- Open Access
Dopaminergic neurons show increased low-molecular-mass protein 7 activity induced by 6-hydroxydopamine in vitro and in vivo
- Ming-Shu Mo†1,
- Gui-Hua Li†1,
- Cong-Cong Sun†2,
- Shu-Xuan Huang1,
- Lei Wei1,
- Li-Min Zhang3,
- Miao-Miao Zhou1,
- Zhuo-Hua Wu1,
- Wen-Yuan Guo1,
- Xin-Ling Yang4,
- Chao-Jun Chen5,
- Shao-Gang Qu6,
- Jian-Xing He7Email author and
- Ping-Yi Xu1, 4Email author
© The Author(s). 2018
- Received: 23 January 2018
- Accepted: 30 July 2018
- Published: 17 August 2018
Abnormal expression of major histocompatibility complex class I (MHC-I) is increased in dopaminergic (DA) neurons in the substantia nigra (SN) in Parkinson’s disease (PD). Low-molecular-mass protein 7 (β5i) is a proteolytic subunit of the immunoproteasome that regulates protein degradation and the MHC pathway in immune cells.
In this study, we investigated the role of β5i in DA neurons using a 6-hydroxydopamine (6-OHDA) model in vitro and vivo.
We showed that 6-OHDA upregulated β5i expression in DA neurons in a concentration- and time-dependent manner. Inhibition and downregulation of β5i induced the expression of glucose-regulated protein (Bip) and exacerbated 6-OHDA neurotoxicity in DA neurons. The inhibition of β5i further promoted the activation of Caspase 3-related pathways induced by 6-OHDA. β5i also activated transporter associated with antigen processing 1 (TAP1) and promoted MHC-I expression on DA neurons.
Taken together, our data suggest that β5i is activated in DA neurons under 6-OHDA treatment and may play a neuroprotective role in PD.
- Parkinson’s disease
Oxidative stress, accumulation of aggregated and misfolded protein aggregates, and neuroinflammation have been suggested to play roles in the pathogenesis of Parkinson’s disease (PD) [1, 2] These factors impair the ubiquitin-proteasome system (UPS) which is critical for protein metabolic homeostasis [3–5], and they promote the replacement of constitutive proteasome subunits β1, β2 and β5 by the respective immunoproteasome catalytic subunits β1i/ low-molecular-mass protein 2 (LMP2, PSMB9), β2i/multicatalytic endo- peptidase complex-like 1 (MECL1, PSMB10) and β5i (LMP7, PSMB8) [6–8]. The immunoproteasome helps to degrade abnormal proteins, present cleaved peptides as antigens to major histocompatibility complex (MHC) molecules and regulate neuroinflammation [9, 10].
Immunoproteasome expression is low in normal young human brains but higher in brain specimens from older normal subjects and Alzheimer’s disease (AD) patients [11, 12]. LMP2 knockout mice show classic AD-like symptoms and severe oxidative stress involved in Aβ aggregation [12, 13]. In Huntington’s disease (HD), immunoproteasomes may contribute to the metabolism of huntingtin protein, which is not easily degraded by classical proteasomes . β5i also plays an important role in the regulation of oxidative stress in chronic epilepsy and stroke [15, 16]. β5i expression and changes in proteasomal structure have been found in tyrosine hydroxylase (TH+) cells in postmortem brains of people with PD-like synucleinopathies such as multiple system atrophy (MSA) and progressive supranuclear palsy (PSP) . β5i is known to shape the antigenic repertoire presented on MHC-I. A recent study demonstrated that catecholamine neurons were more responsive to MHC-I expression under γ-interferon (IFN) treatment and that these neurons were more susceptible to neurotoxicity in neuroinflammatory conditions than in control conditions [18, 19]. However, whether β5i contributes to DA neuronal neurotoxicity remains unclear. In this study, we further explored the role of β5i in the loss of dopaminergic (DA) neurons under 6-hydroxydopamine (6-OHDA) insult in vitro and vivo.
SN4741 cells derived from embryonic substantia nigra and maintained in Dulbecco’s-modified Eagle’s high-glucose medium (DMEM, Life Technologies, Rockville, MD, USA) supplemented with 10% fetal calf serum (FCS, Irvine Scientific, Santa Ana, CA, USA), 1% glucose (Sigma, St. Louis, MO, USA), 1% penicillin–streptomycin (Gibco™, Invitrogen, China) and 2 mmol/L l-glutamine (Gibco™, Invitrogen, China). SN4741 cells were kindly provided by Prof. Qian-Yang of the Fourth Military Medical University . Cells were grown at 37 °C in 5% CO2 and subcultured every 3 days as described previously .
Cell viability, reactive oxygen species (ROS) and chymotrypsin-like function
CCK-8 (Dojindo, Kumamoto, Japan) was used to analyze cell viability under different treatment conditions according to the manufacturer’s recommendations. Cells were trypsinized, suspended and cultured in 96-well plates at a concentration of 5× 103 cells/well. Each sample was made in triplicate. The plate contained blank, positive and negative control wells. PR-957 (Selleck, Houston, CA, USA) was used as a selective inhibitor of β5i in SN4741 cells . Cells were treated with 6-OHDA or PR-957 overnight or for 48 h, respectively, or with control solution. At different time points, 10 μl cell counting kit-8 (CCK-8) (Dojindo, Kumamoto, Japan) solution dissolved in 100 μl DMEM (Life Technologies, Rockville, MD, USA) replaced the drug in each well. The incubation continued for another 0.5, 1, or 2 h at 37 °C following the manufacturer’s instructions. The optical density (OD) value at 450 nm was measured to calculate cell viability using the formula: cell viability (%) = [OD (Sample)-OD (blank control)]/ [OD (negative control)-OD (blank control)] by an ELISA microplate reader (ELX800, BioTeK, USA).
Rhodamine 123 (Sigma-Aldrich, St Louis, MO, USA) was used to measure the mitochondrial membrane potential disruption. Cells were suspended and cultured in 6-well plates. After overnight incubation, groups were exposed to 6-OHDA, PR-957 or control solution. Cells were washed 3 times with PBS and reincubated with 100 μl DMEM (Life Technologies, Rockville, MD, USA) containing 10 μg/mL rhodamine 123 at 37 °C for 30 mins. The fluorescence of rhodamine 123 was detected by a fluorescence spectrophotometer (Shimadzu, Matsuyama, Japan, RF5000U) at 490 nm excitation (Ex) and 520 nm emission (Em).
2′, 7′-Dichlorofluorescin diacetate (DCFH-DA; Sigma-Aldrich, St Louis, MO, USA) was used to measure ROS level following manufacturer’s recommendations. Cells were treated with 6-OHDA or PR-957 at different concentrations and exposure durations. After the cells were washed 3 times with PBS, DCFH-DA diluted in DMEM to 10 μM was added and incubated at 37 °C for 20 min. Cells were washed 3 times with DMEM, and the resultant optical density was measured at 488 nm excitation and 525 nm emission by a microplate reader (Spectramax Gemini XS, Molecular Devices, Pennsylvania, USA). The amount of generated ROS was calculated using the formula: [OD (Sample)-OD (Negative control)]/ OD (Negative control).
The chymotrypsin-like activity (CTL) of the immunoproteasome was assayed with Suc-LLVY-AMC . Cells were seeded at a concentration of 1× 104 cells/well in 96-well plates. Each test was performed in 4 replicates. After treatment with different concentrations of 6-OHDA, cells were harvested and lysed in proteolysis buffer (50 mM Tris-HCl pH 7.4, 5 mM MgCl2, 1 mM DTT ± 0.25 mM ATP). Then, 100 μl containing 2 μg cell lysate was mixed with 50 μM Suc-LLVY-AMC (Sigma-Aldrich, St Louis, MO, USA). After 1 h of equilibration, fluorescence was monitored for 3 h using a SpectraMax M5 plate reader (Molecular Devices, Pennsylvania, USA, Ex/Em: 370 nm/460 nm).
Overexpression plasmid and shRNA transfection
The β5i overexpression plasmid was synthesized by GeneCopoeia (Product ID: EX-Mm34282-M29, GeneCopoeia, Guangzhou, China). This sequence was inserted into a p-EZ-M29 vector containing neomycin as a stable selection marker. The insertion was confirmed by sequencing. The mU6 vector contained the mCherryFP gene as a marker to identify transfection efficiency (Product ID: CSHCTR001, GeneCopoeia, Guangzhou, China). PSMB8 was suppressed by specific shRNA in the mU6 vector (Product ID: RSH052242-mU6, GeneCopoeia, Guangzhou, China) with target sequences GGAATGCAGCCCACTGAATTC, GGAAGGTTCAGATTGAAATGG, GCAGGAAGTTACATTGCTACC and GCCAAGGAATGCAGGCTATAC and the hairpin loop sequence TCAAGAG. The mU6-pri vector (Product ID: CSHCTR001-mU6, GeneCopoeia, Guangzhou, China) without the target gene and an empty plasmid were used in the negative control (NC) and mock (M) groups, respectively. First, we detected β5i mRNA by qQT-PCR and then confirmed β5i protein expression by Western blot.
Transfection was performed based on manufacturer’s instructions (Invitrogen, Grand Island, NY, USA). Cells were suspended and seeded in 24-well plates at a 50% cell density after counting. After 24 h of culture, transfection was performed as follows. Solution A contained 20 pmol shRNA dissolved in 50 μl Opti-MEM without serum, and B solution contained 1 μl lipofectamine 3000 (Invitrogen, Grand Island, NY, USA) dissolved in 50 μl Opti-MEM without serum. Solution A and B were mixed and kept at room temperature for 20 min. The culture medium for each well was replaced with 400 μl serum-free medium. Cells were incubated in this mixture (serum-free medium containing solutions A and B) for 6 h for transfection, which was then replaced with serum medium. Transfection efficiency was assessed by fluorescence on the following day.
Partial 6-OHDA lesion and behavioral test
Forty male Sprague Dawley (SD) rats, ranging from 280 to 300 g in weight, were bred and maintained in the Specific Pathogen-Free Laboratory Animal Center at Guangzhou Medical University (Guangzhou, China). Weight-matched rats were randomly assigned to four groups: the sham group, 6-OHDA (Sigma-Aldrich, St Louis, MO, USA) group, PR-957 (Adooq Bioscience, CA, USA) group and 6-OHDA plus PR-957 group. Rats were anesthetized with ketamine (10%) /xylazine (2%) (Sigma Aldrich, St Louis, MO, USA) and injected with 8 μg 6-OHDA in 4 μl solvent [0.9% w/v NaCl with 0.1% ascorbic acid (Sigma-Aldrich, St Louis, MO, USA)] into the left anterior medial bundle (Coordinates: AP: - 4.0 mm, ML: - 1.5 mm, DV: - 7.8 mm). Animals in the 6-OHDA plus PR-957 group were given the same dose of 6-OHDA followed by 4 μl PR-957 (50 nM) injected into the lateral ventricle. The sham group was given the same volume of solvent [0.9% w/v NaCl with 0.1% ascorbic acid]. At 4 weeks after the 6-OHDA injection, rats were tested in the rotation test. Rotation asymmetry was calculated for 30 min after intraperitoneal injection of 0.6 mg/kg apomorphine (Sigma-Aldrich, St Louis, MO, USA) as described previously . All animal studies followed the institutional guidelines for animal experiments of Guangzhou Medical University. All procedures were approved by the Institutional Animal Care and Use Committee of Guangzhou Medical University.
After electrophoresis of proteins from SN4141 cells or the midbrain of rats and blocking with 0.5% BSA in PBS, the PVDF membranes (Pall Corporation, Pensacola, FL, USA) were incubated with primary antibodies such as anti-β5i (1:800, Abcam, Cambridge, MA, USA), anti-β5 (1:1000, Abcam, Cambridge, MA, USA) or anti-β-actin (1:2000, CST, Danvers, MA, USA) at 4 °C overnight. The primary antibodies were diluted in blocking solution (LI-COR Biosciences, Lincoln, NE, USA). After the membranes were washed, they were incubated with fluorescent-conjugated secondary antibodies (1: 15000; LI-COR Biosciences, Lincoln, NE, USA) for 1 h in the dark. The Odyssey infrared fluorescence detection system (LI-COR Biosciences, Lincoln, NE, USA) was used for scanning and analysis. For traditional Western blot, secondary antibodies conjugated with horseradish peroxidase (HRP, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and the chemical luminescence detection method (ECL, Pierce Biotechnology, Rockford, IL, USA) were used. Data were scanned and analyzed using the GE 600 system (GE Healthcare, Piscataway, NJ, USA).
Following the protocol used by Goyal et al. , 1.0 × 104 cells were inoculated in 96-well plates (Corning, Sigma-Aldrich, Dorset, UK) for the in-cell western assay. Cells were cultured in DMEM with 10% FCS (Irvine Scientific, Santa Ana, CA, USA) for 48 h, which was then replaced with 6-OHDA dissolved in FCS-free DMEM, but the control group was cultured in FCS-free DMEM. Then, each well was washed with PBS and fixed in 4% formaldehyde for 1 h. Formaldehyde was washed away with PBS, and cells were incubated with 0.1% Triton X-100 in PBS (3 times, 5 min each). Then, cells were treated with blocking solution (LI-COR Biosciences, Lincoln, NE, USA) and incubated with mouse anti-β5i (1:800, Abcam, Cambridge, MA, USA) and rabbit anti-β5i (1:800, Abcam, Cambridge, MA, USA) overnight at 4 °C. After the cells were washed, fluorescent-conjugated secondary antibodies (LI-COR Biosciences, Lincoln, NE, USA), diluted at 1: 1000 in PBS, were added, and the cells were incubated for 1 h in the dark at room temperature. Cells were with PBS three times in the dark. Then, plates were imaged on an Odyssey infrared scanner (LI-COR Biosciences, Lincoln, NE, USA).
Immunofluorescence staining and immunohistochemistry
Brain tissue was cut at a thickness of 15 μm and stored at − 20 °C. Primary antibodies used for immunohistochemistry included mouse monoclonal anti-tyrosine hydroxylase (TH) (1:500, MAB318, Merck Millipore, Billerica, MA, USA), anti-β5i (1:500, Abcam, Cambridge, MA, USA) and anti-TAP-1 (1:500, ab10356; Abcam, Cambridge, MA, USA). TAP-1 is a downstream protein that receives peptides provided by the immunoproteasome . After overnight incubation with primary antibodies, the tissue or cells were incubated with secondary antibodies such as Cy3-conjugated anti-mouse IgG (1:400, Jackson Immuno-research laboratory, PA, USA) and/or Alexa 488-conjugated anti-rabbit IgG (1:400, Molecular Probes, Eugene, OR, USA). Images were acquired using a fluorescence microscope (BX51, Olympus, Fujinon, Japan). For immunohistochemistry, the secondary antibody used was a horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:1000, Kangcheng, Shanghai, China). Sections were stained with 3, 3′-diaminobenzidine (DAB) kits (Wuhan Boster Bioengineering Co., Ltd., Wuhan, China). Images were acquired under a microscope (Olympus AX70; Olympus, Tokyo, Japan). Four images at 200× magnification were taken, with each image covering an area of the SN or striatum, and combined into one figure. Images were analyzed by ImageJ software (version 1.45; National Institutes of Health, Bethesda, Maryland, USA).
Fast TH staining and laser capture microdissection (LCM)
To reduce RNA degradation, we used fast TH staining to detect DA neurons. Slices were fixed in acetone-methanol solution at − 20 °C for 10 min, washed with PBS containing 1% Triton X-100, incubated with the TH antibody (MAB318, Merck Millipore, Billerica, MA, USA) at a 1:100 dilution for 10 min, rinsed in PBS with Triton twice, and incubated with the goat antirabbit antibody with HRP (1:100, Kangcheng, Shanghai, China) for 5 min. Immunohistochemistry staining was done by DAB kits (Wuhan Boster Bioengineering Co., Ltd., Wuhan, China). The stained slices were dehydrated in RNase-free solutions as follows: 100% acetone for 5 min, 75% ethanol, 95 and 100% ethanol for 1 min each, and then xylene twice for 1 min and 5 min.
As described previously [26, 27], nonfixed fresh brain tissue was rapidly frozen and cut into 8-μm-thick slices. Slices were collected on to polyethylene naphthalate membrane-coated glass slides (Life Technologies, Grand Island, NY, USA). After fast tyrosine hydroxylase staining, TH+ neurons in the substantia nigra were captured by the Arcturus XT system (Life Technologies, CA, USA). Laser power was set at 70 mW and 150 mV. Approximately 300–450 TH+ neurons were collected, and total RNA was extracted using the mirVana PARIS Kit (PN AM1556, Austin, TX, Ambion, USA) and converted into cDNA by a Reverse Transcription Kit (Takara, Shiga, Japan). RT1A (rat monomorphic MHC class I antigen) binds the peptide or antigens translocated by TAP into the ER, and its mRNA level in DA neurons was detected by qRT-PCR. The PCR primers (TIANGEN Biotech, China) used were as follows: GAPDH-F: 5’-TACTAGCGGTTTTACGGGCG-3′ and GAPDH-R: 5’-TCG-AACAGGAGGAGCAGAGAGCGA-3′; TAP-1-F: 5’-GGCAGACTCAGTTC-CTCTCAC-3′ and TAP-1-R: 5’-CAGAACGGGTTGGGGATCAA-3′; RT1A (Rat monomorphic MHC class I antigen) -F: 5’-GCTCACACTCGCTGCGGTAT-3′ and RT1A-R: 5’-GCCATACATCTCCTGGATGG-3′. GAPDH was used as an internal control, and mRNA expression was analyzed using the 2−ΔΔCT method .
All experiments were repeated at least 3 times. Data are shown as the mean ± SD. ANOVA was followed by Tukey’s or Student-Newman-Keuls (SNK) post hoc testing. P < 0.05 was considered statistically significant. All analyses were performed using SPSS.13 and STATA software (Version 14; StataCorp, College Station, TX, USA).
6-Hydroxydopamine upregulates immunoproteasome expression in DA neurons
β5i dysfunction inhibits antigen presentation in DA neurons
β5i inhibition exacerbates 6-hydroxydopamine-induced DA neuronal damage
Downregulated β5i expression impairs antigen presentation in DA neurons under 6-OHDA treatment
Immunoproteasome and MHC molecules are minimally expressed in the healthy brain, and their activation and upregulation are indicative of a pathological status in the central nervous system (CNS) [12, 19, 31, 32]. The examination of brain specimens from people with neurodegenerative disorders such as AD, HD, and amyotrophic lateral sclerosis (ALS) have revealed dysfunctions in immunoproteasome activity [12, 33, 34], and these phenomena have also been found in those with autoimmune encephalomyelitis (EAE) and epilepsy [8, 15]. A similar failure in proteolytic mechanisms, as well as increases in oxidative stress and neuroinflammation, have also been reported in PD . We propose the following scenario to describe the role of β5i in PD pathogenesis. Initially, ROS and the unfolded protein response (UPR) induce ER stress and neuronal damage in DA neurons. Then, Bip is activated and ships abnormal proteins from the ER to the UPS. The overloaded proteins induce the activation of β5i and transformation of the immunoproteasome to have an updated and expanded proteasome capacity. Consequently, the ubiquitinated proteins are degraded to peptides by β5i and recognized by MHC-I. Here, our study revealed that β5i is activated in DA neurons exposed to 6-OHDA, and upregulation of β5i or other immunoproteasome components might play a neuroprotective role against ROS-mediated damage in PD.
The UPS is a key factor in the proteostasis network . Different subunit configurations of the UPS, such as the standard proteasome, immuneoproteasome, mixed-type proteasome, thymoproteasome and spermatoproteasome, acquire different proteolytic capacities . The immunoproteasomes activated by ROS, lipopolysaccharide (LPS) and IFN-γ have strong capabilities to clear protein deposits and alleviate ROS impairment [8, 38]. The overloading of damaged proteins and insufficient proteolytic capacity may trigger immunoproteasomes to replace impaired proteasome subunits . X-ray crystallography studies have shown that the enzyme active center of the immunoproteasome (iUPS) provides a larger space than the enzyme active center of other proteasomes to accommodate and degrade misfolded or oxidized proteins . For example, the immunoproteasome eliminates the extended huntingtin proteins of HD, Aβ aggregates of AD and mutant SOD1 deposition of ALS more efficiently [14, 41, 42]. Under normal conditions, oxidized cytoplasmic and nuclear proteins are generally degraded by the proteasome . The 20S proteasome, immunoproteasome and PA28αβ regulator are all upregulated under H2O2-induced oxidative stress, and the immunoproteasome may degrade oxidized proteins more selectively than the other proteasomes . Some studies have suggested that the enhanced proteolytic activity of the immunoproteasome more efficiently clears aggregated proteins and is important for cell viability under IFN-γ treatment . Others have suggested that the function of the immunoproteasome to bind and degrade ubiquitin conjugates is similar to that of constitutive proteasomes . Recently, we reported that Chinese females carrying the rs17587-G/G mutation of PSMB9 are at a higher risk of PD . The rs17587 variation at exon 4 of PSMB9 affects the glutamyl peptide hydrolyzing activity associated with proteolytic function . As an immunoproteasome subunit, β5i has been found to be involved in proteinopathies and the innate immune response . In this study, we further explored the role of β5i in the 6-OHDA model of PD. Our results showed that β5i was activated and upregulated in a dose- and time-dependent manner after 6-OHDA treatment in a DA neuron cell line, and this was further confirmed in the 6-OHDA hemilesioned rat model of PD. ER stress and oxidative stress have been suggested to contribute to the loss of DA neurons in PD . Compared to the standard proteasome, the immunoproteasome is thought to be more resistant to oxidative stress and ER stress . When protein homeostasis is impaired in neurons, misfolded proteins aggregate in the ER and induce ER stress . Bip is upregulated and binds aggregated proteins for transportation from the ER to the UPS . If the UPS and immunoproteasome system are deficient, neurons are more susceptible to apoptosis due to the stress from the accumulation of oxidized proteins [8, 48]. In aging-related sporadic inclusion body myositis, intracellular protein aggregation was accompanied by ER stress and proteasome dysfunction . A study from X-linked adrenoleukodystrophy revealed that β5i was significantly elevated and recruited to mitochondria in response to oxidative stress where it participated in mitochondrial protein quality control . Recently, IFN-γ-induced oxidative stress was found to upregulate β5i expression with increased poly-Ub substrate degradation efficiency . In this study, we used a 6-OHDA model to induce massive oxidative stress and the unfolded protein response in DA neurons . ROS and ER stress occurred in a dose- and time-dependent manner following 6-OHDA treatment . We found that inhibition and downregulation of β5i resulted in DA neurons with increased sensitivity to 6-OHDA toxicity, suggesting that the neuroprotective effect of β5i may be related to ROS regulation and ER stress at the early stage of PD.
Immunoproteasomes still play an important role in the regulation of neuroinflammation [13, 52]. In the peripheral immune system, immunoproteasome subunits degrade proteins to peptides, which present to TAP1 as antigens . As a peptide transporter protein, TAP1 loads antigenic peptides into the ER where MHC molecules recognize antigens and present them to the cell membrane [30, 53]. IFN-γ-signaling has been proven to promote MHC class I antigen presentation, and IFN-γ-regulated inflammation in proteasome-associated autoinflammatory syndromes (PRAAS) was partly reduced after inhibition of proteolytic function [54, 55]. As a highly selective inhibitor of β5i, PR-957 was shown to reduce the release of IL-23 and TNF-α from inflammatory cells by 90 and 50%, respectively . PR-957 also inhibits inflammation in MOG35–55-induced experimental autoimmune encephalomyelitis . Notably, neurodegenerative diseases predominantly display disorders of neuroinflammation. In transgenic mouse models of AD and human postmortem tissue, immunoproteasome activities and HLA-DR expression are strongly increased and accompanied by overactivated microglia in the cortex. [57, 58] Previously, neurons were considered to be ‘immunoprivileged’ without antigen presentation capabilities [59, 60]; now MHC-1 expression has been demonstrated on DA neurons in the rodent and human brain [18, 19]. The catecholaminergic neurons expressing MHC-1 have been shown to be more susceptible to apoptosis induction, suggesting that these neurons may be targeted by ROS during the development of PD . In this study, our results revealed significant upregulation of MHC-I and TAP1 accompanied by increased expression of β5i on DA neurons under 6-OHDA treatment and that MHC-I and TAP1 mRNA levels were decreased after β5i inhibition. These findings suggest that β5i may regulate the TAP1/MHC-I pathway in DA neurons under oxidative stress.
In conclusion, our data showed that β5i was activated by 6-OHDA-induced oxidative stress in DA neurons both in vitro and in vivo and may play a neuroprotective role in the survival of DA neurons. Our data might provide new evidence for the consideration of the immunoproteasome as a potential therapeutic target for PD.
This work was supported by research grants from National Key R&D Program of China (2016YFC1306600, SQ2017YFSF110116), National Natural Science Foundation of China (81701254, 81471292, U1603281, U1503222, 81430021, 81501100, NO.8187050204), Science Foundation of Guangdong of China (2015A030311021, 2018A030313649), a technology project of Guangzhou (201504281820463), Shandong Provincial Natural Science Foundation (BS2015YY041), International Project of Science and Technology for Guangdong (2016A050502025), Science and Technology of Guangdong of China (2013B022000026) and Collaborative Innovation Foundation of Guangzhou Science and Technology Bureau (2018-1202-SF-0019).
We thank Dr. Madhuvika Murugan, You-Sheng Xiao, Luan Chen, Li Zhang, Ming Lei, Chao-Hao Yang, Xiang Chen, Qin-Hui Huang, Wei-Guo Liu, Long-Jun Wu and Prof. Kai-Ping Li for their help to improve this manuscript. We thank Prof. Qian Yang provide SN4741 cells.
Availability of supporting data
All data mentioned in this article are available on published article.
Ethical approval and consent to participate
All procedures were approved by Institutional Animal Care and Use Committee of Guangzhou Medical University (NO.2017341).
National Key R&D Program of China, National Natural Science Foundation of China, Science Foundation of Guangdong of China, Technology project of Guangzhou, Shandong Provincial Natural Science Foundation, International Project of Science and Technology for Guangdong, Science and Technology of Guangdong of China and the Collaborative Innovation Foundation of Guangzhou Science and Technology Bureau.
M-MS, H-JX and X-PY mainly conceived, designed study and drafted manuscript. The major experiments included the 6-OH DA model in vivo was performed by L-GH and S-CC, and in vitro was performed by H-SX. Z-MM provided technical supports on in-cell western assay and color western blot, W-ZH and G-WY helped to the part on animal behavior and TH staining, Y-XL provided technical supports on LCM and qRT-PCR, Z-LM and W-L contributed to data analysis. Q-SG and C-CJ were involved in revising manuscript and experiment guide. Q-SG drew figures and tables, modified the manuscript. All authors read and approved final manuscript to be published.
Consent for publication
Authors have no conflict of interest to declare.
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- Chu Y, Kordower JH. Age-associated increases of α-synuclein in monkeys and humans are associated with nigrostriatal dopamine depletion: is this the target for Parkinson's disease? Neurobiol Dis. 2007;25(1):134–49.View ArticlePubMedGoogle Scholar
- Taylor JM, Main BS, Crack PJ. Neuroinflammation and oxidative stress: co-conspirators in the pathology of Parkinson’s disease. Neurochem Int. 2013;62(5):803–19.View ArticlePubMedGoogle Scholar
- Launay N, Ruiz M, Fourcade S, Schlüter A, Guilera C, Ferrer I, et al. Oxidative stress regulates the ubiquitin–proteasome system and immunoproteasome functioning in a mouse model of X-adrenoleukodystrophy. Brain. 2013;136(3):891–904.View ArticlePubMedGoogle Scholar
- Bence NF, Sampat RM, Kopito RR. Impairment of the ubiquitin-proteasome system by protein aggregation. Science. 2001;292(5521):1552–5.View ArticlePubMedGoogle Scholar
- Wang J, Maldonado MA. The ubiquitin-proteasome system and its role in inflammatory and autoimmune diseases. Cell Mol Immunol. 2006;3(4):255–61.PubMedGoogle Scholar
- Ciechanover A, Brundin P. The ubiquitin proteasome system in neurodegenerative diseases: sometimes the chicken, sometimes the egg. Neuron. 2003;40(2):427–46.View ArticlePubMedGoogle Scholar
- Dasuri K, Zhang L, Keller JN. Oxidative stress, neurodegeneration, and the balance of protein degradation and protein synthesis. Free Radic Biol Med. 2013;62:170–85.View ArticlePubMedGoogle Scholar
- Seifert U, Bialy LP, Ebstein F, Bech-Otschir D, Voigt A, Schröter F, et al. Immunoproteasomes preserve protein homeostasis upon interferon-induced oxidative stress. Cell. 2010;142(4):613–24.View ArticlePubMedGoogle Scholar
- Kincaid EZ, Che JW, York I, Escobar H, Reyes-Vargas E, Delgado JC, et al. Mice completely lacking immunoproteasomes show major changes in antigen presentation. Nat Immunol. 2012;13(2):129–35.View ArticleGoogle Scholar
- Chen S, Kammerl IE, Vosyka O, Baumann T, Yu Y, Wu Y, et al. Immunoproteasome dysfunction augments alternative polarization of alveolar macrophages. Cell Death Differ. 2016;23(6):1026.View ArticlePubMedPubMed CentralGoogle Scholar
- Vilchez D, Saez I, Dillin A. The role of protein clearance mechanisms in organismal ageing and age-related diseases. Nat Commun. 2014;5(5):5659.Google Scholar
- Mishto M, Bellavista E, Santoro A, Stolzing A, Ligorio C, Nacmias B, et al. Immunoproteasome and LMP2 polymorphism in aged and Alzheimer's disease brains. Neurobiol Aging. 2006;27(1):54–66.View ArticlePubMedGoogle Scholar
- Orre M, Kamphuis W, Dooves S, Kooijman L, Chan ET, Kirk CJ, et al. Reactive glia show increased immunoproteasome activity in Alzheimer’s disease. Brain. 2013;136(5):1415–31.View ArticlePubMedGoogle Scholar
- Díaz-Hernández M, Martín-Aparicio E, Avila J, Hernández F, Lucas JJ. Enhaced induction of the immunoproteasome by interferon gamma in neurons expressing mutant huntingtin. Neurotox Res. 2004;6(6):463–8.View ArticlePubMedGoogle Scholar
- Mishto M, Raza ML, de Biase D, Ravizza T, Vasuri F, Martucci M, et al. The immunoproteasome β5i subunit is a key contributor to ictogenesis in a rat model of chronic epilepsy. Brain Behav Immun. 2015;49:188–96.View ArticlePubMedGoogle Scholar
- Chen X, Zhang X, Wang Y, Lei H, Su H, Zeng J, et al. Inhibition of immunoproteasome reduces infarction volume and attenuates inflammatory reaction in a rat model of ischemic stroke. Cell Death Dis. 2015;6(1):e1626.View ArticlePubMedPubMed CentralGoogle Scholar
- Bukhatwa S, Zeng B-Y, Rose S, Jenner P. A comparison of changes in proteasomal subunit expression in the substantia nigra in Parkinson's disease, multiple system atrophy and progressive supranuclear palsy. Brain Res. 2010;1326:174–83.View ArticlePubMedGoogle Scholar
- Nardo G, Trolese MC, Bendotti C. Major histocompatibility complex I expression by motor neurons and its implication in amyotrophic lateral sclerosis. Front Neurol. 2016;7:89.View ArticlePubMedPubMed CentralGoogle Scholar
- Cebrián C, Zucca FA, Mauri P, Steinbeck JA, Studer L, Scherzer CR, et al. MHC-I expression renders catecholaminergic neurons susceptible to T-cell-mediated degeneration. Nat Commun. 2014;5:3633.View ArticlePubMedPubMed CentralGoogle Scholar
- Huang L, Xue Y, Feng DY, Yang RX, Nie T, Zhu G, et al. Blockade of RyRs in the ER attenuates 6-OHDA-induced calcium overload, Cellular Hypo-Excitability and Apoptosis in Dopaminergic Neurons. Frontiers in Cellular Neuroscience. 2017;11:52.PubMedPubMed CentralGoogle Scholar
- Zhang S, Gui X-H, Huang L-P, Deng M-Z, Fang R-M, Ke X-H, et al. Neuroprotective effects of β-asarone against 6-hydroxy dopamine-induced parkinsonism via JNK/Bcl-2/Beclin-1 pathway. Mol Neurobiol. 2016;53(1):83–94.View ArticlePubMedGoogle Scholar
- Nijholt DA, De Kimpe L, Elfrink HL, Hoozemans JJ, Scheper W, et al. A selective inhibitor of the immunoproteasome subunit LMP7 blocks cytokine production and attenuates progression of experimental arthritis. Nat Med. 2009;15(7):781–7.View ArticleGoogle Scholar
- Ungerstedt U. Postsynaptic supersensitivity after 6-hydroxy-dopamine induced degeneration of the nigro-striatal dopamine system. Acta Physiol Scand. 1971;82(S367):69–93.View ArticleGoogle Scholar
- Goyal A, Pal N, Concannon M, Paul M, Doran M, Poluzzi C, et al. Endorepellin, the Angiostatic module of Perlecan, interacts with both the α2β1 integrin and vascular endothelial growth factor Receptor 2 (VEGFR2) a DUAL RECEPTOR ANTAGONISM. J Biol Chem. 2011;286(29):25947–62.View ArticlePubMedPubMed CentralGoogle Scholar
- Hwang L-Y, Lieu PT, Peterson PA, Yang Y. Functional regulation of immunoproteasomes and transporter associated with antigen processing. Immunol Res. 2001;24(3):245–72.View ArticlePubMedGoogle Scholar
- Kummari E, Guo-Ross SX, Eells JB. Laser capture microdissection-a demonstration of the isolation of individual dopamine neurons and the entire ventral tegmental area. J Vi. Exp. 2015;96(96):e52336–e52336.Google Scholar
- Fend F, Emmert-Buck MR, Chuaqui R, Cole K, Lee J, Liotta LA, et al. Immuno-LCM: laser capture microdissection of immunostained frozen sections for mRNA analysis. Am J Pathol. 1999;154(1):61–6.View ArticlePubMedPubMed CentralGoogle Scholar
- Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method. methods. 2001;25(4):402–8.View ArticlePubMedGoogle Scholar
- Wang T, Yuan Y, Zou H, Yang J, Zhao S, Ma Y, et al. The ER stress regulator Bip mediates cadmium-induced autophagy and neuronal senescence. Sci Rep. 2016;6:38091.View ArticlePubMedPubMed CentralGoogle Scholar
- Lawand M, Abramova A, Manceau V, Springer S, van Endert P. TAP-dependent and-independent peptide import into dendritic cell Phagosomes. J Immunol. 2016;197(9):3454–63.View ArticlePubMedGoogle Scholar
- Lindå H, Hammarberg H, Piehl F, Khademi M, Olsson T. Expression of MHC class I heavy chain and β2-microglobulin in rat brainstem motoneurons and nigral dopaminergic neurons. J Neuroimmunol. 1999;101(1):76–86.View ArticlePubMedGoogle Scholar
- Ferrington DA, Hussong SA, Roehrich H, Kapphahn RJ, Kavanaugh SM, Heuss ND, et al. Immunoproteasome responds to injury in the retina and brain. J Neurochem. 2008;106(1):158–69.View ArticlePubMedPubMed CentralGoogle Scholar
- Song S, Miranda CJ, Braun L, Meyer K, Frakes AE, Ferraiuolo L, et al. MHC class I protects motor neurons from astrocyte-induced toxicity in amyotrophic lateral sclerosis (ALS). Nat Med. 2016;22(4):397.View ArticlePubMedPubMed CentralGoogle Scholar
- Díaz-Hernández M, Hernández F, Martín-Aparicio E, Gómez-Ramos P, Morán MA, Castaño JG, et al. Neuronal induction of the immunoproteasome in Huntington's disease. J Neurosci. 2003;23(37):11653–61.View ArticlePubMedGoogle Scholar
- Mo M-S, Huang W, Sun C-C, Zhang L-M, Cen L, Xiao Y-S, et al. Association analysis of proteasome subunits and transporter associated with antigen processing on Chinese patients with Parkinson's disease. Chin Med J. 2016;129(9):1053.View ArticlePubMedPubMed CentralGoogle Scholar
- Nijholt D, De Kimpe L, Elfrink H L, Hoozemans J JM, Scheper W. Removing protein aggregates: the role of proteolysis in neurodegeneration. Curr Med Chem. 2011;18(16):2459–76.View ArticlePubMedGoogle Scholar
- Brehm A, Krüger E. Dysfunction in protein clearance by the proteasome: impact on autoinflammatory diseases. Semin Immunopathol. 2015: Springer; 2015:323–33.Google Scholar
- Reis J, Hassan F, Guan XQ, Shen J, Monaco JJ, Papasian CJ, et al. The immunoproteasomes regulate LPS-induced TRIF/TRAM signaling pathway in murine macrophages. Cell Biochem Biophys. 2011;60(1–2):119–26.View ArticlePubMedPubMed CentralGoogle Scholar
- Aiken CT, Kaake RM, Wang X, Huang L. Oxidative stress-mediated regulation of proteasome complexes. Molecular & Cellular Proteomics. 2011;10(5):R110. 006924.View ArticleGoogle Scholar
- Unno M, Mizushima T, Morimoto Y, Tomisugi Y, Tanaka K, Yasuoka N, et al. The structure of the mammalian 20S proteasome at 2.75 Å resolution. Structure. 2002;10(5):609–18.View ArticlePubMedGoogle Scholar
- Aso E, Lomoio S, López-González I, Joda L, Carmona M, Fernández-Yagüe N, et al. Amyloid generation and dysfunctional immunoproteasome activation with disease progression in animal model of familial Alzheimer's disease. Brain Pathol. 2012;22(5):636–53.View ArticlePubMedGoogle Scholar
- Cheroni C, Marino M, Tortarolo M, Veglianese P, De Biasi S, Fontana E, et al. Functional alterations of the ubiquitin-proteasome system in motor neurons of a mouse model of familial amyotrophic lateral sclerosis†. Hum Mol Genet. 2009;18(1):82–96.View ArticlePubMedGoogle Scholar
- Pickering AM, Koop AL, Teoh CY, et al. The immunoproteasome, the 20S proteasome and the PA28αβ proteasome regulator are oxidative-stress-adaptive proteolytic complexes. Biochem J. 2010;432(3):585–95.View ArticlePubMedPubMed CentralGoogle Scholar
- Nathan JA, Spinnenhirn V, Schmidtke G, et al. Immuno-and constitutive proteasomes do not differ in their abilities to degrade ubiquitinated proteins. Cell. 2013;152(5):1184–94.View ArticlePubMedPubMed CentralGoogle Scholar
- Haroon N, Maksymowych WP, Rahman P, Tsui FW, O'Shea FD, Inman RD. Radiographic severity of ankylosing spondylitis is associated with polymorphism of the large multifunctional peptidase 2 gene in the Spondyloarthritis research consortium of Canada cohort. Arthritis Rheum. 2012;64(4):1119–26.View ArticlePubMedGoogle Scholar
- Mercado G, Castillo V, Vidal R, Hetz C. ER proteostasis disturbances in Parkinson's disease: novel insights. Front Aging Neurosci. 2015;7:39.Google Scholar
- Ugunklusek A, Tatham MH, Elkharaz J, Constantinteodosiu D, Lawler K, Mohamed H, et al. Continued 26S proteasome dysfunction in mouse brain cortical neurons impairs autophagy and the Keap1-Nrf2 oxidative defence pathway. Cell Death Dis. 2017;8(1):e2531.View ArticleGoogle Scholar
- Ebstein F, Kloetzel P-M, Krüger E, Seifert U. Emerging roles of immunoproteasomes beyond MHC class I antigen processing. Cell Mol Life Sci. 2012;69(15):2543–58.View ArticlePubMedGoogle Scholar
- Askanas V, Engel WK. Inclusion-body myositis a myodegenerative conformational disorder associated with Aβ, protein misfolding, and proteasome inhibition. Neurology. 2006;66(1 suppl 1):S39–48.View ArticlePubMedGoogle Scholar
- Ryu EJ, Harding HP, Angelastro JM, Vitolo OV, Ron D, Greene LA. Endoplasmic reticulum stress and the unfolded protein response in cellular models of Parkinson's disease. J Neurosci. 2002;22(24):10690–8.View ArticlePubMedGoogle Scholar
- Tao K, Wang B, Feng D, Zhang W, Lu F, Lai J, et al. Salidroside protects against 6-Hydroxydopamine-induced cytotoxicity by attenuating ER stress. Neurosci Bull. 2016;32(1):61–9.View ArticlePubMedPubMed CentralGoogle Scholar
- Basler M, Mundt S, Muchamuel T, Moll C, Jiang J, Groettrup M, et al. Inhibition of the immunoproteasome ameliorates experimental autoimmune encephalomyelitis. EMBO Mol Med. 2014;6(2):226–38. e201303543PubMedPubMed CentralGoogle Scholar
- Shastri N, Nagarajan N, Lind KC, Kanaseki T. Monitoring peptide processing for MHC class I molecules in the endoplasmic reticulum. Curr Opin Immunol. 2014;26:123–7.View ArticlePubMedGoogle Scholar
- McDermott A, Jacks J, Kessler M, Emanuel PD, Gao L. Proteasome-associated autoinflammatory syndromes: advances in pathogeneses, clinical presentations, diagnosis, and management. Int J Dermatol. 2015;54(2):121–9.View ArticlePubMedGoogle Scholar
- McDermott A, de Jesus AA, Liu Y, Kim P, Jacks J, Sanchez GAM, et al. A case of proteasome-associated auto-inflammatory syndrome with compound heterozygous mutations in PSMB8. J Am Acad Dermatol. 2013;69(1):e29.View ArticlePubMedPubMed CentralGoogle Scholar
- Basler M, Mundt S, Bitzer A, Schmidt C, Groettrup M. The immunoproteasome: a novel drug target for autoimmune diseases. Clin Exp Rheumatol. 2015;33:74–9.Google Scholar
- Mangold CA, Masser DR, Stanford DR, Bixler GV, Pisupati A, Giles CB, et al. CNS-wide sexually dimorphic induction of the major histocompatibility complex 1 pathway with aging. J Gerontol A Biol Sci Med Sci. 2016;72(1):16–29. glv232View ArticlePubMedPubMed CentralGoogle Scholar
- McGeer P, Itagaki S, Boyes B, McGeer E. Reactive microglia are positive for HLA-DR in the substantia nigra of Parkinson's and Alzheimer's disease brains. Neurology. 1988;38(8):1285.View ArticlePubMedGoogle Scholar
- Meuth SG, Herrmann AM, Simon OJ, Siffrin V, Melzer N, Bittner S, et al. Cytotoxic CD8+ T cell–neuron interactions: perforin-dependent electrical silencing precedes but is not causally linked to neuronal cell death. J Neurosci. 2009;29(49):15397–409.View ArticlePubMedGoogle Scholar
- Neumann H, Cavalie A, Jenne DE, Wekerle H. Induction of MHC class I genes in neurons. Science. 1995;269(5223):549.View ArticlePubMedGoogle Scholar