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. 2015 Jun 26;147(1):222–234. doi: 10.1093/toxsci/kfv124

Cyclic Ozone Exposure Induces Gender-Dependent Neuropathology and Memory Decline in an Animal Model of Alzheimer’s Disease

Hasina Akhter *, Carol Ballinger *, Nianjun Liu †, Thomas van Groen ‡, Edward M Postlethwait *, Rui-Ming Liu *,§,1
PMCID: PMC4607745  PMID: 26116027

Abstract

Alzheimer’s disease (AD) is a major cause of dementia in the elderly. Although early-onset (familial) AD is attributed to gene mutations, the cause for late-onset (sporadic) AD, which accounts for 95% of AD cases, is unknown. In this study, we show that exposure of 6-week-old amyloid beta precursor protein (APP)/presenilin (PS1) overexpressing mice, a well-established animal model of AD, and nontransgenic littermates to a cyclic O3 exposure protocol, which mimics environmental exposure episodes, accelerated learning/memory function loss in male APP/PS1 mice but not in female APP/PS1 mice or nontransgenic littermates. Female APP/PS1 mice had higher brain levels of amyloid beta peptide (Aβ42) and Aβ40, compared with male APP/PS1 mice; O3 exposure, however, had no significant effect on brain Aβ load in either male or female mice. Our results further show that male APP/PS1 mice had lower levels of antioxidants (glutathione and ascorbate) and experienced augmented induction of NADPH oxidases, lipid peroxidation, and neuronal apoptosis upon O3 exposure, compared with female APP/PS1 mice. No significant effect of O3 on any of these parameters was detected in nontransgenic littermates. In vitro studies further show that 4-hydroxynonenal, a lipid peroxidation product which was increased in the plasma and cortex/hippocampus of O3-exposed male APP/PS1 mice, induced neuroblastoma cell apoptosis. Together, the results suggest that O3 exposure per se may not cause AD but can synergize with genetic risk factors to accelerate the pathophysiology of AD in genetically predisposed populations. The results also suggest that males may be more sensitive to O3-induced neuropathophysiology than females due to lower levels of antioxidants.

Keywords: ozone, Alzheimer’s disease, oxidative stress, gender

Alzheimer’s disease (AD), a neurodegenerative disease, is a major cause of dementia in the elderly. Despite extensive studies, there is no effective treatment for this devastating disease due to an incomplete understanding of its etiology and pathogenesis. Early-onset (familial) AD, which accounts for < 5% of the cases, is attributed to mutations in the amyloid beta precursor protein (APP) or presenilin (PS) genes. The cause for the majority (95%) of AD cases (sporadic form), which occurs after 65 years, however, remains unclear and likely involves a combination of genetic and environmental factors. Identification of environmental factors that interact with genetic factors, leading to the development of late-onset AD, therefore, will be critical not only for understanding the etiology of AD but also for developing strategies for its prevention and treatment.

Ozone (O3) is one of the most abundant urban pollutants that impacts over 30% of the U.S. population due to living in geographic locales that experience unhealthful levels. In addition, workers (eg, pulp mills, outdoor construction, and copy machine operation) may be intermittently exposed to relatively high levels of O3 (Chan and Wu, 2005; Henneberger et al., 2005; Zhou et al., 2003). Although the respiratory system is a major target of O3, emerging evidence indicates that O3 exposure causes myriad pathophysiologic effects in many other organ systems including the cardiovascular system, spleen, liver, and brain (Aibo et al., 2010; Chuang et al., 2009; Hassett et al., 1985; Maniar-Hew et al., 2011; Thomson et al., 2013). It has been reported that O3 exposure induces mitochondrial damage and oxidative stress in vascular tissues (Chuang et al., 2009), influences immune cell trafficking/function (Maniar-Hew et al., 2011), and enhances hepatic susceptibility to other toxicants (Aibo et al., 2010). It has also been shown that O3 exposure induces lipid peroxidation in the hippocampus and cortex in rats (Dorado-Martinez et al., 2001; Martinez-Canabal et al., 2008). Importantly, it has been reported that children and young adults who lived in areas with high levels of air pollutants including O3 exhibited AD-like pathologies in their brains (Calderon-Garciduenas et al., 2004, 2008). An epidemiology study conducted in Taiwan and involving 95 690 individuals aged 65 or older further show that the risk for AD increased by 211% with every 10.91 ppb increase in atmospheric O3 concentration during the 10-year follow-up period from 2001 to 2010 (Jung et al., 2015). Nonetheless, although these case and epidemiology studies show an association between exposure to air pollutants and AD, whether exposure to O3 alone, especially under the conditions that mimic human exposure scenarios, contributes to the development of AD, whether males and females are equally sensitive to O3-induced neuropathology, and how inhaled O3 affects brain structure/function are unknown and warrant further investigation.

To begin our understanding of the potential contribution of environmental factors to the development of AD, we exposed APP/PS1 mice, a well-established animal model of AD, and nontransgenic littermates to a cyclic O3 exposure regimen for 4 months, which mimics environmental exposure scenarios. The results show that cyclic O3 exposure accelerated memory loss in male APP/PS1 mice, although it had no significant effect on the cognitive function of female APP/PS1 mice or wild Type (WT) mice (nontransgenic littermates). Our results suggest that O3 exposure per se may not cause AD but it can synergize with genetic risk factors to accelerate the pathophysiology of AD, especially in males, probably due to a lower antioxidant capacity.

MATERIALS AND METHODS

Animals and ozone exposure

APP/PS1 double transgenic mice, purchased from Jackson Laboratory MICE (JAXMICE) which bear 2 AD mutations (a mutant human PS1 [DeltaE9] and a chimeric mouse/human APP), were maintained on a C57BL/6 genetic background. The mice used in the experiments, both APP/PS1 mice and nontransgenic littermates, were derived from more than 15 breeding pairs and were randomly assigned to O3 or filtered air (FA) treatment group. APP/PS1 transgenic and nontransgenic littermates had comparable body weights. When reaching 6 weeks of age, APP/PS1 mice and age-matched nontransgenic littermates underwent a series of O3 exposure cycles, consisting of 5 days of O3 exposure (0.8 ppm, 7 h/day) followed by 9 days of filtered air (FA) recovery, for 8 cycles at the University of Alabama at Birmingham (UAB) Environmental Exposure Facility as we have described before (Katre et al., 2011). FA controls were treated similarly in all aspects, except for the absence of O3 in the chambers, and were done in parallel. Animals were allowed free access to water while food was withheld during exposures to prevent ingestion of constituents oxidized by O3, which could introduce confounders.

The open field and elevated plus maze test

After exposure, the open field test (day 1) and then the elevated plus maze test (day 2) were conducted to assess general activity levels and fear, ie, time spent in the “open” center or open arm versus the “safe” sides or close arm (Liu et al., 2002) at UAB animal behavior core facility. The open field maze consists of an arena of 42 × 42 cm2 with clear plexiglass sides (20 cm high). The arena is subdivided into 3 areas, the open center area, the sides, and the wall (ie, rearing). The animal was put in the arena, and observed for 4 min, with a camera-driven tracker system, ie, Ethovision (Noldus, The Netherlands), which recorded the position of the animal in the arena at 5 frames/s. The elevated plus maze consists of 4 arms (31 × 5 cm) that are raised 40 cm above the table with 2 closed arms (15 cm high sides of non–see-through material), 2 open arms (sided with transparent material), and the center area where the arms meet. The animal was put in the arena and time each mouse spent in 2 arms and center area during 4-min observation was recorded with a camera as described earlier. The data were analyzed as time spent in each area, speed of locomotion, rearing, defecation, etc.

Water maze test

After the open field and elevated plus maze tests, learning and memory function was assessed at UAB animal behavior core facility by the well-established Morris water maze (Liu et al., 2002), which is consisting of a blue plastic round basin (diameter, 112 cm) filled with water (22°C, controlled by room temperature) to a height of 31 cm and a see-through round platform, 10 cm in diameter, located 0.5 cm below the water surface in the middle of northeast quadrant. A ventilation system was located above the swimming pool to remove any potential odd smell. During day 1 to day 5 of the testing period, the mice were placed in the water next to and facing the wall successively in north (N), east (E), south (S), and west (W) positions (4 trials/day/mouse with the intertrial interval 2 min). All of the mice were tested on the same day in a counterbalanced order. In each trial, the mouse was allowed to swim until it found the hidden platform or until 60 s had elapsed, at which point the mouse was guided to the platform. The mouse was then allowed to sit on the platform for 10 s before being picked up. The escape latency (from the time a mouse was placed into the water till it found the platform), swim path-length (distance), and swim speed were recorded simultaneously and blinded with a camera-driven tracker system, ie, the Noldus Ethovision system (version 7.1). On day 5, a probe trial was further conducted after Morris water maze test by removing the platform and recoding the time of each mouse spent in each pool quadrant in a 1-min trial.

Tissue collection

After memory function tests, mice were euthanized, blood withdrawn from the heart, and transcardial perfusion performed (Liu et al., 2011). The brain was dissected sagittally into right and left hemispheres with the right hemisphere fixed in 10% PBS buffered formalin and the left hemisphere dissected and the hippocampus and cerebral cortex frozen in liquid nitrogen immediately for subsequent biochemical analyses. All procedures involving animals were approved by the Institutional Animal Care and Use Committee at the University of Alabama at Birmingham.

Analysis of amyloid beta peptide accumulation in the cortex and hippocampus

The amounts of sodium dodecyl sulfate soluble (SDS) and insoluble amyloid beta peptide (Aβ42) and Aβ40 in the cortex and hippocampus were quantified using the ELISA kits from Covance (Emeryville, California) as we have described previously (Liu et al., 2011). Briefly, the brain tissues were homogenized in an Aβ extraction buffer containing 20 mM Tris-HCl (pH 7.6), 137 mM NaCl, 1% Triton X-100, 2% SDS, and protease inhibitors (complete protease inhibitor cocktail, Boehringer Mannheim, Mannheim, Germany), and centrifuged at 100 000× g for 1 h. The supernatant was collected (SDS soluble) and stored at −80°C until analysis. Pellets were dissolved in 70% FA, incubated at room temperature with gentle shaking for 2 h, and then centrifuged at 100 000× g for 1 h. The supernatants were collected (SDS insoluble) and neutralized/diluted with a neutralizing buffer containing 1 M Tris, 0.5 M Na2HPO4, and 0.05% NaN3 before analysis by ELISA. Brain Aβ deposits (plaques) were assessed by immunohistochemical staining techniques using monoclonal anti-human Aβ antibody 6E10 (Covance) and semiquantified by determining the percentage of the total section positively stained with Aβ antibody using Axiovision automatic measurement software (Zeiss, Germany) as we have described previously (Akhter et al., 2011; Liu et al., 2011).

Western analysis

For analysis of the proteins that were modified by 4-hydroxynonenone (4HNE), a lipid peroxidation product, the cortex and hippocampus were homogenated in a buffer containing 0.005% diethylenetriaminepentaacetic acid to limit adventitious iron-mediated lipid auto-oxidation. Fifty micrograms of protein from each sample were subjected to 10% SDS-PAGE gel electrophoresis using nonreducing buffers. 4-HNE-protein adducts were revealed by anti-4HNE antibody (alpha Diagnostic, HNE-11S). For Western analyses of other proteins, the following antibodies were used as we have described previously (Akhter et al., 2011; Liu et al., 2011): Caspase-3 (Cell Signaling, Cat No. 9662X), p53 and Bax (Santa Cruz, Cat No. sc6243 and sc6236), Synaptophysin (Sigma, Cat No. S5768), growth associated protein 43 (GAP-43) (Santa Cruz, Cat No. sc17790), Nicotinamide Adenine Dinucleotide Phosphate (NADPH) oxidase 2 (Nox2) (Santa Cruz, Cat No. sc5827), and Nox4 (Dr David Lambeth, Emory University). Semiquantification of the bands was performed using Image J software and normalized to β-actin.

HPLC analyses of low-molecular-weight antioxidants in the cortex and hippocampus

The concentrations of glutathione (GSH), glutathione disulfide (GSSG), and reduced ascorbate (AH2) in mouse brain were determined using a well-established High Performance Liquid Chromatography (HPLC) method as we have described previously (Liu et al., 2012). Briefly, mouse brain tissues were homogenized in m-phosphoric acid and centrifuged at 13 000× g for 30 min. Protein concentrations in centrifuged pellets were measured using the Pierce BCA Protein Assay (Pierce Biotechnology, Rockford, Illinois). Supernatants were filtered through a 0.22 -µm syringe filter, and samples were fractioned in triplicate on a Shimadzu LC-10Ai HPLC (Shimadzu Scientific Instruments, Columbia, Maryland) using a Phenomenex Luna C18(2) 250 × 4.6 mm, 5 -µm reversed-phase column. The concentrations of GSH, GSSG, and AH2 were determined by standard curves run simultaneously with samples and normalized by protein content.

Detection of apoptotic cells in the cortex and hippocampus

Apoptotic cells in the cortex and hippocampus were revealed by Terminal deoxynucleotidyl transferase dUTP Nick End Labeling (TUNEL) assay (Roche Diagnostics GmbH, Mannheim, Germany) as we have described previously (Liu et al., 2012). Briefly, tissue slides were deparaffinized and rehydrated through graded concentrations of ethanol and then boiled in antigen unmasking solution (Vector). Slides were blocked with 5% BSA for 1 h at room temperature and then incubated with the reaction mix containing deoxynucleotidyl transferase and Fluorescein Isothiocyanate-deoxy Uridine Diphosphate (FITC-dUDP) at 37°C for 1 h. Nuclei were stained with propidium iodide (PI, Vector). Apoptotic cells were revealed with epifluoresecent microscope (Nikon TE2000E-2) and analyzed using Image Pro Plus version 5.1.2 (Media Cybernetics). The number of PI (Vector)-stained TUNEL-positive cells were counted in 5 different areas (covering 70% of cortex and hippocampus areas in total) of the cortex and hippocampus in each mouse, 5–6 mice per group, and expressed as percentages of total cells.

Flow cytometry analysis of apoptotic neuroblastoma cells

Human neuroblastoma cells (SHSY5Y), purchased from American Tissue Culture Collection (ATCC), cultured in a 1: 1 mixture of Eagle’s Minimum Essential Medium (ATCC) and F-12 medium (Grand Island Biological Company-GIBCO) supplemented with 10% non–heat-treated FBS and 1% penicillin-streptomycin, were treated with 0, 5, and 10 µM of 4-HNE for 24 h. Apoptotic cell death was analyzed by Flow Cytometry techniques using Alexa FluorR 488 annexin V Kit (Invitrogen) as we have described previously (Huang et al., 2012).

Caspase-3/7 activity assay

SHSY5Y cells were seeded in 96-well plates at the density of 2 × 104/well for 24 h and then treated with 0, 5, and 10 µM 4-HNE for 24 h. Caspase-3/7 activity in SHSY5Y cells was determined using Apo-ONE Homogenous Caspase-3/7 Assay kit (Promega) and normalized with protein content as we have described previously (Huang et al., 2012) to confirm the activation of apoptotic pathways by 4HNE.

Statistical analysis

For memory function data (Fig. 1A), R and lme4 software was used to perform a linear mixed-effects analysis of the relationship between escape latencies and genotype and treatment (exposure) for male and female mice separately. As fixed effects, we included treatment, genotype, time, the escape latency of the first day (baseline), their 2-way interactions, and genotype-treatment-time 3-way interactions into the model. As random effects, we had intercepts for subjects as well as by-subject random slopes for the effect of time. Because memory deficit usually becomes evident at day 5, we have also conducted 3-way Analysis Of Variance (ANOVA) (3 factors: genotype, gender, and treatment) on water maze data (escape latencies, swimming speeds, and swimming distances) at day 5 and day 1 (baseline). Tukey’s Honestly Significant Difference test was used for post hoc analysis. For the data presented in Figures 2, 4, and 6A, 2-way ANOVA were performed (no genotype factor). The statistical analyses for the data presented in Figures 3, 5, 6B, and 7 were performed with male and female mice separately by t test.

FIG. 1.

FIG. 1.

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Water maze test of memory function. Memory function was assessed by a well-established Morris Water Maze test as described in the Materials and Methods section. A, Time-dependent changes in the escape latencies of filtered air (FA) and O3 exposed male and female nontransgenic (Top panels) and amyloid beta precursor protein (APP)/presenilin (PS1) transgenic mice (Bottom panels). B, The escape latencies (top panels), swimming distances (middle panels), and swimming speeds (bottom panels) at day 1 and day 5 of water maze test. The statistical analyses were conducted using linear mixed effects model and 3-way ANOVA as described in the Materials and Methods section. The P values were from Tukey’s Honestly Significant Difference post hoc analyses (n = 6–7). C, Open field test and elevated plus maze test results in APP/PS1 mice.

FIG. 2.

FIG. 2.

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Effect of cyclic O3 exposure on Aβ deposition in the cortex and hippocampus of APP/PS1 mice. A, Representative immunohistochemical staining pictures of amyloid beta peptide (Aβ) plaques in the cortex and hippocampus of APP/PS1 mice; B, Quantitative data of Aβ plaques expressed as percentage of total area of the cortex and hippocampus. C, The amounts of sodium dodecyl sulfate (SDS)-soluble and -insoluble Aβ40 and D, the amounts of SDS-soluble and -insoluble Aβ42 in the cortex and hippocampus of APP/PS1 mice. The results were compared between male and female APP/PS1 mice treated with FA or O3 by 2-way ANOVA. The P values were from Tukey post hoc analyses (n = 5–6).

FIG. 4.

FIG. 4.

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Effect of cyclic O3 exposure on the levels of glutathione (GSH) and ascorbate in the cortex and hippocampus of APP/PS1 mice. The amounts of GSH (A), ascorbate (B), and glutathione disulfide (GSSG) (C) in the cortex and hippocampus of APP/PS1 mice were measured by HPLC and normalized by protein as described in Materials and Methods section. The ratios of GSSG to total GSH (D) were calculated by the formula of GSSGx2/(GSSGx2 + GSH). The results were compared between male and female APP/PS1 mice treated with FA or O3 by 2-way ANOVA and the P values were from Tukey post hoc analyses (n = 5–6).

FIG. 3.

FIG. 3.

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Effect of cyclic O3 exposure on lipid peroxidation in the plasma and cortex/hippocampus of APP/PS1 mice. A and B, 4-hydroxynonenone (4HNE) modified proteins in the plasma. Ponceau S staining pictures were used to show equal protein loading (50 µg protein/lane). C and D, 4HNE modified proteins in the cortex and hippocampus. β-Actin was used to show equal protein loading. Top Panels: Representative Western blots; Bottom panels: Semi-quantitative data of the bands indicated by the arrows. The relative densities of the corresponding bands were compared between FA and O3 exposed same gender APP/PS1 mice by t test (n = 6).

FIG. 5.

FIG. 5.

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Effect of cyclic O3 exposure on the expression of NADPH oxidases in the cortex and hippocampus of APP/PS1 mice. Top panels: Representative Western blots of NADPH oxidase 2 (Nox2) and 4 (Nox4) proteins in the cortex and hippocampus of male and female APP/PS1 mice; Bottom panels: Semiquantitative data of Nox2 and Nox4 Westerns. The relative densities of the corresponding bands were compared between FA and O3 exposed same gender APP/PS1 mice by t test (n = 6).

FIG. 6.

FIG. 6.

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Effect of cyclic O3 exposure on the apoptotic cell death of neuronal cells in the cortex and hippocampus of APP/PS1 mice. A, Top panels, representative pictures of apoptotic cell staining (TUNEL assays) in the cortex and hippocampus of FA or O3 exposed APP/PS1 mice. Red color represents nuclei staining (propidium iodide); yellow color, apoptotic cells. Bottom panel, numbers of apoptotic cells, quantified as described in Materials and Methods section and expressed as percentages of total cells. The results were compared between male and female APP/PS1 mice treated with FA or O3 by 2-way ANOVA and the P values were from Tukey post hoc analyses (n = 5–6). B, Top panels, representative Western blots of Caspase-3, cleaved Caspase-3, and Bax (β-actin was used as protein loading control); Bottom panels, semi-quantitative data of Western blots. The relative densities of the corresponding bands were compared between FA and O3 exposed same gender APP/PS1 mice by t test (n = 6). Full color version available online.

FIG. 7.

FIG. 7.

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Effect of cyclic O3 exposure on the expression of synaptophysin and Growth associated protein 43 (GAP-43) in the cortex and hippocampus of APP/PS1 mice. A and B, Representative Western blots of Synaptophysin and GAP-43 in male and female APP/PS1 mice. β-Actin was used to show equal protein loading. C, Semi-quantitative data of Synaptophysin and GAP-43 Western blots. The relative densities of the corresponding bands were compared between FA and O3 exposed same gender APP/PS1 mice by t test (n = 6).

RESULTS

Effects of Cyclic O3 Exposure on the Memory Function in Mice

To explore whether exposure to O3 contributes to the development of AD, 6-week-old APP/PS1 mice and nontransgenic littermates were exposed to a cyclic O3 exposure regimen or filtered air (FA) for 4 months (8 cycles). Learning and memory function was then assessed by Morris water maze. Figure 1A shows the time-dependent changes in the escape latencies of different groups of mice during 5 days of water maze test. Statistical analyses using a linear mixed-effects model show that, for male, the genotype-treatment-time 3-way interaction was significant [F(1, 24) = 4.988, P = .035], suggesting that, for male mice, the effects of genotype and exposure on escape latency interact with each other and vary over time. For female, on the other hand, the genotype-treatment-time 3-way interaction was not significant [F(1, 65.603) = 0.014, P = .906], suggesting that O3 exposure does not worsen memory of female APP/PS1 mice. As memory deficit usually becomes evident at day 5, we further analyzed the day 5 water maze data (escape latencies, swimming speeds, and swimming distances) by 3-way ANOVA (genotype, gender, and treatment 3 factors) (the day 1 data were analyzed as baseline). The results from 3-way ANOVA of the day 5 escape latencies show that there was an overall statistically significant difference in the escape latencies among 8 groups (F7, 184 = 12.0174, P < .0001). Post hoc analyses further show that female APP/PS1 mice had significantly longer escape latencies than female nontransgenic littermates with or without O3 exposure (Fig. 1B, top right panel). Cyclic O3 exposure, on the other hand, significantly increased the escape latencies of male APP/PS1 mice (39.4 ± 4.8 s for O3-exposed vs 14. 8 ± 5.4 s for FA-exposed male APP/PS1 mice, P < .001), although it had no significant effect on the memory of female APP/PS1 mice or nontransgenic littermates (Fig. 1B, top right panel). The results from 3-way ANONA of the day 1 escape latencies show no significant difference overall between different groups (Fig. 1B, top left panel). There was no significant difference overall in swim distances (Fig. 1B, middle panels) or swimming speeds (Fig. 1B, bottom panels) between 8 groups at either day 1 or day 5, although there was a trend of increase in swimming distances, associated with increased escape latencies (Fig. 1B, middle panels). O3 exposure had no significant effect on the performance of mice at the elevated plus maze or open field maze (Fig. 1C). Together, the data suggest that the increase in the escape latencies in O3-exposed male APP/PS1 mice results from a decline in learning/memory function rather than a change in the motor activity or anxiety level.

Effects of Cyclic O3 Exposure on the Aβ Load in the Cortex and Hippocampus

To elucidate whether O3 exposure increases brain Aβ load, we quantified the amounts of SDS-soluble and -insoluble Aβ42 and Aβ40 as well as Aβ plaques in the cortex and hippocampus by ELISA and immunohistochemistry staining, respectively. The results show that there were more Aβ plaques in the cortex and hippocampus of female APP/PS1 mice, compared with male APP/PS1 mice with or without O3 exposure, although the differences were not statistically significant (Fig. 2A and 2B). Two-way ANOVA results further show that there was an overall significant difference between male and female APP/PS1 mice in their brain levels of SDS-insoluble Aβ40 (F = 38.264, P ≤ .001), SDS-soluble Aβ42 (F = 14.817, P < .001), and SDS-insoluble Aβ42 (F = 29.078, P < .001). Post hoc analyses further show that female APP/PS1 mice had significant higher level of SDS-insoluble Aβ40 with (P = .001) or without (P < .0001) O3 exposure (Fig. 2C right side), higher level of SDS-soluble Aβ42 with (P = .014) or without (P = .011) O3 exposure (Fig. 2D, left side), and higher level of SDS-insoluble Aβ42 with (P = .022) or without (P = .001) O3 exposure (Fig. 2D, right side). O3 exposure, however, had no significant effect on the amounts of Aβ plaques or Aβ40 and Aβ42 in either female or male APP/PS1 mice. No Aβ plaque or Aβ40/Aβ42 accumulation was detected in the brain of nontransgenic littermates with or without O3 exposure (data not shown).

Effects of Cyclic O3 Exposure on Lipid Peroxidation in the Plasma and the Cortex/Hippocampus

O3 is a highly reactive oxidant. To further explore the mechanism whereby cyclic O3 exposure accelerated memory loss in male APP/PS1 mice, we analyzed the amounts of protein adducts of 4-hydroxynonenonal (4HNE), a lipid peroxidation product, in the plasma and cortex/hippocampus of APP/PS1 mice and nontransgenic littermates. The results show that cyclic O3 exposure significantly increased the levels of 4-HNE-protein adducts in the plasma (Fig. 3A) and cortex/hippocampus (Fig. 3C) of male, but not female (Fig. 3B and 3D), APP/PS1 mice. No significant change in the amounts of 4HNE-protein adducts was detected in O3-exposed, compared with FA-exposed, nontransgenic littermates (data not shown).

Effect of Cyclic O3 Exposure on the Concentrations of Small Molecule Antioxidants in the Cortex and Hippocampus

GSH is the most abundant intracellular free thiol and an important antioxidant. The ratio of GSH and GSSG, an oxidized form of GSH, determines cell redox status (Schafer and Buettner, 2001). To better understand the mechanism underlying selective increase in lipid peroxidation in O3-exposed male APP/PS1 mice, we measured the concentrations of GSH, reduced ascorbate, another important antioxidant, and GSSG in the cortex and hippocampus of male and female APP/PS1 mice by HPLC. Two-way ANOVA results show that there was an overall significant difference between male and female mice in GSH (F = 26.053, P < .001), reduced ascorbate (F = 14.484, P < .001), and the ratios of GSSG to GSH (F = 21.2, P < .001). Post hoc analyses (Tukey test) further show that the basal (FA-exposed) levels of GSH (P = .003), reduced ascorbate (P = .002), and GSSG (P < .05) were significantly higher in female than in male APP/PS1 mice (Fig. 4A–C). Cyclic O3 exposure, on the other hand, significantly increased the levels of ascorbate (11.33 ± 0.48 for O3 vs 8.36 ± 0.48 for FA, P < .001) and GSSG (0.192 ± 0.016 for O3 vs 0.129 ± 0.016 for FA, P = .04) in male, but not female, APP/PS1 mice (Fig. 4B–D). The results indicate that male APP/PS1 mice had lower levels of antioxidants in their brain and experienced an augmented oxidative stress upon O3 challenge, compared with female APP/PS1 mice.

Effects of Cyclic O3 Exposure on the Expression of NADPH Oxidases in the Cortex and Hippocampus

Nox2 and Nox4 are 2 important producers of reactive oxygen species (ROS) for various types of cells including neurons. The expression of Nox enzymes is also redox regulated (Pendyala et al., 2009). To determine whether O3 exposure induces these 2 ROS-generating enzymes, Nox2 and Nox4 protein levels were analyzed by Western blotting. The results show that cyclic O3 exposure significantly increased protein levels of Nox2 (by 2.4-folds, P < .001) and Nox4 (by 2.1-folds, P < .001) in the cortex and hippocampus of male APP/PS1 mice but only Nox4 (by 1.8-folds, P = .004) in female APP/PS1 mice (Fig. 5). No significant change in either Nox2 or Nox4 expression was detected in nontransgenic littermates after O3 exposure (data not shown).

Cyclic O3 Exposure Induced Apoptotic Cell Death in the Cortex and Hippocampus of Male APP/PS1 Mice

To further explore the mechanism underlying the acceleration of memory loss by O3 in male APP/PS1 mice, we tested whether cyclic O3 exposure induced neuronal apoptosis in the cortex and hippocampus by TUNEL assay. Two-way ANOVA results show that cyclic O3 exposure significantly increased the number of apoptotic cells in male (10.88 ± 0.51 for O3 vs 1.24 ± 0.71 for FA, P < 0.001), but not female, APP/PS1 mice (Fig. 6A). Consistent with the TUNEL results, Western analyses show that cyclic O3 exposure increased the amounts of caspase-3 by 3.4-folds (P < .001), cleaved caspase-3 by 1.8-folds (P < .001), and Bax by 1.6-folds (P = .022) in the cortex and hippocampus of male, but not female, APP/PS1 mice (Fig. 6B). No significant increase in neuronal cell apoptosis or the activity/expression of Caspase-3 and Bax was detected in nontransgenic littermates upon O3 exposure (data not shown).

Effect of Cyclic O3 Exposure on Synaptophysin and GAP-43 Expression in the Cortex and Hippocampus

Synaptophysin is expressed mainly in presynaptical neurons and participates in synaptic transmission. GAP-43, on the other hand, is used as a marker of neuronal cell growth. Therefore, we further examined whether acceleration of memory loss by O3 was associated with suppression of synaptophysin and/or GAP-43 expression. Western analysis results show that there was no significant effect of cyclic O3 exposure on synaptophysin expression in either male or female APP/PS1 mice (Fig. 7A and 7C) or nontransgenic littermates (data not shown). The expression of GAP-43, on the other hand, was significantly decreased in O3-exposed female APP/PS1 mice, compared with FA-exposed female APP/PS1 mice (100% ± 7.3% for FA vs 58.7% ± 5.2% for O3, P = .001), but not male, APP/PS1 mice (Fig. 7B and 7C) or nontransgenic littermate (data not shown).

4-HNE Induced Apoptosis in Cultured Neuroblastoma Cells

To determine whether increased levels of 4-HNE contribute to neural cell death observed in the brain of O3-exposed male APP/PS1 mice, effects of 4HNE on cultured neuroblastoma cells (SHSY5Y), a commonly used neuronal cell model, were studied. The results show that treatment of SHSY5Y cells with 10 µM 4HNE significantly increased apoptotic cell number (Fig. 8A, P < .001), which was accompanied by an increase in the activity of Caspase-3/7 (Fig. 8B, P < .05) as well as induction of p53 and Bax (Fig. 8C–E, P ≤ .002).

FIG. 8.

FIG. 8.

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Induction of neuroblastoma cell apoptosis by 4HNE. A, Apoptotic cell death of neuroblastoma (SHSY5Y) cells was analyzed by flow cytometry techniques as described in Materials and Methods section; B, The activity of Caspase-3/7 was determined by Apo-ONE Homogenous Caspase-3/7 Assay kit. RFU, relative fluorescence units. C, Representative Western blots of p53 and Bax. β-Actin was used to show equal protein loading. D, Semi-quantitative data of p53 and Bax Western blots (band densities relative to β-actin). One-way ANOVA was conducted to analyze the data with the P values from Tukey post hoc analysis (n = 3–8).

DISCUSSION

The etiology for the majority of AD (sporadic form) is unclear. Emerging evidence suggests that exposure to environmental toxic chemicals including lead, arsenic, pesticides, and air pollutants may contribute to the development of AD (Calderon-Garciduenas et al., 2008, 2012; Jung et al., 2015; Landrigan et al., 2005; Levesque et al., 2011; O’Bryant et al., 2011; Wu et al., 2008). O3 is a highly reactive oxidant and one of the most abundant ambient urban pollutants. Tropospheric O3 is formed when nitrogen oxides and volatile organic compounds, released mainly from fossil fuel combustion, react in the presence of sunlight. O3 concentrations in the atmosphere, therefore, are high during the day (sunlight) and low in the evening. In urban settings, several days of elevated O3 are usually followed by longer periods of “clean” air. Although it has been reported that continuous exposure to unhealthy levels of O3 induces a variety of neuropathological changes as well as memory deficits/behavior change in rats (Avila-Costa et al., 1999; Rivas-Arancibia et al., 2010) whether exposure to O3 in such a cyclic intermittent pattern affects memory function, however, is unknown. This study provides the first line of evidence that cyclic O3 exposure had no significant effect on memory function of wild-type mice (nontransgenic littermates); however, it accelerated learning/memory loss in male APP/PS1 mice. As O3 exposure in humans most likely follows a cyclic pattern, the results from our studies are clinically relevant. We would like to mention, however, that the concentration of O3 used in this study is high compared with EPA National Ambient Air Quality Standard (0.075 ppm) as mice are recognized to be less susceptible to O3 insult than humans due to obligatory nose breathing and other intrinsic factors (Air Quality Criteria for Ozone and Related Photochemical Oxidants [Final]. U.S. Environmental Protection Agency, Washington, DC. EPA/600/R-05/004aF-cF, 2006) (Chuang et al., 2009). Therefore, the employed exposure paradigm represents an approach useful for delineating potential pathobiological sequelae in humans. Nonetheless, our results suggest that cyclic O3 exposure may synergize with genetic risk factors to accelerate the pathophysiology of AD in genetically predisposed populations, although it may not cause AD per se.

It has been long debated whether women are in higher risk than men for AD. Although some studies suggested so (Andersen et al., 1999; Di Carlo et al., 2002; Gao et al., 1998), other studies found no gender difference (Barnes et al., 2003; Jorm and Jolley, 1998; Mielke et al., 2014; Ruitenberg et al., 2001). It was also speculated that low participation rate in survey, which led to the underrepresentation of older and demented men and thereby a selection bias, might have contributed to the observed gender difference in the risk of AD (Bonsignore et al., 2002). A recent study further shows, although more women are affected by AD, men have a higher risk of developing mild cognitive impairment, an early stage of AD (Mielke et al., 2014). This study also suggests that a relative lower incidence of AD in men is probably due to a relative longer lifespan of women. Nonetheless, it has become clear that different risk factors may contribute to the development of AD in men and women (Rocca et al., 2014). In this study, we show that exposure to unhealthy level of O3 accelerated memory loss in male but not female APP/PS1 mice. Our data further support the notion that different risk factors may contribute to the development of AD in men and women. It should be stressed, however, that this study investigated the effect of one environmental factor, O3. People are normally exposed to a mixture of different chemicals simultaneously. Interactions between chemicals and their biological functions may lead to enhancement or decrease of the toxicities or biological effects. Therefore, the conclusion from this study is only applied to the conditions used in this study and may not represent real life situation. Nonetheless, the results from this study suggest that males may be more sensitive than females to O3-induced neuropathophysiology.

Accumulation of Aβ in the brain is one of the major pathological features of AD, although the mechanism underlying Aβ accumulation in sporadic AD patients remains unclear. Studies have shown that spatial learning deficits are correlated with hippocampal Aβ42 levels and that reduction of Aβ burden in the brain improves memory, suggesting that brain Aβ accumulation contributes importantly to memory loss in AD (Hardy, 2009). Emerging evidence, especially the results from numbers of large Phase III clinical trials, which show no beneficial effects of Aβ targeting drugs on cognitive function, however, suggests that accumulation of Aβ may not be the cause of memory deterioration in AD (Doody et al., 2013; Karran and Hardy, 2014; Salloway et al., 2014). In this study, we show that female APP/PS1 mice had significant higher levels of Aβ40 and Aβ42 in their brain, compared with male APP/PS1 mice; O3 exposure, however, had no significant effect on brain Aβ load in either male or female mice, APP/PS1 transgenic or nontransgenic (Fig. 2). Furthermore, we show that O3 exposure impaired memory in male APP/PS1 mice but had no significant effect on memory in female APP/PS1 mice or nontransgenic littermates. The correlation results (data not shown) indicate that there is no relation between brain Aβ loads and escape latencies. Our data support the notion that brain Aβ accumulation may not play a critical role in memory decline in AD and suggest that O3 exposure accelerated memory loss in male APP/PS1 mice through other mechanism(s) rather than increasing brain Aβ load. Our results also suggest that Aβ plaque like pathologies observed in the brain of children and young adults living in urban areas with high levels of air pollutants (Calderon-Garciduenas et al., 2008, 2012) may result from exposure to other air pollutants or a mixture of pollutants rather than O3 alone.

The mechanism underlying the different sensitivity of male and female APP/PS1 mice to O3 is unclear. GSH is the most abundant intracellular free thiol and an important antioxidant. Previous studies from this laboratory have shown that GSH concentrations decrease with age in various organs including the brain in rodents and that male mice experience more dramatic aging-related decline in GSH in many organs than female mice (Wang et al., 2003). Our previous studies have also shown that GSH concentration was decreased in red blood cells from male, but not female, AD patients (Liu et al., 2005). Consistent with our previous findings, we show in this study that the concentrations of GSH and ascorbate, another important antioxidant, in the cortex and hippocampus were significantly lower in male than in female APP/PS1 mice under unchallenged conditions. Such a lower antioxidant capacity rendered male APP/PS1 mice higher sensitivity to O3-induced oxidative stress (higher ratio of GSSG to GSH and more 4HNE-protein adducts). Our data suggest that higher sensitivity of male APP/PS1 mice to O3-induced neuropathophysiology is at least in part due to a lower antioxidant capacity in their brain. The mechanism underlying the differential regulation of GSH and ascorbate homeostasis in male and female AD patients and AD mice is unclear at the moment, although different estrogen levels have been suggested (Foster, 2012; Simpkins et al., 2013). More studies are needed to address this important question.

Neuronal cell death is believed to contribute importantly to memory loss in AD. Interestingly, we show in this study that O3 exposure activated apoptotic pathways (Caspase-3 and Bax) and induced neuronal cell apoptosis in the brain of male, but not female, APP/PS1 mice. As oxidative stress has been shown to cause neuronal cell apoptosis, increased sensitivity of male APP/PS1 mice to O3-induced neuronal apoptosis is most likely due to lower concentrations of antioxidants and thus augmented sensitivity to O3-induced oxidative stress. Nonetheless, as increased neuronal cell apoptosis was associated with memory decline in O3-exposed male APP/PS1 mice, whereas no neuronal apoptosis or memory decline was detected in female APP/PS1 mice, it is suggested that neuronal cell apoptosis contributes importantly to O3-induced memory function decline in male APP/PS1 mice.

GAP-43 is expressed at high levels in neuronal growth cones during development and axonal regeneration and is used as a marker of neuronal growth. It has been reported that downregulation of GAP-43 gene expression proceeded and progressed with the widespread synaptic disconnection and dementia in AD (de la Monte et al., 1995). It has also been shown that heterozygous GAP-43 knockout mice have significant memory impairment (Rekart et al., 2005), further supporting the critical role of GAP-43 in memory. In this study, we show that O3 exposure reduced GAP-43 protein level in the cortex and hippocampus of female, but not male, APP/PS1 mice. The mechanism underlying O3-induced suppression of GAP-43 in female APP/PS1 mice is unknown at the moment. ROS have been shown to inhibit GAP-43 expression in neonatal rat brain (Kaewsuk et al., 2009). However, it was male, not female, APP/PS1 mice that experienced a significant increase in oxidative stress upon O3 challenge. Therefore, it is suggested that O3-mediated suppression of GAP-43 in female APP/PS1 mice may not result from increased oxidative stress. As we did not detect any significant effect of O3 on memory function in female APP/PS1 mice, the biological significance of GAP-43 suppression in O3-exposed female APP/PS1 mice remains to be further investigated.

The most challenging question that remains to be answered is how inhaled O3 affects brain structure and function. In other words, what is (are) the signaling molecule(s) that mediates lung-brain effect of inhaled O3 in APP/PS1 mice? As a highly reactive oxidant, O3 reacts quickly with multiple substrates in airway surface lining fluid and produces secondary reactive species such as ozonide radical, singlet oxygen, antioxidant radical intermediates, and lipid ozonation products (Postlethwait et al., 1998; Pryor et al., 1995). The extrapulmonary effects are most likely induced by these secondary reactive species rather than by O3 itself. Several hypotheses have been proposed to explain the extrapulmonary effects of O3 including inflammatory mediators (Kafoury et al., 1999), nitric oxide (Laskin et al., 1994), and reactive lipid peroxidation product(s) (Santiago-López et al., 2010). Interestingly, we show, in this study, that O3 inhalation increased the levels of 4HNE, an end product of lipid peroxidation, in the plasma and in the cortex/hippocampus of male APP/PS1 mice. Where 4HNE was generated and whether 4HNE functions as a signaling molecule mediating the lung-brain effects of O3, however, remains to be determined.

In summary, the results from this study suggest that O3 exposure per se may not cause AD but it may synergize with genetic risk factors to accelerate the pathophysiology of AD in genetically predisposed populations. The results also suggest that males and females may have different sensitivity to O3-induced neuropathophysiology, probably due to different antioxidant capacity.

FUNDING

National Heart, Lung, and Blood Institute (HL088141), National Institute of Aging (R21AG046701), and an Envelope Award from the School of Public Health, University of Alabama at Birmingham (to R.-M.L.); National Institute of Environmental Health Sciences (P01, ES011617 to E.M.P.); UAB Animal Behavioral Assessment Core Facility (P30 NS 47466 to John Hablitz); an award from Alzheimer’s of Central Alabama (to H.A.).

ACKNOWLEDGMENTS

The authors to thank Dr David Lambeth (Emory University) for providing Nox4 antibody; Dr Namasivayam Ambalavanan, Dr Jo Anne Balanay, and Dr Brian A. Halloran for their technical assistance, and Dr Hui-Chien Kuo for her advice in statistical analysis.

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