Chronic Arsenic Exposure Increases Aβ(1−42) - ACS Publications

Article Cite This: Chem. Res. Toxicol. 2018, 31, 13−21

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Chronic Arsenic Exposure Increases Aβ(1−42) Production and Receptor for Advanced Glycation End Products Expression in Rat Brain Sandra Aurora Niño,† Guadalupe Martel-Gallegos,† Adriana Castro-Zavala,‡ Benita Ortega-Berlanga,†,⊥ Juan Manuel Delgado,‡ Héctor Hernández-Mendoza,¶,# Elizabeth Romero-Guzmán,¶ Judith Ríos-Lugo,§ Sergio Rosales-Mendoza,⊥ María E. Jiménez-Capdeville,‡ and Sergio Zarazúa*,† †

Laboratorio de Neurotoxicología, Facultad de Ciencias Químicas, ‡Departamento de Bioquímica, Facultad de Medicina, §Unidad de Posgrado, Facultad de Enfermería y Nutrición, and ⊥Laboratorio de Biofarmacéuticos Recombinantes, Facultad de Ciencias Químicas, Universidad Autónoma de San Luis Potosí, CP 78210 San Luis Potosí, San Luis Potosí, México ¶ Laboratorio Nacional Forense Nuclear, Instituto Nacional de Investigaciones Nucleares, Carretera México-Toluca s/n, CP 52750 La Marquesa Ocoyoacac, México # Centro de Biociencias, Universidad Autónoma de San Luis Potosí, Km. 14.5 carretera San Luis Potosí − Matehuala, Ejido “Palma de la Cruz”, CP 78321 Soledad de Graciano Sánchez, San Luis Potosí, México ABSTRACT: Chronic arsenic exposure during development is associated with alterations of chemical transmission and demyelination, which result in cognitive deficits and peripheral neuropathies. At the cellular level, arsenic toxicity involves increased generation of reactive species that induce severe cellular alterations such as DNA fragmentation, apoptosis, and lipid peroxidation. It has been proposed that arsenic-associated neurodegeneration could evolve to Alzheimer disease in later life.1,2 In this study, the effects of chronic exposure to inorganic arsenic (3 ppm by drinking water) in Wistar rats on the production and elimination of Amyloid-β (Aβ) were evaluated. Male Wistar rats were exposed to 3 ppm of arsenic in drinking water from fetal development until 4 months of age. After behavioral deficits induced by arsenic exposure through contextual fear conditioning were verified, the brains were collected for the determination of total arsenic by inductively coupled plasma-mass spectrometry, the levels of amyloid precursor protein and receptor for advanced glycation end products (RAGE) by Western blot analysis as well as their transcript levels by RT-qPCR, Aβ(1−42) estimation by ELISA assay and the enzymatic activity of β-secretase (BACE1). Our results demonstrate that chronic arsenic exposure induces behavioral deficits accompanied of higher levels of soluble and membranal RAGE and the increase of Aβ(1−42) cleaved. In addition, BACE1 enzymatic activity was increased, while immunoblot assays showed no differences in the low-density lipoprotein receptor-related protein 1 (LRP1) receptor among groups. These results provide evidence of the effects of arsenic exposure on the production of Aβ(1−42) and cerebral amyloid clearance through RAGE in an in vivo model that displays behavioral alterations. This work supports the hypothesis that early exposure to metals may contribute to neurodegeneration associated with amyloid accumulation.

1. INTRODUCTION

In addition to the neurotoxic damage described above, a report from Vahidnia and co-workers21 indicates that exposure to sodium arsenite produces pathological tau hyperphosphorylation in sciatic nerves of subchronically exposed rats. Furthermore, it has been proposed that arsenic exposure may be related to the development of neurodegenerative diseases such as Alzheimer’s disease (AD).1 Inorganic arsenic and its metabolite DMAV have important effects in brain primary cultures derived from transgenic Tg2576 mice, which overexpress amyloid precursor protein (APP) and are related to the early development of Alzheimer’s disease. Results obtained in our group demonstrated that in vitro exposure to DMAV (10 μM, 12 h) modifies APP processing by increasing levels of Aβ(1−40) and a tendency to

Arsenic exposure from early stages of development generates damage in neurological function. Epidemiological studies demonstrate that arsenic induces learning impairment in children as well as deterioration of cognitive abilities.3−5 Similarly, chronic arsenic exposure in adults is associated with memory decline, decreased speed of information processing, deterioration of cognitive activities, and mood disorders such as anxiety and depression.6,7 Alterations of neuronal plasticity and myelination that could underlie the behavioral deficits induced by arsenic exposure have been demonstrated in animal models.8−12 The detailed exploration of intra and intercellular events affected by arsenic in the central nervous system (CNS) has revealed disturbances on neurotransmitters.11,13−16 intracellular calcium17,18 and other signaling systems19 as well as inhibition of long-term potentiation.20 © 2017 American Chemical Society

Received: July 30, 2017 Published: November 20, 2017 13

DOI: 10.1021/acs.chemrestox.7b00215 Chem. Res. Toxicol. 2018, 31, 13−21

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Chemical Research in Toxicology increase in Aβ(1−42). On the other hand, sodium arsenite (5 μM, 12 h) incubation induced a reduction in release of Aβ(1−40) and Aβ(1−42), perhaps as an inhibitory effect over α-secretase.2 These results suggest that DMAV stimulates the amyloid pathway, besides it is the main arsenic metabolite located in rodent brain after arsenic exposure.22−24 Other possibilities to explain the increase in Aβ(1−40) levels are the stimulatory effect of DMAV on β-secretase or an effect over Aβ clearance mechanism from the brain across the blood−brain barrier (BBB). There are two amyloid removal systems in the brain: (i) proteolytic degradation and (ii) cerebral clearance, mainly mediated by the receptor for receptor for advanced glycation end products (RAGE) and low-density lipoprotein receptorrelated protein 1 (LRP1).25 RAGE mediates Aβ intake from monocytes using endothelial cells through the BBB by transcytosis−endocytosis. Furthermore, RAGE is involved in the expression of proinflammatory citokines in BBB, activation of apoptosis, and decreased cerebral blood flow.26−29 In contrast, LRP1 is a family member of low-density lipoprotein (LDL) involved in the transport of Aβ through BBB from the brain to the bloodstream.30 Studies in rodents and primates indicate that LRP1 expression on capillary endothelium of the brain is reduced during aging in normal conditions.31,32 Moreover, evidence indicates that RAGE is overexpressed in brain of AD patients.33 On the basis of the in vitro effects observed of arsenic in the amyloid cascade, this work explores whether the presence of arsenic modifies Aβ clearance in an established in vivo model of chronic arsenic exposure associated with behavioral deficits. For this purpose, brain arsenic concentrations, fear-conditioning test, and Aβ clearance proteins were assayed in adult Wistar rats exposed to arsenic (3 ppm) from intrauterine development until 4 months of age. Although an exposure of 3 ppm of arsenic in drinking water is higher than any environmental exposure for humans, this is a no-observed-adverse-effect-level (NOAEL) for rats according to the United States Agency for Toxic Substances and Disease Registry (ATSDR).34 This animal model of chronic arsenic exposure presents alterations in behavior, neurotransmitter levels, oxidative status, and myelin damage in absence of body weight changes, litter size, and mortality of dams or pups.35,36

deficits, and changes in DNA methylation;39−41 however, it does not result in weight loss and signs of over toxicity. After 10 days, males were removed and arsenic exposure continued in the pregnant rats throughout gestation and lactation. Offspring were weaned at 4 weeks, separated by sex, and subjected to continued with the arsenic exposure until 4 months of age. At the end of this time, the following experiments were performed: (a) groups of six male rats each, control and arsenic exposed, were analyzed by Western blot, RTqPCR, and enzymatic activity analyses; (b) groups of four female rats and four males of each treatment were assessed by contextual fear conditioning. 2.3. Behavioral Test: Contextual Fear Conditioning (CFC). The behavioral test was performed in 4 month-old female and male rats. Four animals of each sex were employed for each postconditioning time-point. The instrument designed ad hoc to measure CFC consisted on an acrylic box (20 cm × 25 cm × 25 cm) with a metal grid floor for administration of electric shocks of different duration and intensity, and a sound-generating device on the wall of the chamber. The instrument was placed in a noise-isolated room. The conditioning stimulus (CS) consisted on a 40 dB tone for 4 s, while the unconditioned stimulus (US) was an electric shock of 0.75 mA for 2 s. For conditioning, the animal was placed in the training box for 2 min to allow exploration; subsequently, the CS was given followed by US, and this was repeated twice, for a total of three pairs of shock-stimulus. The CS/US were applied at 30 s intervals. After completing the three stimuli, the animal was kept for an additional 30 s period before being returned to the cage. Three postconditioning times were evaluated (1, 24, and 48 h). At these time-points, the animal was placed in the box, it was allowed to explore for 30 s, and then the CS was applied, which resulted in the immobilization behavior (freezing). To obtain the total frozen time, the animal was observed for 5 min. The observer was trained and a validation test was performed to evaluate repeatability and reproducibility. 2.4. Animal Sacrifice and Sample Collection. To obtain the brain tissue, animals were sacrificed by decapitation and the brains were immediately removed from skull and placed in a cold plaque. Cerebellum and olfactory bulbs were eliminated and cerebral hemispheres were separated through a dissection in the medial sagittal line. For arsenic quantification, Western blot analysis, RT-qPCR, and enzymatic activity, each hemisphere was placed into a microtube. All samples were weighted and frozen in liquid nitrogen and then stored at −80 °C until posterior analysis. 2.5. Arsenic Quantification. Total arsenic was quantified in brain samples as an exposure marker. Approximately 100 mg of brain tissue was digested with 10 mL of high purity HNO3 using a microwave MARS6 (CEM, Matthews, North Carolina). The digestion was performed for 20 min at 180 °C. All the samples were evaporated to dryness in a hot plate and recovered in 10 mL of high purity 2% v/v HNO3 and internal standard was added (1 μg L−1 of indium). Arsenic measurements were performed with inductively coupled plasma-sector field mass spectrometry (ICP-SFMS, Element 2/XR from Thermo Fisher Scientific Germany) in high-resolution mode. Aqueous samples were introduced with an SC-2 DX autosampler from Element Scientific Inc. (ESI) and a micro concentric nebulizer coupled to a Twister with a Helix 50 mL cyclonic borosilicate glass spray chamber (Elemental Scientific Inc., USA). The torch of the ICP-SFMS instrument (Elemental Scientific Inc., USA) was shielded with a grounded platinum electrode (Guard Electrode TM, Thermo Scientific). 2.6. Expression of APP or RAGE. 2.6.1. Western Blot Analysis. To evaluate the expression of APP, RAGE, and LRP1 proteins, whole brain samples were homogenized with a glass-Teflon in cold extraction buffer (in mM: sucrose 320, HEPES 10, EDTA 2) supplemented with protease inhibitors (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). APP was measured directly in brain homogenates. For RAGE estimation, a fraction of total lysate was centrifuged at 20 000 × g for 1 h to obtain the pellet and the supernatant corresponding to membranal and soluble fractions, respectively. Proteins were quantified by the bicinchoninic acid method (BCA; Sigma-Aldrich, St. Louis MO, USA). Protein samples (40−60 μg) from all the fractions were loaded

2. MATERIALS AND METHODS 2.1. Reagents. Sodium arsenite was obtained from Sigma (St. Louis, MO, USA) and is a highly toxic reactant that should be handled with extreme caution. Reagents were obtained from IBI Scientific, Santa Cruz Biotechnology, Thermo Scientific, Sigma-Aldrich, Bio-Rad, and Merck Millipore. Solvents were obtained from Karal (León, Gto., Mexico) and Trizol reagent from Invitrogen (Carlsbad, CA, USA). ELISA kit for Aβ(1−42) was obtained from Thermo Fisher Scientific Inc. (Waltham, MA, USA). All the RT-qPCR experiments were performed using the SYBR reaction mix (Bio-Rad Laboratories, Hercules, CA, USA). 2.2. Animal Model. The experiments were performed according to the Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research (NRCNA, USA, 2003).37 Twelve female and six male Wistar rats (200−250 g) were placed in cages at a proportion of 2 female:1 male per cage and maintained under a 12 h light/dark cycle with access to food and water ad libitum.38 One group of six females/three males received arsenic-free drinking water; the second group with the same number of animals received drinking water containing 3 ppm of arsenic. Considering the daily water intake of adult rats, this dose corresponds to approximately 0.3 to 0.4 mg/kg/ day, which is associated with demyelination, behavioral performance 14


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Figure 1. Validation of the animal model. (A) Total arsenic in the brain of exposed animals is significantly higher than in control rats. (B) Behavioral testing in control (white circles) and arsenic exposed (black circles) rats. Arsenic exposed group exhibited impaired performance compared to the control group (p < 0.05). # and ‡ mean a difference between 48 and 1 h for control (p < 0.01) and arsenic group (p < 0.001), respectively. ∗∗∗∗p < 0.0001 versus control. under standard conditions from 100 to 50 °C. PCRs were performed on an Applied Biosystems Step One Real-Time PCR System, and the data were analyzed with the Applied Biosystems 7500 software V.2.0. Samples were analyzed by triplicate. 2.7. Analysis of Enzymatic Activity for BACE1. The enzymatic activity of β-secretase (BACE1) was determined by a fluorescencebased assay according to the manufacturer’s instructions (Calbiochem EMD Chemicals, Inc. Port Wentworth, USA). Briefly, 200 μg of total lysate was incubated at 37 °C for 90 min with the conjugated peptide in complete darkness in a 96-well plate; next, the reaction was stopped on ice and fluorescence at 340/503 nm was measured in a FlexStation II fluorometer (Molecular Devices, Sunnyvale, CA, USA) at 25 °C. The active enzyme BACE1 was employed as positive control and BACE inhibitor as negative control. Results are presented as relative fluorescence units (RFUs) calculated as RFUs = 100(sample fluorescence − negative control/positive control − negative control). 2.8. Enzyme-Linked Immunosorbent Assay (ELISA) for Aβ(1−42). The amyloidogenic form of Aβ was selectively detected in protein soluble fractions by using a solid phase sandwich ELISA kit according to manufacturer’s instructions (Thermo Fisher Scientific Inc.). Briefly, 200 μg of protein from the soluble fraction was incubated in the microplate coated with the anti-Aβ(1−42) NH3terminus monoclonal antibody (2 h, at room temperature). Wells were washed and then incubated with the detector rabbit antibody targeting the COOH-terminus of Aβ(1−42) for 1 h at room temperature. Samples were then incubated with an HRP-labeled antirabbit antibody and finally washed and developed with a chromogenic solution. Plates were read in a microplate reader (Multiskan FC Microplate Photometer, ThermoFisher Sci.) at 450 nm. Results are expressed as fold-changes respect to control. 2.9. Immunoprecipitation of LRP1. For the LRP1 protein detection, immunoprecipitation was performed before Western blot analysis. Briefly 500 μg of protein from brain lysate was mixed with 0.5 mL of IP buffer (in mM: HEPES 10, NaCl 150, EGTA 1, MgCl2 0.1, and 0.1% Triton X-100) at pH 7.4. Samples were precleared with protein A/G PLUS-Agarose (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) during 1 h at 4 °C. After centrifugation, the supernatants were recovered and incubated overnight with 2 μg of anti-LRP1 antibody in a rocking platform. Then protein−antibody immunocomplexes were precipitated by adding 20 μL of protein A/G PLUS-Agarose to samples followed by rocking incubation during 1 h. After centrifugation, pellets were washed three times with IP buffer, resuspended in 35 μL of Laemmli 1X buffer, boiled for 10 min at 90 °C, and subjected to Western blot analysis on SDS-PAGE 12% and immunoblotting against LRP1 as described above. 2.10. Statistical Analysis. First, the normality of the data was assessed using the Shapiro-Wilk test, the level of slant and kurtosis, as well as homoscedasticity using the Brown-Forsythe test. If the results were parametric, data were expressed as mean ± standard deviation (mean ± SD) and the statistical comparison of groups was performed

and run on SDS-PAGE 10% and transferred to PVDF membranes using a Trans-blotTurbo TM (Bio-Rad, CA, USA). Membranes were incubated with anti-APP, anti-RAGE, anti-LRP1, and anti-β-actin (loading control) antibodies. All antibodies were used at 1:500 dilutions. Primary antibodies were detected by quimioluminiscence with a secondary antibody coupled to peroxidase (HRP). The images obtained by autoradiography were digitalized for densitometry analysis using the ImageJ software version 1.5 (NIH, USA). 2.6.2. Immunofluorescence for RAGE. Rats were intracardiacally perfused with 0.1 mol/L phosphate buffer containing 4% paraformaldehyde. Fixed brains were removed and embedded in paraffin. Tissues were cut in 5 μm sections (n = 3 animals/group) and mounted in electro-charged slides. Epitope recovery was performed by immersion in Diva decloaker solution (Biocare Medical LLC, Concord, CA) in conditions of high temperature and pressure. Tissue slices from the hippocampus and cerebral cortex were treated with 0.3% hydrogen peroxide for 15 min and incubated in humidity chamber at room temperature to block nonspecific background staining (background sniper; Biocare Medical, LLC, Concord, CA) and endogenous biotin and biotin-binding proteins (avidin/biotin blocking kit; Vector Laboratories Inc., Burlingame, CA) followed by rinses with TBStween. RAGE primary antibody was incubated overnight at 4 °C, and the secondary antibody used was a goat antimouse IgG antibody marked with Alexa Fluor 488 (Thermo Scientific, Rockford, IL). AntiGFAP was used to stain astrocytes. Nuclei were visualized by ytox staining (Molecular Probes, Eugene, OR). Samples were examined with a confocal microscope (LEICA TCS SP2; Leica Microsystems GmbH, Heidelberg, Germany). 2.6.3. qPCR Analysis for Gene Expression. Total RNA was isolated from each group using Trizol (Invitrogen, Carlsbad, CA, USA). After treatment with DNase I (Invitrogen, Carlsbad, CA, USA), RNA was quantified using an Eppendorf BioSpectrometer basic (Eppendorf AG, Hamburg, Germany). cDNA synthesis was performed using the GoScript Reverse Transcription System kit (Promega Corporation, Madison, WI, USA), according to the manufacturer’s instructions. Quantitative real-time PCR analysis was performed using SYBR reaction mix. The 10 μL-reactions contained 100 ng of total cDNA, 5 μL of 1x SYBR Green mix, and 200 nM of each primer (for APP: forward primer, 5′-CACTACCCATCGGTGTTCATT-3′ and reverse primer, 5′-GAGTTCAGGCATCTACTTGTGT-3′; for RAGE: forward primer, 5′-AGTCAGAGGAAGCGGAAATG-3′ and reverse primer, 5′-AGGTTGAATTGGGATCGTAGAG-3′). Quantification was based on a cycle threshold value for APP and RAGE transcripts, which were normalized respect to β-actin transcripts. β-Actin was amplified using the following primers: forward 5′-GTGTGGATTG G T G G C T C T A T C - 3′ a n d r e v e r s e , 5 ′ - C A G T C C G C C TAGAAGCATTT-3′. Real time PCR conditions were 5 min at 95 °C followed by 40 PCR cycles of 10 s at 95 °C and 30 s at 60 °C. Absence of contaminating genomic DNA was confirmed by PCR analysis having RNA samples as template. Melting curves were done 15


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Figure 2. Arsenic rises the expression of APP and the activity of BACE1 resulting in the increase of Aβ(1−42) released in brain tissue respect to the control group; (A) Western blot assay for APP protein normalized by β-actin (p = 0.0135). (B) Transcript quantification by RT-qPCR normalized as folds of control (p = 0.0011). (C) Representation of BACE1 activity assay (Mann−Whitney U = 14, n1 = 5, IQR1 = 11.49 vsn2 = 6, IQR2 = 9.85, p = 0.0028 two-tailed); (D) Aβ(1−42) estimation by ELISA (p = 0.0165). Results are displayed as graphic representation and expressed as percentage change respect to the control group (mean ± SD). For BACE1 activity, data are presented as relative fluorescence units (RFUs) and represent median ± IQR. ∗p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.0001 versus control. using the Student-t test or two-way ANOVA with post hoc Tukey test; if the results were nonparametric, a Mann−Whitney U was performed and the data were expressed as median ± interquartile range (median ± IQR). The statistical analysis was performed in GraphPad In Stat program version 3.06. A value of p less than 0.05 was considered as significant.

Arsenic exposed group showed a 32% higher expression of APP than that of the control group (Figure 2A, 100.00 ± 4.50 vs 132.30 ± 2.69% of control for untreated and exposed groups, respectively; p = 0.0135 two-tailed). The representative comparison of APP Western blot images (n = 5−6) in control and exposed groups with their respective actin is exhibited in Figure 2A. Fold-change values on the APP transcript levels respect to control group are shown in Figure 2B. The arsenic exposed group presents a three-fold increase on APP expression (Figure 2B; 3.158 ± 0.89 vs 1.00 ± 0.067; p = 0.0011 two-tailed). 3.4. Determination of Enzymatic Activity of BACE1. We had previously reported that arsenic modifies the activity of β-secretase, which could lead to increased Aβ levels in primary cell cultures obtained from transgenic Tg2576 mice brains.2 Therefore, an objective on this work was to measure the activity of BACE1. Results of BACE1 activity measurements are shown in Figure 2C. Enzymatic activity is directly proportional to fluorescence emitted (in RFUs). The arsenic exposed group presented a significant increase in BACE1 activity compared to the control group (medians: 113.7 and 119.3 for control and arsenic exposed group, respectively; Mann−Whitney U = 14, n1 = 5, n2 = 6, IQR1 = 11.49, IQR2 = 9.85, p = 0.0028 two-tailed). 3.5. Aβ(1−42) Determination. To evaluate if arsenic exposure induces abnormal deposition of Aβ, we measured the levels of Aβ(1−42) since it represents the major component of amyloid plaques. Our results showed that brain homogenates from the arsenic exposed group denotes a 125% increase in Aβ(1−42) levels respect to control as shown in Figure 2D (p = 0.0165, two-tailed). 3.6. RAGE Expression. RAGE receptor levels are a key parameter since they represent the main influx/re-entry pathways through BBB for circulating Aβ. RAGE levels were estimated in total lysate, membranal and soluble fractions, since

3. RESULTS 3.1. Validation of Animal Model. The parameters considered to validate the model of arsenic exposure were arsenic brain levels and behavioral deficits. Total arsenic levels were evaluated in control and arsenic exposed groups; the results are represented in Figure 1A. At the end of the exposure period, arsenic in brain of control group remained at very low levels. In contrast, the group exposed to 3 ppm of arsenic reached levels of 164.5 (111−223) ng/g tissue. These values are consistent with previous reports from our group.2 No changes in body weight or mortality were found neither in control nor in arsenic exposed groups (data not shown). 3.2. Behavioral Test. Exposed animals showed a significant difference in freezing time compared to the control group only at 48 h (p = 0.0192). For behavior extinction, a significant difference was observed when the freezing time measured at 48 h was compared to that observed at 1 h in each group (p = 0.0039 in control group and p = 0.0006 in the exposed group, Figure 1B). No statistical difference was found between sexes (data not shown). These results are consistent with previous reports of our group that demonstrate behavioral alterations associated with a 3 ppm arsenic exposure.39,42 3.3. APP Expression. Protein samples were analyzed by Western blot and the obtained bands were digitized, analyzed by optical densitometry (OD), and normalized according to βactin expression. Data from the exposed group were calculated as a percentage of the values obtained from the control group. 16


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Figure 3. Arsenic exposure increases RAGE expression with no changes in LRP1. (A) Representative immunoblots for RAGE in total lysate, membranal, and soluble fractions, normalized by β-actin (p = 0.0195, p < 0.0152 and p < 0.0164 respectively); (B) RT-qPCR results for RAGE mRNA normalized as folds of control (p < 0.0001); (C) levels of LRP1 in brain of control and arsenic exposed groups. White bars correspond to control group and black bars to arsenic exposed group. Data are presented as percentage change of control and represent mean ± SD ∗p < 0.05 and ∗∗∗ p < 0.0001 versus control.

it may be present as soluble and/or membrane-anchored protein. RAGE levels were normalized by β-actin and the results are expressed as percentage of control. Both representative Western blot images with their respective βactin control and grouped data represented as bar graphic are presented in Figure 3A. The arsenic exposed group showed increased levels of RAGE protein in the total lysate respect to the control group (100.00 ± 8.253 vs 120.80 ± 3.12 for untreated and exposed groups respectively; p = 0.0195 twotailed). In membranal fraction, arsenic exposed group displayed an increase of RAGE respect to control (100.00 ± 7.16 vs 175.10 ± 22.14 for control and exposed groups respectively; p = 0.0152). Likewise, in total lysate and membranal fraction, soluble RAGE levels are increased in the arsenic exposed group when compared to the control group (100.00 ± 5.42 OD and 244.20 ± 48.54 for control and exposed groups, respectively; p = 0.0164 two-tailed). Interestingly, levels of RAGE transcripts increased importantly in the arsenic exposed group, reaching almost a 220-fold change (Figure 3B; 218.10 ± 42.97 vs 1.00 ± 0.08; p < 0.0001 two tailed). These results are consistent with Western blot analysis data. Values of RAGE transcript levels respect to control group are represented in Figure 3B, respectively. Immunofluorescence analysis using confocal microscopy was performed to confirm RAGE overexpression and localization. Our results revealed a higher immunopositivity for RAGE in cytosol and neuropile of hippocampus and cortex from arsenic exposed rats compared with the control group (Figure 4). The increase of RAGE immunopositivity associated with arsenic exposure was higher in the hippocampus than in the cortex (panels B and D), and both regions also showed the presence of RAGE in some astrocytes.

Figure 4. Immunofluorescence analysis to evaluate RAGE localization. (A, B) Hippocampus and (C, D) cortex slides from control and arsenic exposed rats were double labeled. Blue signal (Anti-GFAP) indicates the presence of astrocytes, whereas green signal indicates RAGE. Notably higher positivity to RAGE is found in cytosol and neuropile of (B) hippocamus and (D) cortex cells from arsenic exposed rats than in (A, C) controls. Confocal microscopy, 100x fields, nuclei stained with Sytox (red). Scale bar: 10 μm.

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arsenite and DMAV.2 These differences might be explained by the dissimilarity in chronology of arsenic exposure among the models. An extended arsenic exposure ensures the generation of long-lasting oxidative stress and the subsequent negative effects described above. Previous reports indicate that the presence of Aβ overstresses the oxidative environment leading to the development of chronic inflammation, which in turn induces transcription of RAGE by the interaction with ligands such as Aβ, fibrillar Aβ, among others.29 This binding induces the generation of ROS possibly through the activation of NADPH oxidase and consequently results in the activation of signal pathways that lead to phosphorylation of ERK1/2 and phospholipase A2, involved in the progression of neurodegenerative diseases.54,55 Additionally, Aβ−RAGE interaction induces oxidative stress in the vascular unit by stimulating continuous expression of various transcription factors such as NF-κB, extending and amplifying the chronic inflammatory response.56,57 In addition, the presence of Aβ has been related to the activation of JNK kinase and phosphorylation of tau protein.58 The vascular hypothesis of AD proposes that the imbalance between production and removal of Aβ is a contributing factor to the impaired brain homeostasis.59 Clearance system is located at the BBB in a homeostasis-regulating environment, responsible for intake of energy metabolites and elimination of toxic products between brain interstitial fluid and peripheral blood.60,61 Several research groups have reported that the changes in BBB clearance system significantly contribute to increase the concentration of Aβ in the brain.29,33,62,63 Normally, the presence of continuous endothelial cell monolayers in the BBB do not allow the free flow of polar solutes between the brain and the bloodstream, and thus, the exchange is regulated by transport proteins, such as apoE and apoJ, as well as transmembrane receptors like LRP1 and pglycoprotein. On the other hand, RAGE is involved in the entry of molecules from the bloodstream into the brain.64,65 So far, it is known that RAGE is expressed on endothelial and glial cells and it binds several ligands as AGEs, S100 and Aβ. For example, it participates in the resolubilization process of Aβ that contributes to the reduction of cerebral amyloid burden.26 Since the presence of RAGE in the brain depends on the ligand concentration in the medium, under normal conditions there is a low expression of RAGE, whereas in pathologic conditions (i.e., inflammation), ligand accumulation triggers its expression.66 Clearance of Aβ involves mainly two RAGE isoforms: (i) soluble RAGE (sRAGE), which lacks the transmembrane region and is used as ligand decoy in the bloodstream; and (ii) membranal RAGE (mRAGE), which has a cytosolic stem that activates several signaling pathways involved in the production of ROS, activation of p38 and JNK3, among others, related to Aβ neurotoxicity.67 Additionally, cytosolic RAGE is increased in Alzheimer’s disease triple transgenic mouse model (3XTgAD).68 It has been reported that overexpressed RAGE is localized close to Aβ deposits, contributing to the pathophysiology of AD.69 According to Western blot, PCR, and confocal microscopy data, the significant increase of total, membranal, and soluble RAGE isoforms in the brain of arsenic exposed animals suggests a response to increased Aβ levels, and hence, to a higher interaction with Aβ. It is known that RAGE is involved in (i) increased amyloid burden in the brain parenchyma and (ii) transport of circulating amyloid across the BBB for resolubilization of Aβ. Both mechanisms alter the clearance system and contribute to the neurovascular

3.7. LRP1 Expression. Since LRP1 is the main receptor mediating the outflow of Aβ through BBB, levels of this protein were evaluated in total lysate to establish if this receptor has a participation in the increase of Aβ levels in brain. The levels of LRP1 exhibited no significant differences among control and arsenic treated groups as observed in Figure 3C (100.00 ± 17.83 vs 89.71 ± 9.15 control and treated group respectively; p = 0.6347 two-tailed).

4. DISCUSSION This study demonstrates that chronic arsenic exposure during development affects several regulation points of Aβ levels in rat brain and that these effects are accompanied by behavioral deficits. Furthermore, arsenic exposure affects BBB clearance system by increasing expression of RAGE. Assessment of APP levels was performed in brain total lysate by Western blot assay and its gene expression estimated by RTqPCR. Our results show that arsenic exposure results in a 25% increase of APP levels expressed as membrane-bound Aβ protein and a three-fold increase in APP transcript levels when compared to the control group. Additionally, we found that arsenic exposure is able to increase BACE1 activity and consequently, the levels of soluble Aβ(1−42) in 1.25-times over to the control group. The foregoing suggests a stimulatory action of arsenic over BACE1, which would favor the amyloidogenic pathway by raising Aβ(1−42) levels. Our group previously reported that acute DMAV exposure in primary cultures of Tg2576 embryo brains results in Aβ overproduction and increase of membranal APP with no statistical changes in BACE1 activity. In contrast, exposure to arsenite results in decrease of sAPPα, Aβ, and sAPPβ suggesting inhibition of the activity of γ- and α-secretase on APP.2 Since it is documented that following administration of oral inorganic arsenic in animal models, DMAV is the major arsenic metabolite in the brain,22,24 and it could be the main responsible for the effects found in this study. Arsenic has been described as an inducer of inflammatory responses by activating ERK1/2, JNK, and p38 mitogenactivated protein kinases (MAPKs) pathways, and in consequence induces the expression of nuclear factor kappa B (NF-kB) and nuclear factor E2-related factor (Nrf2).43−45 According to our findings, arsenic or its metabolite DMAV increase the activity of BACE1. A possible explanation to this fact is that BACE1 expression is mediated by activation of MAPKs pathways and eukaryotic translation initiation factor 2α (eIF2α). In this sense, NF-κB may influence BACE1 activity in astrocytes and Aβ-exposed neurons from CNS under stress conditions.46,47 On the other hand, a correlation between oxidative damage derived from ischemic injury and the increase of BACE1 activity in differentiated neuroblastoma cells has been established.48 Furthermore, evidence supports the fact that hydrogen peroxide is able to induce the expression of BACE1 gene.49 Several studies have revealed that arsenic exposure in animals and humans results in the increase of oxidative stress and the levels of inflammatory factors such as NF-κB.50−52 Particularly, it has been reported that arsenic is able to increase oxidative stress in the brain of rats exposed to similar doses of arsenic used in the present study.53 Further research is required to establish the role of arsenic-induced oxidative stress on BACE1 activity in cultured cells. In contrast to the present study, previous results of our group showed that BACE1 activity is not modified in primary cell cultured neurons from T2576 mice acutely exposed to sodium 18


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precursor protein α; sAPPβ, secreted amyloid precursor protein β; sRAGE, soluble form of RAGE

dysfunction in AD.70−72 It has been proposed that Aβ elimination from brain results from an equilibrium of two processes through the BBB, where the transporter LRP1 conducts outflow and, RAGE intakes Aβ from blood flow. In normal conditions, this mechanism is balanced in such way that outflow of Aβ from brain parenchyma is higher than intake, leading to the elimination of the peptide from brain by a dilution (clearance) process. It has also been reported that LRP1 is increased during inflammatory processes, which might attenuate inflammation since LRP1 regulates NF-κB production.73 According to our results, arsenic exposure induces no changes in LRP1 expression, whereas RAGE is overexpressed, suggesting that the neural damage induced by arsenic exposure is a consequence of a disturbance in the equilibrium of Aβ clearance that leads to an increased Aβ accumulation in brain tissue and the lack of a strong LRP1-mediated antiinflammatory response. In conclusion, inorganic arsenic exposure increases the production of Aβ(1−42) by increasing BACE1 activity and by significantly raising the expression of APP and RAGE in brain tissue from an animal model with no genetic predisposition to AD. Moreover, our data indicate that arsenic is an environmental factor that increases neuronal dysfunction and decreases the ability of cerebral clearance, supporting the hypothesis that early exposure to metals may contribute to trigger the development of neurodegenerative diseases.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +52 (444) 826 2300 ext 6425. ORCID

Sergio Zarazúa: 0000-0002-8881-1742 Funding

The present study was supported by grants from PROMEP (103.5/12/3953) and CONACYT (241009) for S.Z.; CONACYT (105937), UASLP (CO9-FAI-03-21.21), and P/PIFI2012−24MSU0011 × 10 −13 for M.E.J.-C.; CONACYT (232762) for H.H.-M., and fellowships 503319 for S.A.N., 276577 for A.C.-Z., and DSA/103.5/16/7283 for B.O.B. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors wish to thank Erika Chi for the valuable support in histological techniques.

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ABBREVIATIONS 3XTgAD, triple transgenic model of Alzheimer’s disease; Aβ, amyloid-β; APP, amyloid precursor protein; AGEs, advanced glycation end products; BACE-1, beta-site amyloid precursor protein cleaving enzyme 1; BBB, blood−brain barrier; DMAV, dimethylarsinic acid; eIF2α, eukaryotic translation initiation factor 2α, ERK 1/2, extracellular signal-regulated kinases 1/2; ICP, inductively coupled plasma-mass spectrometry; JNK, c-Jun N-terminal kinase; LRP-1, low-density lipoprotein receptorrelated protein 1; mRAGE, membranal form of RAGE; NF-kB, nuclear factor kappa B; NOAEL, no-observed-adverse-effectlevel; Nrf2, nuclear factor E2-related factor; RAGE, receptor for advanced glycation end products; RFU, relative fluorescence units; ROS, reactive oxygen species; sAPPα, secreted amyloid 19


Article

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