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Abnormal energy metabolism and tau phosphorylation in the brains of middle-aged mice in response to atmospheric PM2.5 exposure
JES-01254; No of Pages 9 J O U RN A L OF E N V I RO N ME N TA L S CI EN CE S X X (2 0 1 7 ) XX X–XXX
Available online at www.sciencedirect.com
ScienceDirect www.elsevier.com/locate/jes
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Abnormal energy metabolism and tau phosphorylation in the brains of middle-aged mice in response to atmospheric PM2.5 exposure Rui Gao, Tingting Ku, Xiaotong Ji, Yingying Zhang, Guangke Li⁎, Nan Sang⁎
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College of Environment and Resource, Research Center of Environment and Health, Shanxi University, Taiyuan, Shanxi 030006, PR China
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Article history:
In light of the accelerated aging of the global population and the deterioration of the 15
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Received 30 March 2017
atmosphere pollution, we sought to clarify the potential mechanisms by which fine 16
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Revised 28 June 2017
particulate matter (PM2.5) can cause cognitive impairment and neurodegeneration through 17
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Accepted 29 June 2017
the alteration of mitochondrial structure and function. The results indicate that PM2.5 18
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inhalation reduces ATP production by disrupting the aerobic tricarboxylic acid (TCA) cycle and 19
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Keywords:
middle-aged mice. Furthermore, excessive ROS generation was involved in the impairment. 21
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Fine particulate matter (PM2.5)
Interestingly, these alterations were partially reversed after exposure to PM2.5 ended. These 22
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Mitochondrial structure
findings clarify the mechanism involved in mitochondrial abnormality-related neuropatho- 23
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and function
logical dysfunction in response to atmospheric PM2.5 inhalation and provide an optimistic 24
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Energy metabolism
sight for alleviating the adverse health outcomes in polluted areas.
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Reactive oxygen species (ROS)
© 2017 The Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences. 26
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Tau phosphorylation
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oxidative phosphorylation, thereby causing the hypophosphorylation of tau in the cortices of 20
Introduction
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Population aging is a pervasive and unprecedented phenomenon that cannot be overlooked. People aged 60 years and older are estimated to become approximately 22% of the global population and to exceed the number of young people for the first time in history by 2050 (World Population Ageing: 1950–2050. Population Division, DESA, United Nations, 2006). As a result, the health problems of aging individuals have become an important social issue. Among these, the number of individuals with illnesses associated with cognitive impairment such as dementia, including Alzheimer's disease (AD) and Parkinson's disease (PD), has reached 24 million and is predicted to quadruple by the year 2050 (Reitz and Mayeux, 2014). An epidemiological investigation showed that approximately 40% of people aged 85 years and over suffer from AD
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and 10% suffer from PD (de Lau and Breteler, 2006; Ferri et al., 2005). While the majority of AD and PD cases are sporadic, inheritable genetic mutations play a small role in their etiologies (Dosunmu et al., 2007; Heusinkveld et al., 2016). Environmental factors such as pesticides, metals, and atmospheric pollutants have been postulated to contribute to the pathogenesis of AD and PD (Yan et al., 2016a, 2016b; McAllum and Finkelstein, 2016; Block and Calderón-Garcidueñas, 2009). Exposure to atmospheric fine particles (PM2.5) has been linked with numerous diseases, both local (in the lung) and systemic (in extra-pulmonary sites, such as cardiovascular and neuronal tissues) (Liu et al., 2017; Kioumourtzoglou et al., 2016). A lifespan study found that atmospheric pollution is associated with the quantifiable impairment of brain development in young people and with cognitive decline in the elderly (Clifford et al., 2016). A meta-analysis based on 108 papers
⁎ Corresponding authors. E-mail: [email protected] (Nan Sang).
http://dx.doi.org/10.1016/j.jes.2017.06.037 1001-0742/© 2017 The Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences. Published by Elsevier B.V.
Please cite this article as: Gao, R., et al., Abnormal energy metabolism and tau phosphorylation in the brains of middle-aged mice in response to atmospheric PM2.5 exposure, J. Environ. Sci. (2017), http://dx.doi.org/10.1016/j.jes.2017.06.037
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1. Materials and methods
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1.1. PM2.5 sampling and preparation
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PM2.5 was collected between November 2014 and February 2015 at Shanxi University (112°21–34′E longitude, 37°47–48′N latitude), Taiyuan City, Shanxi Province, China. A mediumvolume air sampler (TH-150CIII, Wuhan, China) with a flow rate of 100 L/min was placed on the top of a building far from obstacles to obtain free-moving air. Samples were collected on quartz filter membranes (Φ90 mm, Munktell, Sweden) for 22 hr/d and then extracted in Milli-Q deionized water with ultrasonic processing more than three times. After vacuum freeze-drying, the samples were resuspended in sterilized 0.9% physiological saline. The aqueous suspension was pooled, frozen at −20°C and swirled for 10 min before being used for animal experiments.
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1.2. Animals and PM2.5 exposure
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Female 10-month-old C57BL/6 mice were purchased from the Junke Biological Engineering Co., LTD (Nanjing, China). These
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1.3. Transmission electron microscope (TEM) observation
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The brain cortex tissues in mice were rapidly cut into pieces approximately 1 mm3 and was fixed in stationary liquid. Then pieces were washed and en bloc stained with 1% uranyl acetate in 50% ethanol at room temperature in the dark, dehydrated by graded ethanol, and embedded in beam capsules. The 70– 80-nm-thick ultrathin sections cut from the embedded tissue were collected onto grids, and then were stained with uranyl acetate and lead citrate. The mitochondrial structural were observed with an electron microscope (JEOL, JEM 1400, Japan).
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1.4. Determination of the ATP content
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The ATP levels were determined by the ATP detection kit according to the manufacturer's instructions (Beyotime, China). Approximately 30 mg cortex tissues were used with 240 μL lysis buffer for ATP releasing. After high speed centrifuged, the ATP content in supernatant was measured by the luciferin-luciferase method with a Thermo Scientific Varioskan Flash (Thermo Fisher Scientific, USA). The protein concentration was determined with Coomassie light blue according to the Bradford (1976). Finally, the ATP content normalized to protein concentrations were used for comparative analysis.
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mice were housed under standard conditions (24 ± 2°C, 50 ± 5% humidity, 12:12 hr light:dark cycle) and randomly divided into a PM2.5-treated group and a control group. Mice in the PM2.5 treatment group received oropharyngeal aspirations of PM2.5 at 3 mg/kg after anesthetization with isoflurane (Yi Pin Pharmaceutical Co., Ltd., Hebei, China) every other day at different times. The four treatment groups included groups exposed for 2 weeks, exposed for 4 weeks, allowed to recover for 1 week after being exposed for 4 weeks, and allowed to recover for 2 weeks after being exposed for 4 weeks. Mice in the control group were treated with 0.9% saline (processed by the ultrasonic oscillation of the control membrane filter) using the same method. Mice were sacrificed, and their brain cortices were separated 24 hr after the last exposure. The cortices were immediately frozen in liquid nitrogen and then stored at −80°C until further use. All animal experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of Shanxi University.
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revealed a higher risk of death for older populations (0.64%) than for younger populations (0.34%) per 10 μg/m3 increase in particulate matter (PM) (Bell et al., 2013). Although many epidemiological studies have described the adverse effects of PM2.5 on cognitive function in middle-aged people (Ailshire and Crimmins, 2014; Schikowski et al., 2015), there have been limited laboratory investigations into the mechanism of injury. Casanova reported that long-term PM2.5 exposure might accelerate the loss of both gray matter and white matter in the brains of older women (Casanova et al., 2016). However, whether the molecular targets of neurological degenerative damage are influenced by PM2.5 and whether these effects can be reversed remain unclear. Tau is a microtubule-associated protein (MAP) primarily responsible for the assembly, maintenance and stability of microtubules. The abnormal hyperphosphorylation of tau, a hallmark of neurodegenerative illnesses (particularly AD), can decrease the stability of microtubules and disrupt cytoskeletal integrity and axonal transport (Lovestone and Reynolds, 1997; Cowan et al., 2010). Recent studies have revealed an increased trend of tauopathy not only in older patients with AD (Kehoe et al., 2016; Hamasaki et al., 2016) but also in cognitively normal middle-aged people (potentially primary age-related tauopathy) (Crary et al., 2014; Lockhart et al., 2016). Emerging evidence has revealed that abnormal glucose metabolism and decreased adenosine triphosphate (ATP) production are correlated with tau hyperphosphorylation in AD patients (Szablewski, 2017; Hoyer and Lannert, 2007). Due to the high energy demands of the brain, central nervous system (CNS) functions are strongly dependent on sufficient mitochondrial production of ATP. The aging mitochondrial theory suggests that ATP deficiencies play a critical role in the accelerated aging disorders of the elderly (Scheibye-Knudsen, 2016). Thus, the aim of our present study was to clarify the possible mechanisms by which PM2.5 causes tau phosphorylation-related cognitive impairment and neurodegeneration by affecting mitochondrial structure and function.
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1.5. Real-time quantitative reverse transcription-polymerase 169 chain reaction(PCR) 170 Q8 Approximately 30 mg of cortex tissue were used for total RNA extraction with TRIzol reagent (Invitrogen, USA). The RNA concentration was quantified with a NanoDropTM 2000C (Thermo, USA). Then the extracted RNA completes reverse transcription to form the first-strand complementary DNA (cDNA) using a reverse transcription kit (TaKaRa, China). The mRNA expression levels of tricarboxylic acid (TCA) cycle- and oxidative phosphorylation-related genes were determined by real-time polymerase chain reaction (PCR) on a qTOWER 2.2 real-time PCR system (Analytik Jena AG, Jena, Germany). The
Please cite this article as: Gao, R., et al., Abnormal energy metabolism and tau phosphorylation in the brains of middle-aged mice in response to atmospheric PM2.5 exposure, J. Environ. Sci. (2017), http://dx.doi.org/10.1016/j.jes.2017.06.037
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secondary antibody (1:5000) (IRDye 800CW goat anti-rabbit 211 IgG, LI-COR) and imaged with a LI-COR Odyssey Infrared 212 Fluorescent System. 213
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1.6. Reactive oxygen species (ROS) detection
1.8. Statistical analysis
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Data were analyzed using SPSS 17.0 software (SPSS Inc., Chicago, IL, USA), and results are presented as the mean ± SE. Significant differences were identified via one-way analysis of variance (ANOVA) followed by Fisher's least significant difference (LSD) test and were established at p < 0.05.
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Approximately 30 mg of cortex tissue was homogenized with 600 μL of phosphate buffered solution, and the supernatant was collected after centrifugation. The ROS content in the supernatant was detected by incubation with 2′, 7′-dichlorofluorescein-diacetate (DCFH-DA, 50 μM) at 37°C for 30 min in the dark. The formation of the fluorescent-oxidized derivative of 2′, 7′-dichlorofluorescein (DCF) was measured with a Thermo Scientific Varioskan Flash (Thermo Fisher Scientific, USA) at an excitation wavelength of 488 nm and an emission wavelength of 530 nm. The protein concentration was determined with Coomassie light blue according to the Bradford (1976). Finally, the ROS content normalized to the protein concentration was used for comparative analysis.
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1.7. Western blot assays
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Approximately 30 mg of cortex tissue was used for total protein extraction with lysis buffer. The protein concentration was determined with Coomassie light blue according to the Bradford (1976). Then, 50 μg of total proteins were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), transferred to a polyvinylidene difluoride (PVDF) membrane, and blocked with 3% bovine albumin. The blocked membranes were incubated overnight at 4°C with monoclonal antibodies for β-actin (1:1000, Cell Signaling, USA), polyclonal antibodies for tau (1:200, Bioss, China) and polyclonal antibodies for p-tau (1:200, Bioss, China). The membranes were then incubated with fluorescently labeled
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Table 1 – The primer sequences and reaction conditions used for real-time PCR. Sequence (5′ to 3′)
GAPDH sense GAPDH anti-sense PDHA1 sense PDHA1 anti-sense CS sense CS anti-sense OGDH sense OGDH anti-sense IDH2 sense IDH2 anti-sense FH sense FH anti-sense ATP6 sense ATP6 anti-sense CO1 sense CO1 anti-sense CO4 sense CO4 anti-sense
CTTTGGCATTGTGGAAGGGC CAGGGATGATGTTCTGGGCA TGTGAGAACAACCGCTAT ATCCATTCCATCTACCCT CCTGGTCGTTTGGCTTTA TGTGCTATGGGCTCTTAC CAACAGATTCGGTGCTAT AGTGGTGGTGGGTAAGTG GCAGCAGTGCCAAGGAGT AGCCGATGTTCATACCAGAT AATCCCAGTCATTCAAGC CAGTCTGCCAAACCACCA TTCCCATCCTCAAAACGCCT GTTCGTCCTTTTGGTGTGTGG CTATGTTCTATCAATGGGAGC TCTGAGTAGCGTCGTGGT TCTACTTCGGTGTGCCTTCG CGATCAGCGTAAGTGGGGAA
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2. Results
PCR: polymerase chain reaction.
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2.1. Abnormal mitochondrial morphology and deficient ATP 222 production in the cortex 223
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Due to the high energy demands of the brain, CNS functions are strongly dependent on the sufficient production of mitochondrial ATP. To evaluate the mitochondrial signaling pathway after PM2.5 exposure, mitochondrial ultra-structures were observed using transmission electron microscopy (TEM). Broken mitochondrial membranes and fractured and lost mitochondrial cristae appeared in response to PM2.5 exposure for 4 weeks (Fig. 1). Together, these images demonstrate that PM2.5 exposure damages the integrity and function of mitochondria in the brain. Mitochondria produce ATP, which, as a direct source of cellular energy, plays a critical role in accelerated aging disorders in the elderly (Gibson et al., 2010). Abnormal mitochondrial morphology is suggested to be directly related to the production of ATP. To provide further evidence for this hypothesis, we detected the concentration of ATP in the cortex after PM2.5 exposure and following restorative treatment. As shown in Fig. 2, PM2.5 exposure led to a time-dependent decrease in ATP levels, which reached 50% of those in the control group at 4 weeks after treatment. Interestingly, the reduction in ATP levels was reversed to 10% that of the control group after 2 weeks of recovery. These results suggest that ambient PM2.5 exposure has the potential to cause mitochondrial deterioration in the middle-aged mice and that recovery is incomplete.
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relative quantification of gene expression was determined using GAPDH as an internal control. The primer sequences and reaction conditions used for RT-PCR are listed in Table 1.
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2.2. Changes of gene expression involved in the tricarboxylic 248 acid cyclein (TCA) in the cortex 249 The tricarboxylic acid cyclein (TCA) cycle is the main pathway for the oxidation of glucose in the brain and is a crucial intermediate step for the production of ATP (Chen and Zhong, 2013). To test whether the TCA cycle was involved in the depletion of ATP caused by ambient PM2.5, we determined the expression levels of five key TCA cycle genes. As presented in Fig. 3, PM2.5 treatment significantly reduced the expression of pyruvate dehydrogenase (lipoamide) alpha 1 (PDHA1) in the second step of TCA cycle as well as the expression of rate-limiting enzymes in the TCA cycle such as citrate synthase (CS), isocitrate dehydrogenase 2 (NADP+) (IDH2), and oxoglutarate (alpha-ketoglutarate) dehydrogenase (lipoamide) (OGDH), but not fumarase (FH). After the cessation of PM2.5 treatment, the mRNA levels of CS and IDH2 increased considerably, while other genes maintained their
Please cite this article as: Gao, R., et al., Abnormal energy metabolism and tau phosphorylation in the brains of middle-aged mice in response to atmospheric PM2.5 exposure, J. Environ. Sci. (2017), http://dx.doi.org/10.1016/j.jes.2017.06.037
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elevations, a potential reason for the incomplete reversal of ATP production.
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2.3. Changes in oxidative phosphorylation in the cortex
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Oxidative phosphorylation, as the final step in the production of ATP from adenosine diphosphate (ADP), accompanies the process of electron transfer in the mitochondrial respiratory chain. The oxidative phosphorylation system is composed of five multisubunit complexes (complexes I–V). In our study,
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the mRNA levels of complex IV subunits (CO1 and CO4) and complex V subunits (ATP6) were determined (Fig. 4). The expression of CO1 and ATP6, encoded by mitochondrial DNA (mtDNA), decreased after PM2.5 exposure in a time-dependent manner and recovered to normal levels when PM2.5 treatment stopped. The mRNA expression level of CO4, encoded by nuclear DNA (nDNA), decreased weakly after 4 weeks of exposure. Consistent with the TCA cycle, changes in oxidative phosphorylation further clarified the decrease in ATP production following PM2.5 exposure.
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Fig. 1 – Electron micrographs of neuronal mitochondria from the cortical regions of mouse brains after exposure to PM2.5. After PM2.5 treatment, cristae within the mitochondria were broken, fragmented and in some cases lost, and the mitochondrial membrane was ruptured. The red arrows represent these broken structures. 40,000× magnification, scale bars = 500 nm. PM2.5: fine particulate matter.
Fig. 2 – ATP levels in the cortical regions of mouse brains after exposure to PM2.5. Values are recorded as the mean ± SE (n = 8). *p < 0.05 compared with the corresponding control groups. ATP: adenosine triphosphate; PM2.5: fine particulate matter. Please cite this article as: Gao, R., et al., Abnormal energy metabolism and tau phosphorylation in the brains of middle-aged mice in response to atmospheric PM2.5 exposure, J. Environ. Sci. (2017), http://dx.doi.org/10.1016/j.jes.2017.06.037
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Fig. 3 – The expression of genes involved in the TCA cycle in the cortical regions of mouse brains after exposure to PM2.5. The levels of pyruvate dehydrogenase (lipoamide) alpha 1 (PDHA1), citrate synthase (CS), isocitrate dehydrogenase 2 (NADP+) (IDH2), oxoglutarate (alpha-ketoglutarate) dehydrogenase (lipoamide) (OGDH), and fumarase (FH) mRNA expression in mouse brains after exposure to PM2.5 were determined through qRT-PCR analyses. Values are recorded as the mean ± SE (n = 8). *p < 0.05, **p < 0.01 compared with the corresponding control groups; #p < 0.05, ##p < 0.01 vs. the treatment groups. TCA: tricarboxylic acid; PCR: polymerase chain reaction; PM2.5: fine particulate matter.
Fig. 4 – The expression of genes involved in oxidative phosphorylation in the cortical regions of mouse brains after exposure to PM2.5. The mRNA levels of complex IV subunits (CO1 and CO4) and complex V subunits (ATP6) were determined in mouse brains after exposure to PM2.5 through qRT-PCR analyses. Values are recorded as the mean ± SE (n = 8). *p < 0.05, **p < 0.01 compared with the corresponding control groups; #p < 0.05, ##p < 0.01 vs. the treatment groups. ATP: adenosine triphosphate; PCR: polymerase chain reaction; PM2.5: fine particulate matter. Please cite this article as: Gao, R., et al., Abnormal energy metabolism and tau phosphorylation in the brains of middle-aged mice in response to atmospheric PM2.5 exposure, J. Environ. Sci. (2017), http://dx.doi.org/10.1016/j.jes.2017.06.037
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To further determine whether ATP deficiency is linked to the alteration of microtubule-associated tau protein, the phosphorylation of tau was detected after PM2.5 treatment. Compared to the control group, the phosphorylation of tau was increased after PM2.5 exposure, and a significant change was detected after 4 weeks of treatment (Fig. 6). After the cessation of PM2.5 treatment, p-tau reverted to a relatively low level.
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3. Discussion
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In the present study, we analyzed ROS-associated energy deficiencies and tau phosphorylation in the cortices of middle-aged mice in response to ambient PM2.5 exposure. Due to the high energy demands of the brain, neurological functions are strongly dependent on sufficient mitochondrial
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2.5. Phosphorylation of tau and recovery in the cortex
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An accumulating body of evidence has linked the generation of ROS induced by PM2.5 exposure to toxicity processes (Hong et al., 2016; Bo et al., 2016). Oxidative stress is directly responsible for mitochondrial dysfunction (Sumegi et al., 2017), making it important to determine whether oxidative stress is an intermediate process between PM2.5 exposure and mitochondrial changes. As shown in Fig. 5, ROS levels increased in a time-dependent manner after PM2.5 treatment, and these effects were completely reversed 2 weeks after treatment stopped. This result is consistent with the measured changes in ATP content and indicates that ATP deficiencies after PM2.5 exposure could be attributed to the oxidative stress caused by excessive ROS generation.
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ATP production. Indeed, mitochondrial dysfunction has been related to neurodegenerative and age-related disorders (Ingram and Chakrabarti, 2016), and the aging mitochondrial theory suggests that ATP deficiency plays a critical role in the accelerated aging disorders of the elderly (Scheibye-Knudsen, 2016). Perturbations of mitochondrial energetics have been found in many chemical exposure studies. Our previous studies demonstrated that PM2.5, SO2 and NO2 exposure could induce mitochondrial injury and impair neurological behavior in mice (Ku et al., 2016). In the current study, we observed broken mitochondrial membranes and fractured and lost mitochondrial cristae after PM2.5 exposure for 4 weeks. As mitochondrial damage directly results in a reduction of available energy, we also found decreased ATP levels in the cortex, and these injuries were partially alleviated after exposure stopped. The aerobic metabolism of glucose, as the major source of ATP in vivo, is composed of three steps: glycolysis, the TCA cycle and oxidative phosphorylation. Mitochondria are mainly responsible for the TCA cycle and oxidative phosphorylation, corresponding to the intermediate and final stages of aerobic respiration (Weinberg et al., 2015; Pike Winer and Wu, 2014). To further clarify whether the TCA cycle and oxidative phosphorylation are affected by PM2.5 exposure accompanied by mitochondrial damage, five key TCA cycle genes and the mitochondrial respiratory complexes IV and V were selected as indicators. The TCA cycle is the main pathway for the oxidation of glucose in the brain and is a crucial intermediate step for the production of ATP (Chen and Zhong, 2013). Pyruvate, a product of glycolysis, is first decarboxylated to acetyl CoA by the pyruvate dehydrogenase complex. Following this process, the conversion of acetyl CoA to CO2 is regulated by rate-limiting enzymes in the TCA cycle, allowing the production of large quantities of reducing equivalents (e.g., NADH) through oxidative phosphorylation. The reduced expression
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Fig. 5 – The ROS contents in cortical regions of mouse brains after exposure to PM2.5. Values are recorded as the mean ± SE (n = 8). *p < 0.05, **p < 0.01 compared with the corresponding control groups; #p < 0.05, ##p < 0.01 vs. the treatment groups. ROS: reactive oxygen species; PM2.5: fine particulate matter. Please cite this article as: Gao, R., et al., Abnormal energy metabolism and tau phosphorylation in the brains of middle-aged mice in response to atmospheric PM2.5 exposure, J. Environ. Sci. (2017), http://dx.doi.org/10.1016/j.jes.2017.06.037
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Fig. 6 – PM2.5 exposure potentiates tau phosphorylation in the mouse brain. Western blot analysis of tau and phosphorylated tau (p-tau) expression in the cortex. Values are recorded as the means ± SEs (n = 8). *p < 0.05, **p < 0.01 compared with the corresponding control groups; #p < 0.05, ##p < 0.01 vs. the treatment groups. PM2.5: fine particulate matter.
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of genes involved in the TCA cycle and their inconsistent recovery suggest that a disordered TCA cycle contributes to 349 the abnormal mitochondrial structures and functions ob350 served after PM2.5 exposure and that this process could be 351 reversible if pollution levels are controlled. Mitochondrial 352 oxidative phosphorylation, the final step in the production of 353 ATP from ADP, accounts for more than 90% of cellular ATP 354 Q12 production in most cells and tissues (Satish and Narayan, 355 2012). Oxidative phosphorylation is controlled by five mito356 chondrial respiratory chain complexes (complexes I, II, III, IV 357 and V). The reduced levels of genes associated with complex 358 IV subunits and complex V subunits involved in oxidative 359 phosphorylation, consistent with our observations of TCA 360 cycle effects, further demonstrate that PM2.5 pollution injures 361 mitochondria and weakens ATP production. 362 Mitochondrially derived ROS play diverse and critical roles 363 in the metabolic adaptation of cells to atmospheric pollution Q14 364 Q13 such as PM (Saul et al. 2012; Jiang et al., 2016; Reboul et al., Q15365 2017; Lakey et al., 2016). ROS-dependent mitochondrial 366 dysfunction and the loss of ATP have been demonstrated in 367 Q16 previous studies (Jiang et al., 2015; Cheng et al. 2015). To 368 determine whether ROS were the source of the mitochondrial 369 dysfunction observed in this study, we measured the ROS 370 content, and the consistent relationship between changes in 371 ROS production and ATP decline implies that the excessive 372 production of ROS induced by PM2.5 may be related to 373 mitochondrial dysfunction. 374 Declined production of ATP and the disruption of glucose 375 metabolism homeostasis were linked with tau phosphorylation
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in numerous studies (Sato and Morishita, 2015; Joly-Amado et al., 2016; Szablewski, 2017). The microtubule system, consisting of tubulin and MAPs, are the major ingredients of nerve cell skeleton. Tau protein, as the most prevalent MAP, participates in a variety of cellular functions. The abnormal hyperphosphorylation of tau, as a hallmark of neurodegenerative diseases (particularly AD), can decrease the stability of microtubules and disrupt cytoskeletal integrity and axonal transport (Lovestone and Reynolds, 1997; Cowan et al., 2010). Air pollution including diesel exhaust, cigarette smoke and NO2 are speculated to lead to cognitive deficiencies and neurodegeneration (Levesque et al., 2011; Ho et al., 2012; Yan et al., 2016a, 2016b) in children and middle-aged people. In the present study, the hypophosphorylation of tau, its relation to neurodegeneration and its restoration were observed after PM2.5 treatment and recovery. As atmospheric PM2.5 pollution occurs uninterruptedly and sources of pollution are widespread, 83% of the Chinese population is exposed to PM2.5 (>35 μg/m3), a level of pollution classified as “unhealthy for sensitive groups” (Rohde and Muller, 2015), and may be at risk of cognitive impairment and neurodegeneration.
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4. Conclusions
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We found that energy metabolism abnormalities and tau phosphorylation after PM2.5 exposure in the cortices of middle-aged mice are reversible and that the mechanism is associated with excessive ROS-mediated mitochondrial injury
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Please cite this article as: Gao, R., et al., Abnormal energy metabolism and tau phosphorylation in the brains of middle-aged mice in response to atmospheric PM2.5 exposure, J. Environ. Sci. (2017), http://dx.doi.org/10.1016/j.jes.2017.06.037
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Uncited references
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Soberanes et al., 2012 Srinivasan and Avadhani, 2012 Zhang et al., 2015
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Acknowledgments
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This work was supported by National Science Foundation of China (NSFC, Nos. 91543203, 21477070, 21377076, 21222701), 417 Specialized Research Fund for the Doctoral Program of 418 Higher Education of China (SRFDP, Nos. 20121401110003, 419 20131401110005), Project Supported by Shanxi Young Sanjin 420 Scholarship of China, Program for the Outstanding Innovative 421 Teams of Higher Learning Institutions of Shanxi, and Re422 Q18 search Project Supported by Shanxi Scholarship Council of 423 China (No. 2015-006).
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REFERENCES
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Ailshire, J.A., Crimmins, E.M., 2014. Fine particulate matter air pollution and cognitive function among older US adults. Am. J. Epidemiol. 180 (4), 359–366. Bell, M.L., Zanobetti, A., Dominici, F., 2013. Evidence on vulnerability and susceptibility to health risks associated with short-term exposure to particulate matter: a systematic review and meta-analysis. Am. J. Epidemiol. 178 (6), 865–876. Block, M.L., Calderón-Garcidueñas, L., 2009. Air pollution: mechanisms of neuroinflammation and CNS disease. Trends Neurosci. 32 (9), 506–516. Bo, L., Jiang, S., Xie, Y., Kan, H., Song, W., Zhao, J., 2016. Effect of vitamin E and omega-3 fatty acids on protecting ambient PM2.5-induced inflammatory response and oxidative stress in vascular endothelial cells. PLoS One 11 (3), e0152216. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Casanova, R., Wang, X., Reyes, J., Akita, Y., Serre, M.L., Vizuete, W., et al., 2016. A voxel-based morphometry study reveals local brain structural alterations associated with ambient fine particles in older women. Front. Hum. Neurosci. 10, 495. Chen, Z., Zhong, C., 2013. Decoding Alzheimer's disease from perturbed cerebral glucose metabolism: implications for diagnostic and therapeutic strategies. Prog. Neurobiol. 108, 21–43. Clifford, A., Lang, L., Chen, R., Anstey, K.J., Seaton, A., 2016. Exposure to air pollution and cognitive functioning across the life course – a systematic literature review. Environ. Res. 147, 383–398. Cowan, C.M., Bossing, T., Page, A., Shepherd, D., Mudher, A., 2010. Soluble hyper-phosphorylated tau causes microtubule breakdown and functionally compromises normal tau in vivo. Acta Neuropathol. 120 (5), 593–604. Crary, J.F., Trojanowski, J.Q., Schneider, J.A., Abisambra, J.F., Abner, E.L., Alafuzoff, I., et al., 2014. Primary age-related
N
C
O
R
R
E
C
T
42 4
U
R O
406
P
405
tauopathy (PART): a common pathology associated with human aging. Acta Neuropathol. 128 (6), 755–766. Dosunmu, R., Wu, J., Basha, M.R., Zawia, N.H., 2007. Environmental and dietary risk factors in Alzheimer's disease. Expert. Rev. Neurother. 7 (7), 887–900. Ferri, C.P., Prince, M., Brayne, C., Brodaty, H., Fratiglioni, L., Ganguli, M., et al., 2005. Global prevalence of dementia: a Delphi consensus study. Lancet 366 (9503), 2112–2117. Gibson, G.E., Starkov, A., Blass, J.P., Ratan, R.R., Beal, M.F., 2010. Cause and consequence: mitochondrial dysfunction initiates and propagates neuronal dysfunction, neuronal death and behavioral abnormalities in age-associated neurodegenerative diseases. Biochim. Biophys. Acta 1802 (1), 122–134. Hamasaki, H., Honda, H., Okamoto, T., Koyama, S., Suzuki, S.O., Ohara, T., et al., 2016. Recent increases in hippocampal tau pathology in the aging Japanese population: the Hisayama study. J. Alzheimers Dis. 55 (2), 613–624. Heusinkveld, H.J., Wahle, T., Campbell, A., Westerink, R.H., Tran, L., Johnston, H., et al., 2016. Neurodegenerative and neurological disorders by small inhaled particles. Neurotoxicology 56, 94–106. Ho, Y.S., Yang, X., Yeung, S.C., Chiu, K., Lau, C.F., Tsang, A.W., et al., 2012. Cigarette smoking accelerated brain aging and induced pre-Alzheimer-like neuropathology in rats. PLoS One 7 (5), e36752. Hong, Z., Guo, Z., Zhang, R., Xu, J., Dong, W., Zhuang, G., et al., 2016. Airborne fine particulate matter induces oxidative stress and inflammation in human nasal epithelial cells. Tohoku J. Exp. Med. 239 (2), 117–125. Hoyer, S., Lannert, H., 2007. Long-term abnormalities in brain glucose/energy metabolism after inhibition of the neuronal insulin receptor: implication of tau-protein. J. Neural Transm. Suppl. 72 (72), 195–202. Ingram, T., Chakrabarti, L., 2016. Proteomic profiling of mitochondria: what does it tell us about the ageing brain? Aging (Albany NY) 8 (12), 3161–3179. Jiang, K., Wang, W., Jin, X., Wang, Z., Ji, Z., Meng, G., 2015. Silibinin, a natural flavonoid, induces autophagy via ROS-dependent mitochondrial dysfunction and loss of ATP involving BNIP3 in human MCF7 breast cancer cells. Oncol. Rep. 33 (6), 2711–2718. Jiang, L., Yu, Y., Li, Y., Yu, Y., Duan, J., Zou, Y., et al., 2016. Oxidative damage and energy metabolism disorder contribute to the hemolytic effect of amorphous silica nanoparticles. Nanoscale Res. Lett. 11 (1), 57. Joly-Amado, A., Serraneau, K.S., Brownlow, M., Marín de Evsikova, C., Speakman, J.R., Gordon, M.N., et al., 2016. Metabolic changes over the course of aging in a mouse model of tau deposition. Neurobiol. Aging 44, 62–73. Kehoe, P.G., Wong, S., Al Mulhim, N., Palmer, L.E., Miners, J.S., 2016. Angiotensin-converting enzyme 2 is reduced in Alzheimer's disease in association with increasing amyloid-β and tau pathology. Alzheimers Res. Ther. 8 (1), 50. Kioumourtzoglou, M.A., Schwartz, J.D., Weisskopf, M.G., Melly, S.J., Wang, Y., Dominici, F., et al., 2016. Long-term PM2.5 exposure and neurological hospital admissions in the northeastern United States. Environ. Health Perspect. 124 (1), 23–29. Ku, T., Ji, X., Zhang, Y., Li, G., Sang, N., 2016. PM2.5, SO2 and NO2 co-exposure impairs neurobehavior and induces mitochondrial injuries in the mousebrain. Chemosphere 163, 27–34. Lakey, P.S., Berkemeier, T., Tong, H., Arangio, A.M., Lucas, K., Pöschl, U., et al., 2016. Chemical exposure-response relationship between air pollutants and reactive oxygen species in the human respiratory tract. Sci Rep 6, 32916. de Lau, L.M., Breteler, M.M., 2006. Epidemiology of parkinson's disease. Lancet Neurol. 5 (6), 525–535. Levesque, S., Surace, M.J., McDonald, J., Block, M.L., 2011. Air pollution & the brain: subchronic diesel exhaust exposure causes neuroinflammation and elevates early markers of neurodegenerative disease. J. Neuroinflammation 8, 105.
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and decreased ATP production. These findings clarify the mechanism of mitochondrial abnormality-related neuropathological dysfunction in response to atmospheric PM2.5 inhalation and provide a potential opportunity to alleviate adverse health outcomes in polluted areas.
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cognitive functions and its modification by APOE gene variants in elderly women. Environ. Res. 142, 10–16. Soberanes, S., Gonzalez, A., Urich, D., Chiarella, S.E., Radigan, K.A., et al., 2012. Particulate matter air pollution induces hypermethylation of the p16 promoter via a mitochondrial ROS-JNK-DNMT1 pathway. Sci Rep 2, 275. Srinivasan, S., Avadhani, N.G., 2012. Cytochrome c oxidase dysfunction in oxidative stress. Free Radic. Biol. Med. 53 (6), 1252–1263. Sumegi, K., Fekete, K., Antus, C., Debreceni, B., Hocsak, E., Gallyas Jr., F., et al., 2017. Protects against oxidative stress- or lipopolysaccharide-induced mitochondrial destabilization and reduces mitochondrial production of reactive oxygen species. PLoS One 12 (1), e0169372. Szablewski, L., 2017. Glucose transporters in brain: in health and in Alzheimer's disease. J. Alzheimers Dis. 55 (4), 1307. Weinberg, S.E., Sena, L.A., Chandel, N.S., 2015. Mitochondria in the regulation of innate and adaptive immunity. Immunity 42 (3), 406–417. World Population Ageing: 1950–2050. Population Division, DESA, United Nations, 2006. Qual. Life Res. 6 (7–8), 54. Yan, W., Ku, T., Yue, H., Li, G., Sang, N., 2016a. NO2 inhalation causes tauopathy by disturbing the insulin signaling pathway. Chemosphere 165, 248–256. Yan, D., Zhang, Y., Liu, L., Yan, H., 2016b. Pesticide exposure and risk of Alzheimer's disease: a systematic review and meta-analysis. Sci Rep 6, 32222. Zhang, C., Rissman, R.A., Feng, J., 2015. Characterization of ATP alternations in an Alzheimer's transgenic mouse model. J. Alzheimers Dis. 44 (2), 375–378.
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Liu, M., Huang, Y., Ma, Z., Jin, Z., Liu, X., Wang, H., et al., 2017. Spatial and temporal trends in the mortality burden of air pollution in China: 2004–2012. Environ. Int. 98, 75–81. Lockhart, S.N., Baker, S.L., Okamura, N., Furukawa, K., Ishiki, A., Furumoto, S., et al., 2016. Dynamic PET measures of tau accumulation in cognitively normal older adults and Alzheimer's disease patients measured using [18F] THK-5351. PLoS One 11 (6), e0158460. Lovestone, S., Reynolds, C.H., 1997. The phosphorylation of tau: a critical stage in neurodevelopment and neurodegenerative processes. Neuroscience 78 (2), 309–324. McAllum, E.J., Finkelstein, D.I., 2016. Metals in Alzheimer's and Parkinson's diseases: relevance to dementia with lewy bodies. J. Mol. Neurosci. 60 (3), 279–288. Pike Winer, L.S., Wu, M., 2014. Rapid analysis of glycolytic and oxidative substrate flux of cancer cells in a microplate. PLoS One 9 (10), e109916. Reboul, C., Boissière, J., André, L., Meyer, G., Bideaux, P., Fouret, G., et al., 2017. Carbon monoxide pollution aggravates ischemic heart failure through oxidative stress pathway. Sci Rep 7, 39715. Reitz, C., Mayeux, R., 2014. Alzheimer disease: epidemiology, diagnostic criteria, risk factors and biomarkers. Biochem. Pharmacol. 88 (4), 640–651. Rohde, R.A., Muller, R.A., 2015. Air pollution in China: mapping of concentrations and sources. PLoS One 10 (8), e0135749. Sato, N., Morishita, R., 2015. The roles of lipid and glucose metabolism in modulation of β-amyloid, tau, and neurodegeneration in the pathogenesis of Alzheimer disease. Front. Aging Neurosci. 7, 199. Scheibye-Knudsen, M., 2016. Neurodegeneration in accelerated aging. Dan. Med. J. 63 (11) (pii: B5308). Schikowski, T., Vossoughi, M., Vierkötter, A., Schulte, T., Teichert, T., Sugiri, D., et al., 2015. Association of air pollution with
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Abnormal energy metabolism and tau phosphorylation in the brains of middle-aged mice in response to atmospheric PM2.5 exposure Rui Gao, Tingting Ku, Xiaotong Ji, Yingying Zhang, Guangke Li⁎, Nan Sang⁎
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College of Environment and Resource, Research Center of Environment and Health, Shanxi University, Taiyuan, Shanxi 030006, PR China
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Article history:
In light of the accelerated aging of the global population and the deterioration of the 15
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Received 30 March 2017
atmosphere pollution, we sought to clarify the potential mechanisms by which fine 16
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Revised 28 June 2017
particulate matter (PM2.5) can cause cognitive impairment and neurodegeneration through 17
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Accepted 29 June 2017
the alteration of mitochondrial structure and function. The results indicate that PM2.5 18
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Available online xxxx
inhalation reduces ATP production by disrupting the aerobic tricarboxylic acid (TCA) cycle and 19
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Keywords:
middle-aged mice. Furthermore, excessive ROS generation was involved in the impairment. 21
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Fine particulate matter (PM2.5)
Interestingly, these alterations were partially reversed after exposure to PM2.5 ended. These 22
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Mitochondrial structure
findings clarify the mechanism involved in mitochondrial abnormality-related neuropatho- 23
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and function
logical dysfunction in response to atmospheric PM2.5 inhalation and provide an optimistic 24
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Energy metabolism
sight for alleviating the adverse health outcomes in polluted areas.
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Reactive oxygen species (ROS)
© 2017 The Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences. 26
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Tau phosphorylation
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oxidative phosphorylation, thereby causing the hypophosphorylation of tau in the cortices of 20
Introduction
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Population aging is a pervasive and unprecedented phenomenon that cannot be overlooked. People aged 60 years and older are estimated to become approximately 22% of the global population and to exceed the number of young people for the first time in history by 2050 (World Population Ageing: 1950–2050. Population Division, DESA, United Nations, 2006). As a result, the health problems of aging individuals have become an important social issue. Among these, the number of individuals with illnesses associated with cognitive impairment such as dementia, including Alzheimer's disease (AD) and Parkinson's disease (PD), has reached 24 million and is predicted to quadruple by the year 2050 (Reitz and Mayeux, 2014). An epidemiological investigation showed that approximately 40% of people aged 85 years and over suffer from AD
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and 10% suffer from PD (de Lau and Breteler, 2006; Ferri et al., 2005). While the majority of AD and PD cases are sporadic, inheritable genetic mutations play a small role in their etiologies (Dosunmu et al., 2007; Heusinkveld et al., 2016). Environmental factors such as pesticides, metals, and atmospheric pollutants have been postulated to contribute to the pathogenesis of AD and PD (Yan et al., 2016a, 2016b; McAllum and Finkelstein, 2016; Block and Calderón-Garcidueñas, 2009). Exposure to atmospheric fine particles (PM2.5) has been linked with numerous diseases, both local (in the lung) and systemic (in extra-pulmonary sites, such as cardiovascular and neuronal tissues) (Liu et al., 2017; Kioumourtzoglou et al., 2016). A lifespan study found that atmospheric pollution is associated with the quantifiable impairment of brain development in young people and with cognitive decline in the elderly (Clifford et al., 2016). A meta-analysis based on 108 papers
⁎ Corresponding authors. E-mail: [email protected] (Nan Sang).
http://dx.doi.org/10.1016/j.jes.2017.06.037 1001-0742/© 2017 The Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences. Published by Elsevier B.V.
Please cite this article as: Gao, R., et al., Abnormal energy metabolism and tau phosphorylation in the brains of middle-aged mice in response to atmospheric PM2.5 exposure, J. Environ. Sci. (2017), http://dx.doi.org/10.1016/j.jes.2017.06.037
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1. Materials and methods
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1.1. PM2.5 sampling and preparation
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PM2.5 was collected between November 2014 and February 2015 at Shanxi University (112°21–34′E longitude, 37°47–48′N latitude), Taiyuan City, Shanxi Province, China. A mediumvolume air sampler (TH-150CIII, Wuhan, China) with a flow rate of 100 L/min was placed on the top of a building far from obstacles to obtain free-moving air. Samples were collected on quartz filter membranes (Φ90 mm, Munktell, Sweden) for 22 hr/d and then extracted in Milli-Q deionized water with ultrasonic processing more than three times. After vacuum freeze-drying, the samples were resuspended in sterilized 0.9% physiological saline. The aqueous suspension was pooled, frozen at −20°C and swirled for 10 min before being used for animal experiments.
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1.2. Animals and PM2.5 exposure
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Female 10-month-old C57BL/6 mice were purchased from the Junke Biological Engineering Co., LTD (Nanjing, China). These
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1.3. Transmission electron microscope (TEM) observation
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The brain cortex tissues in mice were rapidly cut into pieces approximately 1 mm3 and was fixed in stationary liquid. Then pieces were washed and en bloc stained with 1% uranyl acetate in 50% ethanol at room temperature in the dark, dehydrated by graded ethanol, and embedded in beam capsules. The 70– 80-nm-thick ultrathin sections cut from the embedded tissue were collected onto grids, and then were stained with uranyl acetate and lead citrate. The mitochondrial structural were observed with an electron microscope (JEOL, JEM 1400, Japan).
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1.4. Determination of the ATP content
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The ATP levels were determined by the ATP detection kit according to the manufacturer's instructions (Beyotime, China). Approximately 30 mg cortex tissues were used with 240 μL lysis buffer for ATP releasing. After high speed centrifuged, the ATP content in supernatant was measured by the luciferin-luciferase method with a Thermo Scientific Varioskan Flash (Thermo Fisher Scientific, USA). The protein concentration was determined with Coomassie light blue according to the Bradford (1976). Finally, the ATP content normalized to protein concentrations were used for comparative analysis.
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mice were housed under standard conditions (24 ± 2°C, 50 ± 5% humidity, 12:12 hr light:dark cycle) and randomly divided into a PM2.5-treated group and a control group. Mice in the PM2.5 treatment group received oropharyngeal aspirations of PM2.5 at 3 mg/kg after anesthetization with isoflurane (Yi Pin Pharmaceutical Co., Ltd., Hebei, China) every other day at different times. The four treatment groups included groups exposed for 2 weeks, exposed for 4 weeks, allowed to recover for 1 week after being exposed for 4 weeks, and allowed to recover for 2 weeks after being exposed for 4 weeks. Mice in the control group were treated with 0.9% saline (processed by the ultrasonic oscillation of the control membrane filter) using the same method. Mice were sacrificed, and their brain cortices were separated 24 hr after the last exposure. The cortices were immediately frozen in liquid nitrogen and then stored at −80°C until further use. All animal experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of Shanxi University.
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revealed a higher risk of death for older populations (0.64%) than for younger populations (0.34%) per 10 μg/m3 increase in particulate matter (PM) (Bell et al., 2013). Although many epidemiological studies have described the adverse effects of PM2.5 on cognitive function in middle-aged people (Ailshire and Crimmins, 2014; Schikowski et al., 2015), there have been limited laboratory investigations into the mechanism of injury. Casanova reported that long-term PM2.5 exposure might accelerate the loss of both gray matter and white matter in the brains of older women (Casanova et al., 2016). However, whether the molecular targets of neurological degenerative damage are influenced by PM2.5 and whether these effects can be reversed remain unclear. Tau is a microtubule-associated protein (MAP) primarily responsible for the assembly, maintenance and stability of microtubules. The abnormal hyperphosphorylation of tau, a hallmark of neurodegenerative illnesses (particularly AD), can decrease the stability of microtubules and disrupt cytoskeletal integrity and axonal transport (Lovestone and Reynolds, 1997; Cowan et al., 2010). Recent studies have revealed an increased trend of tauopathy not only in older patients with AD (Kehoe et al., 2016; Hamasaki et al., 2016) but also in cognitively normal middle-aged people (potentially primary age-related tauopathy) (Crary et al., 2014; Lockhart et al., 2016). Emerging evidence has revealed that abnormal glucose metabolism and decreased adenosine triphosphate (ATP) production are correlated with tau hyperphosphorylation in AD patients (Szablewski, 2017; Hoyer and Lannert, 2007). Due to the high energy demands of the brain, central nervous system (CNS) functions are strongly dependent on sufficient mitochondrial production of ATP. The aging mitochondrial theory suggests that ATP deficiencies play a critical role in the accelerated aging disorders of the elderly (Scheibye-Knudsen, 2016). Thus, the aim of our present study was to clarify the possible mechanisms by which PM2.5 causes tau phosphorylation-related cognitive impairment and neurodegeneration by affecting mitochondrial structure and function.
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1.5. Real-time quantitative reverse transcription-polymerase 169 chain reaction(PCR) 170 Q8 Approximately 30 mg of cortex tissue were used for total RNA extraction with TRIzol reagent (Invitrogen, USA). The RNA concentration was quantified with a NanoDropTM 2000C (Thermo, USA). Then the extracted RNA completes reverse transcription to form the first-strand complementary DNA (cDNA) using a reverse transcription kit (TaKaRa, China). The mRNA expression levels of tricarboxylic acid (TCA) cycle- and oxidative phosphorylation-related genes were determined by real-time polymerase chain reaction (PCR) on a qTOWER 2.2 real-time PCR system (Analytik Jena AG, Jena, Germany). The
Please cite this article as: Gao, R., et al., Abnormal energy metabolism and tau phosphorylation in the brains of middle-aged mice in response to atmospheric PM2.5 exposure, J. Environ. Sci. (2017), http://dx.doi.org/10.1016/j.jes.2017.06.037
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secondary antibody (1:5000) (IRDye 800CW goat anti-rabbit 211 IgG, LI-COR) and imaged with a LI-COR Odyssey Infrared 212 Fluorescent System. 213
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1.8. Statistical analysis
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Data were analyzed using SPSS 17.0 software (SPSS Inc., Chicago, IL, USA), and results are presented as the mean ± SE. Significant differences were identified via one-way analysis of variance (ANOVA) followed by Fisher's least significant difference (LSD) test and were established at p < 0.05.
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Approximately 30 mg of cortex tissue was homogenized with 600 μL of phosphate buffered solution, and the supernatant was collected after centrifugation. The ROS content in the supernatant was detected by incubation with 2′, 7′-dichlorofluorescein-diacetate (DCFH-DA, 50 μM) at 37°C for 30 min in the dark. The formation of the fluorescent-oxidized derivative of 2′, 7′-dichlorofluorescein (DCF) was measured with a Thermo Scientific Varioskan Flash (Thermo Fisher Scientific, USA) at an excitation wavelength of 488 nm and an emission wavelength of 530 nm. The protein concentration was determined with Coomassie light blue according to the Bradford (1976). Finally, the ROS content normalized to the protein concentration was used for comparative analysis.
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Approximately 30 mg of cortex tissue was used for total protein extraction with lysis buffer. The protein concentration was determined with Coomassie light blue according to the Bradford (1976). Then, 50 μg of total proteins were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), transferred to a polyvinylidene difluoride (PVDF) membrane, and blocked with 3% bovine albumin. The blocked membranes were incubated overnight at 4°C with monoclonal antibodies for β-actin (1:1000, Cell Signaling, USA), polyclonal antibodies for tau (1:200, Bioss, China) and polyclonal antibodies for p-tau (1:200, Bioss, China). The membranes were then incubated with fluorescently labeled
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Table 1 – The primer sequences and reaction conditions used for real-time PCR. Sequence (5′ to 3′)
GAPDH sense GAPDH anti-sense PDHA1 sense PDHA1 anti-sense CS sense CS anti-sense OGDH sense OGDH anti-sense IDH2 sense IDH2 anti-sense FH sense FH anti-sense ATP6 sense ATP6 anti-sense CO1 sense CO1 anti-sense CO4 sense CO4 anti-sense
CTTTGGCATTGTGGAAGGGC CAGGGATGATGTTCTGGGCA TGTGAGAACAACCGCTAT ATCCATTCCATCTACCCT CCTGGTCGTTTGGCTTTA TGTGCTATGGGCTCTTAC CAACAGATTCGGTGCTAT AGTGGTGGTGGGTAAGTG GCAGCAGTGCCAAGGAGT AGCCGATGTTCATACCAGAT AATCCCAGTCATTCAAGC CAGTCTGCCAAACCACCA TTCCCATCCTCAAAACGCCT GTTCGTCCTTTTGGTGTGTGG CTATGTTCTATCAATGGGAGC TCTGAGTAGCGTCGTGGT TCTACTTCGGTGTGCCTTCG CGATCAGCGTAAGTGGGGAA
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PCR: polymerase chain reaction.
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2.1. Abnormal mitochondrial morphology and deficient ATP 222 production in the cortex 223
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Due to the high energy demands of the brain, CNS functions are strongly dependent on the sufficient production of mitochondrial ATP. To evaluate the mitochondrial signaling pathway after PM2.5 exposure, mitochondrial ultra-structures were observed using transmission electron microscopy (TEM). Broken mitochondrial membranes and fractured and lost mitochondrial cristae appeared in response to PM2.5 exposure for 4 weeks (Fig. 1). Together, these images demonstrate that PM2.5 exposure damages the integrity and function of mitochondria in the brain. Mitochondria produce ATP, which, as a direct source of cellular energy, plays a critical role in accelerated aging disorders in the elderly (Gibson et al., 2010). Abnormal mitochondrial morphology is suggested to be directly related to the production of ATP. To provide further evidence for this hypothesis, we detected the concentration of ATP in the cortex after PM2.5 exposure and following restorative treatment. As shown in Fig. 2, PM2.5 exposure led to a time-dependent decrease in ATP levels, which reached 50% of those in the control group at 4 weeks after treatment. Interestingly, the reduction in ATP levels was reversed to 10% that of the control group after 2 weeks of recovery. These results suggest that ambient PM2.5 exposure has the potential to cause mitochondrial deterioration in the middle-aged mice and that recovery is incomplete.
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relative quantification of gene expression was determined using GAPDH as an internal control. The primer sequences and reaction conditions used for RT-PCR are listed in Table 1.
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2.2. Changes of gene expression involved in the tricarboxylic 248 acid cyclein (TCA) in the cortex 249 The tricarboxylic acid cyclein (TCA) cycle is the main pathway for the oxidation of glucose in the brain and is a crucial intermediate step for the production of ATP (Chen and Zhong, 2013). To test whether the TCA cycle was involved in the depletion of ATP caused by ambient PM2.5, we determined the expression levels of five key TCA cycle genes. As presented in Fig. 3, PM2.5 treatment significantly reduced the expression of pyruvate dehydrogenase (lipoamide) alpha 1 (PDHA1) in the second step of TCA cycle as well as the expression of rate-limiting enzymes in the TCA cycle such as citrate synthase (CS), isocitrate dehydrogenase 2 (NADP+) (IDH2), and oxoglutarate (alpha-ketoglutarate) dehydrogenase (lipoamide) (OGDH), but not fumarase (FH). After the cessation of PM2.5 treatment, the mRNA levels of CS and IDH2 increased considerably, while other genes maintained their
Please cite this article as: Gao, R., et al., Abnormal energy metabolism and tau phosphorylation in the brains of middle-aged mice in response to atmospheric PM2.5 exposure, J. Environ. Sci. (2017), http://dx.doi.org/10.1016/j.jes.2017.06.037
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elevations, a potential reason for the incomplete reversal of ATP production.
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Oxidative phosphorylation, as the final step in the production of ATP from adenosine diphosphate (ADP), accompanies the process of electron transfer in the mitochondrial respiratory chain. The oxidative phosphorylation system is composed of five multisubunit complexes (complexes I–V). In our study,
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the mRNA levels of complex IV subunits (CO1 and CO4) and complex V subunits (ATP6) were determined (Fig. 4). The expression of CO1 and ATP6, encoded by mitochondrial DNA (mtDNA), decreased after PM2.5 exposure in a time-dependent manner and recovered to normal levels when PM2.5 treatment stopped. The mRNA expression level of CO4, encoded by nuclear DNA (nDNA), decreased weakly after 4 weeks of exposure. Consistent with the TCA cycle, changes in oxidative phosphorylation further clarified the decrease in ATP production following PM2.5 exposure.
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Fig. 1 – Electron micrographs of neuronal mitochondria from the cortical regions of mouse brains after exposure to PM2.5. After PM2.5 treatment, cristae within the mitochondria were broken, fragmented and in some cases lost, and the mitochondrial membrane was ruptured. The red arrows represent these broken structures. 40,000× magnification, scale bars = 500 nm. PM2.5: fine particulate matter.
Fig. 2 – ATP levels in the cortical regions of mouse brains after exposure to PM2.5. Values are recorded as the mean ± SE (n = 8). *p < 0.05 compared with the corresponding control groups. ATP: adenosine triphosphate; PM2.5: fine particulate matter. Please cite this article as: Gao, R., et al., Abnormal energy metabolism and tau phosphorylation in the brains of middle-aged mice in response to atmospheric PM2.5 exposure, J. Environ. Sci. (2017), http://dx.doi.org/10.1016/j.jes.2017.06.037
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Fig. 3 – The expression of genes involved in the TCA cycle in the cortical regions of mouse brains after exposure to PM2.5. The levels of pyruvate dehydrogenase (lipoamide) alpha 1 (PDHA1), citrate synthase (CS), isocitrate dehydrogenase 2 (NADP+) (IDH2), oxoglutarate (alpha-ketoglutarate) dehydrogenase (lipoamide) (OGDH), and fumarase (FH) mRNA expression in mouse brains after exposure to PM2.5 were determined through qRT-PCR analyses. Values are recorded as the mean ± SE (n = 8). *p < 0.05, **p < 0.01 compared with the corresponding control groups; #p < 0.05, ##p < 0.01 vs. the treatment groups. TCA: tricarboxylic acid; PCR: polymerase chain reaction; PM2.5: fine particulate matter.
Fig. 4 – The expression of genes involved in oxidative phosphorylation in the cortical regions of mouse brains after exposure to PM2.5. The mRNA levels of complex IV subunits (CO1 and CO4) and complex V subunits (ATP6) were determined in mouse brains after exposure to PM2.5 through qRT-PCR analyses. Values are recorded as the mean ± SE (n = 8). *p < 0.05, **p < 0.01 compared with the corresponding control groups; #p < 0.05, ##p < 0.01 vs. the treatment groups. ATP: adenosine triphosphate; PCR: polymerase chain reaction; PM2.5: fine particulate matter. Please cite this article as: Gao, R., et al., Abnormal energy metabolism and tau phosphorylation in the brains of middle-aged mice in response to atmospheric PM2.5 exposure, J. Environ. Sci. (2017), http://dx.doi.org/10.1016/j.jes.2017.06.037
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To further determine whether ATP deficiency is linked to the alteration of microtubule-associated tau protein, the phosphorylation of tau was detected after PM2.5 treatment. Compared to the control group, the phosphorylation of tau was increased after PM2.5 exposure, and a significant change was detected after 4 weeks of treatment (Fig. 6). After the cessation of PM2.5 treatment, p-tau reverted to a relatively low level.
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In the present study, we analyzed ROS-associated energy deficiencies and tau phosphorylation in the cortices of middle-aged mice in response to ambient PM2.5 exposure. Due to the high energy demands of the brain, neurological functions are strongly dependent on sufficient mitochondrial
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An accumulating body of evidence has linked the generation of ROS induced by PM2.5 exposure to toxicity processes (Hong et al., 2016; Bo et al., 2016). Oxidative stress is directly responsible for mitochondrial dysfunction (Sumegi et al., 2017), making it important to determine whether oxidative stress is an intermediate process between PM2.5 exposure and mitochondrial changes. As shown in Fig. 5, ROS levels increased in a time-dependent manner after PM2.5 treatment, and these effects were completely reversed 2 weeks after treatment stopped. This result is consistent with the measured changes in ATP content and indicates that ATP deficiencies after PM2.5 exposure could be attributed to the oxidative stress caused by excessive ROS generation.
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ATP production. Indeed, mitochondrial dysfunction has been related to neurodegenerative and age-related disorders (Ingram and Chakrabarti, 2016), and the aging mitochondrial theory suggests that ATP deficiency plays a critical role in the accelerated aging disorders of the elderly (Scheibye-Knudsen, 2016). Perturbations of mitochondrial energetics have been found in many chemical exposure studies. Our previous studies demonstrated that PM2.5, SO2 and NO2 exposure could induce mitochondrial injury and impair neurological behavior in mice (Ku et al., 2016). In the current study, we observed broken mitochondrial membranes and fractured and lost mitochondrial cristae after PM2.5 exposure for 4 weeks. As mitochondrial damage directly results in a reduction of available energy, we also found decreased ATP levels in the cortex, and these injuries were partially alleviated after exposure stopped. The aerobic metabolism of glucose, as the major source of ATP in vivo, is composed of three steps: glycolysis, the TCA cycle and oxidative phosphorylation. Mitochondria are mainly responsible for the TCA cycle and oxidative phosphorylation, corresponding to the intermediate and final stages of aerobic respiration (Weinberg et al., 2015; Pike Winer and Wu, 2014). To further clarify whether the TCA cycle and oxidative phosphorylation are affected by PM2.5 exposure accompanied by mitochondrial damage, five key TCA cycle genes and the mitochondrial respiratory complexes IV and V were selected as indicators. The TCA cycle is the main pathway for the oxidation of glucose in the brain and is a crucial intermediate step for the production of ATP (Chen and Zhong, 2013). Pyruvate, a product of glycolysis, is first decarboxylated to acetyl CoA by the pyruvate dehydrogenase complex. Following this process, the conversion of acetyl CoA to CO2 is regulated by rate-limiting enzymes in the TCA cycle, allowing the production of large quantities of reducing equivalents (e.g., NADH) through oxidative phosphorylation. The reduced expression
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Fig. 5 – The ROS contents in cortical regions of mouse brains after exposure to PM2.5. Values are recorded as the mean ± SE (n = 8). *p < 0.05, **p < 0.01 compared with the corresponding control groups; #p < 0.05, ##p < 0.01 vs. the treatment groups. ROS: reactive oxygen species; PM2.5: fine particulate matter. Please cite this article as: Gao, R., et al., Abnormal energy metabolism and tau phosphorylation in the brains of middle-aged mice in response to atmospheric PM2.5 exposure, J. Environ. Sci. (2017), http://dx.doi.org/10.1016/j.jes.2017.06.037
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Fig. 6 – PM2.5 exposure potentiates tau phosphorylation in the mouse brain. Western blot analysis of tau and phosphorylated tau (p-tau) expression in the cortex. Values are recorded as the means ± SEs (n = 8). *p < 0.05, **p < 0.01 compared with the corresponding control groups; #p < 0.05, ##p < 0.01 vs. the treatment groups. PM2.5: fine particulate matter.
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of genes involved in the TCA cycle and their inconsistent recovery suggest that a disordered TCA cycle contributes to 349 the abnormal mitochondrial structures and functions ob350 served after PM2.5 exposure and that this process could be 351 reversible if pollution levels are controlled. Mitochondrial 352 oxidative phosphorylation, the final step in the production of 353 ATP from ADP, accounts for more than 90% of cellular ATP 354 Q12 production in most cells and tissues (Satish and Narayan, 355 2012). Oxidative phosphorylation is controlled by five mito356 chondrial respiratory chain complexes (complexes I, II, III, IV 357 and V). The reduced levels of genes associated with complex 358 IV subunits and complex V subunits involved in oxidative 359 phosphorylation, consistent with our observations of TCA 360 cycle effects, further demonstrate that PM2.5 pollution injures 361 mitochondria and weakens ATP production. 362 Mitochondrially derived ROS play diverse and critical roles 363 in the metabolic adaptation of cells to atmospheric pollution Q14 364 Q13 such as PM (Saul et al. 2012; Jiang et al., 2016; Reboul et al., Q15365 2017; Lakey et al., 2016). ROS-dependent mitochondrial 366 dysfunction and the loss of ATP have been demonstrated in 367 Q16 previous studies (Jiang et al., 2015; Cheng et al. 2015). To 368 determine whether ROS were the source of the mitochondrial 369 dysfunction observed in this study, we measured the ROS 370 content, and the consistent relationship between changes in 371 ROS production and ATP decline implies that the excessive 372 production of ROS induced by PM2.5 may be related to 373 mitochondrial dysfunction. 374 Declined production of ATP and the disruption of glucose 375 metabolism homeostasis were linked with tau phosphorylation
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in numerous studies (Sato and Morishita, 2015; Joly-Amado et al., 2016; Szablewski, 2017). The microtubule system, consisting of tubulin and MAPs, are the major ingredients of nerve cell skeleton. Tau protein, as the most prevalent MAP, participates in a variety of cellular functions. The abnormal hyperphosphorylation of tau, as a hallmark of neurodegenerative diseases (particularly AD), can decrease the stability of microtubules and disrupt cytoskeletal integrity and axonal transport (Lovestone and Reynolds, 1997; Cowan et al., 2010). Air pollution including diesel exhaust, cigarette smoke and NO2 are speculated to lead to cognitive deficiencies and neurodegeneration (Levesque et al., 2011; Ho et al., 2012; Yan et al., 2016a, 2016b) in children and middle-aged people. In the present study, the hypophosphorylation of tau, its relation to neurodegeneration and its restoration were observed after PM2.5 treatment and recovery. As atmospheric PM2.5 pollution occurs uninterruptedly and sources of pollution are widespread, 83% of the Chinese population is exposed to PM2.5 (>35 μg/m3), a level of pollution classified as “unhealthy for sensitive groups” (Rohde and Muller, 2015), and may be at risk of cognitive impairment and neurodegeneration.
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4. Conclusions
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We found that energy metabolism abnormalities and tau phosphorylation after PM2.5 exposure in the cortices of middle-aged mice are reversible and that the mechanism is associated with excessive ROS-mediated mitochondrial injury
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Please cite this article as: Gao, R., et al., Abnormal energy metabolism and tau phosphorylation in the brains of middle-aged mice in response to atmospheric PM2.5 exposure, J. Environ. Sci. (2017), http://dx.doi.org/10.1016/j.jes.2017.06.037
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Uncited references
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Soberanes et al., 2012 Srinivasan and Avadhani, 2012 Zhang et al., 2015
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Acknowledgments
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This work was supported by National Science Foundation of China (NSFC, Nos. 91543203, 21477070, 21377076, 21222701), 417 Specialized Research Fund for the Doctoral Program of 418 Higher Education of China (SRFDP, Nos. 20121401110003, 419 20131401110005), Project Supported by Shanxi Young Sanjin 420 Scholarship of China, Program for the Outstanding Innovative 421 Teams of Higher Learning Institutions of Shanxi, and Re422 Q18 search Project Supported by Shanxi Scholarship Council of 423 China (No. 2015-006).
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REFERENCES
425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459
Ailshire, J.A., Crimmins, E.M., 2014. Fine particulate matter air pollution and cognitive function among older US adults. Am. J. Epidemiol. 180 (4), 359–366. Bell, M.L., Zanobetti, A., Dominici, F., 2013. Evidence on vulnerability and susceptibility to health risks associated with short-term exposure to particulate matter: a systematic review and meta-analysis. Am. J. Epidemiol. 178 (6), 865–876. Block, M.L., Calderón-Garcidueñas, L., 2009. Air pollution: mechanisms of neuroinflammation and CNS disease. Trends Neurosci. 32 (9), 506–516. Bo, L., Jiang, S., Xie, Y., Kan, H., Song, W., Zhao, J., 2016. Effect of vitamin E and omega-3 fatty acids on protecting ambient PM2.5-induced inflammatory response and oxidative stress in vascular endothelial cells. PLoS One 11 (3), e0152216. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Casanova, R., Wang, X., Reyes, J., Akita, Y., Serre, M.L., Vizuete, W., et al., 2016. A voxel-based morphometry study reveals local brain structural alterations associated with ambient fine particles in older women. Front. Hum. Neurosci. 10, 495. Chen, Z., Zhong, C., 2013. Decoding Alzheimer's disease from perturbed cerebral glucose metabolism: implications for diagnostic and therapeutic strategies. Prog. Neurobiol. 108, 21–43. Clifford, A., Lang, L., Chen, R., Anstey, K.J., Seaton, A., 2016. Exposure to air pollution and cognitive functioning across the life course – a systematic literature review. Environ. Res. 147, 383–398. Cowan, C.M., Bossing, T., Page, A., Shepherd, D., Mudher, A., 2010. Soluble hyper-phosphorylated tau causes microtubule breakdown and functionally compromises normal tau in vivo. Acta Neuropathol. 120 (5), 593–604. Crary, J.F., Trojanowski, J.Q., Schneider, J.A., Abisambra, J.F., Abner, E.L., Alafuzoff, I., et al., 2014. Primary age-related
N
C
O
R
R
E
C
T
42 4
U
R O
406
P
405
tauopathy (PART): a common pathology associated with human aging. Acta Neuropathol. 128 (6), 755–766. Dosunmu, R., Wu, J., Basha, M.R., Zawia, N.H., 2007. Environmental and dietary risk factors in Alzheimer's disease. Expert. Rev. Neurother. 7 (7), 887–900. Ferri, C.P., Prince, M., Brayne, C., Brodaty, H., Fratiglioni, L., Ganguli, M., et al., 2005. Global prevalence of dementia: a Delphi consensus study. Lancet 366 (9503), 2112–2117. Gibson, G.E., Starkov, A., Blass, J.P., Ratan, R.R., Beal, M.F., 2010. Cause and consequence: mitochondrial dysfunction initiates and propagates neuronal dysfunction, neuronal death and behavioral abnormalities in age-associated neurodegenerative diseases. Biochim. Biophys. Acta 1802 (1), 122–134. Hamasaki, H., Honda, H., Okamoto, T., Koyama, S., Suzuki, S.O., Ohara, T., et al., 2016. Recent increases in hippocampal tau pathology in the aging Japanese population: the Hisayama study. J. Alzheimers Dis. 55 (2), 613–624. Heusinkveld, H.J., Wahle, T., Campbell, A., Westerink, R.H., Tran, L., Johnston, H., et al., 2016. Neurodegenerative and neurological disorders by small inhaled particles. Neurotoxicology 56, 94–106. Ho, Y.S., Yang, X., Yeung, S.C., Chiu, K., Lau, C.F., Tsang, A.W., et al., 2012. Cigarette smoking accelerated brain aging and induced pre-Alzheimer-like neuropathology in rats. PLoS One 7 (5), e36752. Hong, Z., Guo, Z., Zhang, R., Xu, J., Dong, W., Zhuang, G., et al., 2016. Airborne fine particulate matter induces oxidative stress and inflammation in human nasal epithelial cells. Tohoku J. Exp. Med. 239 (2), 117–125. Hoyer, S., Lannert, H., 2007. Long-term abnormalities in brain glucose/energy metabolism after inhibition of the neuronal insulin receptor: implication of tau-protein. J. Neural Transm. Suppl. 72 (72), 195–202. Ingram, T., Chakrabarti, L., 2016. Proteomic profiling of mitochondria: what does it tell us about the ageing brain? Aging (Albany NY) 8 (12), 3161–3179. Jiang, K., Wang, W., Jin, X., Wang, Z., Ji, Z., Meng, G., 2015. Silibinin, a natural flavonoid, induces autophagy via ROS-dependent mitochondrial dysfunction and loss of ATP involving BNIP3 in human MCF7 breast cancer cells. Oncol. Rep. 33 (6), 2711–2718. Jiang, L., Yu, Y., Li, Y., Yu, Y., Duan, J., Zou, Y., et al., 2016. Oxidative damage and energy metabolism disorder contribute to the hemolytic effect of amorphous silica nanoparticles. Nanoscale Res. Lett. 11 (1), 57. Joly-Amado, A., Serraneau, K.S., Brownlow, M., Marín de Evsikova, C., Speakman, J.R., Gordon, M.N., et al., 2016. Metabolic changes over the course of aging in a mouse model of tau deposition. Neurobiol. Aging 44, 62–73. Kehoe, P.G., Wong, S., Al Mulhim, N., Palmer, L.E., Miners, J.S., 2016. Angiotensin-converting enzyme 2 is reduced in Alzheimer's disease in association with increasing amyloid-β and tau pathology. Alzheimers Res. Ther. 8 (1), 50. Kioumourtzoglou, M.A., Schwartz, J.D., Weisskopf, M.G., Melly, S.J., Wang, Y., Dominici, F., et al., 2016. Long-term PM2.5 exposure and neurological hospital admissions in the northeastern United States. Environ. Health Perspect. 124 (1), 23–29. Ku, T., Ji, X., Zhang, Y., Li, G., Sang, N., 2016. PM2.5, SO2 and NO2 co-exposure impairs neurobehavior and induces mitochondrial injuries in the mousebrain. Chemosphere 163, 27–34. Lakey, P.S., Berkemeier, T., Tong, H., Arangio, A.M., Lucas, K., Pöschl, U., et al., 2016. Chemical exposure-response relationship between air pollutants and reactive oxygen species in the human respiratory tract. Sci Rep 6, 32916. de Lau, L.M., Breteler, M.M., 2006. Epidemiology of parkinson's disease. Lancet Neurol. 5 (6), 525–535. Levesque, S., Surace, M.J., McDonald, J., Block, M.L., 2011. Air pollution & the brain: subchronic diesel exhaust exposure causes neuroinflammation and elevates early markers of neurodegenerative disease. J. Neuroinflammation 8, 105.
F
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and decreased ATP production. These findings clarify the mechanism of mitochondrial abnormality-related neuropathological dysfunction in response to atmospheric PM2.5 inhalation and provide a potential opportunity to alleviate adverse health outcomes in polluted areas.
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P
R O
O
F
cognitive functions and its modification by APOE gene variants in elderly women. Environ. Res. 142, 10–16. Soberanes, S., Gonzalez, A., Urich, D., Chiarella, S.E., Radigan, K.A., et al., 2012. Particulate matter air pollution induces hypermethylation of the p16 promoter via a mitochondrial ROS-JNK-DNMT1 pathway. Sci Rep 2, 275. Srinivasan, S., Avadhani, N.G., 2012. Cytochrome c oxidase dysfunction in oxidative stress. Free Radic. Biol. Med. 53 (6), 1252–1263. Sumegi, K., Fekete, K., Antus, C., Debreceni, B., Hocsak, E., Gallyas Jr., F., et al., 2017. Protects against oxidative stress- or lipopolysaccharide-induced mitochondrial destabilization and reduces mitochondrial production of reactive oxygen species. PLoS One 12 (1), e0169372. Szablewski, L., 2017. Glucose transporters in brain: in health and in Alzheimer's disease. J. Alzheimers Dis. 55 (4), 1307. Weinberg, S.E., Sena, L.A., Chandel, N.S., 2015. Mitochondria in the regulation of innate and adaptive immunity. Immunity 42 (3), 406–417. World Population Ageing: 1950–2050. Population Division, DESA, United Nations, 2006. Qual. Life Res. 6 (7–8), 54. Yan, W., Ku, T., Yue, H., Li, G., Sang, N., 2016a. NO2 inhalation causes tauopathy by disturbing the insulin signaling pathway. Chemosphere 165, 248–256. Yan, D., Zhang, Y., Liu, L., Yan, H., 2016b. Pesticide exposure and risk of Alzheimer's disease: a systematic review and meta-analysis. Sci Rep 6, 32222. Zhang, C., Rissman, R.A., Feng, J., 2015. Characterization of ATP alternations in an Alzheimer's transgenic mouse model. J. Alzheimers Dis. 44 (2), 375–378.
E
Liu, M., Huang, Y., Ma, Z., Jin, Z., Liu, X., Wang, H., et al., 2017. Spatial and temporal trends in the mortality burden of air pollution in China: 2004–2012. Environ. Int. 98, 75–81. Lockhart, S.N., Baker, S.L., Okamura, N., Furukawa, K., Ishiki, A., Furumoto, S., et al., 2016. Dynamic PET measures of tau accumulation in cognitively normal older adults and Alzheimer's disease patients measured using [18F] THK-5351. PLoS One 11 (6), e0158460. Lovestone, S., Reynolds, C.H., 1997. The phosphorylation of tau: a critical stage in neurodevelopment and neurodegenerative processes. Neuroscience 78 (2), 309–324. McAllum, E.J., Finkelstein, D.I., 2016. Metals in Alzheimer's and Parkinson's diseases: relevance to dementia with lewy bodies. J. Mol. Neurosci. 60 (3), 279–288. Pike Winer, L.S., Wu, M., 2014. Rapid analysis of glycolytic and oxidative substrate flux of cancer cells in a microplate. PLoS One 9 (10), e109916. Reboul, C., Boissière, J., André, L., Meyer, G., Bideaux, P., Fouret, G., et al., 2017. Carbon monoxide pollution aggravates ischemic heart failure through oxidative stress pathway. Sci Rep 7, 39715. Reitz, C., Mayeux, R., 2014. Alzheimer disease: epidemiology, diagnostic criteria, risk factors and biomarkers. Biochem. Pharmacol. 88 (4), 640–651. Rohde, R.A., Muller, R.A., 2015. Air pollution in China: mapping of concentrations and sources. PLoS One 10 (8), e0135749. Sato, N., Morishita, R., 2015. The roles of lipid and glucose metabolism in modulation of β-amyloid, tau, and neurodegeneration in the pathogenesis of Alzheimer disease. Front. Aging Neurosci. 7, 199. Scheibye-Knudsen, M., 2016. Neurodegeneration in accelerated aging. Dan. Med. J. 63 (11) (pii: B5308). Schikowski, T., Vossoughi, M., Vierkötter, A., Schulte, T., Teichert, T., Sugiri, D., et al., 2015. Association of air pollution with
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