Neurotoxicology 73 (2019) 112–119 Contents lists available at ScienceDirect Neurotoxicology journal homepage: www.elsevier.com/locate/neuro Full Length Article Activation of the immunoproteasome protects SH-SY5Y cells from the toxicity of rotenone T Congcong Suna, Mingshu Mob, Yun Wangc, Wenfei Yua, Chengyuan Songa, Xingbang Wanga, ⁎ Si Chena, Yiming Liua,d, a Department of Neurology, Qilu Hospital of Shandong University, Jinan, 250012, China b Department of Neurology, First Affiliated Hospital of Guangzhou Medical University, Guangzhou, 510120, China c Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, Xuzhou Medical University, Xuzhou, 221000, China d Brain Science Research Institute, Shandong University, Jinan, 250012, China ARTICLE INFO ABSTRACT Keywords: This study investigated the expression and role of immunoproteasome (i-proteasome) in a cell model of Immunoproteasome Parkinson’s disease (PD). The cytotoxicity of rotenone was measured by CCK-8 assay. The i-proteasome β1i PSMB9 subunit PSMB9 was suppressed by a specific shRNA or transfected with an overexpression plasmid in the SH- ’ Parkinson s disease SY5Y cells. Under the exposure to rotenone or not, the expression of constitutive proteasome β subunits, i- Rotenone proteasome βi subunits, antigen presentation related proteins, α-syn and TH were detected by Western blot in PSMB9-silenced or -overexpressed cells, and the proteasomal activities were detected by fluorogenic peptide substrates. The location of i-proteasome βi subunits and α-syn were detected by immunofluorescence staining. The levels of ROS, GSH and MDA were measured by commercial kits. Cell apoptosis was detected by flow cytometry. Besides impairing the constitutive proteasomes, rotenone induced the expression of βi subunits of i- proteasome and antigen presentation related proteins such as TAP1, TAP2 and MHC-I. Silencing or over- expressing PSMB9 had no obvious effect on the levels of other subunits, but could regulate the chymotrypsin-like activity of 20S proteasome and the expression of TAP1, TAP2 and MHC-I. Three βi subunits (PSMB9, PSMB10, PSMB8) of i-proteasome were all co-localized with α-syn. PSMB9 knockdown aggravated accumulation of α-syn, degradation of TH, release of ROS, increased level of MDA, decreased level of GSH and eventually promoted apoptosis in SH-SY5Y cells after rotenone treatment, while over-expression of PSMB9 could attenuate these toxic effects of rotenone. I-proteasome is activated in SH-SY5Y cells treated with rotenone and may play a neuro- protective role. 1. Introduction particle and the 19S regulator particle. The catalytic 20S core particle contains three active subunits of β1, β2 and β5, which have caspase- Parkinson’s disease (PD) is the second most common neurodegen- like, trypsin-like and chymotrypsin-like peptidase activities, respec- erative disease in elderly people (Goedert and Compston, 2018). The tively (Eskandari et al., 2017). These subunits could be replaced by pathologic changes, which are characterized by loss of dopaminergic homologous subunits β1i (PSMB9), β2i (PSMB10) and β5i (PSMB8), neurons and deposition of abnormal α-synuclein (α-syn) in the sur- and the constitutive proteasome transforms into an immunoproteasome viving neurons, appear long before the onset of the classic movement (i-proteasome) under oxidative stress or inflammation conditions (Kaur disorders (Surmeier et al., 2017). Ubiquitin-proteasome pathway, and Batra, 2016). I-proteasome is a subtype of proteasome which could which plays a key role in maintaining protein homeostasis, is impaired degrade proteins and present optimal peptides to antigen presenting under these stresses, and subsequently induces the formation of harmful cells. Genetic polymorphisms in i-proteasome have been linked to protein aggregates (Mckinnon et al., 2015). neurodegenerative disease. For example, single nucleotide poly- Proteasomes consist of two main components: the catalytic 20S core morphisms located in PSMB9 could influence the proteasome activity in Abbreviations: i-proteasome, immunoproteasome; PD, Parkinson’s disease; α-syn, α-synuclein; AD, Alzheimer’s disease; SD, standard deviation ⁎ Corresponding author at: Department of Neurology, Qilu Hospital of Shandong University and Brain Science Research Institute, Shandong University, No.107, Wenhua West Road., Jinan, 250012, China. E-mail address: [email protected] (Y. Liu). https://doi.org/10.1016/j.neuro.2019.03.004 Received 11 September 2018; Received in revised form 13 March 2019; Accepted 18 March 2019 Available online 20 March 2019 0161-813X/ © 2019 Elsevier B.V. All rights reserved. C. Sun, et al. Neurotoxicology 73 (2019) 112–119 Alzheimer’s disease (AD) (Mishto et al., 2006). A previous study has Bioscience & Technology Co., Ltd, China), which contains the neomycin reported that rs17587 variants in PSMB9 gene might contribute to the selection marker. The vectors without the target gene and target of susceptibility of sporadic PD in Chinese females (Mo et al., 2016). βeta-actin were used as the negative control and positive control, re- Growing evidence shows that i-proteasome could be activated and spectively. Empty plasmids were used as the mock control. All plasmids might be involved in the pathogenesis of neurodegenerative disease were verified by DNA sequencing. The plasmids were transfected with with abnormal protein aggregation. For example, increased levels of Lipofectamine 2000 (Invitrogen, Grand Island, NY, USA) following the PSMB9 and PSMB8 have been detected in cortex and striatum of pa- manufacturer’s instructions. tients with Huntington’s disease (Díaz-Hernández et al., 2003). In- creased i-proteasome activity is found in human AD samples (Orre 2.4. Western blot et al., 2013). A trend of increased PSMB8 expression in remaining TH positive cells in Multiple system atrophy and Progressive supranuclear After treating with vehicles or 500 nM rotenone for 24 h, the whole- palsy has been reported (Bukhatwa et al., 2010). However, the ex- cell lysate was prepared by a Mammalian Cell Extraction Kit (Biovision, pression of i-proteasomes in PD is less studied and the results are CA, USA). The protein concentration was measured with a controversial as yet. Bukhatwa et al detected no obvious change of Bicinchoninic Acid (BCA) protein assay kit (Thermo Fisher Scientific PSMB8 expression in remaining TH positive cells in PD samples com- Inc., MA, USA). Soluble proteins (50 μg) were separated by 8–12% SDS- pared to control (Bukhatwa et al., 2010), while Ugras et al found in- polyacrylamide gels, and then transferred onto polyvinylidene di- creased levels and activity of PSMB8 in postmortem human brains with fluoride (PVDF) membranes (Millipore, MA, USA). Primary antibodies PD (Ugras et al., 2018). were as follows: rabbit anti-20 S proteasome β1(1: 1000, Abcam, Besides modulating inflammation, i-proteasome possess antioxidant Cambridge, MA, USA), mouse anti-20 S proteasome β2 (1: 1,000, capacity and proteolytic activity (Johnston-Carey et al., 2015; Kimura Abcam, Cambridge, MA, USA), rabbit anti-20 S proteasome β5 (1: et al., 2015). Augmented immunoproteasome function may contribute 1000, Abcam, Cambridge, MA, USA), rabbit anti-PSMB9 (1: 1,000, to lifespan diff ;erences in mice and among primate species (Pickering Abcam, Cambridge, MA, USA), rabbit anti-PSMB10 (1: 1,000, Abcam, et al., 2015). To investigate the role of i-proteasome in the pathophy- Cambridge, MA, USA), mouse anti-PSMB8 (1: 200, Santa Cruz siology of PD, we detected the exprssion and founction of βi subunits of Biotechnology, Inc., Santa Cruz, CA, USA), mouse anti-TAP1 (1: 2,00, i-proteasome in SH-SY5Y cells with rotenone toxicty, which is widely Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and rabbit anti- used to imitate the pathogenesis of PD (Johnson and Bobrovskaya, TAP2 (1: 1,000, Abcam, Cambridge, MA, USA), rabbit anti-rat MHC-I 2015). (1:1000, Abcam, Cambridge, MA, USA), rabbit anti-TH (1: 1,000, Abcam, Cambridge, MA, USA), mouse anti-Alpha-synuclein (1: 1,000, 2. Materials and methods Abcam, Cambridge, MA, USA) and mouse anti- β-actin(1:10,000, Proteintech Group Inc., Chicago, IL, USA). Subsequently, membranes 2.1. Cell culture and rotenone treatment were washed and incubated with horseradish peroxidase (HRP)-con- jugated anti-mouse IgG or anti-rabbit IgG (KPL, MD, USA) secondary ® The SH-SY5Y cell line was obtained from American Type Culture antibodies. Proteins were detected by SuperSignal West Pico Collection (ATCC, Manassas, VA, USA), and was cultured in Dulbecco's Chemiluminescent Substrate (Thermo Fisher Scientific Inc., MA, USA). modified Eagle's high glucose medium (DMEM, Life Technologies, The densities were calculated as target protein expression/ β-actin ex- Rockville, MD, USA), supplemented with 10% fetal bovine serum (FBS, pression ratios with Image J 1.42q software (U.S. National Institutes of Invitrogen, CA, USA) in a humidified incubator with 5% CO2 at 37 °C. Health). Rotenone (Sigma-Aldrich, St Louis, MO, USA) was prepared in DMSO (Sigma-Aldrich, St Louis, MO, USA) and stocked at a con- 2.5. Proteasome activity measurement centration of 50 mM. Different concentrations of rotenone (0 nM, 10 nM, 100 nM, 500 nM, 1 u M, 10 u M) were diluted with DMEM re- Cells were treated with vehicles or 500 nM rotenone for 24 h. Then, spectively. Different incubation time (0 h, 3 h, 6 h, 12 h, 24 h, 48 h) was the proteasomal activity was measured with 20S proteasome activity carried out according to corresponding experiments and with vehicle as assay kit (Chemicon-Millipore, Billerica, MA, USA). Briefly, the cells control. were sonicated in cell lysis buffer (Thermo Scientific Pierce, Rockford, IL, USA). A BCA Protein Assay Kit (Thermo Fisher Scientifc, Rockford, 2.2. CCK-8 assay USA) was used to determine protein concentration. The isolated protein (50 μg) was incubated with buffer and 50 μM fluorogenic substrates (Z- Cells (5 × 103 cells/well) were seeded in a 96-well plate and in- LLE-AMC for caspase-like activity, Suc-LLVY-AMC for chymotrypsin- cubated overnight. Then cells were treated with vehicles or different like activity, and Z-ARR-AMC for trypsin-like activity) at 37 °C for 1 h.