The SUMO Conjugase Ubc9 Protects Dopaminergic Cells from Cytotoxicity and Enhances the Stability of α-Synuclein in Parkinson’s Disease Models

Animals

Ubc9-overexpressing transgenic (Ubc9-Tg) C57BL/6 background mice (30–45 g) were received as a gift from John Hallenbeck at NINDS (Lee et al., 2011, 2014). All animal protocols were conducted in accordance with the United States Public Health Service Guide for the Care and Use of Laboratory Animals; all procedures were approved by the Institutional Animal Care and Use Committee (IACUC). Four or five animals per polyacrylic cage were housed with access to food and water ad libitum and were maintained in standard housing conditions, i.e., at room temperature (RT) 24 ± 1°C and humidity 60–65% with 12/12 h light/dark cycle.

Animal groups and treatment

All the Ubc9-Tg mice (males and females) are hemizygous and their wild-type (WT) siblings were used as controls. When mice were aged up to 11–12 months old, 25 μg/g body weight of MPTP (dissolved in 0.9% saline, Sigma) was injected once a day for seven consecutive days (Lazzara et al., 2015). Since MPTP toxicity is strain and age sensitive, the chronic injection to the same age mice was performed at the same time for minimizing the variation of MPTP toxicity (n = 10 per group). In the vehicle group, the same volume of 0.9% saline was injected (n = 10). Seven days after the last injection, mice were deeply anesthetized in an isoflurane chamber and intra-cardiac perfusion was done by using ice-cold 0.9% saline, followed by 150 mm NaCl/70% ethanol. After decapitation, whole brains were isolated. Half of each brain was isolated by brain-region, e.g., the striatum and brain stem, and stored at −80°C for molecular/biochemical analyses including reactive oxygen species (ROS) measurement; the other half brain was postfixed in 4% paraformaldehyde (PFA) for immunohistochemical analysis.

Cell lines with plasmids or RNAi treatment

N27 rat dopaminergic parental cell line (SCC048, EMD Millipore) was used to generate enhanced green fluorescent protein (EGFP) or Ubc9-EGFP-overexpressing (Ubc9-OE) stable cell lines by transfection. Neomycin resistant plasmids containing EGFP (based on pEGFP vectors, Clontech; now Takara Bio USA) or Ubc9-EGFP (a gift from Jo Morris, King’s College, London) were transfected to N27 cells using Lipofectamine 3000 (Thermo) as per the manufacturer’s instructions. All cell lines, including EGFP, Ubc9-OE, and rat dopaminergic parental cell line (N27p), were cultured in RPMI 1640 (Invitrogen) cell culture media along with 10% FBS (Atlanta Bio) and 1% penicillin-streptomycin (Invitrogen) in a 5% CO₂ incubator at 37°C. Forty-eight hours after transfection, G418 (Geneticin, Invitrogen) was added as a selection marker at the concentration of 500–1000 μg/ml for two weeks. Clusters of green fluorescent-positive (EGFP) cells were isolated and plated into 60 mm dishes. G418 (500 μg/ml) was added for another two weeks and then lowered to 200 μg/ml as previously reported (Cartier et al., 2019). To knockdown Ubc9 expression, N27p cells were transfected with dicer substrate siRNAs (RNAi-N013050.12.1 5′-UCCGUACAGUUACUAGUA-3′ 3′-UCAAUGAUCAUCGGACCC-5′, RNAi-N013050.12.3 5′-GUACGAUGAACGUGAUGA-3′ 3′-CUVGGACUACUUGACCCU-5′ or negative control NC1 purchased from IDT using Lipofectamine 3000 (Thermo), according to the manufacturer’s directions. After the exposure overnight, NC1 or Ubc9-RNAi-treated cells were exposed to MPP+ or PFF for 24 h, followed by cellular measurements including cell viability, cytotoxicity, ROS, and thioflavin T staining, etc.

Cell culture and treatment

MPP+ (1-methyl-4-phenylpyridinium, Sigma) treatment was applied at concentrations of 160, 320, 640, and 1280 μm for 24 h. The PFFs of α-syn was purchased from StressMarq Biosciences (SPR-324C) and its treatment was tested at 1, 2, 3, 4, and 5 μg/ml for 24 h. Cells were also treated with cycloheximide (Chx; Sigma), protein synthesis inhibitor (100, 150, and 200 μg/ml) for 24 h and PMA, PKC-mediated lysosomal inducer (2, 4, 6, 8, and 10 μm) for 2 h. Additional experiments were performed using 640 μm MPP+, 150 μg/ml of Chx, and 5 μm PMA for 24-h exposure. There were also co-treatments of chloroquine (lysosomal inhibitor; 10 μm) and MG132 (proteasomal inhibitor; 10 μm), also known as Z-Leu-Leu-Leu-al for 24 h (Cartier et al., 2019).

Cell viability assay

The conversion of MTT dye to soluble formazan is caused by mitochondrial dehydrogenase enzymes in live cells. The formazan is soluble in DMSO and produces purple/blue color that indicates the level of cell viability. The cell viability in the vehicle group of EGFP and Ubc9 cells was calculated as 100% of control. Cell viability was estimated by using the dye 3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide (MTT), following the protocol described by Janezic et al. (2016). A total of 5000 cells were seeded in 96-well plates. After 1 h, cells were treated with different concentrations of MPP+ or PFF (1 μg/ml) for 24 h. MTT (5 μm) dye was added into the culture media and incubated at 37°C for 2 h. The culture medium was removed, and 200 μl of DMSO was added to each well. The purple-colored formazan was dissolved in DMSO and color intensity was measured at 570 nm with a reference wavelength of 630 nm by using a spectrophotometer (SpectraMax M5^e, Molecular devices). The measured optical density is directly correlated with cell viability.

Cytotoxicity assay

By measuring the level of lactate dehydrogenase (LDH; a soluble cytosolic enzyme), released into the culture medium on cell lysis, the level of cytotoxicity was assessed. The cytotoxicity assay was performed by using Pierce LDH cytotoxicity assay kit (88954, Thermo), as per the manufacturer’s protocol. Briefly, after treatment with different concentrations of MPP+ or PFF for 24 h, the cell culture media were collected for LDH assay, and cells were further used for cell viability (MTT) assay. The protein concentration was measured by using Pierce Rapid Gold BCA protein assay kit (A5225, Thermo Fisher Scientific) as per the manufacturer’s protocol. For LDH assay, 50 μl of cell culture media per well was transferred into a new 96-well plate. Then, 50 μl of reaction mixture was added to each well, and a plate was incubated at RT for 30 min. After incubation, 50 μl of stop solution was added to each well. Absorbance changes were measured at 490 and 680 nm by a spectrophotometer (SpectraMax M5e, Molecular Devices). To determine LDH activity (%), 680-nm absorbance values were subtracted from the readings at 490-nm absorbance, and then the normalized values were calculated in the scale of vehicle-treated control.

ROS level measurement

The level of ROS was assessed by using CellROX deep red reagent (C10422, Thermo Fisher Scientific) as the manufacturer’s protocol instructed. Briefly, after treatment of MPP+ or PFF, CellROX (5 μm) was added into the cell culture medium in duplicate and incubated at 37°C for 30 min. The cells were then washed with PBS and images were randomly captured using the EVOS FL Cell Imaging Systems (Invitrogen). After four to five independent experiments (total replicates: n = 8–10), the intensity of red color was analyzed by a blinded rater using ImageJ.

Thioflavin T and α-syn staining

For double staining, up to 4.5 × 104 N27p cells were seeded per well on poly-D-lysine-coated glass coverslips in six-well plates. After transient transfection using Lipofectamine 3000 with Ubc9-RNAi or NC1 treatment overnight, cells were treated with wheat germ agglutinin/N-acetylglucosamine (WGA/GluNAc, Sigma) for PFF (1 μg/ml) penetration as described by Volpicelli-Daley et al. (2011). Briefly, cells were fixed with 4% PFA/PBS solution for 20 min and washed three times in PBS. Blocking and permeabilization was done using 2% bovine serum albumin (BSA) prepared in PBS-T (0.25% Triton X-100) at RT for 1 h. Then, cells were incubated with anti-α-syn primary antibodies (1:250, MABN1817, Sigma-Millipore) at RT for 2 h and washed in PBS three times. The cells were treated with Alexa Fluor 647-conjugated secondary antibodies (1:1000; Molecular probes, Thermo Scientific) at RT for 1 h and washed in PBS three times. For thioflavin T staining, we have followed the procedures from the recent publication (Polinski et al., 2018) with minor modifications. Cells were incubated with 20 μm thioflavin T (Sigma-Aldrich) solution at RT for 30 min and washed in PBS three times. After staining, cell images were randomly captured using fluorescence microscopy (EVOS, Invitrogen) using proper optical filters. As a negative control, the secondary antibody step or the thioflavin T step was omitted. After capturing random cell images, the relative fluorescent intensity of thioflavin T (green) or α-syn (red) was compared with control (WGA/NAcGlu, 100%) for assessing the effect of PFF or Ubc9-RNAi. All the experiments were independently run at least four times in duplicate (n ≥ 8 per group) and three to five images per well were captured for thioflavin T or α-syn. Thus, total images (n = 24–40) per group were analyzed by a blinded rater.

Immunocytochemistry

N27 cells were seeded on poly-D-lysine-coated glass coverslips per well in 6-well plates. Cells were washed in ice-cold PBS without Ca²⁺ or Mg²⁺ (137 mm NaCl, 2.7 mm KCl, 4.3 mm Na₂HPO₄, and 1.47 mm KH₂PO₄) for 5 min three times, followed by fixation with 4% PFA at RT for 20 min and washed in PBS for 5 min three times. Fixed cells were permeabilized with 0.1% Triton X-100 and 0.5% BSA for 3 min, followed by blocking with 5% BSA at RT for 2 h. Then, cells were incubated with mouse anti-α-syn primary antibodies (1:500, MABN1817, Sigma-Millipore) in 1% BSA/PBS at 4°C overnight. On the following day, cells were washed in 0.1% PBS-T (Triton X-100) three times for 5 min each and incubated with secondary goat anti-mouse Alexa Fluor 594 (1:1000; Invitrogen, Thermo Fisher Scientific) at RT for 1 h. Cells were washed in PBS for 3 × 5 min, followed by a brief washing in ultrapure water. Coverslips were mounted on glass slides using ProLong Diamond antifade mounting medium (P36961, Thermo). Images were obtained using Zeiss 780 Multiphoton Confocal microscopy at the imaging core in our institution. All images were acquired with a Plan-Apochromat 40×/1.4 NA Oil DIC objective (Zeiss). Images were processed using Zeiss ZEN software and sampled at optimal pixel density in the X and Y with a line average of 4, and a zoom factor of 1.2. Z-stacks were set up with optimal z-sectioning for optimal overlap and processed into maximum intensity projections.

Immunohistochemistry

A series of total coronal sections throughout the striatum and substantia nigra were sectioned in 16-μm thickness and collected in total four sets for mounting onto positively charged slides (Midwest Sci). For rehydration, slides were immersed into xylene for 5 min twice, followed by washing in serially diluted ethanol (100% for 5 min twice; 95% for 5 min; 70% for 5 min; 50% for 5 min) and rinsed with deionized water. Slides were washed in PBS for 5 min twice. Antigen unmasking was done by using Tris-EDTA Buffer (10 mm Tris-base, 1 mm EDTA solution, and 0.05% Tween 20; pH 9.0) at RT for 15 min and washed in PBS for 5 min three times. After blocking in 2% BSA at RT for 30 min, slides were incubated with anti-tyrosine hydroxylase (TH) antibody (1:1000; AB152, EMD-Millipore) at 4°C overnight and washed in PBS-T for 5 min three times. Then slides were incubated with Alexa Fluor 647-conjugated goat secondary anti-mouse antibody (1:1000; Invitrogen, Thermo Fisher Scientific) at RT for 1 h and washed in PBS-T for 3 × 5 min. As a negative control, the primary antibody step was omitted. Coverslips were mounted on slides using ProLong Diamond antifade mounting medium (Thermo). Images were acquired using LSM 510 confocal microscope with Z-stacks (0.5 μm) which were set up with optimal z-sectioning for optimal overlap at the internal imaging core. All images were acquired with a Plan-Apochromat 40×/1.4 NA oil DIC objective. Images were processed into maximum intensity projections using Zeiss ZEN software. Every fourth section was analyzed for measuring the intensity of TH staining in the striatum and for counting TH+ neurons in the substantia nigra compacta (SNc) in a blinded manner.

α-Syn quantitative mRNA measurement

Using quantitative real-time PCR (qRT-PCR; LightCyler 480II, Roche), the level of α-syn mRNA was determined with β-actin as a housekeeping gene. An equal number of N27 cells overexpressing either Ubc9 or EGFP was collected for RNA extraction using the TRIzol Reagent (Life Technologies). The experimental procedures were repeated, according to the recent report (Cartier et al., 2019) except for the primer information below: the parameters for the reactions were: 95°C for 10 min, followed by 50 cycles of 95°C for 30 s, 60°C for 10 s, and 72°C for 10 s. The fluorescence was recorded during the 72°C step to determine the Crossing point (Cp) value. The primers were chosen based on Primer3Plus software and synthesized by the Integrated DNA Technologies (IDT). The primers are: α-syn forward 5′-GCAGTGAGGCTTATGAAATGC-3′; α-synuclein reverse 5′-AGGCTTCAGGCTCATAGTCTTG-3′; β-actin forward 5′-AGCCATGTACGTAGCCATCC-3′; β-actin reverse 5′-CTCTCAGCTGTGGTGGTGAA-3′. Each trial was performed in triplicate, and three trials were collected for total of nine independent samples for each gene.

Chx protein chase

EGFP or Ubc9 cells were equally plated (1 × 105 cells/plate) on 60-mm dishes and incubated with different concentrations of Chx (100, 150, and 200 μg/ml) in the culture media overnight. Additional experiments were performed using 150 μg/ml Chx at different time points (T = 0, 6, 12, 18, or 24 h). Cells were lysed using 250-μl RIPA buffer [50 mm Tris, 100 nm NaCl, 1% NP-40, 1 mm sodium fluoride (NaF), 2 mm sodium orthovandate (Na₃VO₄), 10 mm NEM, 1 mm PMSF, and 1% protease phosphatase inhibitors] and sonicated briefly on ice for solubilization (3 s on and 10 s off for five cycles). Treatment or vehicle was added to the media at T = 0 h and remained for the rest of chase studies, which was terminated as described previously (Cartier et al., 2019). Cell lysate was centrifuged at 15,000 × g for 10 min, and supernatant was collected and separated by 4–20% SDS-PAGE gels (GenScript). α-Syn was detected by immunoblotting as described below in Immunoblot analyses.

Immunoprecipitation (IP)

After treatment, cells were washed in 1× PBS twice and lysed in 250 μl of RIPA buffer with incubation for 15–20 min. After brief sonication, cell extracts were centrifuged at 15,000 × g for 10 min. The supernatant was transferred into a fresh vial and protein concentration was measured by using Pierce Rapid Gold BCA Protein Assay kit (A53225, Thermo Fisher Scientific). An equal amount of protein (500 μg) was taken for IP assay, and samples were precleared with A/G beads (Santa Cruz Biotech) at 4°C for 1 h. To remove A/G beads, brief centrifugation was followed by collecting the supernatant for transferring to a fresh vial. Then, equal amounts of anti-α-syn antibody (MABN1817, EMD-Millipore) were added to each sample and incubated at 4°C overnight. On the following day, equal amounts of A/G beads were added to each sample. After 2 h of incubation, samples were collected and mixed with non-reducing sample loading buffer (39001, Thermo Fisher Scientific). All Immunoprecipitated samples were separated by 4–20% SDS-PAGE gels (GenScript) along with 5% total lysate as input. SUMO1, ubiquitin and α-syn proteins were detected by immunoblotting as described below in Immunoblot analyses.

Immunoblot analyses

Protein samples were loaded on precast polyacrylamide gel (SurePAGE Bis-Tris, 4–20%, 12 wells; GenScript) and transferred to PVDF membrane (Immobilon-P, EMD-Millipore) using Bio-Rad transfer apparatus. Membranes were incubated with anti-α-syn antibody (1:2000; MABN1817, EMD-Millipore), anti-α-syn antibody for IP samples (1:1000; 610787, BD Biosciences), anti-SUMO1 (1:1000, sc-5308, Santa Cruz Biotech), or anti-ubiquitin (1:1000, sc-8017, Santa Cruz Biotech) at 4°C overnight. Equal loading was determined by stripping and re-probing against glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:10,000; AM4300, Thermo Fisher Scientific). After washing, membranes were incubated with secondary anti-mouse IgG antibodies conjugated with HRP (1:10,000) at RT for 2 h. The PageRuler-prestained protein ladder was used to estimate protein molecular weights on immunoblots (Thermo 26 616). Signals were developed in Immobilon Forte Western HRP substrate (EMD-Millipore) and detected under ChemiDoc iBright CL1000 (Invitrogen). The integrated density (intensity/area) of each band was measured and normalized by GAPDH (or α-syn in IPs) and/or total protein loading labeled by Reversible protein stain kit for PVDF membranes (Thermo, 24585) as a loading control. Immunoblot images were converted into eight-bit gray-scale images and avoided over-saturation. Equal areas corresponding to selected lanes were analyzed on each blot image using ImageJ software (NIH). Splicing was implemented only for clarity purposes and the adjustment was performed using the original larger image.

Sample preparation and data analysis for mass spectrometry

Cell extracts from Ubc9-OE or EGFP N27 cells were IP’ed by α-syn antibodies (MABN1817, EMD Millipore) as described above and then run in 10% SDS-PAGE gel (GenScript). Three pieces per well were dissected based on the size of bands and each piece was separately rinsed once in 200 μl of ddH₂O, twice in 200 μl of 25 mM ammonium bicarbonate in 50% (v/v) acetonitrile, followed by 100 μl of acetonitrile to dehydrate the isolated gel pieces, which were then lyophilized. The dry gel pieces were rehydrated in 150 μl of 25 mM ammonium bicarbonate containing 25 ng/μl trypsin (pH 8.0). After rehydration, additional 100 μl of 25 mm ammonium bicarbonate was added and the gel pieces were incubated at 37°C overnight. Digested samples were desalted using a Harvard apparatus micro-spin column containing C18 resin. The pooled extracts were reduced to dryness and reconstituted in 80% acetonitrile/0.1% formic acid for tims-TOF Pro mass spectrometry (Bruker), which was operated in PASEF mode using Compass Hystar 5.0.37.1. The experiments were repeated three times for quantification (n = 3).

Data files were uploaded to PEAKS X (Bioinformatics Solutions) for processing, de novo sequencing and database search. The sequences were searched based on the UniProt Rattus norvegicus database (37,157 entries) since N27 cells are rat originated. We searched with mass error tolerances of 20 ppm and 0.05 Da for parent and fragment, respectively. Trypsin enzyme specificity, acetylation (N-term), oxidation, and phosphorylation were adopted as variable modifications. Peptides were filtered at an 1% false discovery rate (p < 0.01) in the peptide spectrum match level, and protein filtering was disabled by setting proteins as −log10[P] score at two unique peptides with a requirement for significant peptides.

Chemicals

Most chemicals in this study, such as phorbol 12-myristate 13-acetate (PMA; Sigma P1585), N-ethylmaleimide (NEM; Sigma E3876), Chx (Sigma C7698), chloroquine diphosphate (Sigma C6628), MG132 Z-Leu-Leu-Leu-al (Sigma C2211), lectin also known as WGA (Sigma L9640), N-acetylglucosamine (Sigma PHR1432), MPP+ (Sigma D048), thioflavin T (T3516), MPTP (M0896), NaF (201154), and Na₃VO₄ (567540) were purchased from EMD-Sigma. PFF of α-syn was obtained from StressMarq (SPR-324C) and PMSF (Roche 11359061001), BSA (Fisher bioreagent BP9703), and PBS (Invitrogen 10010031) were used as well. Protease inhibitor used was EDTA-free Halt protease inhibitor cocktail (Thermo Scientific 87785).

Statistical analyses

In most statistical analyses, two-way ANOVA was applied to assess the potential interaction between Ubc9-OE (or Ubc9-RNAi) and toxic treatment (MPP+ or PFF) in Figures 1-4, 9. In Figures 6-8, two-way ANOVA, Tukey’s multiple comparison was also applied to analyze the interaction between Ubc9 and MG132 (or PMA), related to protein degradation. Student’s unpaired t test was applied to assess the Ubc9 effects, compared with EGFP only treatment (Fig. 5B,C). One-way ANOVA, Tukey’s post hoc test was applied to Figure 5E, F. In Extended Data Figure 1-1, one-way ANOVA, Dunnett’s test was adopted to compare different concentrations of treatment with vehicle control. For all studies, p < 0.05 was considered statistically significant (*); GraphPad Prism 8.03 software was used for all data analyses and display. Values are presented as mean ± SEM.

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Figure 1.

Ubc9 expression protects N27 cells from MPP+ or PFF-induced toxicity, enhancing cell viability and reducing cytotoxicity. A, MPP+ exposure reduces the number of viable cells in EGFP cells in a dose-dependent manner, whereas Ubc9 overexpression protects N27 cells from MPP+ toxicity in MTT assay. B, In LDH assay, Ubc9 overexpression reduces the toxic effect derived from MPP+ exposure. C, In MTT assay, Ubc9 knock-down by RNAi exacerbates the cell viability induced by MPP+ exposure, compared with NC1 random cocktail control. D, In LDH assay, Ubc9-RNAi significantly increases the cytotoxicity derived from MPP+ treatment. E, In MTT assay, PFF treatment reduces cell viability in EGFP cells compared with WGA/GluNAc-treated control, while Ubc9 overexpression ameliorates the toxic effect from PFF. F, In LDH assay, PFF-induced cytotoxicity was suppressed by Ubc9 overexpression. G, In MTT assay, Ubc9-RNAi further exacerbates the cell viability induced by PFF treatment, compared with NC1 random cocktail control with WGA/GluNAc. H, In LDH assay, Ubc9 knock-down substantially enhances the cytotoxicity derived from PFF treatment compared with NC1 control. All the treatments were exposed for 24 h, and each dot represents the number of experiments and each experiment was performed in triplicate. Statistical analysis was applied using two-way ANOVA, Tukey’s post hoc test. Scattered dot plots represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (n = 6–15 per group).