Antibodies and reagents
Protein phosphatase inhibitor and protease inhibitor cocktails were purchased from Sigma–Aldrich. Antibodies for ClpX (ab168338, 1:2000), ClpP (ab124822, 1:1000), VDAC (ab34726, 1:1000), α-Synuclein (ab27766, 1:1000), α-Synuclein (ab138501, 1:3000) and α-Synuclein phosphor S129 (ab168381, 1:1000) were from Abcam. GFP (sc-9996, 1:1000), c-Myc (sc-40, 1:1000), α-Synuclein (sc-12767, 1:1000), Enolase (sc-15343, 1:1000) and HSP60 (sc-13115, 1:2000) were from Santa Cruz Biotechnology. β-Actin (A1978, 1:10000) was from Sigma–Aldrich. TH (MAB318, 1:1000) was from Millipore. ClpP (GTX115070, 1:200) was from Genetex. ClpP (NBP1-89557, 1:200) was from Novus. LonP (15440-1-AP, 1:2000), ERAL1 (11478-1-AP, 1:2000), CHCHD3 (25624-1-AP, 1:1000) and WFS1 (11558-1-AP, 1:1000) were from Proteintech. SOD2 (611580, 1:1000) was from BD bioscience. MFN1 (H00055669-M04, 1:1000) was from Abnova. Sirt3 (5490S, 1:1000) was from cell signaling technology.
SH-SY5Y cells stably expressing GFP, GFP-αSyn WT or GFP-αSyn A53T were cultured in DMEM/F12 (1:1) supplemented with 10% FBS, 100 µg/ml penicillin, 100 µg/ml streptomycin, and 400 µg/ml G418. Cells were grown at 37 °C in a 5% CO2 incubator. HEK293 and HeLa cells were maintained in DMEM supplemented with 10% FBS and 1% (v/v) penicillin/streptomycin.
Induced pluripotent stem cells and neuronal differentiation
PD iPS cells lines (αSyn A53T, NN0004337) and its isogenic corrected control line (NN0004344) were obtained from RUCDR Infinite Biologics. The iPS cells were differentiated into dopaminergic neuron-enriched neuronal culture with the protocol described previously [60, 80]. Briefly, iPS cell colonies were disassociated with accutase (Invitrogen), plated onto six-well plates pre-coated with 2.5% Matrigel (BD Biosciences) and allowed to reach 80% confluence in mTeSR medium (Stem Cell Technology). For the first 7 days, cells were treated with SB431542 (10 uM; Tocris Bioscience) and Noggin (100 ng/ml) in Neural Media (NM) with FGF2 (20 ng/μl) and EGF (20 ng/μl). NM media contained: Neurobasal and DMEM/F12 (1:1), B-27 Supplement Minus Vitamin A (50×, Invitrogen), N2 Supplement (100×, Invitrogen), GlutaMAX (Invitrogen, 100×), 100 units/ml penicillin and 100 μg/ml streptomycin (Fisher); for the next 4 days, cells were treated with human recombinant Sonic hedgehog (SHH, 200 ng/ml) in neuronal differentiation medium. Neuronal differentiation medium contained Neurobasal and DMEM/F12 (1:3), B27, N2, GlutaMax and PS. In the following 3 days, cells were switched to BDNF (20 ng/ml), ascorbic acid (200 uM, Sigma–Alderich), SHH (200 ng/ml), and FGF8b (100 ng/ml) in neuronal differentiation medium. Thereafter, cells were treated with BDNF, ascorbic acid, GDNF (10 ng/ml), TGF-b (1 ng/ml), and cAMP (500 uM, Sigma–Aldrich). All growth factors were purchased from Pepro Tech (Rocky Hill, NJ, USA). Neurons were passed onto fresh plates after 20 days of induction, and at 40 days after differentiation, the cells were passed on the cover slides coated with poly-d-lysine (50 ug/ml) and Laminin (5 ug/ml), and were fixed for immunostaining or mitochondrial function assays.
For immunostaining assay, the cells were fixed and stained with TH (AB152, 1:500, Millipore) (MAB318, 1:500, Millipore), Tuj1 (MMS-435P, 1:500, Covance/801201, 1:500, Biolegend), MAP2 (4542, 1:1000, Cell Signaling), and αSyn pS129 (825701, 1:5000, Biolegend). The imaging was observed by confocal microscope (Fluoview FV100, Olympus). To analyze general neurite length, cells were stained with anti-Tuj1 (a mature neuronal marker) and anti-TH (a DA neuronal marker). To analyze dendrite length of neurons, cells were stained with anti-MAP2 (a dendritic marker) and anti-TH. The length of MAP2+ dendrite and Tuj1+ neurite in the neurons immuno-positive for TH was measured by NIH Image J with plugins simple neurite tracer.
Isolation of subcellular fractions
Cells were washed with cold PBS and incubated on ice for 30 min in a lysis buffer (250 mM sucrose, 20 mM HEPES–NaOH, pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, protease inhibitor cocktail and phosphatase inhibitor cocktail). Mouse brains were minced and homogenized in the lysis buffer and then placed on ice for 30 min. Collected cells or tissue were disrupted 20 times by repeated aspiration through a 25-gauge needle, followed by a 30-gauge needle. The homogenates were spun at 800 g for 10 min at 4 °C, and the resulting supernatants were spun at 10,000 g for 20 min at 4 °C. The pellets were washed with lysis buffer and spun at 10,000 g again for 20 min at 4 °C. The final pellets were suspended in lysis buffer containing 1% Triton X-100 and were mitochondrial-rich lysate fractions. The supernatant was cytosolic fractions. The mitochondrial proteins VDAC, the ER protein WFS1, and the cytosolic protein Enolase were used as loading controls for mitochondrial, ER, and cytosolic fractions, respectively. The mitochondrial-rich lysate fractions were then spun at 10,000 g for 20 min and the supernatant was triton-soluble mitochondrial fraction. The pellets were washed twice using mitochondrial lysis buffer containing 1% Triton X-100 and suspended in the lysis buffer containing 1% Triton X-100 and 1% SDS, followed by incubation at 100 °C for 5 min. The final solution was triton-insoluble mitochondrial fraction.
Preparation of triton-soluble and -insoluble fraction
Cells were washed with cold PBS and incubated on ice for 30 min in total cell lysate buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, and protease inhibitor). Collected cells were spun at 12,000 rpm for 10 min at 4 °C; the resulting supernatant was triton-soluble fraction. The pellet was further suspended in total lysis buffer with 1% SDS, and incubated at 100 °C for 3 min, and followed by 20-s vortex. After two more repeats of 100 °C incubation and vortex, the solution was spun at 12,000 rpm for 10 min at 4 °C; the resulting supernatant was triton-insoluble fraction.
Preparation of digitonin-soluble and -insoluble fraction
Cells were washed with cold PBS and incubated on ice for 30 min in lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, protease inhibitor cocktail) containing 0.5% or 1% digitonin. Mouse brains were minced and homogenized in the lysis buffer and then placed on ice for 30 min. Collected cells or tissue were spun at 20,000 g for 20 min at 4 °C; the resulting supernatant was digitonin-soluble fraction. The pellet was further suspended in digitonin lysis buffer with 1% SDS, and incubated at 100 °C for 3 min, and followed by 20-s vortex. After two more repeats of 100 °C incubation and vortex, the solution was spun at 20,000 g for 20 min at 4 °C; the resulting supernatant was digitonin-insoluble fraction.
In vitro fractionation of soluble and insoluble proteins
Recombinant ClpP protein is a gift from Dr. Aaron Schimmer (Princes Margartet Cancer Centre, Canada), and recombinant WT and A53T α-synuclein were purchased from rPeptide. Recombinant ClpP protein (0.5 mg/ml) was incubated with either WT or A53T α-synuclein or protein CHCHD3 (3 mg/ml) in PBS buffer containing 20 mM NaPO4 pH 7.4, 140 mM NaCl, at 37 °C for 8 h. Insoluble aggregates were separated by centrifugation at 10,000 g for 10 min. The supernatant containing soluble protein was transferred to a fresh Eppendorf tube. The pellet fraction was resuspended in PBS and centrifuged twice more. The pellet fraction was finally resuspended in PBS containing 1% SDS.
Constructs and transfection
GFP-tagged αSyn-WT and -A53T plasmids (#40822, #40823), pBI-EGFP-MnSOD (#16612) were obtained from Addgene. To construct Myc-tagged αSyn-WT and –A53T, pCMV-Myc was digested with SalI and KpnI, and αSyn-WT or –A53T was PCR-amplified and inserted into the plasmid backbone. To construct Myc-tagged ClpP plasmid, pCMV-Myc was digested with EcoR1 and Xho1, and ClpP was PCR-amplified and inserted into the plasmid backbone. ClpP point mutant (S153A) was constructed using QuickChange II Site-Directed Mutagenesis Kit (200524, Agilent Technology). Cells were transfected with TransIT-2020 (Mirus Bio, LLC) following the manufacturer’s protocol.
To construct AAV-ClpP, human ClpP cDNA was inserted into the plasmid backbone AAV5.hSyn.eGFP.WPRE.bGH (Cat# AV-5-PV1696) which was obtained from Penn Vector Core, University of Pennsylvania. AAV-GFP-ClpP and AAV-GFP control were then packed to obtain AAVs in the same core facility.
To construct Lenti-ClpP, human ClpP cDNA was inserted into the plasmid backbone pHR-IG (pHR’tripCMV-IRES-eGFP) (53597, Addgene). Lenti-ClpP and lenti-control were then packed to obtain lentivirus for the infection as previously described .
Measurement of cell viability
SH-SY5Y cells were transfected with human ClpP siRNA or control siRNA for 3 days. After then, the cells were cultured in FBS-free DMEM/F12 (1:1) medium for 24 h. Neurons derived from iPS cells carrying αSyn A53T and its corrected isogenic control were cultured in neuronal differentiation medium (GDNF, ascorbic acid, TGFβ, cAMP) without BDNF for 24 h. Cell death was determined by measuring LDH release into the culture medium, using LDH-Cytotoxicity Assay Kit II (Roche, USA, 04744926001) by following the manufacturer’s instruction.
For silencing ClpP in cells, control siRNA and ClpP siRNA were purchased from Thermo Fisher Scientific. HEK293, HeLa and SH-SY5Y cells were transfected either with control siRNA or Clpp siRNA using TransIT-TKO Transfection Reagent (Mirus Bio, LLC, MIR 2154), according to the manufacturer’s instructions. The sequences for the siRNAs used in this study are as follows: ClpP siRNA, 5′-AAACAGAGCCUGCAGGUGA-3′; non-targeting control siRNA, 5′-TTCTCCGAACGTGTCACGT-3′.
ClpP peptidase activity
Human recombinant ClpP (10 µM, obtained from Dr. Aaron Schimmer, Princes Margartet Cancer Centre, Canada) was incubated in the reaction buffer (50 mM Tris pH 8.0, 200 mM KCl, 1 mM DTT, 2 mM ATP) under 37 °C for 10 min. For co-incubation with α-synuclein, recombinant ClpP and αSyn-WT or -A53T were pre-incubated for 30 min under room temperature. Fluorescent substrate of ClpP, ac-WLA-AMC (50 µM), was then added in the reaction buffer. The fluorescence signal was read using TECAN infinite M1000 up for 30 min at excitation/emission wavelength 345/445. ClpP peptidase activity was determined as the slope of the regression line.
Measurement of the amount of mitochondrial misfolded/unfolded proteins
Mitochondrial fractions were isolated as described above. Isolated mitochondria were suspended in PBS with 1% Triton X-100. A mixture of 40 µg mitochondrial protein and 50 µM TPE-MI dye (obtained from Dr. Yuning Hong, La Trobe University, Australia) in a final volume of 50 µl was added into a 96-well plate for fluorescence reading up to 2 h. The load of mitochondrial unfolded protein was represented as the highest relative fluorescence unit (RFU) in each group, as described in .
Measurement of mitochondrial protein oxidation
Mitochondrial fractions were isolated and prepared as described above. 40 µg of mitochondrial protein in each group was subjected to protein oxidation determination using OxyBlot™ Protein Oxidation Detection Kit (Millipore, S7150) by following the manufacturer’s instruction.
Measurement of mitochondrial respiratory activity
The SH-SY5Y cells, iPS cell colonies or neurons derived from patient iPS cells or isogenic control were seeded in XFp 8-well miniplates at 2000 cells per well in 50 µl of growth medium. Three days after transfection of ClpP siRNA or control siRNA, mitochondrial respiration activity in intact cells was analyzed using a Seahorse Bioscience XFp Extracellular Flux Analyzer. Briefly, 1 h prior to measuring oxygen consumption, the cell culture media were replaced with XF assay medium and maintained in a non-CO2 incubator for 1 h. The sensor cartridges were placed in the XFp Analyzer according to the manufacturer’s instructions for the Mito Stress Test kit. Mitochondrial function was determined by the sequential injection of oligomycin A (1 µM), FCCP (1 µM) and antimycin A (0.5 µM).
Mitochondrial ROS measurement
Cells cultured on coverslips or 24-well plates were washed with PBS and then incubated with 5 µM MitoSOX™ Red (Invitrogen, M36008), a mitochondrial superoxide indicator, for 10 min at 37 °C. For cells cultured on coverslips, the images were visualized by microscope, and quantification of images was then carried out using NIH ImageJ software. At least 100 cells per group were counted in the analysis. For cells cultured on 24-well plates, MitoSOX intensity was measured (510 nm excitation/580 nm emission) by infinite M1000 multimode fluorescence plate reader (Tecan, Switzerland). The MitoSOX fluorescence density was normalized to the fluorescence density of DAPI (355 nm excitation/460 nm emission) which stained the nuclei.
Total RNA was isolated using RNeasy Mini Kit (QIAGEN), and 0.5–1 µg of total RNA was used to synthesize cDNA using QuantiTect Reverse Transcription Kit (QIAGEN). qRT-PCR was performed using QuantiTect SYBR Green (QIAGEN) and analyzed with the StepOnePlus Real-Time PCR System (Thermo Fisher Scientific). Three replicates were performed for each biological sample, and the expression values of each replicate were normalized against GAPDH cDNA using the 2−ΔΔCTmethod. The primers used were as follows: ClpP forward 5′-TTGCCAGCCTTGTTATCGCA-3′, ClpP Reverse 5′-GGTTGAGGATGTACTGCATCG-3′; GAPDH forward 5′-GGAGCGAGATCCCTCCAAAAT-3′, GAPDH reverse 5′-GGCTGTTGTCATACTTCTCATGG-3′.
Cells were lysed in a total cell lysate buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, and protease inhibitor). Total lysates were incubated with the indicated antibodies overnight at 4 °C followed by the addition of protein A/G beads for 2 h at 4 °C.
Recombinant α-Synuclein and Clpp (500 ng) were incubated in in vitro interaction buffer (20 mM Tris–HCl pH 7.5, 100 mM KCl, 2 mM MgCl2 and 0.1% Triton-X100) for 30 min at room temperature, and then incubated with indicated antibodies overnight at 4 °C followed by the addition of protein A/G beads for 2 h. Immunoprecipitates were washed four times with cell lysate buffer/in vitro interaction buffer in the presence of 0.1% Triton X-100 and were analyzed by western blot analysis.
Western blot analysis
Protein concentrations were determined by Bradford assay. Protein was resuspended in Laemmli buffer, loaded on SDS–PAGE, and transferred onto nitrocellulose membranes. Membranes were probed with the indicated antibodies, followed by visualization with ECL.
Animal model of PD
All animal experiments were conducted in accordance with protocols approved by the Institutional Animal Care and Use Committee of Case Western Reserve University and were performed based on the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Sufficient procedures were employed for reduction of pain or discomfort of subjects during the experiments.
αSyn A53T [B6.Cg-Tg(Prnp-SNCA*A53T)23Mkle/J, JAX Stock No: 006823] breeders (C57Bl/6J genetic background) were purchased from Jackson Laboratories. The mice were mated, bred, and genotyped in the animal facility of Case Western Reserve University. Male mice at the ages of 4, 6, 8, and 10 months were used in the study. All mice were maintained at a 12-h light/dark cycle (on 6 am, off 6 pm).
Stereotaxic surgery was performed using a model 1900 stereotax (Kopf) under isoflurane anesthesia. Briefly, a small craniotomy was made using a 33-gauge drill bit above the desired coordinate. A small pulled glass pipette containing AAV was attached to a Nanoject II (Drummond) and was then inserted to the appropriate depth. Injections were performed at a rate of 90 nl/min. The coordinates used for substantia nigra injections were anteroposterior, − 3.0 mm from bregma; mediolateral, 1.2 mm; dorsoventral, 4.3 mm). The concentration (ddTiter) of the virus is 6.49e13 GC/ml. A volume of 200 nL of AAV5.hSyn.eGFP.ClpP.WPRE.bGH or AAV5.hSyn.eGFP.WPRE.bGH was injected into two hemispheres of mice. As a result, the number of viral particles of AAV is 1.298e7.
Mice were deeply anesthetized and transcardially perfused with 4% paraformaldehyde in PBS. Frozen brain sections (10 μm, coronal) were used for immunofluorescence staining of ClpP (1:200, Novus, NBP1-89557), TH (1:1000, Milliplore, MAB318), αSyn pS129 (1:10,000, BioLengend, 825701). For histochemical analysis of postmortem brains of normal subjects and PD patients, paraffin-embedded sections (5 µm) were stained with anti-ClpP and/or anti-TH antibodies. Quantitation of immunostaining was conducted using NIH image J software. The same image exposure times and threshold settings were used for sections from all groups. An experimenter blinded to the experimental groups conducted the quantitation.
Behavioral analysis in PD mice
All behavioral analyses were conducted by an experimenter who was blinded to genotypes and treatment groups. Open-field locomotion movement activity was assessed in αSyn A53T mice and age-matched wild-type littermates at the age of 6 months prior to AAV injection, and the age of 7–10 months after AAV injection (once per month). The mice were placed in the center of an open-field chamber (Omnitech Electronics, Inc) and allowed to explore while being tracked by an automated beam system (Vertax, Omnitech Electronics Inc). A 24-h locomotor activity analysis was performed. The body weight and survival rate of αSyn A53T mice and wild-type littermates were recorded throughout the study period.
Sample sizes were determined by a power analysis based on pilot data collected by our labs or published studies. In the cell culture studies, we performed each study with at least three independent replications. For all of the animal studies, we ensured randomization and blinded conduct in experiments. For all imaging analyses, an observer who was blind to the experimental groups conducted the quantitation.
Data were analyzed by Student’s t test or one-way ANOVA with post hoc Tukey’s test for comparison of multiple groups. Data are expressed as mean ± SEM. Statistical significance was considered achieved when the value of p was < 0.05.