Chronic Arsenic Exposure Increases AÎ²(1â42) Production and

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Chronic arsenic exposure increases A#(1-42) production and RAGE expression in rat brain Sandra Aurora Niño, Guadalupe Martel-Gallegos, Adriana Castro-Zavala, Benita OrtegaBerlanga, Juan Manuel Delgado, Hector Hernandez-Mendoza, E Romero-Guzman, Judith Rios-Lugo, Sergio Rosales-Mendoza, María Esther Jiménez-Capdeville, and Sergio Zarazua Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.7b00215 • Publication Date (Web): 20 Nov 2017 Downloaded from http://pubs.acs.org on November 21, 2017

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Chemical Research in Toxicology

Chronic arsenic exposure increases Aβ(1-42) production and RAGE expression in rat brain Sandra Aurora Niño1, Guadalupe Martel-Gallegos1, Adriana Castro-Zavala2, Benita Ortega-Berlanga1,6, Juan Manuel Delgado2, Héctor Hernández-Mendoza3,4, Elizabeth Romero-Guzmán3, Judith Ríos-Lugo5, Sergio Rosales-Mendoza6, María E. Jiménez-Capdeville2 and Sergio Zarazúa1* 1

Laboratorio de Neurotoxicología, Facultad de Ciencias Químicas, Universidad Autónoma de San

Luis Potosí, Av. Manuel Nava 6, CP 78210, San Luis Potosí, S.L.P., México. 2

Departamento de Bioquímica, Facultad de Medicina, Universidad Autónoma de San Luis Potosí,

Av. V. Carranza 2405, CP 78210, San Luis Potosí, S.L.P., México. 3

Laboratorio Nacional Forense Nuclear, Instituto Nacional de Investigaciones Nucleares, Carretera

México-Toluca s/n, CP 52750, La Marquesa Ocoyoacac, México. 4

Centro de Biociencias, Universidad Autónoma de San Luis Potosí, Km. 14.5 carretera San Luis

Potosí – Matehuala, Ejido "Palma de la Cruz", Soledad de Graciano Sánchez S.L.P., CP 78321, México. 5

Unidad de Posgrado, Facultad de Enfermería y Nutrición, Universidad Autónoma de San Luis

Potosí, Avenida Niño Artillero 130, CP 78210, San Luis Potosí, S.L.P., México. 6

Laboratorio de Biofarmacéuticos Recombinantes, Facultad de Ciencias Químicas, Universidad

Autónoma de San Luis Potosí, Av. Dr. Manuel Nava 6, SLP, 78210, México ∗Corresponding author: Sergio Zarazúa Laboratorio de Neurotoxicología Facultad de Ciencias Químicas Av. Manuel Nava 6, CP 78210 San Luis Potosí, S.L.P, México. Phone: +52 (444) 826 2300 Ext. 6425 E-mail: [email protected]

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Abstract Chronic arsenic exposure during development is associated with alterations of chemical transmission and demyelination, which result in cognitive deficits and peripheral neuropathies. At 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 verifying behavioral deficits induced by arsenic exposure through contextual fear conditioning, the brains were collected for the determination of total arsenic by ICP-MS, the levels of amyloid precursor protein (APP) 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 to amyloid accumulation. Keywords: Arsenic (As), Contextual Fear Conditioning (CFC), Amyloid Precursor Protein (APP), Amyloid Beta (Aβ), Receptor for Advanced Glycation End Products (RAGE).


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1. Introduction Arsenic exposure from early stages of development generates damage in neurological function. Epidemiological studies demonstrated that arsenic induces learning impairment in children as well as deterioration of cognitive abilities.3,4,5 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.8,9,10,11,12 The detailed exploration of intra and intercellular events affected by arsenic in the central nervous system (CNS) has revealed disturbances on neurotransmitters.11,13,14,15,16 intracellular calcium17,18 and other signaling systems19 as well as inhibition of longterm potentiation.20 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 over-express amyloid precursor protein (APP) and is related to the early development of Alzheimer's disease. Results obtained in our group demonstrated that in vitro exposure to DMAV (10 µM, 12h) modifies APP processing by increasing levels of Aβ(1-40) and a tendency to increase in Aβ(1-42). On the other hand, sodium arsenite (5 µM, 12h)



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.22,23,24 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 Advanced Glycation Products Receptor (RAGE) and low-density lipoprotein receptor-related 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.26,27,28,29 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 blood stream.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 over-expressed in brain of AD patients.33 Based on 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 (3ppm) from intrauterine development until 4 months of age. Although an exposure of 3 ppm of


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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 and/or pups.35,36

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, SigmaAldrich, 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 (BioRad 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 deficits and changes in DNA methylation;,39,40,41 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, RT-qPCR 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 post-conditioning time-point. The instrument designed ad hoc to measure CFC consisted on an acrylic box (20 cm x 25 cm x 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


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(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 post-conditioning 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, resulting 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 ºC until posterior analysis.

2.5. Arsenic quantification Total arsenic was quantified in brain samples as an exposure marker. Approximately 100 mg of brain tissue were 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 an 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 xg 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 and run on SDS-PAGE 10%,


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and transferred to PVDF membranes using a Trans-blot®Turbo TM (Bio-Rad, CA, USA). Membranes were incubated with anti-APP, anti-RAGE, anti-LRP1 and antiβ-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 Image J software version 1.5 (NIH, USA).

2.6.2. Inmunofluorescence 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 TBS-tween. RAGE primary antibody was incubated overnight at 4ºC, and the secondary antibody used was a goat anti-mouse IgG antibody marked with Alexa Fluor 488 (Thermo Scientific, Rockford, IL). Anti-GFAP was used to stain astrocytes. Nuclei were visualized by ytox staining (Molecular



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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, 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

GAGTTCAGGCATCTACTTGTGT-3′;

for

AGTCAGAGGAAGCGGAAATG-3′

and

reverse RAGE:

primer,

forward

reverse

primer, primer,

5′5′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'GTGTGGATTGGTGGCTCTATC-3´

and

reverse,

5'-

CAGTCCGCCTAGAAGCATTT-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 under standard conditions from 100°C to 50°C. PCRs were performed on an Applied Biosystems Step One Real-Time PCR


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System, and the data was 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 were incubated at 37ºC 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 x (sample fluorescence negative control / positive control - negative control).

2.8. ELISA assay 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 were incubated in the microplate coated with the anti-Aβ(1-42) NH3-terminus monoclonal antibody (2h, 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 anti-rabbit antibody, and finally washed and developed with a chromogenic



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solution. Plates were read in a microplate reader (MultiskanTM 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 were 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 pre-cleared with protein A/G PLUS-Agarose (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) during 1 h at 4ºC. 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 Laemmli 1X buffer, boiled for 10 min at 90ºC 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 using the Student-t test or two-way ANOVA with post hoc Tukey test; if the results were non-


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parametric 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.

3. Results

3.1 Validation of the 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, Fig. 1 B). 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 to 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. 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 3-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 the 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


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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 In order 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/reentry pathways through blood-brain barrier (BBB) for circulating Aβ. RAGE levels were estimated in total lysate, membranal and soluble fractions, since 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 β-actin 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 two-tailed). 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



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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

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