated from hippocampus tissues using TRIzol Reagent (TaKaRa, China) according to the manufacturer’s instructions. RNA quality was insured by 1% gel electrophoresis (28S/18S RNA). The OD260/OD280 ratio was in the range of 1.9–2.1. Total RNA was then treated with DNase I (TaKaRa, China), and cDNA was synthesized using the First Strand cDNA Synthesis kit (Fermentas, Glen Burnie, MD). The cDNA product was stored at 280 C until used. Primer Designer software was used to design all of the PCR primers. The sequences of primers were as Table I. The real-time quantitative PCR (qPCR) was performed using a Rotor-Gene 3000 Real-Time Cycler (Corbett Research, Sydney, Australia) and Maxima SYBR Green qPCR Master Mix kit (Takara, Dalian, China). A melting curve was established for each sample. Cycling conditions were as follows: 3 min at 95 C, 45 cycles of 20 s at 94 C, 20 s at 55–60 C, and 20 s at 72 C. Fluorescence data were acquired at the 72 C step. The threshold cycle (Ct) value for each dilution was then plotted against the log of its concentration, and Ct values for the experimental samples were plotted onto this dilution series standard curve. Target quantities were calculated from separate standard curves generated for each experiment. Relative expression values (REVs) were then determined by dividing the quantities of the target sequence of interest with the quantity obtained for b-actin as an internal reference gene. The qPCR was repeated three times for each gene.
Transmission Electron Microscope (TEM) Observation About 1 3 1 3 1 mm3 pieces were rapidly cut from hippocampus in brain, fixed in 4% formaldehyde and 1% glutaraldehyde in 0.1M Phosphate Buffer (PB) (pH 7.4) for 2 h at room temperature and then postfixed in 2.5% osmium tetroxide in 0.1M PB. After that, En bloc stain with 2% aqueous
Environmental Toxicology DOI 10.1002/tox
Statistical Analysis Results were expressed as mean 6 SE. Unless stated otherwise, one-way ANOVA followed by the Fisher’s least significant difference (LSD) test was used for statistical comparison when appropriate, using SPSS software. Differences were considered significant when P < 0.05.
RESULTS Differential Effects of SO2 on Expression of ARC mRNA in Short-Term and Long-Term Cases The activity-regulated cytoskeletal associated gene Arc is necessary for the stabilization of the synaptic plasticity. As shown in Figure 1, in short-term exposure case, the expression of Arc mRNA in the hippocampus was significantly reduced in SO2 rats as compared with control (0.58- and 0.47-fold of control for 14 and 28 mg/m3). Similar significant results were shown in long-term exposure case (0.61and 0.50-fold of control for 3.5 and 7 mg/m3), whereas the severity of the decrement was alleviated.
Differential Effects of SO2 on Expression of Glutamate Receptors mRNA in Short-Term and Long-Term Cases AMPA and NMDA receptors in excitatory synapses are critical for synaptic transmission and plasticity. As shown in Figure 2, in short-term exposure case, the expression of GRIA1, GRIA2, GRIN1, GRIN2A,and GRIN2B mRNA in the hippocampus was significantly reduced in SO2 rats as compared with control rats. The similar results were obtained in long-term exposure case, SO2 rats showed significantly down-regulated expression of GRIA1 mRNA, on the
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Fig. 1. Differential effects on ARC mRNA in hippocampus of rats exposure to SO2 in short-term (a) and long-term (b) cases. (a) Male Wistar rats were exposed to 14 and 28 mg/m3 SO2 for 6 h/day for 1 week. (b) Male Wistar rats were exposed to 3.5 and 7 mg/m3 SO2 for 6 h/day for 4 weeks. Control group rats were exposed to filtered air using the same schedule, respectively. Value in each treated group was expressed as a fold increase as compared to mean value in control group, which has been ascribed as an arbitrary value of 1. Data were expressed as means 6 SE (n 5 6); *P < 0.05, **P < 0.01 versus negative control; Fisher’s least significant difference (LSD) test.
level of GRIA2 and GRIN2B, significant difference appeared only at relatively high concentration, and no significant change was observed on the level of GRIN1 and GRIN2A.
Differential Effects of SO2 on Expression of Protein Kinases in Short-Term and LongTerm Cases Evidence is emerging that protein kinases, such as protein kinase A (PKA) and protein kinase C (PKC), are
critically involved in synaptic plasticity. As shown in Figure 3, in 1 week exposure case, the expression of PKA and PKC in the hippocampus was significantly increased in SO2 rats as compared with control (1.43and 1.37-fold in PKA and 1.32- and 1.43-fold in PKC of control for 14 and 28 mg/m3, respectively). Opposite significant results were shown in 4 weeks exposure case (0.87- and 0.60-fold in PKA and 0.82- and 0.77fold in PKC of control for 3.5 and 7 mg/m3, respectively).
Fig. 2. Differential effects on glutamate receptors subunits mRNA in hippocampus of rats exposure to SO2 in short-term (a) and long-term (b) cases. (a) Male Wistar rats were exposed to 14 and 28 mg/m3 SO2 for 6 h/day for 1 week. (b) Male Wistar rats were exposed to 3.5 and 7 mg/m3 SO2 for 6 h/day for 4 weeks. Control group rats were exposed to filtered air using the same schedule, respectively. Value in each treated group was expressed as a fold increase as compared to mean value in control group, which has been ascribed as an arbitrary value of 1. Data were expressed as means 6 SE (n 5 6); *P < 0.05, **P < 0.01 versus negative control; Fisher’s least significant difference (LSD) test.
Environmental Toxicology DOI 10.1002/tox
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Fig. 3. Differential effects on PKA, PKC, and p-CaMKga in hippocampus of rats exposure to SO2 in short-term (a and b) and long-term (a and c) cases. (b) Male Wistar rats were exposed to 14 and 28 mg/m3 SO2 for 6 h/day for 1 week. (c) Male Wistar rats were exposed to 3.5 and 7 mg/m3 SO2 for 6 h/day for 4 weeks. Control group rats were exposed to filtered air using the same schedule, respectively. Value in each treated group was expressed as a fold increase as compared to mean value in control group, which has been ascribed as an arbitrary value of 1. Data were expressed as means 6 SE (n 5 6); *P < 0.05, **P < 0.01 versus negative control; Fisher’s least significant difference (LSD) test.
As shown in Figure 3, in short-term exposure case, the expression of p-CaMKga in the hippocampus was significantly decreased in SO2 rats when compared with control. Also, the similar result was obtained in long-term exposure case, SO2 rats exhibited a significant decrease in pCaMKga, but significant difference appeared only at relatively higher concentration.
Differential Effects of SO2 on Expression of Postsynaptic Marker Protein PSD-95 in Short-Term and Long-Term Cases It is reported that postsynaptic density protein 95 (PSD95) is an important postsynaptic scaffolding protein that plays a critical role in anchoring PKA and PKC at postsynaptic density. As shown in Figure 4, in 1 week exposure case, the expression of PDS-95 in the hippocampus was significantly increased in SO2 rats as compared with control (1.19- and 1.17-fold of control for 14 and 28 mg/m3). Opposite significant result was shown in 4 weeks exposure case (0.83- and 0.70-fold of control for 3.5 and 7 mg/m3).
Environmental Toxicology DOI 10.1002/tox
Differential Effects of SO2 on Expression of Presynaptic Marker SYP in Short-Term and Long-Term Cases It is also reported that the protein kinase A plays a critical role for synaptic plasticity in presynaptically by regulating the readily releasable pool of synaptic vesicles. Further to investigate the differential effects of 4 weeks and 1 week exposure of SO2 on presynaptic terminals, we analyzed the expression of Synaptophysin (SYP). As shown in Figure 5, in 1 week exposure case, the expression of SYP in the hippocampus was significantly increased in SO2 rats as compared with control (1.22- and 1.29-fold of control for 14 and 28 mg/m3). Opposite significant results were shown in 4 weeks exposure case (0.76- and 0.60-fold of control for 3.5 and 7 mg/m3).
Synapse Ultrastructural Alterations in Rats Exposure to SO2 in Short-Term and LongTerm Cases To further validate the alterations of presynaptic and postsynaptic marker proteins, the ultrastructural changes of synapses
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Fig. 4. Differential effects on PSD-95 in hippocampus of rats exposure to SO2 in short-term (a and b) and long-term (a and c) cases. (b) Male Wistar rats were exposed to 14 and 28 mg/m3 SO2 for 6 h/day for 1 week. (c) Male Wistar rats were exposed to 3.5 and 7 mg/m3 SO2 for 6 h/day for 4 weeks. Control group rats were exposed to filtered air using the same schedule, respectively. Value in each treated group was expressed as a fold increase compared to mean value in control group, which has been ascribed as an arbitrary value of 1. Data were expressed as means 6 SE (n 5 6); *P < 0.05, **P < 0.01 versus negative control; Fisher’s least significant difference (LSD) test.
in hippocampus of rats after SO2 exposure were studied by TEM (Fig. 6). As shown in Figure 6(a), in 1 week exposure case, SO2 rats showed increased presynaptic vesicles density and thickened/expanded postsynaptic density (PSD). Opposite results, reduced vesicles density and thinned PSD [Fig. 6(b)], were shown in 4 weeks exposure case.
DISCUSSION SO2 is a ubiquitous air pollutant, and the negative effects of SO2 on public health have attracted more attention of public. It is well known that the toxicological effects of chemicals are differential during the two kinds of exposure modes, uniform concentration of lifespan exposure and higher concentration of short-term exposure. The present study reported the differential toxicological effects on synaptic plasticity of long-term and short-term SO2 inhalation. Additionally, in this study the animals were subjected to regular periods of extended exposure, with relief periods between protocols (i.e., 6 h/day, with 18 h between exposures). This may provide a corollary to individuals exposed to the gas in an occupational setting. The OSHA standard for SO2 is 5 ppm (about 13 mg/m3) averaged over an 8 h work shift. The 1
week exposure concentration in this study is 14 or 28 mg/ m3, which is just above or 2 times higher than the OSHA standard. And NIOSH has recommended that the permissible exposure limit be reduced to 2 ppm (about 5 mg/m3) as a time-weighted average concentration. The 4 weeks exposure concentration in this study is 3.5 or 7 mg/m3, which is around the NIOSH standard. Therefore, the exposure model in this study is more in line with the exposure case of occupational groups, and our results provide a series of basic experimental evidences on synaptic damage of SO2 air pollution. Synaptic plasticity is crucial to the development of the nervous system and thereafter to the ability of learning and memory (Martin et al., 2000; Battaglia et al., 2007; Neves et al., 2008). A lot of works have been done in previous studies on SO2 exposure acutely at high concentrations, therefore in order to avoid duplication of these works in this study, the typical indicators of synaptic plasticity and synaptic transmission were select to investigate the differential responses on synaptic plasticity between long-term and short-term exposure cases. The immediate early gene Arc is necessary for the stabilization of the synaptic plasticity and for the formation of long-term memory (Guzowski et al., 2000; Plath et al.,
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Fig. 5. Differential effects on SYP in hippocampus of rats exposure to SO2 in short-term (a and b) and long-term (a and c) cases. (b) Male Wistar rats were exposed to 14 and 28 mg/m3 SO2 for 6 h/day for 1 week. (c) Male Wistar rats were exposed to 3.5 and 7 mg/m3 SO2 for 6 h/day for 4 weeks. Control group rats were exposed to filtered air using the same schedule, respectively. Value in each treated group was expressed as a fold increase compared to mean value in control group, which has been ascribed as an arbitrary value of 1. Data were expressed as means 6 SE (n 5 6); *P < 0.05, **P < 0.01 versus negative control; Fisher’s least significant difference (LSD) test.
Fig. 6. Morphological alterations of hippocampal synapses in rats exposure to SO2 in short-term (a) and long-term (b) cases. Synapses were marked with black arrows. (a) Male Wistar rats were exposed to 14 and 28 mg/m3 SO2 for 6 h/day for 1 week. (b) Male Wistar rats were exposed to 3.5 and 7 mg/m3 SO2 for 6 h/day for 4 weeks. Control group rats were exposed to filtered air using the same schedule, respectively. Scale bars are 1 lm.
Environmental Toxicology DOI 10.1002/tox
SULFUR DIOXIDE AND SYNAPTIC INJURY IN RAT HIPPOCAMPUS
2006). In this study, we demonstrated that SO2 reduced the expression of Arc at mRNA level, suggesting that SO2 disrupted the expression of Arc to change the synaptic plasticity. As glutamate receptors in excitatory synapses play a critical role in synaptic transmission, we examined the mRNA level of glutamate receptors subunits. In present study, SO2 rats showed reduced expression of glutamate receptors at mRNA level, and the influence was serious when rats exposed to SO2 at higher concentration. These results suggested that SO2 inhalation might cause changes of the synaptic structure by impeding the expression of glutamate receptors mRNA. Besides, protein kinases play a crucial role in the process of synaptic transmission involved in synaptic plasticity. Previous studies have shown that activated CaMKga can potentiate and prolong synaptic transmission by its autophosphorylation at Thr-286 when activated (Miller and Kennedy, 1986). In this study, SO2 rats exhibited a significant decrease expression of CaMKga, and this influence was serious when rats exposed to SO2 at higher concentration. These suggest that SO2 suppresses the expression of p-CaMKga, which could repress the phosphorylation of postsynaptic density proteins to induce the interruption of synaptic transmission and result in synaptic dysfunction. Interestingly, in present study, we observed that SO2 rats in 1 week exposure case showed increased expression of PKA, while decreased in 4 weeks exposure case. This may be due to that the protein kinase A is critical for synaptic plasticity not only in postsynaptic mechanisms to coordinate glutamate receptor trafficking or b-adrenergic receptor signaling to mediated synaptic plasticity and memory (Skeberdis et al., 2006; Robertson, et al., 2009; Havekes et al., 2012; Zhang et al., 2013), but also presynaptically regulates the readily releasable pool of synaptic vesicles (Trudeau et al., 1996; Moulder et al., 2008; Park et al., 2014). Further to test the presynaptic role of PKA in the process of synaptic injury induced by SO2 inhalation, we examined the expression of Synaptophysin (SYP), a synaptic vesicle membrane protein involved in synaptic vesicle trafficking and initiation of neurotransmitter release (Valtorta et al., 2004). Also, we obtained similar result in the expression of SYP, SO2 rats showed increased expression of SYP in 1week exposure case, but decreased in 4 weeks exposure case, which were consistent with the results in electron microscopy, increased vesicles density in 1 week exposure case and decreased in 4 weeks exposure case. These data suggested that SO2 may disrupt the expression of SYP to change the neurotransmitter release in synaptic transmission, and indicated that the role of PKA-mediated signal pathway in presynaptic mechanisms is involved in SO2-induced injury in 1 week and 4 weeks exposure cases. In addition, evidences are emerging that the elevated actived PKC, translocating from the cytosolic to membrane compartments (Olds et al., 1989), could enhance synaptic remodeling, synaptogenesis and protein synthesis (Alkon et al., 2005; Sun et al., 2010). Similarly, it is noteworthy that
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SO2 inhalation rats in 1 week exposure case show increased expression of PKC, while the effects were opposite in 4 weeks exposure case. These data suggest that the role of PKCmediated signal pathway might be involved in SO2-induced synaptic injury in 1 week and 4 weeks exposure cases. Numerous studies have shown that the signaling enzymes PKA and PKC at postsynaptic density are binding to Akinase-anchoring protein (AKAP)79/150 (Klauck et al., 1996; Faux, 1999; Dell’Acqua et al., 2006), which linked to glutamate receptors through the postsynaptic marker protein PSD95 (Colledge, 2000). PSD-95 is an important postsynaptic membrane-associated guanylate kinase scaffolding protein that plays a critical role in protein assembly, synaptic development and neural plasticity (Kim and Sheng, 2004; Sheng and Hoogenraad, 2007). In present study, we observed that SO2 rats showed increased expression of PSD-95 in 1 week exposure case, and decreased in 4 weeks exposure case, which was consistent with the results of PKA and PKC. Also, the electron microscopy showed that SO2 inhalation thickened/ expanded postsynaptic density in 1 week exposure case and thinned postsynaptic density in 4 weeks exposure case. These data indicated that the role of PKA- and PKC-mediated signal pathway in postsynaptic mechanisms is also involved in SO2induced injury in 1 week and 4 weeks exposure cases. Our results in present study indicate SO2 inhalationinduced synaptic injury could result not only from postsynaptically inhibiting expression of glutamate receptors and interfering related kinases to mediate synaptic signaling and plasticity, but also from presynaptically regulating the initiation of neurotransmitter release by PKA-mediated signal pathway. It is well known that PKC has high affinity for diacylglycerol (DAG) and it can be activated by DAG (Sun and Alkon, 2009), and in the CNS, most 2-AG is postsynaptically synthesized from DAG via activation of DAG lipase and then across the synaptic cleft to presynaptic side (Murataeva et al., 2014). Both on the presynaptic and postsynaptic side, 2-AG can be broken down into glycerol and arachidonic acid (AA). Our previous studies have confirmed that acutely SO2 exposure at high concentration induced the neurotoxicity via the mechanisms that postsynaptic inducible enzyme cyclooxygenase-2 (COX-2)mediated AA and the metabolite bind to its presynaptic receptors to activate PKA-mediated signal pathway (Sang et al., 2011), these data may imply that DAG-PKC-mediated signal pathway, especially in crosstalk of phospholipid metabolism and endocannabinoids synthesis, might be involved in SO2induced synaptic transmission and synaptic dysfunction, via which mechanism cause differential synaptic responses between high concentration and low concentration exposure cases. However further details are needed in order to work towards preventing CNS damage provoked by polluted air.
CONCLUSIONS In this study, we reported that exposure to same mass of SO2 in both short-term and long-term cases could lead to
Environmental Toxicology DOI 10.1002/tox
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disruption of immediate early gene Arc, alteration of synaptic glutamate receptors (GRIA1, GRIA2, GRIN1, GRIN2A, and GRIN2B) and suppression of CaMKga phosphorylation in rats hippocampal synapses, whereas the expression of presynaptic marker synaptophysin (SYP), postsynaptic density protein 95(PSD-95), Protein kinase A (PKA) and Protein kinase C (PKC) were increased in 1week exposure case, and decreased in 4 weeks exposure case. Furthermore, these adverse effects were more pronounced in rats exposed to SO2 in short-term case with high concentration. These results implied that SO2 inhalation could cause differential synaptic injury participated in synaptic transmission and even might in cognitive impairment.
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Environmental Toxicology DOI 10.1002/tox
