Effects of 1950 MHz radiofrequency electromagnetic fields on Aβ processing in human neuroblastoma and mouse hippocampal neuronal cells

Effects of 1950 MHz radiofrequency electromagnetic fields on Aβ processing in human... Journal of Radiation Research, Vol. 59, No. 1, 2018, pp. 18–26 doi: 10.1093/jrr/rrx045 Advance Access Publication: 6 October 2017 Effects of 1950 MHz radiofrequency electromagnetic fields on Aβ processing in human neuroblastoma and mouse hippocampal neuronal cells 1 2 3 1, Jeongyeon Park , Jong Hwa Kwon , Nam Kim and Kiwon Song Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 03722, Republic of Korea Department of EMF Research Team, Radio and Broadcasting Technology Laboratory, Electronics and Telecommunications Research Institute (ETRI), Daejon, 305-700, Republic of Korea School of Electrical and Computer Engineering, Chungbuk National University, Cheongju, Chungbuk, 362-763, Republic of Korea *Corresponding author. Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 03722, Republic of Korea. Tel: +82-2-2123-2705; Fax: +82-2-362-9897; Email: bc5012@yonsei.ac.kr Received March 24, 2017; Revised May 31, 2017; Editorial Decision July 31, 2017 ABSTRACT Alzheimer’s disease (AD) is a neurodegenerative disease leading to progressive loss of memory and other cogni- tive functions. One of the well-known pathological markers of AD is the accumulation of amyloid-beta protein (Aβ), and its plaques, in the brain. Recent studies using Tg-5XFAD mice as a model of AD have reported that exposure to radiofrequency electromagnetic fields (RF-EMF) from cellular phones reduced Aβ plaques in the brain and showed beneficial effects on AD. In this study, we examined whether exposure to 1950 MHz RF-EMF affects Aβ processing in neural cells. We exposed HT22 mouse hippocampal neuronal cells and SH-SY5Y human neuroblastoma cells to RF-EMF (SAR 6 W/kg) for 2 h per day for 3 days, and analyzed the mRNA and protein expression of the key genes related to Aβ processing. When exposed to RF-EMF, mRNA levels of APP, BACE1, ADAM10 and PSEN1 were decreased in HT22, but the mRNA level of APP was not changed in SH-SY5Y cells. The protein expression of APP and BACE1, as well as the secreted Aβ peptide, was not significantly different between RF-EMF–exposed 7w-PSML, HT22 and SH-SY5Y cells and the unexposed controls. These observa- tions suggest that RF-EMF exposure may not have a significant physiological effect on Aβ processing of neural cells in the short term. However, considering that we only exposed HT22 and SH-SY5Y cells to RF-EMF for 2 h per day for 3 days, we cannot exclude the possibility that 1950 MHz RF-EMF induces physiological change in Aβ processing with long-term and continuous exposure. KEYWORDS: 1950 MHz radiofrequency electromagnetic fields (RF-EMF), Alzheimer’s disease, Aβ processing, mouse hippocampal neuronal cell line, human neuroblastoma cell line, CHO cell–based 7w-PSML cell line INTRODUCTION processing and Aβ formation were simplified and presented in Alzheimer’s disease (AD) is one of the most common neurodegen- Fig. 1. APP is newly synthesized in the endoplasmic reticulum erative disorders, and it leads to progressive loss of memory and (ER), and it traffics through the secretory pathway to the Golgi appar- other cognitive functions (review by Kumar et al. [1]). One of the atus and to the plasma membrane. In the non-amyloidogenic pathway, well-known pathological markers of AD is the accumulation of APP is first cleaved by α-secretase [which is a member of the disinte- amyloid-beta protein (Aβ), and its plaques, in the brain. Aβ is pro- grin and metalloproteinase (ADAM) family, notably ADAM10] and cessed from amyloid precursor protein (APP), a type I then by the γ-secretase complex, to produce an innocuous membrane- transmembrane protein. The pathways and enzymes for APP embedded peptide of 26 amino acids and secreted sAPPα (review by © The Author 2017. Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Radiation Oncology. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re- use, please contact journals.permissions@oup.com � 18 Downloaded from https://academic.oup.com/jrr/article-abstract/59/1/18/4356568 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Effects of 1950 MHz EMF on Aβ processing in neural cells � 19 Fig. 1. A simple diagram of APP processing and Aβ secretion in brain cells. The processing of amyloid precursor protein (APP) occurs in two distinct pathways: amyloidogenic and non-amyloidogenic. In the non-amyloidogenic pathway, APP is cleaved by α-secretase (ADAM10) at the site within the Aβ domain, releasing a soluble APPα (sAPPα) fragment and a C-terminal fragment (C83). The C83 fragment is further cleaved by γ-secretase (PSEN1) to release the APP intracellular domain (AICD) and a 3 kDa (p3) fragment. The amyloidogenic pathway involves the sequential cleavage of APP by β-secretase (BACE1) and γ-secretase (PSEN1). β-secretase generates a soluble APPβ (sAPPβ) fragment and a C-terminal fragment (C99), which is cleaved by γ-secretase (PSEN1) to release AICD and amyloid-β (Aβ) peptide. Secreted Aβ forms oligomers and aggregates, generating the toxic amyloid plaques observed in the AD patient’s brain (drawn by J. Park, and review by Hicks et al. [33]). Jiang et al. [2]). sAPPα has been reported to activate the proliferation Aβ into a toxic aggregated state [12, 14], the Aβ generation through of adult neuroblasts (review by Chasseigneaux et al. [3]), but its physio- endocytosis of APP [11], and the role of neuronal membrane cho- logical role is unknown [4]. The main subunit of the γ-secretase com- lesterol in excessive Aβ peptide production [10]. plex is presenilin 1 encoded by PSEN1, which is mutated in patients Previously, 900 and 1800 MHz RF-EMF radiations, allocated to with AD [5]. APP canalsobeprocessedby β-secretase (encoded by cellular phones, were reported to induce negative effects on brain beta-site amyloid precursor protein cleaving enzyme 1 [BACE1]) and function, such as brain tumors (reviews by Lagorio et al. [15] and γ-secretase, to release the neurotoxic Aβ peptide, which spontaneously Hardell et al. [16]) and memory impairments [17]. However, sev- forms oligomers, and then the larger amyloid plaques found in the eral recent studies using transgenic (Tg)-FAD mice as a model of brain of patients with AD (reviews by Jiang et al. [2], Thinakaran et al. AD have shown that RF-EMFs have beneficial effects on neurode- [6]and Kumar et al. [1]). Diverse genetic and molecular evidence sug- generative disorders: 918 MHz EMF enhanced memory and gests that the abnormal accumulation of Aβ occurs in the early stage of decreased brain Aβ aggregation in AD 3X Tg-mice [18]. RF-EMF the pathophysiological cascade that eventually leads to AD (reviews by with a frequency range from 800 to 2450 MHz improved cognitive Huang et al. [7], Palop et al. [8]and Bertram et al. [9]). However, impairment in AD 2X or 3X Tg-mice [19, 20], and enhanced brain what determines the processing of APP to Aβ,and howAβ impairs mitochondrial function, through the disaggregation of Aβ oligomers neuronal function have not been clearly understood. in Tg-5XFAD and normal mice [21]. In addition, 1950 MHz RF- In neural cells, the endogenous expression of APP is usually very EMF directly affected Aβ pathology by reducing Aβ plaques and low, and the amount of secreted Aβ is insufficient to be detected, BACE1 expression in Tg-5XFAD mice [22]. These studies demon- which has been a technical barrier to studying APP processing and strated the effects of RF-EMF radiation on AD pathology, mainly its mechanism [10]. 7w-PSML is a stably transfected CHO-based with FAD mouse models, but could not elucidate the cellular mech- cell line that expresses both the wild-type human APP and mutant anism involved in these RF-EMF–derived physiological effects. presenilin-1 (M146L), which is an efficient model for the detection In this study, we investigated the effects of RF-EMF on Aβ pro- of APP, and its cleaved and secreted form, Aβ peptide [11–13]. cessing in HT22 mouse hippocampal neuronal cells, SH-SY5Y human Thus, 7w-PSML has been used as a cellular model system for AD neuroblastoma cells and 7w-PSML cells, to examine the mechanism for studying Aβ processing in vitro: the conversion of monomeric for the physiological outcomes of RF-EMF at a cellular level. Downloaded from https://academic.oup.com/jrr/article-abstract/59/1/18/4356568 by Ed 'DeepDyve' Gillespie user on 16 March 2018 20 � J. Park et al. 0.105 ± 0.019 W/kg for the CDMA frequency and 0.262 ± MATERIALS AND METHODS 0.055 W/kg for the WCDMA frequency. Cell culture The exposure system was warmed up for at least 30 min, for HT22 (immortalized mouse hippocampal neuronal) cells and SH- equilibration, before the RF-EMF exposure. The 100-mm culture SY5Y (human neuroblastoma) cells were kindly provided by Dr dishes were placed at 13.6 cm from the conical antenna, which was Inhee Mook-Jung (Seoul National University College of Medicine, located at the center of the exposure chamber, and cells were then Seoul, Korea). These two cell lines have been used as well- exposed to RF-EMF radiation in the culture dishes. This system has established in vitro cellular models for neurodegenerative disorders already been used in other published cellular studies [25, 26]. The such as AD and Parkinson’s disease; HT22 cells have functional variation in RF-EMF exposures on different culture dishes was neg- cholinergic properties related to the cognitive defects of AD [23], ligible, when multiple dishes were exposed at the same time: the and SH-SY5Y cells have synaptic structures, functional axonal ves- variation in the average SAR values for WCDMA frequency was icle transport, and express neurospecific proteins (review by ~7.7% of the single dish exposure when three culture dishes were Carolindah et al. [24]). CHO cell–based 7w-PSML cells [which used, and ~2.0% when six dishes were exposed [25, 26]. overexpress wild-type human APP and the mutant presenilin-1 Cells were exposed to RF-EMF radiation of a single signal (M146L) [13]] were provided by Dr David Kang (Florida State (WCDMA signal at 1950 MHz) at 6 W/kg for 2 h/day for 3 days, University, USA). These cells were maintained in Dulbecco’s modi- with the same fixed timetable for the same intervals. During the fied Eagle’s medium (DMEM) with 10% (v/v) fetal bovine serum exposure period, the temperature was maintained within a range of (FBS; Sigma-Aldrich, MO, USA) and 10 ml/l penicillin–streptomy- 37 ± 0.3°C by circulating water within the cavity, and a 5% CO cin (GIBCO, NY, USA). All cells were grown at 37°C in a humidi- 2 concentration was also maintained in the chamber. After exposure fied atmosphere containing 5% CO . to RF-EMF radiation, the cells were immediately transferred to a cell culture incubator. RF-EMF-treated and untreated cells and each The RF-EMF radiation system for cell exposure culture medium were collected immediately after the last exposure to RF-EMF radiation, for further experiments. We used a radial transmission line (RTL) exposure system as an in vitro multifrequency radiation exposure system for this study [25, 26]. A typical Code–Division Multiple Access (CDMA) signal at 837 MHz and a Wideband Code–Division Multiple Access RNA isolation and quantitative real-time PCR (WCDMA) signal at 1950 MHz were applied to the RTL after amp- Cells were harvested and their total RNA was isolated using the lification. The signals were modulated by real CDMA and WCDMA RNeasy Mini kit (QIAGEN, Germany). cDNA was synthesized signals for cell exposure. The specific absorption rate (SAR) distri- with purified total RNA using the PrimeScript RT reagent kit bution inside the exposed medium was defined by numerical (Takara Bio Inc., Shiga, Japan). Quantitative real-time PCR was per- simulations using the finite-difference time-domain (FDTD) meth- formed using the CFX96 instrument (Bio-Rad, CA, USA), with the od (XFDTD 6.5, Remcom, State College, PA). The simulated and primers in Table 1. The relative expression of genes was normalized measured SAR values for 1 W input power in the entire sample was to that of the control gene encoding β–actin. The normalized fold Table 1. The primers used for quantitative real-time PCR Gene Oligonucleotide primer Product size (bp) Human/Mouse 5′-CATCTTCACTGGCACACCGT-3′ (forward) 115 APP 5′-CAAACTCTACCCCTCGGAAC-3′ (reverse) Human/Mouse 5′-AATTCTGCTCCTCTCCTGGGC-3′ (forward) 211 ADAM10 5′-CCTCTTCATTCGTAGGTTGA-3′ (reverse) Human/Mouse 5′-TGTGGAGATGGTGGACAACCTG-3′ (forward) 168 BACE1 5′-TGCCTCTGGTAGTAGCGATG-3′ (reverse) Human/Mouse 5′-GAGCTGACATTGAAATATGG-3′ (forward) 218 PSEN1 5′-ACAATGACACTGATCATGATGGC-3′ (reverse) Human β-actin 5′-TCCCTGGAGAAGAGCTACGA-3′ (forward) 194 5′-AGCACTGTGTTGGCGTACAG-3′ (reverse) Mouse β-actin 5′-CGCCACCAGTTCGCCATGGA-3′ (forward) 105 5′-TACAGCCCGGGGAGCATCGT-3′ (reverse) Downloaded from https://academic.oup.com/jrr/article-abstract/59/1/18/4356568 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Effects of 1950 MHz EMF on Aβ processing in neural cells � 21 expression of mRNA levels in RF-EMF–exposed and –unexposed indicated statistical significance compared with control. Non- −ΔΔC cells was analyzed using the 2 method as described previously statistically significant results (n.s) represented P > 0.05. [27]. Real-time PCR products were confirmed by electrophoresis on a 2.5% agarose gel. RESULTS The effect of 1950 MHz EMF exposure on the expression of genes associated with Aβ processing Protein analysis in mouse and human neural cells After the cells were exposed to the RF-EMF, the cell culture In order to determine the molecular mechanism of the effect of RF- medium was collected to precipitate secreted peptides by TCA pre- EMF on Aβ processing at the cellular level, we first investigated cipitation. Precipitated samples were separated by the 4–20% gradi- whether exposure to RF-EMF could affect the expression of the ent precast Tris-Glycine SDS gel (KomaBiotech, Seoul, Korea), to genes critical for Aβ processing in neural cells; amyloid precursor detect secreted Aβ peptides. RF-EMF–exposed and unexposed cells protein (APP), α-secretase (ADAM10), β-secretase (BACE1) and γ- were harvested and lysed as described previously by Park et al. [28]. secretase (PSEN1). HT22 (mouse hippocampal neuronal) cells and Cell extracts were analyzed by western blot by 7.5% or 10% SDS- SH-SY5Y (human neuroblastoma) cells were exposed to RF-EMF PAGE with anti-Alzheimer Precursor Protein A4 (clone 22C11; radiation (single 1950 MHz W-CDMA signal at 6 W/kg) for 2 h Millipore, MA, USA), anti-β-amyloid 1–16 (clone 6E10; BioLegend, per day for 3 days. The mRNA level of each gene was measured by CA, USA), anti-BACE1 (Millipore, MA, USA), and anti-β-actin quantitative real-time PCR in the RF-EMF–exposed cells and was (Cell Signaling Technology, Inc., MA, USA) antibodies. To detect compared with that of the unexposed control cells. The mRNA the differentially processed forms of APP in cells, we used two dif- expression of APP was reduced 0.64-fold in RF-EMF–exposed ferent APP antibodies, anti-22C11 and anti-6E10, which detect differ- HT22 cells, when compared with that of unexposed HT22 cells, ent regions of the human APP protein (Fig. 2). Anti-22C11 binds to and the mRNA levels of ADAM10, BACE1 and PSEN1 slightly membrane APP (amino acids 66–81 in the N-terminal domain of decreased in RF-EMF–exposed HT22 cells (Fig. 3A and B). In SH- APP) and can precipitate the full-length APP as well as its N-terminal SY5Y cells exposed to RF-EMF, there was no decrease in the fragments, soluble APPα (sAPPα) and APPβ (sAPPβ)(Fig. 2). Anti- mRNA expression of APP, but the mRNA expression level of 6E10 reacts notonlywithAPP andthe processedsAPPα, but also ADAM10 and BACE1 was slightly decreased and that of PSEN1 was with Aβ by binding between amino acids 597–613 (Fig. 2)[29, 30]. marginally increased compared with those of unexposed cells An enhanced chemiluminescence reagent (Amersham Biosciences, (Fig. 3C and D). These results showed that RF-EMF exposure Buckinghamshire, UK) was used for the blot analysis. The relative decreased the expression of APP mRNA and slightly diminished the band intensity of the samples from RF-EMF–exposed and unexposed expression of genes involved in the processing of APP, ADAM10, cells to the loading control (β-actin) was measured using GelQuantNet BACE1 and PSEN1 in mouse HT22 cells. In human SH-SY5Y cells, (BiochemLabSolutions, CA, USA), and the average result from three RF-EMF exposure induced marginal changes in the expression of independent experiments was plotted. genes involved in Aβ processing, but not on the expression of APP. Statistical analysis 1950 MHz EMF exposure does not affect the processing Statistical analysis was performed using GraphPad Prism 6 (GraphPad Software, Inc., CA, USA). Data are represented with the and secretion of Aβ in 7w-PSML cells mean ± standard error of the mean (S.E.M.) of at least three inde- Several studies have discovered that exposure to RF-EMF induces pendent experiments. We applied t-tests to assess statistically signifi- the reduction of APP, BACE1 and Aβ peptide in the AD transgenic cant differences. P < 0.05 (*), P < 0.01 (**), and P < 0.0001 (****) mouse model [20, 22]. In order to understand whether RF-EMF Fig. 2. A representation of regions of human APP differentially processed and detected with two different antibodies. A schematic structure of human APP protein (695 amino acid form) with the regions that respectively react to two different antibodies: anti-22C11 and anti-6E10 (adapted from [29, 30]). Downloaded from https://academic.oup.com/jrr/article-abstract/59/1/18/4356568 by Ed 'DeepDyve' Gillespie user on 16 March 2018 22 � J. Park et al. Fig. 3. The effect of 1950 MHz EMF exposure on the expression of APP and genes associated with Aβ processing in mouse and human neural cells. (A–D) HT22 (A, B) and SH-SY5Y (C, D) cells were exposed to radio frequency–electromagnetic field (RF-EMF) radiation (1950 MHz at 6 W/kg) for 2 h per day for 3 days. (A, C) The mRNA expression of APP, ADAM10, BACE1 and PSEN1 was analyzed in RF-EMF–treated and –untreated cells by quantitative real-time PCR and normalized to β-actin mRNA expression. Data are represented as mean ± S.E.M. from three independent experiments. P < 0.05 (*); P < 0.01 (**); P < 0.0001(****); P > 0.05 (non-significant, n.s). (B, D) Quantitative real-time PCR products in HT22 (B) and SH-SY5Y (D) were confirmed by electrophoresis on a 2.5% agarose gel. U = unexposed cells, E = RF-EMF–exposed cells, NTC = No Template Control. exposure could affect the processing of APP into Aβ, in neural cells, 7w-PSML cells were exposed to 1950 MHz EMF for 2 h every we first examined the expression of BACE1 and several processed day for 3 days, and both the cells and culture medium were col- forms of APP in 1950 MHz EMF–exposed 7w-PSML cells. lected. Secreted peptides in the culture medium were precipitated by Downloaded from https://academic.oup.com/jrr/article-abstract/59/1/18/4356568 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Effects of 1950 MHz EMF on Aβ processing in neural cells � 23 TCA precipitation, and analyzed by the 4–20% gradient precast Tris- sAPP (α or β)or Aβ peptide in 7w-PSML cells (Fig. 4A and C). Glycine SDS-PAGE. Expression of APP and BACE1 was also These results showed that 1950 MHz EMF exposure does not sig- detected by western blot analysis with the lysates of RF-EMF– nificantly change the pattern of Aβ processing in 7w-PSML cells. exposed cells. No significant difference in the expression of APP and BACE1 was detected in RF-EMF–exposed cells, when compared 1950 MHz EMF exposure has no direct effect on Aβ with the unexposed controls (Fig. 4A). To confirm this result, we normalized the relative band intensity of APP and BACE1 to β-actin processing and secretion in HT22 and SH-SY5Y cells as a loading control, but could not detect a meaningful difference We then investigated whether exposure to 1950 MHz EMF alters between RF-EMF–exposed cells and unexposed controls (Fig. 4B). the endogenous protein expression of APP and BACE1 as well as of In addition, RF-EMF exposure did not affect the secreted level of processed Aβ in HT22 and SH-SY5Y cells. HT22 and SH-SY5Y cells Fig. 4. 1950 MHz EMF exposure does not directly affect the processing and secretion of Aβ in 7w-PSML cells. (A) 7w-PSML cells were exposed to RF-EMF radiation (1950 MHz at 6 W/kg) for 2 h per day for 3 days. Secreted peptides in the culture medium were precipitated by TCA. APP and BACE1 in cell lysates, and sAPP (α or β) in precipitated samples, were detected by western blot analysis. β-actin was used as a loading control. (B) The relative band intensity of APP and BACE1 in cell lysates to the loading control was calculated by GelQuantNet software from the results of three independent experiments, including (A). Data are represented as mean ± S.E.M. from three independent experiments. P > 0.05 (non-significant, n.s). (C) Total 30 and 50 μg proteins of the TCA precipitated medium were separated by a 4–20% gradient precast Tris-Glycine SDS gel, and Aβ peptides were detected with anti-6E10 by western blot analysis. Downloaded from https://academic.oup.com/jrr/article-abstract/59/1/18/4356568 by Ed 'DeepDyve' Gillespie user on 16 March 2018 24 � J. Park et al. were exposed to 1950 MHz EMF radiation for 2 h every day for 3 difference in the amount of secreted Aβ peptide was observed days, and the expression of APP and BACE1 were analyzed by west- between RF-EMF–exposed and unexposed HT22 cells (Fig. 5C). In ern blot. The endogenous expression of APP and BACE1 did not SH-SY5Y cells, we also could not detect any meaningful variation in change in RF-EMF–exposed HT22 cells, compared with in the unex- the expression of APP and BACE1 as well as of the secreted Aβ in posed controls (Fig. 5A). The normalized band intensity of APP and RF-EMF–exposed cells and unexposed controls (Fig. 5D–F). These BACE1 relative to a loading control β-actin in cell lysates of HT22 observations demonstrated that short-term exposure to 1950 MHz showed a marginal decrease of APP in RF-EMF–exposed cells, EMF does not change the expression of proteins for APP processing although it was not statistically meaningful (Fig. 5B). No significant and Aβ secretion in either HT22 or SH-SY5Y cells. Fig. 5. 1950 MHz EMF exposure has no significant effect on the APP protein expression and Aβ secretion in either HT22 or SH-SY5Y cells. (A–F) HT22 (A, B, C) and SH-SY5Y (D, E, F) cells were exposed to 1950 MHz at 6 W/kg for 2 h per day for 3 days. (A, D) Expression of APP and BACE1 in cell extracts of (A) HT22 and (D) SH-SY5Y was analyzed by western blot. β-actin was used as a loading control. (B, E) The relative band intensity of APP and BACE1 in cell lysates of (B) HT22 and (E) SH-SY5Y to the loading control was calculated by GelQuantNet software from the results of three independent experiments, including (A) and (D). Data are represented as mean ± S.E.M. from three independent experiments. P > 0.05 (non-significant, n.s). (C, F) Cell culture medium was precipitated with TCA, and secreted peptides were analyzed by a 4–20% gradient precast Tris-Glycine SDS gel. Secreted Aβ peptide was detected by western blot analysis with anti-6E10 in (C) HT22 and (F) SH-SY5Y cell media. Downloaded from https://academic.oup.com/jrr/article-abstract/59/1/18/4356568 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Effects of 1950 MHz EMF on Aβ processing in neural cells � 25 well as Aβ depositions in the brain? [18, 22]. We believe that a DISCUSSION major reason for the discrepancy in the effect of RF is the exposure Previously, researchers have focused on elucidating the negative effect period. We exposed HT22 and SH-SY5Y cells to RF-EMF for a of RF-EMF radiation emitted by cellular phones on the brain, such as short period (2 h per day for 3 days) only, because 3 days was the brain tumor development [31, 32]. However, several recent studies have reported the beneficial effect of RF-EMF radiation on AD using limitation of the cell culture span. However, the AD mouse models were exposed to RF-EMF for 8 months [22]. Thus, we could con- AD transgenic mouse models [19–21]. In order to evaluate the cellu- clude that a short-term exposure of RF-EMF to neural cells does lar mechanism of the effect of RF-EMF on AD, in this study, we not induce significant changes in the expression or processing of examined the effect of 1950 MHz EMF radiation (single WCDMA APP at the cellular level. However, minute changes caused by RF- signal at SAR 6 W/kg, 2 h per day for 3 days) on the endogenous EMF radiation, accumulated for a long period of exposure in the expression of APP and major proteins for APP processing, as well as AD mouse model, may result in obvious physiological outcomes, the secretion of Aβ in HT22 and SH-SY5Y neural cells. In addition, such as reduced Aβ deposits. Further studies would be necessary to we also investigated the effect of RF-EMF radiation in the 7w-PSML understand whether long-term and/or continuous exposure or cell as it is an efficient model for the detection of APP and Aβ pep- extended exposure time to RF-EMF radiation affects Aβ processing tide [11–13]. We showed in this study that 1950 MHz EMF radiation at the cellular level. for 2 h every day for 3 days did not induce significant changes in the level of APP and processing and secretion of Aβ in mouse and human neural cells as well as in 7w-PSML cells. ACKNOWLEDGEMENTS We exposed these cells with 1950 MHz EMF radiation for 2 h a The authors thank Dr David Kang (at Florida State University) for day, because the Tg-5xFAD mice, which showed ameliorated Aβ providing 7w-PSML cells, and Dr Inhee Mook-Jung (Seoul pathology by RF-EMF, were exposed to 1950 MHz EMF for 2 h National University College of Medicine, Seoul, Korea) for provid- every day [22]. We wanted to examine the cellular effect of RF- ing HT22 cells and SH-SY5Y cells. K.S. conceived the idea, EMF under the same conditions as those used for Tg mice. Also, designed the experiments, and wrote the manuscript. J.P. performed the RF-EMF system we used was originally designed for a maximum the experiments and wrote the manuscript. J.H.K. and N.K. pro- of 2 h exposure per day, based on the assumption that most people vided the 1950 MHz EMF radiation device for exposure of the cells. on average use mobile phones for a total of 2 h a day. With our observations, we could suggest that the RF exposure conditions CONFLICT OF INTEREST applied in this study may not have a significant effect on the expres- The authors have declared that there is no conflict of interest. sion and processing of APP or Aβ in mouse and human neural cells. However, in the RF-EMF–exposed HT22 cells, we repeatedly FUNDING observed that the mRNA expression of APP and genes for APP pro- This work was supported by the National Research Foundation of cessing seems to be decreased with statistically meaningful signifi- Korea (NRF) funded by the Ministry of Science, ICT & Future cance (Fig. 3A), and the expression of APP was marginally reduced, Planning [No. NRF-2016M3A9C6918275] and by Korea Mobile although the secreted Aβ was not much changed (Fig. 5A–C). Thus, EMF consortium. J. Park was partially supported by the Graduate with these results, it would be difficult to define whether 1950 MHz School of YONSEI University Research Scholarship Grants of 2017. EMF radiation has a meaningful effect on decreasing the expression of APP and the secretion of Aβ in mouse neural cells, or not. However, we cannot exclude the possibility that 1950 MHz RF-EMF REFERENCES exposures to mouse neural cells induce the minute decline observed 1. 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Lipid rafts and modifies cerebral blood flow, and provides selected cognitive Alzheimer’s disease: protein–lipid interactions and perturbation benefit. PLoS One 2012;7:e35751. of signaling. Front Physiol 2012;3:189. Downloaded from https://academic.oup.com/jrr/article-abstract/59/1/18/4356568 by Ed 'DeepDyve' Gillespie user on 16 March 2018 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Radiation Research Oxford University Press

Effects of 1950 MHz radiofrequency electromagnetic fields on Aβ processing in human neuroblastoma and mouse hippocampal neuronal cells

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Journal of Radiation Research, Vol. 59, No. 1, 2018, pp. 18–26 doi: 10.1093/jrr/rrx045 Advance Access Publication: 6 October 2017 Effects of 1950 MHz radiofrequency electromagnetic fields on Aβ processing in human neuroblastoma and mouse hippocampal neuronal cells 1 2 3 1, Jeongyeon Park , Jong Hwa Kwon , Nam Kim and Kiwon Song Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 03722, Republic of Korea Department of EMF Research Team, Radio and Broadcasting Technology Laboratory, Electronics and Telecommunications Research Institute (ETRI), Daejon, 305-700, Republic of Korea School of Electrical and Computer Engineering, Chungbuk National University, Cheongju, Chungbuk, 362-763, Republic of Korea *Corresponding author. Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 03722, Republic of Korea. Tel: +82-2-2123-2705; Fax: +82-2-362-9897; Email: bc5012@yonsei.ac.kr Received March 24, 2017; Revised May 31, 2017; Editorial Decision July 31, 2017 ABSTRACT Alzheimer’s disease (AD) is a neurodegenerative disease leading to progressive loss of memory and other cogni- tive functions. One of the well-known pathological markers of AD is the accumulation of amyloid-beta protein (Aβ), and its plaques, in the brain. Recent studies using Tg-5XFAD mice as a model of AD have reported that exposure to radiofrequency electromagnetic fields (RF-EMF) from cellular phones reduced Aβ plaques in the brain and showed beneficial effects on AD. In this study, we examined whether exposure to 1950 MHz RF-EMF affects Aβ processing in neural cells. We exposed HT22 mouse hippocampal neuronal cells and SH-SY5Y human neuroblastoma cells to RF-EMF (SAR 6 W/kg) for 2 h per day for 3 days, and analyzed the mRNA and protein expression of the key genes related to Aβ processing. When exposed to RF-EMF, mRNA levels of APP, BACE1, ADAM10 and PSEN1 were decreased in HT22, but the mRNA level of APP was not changed in SH-SY5Y cells. The protein expression of APP and BACE1, as well as the secreted Aβ peptide, was not significantly different between RF-EMF–exposed 7w-PSML, HT22 and SH-SY5Y cells and the unexposed controls. These observa- tions suggest that RF-EMF exposure may not have a significant physiological effect on Aβ processing of neural cells in the short term. However, considering that we only exposed HT22 and SH-SY5Y cells to RF-EMF for 2 h per day for 3 days, we cannot exclude the possibility that 1950 MHz RF-EMF induces physiological change in Aβ processing with long-term and continuous exposure. KEYWORDS: 1950 MHz radiofrequency electromagnetic fields (RF-EMF), Alzheimer’s disease, Aβ processing, mouse hippocampal neuronal cell line, human neuroblastoma cell line, CHO cell–based 7w-PSML cell line INTRODUCTION processing and Aβ formation were simplified and presented in Alzheimer’s disease (AD) is one of the most common neurodegen- Fig. 1. APP is newly synthesized in the endoplasmic reticulum erative disorders, and it leads to progressive loss of memory and (ER), and it traffics through the secretory pathway to the Golgi appar- other cognitive functions (review by Kumar et al. [1]). One of the atus and to the plasma membrane. In the non-amyloidogenic pathway, well-known pathological markers of AD is the accumulation of APP is first cleaved by α-secretase [which is a member of the disinte- amyloid-beta protein (Aβ), and its plaques, in the brain. Aβ is pro- grin and metalloproteinase (ADAM) family, notably ADAM10] and cessed from amyloid precursor protein (APP), a type I then by the γ-secretase complex, to produce an innocuous membrane- transmembrane protein. The pathways and enzymes for APP embedded peptide of 26 amino acids and secreted sAPPα (review by © The Author 2017. Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Radiation Oncology. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re- use, please contact journals.permissions@oup.com � 18 Downloaded from https://academic.oup.com/jrr/article-abstract/59/1/18/4356568 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Effects of 1950 MHz EMF on Aβ processing in neural cells � 19 Fig. 1. A simple diagram of APP processing and Aβ secretion in brain cells. The processing of amyloid precursor protein (APP) occurs in two distinct pathways: amyloidogenic and non-amyloidogenic. In the non-amyloidogenic pathway, APP is cleaved by α-secretase (ADAM10) at the site within the Aβ domain, releasing a soluble APPα (sAPPα) fragment and a C-terminal fragment (C83). The C83 fragment is further cleaved by γ-secretase (PSEN1) to release the APP intracellular domain (AICD) and a 3 kDa (p3) fragment. The amyloidogenic pathway involves the sequential cleavage of APP by β-secretase (BACE1) and γ-secretase (PSEN1). β-secretase generates a soluble APPβ (sAPPβ) fragment and a C-terminal fragment (C99), which is cleaved by γ-secretase (PSEN1) to release AICD and amyloid-β (Aβ) peptide. Secreted Aβ forms oligomers and aggregates, generating the toxic amyloid plaques observed in the AD patient’s brain (drawn by J. Park, and review by Hicks et al. [33]). Jiang et al. [2]). sAPPα has been reported to activate the proliferation Aβ into a toxic aggregated state [12, 14], the Aβ generation through of adult neuroblasts (review by Chasseigneaux et al. [3]), but its physio- endocytosis of APP [11], and the role of neuronal membrane cho- logical role is unknown [4]. The main subunit of the γ-secretase com- lesterol in excessive Aβ peptide production [10]. plex is presenilin 1 encoded by PSEN1, which is mutated in patients Previously, 900 and 1800 MHz RF-EMF radiations, allocated to with AD [5]. APP canalsobeprocessedby β-secretase (encoded by cellular phones, were reported to induce negative effects on brain beta-site amyloid precursor protein cleaving enzyme 1 [BACE1]) and function, such as brain tumors (reviews by Lagorio et al. [15] and γ-secretase, to release the neurotoxic Aβ peptide, which spontaneously Hardell et al. [16]) and memory impairments [17]. However, sev- forms oligomers, and then the larger amyloid plaques found in the eral recent studies using transgenic (Tg)-FAD mice as a model of brain of patients with AD (reviews by Jiang et al. [2], Thinakaran et al. AD have shown that RF-EMFs have beneficial effects on neurode- [6]and Kumar et al. [1]). Diverse genetic and molecular evidence sug- generative disorders: 918 MHz EMF enhanced memory and gests that the abnormal accumulation of Aβ occurs in the early stage of decreased brain Aβ aggregation in AD 3X Tg-mice [18]. RF-EMF the pathophysiological cascade that eventually leads to AD (reviews by with a frequency range from 800 to 2450 MHz improved cognitive Huang et al. [7], Palop et al. [8]and Bertram et al. [9]). However, impairment in AD 2X or 3X Tg-mice [19, 20], and enhanced brain what determines the processing of APP to Aβ,and howAβ impairs mitochondrial function, through the disaggregation of Aβ oligomers neuronal function have not been clearly understood. in Tg-5XFAD and normal mice [21]. In addition, 1950 MHz RF- In neural cells, the endogenous expression of APP is usually very EMF directly affected Aβ pathology by reducing Aβ plaques and low, and the amount of secreted Aβ is insufficient to be detected, BACE1 expression in Tg-5XFAD mice [22]. These studies demon- which has been a technical barrier to studying APP processing and strated the effects of RF-EMF radiation on AD pathology, mainly its mechanism [10]. 7w-PSML is a stably transfected CHO-based with FAD mouse models, but could not elucidate the cellular mech- cell line that expresses both the wild-type human APP and mutant anism involved in these RF-EMF–derived physiological effects. presenilin-1 (M146L), which is an efficient model for the detection In this study, we investigated the effects of RF-EMF on Aβ pro- of APP, and its cleaved and secreted form, Aβ peptide [11–13]. cessing in HT22 mouse hippocampal neuronal cells, SH-SY5Y human Thus, 7w-PSML has been used as a cellular model system for AD neuroblastoma cells and 7w-PSML cells, to examine the mechanism for studying Aβ processing in vitro: the conversion of monomeric for the physiological outcomes of RF-EMF at a cellular level. Downloaded from https://academic.oup.com/jrr/article-abstract/59/1/18/4356568 by Ed 'DeepDyve' Gillespie user on 16 March 2018 20 � J. Park et al. 0.105 ± 0.019 W/kg for the CDMA frequency and 0.262 ± MATERIALS AND METHODS 0.055 W/kg for the WCDMA frequency. Cell culture The exposure system was warmed up for at least 30 min, for HT22 (immortalized mouse hippocampal neuronal) cells and SH- equilibration, before the RF-EMF exposure. The 100-mm culture SY5Y (human neuroblastoma) cells were kindly provided by Dr dishes were placed at 13.6 cm from the conical antenna, which was Inhee Mook-Jung (Seoul National University College of Medicine, located at the center of the exposure chamber, and cells were then Seoul, Korea). These two cell lines have been used as well- exposed to RF-EMF radiation in the culture dishes. This system has established in vitro cellular models for neurodegenerative disorders already been used in other published cellular studies [25, 26]. The such as AD and Parkinson’s disease; HT22 cells have functional variation in RF-EMF exposures on different culture dishes was neg- cholinergic properties related to the cognitive defects of AD [23], ligible, when multiple dishes were exposed at the same time: the and SH-SY5Y cells have synaptic structures, functional axonal ves- variation in the average SAR values for WCDMA frequency was icle transport, and express neurospecific proteins (review by ~7.7% of the single dish exposure when three culture dishes were Carolindah et al. [24]). CHO cell–based 7w-PSML cells [which used, and ~2.0% when six dishes were exposed [25, 26]. overexpress wild-type human APP and the mutant presenilin-1 Cells were exposed to RF-EMF radiation of a single signal (M146L) [13]] were provided by Dr David Kang (Florida State (WCDMA signal at 1950 MHz) at 6 W/kg for 2 h/day for 3 days, University, USA). These cells were maintained in Dulbecco’s modi- with the same fixed timetable for the same intervals. During the fied Eagle’s medium (DMEM) with 10% (v/v) fetal bovine serum exposure period, the temperature was maintained within a range of (FBS; Sigma-Aldrich, MO, USA) and 10 ml/l penicillin–streptomy- 37 ± 0.3°C by circulating water within the cavity, and a 5% CO cin (GIBCO, NY, USA). All cells were grown at 37°C in a humidi- 2 concentration was also maintained in the chamber. After exposure fied atmosphere containing 5% CO . to RF-EMF radiation, the cells were immediately transferred to a cell culture incubator. RF-EMF-treated and untreated cells and each The RF-EMF radiation system for cell exposure culture medium were collected immediately after the last exposure to RF-EMF radiation, for further experiments. We used a radial transmission line (RTL) exposure system as an in vitro multifrequency radiation exposure system for this study [25, 26]. A typical Code–Division Multiple Access (CDMA) signal at 837 MHz and a Wideband Code–Division Multiple Access RNA isolation and quantitative real-time PCR (WCDMA) signal at 1950 MHz were applied to the RTL after amp- Cells were harvested and their total RNA was isolated using the lification. The signals were modulated by real CDMA and WCDMA RNeasy Mini kit (QIAGEN, Germany). cDNA was synthesized signals for cell exposure. The specific absorption rate (SAR) distri- with purified total RNA using the PrimeScript RT reagent kit bution inside the exposed medium was defined by numerical (Takara Bio Inc., Shiga, Japan). Quantitative real-time PCR was per- simulations using the finite-difference time-domain (FDTD) meth- formed using the CFX96 instrument (Bio-Rad, CA, USA), with the od (XFDTD 6.5, Remcom, State College, PA). The simulated and primers in Table 1. The relative expression of genes was normalized measured SAR values for 1 W input power in the entire sample was to that of the control gene encoding β–actin. The normalized fold Table 1. The primers used for quantitative real-time PCR Gene Oligonucleotide primer Product size (bp) Human/Mouse 5′-CATCTTCACTGGCACACCGT-3′ (forward) 115 APP 5′-CAAACTCTACCCCTCGGAAC-3′ (reverse) Human/Mouse 5′-AATTCTGCTCCTCTCCTGGGC-3′ (forward) 211 ADAM10 5′-CCTCTTCATTCGTAGGTTGA-3′ (reverse) Human/Mouse 5′-TGTGGAGATGGTGGACAACCTG-3′ (forward) 168 BACE1 5′-TGCCTCTGGTAGTAGCGATG-3′ (reverse) Human/Mouse 5′-GAGCTGACATTGAAATATGG-3′ (forward) 218 PSEN1 5′-ACAATGACACTGATCATGATGGC-3′ (reverse) Human β-actin 5′-TCCCTGGAGAAGAGCTACGA-3′ (forward) 194 5′-AGCACTGTGTTGGCGTACAG-3′ (reverse) Mouse β-actin 5′-CGCCACCAGTTCGCCATGGA-3′ (forward) 105 5′-TACAGCCCGGGGAGCATCGT-3′ (reverse) Downloaded from https://academic.oup.com/jrr/article-abstract/59/1/18/4356568 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Effects of 1950 MHz EMF on Aβ processing in neural cells � 21 expression of mRNA levels in RF-EMF–exposed and –unexposed indicated statistical significance compared with control. Non- −ΔΔC cells was analyzed using the 2 method as described previously statistically significant results (n.s) represented P > 0.05. [27]. Real-time PCR products were confirmed by electrophoresis on a 2.5% agarose gel. RESULTS The effect of 1950 MHz EMF exposure on the expression of genes associated with Aβ processing Protein analysis in mouse and human neural cells After the cells were exposed to the RF-EMF, the cell culture In order to determine the molecular mechanism of the effect of RF- medium was collected to precipitate secreted peptides by TCA pre- EMF on Aβ processing at the cellular level, we first investigated cipitation. Precipitated samples were separated by the 4–20% gradi- whether exposure to RF-EMF could affect the expression of the ent precast Tris-Glycine SDS gel (KomaBiotech, Seoul, Korea), to genes critical for Aβ processing in neural cells; amyloid precursor detect secreted Aβ peptides. RF-EMF–exposed and unexposed cells protein (APP), α-secretase (ADAM10), β-secretase (BACE1) and γ- were harvested and lysed as described previously by Park et al. [28]. secretase (PSEN1). HT22 (mouse hippocampal neuronal) cells and Cell extracts were analyzed by western blot by 7.5% or 10% SDS- SH-SY5Y (human neuroblastoma) cells were exposed to RF-EMF PAGE with anti-Alzheimer Precursor Protein A4 (clone 22C11; radiation (single 1950 MHz W-CDMA signal at 6 W/kg) for 2 h Millipore, MA, USA), anti-β-amyloid 1–16 (clone 6E10; BioLegend, per day for 3 days. The mRNA level of each gene was measured by CA, USA), anti-BACE1 (Millipore, MA, USA), and anti-β-actin quantitative real-time PCR in the RF-EMF–exposed cells and was (Cell Signaling Technology, Inc., MA, USA) antibodies. To detect compared with that of the unexposed control cells. The mRNA the differentially processed forms of APP in cells, we used two dif- expression of APP was reduced 0.64-fold in RF-EMF–exposed ferent APP antibodies, anti-22C11 and anti-6E10, which detect differ- HT22 cells, when compared with that of unexposed HT22 cells, ent regions of the human APP protein (Fig. 2). Anti-22C11 binds to and the mRNA levels of ADAM10, BACE1 and PSEN1 slightly membrane APP (amino acids 66–81 in the N-terminal domain of decreased in RF-EMF–exposed HT22 cells (Fig. 3A and B). In SH- APP) and can precipitate the full-length APP as well as its N-terminal SY5Y cells exposed to RF-EMF, there was no decrease in the fragments, soluble APPα (sAPPα) and APPβ (sAPPβ)(Fig. 2). Anti- mRNA expression of APP, but the mRNA expression level of 6E10 reacts notonlywithAPP andthe processedsAPPα, but also ADAM10 and BACE1 was slightly decreased and that of PSEN1 was with Aβ by binding between amino acids 597–613 (Fig. 2)[29, 30]. marginally increased compared with those of unexposed cells An enhanced chemiluminescence reagent (Amersham Biosciences, (Fig. 3C and D). These results showed that RF-EMF exposure Buckinghamshire, UK) was used for the blot analysis. The relative decreased the expression of APP mRNA and slightly diminished the band intensity of the samples from RF-EMF–exposed and unexposed expression of genes involved in the processing of APP, ADAM10, cells to the loading control (β-actin) was measured using GelQuantNet BACE1 and PSEN1 in mouse HT22 cells. In human SH-SY5Y cells, (BiochemLabSolutions, CA, USA), and the average result from three RF-EMF exposure induced marginal changes in the expression of independent experiments was plotted. genes involved in Aβ processing, but not on the expression of APP. Statistical analysis 1950 MHz EMF exposure does not affect the processing Statistical analysis was performed using GraphPad Prism 6 (GraphPad Software, Inc., CA, USA). Data are represented with the and secretion of Aβ in 7w-PSML cells mean ± standard error of the mean (S.E.M.) of at least three inde- Several studies have discovered that exposure to RF-EMF induces pendent experiments. We applied t-tests to assess statistically signifi- the reduction of APP, BACE1 and Aβ peptide in the AD transgenic cant differences. P < 0.05 (*), P < 0.01 (**), and P < 0.0001 (****) mouse model [20, 22]. In order to understand whether RF-EMF Fig. 2. A representation of regions of human APP differentially processed and detected with two different antibodies. A schematic structure of human APP protein (695 amino acid form) with the regions that respectively react to two different antibodies: anti-22C11 and anti-6E10 (adapted from [29, 30]). Downloaded from https://academic.oup.com/jrr/article-abstract/59/1/18/4356568 by Ed 'DeepDyve' Gillespie user on 16 March 2018 22 � J. Park et al. Fig. 3. The effect of 1950 MHz EMF exposure on the expression of APP and genes associated with Aβ processing in mouse and human neural cells. (A–D) HT22 (A, B) and SH-SY5Y (C, D) cells were exposed to radio frequency–electromagnetic field (RF-EMF) radiation (1950 MHz at 6 W/kg) for 2 h per day for 3 days. (A, C) The mRNA expression of APP, ADAM10, BACE1 and PSEN1 was analyzed in RF-EMF–treated and –untreated cells by quantitative real-time PCR and normalized to β-actin mRNA expression. Data are represented as mean ± S.E.M. from three independent experiments. P < 0.05 (*); P < 0.01 (**); P < 0.0001(****); P > 0.05 (non-significant, n.s). (B, D) Quantitative real-time PCR products in HT22 (B) and SH-SY5Y (D) were confirmed by electrophoresis on a 2.5% agarose gel. U = unexposed cells, E = RF-EMF–exposed cells, NTC = No Template Control. exposure could affect the processing of APP into Aβ, in neural cells, 7w-PSML cells were exposed to 1950 MHz EMF for 2 h every we first examined the expression of BACE1 and several processed day for 3 days, and both the cells and culture medium were col- forms of APP in 1950 MHz EMF–exposed 7w-PSML cells. lected. Secreted peptides in the culture medium were precipitated by Downloaded from https://academic.oup.com/jrr/article-abstract/59/1/18/4356568 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Effects of 1950 MHz EMF on Aβ processing in neural cells � 23 TCA precipitation, and analyzed by the 4–20% gradient precast Tris- sAPP (α or β)or Aβ peptide in 7w-PSML cells (Fig. 4A and C). Glycine SDS-PAGE. Expression of APP and BACE1 was also These results showed that 1950 MHz EMF exposure does not sig- detected by western blot analysis with the lysates of RF-EMF– nificantly change the pattern of Aβ processing in 7w-PSML cells. exposed cells. No significant difference in the expression of APP and BACE1 was detected in RF-EMF–exposed cells, when compared 1950 MHz EMF exposure has no direct effect on Aβ with the unexposed controls (Fig. 4A). To confirm this result, we normalized the relative band intensity of APP and BACE1 to β-actin processing and secretion in HT22 and SH-SY5Y cells as a loading control, but could not detect a meaningful difference We then investigated whether exposure to 1950 MHz EMF alters between RF-EMF–exposed cells and unexposed controls (Fig. 4B). the endogenous protein expression of APP and BACE1 as well as of In addition, RF-EMF exposure did not affect the secreted level of processed Aβ in HT22 and SH-SY5Y cells. HT22 and SH-SY5Y cells Fig. 4. 1950 MHz EMF exposure does not directly affect the processing and secretion of Aβ in 7w-PSML cells. (A) 7w-PSML cells were exposed to RF-EMF radiation (1950 MHz at 6 W/kg) for 2 h per day for 3 days. Secreted peptides in the culture medium were precipitated by TCA. APP and BACE1 in cell lysates, and sAPP (α or β) in precipitated samples, were detected by western blot analysis. β-actin was used as a loading control. (B) The relative band intensity of APP and BACE1 in cell lysates to the loading control was calculated by GelQuantNet software from the results of three independent experiments, including (A). Data are represented as mean ± S.E.M. from three independent experiments. P > 0.05 (non-significant, n.s). (C) Total 30 and 50 μg proteins of the TCA precipitated medium were separated by a 4–20% gradient precast Tris-Glycine SDS gel, and Aβ peptides were detected with anti-6E10 by western blot analysis. Downloaded from https://academic.oup.com/jrr/article-abstract/59/1/18/4356568 by Ed 'DeepDyve' Gillespie user on 16 March 2018 24 � J. Park et al. were exposed to 1950 MHz EMF radiation for 2 h every day for 3 difference in the amount of secreted Aβ peptide was observed days, and the expression of APP and BACE1 were analyzed by west- between RF-EMF–exposed and unexposed HT22 cells (Fig. 5C). In ern blot. The endogenous expression of APP and BACE1 did not SH-SY5Y cells, we also could not detect any meaningful variation in change in RF-EMF–exposed HT22 cells, compared with in the unex- the expression of APP and BACE1 as well as of the secreted Aβ in posed controls (Fig. 5A). The normalized band intensity of APP and RF-EMF–exposed cells and unexposed controls (Fig. 5D–F). These BACE1 relative to a loading control β-actin in cell lysates of HT22 observations demonstrated that short-term exposure to 1950 MHz showed a marginal decrease of APP in RF-EMF–exposed cells, EMF does not change the expression of proteins for APP processing although it was not statistically meaningful (Fig. 5B). No significant and Aβ secretion in either HT22 or SH-SY5Y cells. Fig. 5. 1950 MHz EMF exposure has no significant effect on the APP protein expression and Aβ secretion in either HT22 or SH-SY5Y cells. (A–F) HT22 (A, B, C) and SH-SY5Y (D, E, F) cells were exposed to 1950 MHz at 6 W/kg for 2 h per day for 3 days. (A, D) Expression of APP and BACE1 in cell extracts of (A) HT22 and (D) SH-SY5Y was analyzed by western blot. β-actin was used as a loading control. (B, E) The relative band intensity of APP and BACE1 in cell lysates of (B) HT22 and (E) SH-SY5Y to the loading control was calculated by GelQuantNet software from the results of three independent experiments, including (A) and (D). Data are represented as mean ± S.E.M. from three independent experiments. P > 0.05 (non-significant, n.s). (C, F) Cell culture medium was precipitated with TCA, and secreted peptides were analyzed by a 4–20% gradient precast Tris-Glycine SDS gel. Secreted Aβ peptide was detected by western blot analysis with anti-6E10 in (C) HT22 and (F) SH-SY5Y cell media. Downloaded from https://academic.oup.com/jrr/article-abstract/59/1/18/4356568 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Effects of 1950 MHz EMF on Aβ processing in neural cells � 25 well as Aβ depositions in the brain? [18, 22]. We believe that a DISCUSSION major reason for the discrepancy in the effect of RF is the exposure Previously, researchers have focused on elucidating the negative effect period. We exposed HT22 and SH-SY5Y cells to RF-EMF for a of RF-EMF radiation emitted by cellular phones on the brain, such as short period (2 h per day for 3 days) only, because 3 days was the brain tumor development [31, 32]. However, several recent studies have reported the beneficial effect of RF-EMF radiation on AD using limitation of the cell culture span. However, the AD mouse models were exposed to RF-EMF for 8 months [22]. Thus, we could con- AD transgenic mouse models [19–21]. In order to evaluate the cellu- clude that a short-term exposure of RF-EMF to neural cells does lar mechanism of the effect of RF-EMF on AD, in this study, we not induce significant changes in the expression or processing of examined the effect of 1950 MHz EMF radiation (single WCDMA APP at the cellular level. However, minute changes caused by RF- signal at SAR 6 W/kg, 2 h per day for 3 days) on the endogenous EMF radiation, accumulated for a long period of exposure in the expression of APP and major proteins for APP processing, as well as AD mouse model, may result in obvious physiological outcomes, the secretion of Aβ in HT22 and SH-SY5Y neural cells. In addition, such as reduced Aβ deposits. Further studies would be necessary to we also investigated the effect of RF-EMF radiation in the 7w-PSML understand whether long-term and/or continuous exposure or cell as it is an efficient model for the detection of APP and Aβ pep- extended exposure time to RF-EMF radiation affects Aβ processing tide [11–13]. We showed in this study that 1950 MHz EMF radiation at the cellular level. for 2 h every day for 3 days did not induce significant changes in the level of APP and processing and secretion of Aβ in mouse and human neural cells as well as in 7w-PSML cells. ACKNOWLEDGEMENTS We exposed these cells with 1950 MHz EMF radiation for 2 h a The authors thank Dr David Kang (at Florida State University) for day, because the Tg-5xFAD mice, which showed ameliorated Aβ providing 7w-PSML cells, and Dr Inhee Mook-Jung (Seoul pathology by RF-EMF, were exposed to 1950 MHz EMF for 2 h National University College of Medicine, Seoul, Korea) for provid- every day [22]. We wanted to examine the cellular effect of RF- ing HT22 cells and SH-SY5Y cells. K.S. conceived the idea, EMF under the same conditions as those used for Tg mice. Also, designed the experiments, and wrote the manuscript. J.P. performed the RF-EMF system we used was originally designed for a maximum the experiments and wrote the manuscript. J.H.K. and N.K. pro- of 2 h exposure per day, based on the assumption that most people vided the 1950 MHz EMF radiation device for exposure of the cells. on average use mobile phones for a total of 2 h a day. With our observations, we could suggest that the RF exposure conditions CONFLICT OF INTEREST applied in this study may not have a significant effect on the expres- The authors have declared that there is no conflict of interest. sion and processing of APP or Aβ in mouse and human neural cells. However, in the RF-EMF–exposed HT22 cells, we repeatedly FUNDING observed that the mRNA expression of APP and genes for APP pro- This work was supported by the National Research Foundation of cessing seems to be decreased with statistically meaningful signifi- Korea (NRF) funded by the Ministry of Science, ICT & Future cance (Fig. 3A), and the expression of APP was marginally reduced, Planning [No. NRF-2016M3A9C6918275] and by Korea Mobile although the secreted Aβ was not much changed (Fig. 5A–C). Thus, EMF consortium. J. Park was partially supported by the Graduate with these results, it would be difficult to define whether 1950 MHz School of YONSEI University Research Scholarship Grants of 2017. EMF radiation has a meaningful effect on decreasing the expression of APP and the secretion of Aβ in mouse neural cells, or not. However, we cannot exclude the possibility that 1950 MHz RF-EMF REFERENCES exposures to mouse neural cells induce the minute decline observed 1. 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Journal of Radiation ResearchOxford University Press

Published: Jan 1, 2018

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