Exposure to Traffic-Generated Pollutants Exacerbates the Expression of Factors Associated with the Pathophysiology of Alzheimer’s Disease in Aged C57BL/6 Wild-Type Mice

Abstract

Background:

Multiple studies report a strong correlation between traffic-generated air pollution-exposure and detrimental outcomes in the central nervous system (CNS), including Alzheimer’s disease (AD). Incidence of AD is rapidly increasing and, worldwide, many live in regions where pollutants exceed regulatory standards. Thus, it is imperative to identify environmental pollutants that contribute to AD, and the mechanisms involved.

Objective:

We investigated the effects of mixed gasoline and diesel engine emissions (MVE) on the expression of factors involved in progression of AD in the hippocampus and cerebrum in a young versus aged mouse model.

Methods:

Young (2 months old) and aged (18 months old) male C57BL/6 mice were exposed to either MVE (300μg/m³ PM) or filtered air (FA) for 6 h/d, 7 d/wk, for 50 d. Immunofluorescence and RT-qPCR were used to quantify oxidative stress (8-OHdG) and expression of amyloid-β protein precursor (AβPP), β secretase (BACE1), amyloid-β (Aβ), aryl hydrocarbon receptor (AhR), cytochrome P450 (CYP) 1B1, angiotensin-converting enzyme (ACE1), and angiotensin II type 1 (AT1) receptor in the cerebrum and hippocampus, in addition to cerebral microvascular tight junction (TJ) protein expression.

Results:

We observed age-related increases in oxidative stress, AhR, CYP1B1, Aβ, BACE1, and AT1 receptor in the CA1 region of the hippocampus, and elevation of cerebral AβPP, AhR, and CYP1B1 mRNA, associated with decreased cerebral microvascular TJ protein claudin-5. MVE-exposure resulted in further promotion of oxidative stress, and significant increases in AhR, CYP1B1, BACE1, ACE1, and Aβ, compared to the young and aged FA-exposed mice.

Conclusion:

Such findings suggest that MVE-exposure exacerbates the expression of factors in the CNS associated with AD pathogenesis in aged populations.

Keywords

Air pollution Alzheimer’s disease AT1 receptor reactive oxygen species

INTRODUCTION

Alzheimer’s disease (AD) is currently the most common form of dementia, and the 5th leading cause of death worldwide [1, 2]. The incidence of AD has doubled since the year 2000; it is expected to triple by the year 2050 due to multiple factors including increased lifespans, increased incidence of comorbidities that increase risk, such as cardiovascular di-sease, improved diagnosis, as well as increased exposure to harmful environmental contaminants [2, 3]. AD pathology in the brain is characterized by the accumulation of amyloid-β (Aβ) plaques and neuro-fibrillary tangles consisting of aggregates of tau protein, as well as neuroinflammation and neurodegeneration. Aβ is produced in the brain via the proteolytic cleavage of the amyloid-β protein precursor (AβPP) by α-secretase, β-secretase (BACE1), and γ-secretase. Processing of AβPP by BACE1 favors the amyloidogenic pathway and is correlated with the accumulation of Aβ-associated plaques within the brain [4]. There is no cure for AD, only palliative pharmacotherapeutic options to slow the progression and treat memory loss without treating its underlying causes [3].

Due to a lack of viable treatment options, identifying and mitigating the health and environmental risk factors for AD is vital for the world’s aging population. Currently, the most prominent risk factor for AD is aging, but cerebrovascular diseases such as stroke, obesity, dyslipidemia, cardiovascular disease, as well as genetics and smoking have been associated with increased risk of AD [5]. There is strong evidence that there is also a significant contribution of environmental factors to AD pathogenesis. Several recent studies have reported a positive correlation between exposure to air pollution and the incidence and development of AD pathology even in childhood [6 –10]. Although there is a clear association between exposure to urban air pollution and AD, epidemiological and laboratory exposure studies have not yet fully elucidated the contributing pollutants and mechanisms involved. Recent studies have shown a strong correlation bet-ween exposure to the particulate matter (PM) fraction of urban air pollution, a majority of which is generated from motor vehicle emissions, and the incidence of AD [11, 12]. Mechanisms of PM-mediated contributions to AD pathology is associated with microg-lial activation in the CA1 pyramidal neurons of the hippocampus, which are the most vulnerable to AD [11]. PM exposure from motor vehicle emissions has been associated with pathological alterations in the expression of Aβ protein accumulation and phos-phorylated tau protein, both of which represent hallmarks of AD pathology in the brain [13, 14]. While exposure to diesel engine emissions, which comprises a significant portion of traffic-generated ambient air pollution [15], has been shown to mediate neuroinflammation and plaque formation in a mouse model of AD [16, 17]; the contribution of mixed gasoline and diesel engine emissions (MVE) to the promotion of AD pathology has not been thoroughly investigated.

The interactions of traffic-generated air pollutants with the central nervous system (CNS) are vast and complex. Many studies reveal a strong correlation between components of traffic generated air pollution and detrimental outcomes on the CNS, including stroke, neuroinflammation, and neurodegeneration [18 –25]. We, as well as others, have previously reported that exposure to components of urban air pollution results in disruption of the blood-brain barrier (BBB) integrity and increased permeability via the degradation of tight junction (TJ) proteins [19 , 24–27]. The BBB is composed of microvascular endothelial cells joined together by TJ proteins such as occludin and claudin-5, astrocytes, and pericytes that serve as a physical and chemical barrier regulating the transport into and out of the brain parenchyma. The BBB plays a significant role in mitigating the toxicant-induced brain disorders [28], and the disruption of this barrier is associated with contributing to the progression of AD [20, 29].

The renin-angiotensin system (RAS) is a hormonal signaling system that contributes to homeostasis. Angiotensin (Ang) II, the primary active peptide of the RAS, is produced systemically via the conversion of angiotensinogen to Ang I, via renin, and then conversion of Ang I to Ang II via the angiotensin-converting enzyme (ACE1). A local RAS has also been identified in the CNS, where astrocytes secrete angiotensinogen, which is subsequently processed to Ang II [30, 31]. Ang II exerts its effects in the brain via receptors AT1 and AT2, which are expressed differentially in the CNS [32, 33]. Excessive Ang II –AT1 receptor signaling in the brain is associated with increased oxidative stress in neurons and glial cells [34 –38], cerebrovascular dysfunction [39], cognitive impairment [40], and increased Aβ production, which has been confirmed by AT1 receptor antagonist studies [41 –44].

Increased oxidative stress in the brain is also associated with the pathogenesis of AD and is known to occur with age, disease, altered mitochondrial fun-ction, decreased antioxidant defense, environmental exposures, as well as other factors [45]. While reactive oxygen species (ROS) are generated through multiple mechanisms in the brain, one potential con-tributor related to air pollution exposure could be through xenobiotic biotransformation into active ROS metabolites. The aryl hydrocarbon receptor (AhR) is a ligand-activated receptor that is involved in multiple signaling pathways in the brain, but it is classically characterized for the regulation of the transcription of multiple factors involved in xenobi-otic biotransformation, including cytochrome (CYP) P450s family members, namely CYP1A1 and CYP1B1. CYP1B1 is a monooxygenase involved in Phase I biotransformation, and its activity has been associated with metabolic activation of environmental carcinogens, including those found in ambient air pollution [46]. Alterations of AhR signaling have been associated with inflammation in the brain [47] and was recently reported to be upregulated in the aging brain, with even further increases in expression present in the serum and brains of AD patients [48].

It has previously been reported by our laboratory, as well as others, that exposure to MVE results in increased BBB disruption [25 –27], inflammation [27, 49], microglial activation [49], ROS [27], and cerebral microvasculature RAS signaling [25] in C57BL/6 mice; however, the effects of MVE expo-sure on the hippocampal expression of factors associated with AD pathology have not been investigated. Due to variation in expression of RAS components with age, growing evidence that excessive AT1 receptor signaling in the hippocampus region is associated with increased Aβ production, and the correlation between traffic-generated urban air pollution and the pathogenesis of AD; there is a clear gap in knowledge of the mechanisms governing these interactions. As such, we investigated the hypothesis that exposure to traffic-generated air pollution (MVE) in either young or aged mice (C57BL/6 wild-type mice) results in increased Aβ production, associated with AT1 receptor expression.

MATERIALS AND METHODS

Animals and inhalation exposure protocol

Two-month-old and 18-month-old male C57BL/6 mice were fed standard mouse chow and allowed to acclimate 30 days prior to initiation of exposure. Mice were then randomly grouped to be exposed by whole-body inhalation to either 300μg PM/m³ mixture of whole gasoline engine exhaust and diesel engine exhaust (MVE: 50μg PM/m³ gasoline engine emissions + 250μg/m³ PM/m³ diesel engine emissions, n = 16) or filtered air (FA: controls, n = 16) for 6 hours/day, 7 days/week for 50 days, as previously described [26]. MVE was created by combining exhaust from a 1996 GM gasoline engine and a Yanmar diesel generator system and characterized for chemical and PM components, as previously repor-ted [27 , 51]. The chemical characteristics for each exposure chamber have previously been reported in [49]. Mice were housed in standard shoebox cages within an AAALAC International-approved rodent housing facility (2 m³ exposure chambers) throughout the study, which maintained constant tempera-ture (20°C–24°C) and humidity (30% –60% relative humidity). Mice had access to chow and water ad libitum throughout the study period, except during daily exposures. All procedures were approved by the Lovelace Respiratory Research Institute’s Animal Care and Use Committee and conform to the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH Publication No. 85-23, revised 1996).

Tissue collection

Animals were sacrificed within 14–16 h after the final exposure. Mice were anesthetized with Euthasol (0.1 ml per 30 g mouse) and euthanized by exsanguination, brains carefully dissected, and the meninges gently removed. Brains were cut (coronal plane/cut) at ∼ Bregma 0 –Bregma –2.12 mm, and fixed in Histochoice (VWR, Irving, TX) at 4°C overnight. Brain tissue was then rehydrated in 30% sucrose/PBS (weight/vol) at 4°C overnight, embedded in Tissue Freezing Medium (TBS, IMEB Inc., San Marcos, CA) and frozen on dry ice for sectioning. All remaining cerebral tissue was immediately snap-frozen in liquid nitrogen and stored at –80°C until processed for molecular analysis.

Immunofluorescent staining of brain tissue

Brain sections (10μm) were prepared for either single or double immunofluorescence, using techniques previously described by our laboratory [57] with the following primary antibodies: 8-OHdG (1:1000; Santa Cruz Biotechnology, Dallas TX, #sc66036), occludin (1:500; Abcam, Cambridge, MA, #168986), claudin-5 (1:1000; Abcam #15106), AT1 receptor (1:1000; Abcam #18801), AhR (1:100; In-vitrogen, Waltham, MA, #MA1-513), CYP1B1 (1:500; Invitrogen #PA5-28040), von Willebrand factor (vWF) (1:1000; Abcam #11713), Aβ (1:500, Novus Biologicals, Littleton, CO, #NBP2-13075), ACE (1:1000, Santa Cruz Biotechnology #sc-23908), or BACE1 (1:1000, Santa Cruz Biotechnology #sc-33711SS). The secondary antibodies anti-rabbit Alexa Fluor 555, anti-mouse Alexa Fluor 555, anti-mouse Alexa Fluor 488, and anti-sheep Alexa Fluor 488 were used at a concentration that was double that of the primary antibody used (1:200 –1:2000). A minimum of 8–10 vessels on each section (2 sections per slide), two slides, and n = 3 per group were used for analysis, as previously described by our laboratory [57]. Slides were imaged under fluorescent microscopy at 10× and 40× with the appropriate excitation/emission filter, digitally recorded, and analyzed using image densitometry with Image J software (NIH). The fluorescence in the CA1 area of the hippocampus (3–6 sections) was analyzed using beacons to quantify in the specific region outlined in Fig. 1 for consistency across sections and slides.

Fig. 1

Representative image of hippocampus CA1 region used for immunofluorescence analysis.

Real time quantitative reverse transcription polymerase chain reaction (RT-qPCR)

Gene expression of AT1 receptor, AβPP, AhR, and CYP1B1 in cerebral tissue was analyzed using the appropriate forward and reverse primers (Table 1), via real-time RT-qPCR, as previously described [57]. RNA was isolated using a Tissue Lyser system and RNeasy Mini kit (Qiagen) for tissue, following the manufacturer protocol. Real time RT-qPCR was completed and analyzed in the BIORAD CFX96 (Hercules, CA) using the appropriate primers, as previously described by our laboratory [27]. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal control. Results were analyzed from n = 6–8 animals from each group.

Table 1

Mouse primers used for real time RT-qPCR

AhR	FP: 5^′-CGGCTTCTTGCAAAACACAGT-3^′
	RP: 5^′-GTAAATGCTCTCGTCCTTCTTCATC-3^′
AβPP	FP: 5^′-GACCACTCGACCAGGTTCTG-3^′
	RP: 5^′-GATGATGGCGCCTTTGTTCG-3^′
AT1	FP: 5^′-GTTGGGAGGGACTGGATGATG-3^′
	RP: 5^′-CACTTGATCTGGTGATGGGCTTT-3^′
CYP1B1	FP 5^′-AATGAGGAGTTCGGGCGCACA-3^′
	RP 5^′-GGCGTGTGGAATGGTGACAGG-3^′
GAPDH	FP: 5^′-CATGGCCTTCCGTGTTCCTA-3^′
	RP: 5^′-GCGGCACGTCAGATCCA-3^′

AhR, aryl hydrocarbon receptor; AβPP, amyloid β precursor protein; AT1, Angiotensin II receptor type 1; CYP1B1, cytochrome P450 1B1 enzyme; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase.

Statistical analysis

Data are shown as mean±SEM. A 2-way ANOVA with post hoc Tukey’s test was used to analyze statistical significance between age and exposure, and age x exposure interactions for each endpoint. Statistical analyses were conducted using Sigma Plot 12.0 (Systat, San Jose, CA). A p < 0.05 was considered statistically significant for all endpoints.

RESULTS

Exposure to MVE increases oxidative stress in the hippocampus of aged C57BL/6 male mice

As increased oxidative stress is a hallmark of AD pathology, we analyzed the expression of 8-hy-droxy-2’-deoxyguanosine (8-OHdG), a modification of DNA induced by ROS, in the hippocampus of our study animals. Compared to young animals ex-posed to FA (Fig. 2A) or MVE (Fig. 2B), we observed a significant elevation in 8-OHdG expression in the CA1 region of the hippocampus in aged mice exposed to FA (Fig. 2C), which is even further increased in aged mice exposed to MVE (Fig. 2D), as shown in Fig. 2E. The 2-way ANOVA showed that age (F = 34.501, p < 0.001) and exposure (F = 9.701, p = 0.003) mediated the increase in oxidative stress in the in hippocampus; however, there was also a significant age×exposure interaction (F = 8.361; p = 0.005).

Fig. 2

Representative images of 8-hydroxy-2’-deoxyguanosine (8-OHdG, red) in the hippocampus CA1 region in either (A) young (2 months old) male C57BL/6 mice exposed to filtered air (FA), (B) young mice exposed to 300μg PM/m³ of mixed vehicle emissions (MVE) for 6 h/d, for 50 d, (C) aged (18 months old) male C57BL/6 mice exposed to FA, or (D) aged mice exposed to MVE. E) Fluorescence was quantified and represented as mean fluorescence for CA1 region in each animal. n = 3 animals per group, 4–5 sections per animal. Blue fluorescence = nuclear Hoechst stain. 10× magnification (scale bar = 400μm) with 40× magnification (CA1 region) inset (scale bar = 100μm) image. Statistical analyses conducted using a 2-way pairwise ANOVA. ^*p < 0.050 compared to young FA; ^†p < 0.050 compared to young MVE; ^‡p < 0.050 compared to aged FA.

Hippocampus aryl hydrocarbon receptor expression and activity are elevated with MVE-exposure in aged C57Bl/6 male mice

The AhR has recently been reported to be upregulated in the brain with age and is even further increased in the serum and brains of AD patients [48]. Furthermore, AhR activation results in increased expression of CYP enzymes, including CYP1B1, which is associated with increased oxidative stress through mediating Phase I oxidation reactions of xenobiotics. Thus, we quantified the expression of AhR and CYP1B1 in the hippocampus and transcr-ipt levels in the cerebrum of our study animals. Compared to young FA and MVE (Fig. 3A, and B, re-spectively, we observed a significant increase in AhR expression in the CA1 region of the hippocampus in the aged FA mice (Fig. 3C). There was an even further induction of AhR in the CA1 of the hippocampus in the brains of aged MVE-exposed mice (Fig. 3D), compared to the aged FA mice, as quantified in Fig. 3E. The F value for age was 139.209 (p < 0.001), while the F value for exposure was 6.745 (p = 0.015), and there was a significant age x exposure interaction (F = 5.364, p = 0.028). In agreement with the hippocampal expression, we measured an increase in AhR mRNA in the cerebrum of both aged FA and MVE-exposed animals, compared to young FA and MVE brains (Fig. 3F). However, AhR mRNA expression was significantly increased in the cerebrum of aged MVE-exposed mice, compared to the FA exposed aged mice (Fig. 3F); the F value for age was 25.974 (p < 0.001), while the F value for exposure was 5.850 (p = 0.022), with no age x exposure interaction noted.

Fig. 3

Representative images of Aryl hydrocarbon receptor (AhR, green) expression in the CA1 region of the hippocampus in either (A) young (2 months old) male C57BL/6 mice exposed to filtered air (FA), (B) young mice exposed to 300μg PM/m³ of mixed vehicle emissions (MVE) for 6 h/d, for 50 d, (C) aged (18 months old) male C57BL/6 mice exposed to FA, or (D) aged mice exposed to MVE. (E) Fluorescence was quantified and represented as mean fluorescence for the CA1 region in each animal. n = 3 animals per group, 4–5 sections per animal. Blue fluorescence = nuclear Hoechst stain. 10× magnification (scale bar = 400μm) with 40× magnification (CA1 region) inset (scale bar = 100μm) image. (F) Mean normalized gene expression of cerebral AhR expression, as determined by real time RT-qPCR, n = 8 per group. Data are expressed as mean normalized gene expression using ΔΔC_T values, with GAPDH used as the house-keeping gene. ^*p < 0.050 compared to young FA; ^†p < 0.050 compared to young MVE; ^‡p < 0.050 compared to aged FA using a 2-way ANOVA.

Similarly, compared to expression in the young FA (Fig. 4A) and MVE-exposed (Fig. 4B) mice, we observed a significant increase in hippocampal expression of CYP1B1 in the aged FA (Fig. 4C), mice which was exacerbated in the brains of the MVE-exposed mice (Fig. 4D, E). The F value for age was 130.983 (p < 0.001), while the F value for exposure was 19.906 (p < 0.001), and there was a significant age x exposure interaction (F = 5.083, p = 0.032). Un-like expression in the hippocampus, cerebral CYP1B1 mRNA expression was only observed to be increased in the aged mice exposed to MVE, compared to all other study groups (Fig. 4F). The 2-way ANOVA showed that exposure (F = 5.491; p = 0.028), but not age (F = 2.853, p = 0.104) was associated with the induction of cerebral CYP1B1 mRNA transcript expression.

Fig. 4

Representative images of Cytochrome P450 1B1 (CYP1B1, red) expression in the CA1 region of the hippocampus in either (A) young (2 months old) male C57BL/6 mice exposed to filtered air (FA), (B) young mice exposed to 300μg PM/m³ of mixed vehicle emissions (MVE) for 6 h/d, for 50 d, (C) aged (18 months old) male C57BL/6 mice exposed to FA, or (D) aged mice exposed to MVE. E) Fluorescence was quantified and represented as mean fluorescence for the CA1 region in each animal. n = 3 animals per group, 4–5 sections per animal. Blue fluorescence = nuclear Hoechst stain. 10× magnification (scale bar = 400μm) with 40× magnification (CA1 region) inset (scale bar = 100μm) image. F) Mean normalized gene expression of cerebral CYP1B1 expression, as determined by real time RT-qPCR, n = 8 per group. Data are expressed as mean normalized gene expression using ΔΔC_T values, with GAPDH used as the house-keeping gene. ^*p < 0.050 compared to young FA; ^†p < 0.050 compared to young MVE; ^‡p < 0.050 compared to aged FA using a 2-way ANOVA.

MVE-exposure mediates increased cerebral transcript levels of AβPP in aged C57BL/6 male mice

AβPP is the precursor protein for Aβ production in the brain. When AβPP is proteolytically cleaved by secretase enzymes, Aβ is produced and can accumulate in neurotoxic amyloid plaques, which are a hallmark of AD pathology in the brain. Compared to young mice exposed to either FA or MVE, we observed a significant induction of AβPP mRNA expression in the cerebrum of aged mice (Fig. 5). The 2-way ANOVA analysis showed AβPP was strongly associated with age (F = 31.147, p < 0.001), but not statistically associated with exposure (F = 2.773; p = 0.109) or the interaction of age x exposure. As BACE1 is one of the key secretase enzymes involved in proteolytic cleavage of AβPP to Aβ, leading to amyloidogenesis, we analyzed BACE1 expression in the CA1 region of the hippocampus. Compared to young animals exposed to FA (Fig. 6A) or MVE (Fig. 6B), we quantified a statistical elevation in BACE1 in the hippocampus of aged mice. Compared to aged FA mice (Fig. 6C), there was a significant increase in BACE1 expression in the hippocampus of aged MVE-exposed mice (Fig. 6D), as quantified in Fig. 6E. The 2-way ANOVA analysis showed that age (F = 93.741, p < 0.001) and exposure (F = 38.044, p < 0.001) were associated with BACE1 expression; there was also a significant age×exposure interaction noted (F = 37.166, p < 0.001).

Fig. 5

Real time RT-qPCR analysis of cerebral mRNA expression of amyloid-β protein precursor (AβPP) in young (2 months old) or aged (18 months old) male C57BL/6 mice exposed to either filtered air (FA) or mixed vehicle emissions (MVE; 300μg PM/m³) for 6 h/d, 7 d/wk, 50 d. Data are expressed as mean normalized gene expression using ΔΔC_T values, with GAPDH used as the house-keeping gene. ^*p < 0.050 compared to young FA; ^†p < 0.050 compared to young MVE; ^‡p < 0.050 compared to aged FA using a 2-way ANOVA.

Fig. 6

Representative images of β-secretase (BACE1, red) in the hippocampus CA1 region in either (A) young (2 months old) male C57BL/6 mice exposed to filtered air (FA), (B) young mice exposed to 300μg PM/m³ of mixed vehicle emissions (MVE) for 6 h/d, for 50 d, (C) aged (18 months old) male C57BL/6 mice exposed to FA, or (D) aged mice exposed to MVE. E) Fluorescence was quantified and represented as mean fluorescence for the CA1 region in each animal. n = 3 animals per group, 4–5 sections per animal. Blue fluorescence = nuclear Hoescht stain. 10× magnification (scale bar = 400μm) with 40× magnification (CA1) inset (scale bar = 100μm) image. Statistical analyses conducted using a 2-way pairwise ANOVA. ^*p < 0.050 compared to young FA; ^†p < 0.050 compared to young MVE; ^‡p < 0.050 compared to aged FA.

Exposure to MVE induces increased expression of members of the RAS signaling pathway in the hippocampus of aged C57Bl/6 male mice

We have previously reported that MVE-exposure mediates increased cerebrovascular Ang II - AT1 receptor signaling, and increased expression and secretion of ACE1 by astrocytes in a BBB co-culture, in response to factors present in the plasma of MVE-exposed C57BL/6 mice [25]. In the current study, compared to young mice exposed to either FA (Fig. 7A) or MVE (Fig. 7B), we measured an increase in ACE1 expression in the hippocampus of aged mice exposed to MVE (Fig. 7D), which was significantly increased above that observed in aged FA exposed mice (Fig. 7C). The expression of ACE1 in the aged FA was also statistically increased in the hippocampus, compared to young animals (Fig. 7E, p = 0.037). The 2-way ANOVA showed both an age (F = 20.536, p < 0.001) and exposure (F = 7.021, p = 0.011) statistical relationship. Since increased Ang II - AT1 receptor signaling has been associated with AD pathology in the brain [39, 40], we next analyzed expression of the AT1 receptor at the transcript (cerebrum) and protein level (hippocampus) in our study animals. Compared to the expression of AT1 receptor in the hippocampus of the young FA (Fig. 8A) and young MVE exposed (Fig. 8B) animals, we quantified a significant induction of AT1 receptor expression in the hippocampus of the aged FA (Fig. 8C) and aged MVE-exposed (Fig. 8D) mice, as shown in the graph labeled Fig. 8E. The F values from the 2-way ANOVA were F = 19.723 (p < 0.001) for age and F = 1.597 (p = 0.214) for exposure. There was no statistical difference in AT1 receptor expression between the aged FA and aged MVE groups due to large intragroup variability of expression in the aged MVE group. In agreement with these findings, we observed an induction of AT1 receptor mRNA expression in the cerebrum of aged mice, compared to young mice, regardless of the exposure (Fig. 8F: age F value = 15.140, p < 0.001; exposure F value = 2.644; p = 0.188).

Fig. 7

Representative images of Angiotensin converting enzyme-1 (ACE1, red) in the hippocampus CA1 region in either (A) young (2 months old) male C57BL/6 mice exposed to filtered air (FA), (B) young mice exposed to 300μg PM/m³ of mixed vehicle emissions (MVE) for 6 h/d, for 50 d, (C) aged (18 months old) male C57BL/6 mice exposed to FA, or (D) aged mice exposed to MVE. (E) Fluorescence was quantified and represented as mean fluorescence for the CA1 region in each animal. n = 3 animals per group, 4–5 sections per animal. Blue fluorescence = nuclear Hoechst stain. 10× magnification (scale bar = 400μm) with 40× magnification (CA1 region) inset (scale bar = 100μm) image. Statistical analyses conducted using a 2-way pairwise ANOVA. ^*p < 0.050 compared to young FA; ^†p < 0.050 compared to young MVE; ^‡p < 0.050 compared to aged FA.

Fig. 8

Representative images of Angiotensin II type 1 receptor (AT1 receptor, red) expression in the CA1 region of the hippocampus in either (A) young (2 months old) male C57BL/6 mice exposed to filtered air (FA), (B) young mice exposed to 300μg PM/m³ of mixed vehicle emissions (MVE) for 6 h/d, for 50 d, (C) aged (18 months old) male C57BL/6 mice exposed to FA, or (D) aged mice exposed to MVE. E) Fluorescence was quantified and represented as mean fluorescence for the CA1 region in each animal. n = 3 animals per group, 4–5 sections per animal. Blue fluorescence = nuclear Hoechst stain. 10× magnification (scale bar = 400μm) with 40× magnification (CA1 region) inset (scale bar = 100μm) image. (F) Mean normalized gene expression of cerebral AT1 receptor expression, as determined by real time RT-qPCR, n = 8 per group. Data are expressed as mean normalized gene expression using ΔΔC_T values. Statistical analyses conducted using a 2-way pairwise ANOVA. ^#p = 0.057 compared to young FA; ^*p < 0.050 compared to young FA; ^†p < 0.050 compared to young MVE.

As increased AT1 receptor and ROS signaling is associated with increased Aβ production, and since we observed an increase in AβPP and BACE in our aged mice, which was further exacerbated by MVE-exposure, we next quantified Aβ expression in the hippocampus of our study animals. Compared the young FA (Fig. 9A) and MVE (Fig. 9B) mice, we observed an increase of Aβ expression in the CA1 of the hippocampus of the aged FA-exposed (Fig. 9C) mice, with further exacerbation observed in the aged MVE-exposed mice (Fig. 9D), as shown in the gra-ph labeled Fig. 9E. The 2-way ANOVA showed age (F = 25.430, p < 0.001) and exposure (F = 5.829; p =0.047) mediated hippocampal Aβ expression. When looking at an overlay/merged image of AT1 rece-ptor and Aβ protein expression in the hippocampus, there appears to be some colocalization within the brains of aged mice (Supplementary Figure 1).

Fig. 9

Representative images of amyloid-β (Aβ, green) expression in the CA1 region of the hippocampus in either (A) young (2 months old) male C57BL/6 mice exposed to filtered air (FA), (B) young mice exposed to 300μg PM/m³ of mixed vehicle emissions (MVE) for 6 h/d, for 50 d, (C) aged (18 months old) male C57BL/6 mice exposed to FA, or (D) aged mice exposed to MVE. E) Fluorescence was quantified and represented as mean fluorescence for the CA1 region in each animal. n = 3 animals per group, 4–5 sections per animal. Blue fluorescence = nuclear Hoechst stain. 10× magnification (scale bar = 400μm) with 40x magnification (CA1 region) inset (scale bar = 100μm) image. Statistical analyses conducted using a 2-way pairwise ANOVA. ^#p = 0.057 compared to young FA; ^*p < 0.050 compared to young FA; ^†p < 0.050 compared to young MVE; ^‡p < 0.050 compared to aged FA.

MVE-exposure mediates decreased TJ protein, claudin-5, in the cerebral microvasculature of aged C57Bl/6 male mice

We have previously reported that exposure to MVE results in altered cerebral microvascular integrity, including decreased TJ protein expression, associated with ROS, and increased AT1 receptor ex-pression [25 –27]. As vascular disease and altered BBB permeability are strongly associated with AD pathophysiology [20, 29], we analyzed TJ proteins claudin-5 and occludin in the cerebral microvasculature of our study animals. In agreement with our previous findings, we observe a significant decrease in claudin-5 protein expression in cerebral microvasculature in young MVE-exposed mice, compared to young FA exposed mice (Fig. 10). There is a further reduction of claudin-5 in the microvasculature of both aged FA and aged MVE-exposed mice (Fig. 10). The respective F values from the 2-way ANOVA analysis were age F = 30.939, p < 0.001; exposure F = 5.119, p = 0.029; as well as a significant age x exposure interaction (F = 14.481, p < 0.001). Analysis of occludin expression showed no statistical difference across any groups (Supplementary Figure 2).

Fig. 10

Representative images of tight junction protein, claudin-5, staining (red) and endothelial cell marker vonWillebrand factor (vWF, green) expression in the cerebral microvasculature of either young (2 months old) male C57BL/6 mice exposed to filtered air (FA), young mice exposed to 300μg PM/m³ of mixed vehicle emissions (MVE) for 6 h/d, for 50 d, aged (18 months old) male C57BL/6 mice exposed to FA, or aged mice exposed to MVE. Fluorescence was quantified and represented as mean fluorescence from the overlay image for each animal, as shown in the graph. n = 3 animals per group, 2 slides per animals, and a minimum of 8–10 vessels (50μm diameter or less) on each section (2 sections per slide) were used for analysis. Blue fluorescence = nuclear Hoechst stain. 40× magnification, scale bar = 100μm. Statistical analyses conducted using a 2-way pairwise ANOVA. ^*p < 0.050 compared to young FA; ^†p < 0.050 compared to young MVE.

DISCUSSION

Multiple epidemiology studies have reported ambient air pollution, a majority of which is derived from MVE contributes to a cognitive and motor decline in both aging [52 –57] and young [14 , 59] populations. In regions of the world with air pollution levels that exceed the World Health Organization (WHO) and U.S. Environmental Protection Agency (EPA) standards, it is reported that the pathogenic onset of AD is initiated in populations prior to the age of 20 [6, 14]. Traffic-generated pollutants are the major contributing factor to unsafe air pollution levels associated with these detrimental CNS outcomes, including hyperphosphorylated tau protein and Aβ accumulation, which are hallmarks of AD [6 , 24]. Recent literature reported that exposure to traffic-generated PM initiates the expression of factors associated with AD pathogenesis in young (otherwise healthy) wild-type animals [11, 60]. Interestingly, in our study, we did not observe any significant alterations in measured endpoints in our young (2 months old) animals with MVE exposure. The different outcomes in the young animals are likely due to the difference in exposure chemistries and constituents (mixture of emissions versus PM), method of exposure (inhalation versus instillation), concentration, or the duration of the current studies compared to others previously published.

As much of the literature in the field focuses on exposure to one component of traffic-generated air pollution, in the current study, we analyzed the effects of combined gasoline and diesel engine emissions (MVE) exposure that would be more translatable to an occupational or near-road way exposure scenario. We have previously investigated the effects of MVE in the brains of C57BL/6 mice and reported increased inflammation, microglial activation, BBB disruption, and increased RAS signaling, all of which have been linked to the progression of AD [25 , 49]. However, most of these studies were all conducted in young animals (age 2–3 months old). Thus, in the current study, we analyzed age- and exposure-mediated outcomes in the hippocampal CA1 region of young (2 month old) and aged (18 months old) mice exposed to either MVE or FA over 50 days.

Oxidative stress from ROS has also been shown to contribute to the early stages of AD prior to the onset of Aβ accumulation and clinical symptoms [45]. In the current study, we observed an induction of oxidative stress, measured by 8-OHdG, in the hippocampus of the aged FA mice compared to the young FA and MVE-exposed groups; the 8-OHdG expression is further exacerbated in the CA1 region in the aged MVE-exposed animals. This trend in increased ROS production in aged mice is in agreement with previous studies [45], and the exacerbation by MVE-exposure suggests that environmental exposures can contribute significantly to the production of ROS in the CA1 region of the hippocampus. ROS production in the brain can come from multiple sources, including activation of microglial cells and inflammatory signaling. Correlated to the oxidative stress results in the current study, our collaborators have previously shown that microglial activation is increased with age in C57BL/6 mice, with an even further (statistical) increase observed in MVE-exposed aged animals [49]. Furthermore, neuroinflammation, measured by TNFα mRNA levels, was also found to be highest in the MVE-exposed aged animals, and expression was not significantly elevated in FA controls or young mice [49].

MVE contains multiple components such as PM, metals, and polyaromatic hydrocarbons (PAHs), all of which can potentially interact with the brain parenchyma via direct entry through the olfactory bulb or by circulating factors via the lung brain axis [49]. PAHs are ligands for the AhR, which results in transcriptional activation of xenobiotic-metabolizing enzymes, including CYP1B1. AhR signaling participates in the neuroinflammatory response via microglial and astrocyte activation, and evidence from animal studies report that aging is associated with changes in AhR expression [47, 48]. Furthermore, AhR levels are observed to be significantly elevated in postmortem hippocampal tissue and serum of human AD patients [48]. Such observations support a role for the AhR in the aging process, and the progression of neurodegenerative diseases such as AD. In agreement with this premise, we observed a significant increase in cerebral AhR mRNA expression in the aged FA and MVE-exposed mice, compared to their younger counterparts. However, cerebral AhR mRNA expression was found to be more pronounced in the aged MVE-exposed mice, compared to the aged FA-exposed mice. Interestingly, when analyzing cerebral CYP1B1 mRNA expression, only the aged MVE-exposed group showed a statistical increase in expression. A recent study using CYP1B1 knockout mice suggests that CYP1B1 may be involved in AD pathology and cognitive impairment [62]. Furthermore, CYP1B1 deficiency resulted in decreased oxidative stress and Aβ deposition in the hippocampus region in the HF-fed animals, compared to wild-type controls [62]. Taken together, MVE-exposure mediates activation of AhR and CYP1B1 expression, which may contribute to increased oxidative stress and Aβ accumulation in the aged brain.

Multiple studies have reported an increase in the expression of AβPP and the accumulation of Aβ in the brain resulting from air pollution exposure [13 , 63]. In the brain, Aβ is generated from proteolytic cleavage of AβPP by BACE1. As such, increased BACE1 expression can result in an accumulation of Aβ oligomers [4]. We observed a significant increase in the cerebral expression of AβPP mRNA in our aged mice, compared to the young groups. Additionally, we quantified a significant increase in BACE1 in the hippocampus of aged mice, which showed a statistical age x exposure interaction. Associated with the increase in AβPP mRNA, and BACE1, we also observe an increase in Aβ oligomer expression in the CA1 region of the hippocampus in aged mice, which is further exacerbated with MVE-exposure. A previous study reported that exposure to ambient PM_2.5 resulted in increased BACE1 levels in the cerebral cortex, associated with increased Aβ in the temporal cortex, but decreased AβPP expression in C57BL/6 mice [64]. The differences in the results from this study, and our current study, are likely because we analyzed total brain homogenate for AβPP at the transcript level versus protein expression in the cortex; however, further studies are necessary to quantify hippocampal levels resulting from MVE-exposure. Collectively, our findings indicate that age is the main factor mediating AβPP mRNA expression, but MVE-exposure increases the expression of BACE1 and Aβ accumulation in the brains of our aged mice, although additional studies are necessary to analyze BACE1 activity levels.

In the CNS, there is a local RAS, where angiotensinogen is secreted by astrocytes and is converted into its active form, Ang II, via renin and ACE1, which signals through the AT1 and AT2 receptors [30, 31]. Excessive AT1 receptor signaling in the brain is associated with increased ROS product-ion, the progression of cerebrovascular diseases, and contributes to cognitive impairment [37 , 40]. This premise has been further confirmed through the use of AT1 receptor blockers, which show inhibition of AT1 receptor signaling ameliorates the hypertensive effects that contribute to neurovascular disease and is associated with reduced incidence and progression of AD [42, 43]. Ang II-AT1 receptor signaling has recently been associated with AD through increased Aβ production via various mechanisms, including increased AβPP mRNA and increased BACE1 activity [65]. We have previously reported that exposure to MVE in C57BL/6 mice results in increased plasma Ang II levels, and AT1 receptor expression in cerebral microvasculature [25]. In the current study, we characterized AT1 receptor mRNA expression in the brain, as well as AT1 receptor expression in the CA1 region of the hippocampus. We observed a significant increase in AT1 receptor mRNA levels in our aged mice, regardless of exposure, associated with elevated AT1 receptor expression in the CA1 of the hippocampus, compared to that observed in the young mice. Interestingly, we also observed an increase in ACE1 expression in the CA1 region of the hippocampus in the aged MVE-exposed animals, compared to all other groups. While ACE1 has been reported to degrade secreted Aβ in a cell culture model [66], and mediate the conversion of the more pathogenic Aβ_1–42 peptide to the less neurotoxic Aβ_1–40 peptide [67], its role in the accumulation of Aβ in the brain in vivo appears to be more complex. This is likely because the increased ACE1 activity can lead to increased Ang II production in the brain. Ang II in the brain has been shown to promote BACE1 activity and Aβ accumulation in the brain through AT1 receptor signaling [68]. Furthermore, immunohistochemical analysis of brains from postmortem AD patients shows increased levels of Ang II, ACE1, and AT1 receptors in the pyramidal neurons of the cortex (predominately cortical layer V), compared to samples from aged control brains [69]. In our study, we observed an age-related increase in AT1 receptor expression in the hippocampus; however, MVE-exposure in the aged mice mediated the elevation in ACE1 expression and associated exacerbation of Aβ accumulation. Further studies are necessary to characterize whether ACE1 expression and activity are correlated in the brains of our MVE-exposed animals.

Alterations in BBB integrity is associated with the progression and development of neurovascular and CNS disorders, including AD [20]. We have previously reported that exposure to MVE increases BBB permeability through the degradation of tight junction proteins, such as claudin-5 [26, 27]. In agreement with our previous studies, we observed that MVE-exposure was associated with decreased cerebral microvascular claudin-5 expression, regardless of age, although aged FA mice also showed decreased claudin-5 expression. While the BBB integrity is known to diminish with age, the observed alteration in the integrity of the BBB in young MVE-exposed animals may allow for increased xenobiotic transport into the brain that can initiate early signaling events involved in inflammation and the pathogenesis of neurodegenerative disorders. In a separate experiment, we also analyzed cerebral microvascular fibrosis, as increased microvascular fibrosis that occurs with age is believed to contribute to ischemia and dementia [70]. Our results showed no significant alteration in cerebral microvascular fibrosis across FA or MVE groups; however, there was increased perivascular fibrosis in the aged versus young MVE-exposed brains (Supplementary Figure 3), indicating that that age was primarily driving the response when the MVE-exposure insult was present.

Taken together, the results from this study show that inhalation exposure to MVE, in an aged mouse model, leads to elevated AhR expression and signa-ling in the brain, as well as increased BACE1 and Aβ accumulation in the CA1 of the hippocampus, associated with increased expression of RAS sig-naling pathway members (ACE and AT1 receptor expression). Moreover, MVE-exposure also incre-ased oxidative stress and altered cerebral microvascular integrity, which are also associated with the progression of CNS pathologies. While these findings suggest a need for further mechanistic studies to determine regulatory pathways altered by traffic-generated air pollution exposure that may contribute to AD pathogenesis or progression, it is important to note the limitations of the current study. The concentration of MVE used for the current study (300μg/m³ PM), which was chosen for comparison to previous studies from our laboratory, would be considered in the “Unhealthy” Air Quality Index category and is much higher than would be experienced in most human exposure scenarios. However, it is only ∼25% greater than PM levels that have been reported in highly polluted cities worldwide, which are often reporting monthly averages [71]. Additionally, the endpoints analyzed in this study represent only one time point in the study (50 d, subchronic), and thus are not necessarily representative of acute or chronic outcomes of MVE-exposure. Finally, only male mice were used in the current study, and thus further studies are necessary to determine if the exposure-related outcomes are similar in the female brain. As traffic-generated air pollution has been reported to contribute to detrimental outcomes in the CNS, including the promotion of AD pathology as early as childhood [24], it is imperative to identify contributing pollutants for regulatory actions and mechanistic pathways for preventative and therapeutic targets.

Footnotes

ACKNOWLEDGMENTS

We would like to thank Dr. Jacob McDonald and the Chemistry and Inhalation Exposure Group at Lovelace Biomedical Research Institute for the characterization and monitoring of the animal exposures.

This work was supported by National Institute of Environmental Health Sciences at the National Institute of Health grants R00ES016586 and R15ES026795 to A.K.L., and also University of North Texas internal funding to A.K.L.

Authors’ disclosures available online ().

The supplementary material is available in the electronic version of this article: .

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