Microglial Activation and Oxidative Stress in PM2.5-Induced Neurodegenerative Disorders

Fine particulate matter (PM2.5) pollution remains a prominent environmental problem worldwide, posing great threats to human health. The adverse effects of PM2.5 on the respiratory and cardiovascular systems have been extensively studied, while its detrimental effects on the central nervous system (CNS), specifically neurodegenerative disorders, are less investigated. Neurodegenerative disorders are characterized by reduced neurogenesis, activated microglia, and neuroinflammation. A variety of studies involving postmortem examinations, epidemiological investigations, animal experiments, and in vitro cell models have shown that PM2.5 exposure results in neuroinflammation, oxidative stress, mitochondrial dysfunction, neuronal apoptosis, and ultimately neurodegenerative disorders, which are strongly associated with the activation of microglia. Microglia are the major innate immune cells of the brain, surveilling and maintaining the homeostasis of CNS. Upon activation by environmental and endogenous insults, such as PM exposure, microglia can enter an overactivated state that is featured by amoeboid morphology, the over-production of reactive oxygen species, and pro-inflammatory mediators. This review summarizes the evidence of microglial activation and oxidative stress and neurodegenerative disorders following PM2.5 exposure. Moreover, the possible mechanisms underlying PM2.5-induced microglial activation and neurodegenerative disorders are discussed. This knowledge provides certain clues for the development of therapies that may slow or halt the progression of neurodegenerative disorders induced by ambient PM.


Introduction
Air pollution is comprised of particulate matter (PM), gases, organic compounds, and metals derived from both human activity and natural sources. PM is the most widespread health threat and has been strongly implicated in diverse diseases [1]. An important contributor to PM is traffic-related air pollution (TRAP), mostly ascribed to diesel exhaust particles (DEP) [2]. Ambient PM can be split up in several size fractions based on the aerodynamic diameter: coarse PM (2.5-10 µm), fine PM (<2.5 µm, PM 2.5 ), and ultrafine PM (<0.1 µm, UFPM) [3]. PM 2.5 exhibits tempo-spatial variations of complex components, such as bacteria lipopolysaccharides (LPS), carbon-containing particles, sulfate, nitrate, ammonium salt, and heavy metals [4,5]. PM 2.5 and UFPM are of particular concern, as these particles can enter systemic circulation and be distributed in the brain and other organs, posing significant potential danger to human health [6]. Globally, there are approximately 6.5 million excess deaths attributable to ambient PM 2.5 pollution annually [7]. Recent evidence indicates that PM 2.5 air pollution, in addition to causing respiratory and cardiovascular diseases, also negatively affects the brain and contributes to central nervous system (CNS) diseases [1]. that catalyze the generation of ROS and nitric oxide (NO), respectively [36], and a major histocompatibility complex-II that presents antigens, triggers and spreads further inflammatory response in surrounding microglial cells [28], integrins, co-stimulatory molecules, Fc receptors, and intracellular proteins (e.g., ionized calcium binding adapter molecule-1, Iba-1), contributing to neurological damage [37]. In contrast, the M2 phenotype characterized by thin cell bodies and branched processes can be induced by IL-4, IL-13, IL-10, or activated peroxisome proliferator-activated receptors γ (PPARγ) [38], resulting in the release of anti-inflammatory cytokines, such as IL-10, transforming growth factor-β (TGFβ), growth factors, colony stimulating factor−1 (CSF-1), neurotrophic growth factors, such as brain derived neurotrophic factor (BDNF), a neuroprotective status [38][39][40].
From the above, activated microglia show a broad spectrum of phenotypes ranging from the pro-inflammatory, potentially cytotoxic M1 to the anti-inflammatory, scavenging, and regenerative M2. The resting microglia can be activated by toxic mediators, such as IFN-γ, LPS, and PM2.5, and display a M1 phenotype. In this condition, microglia induce neurotoxicity via release of neurotoxic mediators (TNF-α, PGE2, ROS, NO, etc.). The factors secreted by the dead or damaged neurons in turn exacerbate the chronic activation of microglia. Besides the M1 phenotype, in combination with IL-4, IL-10, and TGF-β, microglia could be induced into the M2 phenotype, which plays a neuroprotective role through the generation and release of anti-inflammatory cytokines (IL-13, IL-10, TGF-β, etc.).

Evidence from Postmortem Examinations
The first histopathological evidence for a link between air pollution and neuropathology came from a necropsy study with canine residents naturally exposed to a highly polluted environment in Mexico City [41]. In this study, the increased expression of neuroinflammatory and oxidative stress biomarkers, including nuclear factor-κB (NF-κB) and iNOS in cortical endothelial cells occurred at ages 2 and 4 weeks of dogs with subsequent neurodegenerative changes, such as the impairment of the blood-brain barrier (BBB) and the extracellular deposition of Aβ peptide fibrils and intracellular neurofibrillary tangles (NFTs) in the olfactory epithelium (OE) and the olfactory bulbs (OB), as well as in subcortical and cortical structures [41]. In addition, dogs aged 8 months demonstrated pronounced inflammatory effects, diffuse Aβ plaques, and a significant increase in DNA damage in OB, frontal cortex, and hippocampus with ameboid microglia in the cortex and white matter [8,41]. Moreover, feral dogs inhabiting in Mexico City presented brain tissue damage and accumulated metals (nickel and vanadium) in a gradient fashion from OE > OB > frontal cortex, indicating the nose as portal of entry [41]. Notably, those alterations of the olfactory pathway were similar to the early olfactory pathology observed in AD.
The similar findings were also observed in autopsy examinations of human samples [9]. Adult human residents living in Mexico City presented an increased expression of inflammatory mediator cyclooxygenase-2 (COX-2) and the greater accumulation of neuronal and astrocytic Aβ42, the 42-amino acid form of Aβ, in frontal cortex, hippocampus, The resting microglia can be activated by toxic mediators, such as IFN-γ, LPS, and PM 2.5 , and display a M1 phenotype. In this condition, microglia induce neurotoxicity via release of neurotoxic mediators (TNF-α, PGE2, ROS, NO, etc.). The factors secreted by the dead or damaged neurons in turn exacerbate the chronic activation of microglia. Besides the M1 phenotype, in combination with IL-4, IL-10, and TGF-β, microglia could be induced into the M2 phenotype, which plays a neuroprotective role through the generation and release of anti-inflammatory cytokines (IL-13, IL-10, TGF-β, etc.).
From the above, activated microglia show a broad spectrum of phenotypes ranging from the pro-inflammatory, potentially cytotoxic M1 to the anti-inflammatory, scavenging, and regenerative M2.

Evidence from Postmortem Examinations
The first histopathological evidence for a link between air pollution and neuropathology came from a necropsy study with canine residents naturally exposed to a highly polluted environment in Mexico City [41]. In this study, the increased expression of neuroinflammatory and oxidative stress biomarkers, including nuclear factor-κB (NF-κB) and iNOS in cortical endothelial cells occurred at ages 2 and 4 weeks of dogs with subsequent neurodegenerative changes, such as the impairment of the blood-brain barrier (BBB) and the extracellular deposition of Aβ peptide fibrils and intracellular neurofibrillary tangles (NFTs) in the olfactory epithelium (OE) and the olfactory bulbs (OB), as well as in subcortical and cortical structures [41]. In addition, dogs aged 8 months demonstrated pronounced inflammatory effects, diffuse Aβ plaques, and a significant increase in DNA damage in OB, frontal cortex, and hippocampus with ameboid microglia in the cortex and white matter [8,41]. Moreover, feral dogs inhabiting in Mexico City presented brain tissue damage and accumulated metals (nickel and vanadium) in a gradient fashion from OE > OB > frontal cortex, indicating the nose as portal of entry [41]. Notably, those alterations of the olfactory pathway were similar to the early olfactory pathology observed in AD.
The similar findings were also observed in autopsy examinations of human samples [9]. Adult human residents living in Mexico City presented an increased expression of inflammatory mediator cyclooxygenase-2 (COX-2) and the greater accumulation of neuronal and astrocytic Aβ 42 , the 42-amino acid form of Aβ, in frontal cortex, hippocampus, and OB [9]. In contrast, children and young adults exhibited a wide spectrum of neurodegenerative disorders, such as increases in microglial activation (CD68 and CD163), elevated pro-inflammatory proteins (COX-2 and IL-1β) and the innate immunity receptor CD14, accumulated AD or PD proteins (Aβ 42 and α-synuclein), oxidative stress in frontal and infratentorial neurons and microglia (8-hydroxydeoxyguanosine (8-OHdG) and nitrotyrosine), and frontal BBB impairment, as well as the reduction of the neuroprotective cellular prion protein (PrPC) in the frontal cortex [42]. Intriguingly, metals (manganese, nickel, and chromium) were enriched in the frontal cortex with the high expression of COX-2, TGF-β, CD14, and IL-1β [42]. Additionally, those children in Mexico City had brainstem abnormalities, such as inflammation, α-synuclein and/or Aβ 42 , deposition, activated microglia, and reactive glial fibrillary acidic protein (GFAP) positive astrocytes throughout the brainstem [43]. Early olfactory abnormalities similar to the olfactory pathology in AD were also observed in Mexico City children [44]. It is noteworthy that Apolipoprotein E4 (APOE4) is the strongest genetic risk factor for AD [45], APOE4 carriers exposed to air pollution had greater hyperphosphorylated tau, diffused Aβ plaques, and more pronounced olfactory deficits than APOE3 carriers [42,46,47].
Taken together, the preliminary evidence of postmortem studies indicates that exposure to ambient air pollutants is associated with microglia activation, oxidative stress, and neurodegenerative alterations in brain tissues. However, due to the complex mixture of air pollution, the causative association of air pollution exposure with the observed neurological effects of CNS remains to be specified.

Evidence from Epidemiological Investigations
There is a growing body of epidemiological studies reporting ambient air pollutionassociated neurodegenerative disorders (see Table 1 for details), such as cognitive decline, AD, and PD. Regarding cognitive alteration, an early important aspect of AD, many population-based studies with the elderly [48][49][50] have reported that PM air pollution, particularly TRAP, is consistently associated with declined cognitive abilities [51]. However, controlled animal studies in this aspect are still limited for further verification.
Several other epidemiological studies found that exposure to PM 2.5 was associated with a significant risk for AD [52][53][54][55][56], which was consistent with the pathological findings from the autopsy samples of individuals with AD-like pathologies in the highly polluted Mexico City. These observations were supported by a follow-up study displaying a decrease in Aβ 42 levels in the cerebrospinal fluids (CSF) of Mexico City children [57], a very early change in AD [58]. In addition, Calderon-Garciduenas et al. recently reported elevated levels of non-phosphorylated tau in the CSF, a marker of AD axonal pathology, or increases in hyperphosphorylated tau and amyloid plaques in the OB of children and young adults in Mexico City [51,59]. Given that aging is a risk factor for neurodegenerative diseases [60], the aging brain is assumed to be particularly vulnerable to air pollution-induced neurotoxicity [61]. Therefore, it is not surprising to see an accelerated decline in episodic memory among older females with late-life exposure to PM 2.5 [62].
A few studies have examined the association of ambient PM 2.5 exposure with the risk for PD. However, the epidemiological results appear inconsistent. For example, one study showed opposite associations between ambient PM 2.5 exposure and the incidence of PD among two populations from two locations with different severity of air pollution [63]. Another study found an association between the concentrations of ambient PM 2.5 and PD risk in female never-smokers [64]. However, in a large prospective study of women, Palacios et al. did not see significant associations between PM 10 or PM 2.5 exposure and the incidence of PD [65].
Thus far, the epidemiological evidence specifically linking microglial activation to air pollution-induced neurodegenerative disorders is mainly from the studies with children and young adults in Mexico City [66][67][68]. Overall, these studies demonstrated that those children presented early markers of neurodegeneration, neuroinflammation (e.g., elevated macrophage inhibitory factor (MIF), IL-6, and IL-2 in CSF [69] and increased IL-6 and Toll-like receptors (TLRs) expression in frontal cortex), olfactory dysfunction, and cognitive deficits compared to control children from nearby non-polluted cities. Of these biomarkers examined, MIF is a cytokine essential for microglial activation and the production of IL-6, IL-1β, TNF-α, and iNOS [70]. Song et al. [72] More recently, the association of PM 2.5 -associated microglial activation with neurodegenerative disorders have been investigated among the elderly. In a panel study, Tang et al. examined the air pollutant (including PM) exposure in Chinese people aged 60-69 years, showing that air pollution exposure could induce alterations of neurodegenerative biomarkers, such as Aβ 40 , Aβ 42 , α-synuclein, PrPC, Tau (pThr181), and the activation of microglia represented by the over-expression of S100B and microglial triggering receptor expressed on myeloid cells2 (TREM2) [71]. Our recent findings from a panel study on healthy retired adults demonstrated that PM 2.5 concentration increments were associated with increases in the biomarkers of neural damage, including a neurofilament light chain (NfL), neuronspecific enolase, and the activation of microglia (S100B) in serum [72]. Meanwhile, several constituents of ambient PM 2.5 , such as Cu, Zn, Ni, Mn, Sn, V, Rb, Pb, Al, Be, Cs, Co, Th, U, Cl − , and F − were found to be significantly associated with serum levels of NfL [72].
In summary, the available evidence in humans, albeit limited and variable, is suggestive of the association of PM 2.5 exposure with neurodegenerative disorders.

Evidence from Animal Studies
In vivo studies in general corroborate and expand the major pathophysiological findings in human brain tissue and other accessible tissues [73,74], such as the markers of oxidative stress, neuroinflammation, and neurodegeneration, and help decipher underlying mechanisms linking exposure to the development of neurodegenerative disorders. Most of the animal studies have focused on the effects of PM 2.5 , especially DEP and UFPM, on AD-like pathologies (see Table 2 for details).
In AD condition [75], microglia-mediated neuroinflammation is a critical event characterized by the release of IL-1β, IL-6, and TNF-α. This feature has been verified in animals exposed to PM 2.5 under diverse exposure scenarios, mostly chronic exposure. For example, children and dogs chronically exposed to severely polluted air pollution in Mexico City displayed similar inflammatory neuropathological lesions [10,76]. The chronic inhalation of airborne PM 2.5 caused time-dependent early AD-like changes in mice, such as an increase in Aβ 40 , BACE (beta-site amyloid precursor protein (APP)-cleaving enzyme), and COX-2, as well as a decrease in APP, with a minimal change of phosphorylated tau [77]. The oropharyngeal aspiration of PM 2.5 for 4 weeks induced a dose-dependent increase in IL-1β and TNF-α in the blood and hippocampus of mice [78]. The whole-body exposure of rats to PM 1.0 for 3 and 6 months resulted in microglia activation in the hippocampus [79]. In addition, short-term exposure showed similar neurological effects. For example, mice exposed for 5 d to a traffic-polluted highway tunnel exhibited increased expression of microgliaassociated inflammatory genes (COX-2, iNOS, and nuclear factor) in the hippocampus, and decreased BDNF expression in the OB [80].
DEP are a major constituent of ambient PM 2.5 and are commonly used as a surrogate model of air pollution in health effects studies [81]. The short-or long-term inhalation of DEP can induce the over-expression of pro-inflammatory factors in select brain regions [82,83]. For example, the inhalation of DEP (0.5 and 2 mg/m 3 , for 1 month) increased IL-1β, TNF-α, IL-6, MIP-1α (macrophage inflammatory protein-1α), fractalkine, and Iba-1 in most regions of Sprague Dawley (SD) rats, with the midbrain showing the greatest DEP response [84]. Meanwhile, a single intratracheal administration of DEP increased microglial Iba-1 levels in the substantia nigra and elevated serum and whole-brain TNF-α at 6 h post-treatment [85]. The susceptibility of the midbrain to DEP neuroinflammatory effects was confirmed by another inhalation study over 6 months on male Fischer 344 rats exposed to DEP (35-992 µg/m 3 ), probably due to the most microglia in the midbrain [85]. In addition, TRAP-related PM has also been examined for neurological effects. For example, SD rats exposed to PM 1 (250-300 µg/m 3 ) for 3 and 6 months demonstrated that PM 1 induced cytotoxicity, lipid peroxidation, microglial activation, and inflammation as well as autophagy and caspase-3 up-regulation in microglia [80]. Wistar rats inhaling DEP nanoPM (0.3-1.0 mg/L) for 3 months had higher levels of COX-2 and Aβ 42 in brain regions [86]. An interesting finding came from transgenic mice with human APOE3 and E4 alleles, showing that chronic exposure to nanoPM over 15 weeks increased the cerebral Aβ production and deterioration of hippocampal CA1 neurons, with a more significant effect in APOE4 carriers [87].
The role of microglia in DEP-induced neurodegenerative disorders has been explored mostly in mouse models. The acute exposure of adult mice to DEP (250-300 mg/m 3 for 6 h) caused microglial activation, lipid peroxidation, and reduced neurogenesis in all brain regions, particularly in the hippocampus and the OB [88]. The blockage of microglial activity with the PPAR-γ agonist pioglitazone inhibited the DEP-induced neuroinflammation in cerebral cortex, oxidative stress, and neurogenesis reduction in the hippocampus [89]. The exposure of mice to TRAP-PM 0.2 (300 µg/m 3 ) or of neuronal cells to the same nanoPM (1-10 µg/mL) caused an increase in oxidative stress in lipid rafts associated with an increase in Aβ, the latter was inhibited by the antioxidant N-acetyl cysteine, suggesting that oxidative stress was involved in the pro-amyloidogenic effects of air pollution [90]. Interestingly, Cheng et al. examined the differential time course of oxidative stress and inflammatory responses to UFPM between the OE and the brain. It was found that OE and OB, but not the cerebral cortex and cerebellum, had rapid increases in microglial number, and oxidative and nitrosative protein adducts in the nasal epithelium turbinate after 5 h exposure, which precedes an increase in levels of TNF-α by 45 h [91]. These responses corresponded to in vitro OE and mixed glial responses, with the rapid induction of nitrite and iNOS preceding the induction of TNF-α [91] Furthermore, wild-type (WT) and Nrf2 knockout (Nrf2 −/− ) mice were subjected to the intranasal instillation of 1 mg/kg PM 2.5 for 28 days. Lower levels of antioxidant enzymes, oxidative stress, microglia activation, inflammation, NF-κB activation, and severe nerve injury were detected in the OB of Nrf2 −/− mice compared to the OB of WT mice [92]. In addition, PM 2.5 exposure-induced oxidative stress and microglia activation was attributed to its metal contents and glutaminase-containing extracellular vehicles (EVs) in the OB [93].
In PD condition, α-synuclein is a major component of Lewy bodies, a pathological hallmark of PD [94]. A controlled study found that exposure of male Fischer 344 rats to DEP (311 µg/m 3 or higher) for 6 months increased α-synuclein and Aβ 42 levels in the midbrain [85]. In another study, an increase in α-synuclein levels was also found in the cerebral cortex of C57BL6/J mice exposed to DEP (250 µg/m 3 ) for 3 weeks [1].
In summary, animals exposed to PM ambiently or in controlled experiments reveals the same pattern of neurotoxic effects as in humans although the crosstalk among these events need further clarification. Short-term exposure to traffic-related air pollution induces the differential expression of inflammatory and oxidative genes in different brain regions. The olfactory bulb may display a lower neurotrophic support in response to air pollution.
Elevated levels of whole-brain IL-6, nitrated proteins and Iba-1 (biomarker of microglia activation). The midbrain displayed a higher sensibility to DEP.
Inhalation of DEP causes various degrees of microglia activation and neuroinflammation in different brain regions.

Evidence from In Vitro Studies
In vitro studies provide insights into in-depth cellular and molecular mechanisms by which PM exposure promotes cellular damage and abnormality, linking to neurodegenerative disorders, such as the alterations of cell viability and apoptosis, the dysfunction of mitochondria, the production of ROS, or the release of pro-inflammatory mediators [1,17]. Thus far, neuronal and microglial cell lines, primary cultures or co-culture of those cells have been introduced for exposure to concentrated ambient air particles, DEP, and LPS, among others [95] (Table 3).
A mouse microglial cell line (BV2) exposed to concentrated ambient PM 2.5 displayed the upregulated mRNA of pro-inflammatory cytokines, such as IL-1β and TNF-α [96]. Moreover, the inhibition of Nrf2 activity significantly blocked the PM 2.5 -induced decrease in cell viability, the increase in the intracellular ROS generation, and the NFκB phosphorylation in BV2 cells [92]. The acute exposure of microglial cells to high-dose PM 2.5 decreased cell survival as a result of neuroinflammation and the production of ROS [97,98]. In addition, an in vitro neuron-microglia culture model exposed to PM 2.5 presented elevated apoptosis, IL-1β, and caspase-1 activity, which could be alleviated by the addition of IL-1 receptor antagonists and ROS inhibitors [98]. Together, these findings suggest that PM 2.5 has a role in AD pathogenesis, the underlying mechanisms possibly being PM 2.5induced microglial activation, neuroinflammation, increased ROS activity, and even Aβ production [95]. Notably, there is evidence that metals associated with PM 2.5 may activate microglia, since microglia can be activated in vitro by manganese [99].
Microglia were first shown to recognize and respond to PM in an in vitro study using DEP [100]. Generally, in vitro studies support in vivo observations by showing that DEP can activate microglia, resulting in oxidative stress and neuroinflammation [86,100,101]. With BV2 cells, DEP were shown to reduce cell viability and increase microglial activation, lipid peroxidation, the production of pro-inflammatory mediators, including IL-6, TNF-α and prosglandin E2 (PGE2), and cytotoxicity [80]. Intriguingly, exposure to DEP (25-100 µg/cm 2 ) did not affect the viability of mouse primary cerebellar granule neurons in vitro. However, the death of these neurons was increased two-or three-fold with simultaneous exposure to DEP and microglia [101]), suggesting that microglia play an essential role in DEP-induced neurotoxicity.
In the PD condition, primary neuron-glia co-cultures and the HAPI (highly aggressively proliferating immortalized) microglial cell line were pre-treated with DEP (5 µg/mL) followed by LPS (2.5 ng/mL) and synergistically amplified NO production, TNF-α release, and DA neurotoxicity [84]. Pre-treatment with fractalkine (50 pg/mL), a chemokine from neurons as a soluble anti-inflammatory signal for microglia [102], ameliorated DEP (50 µg/mL)-induced H 2 O 2 production from microglia and protected against DEP-induced DA neurotoxicity in midbrain neuron-glia cultures [84]. Another study with mesencephalic neuron-glia cultures treated with DEP (5-50 µg/mL) resulted in a dose-dependent microglial activation determined by changes in morphology and increase in superoxide production and a decrease in DA neurons, with no TNF-α, NO, or prostaglandin PGE2 detected [100]. Noticeably, the selective DA neurotoxicity only occurred in the presence of microglia, indicating that microglia mediated the neuron damage. In addition, a study revealed that microglia cultures derived from mice missing functional NADPH oxidase, the enzyme responsible for microglial extracellular superoxide production, were insensitive to DEP-induced neurotoxicity, indicating that microglia-derived ROS are key for DEP-induced DA neurotoxicity [100]. Thus, it is assumed that the neurotoxic effects of DEP on DA neurons could be either direct or indirect via the release of inflammatory mediators and ROS from activated microglial cells [84,100,102].
Overall, in vitro studies suggest PM-induced oxidative stress and microglial cell mediated inflammatory and/or oxidative responses as potential mechanisms leading to neurotoxicity, and the increased risk of neurodegenerative disease as seen in epidemiological and animal studies.  DEP treatment did not affect the viability of CGNs. Neuronal cell death increased by 2-3-fold after co-treatment with microglia. Elevated level of ROS genes expression of IL-1β and IL-6 in microglia.
Microglia may mediate DEP-induced neuronal toxicity through oxidative stress and neuroinflammatory mechanisms.

Potential Mechanisms for PM 2.5 -Induced Microglial Oxidative Stress and Neuronal Toxicity
The aforementioned evidence suggests that exposure to PM 2.5 has a crucial role in neurodegenerative disorders possible through the activation of microglial cells [95]. However, the responsible mechanisms remain large unknown. The four critical issues are of significant relevance and need to be addressed: (1) The routes through which PM 2.5 access CNS; (2) the receptors microglia use to relay PM 2.5 neurotoxic signals; (3) PM 2.5 -induced microglial oxidative stress; and (4) the interactions between microglia and neurons.
Route of CNS Effects: Multiple routes for PM 2.5 impact on the CNS have been proposed, two of them are predominant (Figure 2). First, PM 2.5 , UFPM, and their components can enter the olfactory receptor neurons that extend their dendrites into the mucous layer covering the OE through pinocytosis, simple diffusion, or receptor-mediated endocytosis, and is further transported along the axons to the OB and olfactory cortex [9,103]. In addition, UFPM exposure has been shown to rapidly increase the products of lipid peroxidation, such as 4-hydroxy-2-nonenal (4-HNE) and 3-nitrotyrosine (3-NT) protein adducts in the OE and OB [91], further leading to oxidative inflammation from nose to brain. Second, PM 2.5 inhaled into the deep lung successively penetrates the alveolar-blood barrier and the BBB and finally reaches brain regions. Meanwhile, lung-derived circulating cytokines induced by PM 2.5 exposure could also enter the brain [18]. In both cases, PM 2.5 directly or indirectly activates microglia and induces the release of pro-inflammatory cytokines and ROS, leading to neurodegeneration [66,104]. Alternatively, PM 2.5 may potentially affect the CNS via the gut microbiota-brain axis [1,105] or lung microbiota [106].

Potential Mechanisms for PM2.5-Induced Microglial Oxidative Stress and Neuronal Toxicity
The aforementioned evidence suggests that exposure to PM2.5 has a crucial role in neurodegenerative disorders possible through the activation of microglial cells [95]. However, the responsible mechanisms remain large unknown. The four critical issues are of significant relevance and need to be addressed: (1) The routes through which PM2.5 access CNS; (2) the receptors microglia use to relay PM2.5 neurotoxic signals; (3) PM2.5-induced microglial oxidative stress; and 4) the interactions between microglia and neurons.
Route of CNS Effects: Multiple routes for PM2.5 impact on the CNS have been proposed, two of them are predominant ( Figure 2). First, PM2.5, UFPM, and their components can enter the olfactory receptor neurons that extend their dendrites into the mucous layer covering the OE through pinocytosis, simple diffusion, or receptor-mediated endocytosis, and is further transported along the axons to the OB and olfactory cortex [9,103]. In addition, UFPM exposure has been shown to rapidly increase the products of lipid peroxidation, such as 4-hydroxy-2-nonenal (4-HNE) and 3-nitrotyrosine (3-NT) protein adducts in the OE and OB [91], further leading to oxidative inflammation from nose to brain. Second, PM2.5 inhaled into the deep lung successively penetrates the alveolar-blood barrier and the BBB and finally reaches brain regions. Meanwhile, lung-derived circulating cytokines induced by PM2.5 exposure could also enter the brain [18]. In both cases, PM2.5 directly or indirectly activates microglia and induces the release of pro-inflammatory cytokines and ROS, leading to neurodegeneration [66,104]. Alternatively, PM2.5 may potentially affect the CNS via the gut microbiota-brain axis [1,105] or lung microbiota [106]. The routes that PM2.5 enters the brain. The lung-brain axis and olfactory pathway are two recognized predominant routes that PM2.5 takes into the brain. Once inhaled, PM2.5 can quickly diffuse throughout the alveoli and lead to lung inflammation. These circulating cytokines (IL-6, TNFα, IL-1β, etc.), in combination with soluble components of PM2.5, cross the BBB directly or via a disruption to the permeability of the BBB, and then induce microglia activation and neurotoxicity. Meanwhile, with a consequence of lipid peroxidation (4-HNE and 3-NT protein adduction), PM2.5 could also gain access to the olfactory bulb through the olfactory epithelium and then move into the deep regions of the brain. Moreover, the gut-brain axis is potentially another route through which PM2.5 exerts its neurotoxicity, which is probably associated with the dysbiosis of gut microbiota. The routes that PM 2.5 enters the brain. The lung-brain axis and olfactory pathway are two recognized predominant routes that PM 2.5 takes into the brain. Once inhaled, PM 2.5 can quickly diffuse throughout the alveoli and lead to lung inflammation. These circulating cytokines (IL-6, TNF-α, IL-1β, etc.), in combination with soluble components of PM 2.5 , cross the BBB directly or via a disruption to the permeability of the BBB, and then induce microglia activation and neurotoxicity. Meanwhile, with a consequence of lipid peroxidation (4-HNE and 3-NT protein adduction), PM 2.5 could also gain access to the olfactory bulb through the olfactory epithelium and then move into the deep regions of the brain. Moreover, the gut-brain axis is potentially another route through which PM 2.5 exerts its neurotoxicity, which is probably associated with the dysbiosis of gut microbiota.

Sensing receptors on microglia:
Microglia monitor the brain environment by interpreting and processing stimuli through pattern recognition receptors (PRRs) (Figure 3), which mainly include TLRs [107,108], scavenger receptors (e.g., SR-A1 and SR-B1) [109], macrophage antigen complex 1 (MAC1), and receptor complexes (CD36, α6β1 integrin, and CD47) [16], for diverse neurotoxic and pro-inflammatory ligands, respectively. Both nanoPM and LPS have been shown to strongly activate TLR4 and NF-κB in mixed glial cultures. TLR4 siRNA attenuated TNF-α and other inflammatory responses to nanoPM via the MyD88-dependent pathway [110]. Thus, PPRs expressed on the microglial surface seem to be one of the primary common pathways by which ambient PM signals are transduced into ROS production [16].
Antioxidants 2022, 11, x FOR PEER REVIEW 19 of 25 Figure 3. Proposed mechanisms for PPR-mediated microglia activation and neuronal toxicity induced by PM2.5. Microglia monitor the brain environment by interpreting and processing stimuli through pattern recognition receptors (PRRs), which mainly include TLRs, scavenger receptors, MAC1, and receptor complex for diverse neurotoxic and pro-inflammatory ligands, respectively. Exogenous and endogenous insults bind to diverse PPRs and result in microglial activation and release of soluble factors, such as cytokines, PGE2, and neurotrophins (BDNF), which bind to neuronal receptors. Meanwhile, neuronal metabolites and damaged neuron components could also activate microglia. Together, microglia-neuron interactions further promote the pathogenesis of neurodegenerative disorders.

Conclusions and Future Directions
In summary, air pollution, together with the increasing age of the global population, pose great threats to public health. Thus far, the mechanisms responsible for PM2.5-induced neurodegenerative diseases remain largely unknown. The CNS effects are chronic, beginning in childhood, and may take time to accumulate pathology. Specifically, air pollution has been shown to cause neuroinflammation, oxidative stress, cerebral vascular damage, and neurodegenerative pathology, which all involve microglial activation. Evidence from epidemiological and experimental studies suggests that exposure to ambient Proposed mechanisms for PRR-mediated microglia activation and neuronal toxicity induced by PM 2.5 . Microglia monitor the brain environment by interpreting and processing stimuli through pattern recognition receptors (PRRs), which mainly include TLRs, scavenger receptors, MAC1, and receptor complex for diverse neurotoxic and pro-inflammatory ligands, respectively. Exogenous and endogenous insults bind to diverse PPRs and result in microglial activation and release of soluble factors, such as cytokines, PGE2, and neurotrophins (BDNF), which bind to neuronal receptors. Meanwhile, neuronal metabolites and damaged neuron components could also activate microglia. Together, microglia-neuron interactions further promote the pathogenesis of neurodegenerative disorders.
Microglia-associated oxidative stress: ROS are critical components of the proinflammatory signaling pathway in microglia [110]. Activated microglia by exogenous and endogenous insults can become a chronic source of pro-inflammatory factors and oxidative stress in the brain, driving neurodegenerative diseases [16]. In microglia, ROS primarily from both NOX and the mitochondria, may act as second messengers to propagate immune activation, excessive inflammation, and oxidative stress [111]. Ambient particles transported into the brain could be phagocytized by microglia, leading to NOX and microglial activation, and ROS production [84]. Mitochondrial dysfunction in microglia has been proposed to play a role in the progression of neurodegenerative diseases [112]. The elevated generation of ROS and the loss of mitochondrial membrane potential through various mechanisms have been observed in AD. Aβ interacts with microglial receptors, such as TREM2, activating downstream pathways, causing mitochondrial damage, and aggravating inflammation and cytotoxicity. Fibrillar Aβ activates NOX in microglia leading to the elevated induction of mitochondrial ROS, which further causes neurotoxicity [112].
Microglia-neuron interactions: The bi-directional communication between microglia and neurons has been recognized to be critical for maintaining a healthy environment in the CNS and also for the chronic development of neuroinflammation [113]. The air pollution-induced loss of neurons has been detected in postmortem and experimental studies, as described earlier, and neuronal cell death may be direct or indirect via microglia activation [12]. Thus far, the mechanisms for microglia-neuron interaction remain elusive. Activated microglia can release soluble factors, such as cytokines (IL-1β; TNF-α), PGE2, and neurotrophins (BDNF), which bind to neuronal receptors [114]. With a primary cerebellar granule neuron (CGN) model, DEP showed minimal effect on neuron viability after 24 h of treatment. In the presence of primary cortical microglia neuronal cell death increased by 2-3 fold after co-treatment with DEP, suggesting that microglia are important contributors to DEP-induced CGN neurotoxicity, possibly due to soluble intermediates since microgliaconditioned medium by DEP treatment was also toxic to CGNs [101]. In addition, Block et al. showed that DEP could damage DA neurons through microglia-derived oxidant species [104]. However, another study reported that DEP caused a significant increase in ROS in microglia, antioxidants failed to protect neurons from DEP/microglia-induced toxicity [105]. From the above, the mechanisms underlying PM 2.5 -induced microglial activation and its interaction with neurons are still unclear and warrant further investigation.

Conclusions and Future Directions
In summary, air pollution, together with the increasing age of the global population, pose great threats to public health. Thus far, the mechanisms responsible for PM 2.5 -induced neurodegenerative diseases remain largely unknown. The CNS effects are chronic, beginning in childhood, and may take time to accumulate pathology. Specifically, air pollution has been shown to cause neuroinflammation, oxidative stress, cerebral vascular damage, and neurodegenerative pathology, which all involve microglial activation. Evidence from epidemiological and experimental studies suggests that exposure to ambient PM, especially PM 2.5 , is associated with neurodegenerative disorders. The interpretation of the intracellular and extracellular pathways participating in the generation of oxidative stress in microglia may be important not only for comprehending the pathophysiological basis for neuron damage in neurodegenerative diseases but also for designing effective strategies to mitigate or even prevent PM 2.5 -induced neural neurodegenerative damage. While epidemiology has linked an increased risk of stroke, AD, and PD with exposure to PM 2.5 , further epidemiological and mechanistic studies regarding the association between the components of air pollution and the development of CNS diseases are of pressing concern for human health.