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Apelin-13 protects dopaminergic neurons in MPTP-induced Parkinson’s disease model mice through inhibiting endoplasmic reticulum stress and promoting autophagy
Brain Research 1715 (2019) 203–212
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Research report
Apelin-13 protects dopaminergic neurons in MPTP-induced Parkinson’s disease model mice through inhibiting endoplasmic reticulum stress and promoting autophagy Junge Zhua, Shanshan Doub, Yunlu Jiangb, Jing Chenb, Chunmei Wangb, , Baohua Chengb, ⁎
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Cheeloo College of Medicine, Shandong University, 250014 Jinan, China Neurobiology Institute, Jining Medical University, 272067 Jining, China
ARTICLE INFO
ABSTRACT
Keywords: Parkinson’s disease α-Synuclein 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridin Apelin-13 Endoplasmic reticulum stress Autophagy
The dopaminergic neurodegeneration in the substantia nigrapars compacta (SNpc) and striatum of the midbrain is the important pathological feature of Parkinson's disease (PD). It has been shown that autophagy and endoplasmic reticulum stress (ERS) are involved in the occurrence and development of PD. The neuropeptide Apelin-13 is neuroprotective in the neurological diseases such as PD, Alzheimer's disease and cerebral ischemic stroke. In the present work, we investigated the neuroprotective effects of Apelin-13 on ERS and autophagy in the dopaminergic neurodegeneration of SNpc of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridin (MPTP)-treated mice. The intranigral injection of Apelin-13 alleviated the behavioral dysfunction and dopaminergic neurodegeneration induced by MPTP. After the exposure to MPTP, the expression of tyrosine hydroxylase (TH) was significantly decreased as well as the increased α-synuclein expression, which was significantly reversed by the intranigral injection of Apelin-13. Also, Apelin-13 significantly reversed the decreasing autophagy induced by MPTP which was indicated by the up-regulation of LC3B-II and Beclin1 and down-regulation of p62. And MPTPinduced ERS such as IRE1α, XBP1s, CHOP and GRP78 was significantly inhibited by Apelin-13. Taken together, Apelin-13 protects dopaminergic neurons in MPTP-induced PD model mice in vivo through inhibiting ERS and promoting autophagy, which contributes to the therapy for PD in the future.
1. Introduction Parkinson's disease (PD) is a common neurodegenerative disease which is characterized by high morbidity rate, high disability rate and chronic course (Steib et al., 2018). The dopaminergic neurodegeneration in the substantia nigrapars compacta (SNpc) and striatum of the midbrain accompanied by abnormal aggregation of a large number of α-synuclein is the important pathological feature of PD. Although the mechanisms are not fully understood, the autophagy and endoplasmic reticulum stress (ERS) are closely associated with PD. Autophagy is the degradation process of long-lived proteins and damaged organelles in cells by lysosomes which is an unique catabolism pathway of eukaryotic cells that can promote energy metabolism and cell survival (Parzych and Klionsky, 2014). It plays a role in the extension and maturation of autophagic vesicles by initiating mammalian target of rapamycin (mTOR) and Beclin1 and ultimately degrading the inclusions in lysosomes (Hwang et al., 2017; Qian et al., 2017). The microtubule-associated protein1 light chain 3B-II (LC3B-II) produced at
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its extension stage is an autophagic biomarker (Meyer et al., 2013). In addition, p62/SQSTM1 is involved in the formation of autophagy as a regulatory factor by coupling to LC3 which is degraded in the late stage of autophagy (Liu et al., 2016). Therefore, the expression of p62 is negatively correlated with autophagy activity (Lee et al., 2011). A series of studies indicate that the autophagy plays an important role in neurodegenerative disease including PD (Kim et al., 2017). In PD model in vivo, MPTP decreases LC3B-II and Beclin1 and increases p62 in SNpc which results in the inhibition of autophagy and induction of α-synuclein aggregation (Guo et al., 2016). It has been reported that the abnormal aggregation of α-synuclein, which is closely associated with the inhibition of autophagy, leads to ERS (Chen et al., 2018; Melo et al., 2018). And then IRE1α/XBP-1/CHOP is activated by the dissociation of glucose regulated protein 78 (GRP78) from IRE1α in PD model in vivo and in vitro (Jiao et al., 2017; Ning et al., 2016). Apelin was extracted and purified from bovine secretion for the first time by Tatemoto et al. in 1998, which was proved to be an endogenous ligand of APJ (Tatemoto et al., 1998). It can be hydrolyzed into several
Corresponding authors. E-mail addresses: [email protected] (C. Wang), [email protected] (B. Cheng).
https://doi.org/10.1016/j.brainres.2019.03.027 Received 24 December 2018; Received in revised form 11 March 2019; Accepted 22 March 2019 Available online 23 March 2019 0006-8993/ © 2019 Elsevier B.V. All rights reserved.
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peptide segments of different lengths such as Apelin-12, Apelin-13, Apelin-17, Apelin-36 and so on, of which Apelin-13 is known to be the most bioactive Apelin receptor activator (Boal et al., 2016). Many experiments have shown that Apelin-13 is neuroprotective against the neuronal damage (Chen et al., 2015). It has been shown that apelin-13 exerts the effects of ameliorating cognitive impairments in 6-OHDAtreated rats (Haghparast et al., 2018). Moreover, Apelin-13 has been proved to inhibit oxidative stress and apoptosis in 6-OHDA-treated SHSY5Y cells (Pouresmaeili-Babaki et al., 2018). And MPP+-induced cytotoxicity was reversed by Apelin-13 in SH-SY5Y cells (Jiang et al., 2018). In the present work, we investigated the effects of intranigral injection of Apelin-13 on MPTP-induced PD model mice including the behavioral dysfunction, dopaminergic neurodegeneration and α-synuclein expression. The role of autophagy and ERS was also investigated.
decreased compared with control group (p < 0.001). The intranigral Apelin-13 injection significantly the loss of dopaminergic neurons in the SNpc compared with MPTP group (p < 0.01). The similar results was found by TH immunofluorescence (Fig. 2C and D). Also, the expression of TH of the SNpc was significantly increased in MPTP group compared with control group (p < 0.001), while the intranigral Apelin-13 injection significantly reversed TH expression compared MPTP group (p < 0.01) (Fig. 2E and F). Taken together, Apelin-13 is neuroprotective against MPTP-induced dopaminergic neurodegeneration. 2.3. Apelin-13 reversed MPTP-induced α-synuclein expression in the SNpc of mice α-synuclein has been identified as the key pathological hallmark of PD, which is increased significantly in MPTP-induced PD model mice (Wang et al., 2015). In the present work, we investigated the effect of intranigral Apelin-13 injection on MPTP-induced α-synuclein expression in the SNpc of mice by western blot (Fig. 3A and B) and double immunostaining fluorescence for TH (green) and α-synuclein (red) (Fig. 3C–E). In MPTP-treated mice, α-synuclein expression in the SNpc of mice was significantly increased compared with control group (p < 0.01) (Fig. 3A and B). The intranigral Apelin-13 injection significantly reversed α-synuclein expression compared with MPTP group (p < 0.01) (Fig. 3A and B). α-synuclein expression in the SNpc of MPTP-treated mice was significantly increased compared with control group (p < 0.001), TH expression in the SNpc of MPTP-treated mice was significantly decreased compared with control group (p < 0.001) (Fig. 3C–E). The intranigral Apelin-13 injection significantly reversed α-synuclein (p < 0.01) and TH (p < 0.05) expression compared with MPTP group (Fig. 3C–E). So Apelin-13 could decrease MPTP-induced αsynuclein expression which is involved in the neuroprotection of Apelin-13.
2. Results 2.1. Apelin-13 alleviated the motor behavioral deficits of MPTP-induced PD model mice To investigate the intranigral Apelin-13 on MPTP-induced the motor behavioral deficits, the time on the rod was measured by rota-rod test. As shown in Fig. 1, the time of latency to fall on the rod was significantly decreased in MPTP group compared with control group (p < 0.001). However, the intranigral injection of Apelin-13 significantly increased the time of latency to fall on the rod in Apelin13 + MPTP group compared with MPTP group (p < 0.001). The data show that Apelin-13 alleviates the motor behavioral deficits of MPTPinduced PD model mice. 2.2. Apelin-13 inhibited MPTP-induced dopaminergic neurodegeration in SNpc of mice To investigate the effects of Apelin-13 on MPTP-induced dopaminergic neurodegeration in SNpc of mice, the dopaminergic neurodegeneration was measured by TH immunohistochemistry and immunofluorescence. As shown in Fig. 2A and B by TH immunohistochemistry, the number of TH-positive neurons in MPTP group was significantly
2.4. Apelin-13 enhanced the autophagic biomarkers of the SNpc in MPTPinduced PD model mice The autophagy plays an important role in the clearance of α-synuclein (Su et al., 2015). In MPTP-induced PD model mice, the expression of autophagy-related proteins, LC3BII/I and Beclin1, were significantly decreased compared with control group (p < 0.01). Conversely, p62 expression of the SNpc was significantly increased due MPTP treatment compared with control group (p < 0.01). The intranigral Apelin-13 injection significantly enhanced the autophagy such as the upregulation of LC3BII/I and Beclin1 and downregulation of p62 compared with MPTP group (p < 0.01) (Fig. 4). Therefore, the enhancement of auphagy is mediated in the neuroprotection of Apelin-13 against MPTP neurotoxicity. 2.5. Apelin-13 alleviated the activation of IRE1α/XBP1/CHOP signaling pathway of the SNpc in MPTP-induced PD model mice The activation of IRE1α/XBP-1/CHOP signaling pathway is found in PD models. In the present work, MPTP induced the activation of IRE1α/ XBP-1/CHOP signaling pathway. In MPTP-treated mice, the phosphorylated-IRE1α (p-IRE1α) was significantly increased, which led to the induction of Xbp1s mRNA and XBP1 protein compared with control group (p < 0.01) (Fig. 5A–E). Then the expression of CHOP and GRP78 was also increased significantly compared with control group (p < 0.01) (Fig. 5F–I). However, the intranigral Apelin-13 injection decreased the levels of p-IRE1α, Xbp1s mRNA and XBP1 protein, CHOP and GRP78 compared MPTP group (p < 0.01) (Fig. 5A–I). The data show that the inhibition of IRE1α/XBP-1/CHOP signaling pathway participates in the neuroprotection of Apelin-13 against MPTP neurotoxicity.
Fig. 1. The effect of Apelin-13 on motor behavioral deficits in MPTP-induced PD model mice assessed by rota-rod test. The time of latency to fall was recorded after the first intranigral Apelin-13 injection for 13 days. The intranigral Apelin-13 injection increased the time on the rod in MPTP-induced PD model mice. Data were represented as mean ± SEM (n = 10), ***p < 0.001 vs control group, ###p < 0.001 vs MPTP group. 204
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Fig. 2. The effect of Apelin-13 on MPTP-induced dopaminergic neurodegeneration in SNpc of mice. A and B: TH Immunohistochemistry for TH (Scale bar, 500 μm). C and D: Immunofluorescence for TH (Scale bar, 250 μm). E and F: Western blot for TH. The intranigral Apelin-13 injection significantly inhibited MPTP-induced the neurodegeneration of dopaminergic neurons in the SNpc. Data were represented as mean ± SEM (n = 4), ***p < 0.001 vs control group, ##p < 0.01, ### p < 0.001 vs MPTP group. 205
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Fig. 3. The effect of Apelin-13 on MPTP-induced α-synuclein expression in the SNpc of mice. A and B: Western blot for α-synuclein. C–E: Double immunostaining fluorescence of TH (green) and α-synuclein (red) in SNpc, DAPI (blue) stained nucleus (Scale bar, 50 μm). The intranigral Apelin-13 injection significantly decreased α-synuclein expression and increased TH expression in the SNpc of MPTP-induced PD model mice. Data were represented as mean ± SEM (n = 4), **p < 0.01, *** p < 0.001 vs control group, #p < 0.05, ##p < 0.01 vs MPTP group. 206
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Fig. 4. The effect of Apelin-13 on the autophagic biomarkers of the SNpc in MPTP-induced PD model mice. A-D: Western blottings and quantitative analyses for Beclin1 (A and B), p62 (A and C) and LC3BII/I (A and D) in brain homogenate. The intranigral Apelin-13 injection significantly inhibited the autophagy indicated by LC3B-II/I, Beclin1 and p62 expression. Data were represented as mean ± SEM (n = 4), **p < 0.01, ***p < 0.001 vs control group, #p < 0.05, ###p < 0.001 vs MPTP group.
3. Discussion
inhibition of autophagy through the regulation of LC3BII/I, Beclin1 and p62, which contributed to the alleviation of α-synuclein aggregation. ERS induced by abnormal accumulation of α-synuclein is an important mechanism of degeneration of dopaminergic neurons in PD (Song et al., 2017). Studies have shown that ERS is involved in the development of PD which results in the activation of IRE1α/XBP1/ CHOP signaling pathway (Janyou et al., 2015). The expression of pIRE1α, XBP1s, GRP78 and CHOP were increased in PD models (Avagliano et al., 2016; Silva et al., 2005). The inhibition of IRE1α/ XBP1/CHOP signaling pathway is involved in the neuroprotection of drugs (Chalmers et al., 2017; Tong et al., 2016). The present work also showed that the intranigral Apelin-13 injection significantly inhibited MPTP-induced the activation of IRE1α/XBP1/CHOP signaling pathway. Taken together, we propose that Apelin-13 is neuroprotective for PD model through the regulation of α-synuclein, autophagy and IRE1α/ XBP1/CHOP signaling pathway (Fig. 6), which contributes to the therapy for PD for the future. The mechanism of DA neurons degeneration is complicated. It has been indicated that oxidative stress, ERS, autophagy, and disruption of calcium homeostasis may be involved in the process of PD (Cali et al., 2014; Fujino et al., 2007; Nixon, 2013; Ryu et al., 2002). However, we only investigated the neuroprotective effect of Apelin-13 on MPTP-induced ERS and autophagy dysfunction. Therefore, other mechanisms for the neuroprotection of apelin-13 in PD need to be further investigated in our future experiments.
It has been shown that the autophagy and ERS play important roles in the dopaminergic neurodegeneration of PD (Anglade et al., 1997; Chun et al., 2001). And Apelin-13 is neuroprotective in the neurological diseases (Pouresmaeili-Babaki et al., 2018; Xu et al., 2018). In the present work, the intranigral Apelin-13 injection alleviated the neurodegeneration of dopaminergic neurons and behavioral dysfunction induced by MPTP. The autophagic inhibition and IRE1α/XBP-1/CHOP signaling pathway activation of SNpc induced by MPTP were significantly reversed by Apelin-13. Autophagy is widespread in eukaryotes which refers to a degradation process of damaged organelles and abnormal aggregation of protein in cells through lysosomes, thus maintaining the body in steady state (Escobar et al., 2019). A series of studies have shown that the autophagy participates in the pathogenesis of PD which is involved in α-synuclein clearance (Wani et al., 2017). In PD models in vitro and in vivo, the impaired autophagy pathways contribute to the pathogenesis of PD and α-synuclein aggregation (Choi et al., 2018; Jiang et al., 2018). It has been reported that the enhancement of autophagy provides the neuroprotective effects of dopaminergic neurons as well as the alleviation of α-synuclein aggregation (Zhang et al., 2018). In MPTP PD model the autophagy is inhibited which induces the increase of α-synuclein expression (Jang et al., 2018). In our work, we found that the intranigral Apelin-13 injection significantly reversed MPTP-induced the
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Fig. 5. The effect of Apelin-13 on the activation of IRE1α/XBP-1/CHOP signaling pathway of the SNpc in MPTP-induced PD model mice. A and B: Western blottings and quantitative analyses for p-IRE1α in brain homogenate; C and E: Western blottings and quantitative analyses for XBP-1 in brain homogenate; D: qRT-PCR for Xbp1s; F and G: Western blottings and quantitative analyses for CHOP in brain homogenate; H and I: Western blottings and quantitative analyses for GRP78 in brain homogenate. The intranigral Apelin-13 injection significantly inhibited the activation of IRE1α/XBP-1/CHOP signaling pathway of the SNpc in MPTP-induced PD model mice indicated by p-IRE1α, XBP-1, CHOP and GRP78 expression. Data were represented as mean ± SEM (n = 4), *p < 0.05, **p < 0.01, ***p < 0.001 vs control group, #p < 0.05, ##p < 0.01 vs MPTP group.
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Fig. 5. (continued)
Fig. 6. Proposed mechanism of Apelin-13 against MPTP neurotoxicity in PD model mice.
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4. Methods and materials
then blocked in goat serum. Next, the sections were incubated with primary rabbit anti-TH antibodies (1:500, NOVUS) overnight at 4℃, followed by the incubation in the secondary goat anti-rabbit IgG for 60 min and an avidin–biotin horseradish peroxidase (HRP) complex
4.1. Outline
(Zhongshan Golden Bridge Inc., Beijing, China) for 20 min at room temperature. Then the sections were developed in diaminobenzidine (DAB) for 6 min. Finally, the sections were dehydrated by alcohol gradient and coversliped. The number of dopaminergic neurons of the SNpc was evaluated under a bright-field inverted microscope (Olympus IX 71, Tokyo, Japan). TH-positive neurons in five continuous sections of SNpc were counted for 3 times and the mean value was presented for the number of TH-positive neurons.
4.2. Animals and rota-rod training Male C57BL/6 mice aged 10–12 weeks (weighting 23–26 g) were purchased from Pengyue experimental animal Ltd. (Jinan, Shangdong, PR China) and housed at a 12/12-h light–dark cycle with free access to water and food ad libitum for 1 week. All experimental protocols and treatments were approved by the Ethics Committee of Jining Medical University. Then the mice were trained on rota-rod apparatus (Anhui Zhenghua Biological Apparatus Company, Anhui, China) with 15 rpm for 20 min/day. After pretraining, all the mice were randomly divided into four groups (n = 10 per group): (1) control group (intranigral and intraperitoneal saline injection), (2) Apelin-13 group (intranigral Apelin-13 and intraperitoneal saline injection), (3) MPTP group (intranigral saline and intraperitoneal MPTP injection), (4) Apelin13 + MPTP group (intranigral Apelin-13 and intraperitoneal MPTP injection).
4.6. Immunofluorescence The similar sections of the SNpc in different groups were incubated with primary mouse anti-TH antibodies (1:500, Bio-tech) and primary rabbit anti-α-synuclein antibodies (1:250, NOVUS) overnight at 4℃ as described above. Then the sections were incubated with Cy3 Goat antirabbit (Boster Biological Technology, Wuhan, China) and Alexa Fluor 488-conjugated Goat anti-mouse (Invotrogen) at room temperature for 1 h. The immunofluorescence was visualized by SP8 microscope (Leica Microsystems, Germany) and the intensity of immunofluorescence was measured by with ImageJ software.
4.3. Drug treatments Apelin-13 dissolved in 2 μl saline (0.3 μg/mice/day) or the same volume of saline was gradually injected into SNpc (stereotactic coordinates: AP: −3.1 mm; ML: 1.3 mm; DV: −4.25 mm) at a rate of 1 μl/ min via guide cannula (RWD, Shenzhen, China) using a 10 μl microsyringe (Shanghai Anting Microsyringe Factory, shanghai, China) for 12 days in C57BL/6 mice. After the intranigral Apelin-13 or saline injection, C57BL/6 mice were administered with MPTP (25 mg/kg/day) (MedChemExpress, Shanghai, China) or saline intraperitoneally for 5 days.
4.7. Western blot The SNpc of mice were dissected and homogenized in RIPA lysis buffer supplemented with PMSF, and protein concentrations were quantified by BCA method (Beyotime, Shanghai, China). Equal amounts of protein (25 μg) from each sample were separated by 10% SDS-PAGE, and then transferred onto PVDF membranes. After the membranes were blocked in 5% non-fat dry milk in TBST for 1 h at room temperature, they were incubated in primary antibodies overnight at 4℃ against GRP78 (1:1000, Cell Signaling Technology), CHOP (1:1000, Wanlei), tyrosine hydroxylase (TH, 1:1000, NOVUS), α-synuclin (1:1000, NOVUS), p-IRE1α (1:1000, ThermoFisher), XBP1 (1:500, ThermoFisher) and β-actin (1:1000, Zhongshan Golden Bridge Biotechnology). Next, the membranes were incubated with secondary antibody at room temperature for 1 h, followed by an enhanced chemiluminescence system (ECL, liankebio, Hangzhou, China). The optical density of bands was quantified by ImageJ program (Media Cybernetics Inc., Bethesda, MD, USA).
4.4. Rota-rod test The motor behavioral deficits were measured by rota-rod. After 13 days of the first intranigral Apelin-13 or saline injection, the motor behavioral deficits were measured and the time spent on rod was recorded for 3 times with 1-hour interval. Then the mean value was presented for the time spent on rod. 4.5. Immunohistochemistry for dopaminergic neurons
4.8. Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR)
After the drugs treatment, all the mice were deeply anesthetized and transcardially perfused with 4% paraformaldehyde (PFA) dissolved in 0.1 M phosphate buffer (PFA) from the left ventricle. The brain was carefully removed from the skull, post-fixed in 4% PFA overnight at 4 °C and then immersed in 30% sucrose solution for storage at 4 °C until they sank. Subsequently, the samples were then cut to 30-µm thick by using Manual Microtome (Thermo scientific, Walldorf, Germany). The brain sections were firstly incubated in endogenous peroxidase inhibitor and
Total RNA was isolated from SNpcof brain using TRIzol reagent (Invitrogen, USA) according to the manufacturer’s guidelines. The concentration of RNA was determined by a Nanodrop spectrophotometer. The purity of total RNA was measured by the absorbance ratios of OD260/280 and OD260/230. cDNA was synthesized from 1 μg RNA using SuperScript® II Reverse Transcriptase (TIANGEN BIOTECH, 210
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Beijing, China) according to the manufacturer’s instructions. The cDNA was diluted 4-fold for subsequent qRT-PCR analysis. Then the total cDNA for qRT-PCR was amplified and analyzed by SYBR Green PCR Master Mix (TIANGEN BIOTECH, Beijing, China) using the Roche Light Cycler 480 (LC480). The following primers synthesized by Shanghai Biotechnology Co., Ltd. of China were following: X-box-binding protein 1 isoform Xbp1(s):
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5′-TTGCCTCTTCAGATTCTGAGTC-3′ (sense) 5′-GGGGAAGGACATTTGAAAAACA-3′(anti-sense), β-actin (internal control): 5′-CTACCTCATGAAGATCCTGACC-3′ (sense) 5′-CACAGCTTCTCTTTGATGTCAC-3′(anti-sense). The conditions of amplification were as following: activation 95 °C for 10 min, 40 cycles of denaturation at 95℃for 15 s and annealing at 60℃ for 1 min. 4.9. Statistical analysis The data were represented mean ± SEM. One-way ANOVA followed by Tukey test was performed by using GraphPad Prism. P < 0.05 was considered as statistically significant. Conflict of interest The authors declare no competing financial interests. Acknowledgements The present work was supported by National Nature Science Foundation of China (Nos. 81671276, and Nos. 81501018), Shandong Province Natural Science Foundation (Nos. ZR2014HL040, Nos. ZR2010HL057 and Nos. ZR2018Pc011), supporting Found for Teachers' Research of Jining Medical University (No. JY2017KJ035), and Undergraduate Innovation Project of Jining Medical University. References Anglade, P., Vyas, S., Javoy-Agid, F., Herrero, M.T., Michel, P.P., Marquez, J., MouattPrigent, A., Ruberg, M., Hirsch, E.C., Agid, Y., 1997. Apoptosis and autophagy in nigral neurons of patients with Parkinson's disease. Histol. Histopathol. 12, 25–31. Avagliano, C., Russo, R., De Caro, C., Cristiano, C., La Rana, G., Piegari, G., Paciello, O., Citraro, R., Russo, E., De Sarro, G., Meli, R., Mattace Raso, G., Calignano, A., 2016. Palmitoylethanolamide protects mice against 6-OHDA-induced neurotoxicity and endoplasmic reticulum stress: in vivo and in vitro evidence. Pharmacol. Res. 113, 276–289. Boal, F., Timotin, A., Roumegoux, J., Alfarano, C., Calise, D., Anesia, R., Parini, A., Valet, P., Tronchere, H., Kunduzova, O., 2016. Apelin-13 administration protects against ischaemia/reperfusion-mediated apoptosis through the FoxO1 pathway in high-fat diet-induced obesity. Br. J. Pharmacol. 173, 1850–1863. Cali, T., Ottolini, D., Brini, M., 2014. Calcium signaling in Parkinson's disease. Cell Tissue Res. 357, 439–454. Chalmers, F., van Lith, M., Sweeney, B., Cain, K., Bulleid, N.J., 2017. Inhibition of IRE1alpha-mediated XBP1 mRNA cleavage by XBP1 reveals a novel regulatory process during the unfolded protein response. Welcome Open Res. 2, 36. Chen, D., Lee, J., Gu, X., Wei, L., Yu, S.P., 2015. Intranasal delivery of apelin-13 is neuroprotective and promotes angiogenesis after ischemic stroke in mice. ASN Neuro. 7. Chen, Y., Zhang, N., Ji, D., Hou, Y., Chen, C., Fu, Y., Ge, R., Zheng, Q., Chen, J., Wang, H., 2018. Dysregulation of bcl-2 enhanced rotenone-induced alpha-synuclein aggregation associated with autophagic pathways. NeuroReport 29, 1201–1208. Choi, D.C., Yoo, M., Kabaria, S., Junn, E., 2018. MicroRNA-7 facilitates the degradation of alpha-synuclein and its aggregates by promoting autophagy. Neurosci. Lett. 678, 118–123. Chun, H.S., Lee, H., Son, J.H., 2001. Manganese induces endoplasmic reticulum (ER) stress and activates multiple caspases in nigral dopaminergic neuronal cells, SN4741. Neurosci. Lett. 316, 5–8. Escobar, K.A., Cole, N.H., Mermier, C.M., VanDusseldorp, T.A., 2019. Autophagy and aging: maintaining the proteome through exercise and caloric restriction. Aging Cell, e12876.
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Research report
Apelin-13 protects dopaminergic neurons in MPTP-induced Parkinson’s disease model mice through inhibiting endoplasmic reticulum stress and promoting autophagy Junge Zhua, Shanshan Doub, Yunlu Jiangb, Jing Chenb, Chunmei Wangb, , Baohua Chengb, ⁎
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Cheeloo College of Medicine, Shandong University, 250014 Jinan, China Neurobiology Institute, Jining Medical University, 272067 Jining, China
ARTICLE INFO
ABSTRACT
Keywords: Parkinson’s disease α-Synuclein 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridin Apelin-13 Endoplasmic reticulum stress Autophagy
The dopaminergic neurodegeneration in the substantia nigrapars compacta (SNpc) and striatum of the midbrain is the important pathological feature of Parkinson's disease (PD). It has been shown that autophagy and endoplasmic reticulum stress (ERS) are involved in the occurrence and development of PD. The neuropeptide Apelin-13 is neuroprotective in the neurological diseases such as PD, Alzheimer's disease and cerebral ischemic stroke. In the present work, we investigated the neuroprotective effects of Apelin-13 on ERS and autophagy in the dopaminergic neurodegeneration of SNpc of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridin (MPTP)-treated mice. The intranigral injection of Apelin-13 alleviated the behavioral dysfunction and dopaminergic neurodegeneration induced by MPTP. After the exposure to MPTP, the expression of tyrosine hydroxylase (TH) was significantly decreased as well as the increased α-synuclein expression, which was significantly reversed by the intranigral injection of Apelin-13. Also, Apelin-13 significantly reversed the decreasing autophagy induced by MPTP which was indicated by the up-regulation of LC3B-II and Beclin1 and down-regulation of p62. And MPTPinduced ERS such as IRE1α, XBP1s, CHOP and GRP78 was significantly inhibited by Apelin-13. Taken together, Apelin-13 protects dopaminergic neurons in MPTP-induced PD model mice in vivo through inhibiting ERS and promoting autophagy, which contributes to the therapy for PD in the future.
1. Introduction Parkinson's disease (PD) is a common neurodegenerative disease which is characterized by high morbidity rate, high disability rate and chronic course (Steib et al., 2018). The dopaminergic neurodegeneration in the substantia nigrapars compacta (SNpc) and striatum of the midbrain accompanied by abnormal aggregation of a large number of α-synuclein is the important pathological feature of PD. Although the mechanisms are not fully understood, the autophagy and endoplasmic reticulum stress (ERS) are closely associated with PD. Autophagy is the degradation process of long-lived proteins and damaged organelles in cells by lysosomes which is an unique catabolism pathway of eukaryotic cells that can promote energy metabolism and cell survival (Parzych and Klionsky, 2014). It plays a role in the extension and maturation of autophagic vesicles by initiating mammalian target of rapamycin (mTOR) and Beclin1 and ultimately degrading the inclusions in lysosomes (Hwang et al., 2017; Qian et al., 2017). The microtubule-associated protein1 light chain 3B-II (LC3B-II) produced at
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its extension stage is an autophagic biomarker (Meyer et al., 2013). In addition, p62/SQSTM1 is involved in the formation of autophagy as a regulatory factor by coupling to LC3 which is degraded in the late stage of autophagy (Liu et al., 2016). Therefore, the expression of p62 is negatively correlated with autophagy activity (Lee et al., 2011). A series of studies indicate that the autophagy plays an important role in neurodegenerative disease including PD (Kim et al., 2017). In PD model in vivo, MPTP decreases LC3B-II and Beclin1 and increases p62 in SNpc which results in the inhibition of autophagy and induction of α-synuclein aggregation (Guo et al., 2016). It has been reported that the abnormal aggregation of α-synuclein, which is closely associated with the inhibition of autophagy, leads to ERS (Chen et al., 2018; Melo et al., 2018). And then IRE1α/XBP-1/CHOP is activated by the dissociation of glucose regulated protein 78 (GRP78) from IRE1α in PD model in vivo and in vitro (Jiao et al., 2017; Ning et al., 2016). Apelin was extracted and purified from bovine secretion for the first time by Tatemoto et al. in 1998, which was proved to be an endogenous ligand of APJ (Tatemoto et al., 1998). It can be hydrolyzed into several
Corresponding authors. E-mail addresses: [email protected] (C. Wang), [email protected] (B. Cheng).
https://doi.org/10.1016/j.brainres.2019.03.027 Received 24 December 2018; Received in revised form 11 March 2019; Accepted 22 March 2019 Available online 23 March 2019 0006-8993/ © 2019 Elsevier B.V. All rights reserved.
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peptide segments of different lengths such as Apelin-12, Apelin-13, Apelin-17, Apelin-36 and so on, of which Apelin-13 is known to be the most bioactive Apelin receptor activator (Boal et al., 2016). Many experiments have shown that Apelin-13 is neuroprotective against the neuronal damage (Chen et al., 2015). It has been shown that apelin-13 exerts the effects of ameliorating cognitive impairments in 6-OHDAtreated rats (Haghparast et al., 2018). Moreover, Apelin-13 has been proved to inhibit oxidative stress and apoptosis in 6-OHDA-treated SHSY5Y cells (Pouresmaeili-Babaki et al., 2018). And MPP+-induced cytotoxicity was reversed by Apelin-13 in SH-SY5Y cells (Jiang et al., 2018). In the present work, we investigated the effects of intranigral injection of Apelin-13 on MPTP-induced PD model mice including the behavioral dysfunction, dopaminergic neurodegeneration and α-synuclein expression. The role of autophagy and ERS was also investigated.
decreased compared with control group (p < 0.001). The intranigral Apelin-13 injection significantly the loss of dopaminergic neurons in the SNpc compared with MPTP group (p < 0.01). The similar results was found by TH immunofluorescence (Fig. 2C and D). Also, the expression of TH of the SNpc was significantly increased in MPTP group compared with control group (p < 0.001), while the intranigral Apelin-13 injection significantly reversed TH expression compared MPTP group (p < 0.01) (Fig. 2E and F). Taken together, Apelin-13 is neuroprotective against MPTP-induced dopaminergic neurodegeneration. 2.3. Apelin-13 reversed MPTP-induced α-synuclein expression in the SNpc of mice α-synuclein has been identified as the key pathological hallmark of PD, which is increased significantly in MPTP-induced PD model mice (Wang et al., 2015). In the present work, we investigated the effect of intranigral Apelin-13 injection on MPTP-induced α-synuclein expression in the SNpc of mice by western blot (Fig. 3A and B) and double immunostaining fluorescence for TH (green) and α-synuclein (red) (Fig. 3C–E). In MPTP-treated mice, α-synuclein expression in the SNpc of mice was significantly increased compared with control group (p < 0.01) (Fig. 3A and B). The intranigral Apelin-13 injection significantly reversed α-synuclein expression compared with MPTP group (p < 0.01) (Fig. 3A and B). α-synuclein expression in the SNpc of MPTP-treated mice was significantly increased compared with control group (p < 0.001), TH expression in the SNpc of MPTP-treated mice was significantly decreased compared with control group (p < 0.001) (Fig. 3C–E). The intranigral Apelin-13 injection significantly reversed α-synuclein (p < 0.01) and TH (p < 0.05) expression compared with MPTP group (Fig. 3C–E). So Apelin-13 could decrease MPTP-induced αsynuclein expression which is involved in the neuroprotection of Apelin-13.
2. Results 2.1. Apelin-13 alleviated the motor behavioral deficits of MPTP-induced PD model mice To investigate the intranigral Apelin-13 on MPTP-induced the motor behavioral deficits, the time on the rod was measured by rota-rod test. As shown in Fig. 1, the time of latency to fall on the rod was significantly decreased in MPTP group compared with control group (p < 0.001). However, the intranigral injection of Apelin-13 significantly increased the time of latency to fall on the rod in Apelin13 + MPTP group compared with MPTP group (p < 0.001). The data show that Apelin-13 alleviates the motor behavioral deficits of MPTPinduced PD model mice. 2.2. Apelin-13 inhibited MPTP-induced dopaminergic neurodegeration in SNpc of mice To investigate the effects of Apelin-13 on MPTP-induced dopaminergic neurodegeration in SNpc of mice, the dopaminergic neurodegeneration was measured by TH immunohistochemistry and immunofluorescence. As shown in Fig. 2A and B by TH immunohistochemistry, the number of TH-positive neurons in MPTP group was significantly
2.4. Apelin-13 enhanced the autophagic biomarkers of the SNpc in MPTPinduced PD model mice The autophagy plays an important role in the clearance of α-synuclein (Su et al., 2015). In MPTP-induced PD model mice, the expression of autophagy-related proteins, LC3BII/I and Beclin1, were significantly decreased compared with control group (p < 0.01). Conversely, p62 expression of the SNpc was significantly increased due MPTP treatment compared with control group (p < 0.01). The intranigral Apelin-13 injection significantly enhanced the autophagy such as the upregulation of LC3BII/I and Beclin1 and downregulation of p62 compared with MPTP group (p < 0.01) (Fig. 4). Therefore, the enhancement of auphagy is mediated in the neuroprotection of Apelin-13 against MPTP neurotoxicity. 2.5. Apelin-13 alleviated the activation of IRE1α/XBP1/CHOP signaling pathway of the SNpc in MPTP-induced PD model mice The activation of IRE1α/XBP-1/CHOP signaling pathway is found in PD models. In the present work, MPTP induced the activation of IRE1α/ XBP-1/CHOP signaling pathway. In MPTP-treated mice, the phosphorylated-IRE1α (p-IRE1α) was significantly increased, which led to the induction of Xbp1s mRNA and XBP1 protein compared with control group (p < 0.01) (Fig. 5A–E). Then the expression of CHOP and GRP78 was also increased significantly compared with control group (p < 0.01) (Fig. 5F–I). However, the intranigral Apelin-13 injection decreased the levels of p-IRE1α, Xbp1s mRNA and XBP1 protein, CHOP and GRP78 compared MPTP group (p < 0.01) (Fig. 5A–I). The data show that the inhibition of IRE1α/XBP-1/CHOP signaling pathway participates in the neuroprotection of Apelin-13 against MPTP neurotoxicity.
Fig. 1. The effect of Apelin-13 on motor behavioral deficits in MPTP-induced PD model mice assessed by rota-rod test. The time of latency to fall was recorded after the first intranigral Apelin-13 injection for 13 days. The intranigral Apelin-13 injection increased the time on the rod in MPTP-induced PD model mice. Data were represented as mean ± SEM (n = 10), ***p < 0.001 vs control group, ###p < 0.001 vs MPTP group. 204
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Fig. 2. The effect of Apelin-13 on MPTP-induced dopaminergic neurodegeneration in SNpc of mice. A and B: TH Immunohistochemistry for TH (Scale bar, 500 μm). C and D: Immunofluorescence for TH (Scale bar, 250 μm). E and F: Western blot for TH. The intranigral Apelin-13 injection significantly inhibited MPTP-induced the neurodegeneration of dopaminergic neurons in the SNpc. Data were represented as mean ± SEM (n = 4), ***p < 0.001 vs control group, ##p < 0.01, ### p < 0.001 vs MPTP group. 205
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Fig. 3. The effect of Apelin-13 on MPTP-induced α-synuclein expression in the SNpc of mice. A and B: Western blot for α-synuclein. C–E: Double immunostaining fluorescence of TH (green) and α-synuclein (red) in SNpc, DAPI (blue) stained nucleus (Scale bar, 50 μm). The intranigral Apelin-13 injection significantly decreased α-synuclein expression and increased TH expression in the SNpc of MPTP-induced PD model mice. Data were represented as mean ± SEM (n = 4), **p < 0.01, *** p < 0.001 vs control group, #p < 0.05, ##p < 0.01 vs MPTP group. 206
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Fig. 4. The effect of Apelin-13 on the autophagic biomarkers of the SNpc in MPTP-induced PD model mice. A-D: Western blottings and quantitative analyses for Beclin1 (A and B), p62 (A and C) and LC3BII/I (A and D) in brain homogenate. The intranigral Apelin-13 injection significantly inhibited the autophagy indicated by LC3B-II/I, Beclin1 and p62 expression. Data were represented as mean ± SEM (n = 4), **p < 0.01, ***p < 0.001 vs control group, #p < 0.05, ###p < 0.001 vs MPTP group.
3. Discussion
inhibition of autophagy through the regulation of LC3BII/I, Beclin1 and p62, which contributed to the alleviation of α-synuclein aggregation. ERS induced by abnormal accumulation of α-synuclein is an important mechanism of degeneration of dopaminergic neurons in PD (Song et al., 2017). Studies have shown that ERS is involved in the development of PD which results in the activation of IRE1α/XBP1/ CHOP signaling pathway (Janyou et al., 2015). The expression of pIRE1α, XBP1s, GRP78 and CHOP were increased in PD models (Avagliano et al., 2016; Silva et al., 2005). The inhibition of IRE1α/ XBP1/CHOP signaling pathway is involved in the neuroprotection of drugs (Chalmers et al., 2017; Tong et al., 2016). The present work also showed that the intranigral Apelin-13 injection significantly inhibited MPTP-induced the activation of IRE1α/XBP1/CHOP signaling pathway. Taken together, we propose that Apelin-13 is neuroprotective for PD model through the regulation of α-synuclein, autophagy and IRE1α/ XBP1/CHOP signaling pathway (Fig. 6), which contributes to the therapy for PD for the future. The mechanism of DA neurons degeneration is complicated. It has been indicated that oxidative stress, ERS, autophagy, and disruption of calcium homeostasis may be involved in the process of PD (Cali et al., 2014; Fujino et al., 2007; Nixon, 2013; Ryu et al., 2002). However, we only investigated the neuroprotective effect of Apelin-13 on MPTP-induced ERS and autophagy dysfunction. Therefore, other mechanisms for the neuroprotection of apelin-13 in PD need to be further investigated in our future experiments.
It has been shown that the autophagy and ERS play important roles in the dopaminergic neurodegeneration of PD (Anglade et al., 1997; Chun et al., 2001). And Apelin-13 is neuroprotective in the neurological diseases (Pouresmaeili-Babaki et al., 2018; Xu et al., 2018). In the present work, the intranigral Apelin-13 injection alleviated the neurodegeneration of dopaminergic neurons and behavioral dysfunction induced by MPTP. The autophagic inhibition and IRE1α/XBP-1/CHOP signaling pathway activation of SNpc induced by MPTP were significantly reversed by Apelin-13. Autophagy is widespread in eukaryotes which refers to a degradation process of damaged organelles and abnormal aggregation of protein in cells through lysosomes, thus maintaining the body in steady state (Escobar et al., 2019). A series of studies have shown that the autophagy participates in the pathogenesis of PD which is involved in α-synuclein clearance (Wani et al., 2017). In PD models in vitro and in vivo, the impaired autophagy pathways contribute to the pathogenesis of PD and α-synuclein aggregation (Choi et al., 2018; Jiang et al., 2018). It has been reported that the enhancement of autophagy provides the neuroprotective effects of dopaminergic neurons as well as the alleviation of α-synuclein aggregation (Zhang et al., 2018). In MPTP PD model the autophagy is inhibited which induces the increase of α-synuclein expression (Jang et al., 2018). In our work, we found that the intranigral Apelin-13 injection significantly reversed MPTP-induced the
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Fig. 5. The effect of Apelin-13 on the activation of IRE1α/XBP-1/CHOP signaling pathway of the SNpc in MPTP-induced PD model mice. A and B: Western blottings and quantitative analyses for p-IRE1α in brain homogenate; C and E: Western blottings and quantitative analyses for XBP-1 in brain homogenate; D: qRT-PCR for Xbp1s; F and G: Western blottings and quantitative analyses for CHOP in brain homogenate; H and I: Western blottings and quantitative analyses for GRP78 in brain homogenate. The intranigral Apelin-13 injection significantly inhibited the activation of IRE1α/XBP-1/CHOP signaling pathway of the SNpc in MPTP-induced PD model mice indicated by p-IRE1α, XBP-1, CHOP and GRP78 expression. Data were represented as mean ± SEM (n = 4), *p < 0.05, **p < 0.01, ***p < 0.001 vs control group, #p < 0.05, ##p < 0.01 vs MPTP group.
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Fig. 6. Proposed mechanism of Apelin-13 against MPTP neurotoxicity in PD model mice.
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4. Methods and materials
then blocked in goat serum. Next, the sections were incubated with primary rabbit anti-TH antibodies (1:500, NOVUS) overnight at 4℃, followed by the incubation in the secondary goat anti-rabbit IgG for 60 min and an avidin–biotin horseradish peroxidase (HRP) complex
4.1. Outline
(Zhongshan Golden Bridge Inc., Beijing, China) for 20 min at room temperature. Then the sections were developed in diaminobenzidine (DAB) for 6 min. Finally, the sections were dehydrated by alcohol gradient and coversliped. The number of dopaminergic neurons of the SNpc was evaluated under a bright-field inverted microscope (Olympus IX 71, Tokyo, Japan). TH-positive neurons in five continuous sections of SNpc were counted for 3 times and the mean value was presented for the number of TH-positive neurons.
4.2. Animals and rota-rod training Male C57BL/6 mice aged 10–12 weeks (weighting 23–26 g) were purchased from Pengyue experimental animal Ltd. (Jinan, Shangdong, PR China) and housed at a 12/12-h light–dark cycle with free access to water and food ad libitum for 1 week. All experimental protocols and treatments were approved by the Ethics Committee of Jining Medical University. Then the mice were trained on rota-rod apparatus (Anhui Zhenghua Biological Apparatus Company, Anhui, China) with 15 rpm for 20 min/day. After pretraining, all the mice were randomly divided into four groups (n = 10 per group): (1) control group (intranigral and intraperitoneal saline injection), (2) Apelin-13 group (intranigral Apelin-13 and intraperitoneal saline injection), (3) MPTP group (intranigral saline and intraperitoneal MPTP injection), (4) Apelin13 + MPTP group (intranigral Apelin-13 and intraperitoneal MPTP injection).
4.6. Immunofluorescence The similar sections of the SNpc in different groups were incubated with primary mouse anti-TH antibodies (1:500, Bio-tech) and primary rabbit anti-α-synuclein antibodies (1:250, NOVUS) overnight at 4℃ as described above. Then the sections were incubated with Cy3 Goat antirabbit (Boster Biological Technology, Wuhan, China) and Alexa Fluor 488-conjugated Goat anti-mouse (Invotrogen) at room temperature for 1 h. The immunofluorescence was visualized by SP8 microscope (Leica Microsystems, Germany) and the intensity of immunofluorescence was measured by with ImageJ software.
4.3. Drug treatments Apelin-13 dissolved in 2 μl saline (0.3 μg/mice/day) or the same volume of saline was gradually injected into SNpc (stereotactic coordinates: AP: −3.1 mm; ML: 1.3 mm; DV: −4.25 mm) at a rate of 1 μl/ min via guide cannula (RWD, Shenzhen, China) using a 10 μl microsyringe (Shanghai Anting Microsyringe Factory, shanghai, China) for 12 days in C57BL/6 mice. After the intranigral Apelin-13 or saline injection, C57BL/6 mice were administered with MPTP (25 mg/kg/day) (MedChemExpress, Shanghai, China) or saline intraperitoneally for 5 days.
4.7. Western blot The SNpc of mice were dissected and homogenized in RIPA lysis buffer supplemented with PMSF, and protein concentrations were quantified by BCA method (Beyotime, Shanghai, China). Equal amounts of protein (25 μg) from each sample were separated by 10% SDS-PAGE, and then transferred onto PVDF membranes. After the membranes were blocked in 5% non-fat dry milk in TBST for 1 h at room temperature, they were incubated in primary antibodies overnight at 4℃ against GRP78 (1:1000, Cell Signaling Technology), CHOP (1:1000, Wanlei), tyrosine hydroxylase (TH, 1:1000, NOVUS), α-synuclin (1:1000, NOVUS), p-IRE1α (1:1000, ThermoFisher), XBP1 (1:500, ThermoFisher) and β-actin (1:1000, Zhongshan Golden Bridge Biotechnology). Next, the membranes were incubated with secondary antibody at room temperature for 1 h, followed by an enhanced chemiluminescence system (ECL, liankebio, Hangzhou, China). The optical density of bands was quantified by ImageJ program (Media Cybernetics Inc., Bethesda, MD, USA).
4.4. Rota-rod test The motor behavioral deficits were measured by rota-rod. After 13 days of the first intranigral Apelin-13 or saline injection, the motor behavioral deficits were measured and the time spent on rod was recorded for 3 times with 1-hour interval. Then the mean value was presented for the time spent on rod. 4.5. Immunohistochemistry for dopaminergic neurons
4.8. Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR)
After the drugs treatment, all the mice were deeply anesthetized and transcardially perfused with 4% paraformaldehyde (PFA) dissolved in 0.1 M phosphate buffer (PFA) from the left ventricle. The brain was carefully removed from the skull, post-fixed in 4% PFA overnight at 4 °C and then immersed in 30% sucrose solution for storage at 4 °C until they sank. Subsequently, the samples were then cut to 30-µm thick by using Manual Microtome (Thermo scientific, Walldorf, Germany). The brain sections were firstly incubated in endogenous peroxidase inhibitor and
Total RNA was isolated from SNpcof brain using TRIzol reagent (Invitrogen, USA) according to the manufacturer’s guidelines. The concentration of RNA was determined by a Nanodrop spectrophotometer. The purity of total RNA was measured by the absorbance ratios of OD260/280 and OD260/230. cDNA was synthesized from 1 μg RNA using SuperScript® II Reverse Transcriptase (TIANGEN BIOTECH, 210
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Beijing, China) according to the manufacturer’s instructions. The cDNA was diluted 4-fold for subsequent qRT-PCR analysis. Then the total cDNA for qRT-PCR was amplified and analyzed by SYBR Green PCR Master Mix (TIANGEN BIOTECH, Beijing, China) using the Roche Light Cycler 480 (LC480). The following primers synthesized by Shanghai Biotechnology Co., Ltd. of China were following: X-box-binding protein 1 isoform Xbp1(s):
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