TY - JOUR
AU - Baixiang, Li,
AB - Abstract Atrazine (2-chloro-4-ethylamino-6-isopropylamine-1,3,5-triazine; ATR) has been demonstrated to regulate autophagy- and apoptosis-related proteins in doparminergic neuronal damage. In our study, we investigated the role of LC3-II in ATR-induced degeneration of dopaminergic neurons. In vivo dopaminergic neuron degeneration model was set up with ATR treatment and confirmed by the behavioral responses and pathological analysis. Dopaminergic neuron cells were transfected with LC3-II siRNA and treated with ATR to observe cell survival and reactive oxygen species release. The process of mitochondrial autophagy and the neurotoxic effects of mitochondrial autophagy were detected by immunofluorescence assay, immunohistochemical analysis, real-time PCR, and western blot analysis. Results showed that after ATR treatment, the grip strength of Wistar rats was significantly decreased, and behavioral signs of anxiety were clearly observed. The mRNA and protein levels of tyrosine hydroxylase, LC3-II, PINK1, and Parkin were significantly decreased in ATR-induced rat dopaminergic neurons and PC-12 cells, while the mRNA expression and protein levels of SQSTM1/p62 and Parl were increased. Exposure to ATR also led to accumulation of autophagic lysosomes and autophagic bodies along with significantly decreased levels of dopaminergic neurons and alterations in mitochondrial homeostasis, which was reversed by LC3-II siRNA. Our results suggest that ATR affects the mitochondria-mediated dopaminergic neuronal death, which may be mediated by LC3-II and other autophagy markers in vivo and in vitro through SQSTM1/p62 signaling pathway. atrazine, dopaminergic neuron, mitochondrial dysfunction, autophagy, LC3-II, PINK1/Parkin Introduction Atrazine (2-chloro-4-ethylamino-6-isopropylamine-1,3,5-triazine; ATR) is a globally used chlorotriazine herbicide with a wide range of applications, including corn, sorghum, sugarcane, and cotton cultivation, owing to its relatively high efficacy and low cost [1,2]. As a result of its wide-spread use as a low-toxicity pesticide, ATR and/or its metabolites are frequently detected in water or soil and other surfaces, displaying long-term residual persistence and chemical stability. The presence of ATR in urine samples from pesticide applicators near farm houses has also been reported [3–5]. Previous studies have demonstrated selective effects of excessive ATR exposure on multiple organs and tissues owing to its potential to disrupt the balance in reproductive, endocrine, and nervous systems [6–11]. ATR has been characterized as an endocrine disruptor that can reduce pituitary-testis hormone levels, quality of spermatogenesis, and semen. Accumulating reports have documented significant ATR-induced hepatic damage in rodents via oxidative stress [6,12–14]. In the diencephalon, ATR has the potential to exert neurotoxicity by selectively affecting the dopaminergic system, leading to reduced levels of dopamine (DA) and monoamine, downregulation of the striatal catecholamine content [15,16], and decreased number of tyrosine hydroxylase (TH)-positive (TH+) neurons in the substantia nigra (SNpc) and corpus striatum [14]. Exposure to ATR has been proposed to influence the absorption of synaptic vesicles and synaptic bodies to a moderate extent. Moreover, ATR treatment has been shown to trigger an imbalance in DA storage and transport as well as neurodegenerative disorders in vivo [7,15,17] and in vitro [16,18–22]. Mitochondrion is significantly involved in energy support and cellular life processes [23]. Accumulation of environmental toxins in mitochondria can induce mitochondrial dysfunction, resulting in the development of neurodegenerative diseases, accompanied by activation of NADPH oxidase and release of reactive oxygen species (ROS) [24]. Autophagy could be used as a defense mechanism to remove damaged organelles and metabolites in the cytoplasm and protect damaged cells. Autophagy participates in cellular bioenergetics and homeostasis in mitochondria to regulate the progress of cellular degeneration programs, a process also known as mitophagy [25–27]. When autophagy is not enough to clear the damaged mitochondria, it can be over-activated and used as a programmed cell death to induce cell apoptosis [23]. Over-activation of autophagy may promote the progression of mitochondrial dysfunction, which plays a role in Parkinson’s disease (PD) pathogenesis [28,29]. Growing evidence has shown that early-onset mitochondrial dysfunction regulates the initial steps of autophagosome biogenesis and promotes the recruitment of autophagy proteins. Microtubule-associated protein 1 light chain 3 (LC3), the mammalian homolog of yeast Atg8, is an important participant in the mechanism of autophagy, which is a protein located in microtubules and interacts with other proteins and organelles, including mitochondria [30]. During the process of conversion of phagophores into autophagosomes, LC3 reacts with phosphatidylethanolamine to form LC3-II which is then again recruited and incorporated into the autophagosomal membrane. SQSTM1/p62 accumulates and combines with LC3 to weaken the signal of aggresomes in autophagy [30]. PINK1, a mitochondrial regulator has been identified as a marker for neuronal degeneration in conjunction with Parkin during autophagy. PINK1/Parkin co-operates with SQSTM1/p62, which accumulates in the absence of Parl, recruits Parkin to damaged mitochondria and contributes to mitochondrial matrix protein ubiquitination, leading to engulfment and degradation via autophagy [29–33]. Parl could stabilize and affect PINK1 kinase activity, which leads to mitochondrial recruitment of the E3 ubiquitinprotein ligase PARK2 and activation of mitophagy. Parl is an important intrinsic player in mitochondrial quality control, which has alternative mitochondrial proteases that have been implicated in PINK1 processing. This provides an important validation of the pivotal impact of Parl on the PINK1 abundance, but Parl has an opposite effect in triggering mitophagy. A number of studies have demonstrated the significance of the PINK1/Parkin/p62 pathway in the pathology of PD [34–36]. SQSTM1/p62 has been implicated in the process of mitophagy downstream of Parkin translocation. They found that following its recruitment, Parkin induces the K63-linked polyubiquitination of mitochondrial substrate(s) and recruits the ubiquitin- and LC3-binding adaptor protein p62. SQSTM1/p62 mediates the aggregation of mitochondria through polymerization via its PB1 domain, in a manner analogous to the aggregation of misfolded proteins. p62-independent mechanisms downstream of ubiquitination may mediate at least some forms of mitophagy in mammalian cells, and that ubiquitination of mitochondrial substrates could mediate Parkin-induced mitophagy. The p62/Parkin-dependent mitophagy pathway uses, at least in part, canonical autophagy machinery in conjunction with the ubiquitin–proteasome system, leading to mitochondrial dynamics and mitochondrial dysfunction [37,38]. Therefore, the LC3-II/SQSTM1/p62 signaling pathway is proposed to play a key role in the autophagic process in PD [33,39,40]. The issue of whether autophagy promotes ATR-induced dopaminergic neuronal death in rats or protects brain neurons from further injury remains to be established. In a previous study, we found that ATR could regulate autophagy- and apoptosis-related proteins in doparminergic neuronal damage [14], but the exact mechanism is not clear. In this study, we investigated the effect of LC3-II on the process of mitochondrial autophagy induced by ATR in dopaminergic neurons in vivo and in vitro. Our results demonstrated that LC3-II could accelerate ATR-induced mitophagy in dopaminergic neurons through SQSTM1/p62 pathway, which will contribute to the development of novel therapeutic strategies and targets for human neurodegenerative disorders. Materials and Methods Reagents ATR (CAS: 1912-24-9) was provided by Trustchem (Shanghai, China). Rat pheochromocytoma PC-12 cells (low-differentiated) were obtained from the China Infrastructure of Cell Line Resources (Wuhan, China) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing fetal bovine serum (FBS) from PAN-Biotech GmbH Industries (Aidenbach, Bavaria, Germany). The Cell Counting Kit (CCK8) was purchased from Dojindo (Tokyo, Japan). Penicillin, streptomycin, ROS detection kit, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) preparation kit, and BCA Protein Assay Kit were acquired from Beyotime Biotechnology (Shanghai, China). The anti-TH antibody was purchased from Millipore (Billerica, USA). Antibodies against Beclin-1 and HDAC-6 were obtained from Santa Cruz Biotechnology (Santa Cruz, USA). Antibodies against LC3-II, SQSTM1/p62, Ambra-1, Atg12, cytochrome c, BNIP3L, Parl, PINK1, and Parkin were obtained from Abcam New Territories (Hong Kong, China). Antibodies against Bcl-2, MIF-1, p53, caspase-9, and ACTB were purchased from Immunoway Biotechnology Company (Newark, USA). Alkaline phosphatase-conjugated goat anti-rabbit IgG, alkaline phosphatase-conjugated rabbit anti-goat IgG and alkaline phosphatase-conjugated rabbit anti-mouse IgG were acquired from ZSGB-BIO (Beijing, China). SYBR Premix Ex Taq II Reagent Kit was from Takara (Otsu, Japan), and primers were from Generay Biological Engineering Co. (Shanghai, China). Lipofectamine 2000 was purchased from Invitrogen (Carlsbad, USA), and PVDF membranes were from Bio-Rad (Hercules, USA). Methylcellulose (1%) was acquired from Sigma (St Louis, USA). Animals and treatments Two hundred male Wistar rats were obtained from Vital River Laboratories (Beijing, China). All rats were acclimatized for 1 week and housed under a 12-h light/dark cycle at a constant temperature of 23 ± 1°C, with water and food provided ad libitum. Body weight (BW) was recorded once a week. All procedures were performed in accordance with the regulations of the Ethical Committee for Research on Laboratory Animals, as assessed and approved by the Medical Ethics Committee of Harbin Medical University (Harbin, China), which complies with National Institutes of Health Guidelines. Rats were randomly divided into control and ATR test groups. All animals were treated with vehicle ATR with 1% methylcellulose at doses of 0, 50, 100, and 200 mg/kg BW per day (five times per week) for 45 days via oral gavage [15,41]. Rats were sacrificed after the behavior tests. Immediately following euthanasia, the entire brain was removed and rinsed with ice-cold physiological saline. The meninges and vasculature were removed, and the whole striatum and midbrain from half of the rats in each group were isolated, weighed, and frozen in liquid nitrogen. The remaining half was divided into two groups, and the midbrains were soaked in glutaraldehyde/paraformaldehyde for either pathological examination or immunohistochemical analysis. Behavioral tests Behavioral tests were performed in succession over 3 days (n = 10/group) after treatment. Specifically, rats were sequentially subject to open field and grip strength tests (~10 min between tests). Animals were initially naïve to behavioral apparatus and testing ambience at each time-point with the experimenter, who was blinded to the treatments. The animals were housed in a separate room near the designated behavioral testing room for test preparation. Open field test The activity of each rat was individually monitored in an open field arena (l × w × h: 25 × 25 × 40 cm, divided into 16 square grids; Coulbourn Instruments, Whitehall, USA) for 10/30 min with Limelight video tracking software (Actimetrics, Wilmette, USA). The parameters evaluated included: (1) total distance traveled (cm) and number of crossings at 5 min intervals (horizontal activity), (2) number of rearings (vertical activity) in the first 5 min, and (3) time spent in the center versus periphery analyzed at 5 min intervals (location parameters) [42]. Grip strength test This test measured the forelimb grip strength using a mouse-specific strength gauge (Bioseb, France) for evaluating neuromuscular function quality [43]. Briefly, rats were carefully placed in front of a wire grid (6 cm × 6 cm) and allowed to grab hold with both forepaws. After the grip was established, maximum grip strength was recorded in Newtons [N] three times (1 min apart). Maximum measurements for each animal were obtained to determine the average for subsequent statistical analysis [44]. Light microscopy Midbrains were dehydrated, made transparent, and embedded in paraffin for preparation after fixation with 10% buffered formalin. Tissues were sliced into 4-μm-thick serial sections and stained with Harris hematoxylin and eosin (HE) for histopathological evaluation. Sections were observed under a BH-2 light microscope (Olympus, Tokyo, Japan) to determine the number of degenerative neurons and neuronal structure. The central one-third area of each slice was selected for counting in five randomly selected fields at 200× magnification. Transmission electron microscopy Tissues of the midbrain were initially fixed and subsequently cut into 1 mm3 cubes and fixed with 2% glutaraldehyde and 2% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 48 h at 4°C. After being washed three times with phosphate-buffered saline (PBS), tissues were postfixed in 1% osmium tetroxide solution for 2 h on ice, followed by dehydration in graded ethanol and embedding in Epon 812 mixture. Ultrathin sections were obtained and stained with 2% uranyl acetate and lead citrate for 15 min. Stained sections were examined under a JEM-2100 transmission electron microscope (TEM; Jeol Electron Inc., Tokyo, Japan). Immunohistochemical analysis All rats were anesthetized and perfused transcardially with 200 ml saline (4°C) and then with 300 ml ice-cold 4% paraformaldehyde (4°C). The ventral midbrains were removed and fixed with 4% paraformaldehyde for 4 h at 4°C. After fixation, tissues were transferred to 30% sucrose in 0.01 M PBS until they sank to the bottom. Brains were mounted onto poly-l-lysine-coated slides and cut into 10-μm sections using a cryostat. Sections were incubated with 2% normal goat serum in 0.01 M PBS for 30 min to block non-specific binding, followed by incubation with rabbit anti-rat TH antibody (dilution, 1:1000) containing 0.2% Triton X-100 and 2% normal goat serum at 4°C overnight for neuronal counting. After five times wash with PBS, HRP-conjugated goat anti-rabbit immunoglobulin G (IgG) secondary antibody was added and incubated for 1 h, followed by detection with the diaminobenzidine kit. Sections were covered and imaged with a BX51 microscope (Olympus) and intensities were measured using Image J v1.50 software. Substantia Nigra pars compacta (SNpc) were determined based on the integrated optical density value. PC-12 cell culture and transient transfection of LC3-II siRNA PC-12 cells were cultured in DMEM containing 10% FBS, 100 IU/ml penicillin and 100 μg/ml streptomycin at 37°C in an incubator under 5% CO2 and 95% air. Cells in the logarithmic growth phase were collected and used for the subsequent experiments. Specific siRNA (Ribobio Biotechnology, Guangzhou, China) was used for LC3-II knockdown in PC-12 cells, and stable transfectants at the gene and protein levels were obtained by using Lipofectamine 2000 [45]. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of total RNA was performed using a SYBR Premix Ex Taq II Reagent Kit and gDNA Eraser reverse transcriptase (Takara), according to the manufacturer’s instructions. Cells treated with non-silencing scrambled siRNA and transfection reagent were used as controls. The LC3-II siRNA target sequence was: 5′-GCAGCTCAATGCTAACCAA-3′. The NC siRNA target sequence was: 5′-UUCUCCGAACGUGUCACGUTT-3 (Ribobio Biotechnology) CCK8 cell viability assay Cells in both the transfected and non-silencing groups were stimulated with 100 μM ATR in 0.1% dimethyl sulfoxide (DMSO) for 24 h at 4°C after filtration and sterilization. As a positive control, 100 μM LPS was added to cells [46]. Cells were collected for the next phase of the experiment, and cell viability was measured by detecting mitochondria in living cells by the CCK8 assay. The CCK8 reagent (0.5 mg/ml) was incubated with culture medium at 37°C for 1 h and absorbance was evaluated at 450 nm using a Bio-Tek Elx800 microplate reader (Bio-Tek, Winooski, USA). ROS assay Intracellular ROS can oxidize non-fluorescent DCFH to generate highly fluorescent DCF that is detectable using the fluorescent probe, 2′-7′-dichlorofluorescin diacetate (DCFH-DA). PC-12 cells were seeded on coverslips and stimulated for 24 h. Then, cells were incubated with 10 mM DCFH-DA at 37°C for 20 min and washed twice with PBS. Fluorescence was measured using a Nikon A1R laser scanning confocal microscope (Nikon, Tokyo, Japan) [6]. Immunofluorescence analysis To investigate the degeneration of DA neurons in the SNpc, sections were subject to immunofluorescence labeling using primary rabbit anti-rat TH antibody (dilution, 1:1000), a marker of neuronal nuclei. To assess the activation of autophagy in mitochondrial dysfunction, double-labeling for subunits LC3-II and SQSTM1/p62 was performed in PC-12 cells using primary rabbit anti-rat LC3-II antibody (dilution, 1:1000) and mouse anti-rat SQSTM1/p62 antibody (dilution, 1:1000). The secondary antibodies used for incubation were DyLight® 488 goat anti-rabbit IgG and DyLight® 594 goat anti-mouse IgG (both 1:400 dilution; Vector Laboratories, Burlingame, USA). PC-12 cell nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). In brief, secondary antibodies were blocked with same-species normal serum, and the sections were incubated with primary antibodies at 4°C overnight. After three times wash with PBS, sections were treated with the appropriate secondary antibodies for 1 h. PC-12 cells were also labeled via nuclear staining with DAPI for 3 min. Finally, the coverslips were sealed with Antifade Mounting Medium and viewed under a BX51 microscope equipped with a DP72 camera (Olympus Corporation). RT-PCR Total RNA was extracted from rat tissues and cultured PC-12 cells using Trizol reagent (Sigma), and used as a template to synthesize cDNA with a SYBR Premix Ex Taq II Reagent Kit and gDNA Eraser reverse transcriptase, according to the manufacturer’s instructions. Rat-specific primers were listed in Supplementary Fig. S1. The relative expression levels of each target gene were determined based on the cycle threshold (Ct) method. For PCR analysis, expression was normalized to that of β-actin using the equation: ΔCT = CTtarget gene − CTβ-actin. The 2−ΔΔCT method was employed to calculate the relative amounts of target mRNA. Western blot analysis Western blot analysis was performed to assess ATR-induced expression of mitochondria-mediated dopaminergic neuronal proteins in SNpc and PC-12 cells. To this end, rat tissues frozen in liquid nitrogen were thawed, and PC-12 cells were subject to ATR stimulation and collected for the next phase of experiment. Following homogenization for 30 min in lysis buffer containing 1 mM phenylmethanesulfonyl fluoride, tissue and cell homogenates were centrifuged at 1500 g for 10 min at 4°C and protein concentrations were determined using the BCA protein assay kit (ZSGB-BIO). Equal amounts of protein (20 μg) were separated by 10% or 12% SDS–PAGE and transferred onto PVDF membranes. Membranes were blocked with 1% bovine serum albumin containing 0.05% Tween 20 in PBS for 30 min. Next, membranes were incubated with antibodies against TH, LC3-II, SQSTM1/p62, Ambra-1, Atg12, cytochrome c, BNIP3L, Parl, PINK1, Parkin, Bcl-2, MIF-1, p53, caspase-9 (1:1000 dilution), Beclin-1, and HDAC-6 (1:200 dilution) at 4°C overnight, using anti-β-actin antibody (1:1000 dilution) as a control, followed by incubation with alkaline phosphatase-conjugated goat anti-rabbit IgG (1:1000 dilution) secondary antibody, alkaline phosphatase-conjugated rabbit anti-goat IgG (1:1000 dilution) secondary antibody, or alkaline phosphatase-conjugated rabbit anti-mouse IgG (1:1000 dilution) secondary antibody for 1 h at room temperature. Immunoreactive signals were detected using Western Blue® Stabilized Substrate for Alkaline Phosphatase (ZSGB-BIO). The air-dried membranes were imaged using an image analyzer (Bio-Rad Laboratories). Band intensities were measured using the Image J v1.50 software. Statistical analysis Data were expressed as the mean ± SEM. Each assay was performed independently at least three times. Differences among groups were analyzed using one-way or two-way ANOVA, followed by the Dunnett’s multiple comparison test. Values of P < 0.05 were considered statistically significant. Results ATR induces dopaminergic neuron degeneration in rats Body characteristics After 45 days of ATR exposure, rats in the 50 mg/kg group did not display any significant decrease in BW or alterations in general appearance. In contrast, the BW was decreased after 45 days in the 100 mg/kg and 200 mg/kg ATR groups (P < 0.05) in a dose-dependent manner (Supplementary Fig. S1). Behavioral tests In the 200 mg/kg ATR treatment groups, vertical activity (number of rearing responses) was significantly decreased after 45 days (P < 0.05; Fig. 1A). Similarly, rats displayed a remarkable decrease in the distance traveled in the 100 and 200 mg/kg ATR treatment groups (P < 0.05; Fig. 1B) and in the number of crossings at 5 min intervals (Fig. 1C) in the 200 mg/kg ATR treatment group after 45 days (P < 0.05). Grip strength displayed a strong decreasing trend in rats exposed to 100 and 200 mg/kg ATR for 45 days (P < 0.05; Fig. 1D). Figure 1. View largeDownload slide Effects of ATR exposure on spontaneous locomotor activity Rats were exposed to ATR for 45 days and spontaneous locomotor activity parameters were assayed. (A) Number of rearings during the first 5 min. (B) Distance traveled per 5 min. (C) Number of crossings per 5 min. (D) Effects of ATR on grip strength. Data are presented as the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. Figure 1. View largeDownload slide Effects of ATR exposure on spontaneous locomotor activity Rats were exposed to ATR for 45 days and spontaneous locomotor activity parameters were assayed. (A) Number of rearings during the first 5 min. (B) Distance traveled per 5 min. (C) Number of crossings per 5 min. (D) Effects of ATR on grip strength. Data are presented as the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. Histopathology effects Histopathological examination of the substantia nigra showed degenerative changes after ATR treatment. Neuronal degeneration symptoms, Nissl body side shift, levels of neuronal pyknosis, and massive nerve cell shrinkage were exacerbated following ATR treatment (Fig. 2A). Compared with the control group, sporadic neuronal pyknosis in the midbrain was observed, and the number of degenerated neurons was increased clearly following exposure to 50, 100, and 200 mg/kg ATR for 45 days. Our findings indicated that ATR promoted neuronal degeneration in a dose-dependent manner (P < 0.05; Fig. 2B). TEM examination of the substantia nigra revealed chromatin margination or disappearance, perinuclear cistern widening, and apoptotic chromatin condensation under the nuclear membrane-fixing ring in the ATR treatment groups after 45 days (Fig. 2C). Additionally, ultrastructural examination revealed mitochondrial autophagic vacuolar degeneration, degenerative autophagosome (a double membrane structure enclosing mitochondria), and mitochondrial autophagy (Fig. 2C). The observed characteristics indicated that cells underwent retrogression and autophagy process was activated by ATR. Figure 2. View largeDownload slide Histopathological examination of substantia nigra (SNpc) in rats after ATR treatment (A) Nerve cell morphology analysis. Tissues were exposed to ATR for 45 days. Sections were examined under a light microscope to assess the number of degenerative neurons and neuronal structure. Scale bar, 10 μm. Nerve cell shrinkage is highlighted with red arrows, swelling in neurons with black arrows, and microglia infiltration with black edge filled white arrows. (B) Quantification of degenerative neurons. (C) Ultrastructural properties of neuronal cells in substantia nigra (SNpc) of rats treated with ATR. Sections were examined under a transmission electron microscope. Scale bar, 2 μm. Black arrows: swelling and mitochondrial vacuolization; red arrows: mitochondrial autophagy. Data are presented as the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. Figure 2. View largeDownload slide Histopathological examination of substantia nigra (SNpc) in rats after ATR treatment (A) Nerve cell morphology analysis. Tissues were exposed to ATR for 45 days. Sections were examined under a light microscope to assess the number of degenerative neurons and neuronal structure. Scale bar, 10 μm. Nerve cell shrinkage is highlighted with red arrows, swelling in neurons with black arrows, and microglia infiltration with black edge filled white arrows. (B) Quantification of degenerative neurons. (C) Ultrastructural properties of neuronal cells in substantia nigra (SNpc) of rats treated with ATR. Sections were examined under a transmission electron microscope. Scale bar, 2 μm. Black arrows: swelling and mitochondrial vacuolization; red arrows: mitochondrial autophagy. Data are presented as the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. ATR inhibits TH expression in rat dopaminergic neurons and PC-12 cells Immunohistochemical analysis was performed to assess the extent of TH-positive DA neuron degeneration in rats. A reduction in the number of TH+ neurons was detected and mild cellular atrophy began to appear after ATR treatment (100 and 200 mg/kg) for 45 days (P < 0.05) compared with the control group (Fig. 3A,B). TH mRNA and protein expressions were significantly decreased by ATR exposure (100 and 200 mg/kg) for 45 days (P < 0.05; Fig. 3C,D). To further study the ATR-induced dysfunction in the dopaminergic nerve system, we used a common nerve cell line pheochromocytoma PC-12 for TH expression analysis. It was found that 100 μM ATR significantly decreased the levels of TH mRNA and protein compared with the control group in PC-12 cells (Fig. 3E,F). These data indicated that ATR reduced TH expression in rat dopaminergic neurons and in pheochromocytoma PC-12 cells to enhance neuron dysfunction. Figure 3. View largeDownload slide Dopaminergic neuron degeneration following ATR treatment with or without LC3-II siRNA in vivo and in vitro (A) Dopaminergic neuron degeneration following ATR treatment for 45 days as shown by immunohistochemical analysis in rats. Scale bar, 10 μm. (B) The number analysis of TH positive dopaminergic neurons in A. (C,D) TH mRNA and protein expressions were detected by real-time PCR and western blotting in rats after ATR treatment. (E,F) TH mRNA and protein levels were measured by real-time PCR and western blot analysis in PC-12 cells after ATR treatment with or without LC3-II siRNA. Data are presented as the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. Figure 3. View largeDownload slide Dopaminergic neuron degeneration following ATR treatment with or without LC3-II siRNA in vivo and in vitro (A) Dopaminergic neuron degeneration following ATR treatment for 45 days as shown by immunohistochemical analysis in rats. Scale bar, 10 μm. (B) The number analysis of TH positive dopaminergic neurons in A. (C,D) TH mRNA and protein expressions were detected by real-time PCR and western blotting in rats after ATR treatment. (E,F) TH mRNA and protein levels were measured by real-time PCR and western blot analysis in PC-12 cells after ATR treatment with or without LC3-II siRNA. Data are presented as the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. LC3-II knockdown decreases the inhibition of ATR on PC-12 cell viability To determine whether LC3-II mediated mitophagy is involved in neuronal death, we knocked down LC3-II by siRNA before ATR treatment and found that LC3-II siRNA could reverse the decreases in the TH mRNA and protein expression levels induced by ATR treatment in PC-12 cells (Fig. 3E,F). It was also found that ATR treatment of PC-12 cells led to significantly decreased cell viability (P < 0.05), and LC3-II siRNA could reverse the decreased cell viability induced by ATR treatment as revealed by CCK8 assay (Fig. 4A). These data indicated that ATR decreased PC-12 cell viability through LC3-II-mediated autophagy over-activation. Figure 4. View largeDownload slide Effects of ATR treatment with or without LC3-II siRNA on PC-12 cell viability (A) Cell viability of PC-12 cells after ATR treatment with or without LC3-II siRNA and LPS was detected. (B) Intracellular ROS levels in PC-12 cells after ATR treatment with or without LC3-II siRNA were detected by DCFH-DA staining after 24 h. Images were obtained with laser scanning confocal microscopy at excitation and emission wavelengths of 485–530 nm, respectively. Scale bar, 40 μm. (C) Relative quantification of ROS generation in B, by using Image Pro Plus analysis software. Data are presented as the mean ± SEM, n ≥ 3. *P < 0.05, **P < 0.01. Figure 4. View largeDownload slide Effects of ATR treatment with or without LC3-II siRNA on PC-12 cell viability (A) Cell viability of PC-12 cells after ATR treatment with or without LC3-II siRNA and LPS was detected. (B) Intracellular ROS levels in PC-12 cells after ATR treatment with or without LC3-II siRNA were detected by DCFH-DA staining after 24 h. Images were obtained with laser scanning confocal microscopy at excitation and emission wavelengths of 485–530 nm, respectively. Scale bar, 40 μm. (C) Relative quantification of ROS generation in B, by using Image Pro Plus analysis software. Data are presented as the mean ± SEM, n ≥ 3. *P < 0.05, **P < 0.01. LC3-II knockdown inhibits the ROS activation induced by ATR in PC-12 cells Normal PC-12 cells displayed small cell bodies and long synapses, indicating a typical resting state with occasional ROS expression. ROS levels were significantly increased upon ATR treatment, indicating autophagy activation, as revealed by the number of fluorescent cells detected, rounder and brighter cell bodies, and thinner and shorter synapses in the presence of LC3-II siRNA. ROS levels were sharply increased because of low activation of mitophagy (Fig. 4B,C), as assessed by confocal laser scanning microscopy. These data indicated that ATR could promote ROS release through LC3-II-mediated autophagy over-activation. ATR influences the mRNA expression of autophagy-related genes To further examine the mechanism of the effect of ATR on dopaminergic neurons, we detected the mRNA levels of LC3-II, Beclin-1, PINK1, Parkin, Parl, SQSTM1/p62, and other autophagic genes (HDAC-6, Ambra-1, Atg12, BNIP3L, and MIF-1), as well as the apoptosis-related genes (such as Bcl-2, caspase-9, p53, and cytochrome c) after ATR treatment. Real-time PCR analysis revealed that the mRNA levels of LC3-II, Beclin-1, PINK1, Parkin, and caspase-9 were significantly increased in ATR treatment groups compared with the control group (Fig. 5). Moreover, the mRNA expressions of SQSTM1/p62, Parl, and Bcl-2 were decreased by ATR treatment in a dose-dependent manner (Fig. 5). The mRNA expressions of other autophagy- and apoptosis-related genes were also greatly influenced in rats as shown in Supplementary Fig. S2. These data indicated that ATR could damage dopaminergic neurons by regulating the expressions of autophagy- and apoptosis-related genes in vivo. Figure 5. View largeDownload slide The mRNA expressions of autophagy and apoptosis-related genes in rats after ATR treatment The mRNA levels of autophagy and apoptosis-related genes in the ventral midbrains of rats after ATR treatment were measure by real-time PCR (A) LC3-II, (B) SQSTM1/p62, (C) Parkin, (D) PINK1, (E) Parl, (F) Beclin-1, (G) Bcl-2, and (H) caspase-9. Each group consisted of eight rats. Data are presented as the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. Figure 5. View largeDownload slide The mRNA expressions of autophagy and apoptosis-related genes in rats after ATR treatment The mRNA levels of autophagy and apoptosis-related genes in the ventral midbrains of rats after ATR treatment were measure by real-time PCR (A) LC3-II, (B) SQSTM1/p62, (C) Parkin, (D) PINK1, (E) Parl, (F) Beclin-1, (G) Bcl-2, and (H) caspase-9. Each group consisted of eight rats. Data are presented as the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. LC3-II knockdown inhibits the influence of ATR on the mRNA level of autophagy-related genes In PC-12 cells, the mRNA levels of LC3-II, Beclin-1, PINK1, Parkin, and caspase-9 were significantly increased after ATR treatment through autophagy activation, while the mRNA levels of Parl, SQSTM1/p62, and Bcl-2 were decreased compared with the control group; and the transfection of LC3-II siRNA with ATR treatment almost reversed the effect of ATR (Fig. 6). What's more, the expression levels of these genes in ATR with LC3-II siRNA group were not significantly different from that in the LC3-II siRNA only group (Fig. 6). The mRNA expressions of other autophagic and apoptotic genes in PC-12 cells were also shown in Supplementary Fig. S3. These data indicated that LC3-II may play a crucial role in regulating the mRNA expressions of autophagy and apoptosis-related genes during ATR-induced neuron degeneration in vitro. Figure 6. View largeDownload slide The mRNA expressions of autophagy and apoptosis-related genes in PC-12 cells after ATR treatment with or without LC3-II siRNA PC-12 cells were exposed to ATR with or without LC3-II siRNA for 24 h and mRNA expression was measured by real-time PCR. (A) LC3-II, (B) SQSTM1/p62, (C) Parkin, (D) PINK1, (E) Parl, (F) Beclin-1, (G) Bcl-2, and (H) caspase-9. Data are presented as the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. Figure 6. View largeDownload slide The mRNA expressions of autophagy and apoptosis-related genes in PC-12 cells after ATR treatment with or without LC3-II siRNA PC-12 cells were exposed to ATR with or without LC3-II siRNA for 24 h and mRNA expression was measured by real-time PCR. (A) LC3-II, (B) SQSTM1/p62, (C) Parkin, (D) PINK1, (E) Parl, (F) Beclin-1, (G) Bcl-2, and (H) caspase-9. Data are presented as the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. ATR influences the protein expression of autophagy-related genes Compared with those in the control group, the protein levels of LC3-II, Beclin-1, PINK1, and Parkin, and caspase-9 were significantly increased by exposure to ATR, and the SQSTM1/p62, Parl, and Bcl-2 protein levels were significantly downregulated following exposure to ATR in dose-dependent manner (Fig. 7). The protein expressions of other related genes in rats were shown in Supplementary Fig. S4. These data indicated that ATR could regulate the protein levels of autophagy- and apoptosis-related genes in vivo. Figure 7. View largeDownload slide Western blot analysis of autophagy- and apoptosis-related proteins in rats after ATR treatment Protein expression in ventral midbrains of rats exposed to ATR for 45 days. (A) LC3-II, (B) SQSTM1/p62, (C) Parkin, (D) PINK1, (E) Parl, (F) Beclin-1, (G) Bcl-2, and (H) caspase-9. Each group consisted of eight rats. β-Actin was detected as the internal control. Data are presented as the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. Figure 7. View largeDownload slide Western blot analysis of autophagy- and apoptosis-related proteins in rats after ATR treatment Protein expression in ventral midbrains of rats exposed to ATR for 45 days. (A) LC3-II, (B) SQSTM1/p62, (C) Parkin, (D) PINK1, (E) Parl, (F) Beclin-1, (G) Bcl-2, and (H) caspase-9. Each group consisted of eight rats. β-Actin was detected as the internal control. Data are presented as the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. LC3-II knockdown inhibits the influence of ATR on the protein level of autophagy-related genes In PC-12 cells, the levels of LC3-II, Beclin-1, PINK1, Parkin, and caspase-9 were significantly increased in the ATR treatment group, while the protein expressions of Parl, SQSTM1/p62, and Bcl-2 were sharply decreased by ATR (Fig. 8). However, the transfection of LC3-II siRNA with ATR treatment significantly inhibited the protein levels of most autophagy- and apoptosis-related genes, which may be due to the complete inhibition of ATR-mediated mitophagy by LC3-II knockdown (Fig. 8 and Supplementary Fig. S5), which was similar to LC3-II knockdown only group (Fig. 8). These data indicated that LC3-II-mediated autophagic protein activation plays a dominant role in ATR-induced neuron degeneration in vitro. Figure 8. View largeDownload slide Western blot analysis of autophagy and apoptosis-related proteins in PC-12 cells after ATR treatment with or without LC3-II siRNA Protein expressions in PC-12 cells exposed to ATR and LPS for 24 h were detected by western bot analysis. (A) LC3-II, (B) SQSTM1/p62, (C) Parkin, (D) PINK1, (E) Parl, (F) Beclin-1, (G) Bcl-2, and (H) caspase-9. β-Actin was detected as the internal control. Data are presented as the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. Figure 8. View largeDownload slide Western blot analysis of autophagy and apoptosis-related proteins in PC-12 cells after ATR treatment with or without LC3-II siRNA Protein expressions in PC-12 cells exposed to ATR and LPS for 24 h were detected by western bot analysis. (A) LC3-II, (B) SQSTM1/p62, (C) Parkin, (D) PINK1, (E) Parl, (F) Beclin-1, (G) Bcl-2, and (H) caspase-9. β-Actin was detected as the internal control. Data are presented as the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. LC3-II knockdown may mediate ATR-induced mitophagy through SQSTM1/p62 pathway To further determine the mechanism of ATR-induced mitophagy in PC-12 cells, we analyzed the expression patterns of LC3-II and p62, which has a close relationship with LC3-II, after ATR treatment by immunofluorescence assay. Autophagy was activated after exposure to 100 μM ATR for 24 h as shown by the cell body morphology. LC3-II level was increased and SQSTM1/p62 level was decreased in PC-12 cells exposed to ATR compared with those in the control group (Fig. 9A,B), which indicated a close association between autophagy and expressions of LC3-II and SQSTM1/p62. Furthermore, the knockdown of LC3-II clearly reversed the effect of ATR on the SQSTM1/p62 level and the autophagy morphology. These data indicated that LC3-II may mediate ATR-induced mitophagy through the SQSTM1/p62 pathway. Figure 9. View largeDownload slide Immunofluorescence analysis of LC3-II and SQSTM1/p62 colocalization in PC-12 cells after ATR treatment with or without LC3-II siRNA PC-12 cells (1.0 × 105 cells) after ATR treatment with or without LC3-II siRNA. (A) Double-labeled immunofluorescence analysis. Green: LC3-II; red: SQSTM1/p62. Scale bar: 40 μm. (B) The combination rate of LC3-II with SQSTM1/p62. Data are presented as the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. Figure 9. View largeDownload slide Immunofluorescence analysis of LC3-II and SQSTM1/p62 colocalization in PC-12 cells after ATR treatment with or without LC3-II siRNA PC-12 cells (1.0 × 105 cells) after ATR treatment with or without LC3-II siRNA. (A) Double-labeled immunofluorescence analysis. Green: LC3-II; red: SQSTM1/p62. Scale bar: 40 μm. (B) The combination rate of LC3-II with SQSTM1/p62. Data are presented as the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. Discussion Mitochondrial dysfunction has been shown to play a significant role in the pathogenesis and progression of PD, and mitophagy may be involved in the initiation and aggravation of neurotoxicity [28,31,47]. Here, we investigated the mechanisms underlying the ATR-induced mitophagy response in vitro and in vivo, and the potential role of ATR in generation of dopaminergic system disorders. ATR-enhanced LC3-II reduced neuronal cell proliferation and accelerated mitochondrial dysfunction, resulting in neuronal cell death. Our results indicate that ATR-induced autophagy influences dopaminergic neurodegeneration and thus regulates mitochondrial proteins and dynamics. In the present study, exposure to 100- or 200-mg/kg ATR triggered short-term behavioral alterations in locomotor activities after 45 days, which is related to the damage in the monoaminergic nigrostriatal system with reduced levels of DA and increased number of dopaminergic neurodegeneration neurons [15,16]. In light of this research, ATR could decrease the immediate behavioral response owing to increased anxiety and tensity, which is in agreement with the hypoactivity observed after ATR treatment [21,48,49]. Meanwhile, we observed karyopyknosis, myelin bodies, mitochondrial vacuoles, and degeneration of autophagosomes by electron microscopy, further confirming that ATR significantly enhances autophagy in mitochondria and accelerates the progress of degeneration programs in cellular bioenergetics and homeostasis [25–27]. In the dopaminergic systems, TH is a limited enzyme marker for DA synthesis. ATR injection was reported to suppress TH expression in neuronal cells and increase dopaminergic neurotoxicity, both in vivo [17,41] and in vitro [11,50,51]. Neural cell pyknosis, neural cell death, and degenerative neurons were increased in a time- and dose-dependent manner in ATR treatment groups, compared with the control group, as revealed by the histopathological HE staining data. We also found that exposure to 100- and 200-mg/kg ATR could lead to significant loss of TH-positive cells in SNpc in 45 days. RT-PCR and western blot analysis from rats demonstrated that TH mRNA and protein levels were clearly decreased. These histopathological alterations indirectly indicate ATR-induced the dysfunction of dopaminergic systems and neurotoxicity in the substantia nigra of Wistar rats. Our data also indicate that mitochondrial dysfunction specifically occurs after chronic exposure to ATR in vitro and at an early stage of ATR treatment in vivo. ATR-activated mitophagy decreased the viability of neuronal PC-12 cells, as revealed by cell viability assay, which showed reduced DA levels and enhanced neurotoxicity. Accumulation of significant quantities of environmental toxins in mitochondria led to mitochondrial dysfunction under conditions of oxidative stress and release of ROS, which was significantly increased in PC-12 neurons treated with 100-μM ATR. When subjected to further ATR injury, irreversible and permanent damage to mitochondrial function systems may be induced owing to autophagy, which fails to regulate the progress of cellular bioenergetics and homeostasis [25]. Following ATR treatment, PINK1 and Parkin mRNA and protein levels were increased significantly, while Parl levels showed a sustained decrease in a dose-dependent manner. These data showed that the over-activated PINK1/Parkin pathway alters mitochondrial dynamics, function and maintenance of mitochondrial homeostasis [37,38]. PINK1/Parkin co-operates with SQSTM1/p62, which accumulates in the absence of Parl, recruits Parkin to the damaged mitochondria and contributes to mitochondrial matrix protein ubiquitination, leading to engulfment and degradation via autophagy [29–33]. We found that following recruitment, Parkin promotes polyubiquitination of mitochondrial substrate(s), which recruits the ubiquitin- and LC3-binding adaptor protein p62. ATR-induced over-activation of the PINK1/Parkin pathway could damage mitochondrial fission and/or fusion and promote mitophagy, suggesting that p62/SQSTM1 is required for Parkin-induced mitochondrial disfunction [32,52] and plays a key role in the mechanism of autophagy-mediated neurodegeneration [53,54]. LC3 was reported to accumulate and combine with SQSTM1/p62 to weaken the signal of aggresomes in autophagy [55–57]. LC3-II combines with AMBRA-1 to regulate mitochondrial depolarization in mitochondrial clearance and participates in a ubiquitin-like system [58]. The mitochondrial outer membrane protein, BNIP3L/Nix, interacts with LC3, which is required for mitophagy [59–61]. The Atg5-Atg12-Atg16 complex also contributes to phagophore development by dissociation of the components [62]. Phosphorylation of SQSTM1/P62 reduces mitochondrial membrane potential, thereby impairing signaling of aggresomes and the ubiquitin–proteasome network [63,64]. The main mediators of aggresome formation, sequestosome-1 (SQSTM1/p62), and histone deacetylase-6 (HDAC-6), have been characterized as a multifunctional scaffold protein participating in autophagic isolation and degradation of mitochondria that is associated with the outer phagophore membrane proteins, ATG5 and LC3, in autophagy [27,39,65]. Immunofluorescence analysis of overexpressed LC3-II and downregulated SQSTM1/p62 in ATR-treated PC-12 cells revealed the activation of these molecules. Cells positive for LC3-II and SQSTM1/p62 receptors appeared more intensively stained, supporting their use as markers of cell body morphology and mitophagy. Compared with those in the naive PC-12 cells, LC3-II mRNA and protein levels were significantly increased and SQSTM1/p62 mRNA and protein levels were significantly decreased in ATR treatment group, as evidenced by real-time PCR and western blot analyses. After exposure to 100 and 200 mg/kg ATR for 45 days, ATR over-activated LC3-II and autophagy, aberrantly decreased SQSTM1/p62 levels, which led to a clear increase in positive regulatory (Ambra-1, MIF-1, Atg12, HDAC-6, BNIP3L, and Beclin-1) mRNA and protein levels. Meanwhile, PC-12 cells with LC3-B RNA knocked-down were treated with ATR to observe cell survival and mitochondrial function. The results showed a significant inhibition in mitophagy, and decrease in PINK1 and Parkin. Based on these results, we propose that LC3-II-dependent mitophagy is triggered, and these proteins may play indispensable roles in the process of phagophore conversion to autophagosomes and cellular activation of autophagy in mitochondria in vivo and in vitro. p62 directly binds to the autophagy proteins LC3, and is believed to serve as an autophagy receptor for ubiquitinated protein aggregates as well as peroxisomes, which indicates that at single cell level mitophagy can still proceed upon acute p62 depletion. These findings suggest that the p62-independence of Parkin-induced mitophagy may not be the result of a compensatory mechanism and may be characteristic of many mammalian cell types. Negative modulation of apoptosis may mediate or collaborate with the mechanism of autophagy under several conditions [66,67]. As an important upstream signaling molecule in apoptosis, Bcl-2 interacts with and/or mutually inhibits the autophagic switching factor Beclin-1 [68]. Here, the mRNA and protein expression levels of Bcl-2 were found to be significantly decreased by ATR, along with Beclin-1 upregulation, in a time- and dose-dependent manner. Upon alterations in the balance between autophagy and apoptotic processes, Bcl-2 is converted into a pro-apoptotic protein and restores the permeability of mitochondrial membrane, which stimulates the release of cytochrome c [57,69]. Binding of cytochrome c to Apaf-1 triggers the activation of caspase-9, which accelerates apoptosis via activation of other caspases [70]. Activated caspases enhance apoptosis via p53 death signaling in the mitochondrial pathway. In our experiments, upregulated mRNA and protein levels of cytochrome c, caspase-9, and p53 after ATR treatment clearly supported ATR-induced neurodegenerative damage. In summary, ATR-induced LC3-II and autophagy over-activation could promote dopaminergic neuron death, which is combined with mitochondrial dysfunction. Over-activation of LC3-II and autophagy by ATR may play a critical role in dopaminergic neurons mitochondrial dysfunction, which is regulated by p62/SQSTM1 and Parkin signaling pathway. ATR-induced mitochondrial dysfunction via autophagy reduces DA levels and enhances neurotoxicity, thereby suppressing the viability of dopaminergic neurons in a dose- and time-dependent manner. ATR may be an environmental risk factor contributing to dopaminergic system toxicity and pathogenesis of neurodegenerative disorders, such as PD. Therefore, further studies are needed to clarify the mechanisms underlying ATR-induced dopaminergic neuronal death as a result of mitophagy and highlight the importance of assessing ATR-induced human health risks. Funding This work was supported by a grant from National Natural Science Foundation of China (No. 81273109). References 1 Eldridge JC , Tennant MK , Wetzel LT , Breckenridge CB , Stevens JT . 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TI - LC3-II may mediate ATR-induced mitophagy in dopaminergic neurons through SQSTM1/p62 pathway
JF - Acta Biochimica et Biophysica Sinica
DO - 10.1093/abbs/gmy091
DA - 2018-10-01
UR - https://www.deepdyve.com/lp/oxford-university-press/lc3-ii-may-mediate-atr-induced-mitophagy-in-dopaminergic-neurons-iP0UlES0Zp
SP - 1047
VL - 50
IS - 10
DP - DeepDyve
ER -