Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2021 Sep 1.
Published in final edited form as: Environ Pollut. 2020 May 1;264:114716. doi: 10.1016/j.envpol.2020.114716

Urban airborne PM2.5-activated microglia mediate neurotoxicity through glutaminase-containing extracellular vesicles in olfactory bulb

Xiaoyu Chen 1,#, Jing Guo 1,#, Yunlong Huang 2,3, Shan Liu 1, Ying Huang 1, Zezhong Zhang 1, Fang Zhang 1, Zhongbing Lu 1, Fang Li 1, Jialin C Zheng 2,3, Wenjun Ding 1,*
PMCID: PMC7364855  NIHMSID: NIHMS1599543  PMID: 32559876

Abstract

Emerging evidence has showed that exposure to airborne particulate matter (PM) with an aerodynamic diameter less than 2.5 μm (PM2.5) is associated with neurodegeneration. Our previous studies in vitro found that PM2.5 exposure causes primary neurons damage through activating microglia. However, the molecular mechanism of microglia-mediated neurotoxicity remains to elucidate. In this study, five groups (N = 13 or 10) of six-week-old male C57BL/6 mice were daily exposed to PM2.5 (0.1 or 1 mg/kg/day body weight), Chelex-treated PM2.5 (1 mg/kg/day body weight), PM2.5 (1 mg/kg/day body weight) plus CB-839 (glutaminase inhibitor), or deionized water by intranasal instillation for 28 days, respectively. Compared with the control groups, We found that PM2.5 triggered reactive oxygen species (ROS) generation and microglia activation evidenced by significant increase of ionized calcium binding adaptor molecule-1 (IBa-1) staining in the mouse olfactory bulbs (OB). Data from transmission electron microscope (TEM) images and Western blot analysis showed that PM2.5 significantly increased extracellular vesicles (EVs) release from OB or murine microglial line BV2 cells, and glutaminase C (GAC) expression and glutamate generation in isolated OB and BV2 cells. However, treatment with N-acetylcysteine (NAC) or CB-839 significantly diminished the number of EVs and the expression of GAC and abolished PM2.5-induced neurotoxicity. These findings provide new insights that PM2.5 induces oxidative stress and microglia activation through its metal contents and glutaminase-containing EVs in OBs, which may serve as a potential pathway/mechanism of excessive glutamate generation in PM2.5-induced neurotoxicity.

Keywords: PM2.5, olfactory bulb, extracellular vesicles, glutaminase, glutamate, Neurotoxicity

Graphical Abstract

graphic file with name nihms-1599543-f0001.jpg

1. Introduction

While research on health impacts of airborne particulate matter (PM) with an aerodynamic diameter less than 2.5 μm (PM2.5) has long focused on cardiovascular and pulmonary systems, emerging evidences have now showed that PM2.5 also adversely affects central nerve system (CNS) (Jung et al., 2015; Maher et al., 2016; Xu et al., 2016; Kulas et al., 2018; Zhang et al., 2018; Li et al., 2019). Recently, a population-based cohort study in Taiwan, a case-control study in Denmark and a longitudinal study in Sweden suggested that long-term exposure to PM is associated with increased the risks of Alzheimer’s disease (AD) (Jung et al., 2015) and Parkinson’s disease (PD) (Ritz et al., 2016) and vascular dementia (Oudin et al., 2016). The early study assessing whether air pollution is culpable in neurodegenerative disease is investigated in feral dog populations naturally exposed to polluted urban environments (Calderón-Garcidueñas et al., 2002). Feral dogs living in regions of high pollution showed enhanced oxidative damage, premature presence of diffuse amyloid plaques, and a significant increase in DNA damage in olfactory bulb, frontal, cortex, and hippocampus 1(Calderón-Garcidueñas et al., 2003). Further, dogs exposed show accumulated metals (nickel and vanadium) at target brain regions in a gradient fashion (olfactory mucosa > olfactory bulb (OB) > frontal cortex), implicating the nasal pathway as a key portal of entry of air pollutants to brain (Calderón-Garcidueñas et al., 2002). Specifically, the nose-to-brain transfer route for small inhaled particles has been reported in rodents and primates (Dorman et al., 2006; Garcia et al., 2015; Hopkins et al., 2014). Magnetite pollution nanoparticles have been identified in the OB and brain by magnetic analyses and electron microscopy (Maher et al., 2016). However, the toxic effect on OB exerted by the chemical composition of PM2.5 at a subcellular level remains to elucidate. Also, very little is known about the intracellular molecular targets of PM2.5.

Microglia act as the specialized macrophages in the CNS and respond to pathogens and injury by becoming “activated”. Activated microglia can release inflammatory cytokines, chemokines and other inflammatory factors, leading to neurotoxicity and even neurodegenerative diseases (Yang et al., 2018). We previously reported that PM2.5 induces release of macrophage glutaminase (GLS) through extracellular vesicles (EVs), which underlies the detrimental effects of macrophage neurotoxicity following PM2.5 exposure (Liu et al., 2015). In the CNS, GLS is an enzyme that catalyzes the hydrolytic deamidation of glutamine to glutamate and is thought to play a critical role in excitotoxic glutamate generation during CNS disorders (Shijie et al., 2009). In the brain, two isoforms of GLS1 exist, which include kidney type glutaminase (KGA) and glutaminase C (GAC) (Campos-Sandoval et al., 2015). Our previous study in vitro also found that PM2.5 exposure promotes KGA and GAC release from macrophages. The release of KGA or GAC into extracellular supernatants contributes to excess glutamate production (Liu et al., 2015). Therefore, it is imperative to understand the molecular mechanism of PM2.5-induced cellular GLS1 release.

Extracellular vesicles (EVs) are membrane-bound particles (30–1000 nm) which involves in the exchange of a broad range of bioactive molecules between cells and the microenvironment (Iraci et al., 2017; Kadiu et al., 2012). In the CNS, EVs are directly derived from microglia, astrocytes, neurons, neural stem cells and oligodendrocytes (Lai and Breakefield, 2012). Emerging evidence indicates that EVs play a key role in cell-to-cell communication through secretion of signaling molecules, such as nucleic acids, lipids, and proteins (Pegtel et al., 2014; Saenz-Cuesta et al., 2014). The release of EVs has been recognized as an important modulator not only in CNS physiology but also in neurodegenerative disease states (Rufino-Ramos et al., 2017). However, the role of EVs in PM2.5-induced neurotoxicity remains to be elucidated. In the present study, we first detected deposition and metal components of PM2.5 in OB of mice. We then evaluated oxidative stress, microglia activation, and EVs release from the activated microglia in the PM2.5-exposed olfactory bulb. We further explore the molecular mechanism of the role of EVs in PM2.5-induced neurotoxicity.

2. Materials and Methods

Five groups (N = 13 or 10) of six-week-old male C57BL/6 mice were daily exposed to PM2.5 (0.1 or 1 mg/kg/day body weight), Chelex-treated PM2.5 (1 mg/kg/day body weight, C-PM2.5), or PM2.5 (1 mg/kg/day body weight) plus CB-839 (glutaminase inhibitor) or deionized water (Control) by intranasal instillation for 28 days, respectively. Murine microglial line BV2 cells were treated with PM2.5 at the final concentration of 50 μg/ml for 24 h, respectively. Isolated mouse cortical neurons (MCN) were cultured in neurobasal medium in a humidified atmosphere of 5% CO2 at 37 °C for 7 days. EVs from the supernatants of PM2.5-treated OB organotypic cultures and BV2 cells were isolated using differential centrifugation, and were observed by transmission electron microscope (TEM). Immunohistochemistry, TUNEL assay and Western blotting analysis were used to evaluate the molecular mechanism by which PM2.5 exposure induces OB injury. Detailed materials and methods were provided in Supplemental section.

3. Results

3.1. PM2.5 exposure induces oxidative stress in the OB of mice

To evaluate whether PM2.5 could induce oxidative stress in the OBs of mice, we determined oxidative markers in the OB of mice after exposure to 0.1 (PM2.5-L) or 1 mg/kg (PM2.5-H) body weight of PM2.5 for 28 days. The images of immunostaining showed that 3’-NT and 4-HNE were mainly distributed in glomerular layer and mitral cell layer of OB (Fig. 1A). PM2.5 enhanced the immunoreactivities of 3’-NT and 4-HNE in the OB of mice in a dose-dependent manner (Fig. 1B–C). The immunoreactivities of 3’-NT and 4-HNE in the OB of PM2.5-H group were approximately 3.8-fold higher than those of control group. Moreover, treatment with PM2.5 significantly increased the concentrations of MDA in the olfactory bulb of the experimental animals in a dose-dependent manner (Fig. 1D). However, the elevated 3’-NT, 4-HNE and MDA levels were significantly decreased in C-PM2.5 group (Fig. 1B–C). Combined with Fig. S1 and Table S1–3, these results suggested that metal components of PM2.5 are responsible for the oxidative stress and oxidative injury in the OB of mice.

Fig. 1. PM2.5 exposure induces oxidative stress and oxidative injury in the OB of mice.

Fig. 1.

Open in a new tab

Mice were daily treated with a dose of PM2.5 (0.1 or 1 mg/kg body weight), or C-PM2.5 (1 mg/kg body weight) by intranasal instillation for 28 days, respectively. (A) 3’-NT and 4-HNE in the OB of PM2.5-L and PM2.5-H groups were determined by immunostaining (Scale bar, 200 μm). GL: glomerular layer, MCL: mitral cell layer, GCL: granule cell layer. (B-C) Quantification image analyses of the image data of 3’-NT and 4-HNE in the OB of PM2.5-L and PM2.5-H groups. (D) The levels of MDA in the OB of PM2.5-treated mice were determined by immunostaining. Data were shown as the mean ± SEM. N = 6. * indicates p<0.05, ** indicates p<0.01.

3.2. PM2.5 exposure activates microglia in the OB of mice

To evaluate the effects of PM2.5 on OB cellular responses, we performed immunohistochemical analysis of Iba-1 and GFAP in microglia and astrocytes using quantitative image analysis, respectively. After exposure to PM2.5, the number of Iba-1 (microglia marker) was significantly increased in a dose-dependent manner (Fig. 2A–B). In PM2.5-H group, the percentage of Iba-1-tracker positive microglia in the OB was 24%. Activated microglia were mainly observed along the glomerular layer rather than adjacent mitral or granule cell layer. In contrast, astrocytes activation in the OB of PM2.5-treated mice was not significant as evidenced by GFAP staining positive cells (Fig. S3), suggesting that PM2.5 mainly triggered microglia activation. Furthermore, we found that the elevated microglial activation levels were significantly declined in the OB of C-PM2.5 group (Fig. 2C). These results suggested that metals in PM2.5 were associated with microglial activation.

Fig. 2. PM2.5 exposure induces microglial activation in the OB of mice.

Fig. 2.

Open in a new tab

(A) Immunohistochemical images of Iba-1 staining in the OB of mice (Scale bar, 200 μm). The images of the right panels were high-magnification images (Scale bar, 100 μm). Blue solid arrows indicate activated microglia. GL: glomerular layer, MCL: mitral cell layer, GCL: granule cell layer. (B) High-magnification images of resting and activated microglia. (C) Quantitative analyses of Iba-1 positive staining microglia. Data were shown as the mean ± SEM. N = 6. * indicates p<0.05, ** indicates p<0.01.

3.3. PM2.5 exposure triggers EVs release from activated microglia in OB organotypic cultures (ex vivo)

To investigate whether PM2.5-activated microglia directly acted on EVs release, ex vivo OB organotypic cultures was carried out and used TEM to quantitatively evaluate EVs release from PM2.5-treated OB of mice. EVs were first isolated from PM2.5-treated OB supernatants by differential centrifugation. The EV pellets were collected and re-suspended for negative staining. The morphology and structure of EVs from twelve random fields were captured by TEM. As shown in Fig. 3A, membranes of EVs exhibited clear structures, presenting as saucer or concave hemisphere. After relative quantification, we found that the numbers of EVs per field in PM2.5-treated olfactory bulbs were significantly higher than those in untreated controls, suggesting that PM2.5 exposure causes increased release of EVs from OB. To validate EVs release, EVs isolated from PM2.5-treated OB were subjected to Western blots for specific EV markers, including ALG-2 interacting protein (Alix) and flotillin-2. The levels of Alix and flotillin-2 were increased in EV lysates from PM2.5-treated OB compared to the untreated controls (Fig. 3B). These results suggested that PM2.5 exposure increased EVs release from the OB and the cultures.

Fig. 3. PM2.5 exposure triggers EVs release from activated microglia in OB organotypic cultures.

Fig. 3.

Open in a new tab

(A) The OB organotypic cultures were incubated in artificial cerebral spinal fluid (ACSF) with or without 50 μg/ml PM2.5 for 12 h. EVs were isolated from ACSF media by differential centrifugation. Representative TEM images of EVs (Magnification, 50000×) were shown. The numbers of EVs were quantified by manually counting from a total of twelve random vision fields. (B) The expression levels of Alix and flotillin-2 in EVs lysates were determined by Western blots, and the density of each band was normalized to the total protein. Data were shown as the mean ± SEM. N = 3. * indicates p<0.05, ** indicates p<0.01.

3.4. PM2.5 exposure promotes GLS1-containing EVs release from the OB of mice

To test whether glutaminase-containing EVs release from the OB of mice following PM2.5 exposure, we first determined the protein levels of GAC and KGA in PM2.5-treated OB by Western blots. As shown in Fig. 4A, the levels of GAC expression in the OB of PM2.5 group were higher than those of control group. In contrast, no significant changes of KGA expression were observed among the groups. Using EVs directly isolated from OB organotypic cultures, we also found significant up-regulation of Alix, flotillin-2, and GAC protein expression in EVs lysates isolated from PM2.5 group as compared to the control group (Fig. 4B), indicating that that PM2.5 promotes release of GLS1-containing EVs. However, the elevated Alix, flotillin-2 and GAC expression were significantly decreased in C-PM2.5 groups (Fig. 4A–B), suggesting that metal components of PM2.5 were responsible for GLS1-containing EVs release.

Fig. 4. PM2.5 exposure induces GLS1-containing EVs release.

Fig. 4.

Open in a new tab

(A) Mice were daily treated with a single dose of PM2.5 or C-PM2.5 (1 mg/kg body weight) by intranasal instillation for 28 days, respectively. The expression of KGA and GAC in OB were determined by Western blots. Densities of the proteins were relative to β-actin. β-actin was used for loading control. (B) The OB organotypic tissues were incubated in ACSF media with 50 μg/ml PM2.5 or C-PM2.5 for 12 h, respectively. EVs were isolated from ACSF media. Alix, flotillin-2, and GAC in EVs lysates were detected by Western blots, and the density of each band was normalized to the total protein. Data were shown as the mean ± SEM. N = 3–4. * indicates p<0.05, ** indicates p<0.01, NS, not significant.

3.5. EVs isolated from PM2.5-treated OB mediate glutamate generation through GLS1

To further determine whether GLS1, such as GAC, released from EVs promote glutamate generation, the mice were treated with PM2.5 or C-PM2.5 in the presence or absence of CB-839, respectively. The levels of glutamate production in both OBs and OB organotypic cultures were determined by glutamate assay kit. As shown in Fig. 5A and B, PM2.5 exposure significantly increased the levels of glutamate production in PM2.5-treated OB and OB supernatants compared to the control group. However, the elevation of glutamate was significantly decreased in C-PM2.5 and CB-839-treated group. These results indicated that both metals of PM2.5 and the released GLS1 promote glutamate generation in the extracellular fluid of PM2.5-treated OB.

Fig. 5. PM2.5 exposure mediates glutamate generation through GLS1 in the OB of mice.

Fig. 5.

Open in a new tab

(A) The mice were daily treated with a single dose of PM2.5 or C-PM2.5 (1 mg/kg body weight), or PM2.5 (1 mg/kg/day body weight) plus CB-839 (40 mg/ml) by intranasal instillation for 28 days, respectively. Glutamate levels in OB were determined by glutamate assay kit. (B) The isolated OB tissues of mice were incubated in ACSF media with or without 50 μg/ml PM2.5 and C-PM2.5 for 12 h. Glutamate concentrations in ACSF media were determined by glutamate assay kit. Data were shown as the mean ± SEM. N = 5–6. * indicates p<0.05, ** indicates p<0.01.

3.6. GLS1-containing EVs is critical in PM2.5-induced apoptosis in the OB of mice

To determine the role of GLS1-containing EVs in PM2.5-induced neurotoxicity, the mice were treated with PM2.5, C-PM2.5, or CB-839+PM2.5 for 28 days, respectively. Apoptotic cells in the OB of mice were detected by TUNEL staining. As shown in Fig. 6A, the levels of TUNEL positive area were significantly higher in PM2.5 group than those of control and C-PM2.5 groups. Consistent with the TUNEL staining results, MAP-2 expression was decreased whereas cleaved-PPAR and caspase-3 expression were increased in PM2.5 group (Fig. 6B). However, the elevation of TUNEL positive area, MAP-2, cleavage of PPAR and caspase-3 were blocked by CB-839 treatment. These data strongly suggested that GLS1 is critical in PM2.5-induced neurotoxicity in the OB of mice.

Fig. 6. PM2.5 induces apoptosis through GLS1-containing EVs in the OB of mice.

Fig. 6.

Open in a new tab

(A) he mice were daily treated with a single dose of PM2.5 or C-PM2.5 (1 mg/kg body weight), or PM2.5 (1 mg/kg/day body weight) plus CB-839 (40 mg/ml) by intranasal instillation for 28 days, respectively. Apoptotic cells in OB were detected by TUNEL staining. Quantitative assessments of apoptosis in OB were determined through quantitative image analysis (Right panel). Scale bar = 200 μm. (B) The expression of MAP-2, PARP and caspase 3 were determined by Western blots. Densities of the proteins were relative to β-actin. β-actin was used for loading control. Data were shown as the mean ± SEM. N = 3–4. * indicates p<0.05, ** indicates p<0.01, NS, not significant.

3.7. EVs released from PM2.5-treated BV2 cells induce oxidative stress and cell death

To investigate the mechanism of glutaminase-mediated neurotoxicity during PM2.5 exposure, we first determined the role of PM2.5-induced ROS in EVs release and glutamate generation, BV2 cells were treated with PM2.5 and C-PM2.5 or pre-treated with NAC before PM2.5 exposure, respectively. As shown in Fig. 7A–E, a significant increase of ROS generation and higher number of EVs release after PM2.5 exposure. Moreover, PM2.5 induced Alix, flotillin-2, and GAC expression in EVs isolated from BV2 cells. Consistent with PM2.5-treated OB, the levels of glutamate generation in PM2.5-treated BV2 cells were significantly increased compared to the untreated controls (Fig. 7E). However, pretreatment with NAC or treatment with C-PM2.5 significantly reduced PM2.5–induced ROS generation, EVs release and GAC expression. The increase of glutamate generation in the PM2.5-treated BV2 cells was blocked after CB-839 treatment. These results suggested that metal content, oxidative stress, EVs, and GLS1 as contributing factors may facilitate glutamate generation during PM2.5 exposure.

Fig. 7. PM2.5-induced GLS1-containing EVs provoke neurotoxicity.

Fig. 7.

Open in a new tab

BV2 cells were treated with 50 μg/ml PM2.5 or C-PM2.5 for 24 h, respectively. (A) The intracellular ROS levels were determined. (B) TEM images of EVs from control and PM2.5-treated BV2 cells were shown. (C) Alix and flotillin-2 in EVs lysates were determined by Western blots. The density of each band was normalized to the total protein. (D) EVs were isolated from the supernatants of BV2 cells in the presence or absence of PM2.5, C-PM2.5, NAC (10 mM), and GW4869 (10 μM). Representative Western blots and quantitative data of Alix, flotillin-2, and GAC in EV lysates. The density of each band was normalized to the total protein. (E) Glutamate levels in cell-free supernatants were determined by glutamate assay kit. (F) EVs were added to MCN cultures with or without 5 μM CB-839. The cell viability was determined by MTT assay. (G) Representative Western blots and quantitative data of MAP-2, cleaved-PARP and caspase3 in MCN. Densities of the proteins were relative to β-actin. β-actin was used for loading control. Data were shown as the mean ± SEM. N = 3–6. * indicates p<0.05, ** indicates p<0.01, NS, not significant.

Furthermore, we collected EVs from PM2.5-treated BV2 cells and re-suspended in neuronal culture medium. The volumes of culture medium used to re-suspend EVs were adjusted based on the whole cell protein concentration in the same culture. After PM2.5 exposure, EV-treated MCN cells exhibited significantly lower cell viability (Fig. 7F) and higher neurotoxicity (Fig. 7G) as indicated by the expression levels of MAP-2 compared with those treated with EVs from untreated control microglia. However, the EV-induced neurotoxicity was rescued at the presence of GW4869 (a selective inhibitor of N-SMase) and CB-839, when 100 μl of EVs were incubated with MCN (Fig. 7G). We also observed that NAC significantly abolished PM2.5-induced apoptosis as evidenced by an increase in the cell viability. Correspondingly, Western blotting data also showed a significant reduction of cleaved-PARP expression in NAC-pretreated cells compared with untreated cells, respectively (Fig. 7G). Taken together, these data suggested that GLS1-containing EVs contribute to neurotoxicity induced by PM2.5.

4. Discussion

PM2.5-induced neurotoxicity has been extensively studied (Kulas et al., 2018; Zhang et al., 2018; Li et al., 2019). However, the underlying toxicity mechanisms still remain to be elucidated. We chose to evaluate the release EVs from PM2.5-activated microglia in the mouse olfactory bulb because olfactory bulb is the first neuronal contact to inhaled PM2.5 (Heusinkveld et al., 2016). Recent study has demonstrated that PM2.5 causes deficiency in barrier integrity in human nasal epithelial cells through degradation of tight junction proteins (Xian et al., 2020). Harmful chemical components of PM2.5 could attach to the olfactory neuroepithelium and reach the brain through a process known as olfactory transfer (Calderón-Garcidueñas et al., 2013; Cheng et al., 2016). Intranasally administered substances could cross the gaps between the olfactory neurons in the olfactory epithelium, which are subsequently transported to olfactory bulb (Mittal et al., 2014). Two doses of PM2.5 used in this study were to simulate human exposure: daily ambient PM2.5 concentrations (75 μg/m3) and heavily polluted air (886 μg/m3) (Li et al., 2015; Seltenrich, 2016). Thus, understanding the interaction of PM2.5 with OB and the subsequent signaling cascade leading to neurodegeneration will shed light on the development of preventative measures to mitigate the detrimental CNS effects of PM2.5. We previously have reported the release of GLS1 from macrophages into the extracellular compartment after PM2.5 treatment (Liu et al., 2015). The current study focused on PM2.5-induced oxidative stress, microglial activation, glutaminase-containing EVs release and neurotoxicity in the olfactory bulb. Our results showed that EVs released from microglia in olfactory bulb play a critical role in PM2.5-induced neurotoxicity by increasing glutamate generation through GLS1. The current study, however, has potential limitations because female mice were excluded in part due to estrous cyclicity.

The chemical composition of airborne PM2.5, particularly metals, could affect bioaccumulation and oxidative damage induced by PM2.5 (Ribeiro et al., 2016). Several individual metals, including Al, Cr, Ti, Mn, Ni, Pb, As, Zn, Cu, and mercury (Hg), have been demonstrated to affect the neurological system (Pohl et al., 2011). These toxic metals incorporated in PM2.5 and general accumulation of metal ions in the brain contributes to heightened oxidative stress and neuronal damage (Bolognin et al., 2009; Zatta et al., 2008). Oxidative stress has long been defined as one of the major contributing factors to many neurodegenerative diseases (Block et al., 2004). In the present study, after exposure to PM2.5, the contents of Pb, Ti, Ni, Mn, Cr, Cu and Zn in the olfactory bulb of mice were significantly increased in a dose-dependent manner, indicating that the metals detected were bioaccumulated (Fig. S2). These results are consistent with an earlier report, which showed high concentrations of Ni and V in the olfactory bulb of dogs in Mexican City (Calderón-Garcidueñas et al., 2002). The change of metal content has measured in olfactory bulb, Cu and Fe may be deposited in other brain regions, such as cerebral cortex and hippocampus (Maher et al., 2016). It has been demonstrated that redox-active transition metals including Cu and Fe can prone to induce ROS production, such as hydrogen peroxide (H2O2), hydroxyl radical (OH•) and superoxide anion (O•2) (Jomova and Valko, 2011; Leonard et al., 2004). Here, we found that PM2.5 exposure induced oxidative stress as evidenced by the increases of 3’-NT, 4-HNE and MDA (Fig. 1). Moreover, PM2.5 exposure activated microglia in the olfactory bulb of mice as evidenced by the increase of Iba-1 staining intensity (microglia marker) (Fig. 2). These results are consistent with a previous study demonstrating that nanoscale particulate matter from urban traffic increased oxidative stress and Iba1-positive macrophages in glomerular layer of olfactory bulb (Calderón-Garcidueñas et al., 2002). In order to further evaluate the biological effect of metals in PM2.5, we used Chelex to remove metals from the PM2.5 extracts. We found that C-PM2.5 significantly decreased PM2.5-inudced oxidative stress and microglia activation (Fig. 1 and 2), suggesting that metal bioaccumulation triggered oxidative stress that leads to activation of microglia in the olfactory bulb. This observation is consistent with the previous reports demonstrating that the ROS is involved in microglia activation (Park et al., 2015; Shafer et al., 2010).

Microglia, the resident innate immune cells in the brain, have been reported to be a crucial factor responsible for cellular damage caused by air pollution (Brown and Neher, 2010). Glutaminase released from microglia, which facilitates glutamate generation, may cause neurotoxicity (Bezzi et al., 2001; Block et al., 2007; Brown and Neher, 2010). A recent study has demonstrated that microvesicles as a primary mechanism of GLS1 release from immune-activated microglial cells, which subsequently mediates excess glutamate generation and neurotoxicity from immune-activated microglial cells (Wu et al., 2015). In the present study, we designed to establish the purity and characterization of the isolated EVs from the olfactory bulb of PM2.5-treated mice. TEM was used to observe the morphology and structure of EVs release from activated microglia in the in OB organotypic cultures of untreated control group and PM2.5-treated group. EVs showed clear structures, presenting as saucer or concave hemisphere. Western blot analysis also showed that PM2.5 induced EVs released from PM2.5-treated olfactory bulb as evidenced by the increase of levels of Alix and flotillin-2 expression in EV lysates (Fig. 3). Furthermore, the glutaminase isoform GAC was found to be higher in the isolated EVs fraction from PM2.5-treated OB, but not KGA (Fig. 4). The GAC isoform is believed to be regulated in an active fashion whereas KGA is constitutively expressed (Wu et al., 2015). Direct EVs treatment recapitulates the neurotoxic effects of activated microglia. The effect of EVs on neurons can be blocked by pre-treatment of microglia with GW4869, which is known to specifically impede EVs biogenesis and reduce EVs release. Therefore, we conclude that EVs are the primary instigator in facilitating GLS1 release and neurotoxicity in PM2.5-activated microglia.

Excessive glutamate generation by glutaminase causes neurotoxicity and has been linked to many neurological diseases, including AD (Hynd et al., 2004), PD (Caudle and Zhang, 2009), and HIV-1-associated neurocognitive disorders (Zhao et al., 2004). Indeed, patients with PD, AD, or other neuronal diseases presented the elevated glutamate levels produced by microglia or macrophages (Wu et al., 2015). In this study, we found that PM2.5 exposure significantly increases the levels of glutamate production both in OB and OB organotypic cultures (Fig. 5), suggesting that GLS1 is released through EVs in PM2.5-treated OB and the EV-mediated GLS1 release promotes glutamate generation. These findings are consistent with our previous report that PM2.5 exposure significantly increases glutamate production by macrophages and microglia (Liu et al., 2015). We have further identified several approaches to block the harmful effects of EVs and their related excess glutamate generation. Pre-treatment with CB-839 significantly reduced PM2.5-induced glutamate production in OB. Furthermore, we have demonstrated that the neurotoxicity by PM2.5-activated microglia BV2 cells can be abolished by pre-treatment with GW4869, CB-839 and NAC, which indicates that GLS1-containing EVs are the neurotoxic factors in PM2.5-activated microglia. These data from the present study showed that PM2.5 exposure induces oxidative stress and microglia activation, leading to neurotoxicity through releasing GLS1-containing EVs.

In conclusion, the results of the present study show that PM2.5 induces oxidative stress by increasing ROS generation and microglial activation, resulting in release of GLS1-containing EVs in PM2.5-treated olfactory bulb. GLS1 from EVs is responsible for increasing glutamate generation, leading to neurotoxicity. Our results suggest that GLS1-containing EVs released from microglia in OB play a key role in PM2.5-induced neurotoxicity by increasing glutamate generation through GLS1. These mechanistic insights may have potentially important implications for PM2.5-induced pathologies and neurologic diseases.

Supplementary Material

1

Highlights.

Intranasal instillation PM2.5 deposits in the mouse olfactory bulb.

PM2.5 induces oxidative stress and microglial activation in the olfactory bulb.

PM2.5-activated microglia release glutaminase-containing extracellular vesicles (EVs).

Soluble metal components of PM2.5 are responsible for EVs-mediated neurotoxicity.

Synopsis.

Intranasal PM2.5 induces oxidative stress and microglial activation in the mouse olfactory bulb. PM2.5 induces glutaminase release and glutaminase-mediated glutamate generation via microglia-derived extracellular vesicles (EVs) and causes glutaminase-mediated glutamate generation. Glutaminase-containing EVs contributes to PM2.5-induced neurotoxicity.

Acknowledgments

This study was supported by the Major Program of National Natural Science Foundation of China (91643206, U1432245 to WD), the State Key Program of the National Natural Science Foundation of China (81830037 to JZ), the National Institutes of Health: R01 NS097195 (JZ) and the Chinese Academy of Sciences/State Administration of Foreign Experts Affairs International Partnership Program for Creative Research Teams. We thank Yinzi Ma and Pengyan Xia from the State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences (CAS) for their technical assistance. We also thank Fang Li, Shasha Zuo and Dandan Sun from University of Chinese Academy of Science for their kindly help in instrument operation.

Abbreviations

3’-NT

3’-nitrotyrosine

4-HNE

4-hydroxynonenal

AD

Alzheimer’s disease

Alix

ALG-2-interacting protein

CNS

central nervous system

DCF

fluorescent compound dichlorofluorescin

DCFH-DA

2′,7′-dichlorofluorescein diacetate

DMEM

Dulbecco’s modified Eagle’s medium

DMSO

dimethyl sulfoxide

ECL

enhanced chemiluminescence

EVs

extracellular vesicles

GAC

glutaminase C

GFAP

glial fibrillary acid protein

GLS

glutaminase

Iba-1

ionized calcium binding adaptor molecule-1

ICP-MS

inductively coupled plasma-mass spectrometry

KGA

kidney-type glutaminase

MDA

malonaldehyde

NAC

N-acetylcysteine

OB

olfactory bulb

PARP

poly(ADP-ribose) polymerase

PD

Parkinson’s disease

ROS

reactive oxygen species

SDS-PAGE

sodium dodecyl sulphate polyacrylamide gel electrophoresis

TBST

tris-buffered saline-Tween

TUNEL

terminal deoxynucleotidyl transferase dUTP nick end labeling

Footnotes

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Main finding

Intranasal instillation PM2.5 induces neurotoxicity through its metal contents and glutaminase-containing extracellular vesicles (EVs) in olfactory bulbs of mice.

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

  1. Bezzi P, Domercq M, Brambilla L, Galli R, Schols D, De Clercq E, Vescovi A, Bagetta G, Kollias G, Meldolesi J, Volterra A, 2001. CXCR4-activated astrocyte glutamate release via TNFα: amplification by microglia triggers neurotoxicity. Nat. Neurosci 4, 702. [DOI] [PubMed] [Google Scholar]
  2. Block ML, Wu X, Pei Z, Li G, Wang T, Qin L, Wilson B, Yang J, Hong JS, Veronesi B, 2004. Nanometer size diesel exhaust particles are selectively toxic to dopaminergic neurons: the role of microglia, phagocytosis, and NADPH oxidase. FASEB J. 18, 1618–1620. [DOI] [PubMed] [Google Scholar]
  3. Block ML, Zecca L, Hong JS, 2007. Microglia-mediated neurotoxicity: uncovering the molecular mechanisms. Nat. Rev. Neurosci 8, 57–69. [DOI] [PubMed] [Google Scholar]
  4. Bolognin S, Messori L, Zatta P, 2009. Metal ion physiopathology in neurodegenerative disorders. Neuromolecular Med. 11, 223–238. [DOI] [PubMed] [Google Scholar]
  5. Brown GC, Neher JJ, 2010. Inflammatory neurodegeneration and mechanisms of microglial killing of neurons. Mol. Neurobiol 41, 242–247. [DOI] [PubMed] [Google Scholar]
  6. Calderón-Garcidueñas L, Azzarelli B, Acuna H, Garcia R, Gambling TM, Osnaya N, Monroy S, MR DELT, Carson JL, Villarreal-Calderon A, Rewcastle B, 2002. Air pollution and brain damage. Toxicol. Pathol 30, 373–389. [DOI] [PubMed] [Google Scholar]
  7. Calderón-Garcidueñas L, Maronpot RR, Torres-Jardon R, Henriquez-Roldan C, Schoonhoven R, Acuna-Ayala H, Villarreal-Calderon A, Nakamura J, Fernando R, Reed W, Azzarelli B, Swenberg JA, 2003. DNA damage in nasal and brain tissues of canines exposed to air pollutants is associated with evidence of chronic brain inflammation and neurodegeneration. Toxicol. Pathol 31, 524–538. [DOI] [PubMed] [Google Scholar]
  8. Calderón-Garcidueñas L, Serrano-Sierra A, Torres-Jardon R, Zhu H, Yuan Y, Smith D, Delgado-Chavez R, Cross JV, Medina-Cortina H, Kavanaugh M, Guilarte TR, 2013. The impact of environmental metals in young urbanites’ brains. Exp. Toxicol. Pathol 65, 503–511. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Campos-Sandoval JA, Martin-Rufian M, Cardona C, Lobo C, Penalver A, Marquez J, 2015. Glutaminases in brain: Multiple isoforms for many purposes. Neurochem. Int 88, 1–5. [DOI] [PubMed] [Google Scholar]
  10. Caudle WM, Zhang J, 2009. Glutamate, excitotoxicity, and programmed cell death in Parkinson disease. Exp. Neurol 220, 230–233. [DOI] [PubMed] [Google Scholar]
  11. Cheng H, Davis DA, Hasheminassab S, Sioutas C, Morgan TE, Finch CE, 2016. Urban traffic-derived nanoparticulate matter reduces neurite outgrowth via TNFα in vitro. J. Neuroinflammation. 13, 19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dorman DC, Struve MF, Wong BA, Dye JA, Robertson ID, 2006. Correlation of brain magnetic resonance imaging changes with pallidal manganese concentrations in rhesus monkeys following subchronic manganese inhalation. Toxicol. Sci 92, 219–227. [DOI] [PubMed] [Google Scholar]
  13. Garcia GJ, Schroeter JD, Kimbell JS, 2015. Olfactory deposition of inhaled nanoparticles in humans. Inhal. Toxicol 27, 394–403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Heusinkveld HJ, Wahle T, Campbell A, Westerink RHS, Tran L, Johnston H, Stone V, Cassee FR, Schins RPF, 2016. Neurodegenerative and neurological disorders by small inhaled particles. Neurotoxicology 56, 94–106. [DOI] [PubMed] [Google Scholar]
  15. Hopkins LE, Patchin ES, Chiu PL, Brandenberger C, Smiley-Jewell S, Pinkerton KE, 2014. Nose-to-brain transport of aerosolised quantum dots following acute exposure. Nanotoxicology 8, 885–893. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hynd MR, Scott HL, Dodd PR, 2004. Glutamate-mediated excitotoxicity and neurodegeneration in Alzheimer’s disease. Neurochem. Int 45, 583–595. [DOI] [PubMed] [Google Scholar]
  17. Iraci N, Gaude E, Leonardi T, Costa ASH, Cossetti C, Peruzzotti-Jametti L, Bernstock JD, Saini HK, Gelati M, Vescovi AL, Bastos C, Faria N, Occhipinti LG, Enright AJ, Frezza C, Pluchino S, 2017. Extracellular vesicles are independent metabolic units with asparaginase activity. Nat. Chem. Biol 13, 951–955. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Jomova K, Valko M, 2011. Advances in metal-induced oxidative stress and human disease. Toxicology 283, 65–87. [DOI] [PubMed] [Google Scholar]
  19. Jung CR, Lin YT, Hwang BF, 2015. Ozone, particulate matter, and newly diagnosed Alzheimer’s disease: a population-based cohort study in Taiwan. J. Alzheimers Dis 44, 573–584. [DOI] [PubMed] [Google Scholar]
  20. Kadiu I, Narayanasamy P, Dash PK, Zhang W, Gendelman HE, 2012. Biochemical and biologic characterization of exosomes and microvesicles as facilitators of HIV-1 infection in macrophages. J. Immunol 189, 744–754. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kulas JA, Hettwer JV, Sohrabi M, Melvin JE, Manocha GD, Puig KL, Gorr MW, Tanwar V, McDonald MP, Wold LE, Combs CK, 2018. In utero exposure to fine particulate matter results in an altered neuroimmune phenotype in adult mice. Environ. Pollut 241, 279–288. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lai CP, Breakefield XO, 2012. Role of exosomes/microvesicles in the nervous system and use in emerging therapies. Front. Physiol 3, 228. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Leonard SS, Harris GK, Shi XL, 2004. Metal-induced oxidative stress and signal transduction. Free Radic. Biol. Med 37, 1921–1942. [DOI] [PubMed] [Google Scholar]
  24. Li D, Zhang R, Cui L, Chu C, Zhang H, Sun H, Luo J, Zhou L, Chen L, Cui J, Chen S, Mai B, Chen S, Yu J, Cai Z, Zhang J, Jiang Y, Aschner M, Chen R, Zheng Y, Chen W, 2019. Multiple organ injury in male C57BL/6J mice exposed to ambient particulate matter in a real-ambient PM exposure system in Shijiazhuang, China. Environ. Pollut 248, 874–887. [DOI] [PubMed] [Google Scholar]
  25. Li R, Gong J, Chen L, Wang Z, 2015. Estimating ground-level PM2.5 using fine-resolution satellite data in the megacity of Beijing, China. Aerosol Air Qual. Res 15, 1347–1356. [Google Scholar]
  26. Liu F, Huang YL, Zhang F, Chen Q, Wu BQ, Rui W, Zheng JLC, Ding WJ, 2015. Macrophages treated with particulate matter PM2.5 induce selective neurotoxicity through glutaminase-mediated glutamate generation. J. Neurochem 134, 315–326. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Maher BA, Ahmed IA, Karloukovski V, MacLaren DA, Foulds PG, Allsop D, Mann DM, Torres-Jardon R, Calderón-Garcidueñas L, 2016. Magnetite pollution nanoparticles in the human brain. Proc. Natl. Acad. Sci. U S A 113, 10797–10801. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Mittal D, Ali A, Md S, Baboota S, Sahni JK, Ali J, 2014. Insights into direct nose to brain delivery: current status and future perspective. Drug Deliv. 21, 75–86. [DOI] [PubMed] [Google Scholar]
  29. Oudin A, Forsberg B, Adolfsson AN, Lind N, Modig L, Nordin M, Nordin S, Adolfsson R, Nilsson LG, 2016. Traffic-related air pollution and dementia incidence in Northern Sweden: A longitudinal study. Environ. Health Perspect 124, 306–312. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Park J, Min JS, Kim B, Chae UB, Yun JW, Choi MS, Kong IK, Chang KT, Lee DS, 2015. Mitochondrial ROS govern the LPS-induced pro-inflammatory response in microglia cells by regulating MAPK and NF-κB pathways. Neurosci. Lett 584, 191–196. [DOI] [PubMed] [Google Scholar]
  31. Pegtel DM, Peferoen L, Amor S, 2014. Extracellular vesicles as modulators of cell-to-cell communication in the healthy and diseased brain. Phil. Trans. R. Soc 369, 20130516. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Pohl HR, Roney N, Abadin HG, 2011. Metal ions affecting the neurological system. Met. Ions Life Sci 8, 247–262. [PubMed] [Google Scholar]
  33. Ribeiro JD, Kalb AC, Campos PP, De la Cruz ARH, Martinez PE, Gioda A, de Souza MM, Gioda CR, 2016. Toxicological effects of particulate matter (PM2.5) on rats: Bioaccumulation, antioxidant alterations, lipid damage, and ABC transporter activity. Chemosphere 163, 569–577. [DOI] [PubMed] [Google Scholar]
  34. Ritz B, Lee PC, Hansen J, Lassen CF, Ketzel M, Sorensen M, Raaschou-Nielsen O, 2016. Traffic-related air pollution and Parkinson’s disease in Denmark: A case–control study. Environ. Health Perspect 124, 351–356. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Rufino-Ramos D, Albuquerque PR, Carmona V, Perfeito R, Nobre RJ, Pereira de Almeida L, 2017. Extracellular vesicles: Novel promising delivery systems for therapy of brain diseases. J. Control Release 262, 247–258. [DOI] [PubMed] [Google Scholar]
  36. Rui W, Guan L, Zhang F, Zhang W, Ding W, 2016. PM2.5-induced oxidative stress increases adhesion molecules expression in human endothelial cells through the ERK/AKT/NF-kappaB-dependent pathway. J. Appl. Toxicol 36, 48–59. [DOI] [PubMed] [Google Scholar]
  37. Sadasivan S, Zanin M, O’Brien K, Schultz-Cherry S, Smeyne RJ, 2015. Induction of microglia activation after infection with the non-neurotropic A/CA/04/2009 H1N1 influenza virus. Plos One 10, e0124047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Saenz-Cuesta M, Osorio-Querejeta I, Otaegui D, 2014. Extracellular vesicles in multiple sclerosis: What are they telling us? Front. Cell Neurosci 8, 100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Seltenrich N, 2016. A clearer picture of China’s air: Using satellite data and ground monitoring to estimate PM2.5 over time. Environ. Health Perspect 124, A38. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Shijie J, Takeuchi H, Yawata I, Harada Y, Sonobe Y, Doi Y, Liang J, Hua L, Yasuoka S, Zhou Y, Noda M, Kawanokuchi J, Mizuno T, Suzumura A, 2009. Blockade of glutamate release from microglia attenuates experimental autoimmune encephalomyelitis in mice. Tohoku J. Exp. Med 217, 87–92. [DOI] [PubMed] [Google Scholar]
  41. Xian M, Ma S, Wang K, Lou H, Wang Y, Zhang L, Wang C, Akdis CA, 2020. Particulate matter 2.5 causes deficiency in barrier integrity in human nasal epithelial cells. Allergy Asthma Immunol. Res 12, 56–71. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Xu X, Ha SU, Basnet R, 2016. A review of epidemiological research on adverse neurological effects of exposure to ambient air pollution. Front. Public Health 4, 157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Yang Y, Boza-Serrano A, Dunning CJR, Clausen BH, Lambertsen KL, Deierborg T, 2018. Inflammation leads to distinct populations of extracellular vesicles from microglia. J. Neuroinflammation 15, 168. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Zatta P, Drago D, Zambenedetti P, Bolognin S, Nogara E, Peruffo A, Cozzi B, 2008. Accumulation of copper and other metal ions, and metallothionein I/II expression in the bovine brain as a function of aging. J. Chem. Neuroanatomy 36, 1–5. [DOI] [PubMed] [Google Scholar]
  45. Zhao J, Lopez AL, Erichsen D, Herek S, Cotter RL, Curthoys NP, Zheng J, 2004. Mitochondrial glutaminase enhances extracellular glutamate production in HIV-1-infected macrophages: linkage to HIV-1 associated dementia. J. Neurochem 88, 169–180. [DOI] [PubMed] [Google Scholar]
  46. Zhang Q, Li Q, Ma J, Zhao Y, 2018. PM2.5 impairs neurobehavior by oxidative stress and myelin sheaths injury of brain in the rat. Environ. Pollut 242(Pt A), 994–1001. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS1599543-supplement-1.docx (1MB, docx)

RESOURCES