Abstract Epidemiological studies indicate that smoking is negatively correlated with the incidence and development of Alzheimer's disease (AD). Nicotine was reported to be the active factor. However, the detailed mechanisms still remain to be fully elucidated. Early growth response gene 1 (EGR-1) plays important roles in several important biological processes such as promoting cell growth, differentiation, anti oxidative stress, and apoptosis, but few in the pathogenesis of AD. In the present study, we show that nicotine can activate the MAPK/ERK/EGR-1 signaling pathway partially through α7 nAChR. In addition, the up-regulation of EGR-1 by nicotine can also increase the phosphorylation of CyclinD1 which contributes to the attenuation of amyloid-β (Aβ 25–35 ) -induced neurotoxicity. Although nicotine and Aβ 25–35 can activate EGR-1, the expression of EGR-1 is down-regulated following treatment with nicotine and Aβ 25–35 . This study demonstrates that low dose nicotine attenuates Aβ 25–35 -induced neurotoxicity in vitro and in vivo through activating EGR-1 pathway.

Citation: Xue M, Zhu L, Zhang J, Qiu J, Du G, Qiao Z, et al. (2015) Low Dose Nicotine Attenuates Aβ Neurotoxicity through Activation Early Growth Response Gene 1 Pathway. PLoS ONE 10(3): e0120267. https://doi.org/10.1371/journal.pone.0120267 Academic Editor: Michelle L. Block, Indiana School of Medicine, UNITED STATES Received: October 27, 2014; Accepted: January 21, 2015; Published: March 27, 2015 Copyright: © 2015 Xue et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited Data Availability: All relevant data are within the paper. Funding: This work was supported by National Science Foundation in China (2014J01375; 81271421). The Natural Science Foundation of Fujian Province of China (Grant: 2013J01147; and 2014J06019). This work was also supported by Program for New Century Excellent Talents in University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction Alzheimer's disease (AD), the most common form of dementia, is characterized by memory loss and cognitive defects [1–3]. Nevertheless, the impact mechanisms of learning and memory deficits in AD remain poorly understood. There are two hallmarks for AD: extracellular amyloid-β (Aβ) plaques and intracellular neurofibrillary tangles formed by hyperphosphorylated Tau. Many factors are attributed to the progression of AD, such as oxygen free radicals and metal ions homeostasis [4, 5]. Epidemiological studies have consistently shown that the incidence of AD is twice lower in smokers than in age-matched controls [6]. Nicotine exposure was reported to improve cognitive function and plays an important role in the prevention of impairment of memory and learning in AD [7, 8]. However, the detailed mechanism is not fully understood. The zinc finger transcription factor EGR-1, which belongs to a group of early response genes, was independently discovered by several laboratories [9–11]. The egr-1 gene can be induced by environmental factors such as ultraviolet (UV), ionizing radiation and injury, and cellular signals including growth factors, neurotransmitters, and cytokines as well as stress signals [12]. Thus, EGR-1 is involved in a variety of cellular processes including growth, differentiation, neurite outgrowth, wound healing and apoptosis. EGR-1 also can regulate neuroendocrine genes to benefit neuroplasticity and perform neuroprotective role to prevent memory loss [13, 14]. A previous study found that EGR-1 directly transactivated bim gene expression to mediate apoptosis of rat cerebellar granule neurons [15]. In addition, microtubules are destabilized by overexpression of egr-1 in rat hippocampus neurons which promotes Tau phosphorylation [16]. EGR-1 is sufficient to promote axonal elongation induced by sAPPs [17]. EGR-1 transcription factor represents a potential mediator for neuroprotection induced by glatiramer acetate in AD [18]. In our study, we found that nicotine can activate EGR-1 through MAPK/ERK signaling pathway by the activation of α7 nAChRs to attenuate the neurotoxicity of Aβ 25–35 . The activation of EGR-1 by nicotine contributes to up-regulating the expression of cyclinD1 and its phosphorylation. Our immunohistochemistry results indicate that Aβ 25–35 can increase the phosphorylation of ERK1/2, and this process can be blocked by nicotine administration. Last but not least, the protein level of EGR-1 was significantly decreased by nicotine treatment which was up-regulated by Aβ 25–35 . In this study, we elucidated the partial mechanisms that underlie the activation of EGR-1 by nicotine, and provide theoretical knowledge for retarding the proceeding of AD.

Materials and Methods Antibodies and Chemical reagents The antibodies against ERK1/2, Thr202/Tyr204 phospho-ERK1/2, cyclinD1, Thr286 phospho-cyclinD1 were from Cell Signaling Technology; Nicotinic acetylcholine receptor α7 was from Abcam; EGR-1, and β-actin were from Santa Cruz Biotechnology. U0126 was from Cell Signaling Technology; α-BTX, nicotine, and Aβ25–35 were from Sigma-Aldrich. Unless specified, all chemicals were obtained from Sigma-Aldrich Co (St. Louis, MO). Cell Culture SH-SY5Y and rat PC12 pheochromocytoma cell lines were purchased from the Shanghai Institute of Material Medical, Chinese Academy of Science. SH-SY5Y cells were cultured in Dulbecco's modified Eagle's medium (Hyclone, Logan, UT, USA), and supplemented with 10% (v/v) fetal bovine serum (Hyclone) and 1×Penicillin-Streptomycin (100 U/ml or 100 mg/ml) (Invitrogen, Carlsbad CA, USA). PC12 cells were cultured in the same manner plus 5% (v/v) serum equinum. Animal model Forty eight four-week-old male C57/BL/L6 mice were randomly divided into four groups, and all the animals are SPF (Specific Pathogen Free) laboratory animals and they are healthy, their weights ranged from 18g to 22g. The four groups included a control group, an AD model group, a standalone nicotine group and a nicotine-treatment AD model group. The standalone nicotine group and nicotine-treatment AD model group were injected 0.15 mg/kg nicotine using a subcutaneous injection every day for 2 weeks. Identical doses of sodium chloride were administered to the other two groups. Then the AD model group and nicotine-treatment AD model group were injected with 5 μL (5mg/ml) Aβ 25–35 using a stereotaxis instrument (RWD, Shenzhen, China) and microinjector (KD Scientific, Holliston, MA, USA) into the left lateral ventricle of C57/BL/L6 mice (coordinates: anteroposterior, -2.0mm from the bregma; lateral, -2.0mm; dorsovental, -3mm), and other two groups were injected with identical doses of sodium chloride into the left lateral ventricle of C57/BL/L6 mice. After 16 days, the control group, the nicotine-treatment AD model group and the AD model group were practiced with the place navigation test and spatial probe test of water maze within 5 days. At the end of this period, all the mice were anesthetized with sodium pentobarbital (50μg/g) and euthanized by transcardial perfusion with ice-cold physiological saline. Brain cortex and hippocampus were collected for immunohistochemistry. Ethics statement All animal procedures were in accordance with the National Institute of Health Guidelines for the Care and Use of Laboratory Animals and were approved by the Animal Ethics Committee of Xiamen University (IACUC #: XMULAC20130066). Plasmid and recombinant retrovirus construction EGR-1 shRNA, the neuronal nicotinic acetylcholine receptors α7 shRNA plasmids and recombinant retroviruses were constructed. The sequences of EGR-1 and α7 shRNA were designed by Clontech RNAi designer (http://bioinfo.clontech.com), and were constructed into the retroviral vector RNAi-Ready pSIREN-RetroQ (Clontech). shRNA targets for hEGR-1: CGCCGAACACTGACATTTT; shRNA targets for hnAChRα7: AACAGTGCTGATGAGCGCTTTGAC. Recombinant retroviruses were packaged using GP2-293 cells according to the BD Retro-XTM Universal Packaging System protocol (BD Biosciences Clontech). MTT Assay for Cell Viability To conduct MTT analysis, exponentially growing SH-SY5Y cells were plated into 96-well plates (5000 cells in 100 μL medium containing 10% fetal bovine serum per well). On the day following the plating of the cells, the culture medium was replaced with the serum free medium. The culture medium was prepared for each respective treatment, and culturing continued for another 48 to 72 hours. Cellular viability was measured in a 96-well plate by a quantitative colorimetric assay with MTT [19]. Real-time Quantitative RT-PCR Regular RT-PCR and quantitative RT-PCR (qRT-PCR) were performed as previously described [20]. For this assay, an ABI PRISM 7300 detection system was used with the primers as followings. The RT-PCR reactions were repeated at least three times. β-actin (Forward: 5'-TCAGAAAGATTCCTACGTGGGCGA-3'; Reverse: 5'-TGTGGTGCCAGATCTTCTCCATGT-3'); EGR-1: primer1 (Forward: 5'-CCAAGGCCGAGATGCAATTGA-3'; Reverse: 5'-CCAGGGAGAAGCGGC CAGTAT-3'); EGR-1: primer2 (Forward: 5'-ATGCTTGCCCTGTCGAGTC-3'; Reverse: 5'-GGTATGCCTCTTGCGTTCATC-3'); nAChRα7 (Forward: 5'-AACAGTGCTGATGAGCGCTTTGAC-3'; Reverse: 5'-AGTGCTGCACATCAAAGGGAAACC-3'). Western blot analysis The Western blotting was carried out as described previously [20]. The antibodies and their dilutions were as following: Thr202/Tyr204 phospho-ERK1/2 (1:1000); Nicotinic acetylcholine receptor α7 (1:500); cyclinD1 (1:2000); Thr286 phospho-cyclinD1 (1:1000); EGR-1 (1:1500); β-actin (1:10000); ERK1/2 (1:2000). The intensities of immunoreactive bands were quantified by using an NIH Image tool. Cell apoptosis assay Quantification of apoptosis by Annexin-V/PI staining was performed as described previously. Briefly, the cells were first collected after 24 hours drug treatment. Using a Beckman Coulter Elite flow cytometer (Beckman Coulter Electronics, FL, USA), 10,000 events per sample were collected into list mode files, and were analyzed by the WinMDI software. Immunohistochemistry and immunofluorescent staining Immunohistochemistry and immunofluorescent staining was performed using an affinity-purified anti-EGR-1 and anti-pERK1/2 antibodies. The empirical method was identical as previously described [20]. After dewaxing, rehydration and washing, antigen retrieval was operated in EDTA buffer solution, and the endogenous peroxidase activity was inhibited by incubation with 3% H 2 O 2 for 30 min. Then the sections were incubated with 5% normal fetal bovine serum at room temperature for 30 min. Then primary antibodies (EGR-1, 1:200; pERK1/2, 1:100) were loaded to brain slices (4°C, 12 h). After washing with PBS, peroxidase activity was developed using DAB as the chromogen (Maixin, Fuzhou, China). The experimental results were photographed under a confocal microscope (×400). Nuclei were counterstained with DAPI, and the stained cells were analyzed and photographed under a confocal microscope(×400). Data and statistics analysis All data were obtained from at least three different preparations. Statistical analysis of the data was performed with a two-tailed Student’s t-test using Graphpad Prism 5. Data are presented as the mean ± SEM. *p<0.05 was taken to represent statistical significance.

Discussion Cigarette smoking has been suggested as a risk factor for chronic airway obstruction and lung cancer [29, 30]. Nicotine is the major addictive agent in tobacco. Interestingly, the regular doses of nicotine may be beneficial for human health, especially for the treatment of AD. A variety of studies have shown that acute treatment with nicotine or nicotinic agonists can improve working memory function in rats [7, 8, 23]. Additionally, chronic nicotine treatment prevented Aβ-induced memory impairment [8]. Nicotine was also reported to enhance learning and memory by activating hippocampal Jun-N terminal kinase pathway [31]. Aβ is the major constituent of the senile plaques in AD. It is produced by proteolytic processing of amyloid-β protein precursor (APP), a transmembrance glycoprotein expressed during normal cellular metabolism [32]. Neuronal apoptosis has been implicated in the pathogenesis of various neurodegenerative disorders including AD [33]. Several studies have indicated that Aβ induces apoptosis and neuronal cell death by increasing the expression of iNOS, which leads to the peroxidation of membrane lipids and oxidative stress [34]. According to the previous literature, Aβ-associated free radical damage to neurons is a fundamental process in connection with AD [34]. In this study, treatment of SH-SY5Y cells with Aβ 25–35 clearly decreased cellular MTT reduction, thereby providing direct evidence for cellular damage induced by Aβ. Nicotine can protect SH-SY5Y cells from Aβ 25–35 -induced cytotoxicity (Fig. 4C). Aβ 25–35 can increase SH-SY5Y apoptosis compared to control, and nicotine can partly attenuate this Aβ 25–35 -induced cytotoxicity (Fig. 4A and 4B). The ERK pathway plays an important role in the processes related to synaptic plasticity, including learning and memory. In vivo, immunohistochemistry indicated that Aβ 25–35 can up-regulate ERK phosphorylation and activated Egr-1, while nicotine blocked this effect which was induced by Aβ 25–35 (Fig. 6). Therefore, it is conceivable that the induction of EGR-1 may direct the transcription of downstream genes that are degenerative processes associated with AD. However, additional studies are necessary to confirm the underlying mechanism. In this work, it was suggested that nicotine activated EGR-1 expression in the human neuroblastoma SH-SY5Y cell line via the stimulation of the MAPK/ERK signaling pathway and the α7 subtype of nAchR. SH-SY5Y cells whose ancestral cell is SK-N-SH were directly subcloned from SH-SY5[35]. The SH-SY5Y cell line expresses α3, α5, α7, β2, and β4 nAChR subunits that assemble to form various α3-nAChR subtypes or homomeric α7 nAChR [36, 37]. The intermediate steps coupling α7 nAChR activation and Ca2+ entry to the activation of MEK are likely to depend on stimulus-specific factor [24, 38]. When α7 nAchR was blocked (Fig. 3A, 3B), nicotine-induced ERK1/2 phosphorylation and EGR-1 expression were reduced compared to control (Fig. 3C). The results presented here provide evidence for a functional role for α7 nAChR on SH-SY5Y cells, which is capable of activating the ERK1/2 signaling cascade. To date, considerable interest has been focused on nAChRs in connection with the treatment of AD due to the role it plays in neurotransmitter release, cell survival, cognitive enhancement, synaptic plasticity and neuroprotection [26, 38]. These results suggest that the effects of nicotine exposure may be due to its effects on gene expression initiated by interactions with nAChR. The regulation by the MEK-ERK1/2 signaling pathway after stimulation of the α7 nAChR constitutes a novel signaling cascade in SH-SY5Y cells. The egr-1 gene has a serum responsive element (SRE) located within its promoter regions [39]. EGR-1 can be activated by the ERK pathway [22]. We found that the presence of U0126, an ERK pathway inhibitor, effectively weakened the signals of EGR-1, which was triggered by nicotine (Figs. 1, 2C). CyclinD1, as the targeted gene of Wnt/β-catenin signaling pathway, plays an important function in AD [40]. The early growth response protein may bind to a cis-regulatory region spanning nucleotides −144 to −104 of the CyclinD1 promoter [41]. The relation between CyclinD1 and EGR-1 in cells treated with 10–6 M nicotine for 45 min was revealed (Fig. 5A). When the EGR-1 was blocked, CyclinD1 and its phosphorylation were up-regulated (Fig. 5B, 5C). Therefore, the hypotheses regarding EGR-1 over-expression in this cell line was confirmed (Fig. 5D). In conclusion, we demonstrated that nicotine activated the EGR-1 via ERK1/2 and the α7 nicotinic acetylcholine receptor in SH-SY5Y cells. Nicotine can protect SH-SY5Y from Aβ-induced cytotoxicity via up-regulating CyclinD1. The results suggested that nicotine could attenuate the effect of Aβ, and it may be beneficial for the prevention of AD. We elucidated the partial mechanisms that underlie the activation of EGR-1 by nicotine, and provide theoretical knowledge for retarding the proceeding of AD.

Acknowledgments We thank Dr. Huaxi Xu from Xiamen University for the careful reading and suggestions to organize the manuscript.

Author Contributions Conceived and designed the experiments: QZ MX JZ GJ. Performed the experiments: MX LZ JQ GD ZQ. Analyzed the data: MX LZ GJ FG. Contributed reagents/materials/analysis tools: JQ GD ZQ GJ FG. Wrote the paper: MX LZ GJ FG QZ.