Priming mesenchymal stem cells with α-synuclein enhances neuroprotective properties through induction of autophagy in Parkinsonian models

Cell culture

Isolation and culture of bone marrow-derived MSCs were performed as described previously. Flow cytometry analysis revealed that MSCs were positive for CD29, CD44, CD73, and CD105 and were negative for CD45 and MHC class type II (Additional file 2: Figure S2). MSCs were cultured in DMEM (Gibco BRL, Grand Island, NY, USA) supplemented with 10% FBS (Merck, Darmstadt, Germany) and 100 U penicillin–streptomycin (Gibco BRL) at 37 °C under 5% CO2 in a humidified incubator. SH-SY5Y cells were cultured in DMEM (Gibco BRL) supplemented with 10% FBS (Gibco BRL), 100 U penicillin–streptomycin (Gibco BRL) under identical conditions.

α-Syn aggregate preparation and priming conditions

Recombinant α-syn (5 mg/ml in phosphate buffered saline (PBS)) was agitated at 37℃ (1000 rpm) for 5 days. An additional file shows α-syn fibrillary form to use in this study (Additional file 2: Figure S1, Additional file 1). Aggregated protein was briefly sonicated. Briefly, aggregate proteins were incubated at room temperature for 1 h. Unless otherwise stated, MSCs were seeded at a density of 5 × 103 cells/cm2 and cultured in complete DMEM medium until 80% confluent. To induce priming, MSCs were treated with different concentrations of α-syn in DMEM for 1, 3, and 6 h.

Cell proliferation assay

Cell viability was measured using the CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA), in accordance with the manufacturer’s protocol. Briefly, after the cells were incubated with various concentrations of α-syn in DMEM, MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) was added to a final concentration of 0.5 mg/mL. After incubation at 37 °C for 1 h, the plates were centrifuged and the medium was aspirated from each well. Absorbance was measured at 490 nm on an ELISA microplate Versa Max reader (Molecular Devices, Sunnyvale, CA, USA).

Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR)

MSCs were seeded at a density of 1 × 105 cells/cm2. Total RNA was extracted from the MSCs using TRIzol® reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. An equal amount of RNA (1 µg), for each experiment, was reverse-transcribed using amfiRivert cDNA Synthesis Premix (GenDEPOT, Barker, TX, USA). Subsequently, 2 µL cDNA was used as template for qPCR with the amfiRivert 1-Step RT-PCR Kit (GenDEPOT). PCR was performed using 10 pmol primers. Primers for amplification are shown in Additional file 1: Table S1, Additional file 2. Real-time PCR was carried out with the 7300 RT-PCR system (Applied Biosystems, USA). Every assay was performed in triplicate, and all experiments included analysis of GAPDH mRNA levels as internal standard. Relative expression was determined by the Ct method, and levels were expressed as folds relative to the GAPDH mRNA levels.

Western blot analysis

Cells and brain tissues were harvested by scraping and lysed in a buffer containing 50 mM Tris–HCl pH 8.0, 150 mM NaCl, 1% Triton X-100, 1 mM Na3VO4, 1% sodium deoxycholate, 0.1% SDS, 1 μg/ml pepstatin A, 50 mM NaF, 0.5 mM EDTA, 1 mM EGTA, and Protease inhibitor cocktail (Roche, Basel, Switzerland). Equal amounts of protein were resolved on an SDS-PAGE gel and transferred to a nitrocellulose membrane. The membranes were blocked by 5% skim milk for 1 h at 37 °C and then incubated with primary antibodies against BECN1-regulated autophagy protein 1(AMBRA1), microtubule-associated proteins 1A/1B light chain 3B (LC3B), α-syn, BECN, pAKT, AKT, lysosomal-associated membrane protein 1 (LAMP1), ras-related protein 7 (RAB7), transcription factor EB (TFEB) (Cell Signaling Technology, Danvers, MA, USA), and β-actin (Sigma) at 4 °C overnight. Then, the membranes were incubated with the respective secondary antibodies (GeneTex, Irvine, CA, USA) for 2 h at 37 °C. Western blot signals were visualized using the Immobilon ECL Ultra Western HRP Substrate (ECL Plus kit, GE Healthcare, Piscataway, NJ, USA) and captured on an iBright CL1000 Imaging System equipped with iBright Analysis Software (Invitrogen, Carlsbad, CA).

Karyotype analysis

We examined the karyotype of primed MSCs and naïve MSCs in passage 8 using G-banded karyotype analysis. Briefly, metaphase chromosome spreads were prepared from cultures at the designated passages during the exponential phase of growth (65–75% confluence). For naïve MSCs and primed MSCs cultures, 0.1 μg /mL colcemid (Gibco BRL) was added directly to the cultures and incubated for two hours at 37 °C. The cells were subsequently trypsinized, fixed, and mounted on glass slides. The chromosomes were visualized by using modified Wright's staining and analyzed under a light microscope at × 10 and × 100 magnifications. Images of the individual metaphase spreads were captured and karyotyped using an automated imaging system for cytogenetics (CytoVision; Applied Imaging Corporation).

Administration of adeno-associated virus (AAV) vector-mediated overexpression of α-syn

The plasmids for AAV vector production included the construct for the AAV2/7-serotype, the AAV viral vector transfer plasmid, and the pAdvDeltaF6 adenoviral helper plasmid. AAV serotype 2/7 expressing human wild type (WT) α-syn was driven by a human synapsin1 promoter and enhanced by a woodchuck hepatitis virus posttranscriptional regulatory element. Virus was produced by Korea Institute of Science and Technology (Seoul, South Korea). The AAV suspension is finally concentrated by iodixanol density gradient ultrafiltration and sterile filtered. The SYBR Green method was used in AAV titration to count the genome-containing particles in AAV preparations. Animals were injected with 2 µl of human WT α-syn AAV2 (~ 1 × 1013 genome copies per milliliter) into the right SN at a flow rate of 0.5 ml/minute.

Animal study

All procedures were performed in accordance with the Laboratory Animals Welfare Act, the Guide for the Care and Use of Laboratory Animals, and the Guidelines and Policies for Rodent Experiments provided by the Institutional Animal Care and Use Committee at the Yonsei University Health System. Animals were acclimated in a climate-controlled room at a constant 12 h light/dark cycle for 1 week prior to the initiation of the drug administration. To evaluate the effects of MSCs and primed MSCs on α-syn modulation, the mice were randomly divided into the following four groups (n = 6 per group): (1) vector-only (control); (2) AAV-WT α-syn; (3) AAV-WT α-syn with MSCs (naïve MSC group) and (4) AAV-WT α-syn with primed MSCs (primed MSC group). Mice were injected with naïve MSCs (1 × 107cells/kg) or primed MSCs (1 × 107cells/kg) via tail vein on postoperative day 7. All mice were killed 1 month postoperative. All mice were killed 1 month postoperative.

Brain sample preparation

For immunochemical analysis, all mice were deeply anesthetized with chloral hydrate (intraperitoneal injection, 0.4 g/kg; Sigma) and then, perfused with 4% paraformaldehyde (Sigma) in 0.1 M phosphate buffer (pH 7.4). The brains were harvested from the skulls, post-fixed overnight in 4% paraformaldehyde, and stored in 30% sucrose solution for 1–2 days at 4℃ until they sank. Finally, 25-μm coronal sections were obtained using cryostat. The sections were stored in tissue storage solution (30% glycerol, 30% ethylene glycol, 30% distilled water, 10% 0.2 M PB) at 4℃ until required.

Immunohistochemistry

Brain sections were washed twice in PBS and incubated in 0.5% Triton X-100 (Sigma) for 15 min at room temperature. They were blocked with 5% bovine serum albumin (BSA; Sigma) for 30 min. After blocking, they were incubated overnight at 4 °C with specific primary antibodies. The primary antibody was used mouse anti-tyrosine hydroxylase (TH) (Sigma, T2928). The TH antibodies were detected using 0.05% diaminobenzidine (DAB, Vector Laboratories, USA). The immune-stained cells were analyzed using bright-field microscopy and viewed under a Zeiss LSM 700 confocal imaging system (Zeiss, Germany).

Glucose uptake measurement

The assay was performed essentially as described previously (Perrini et al. 2004). The cells were washed thrice with PBS, then incubated in Krebs–Ringer phosphate buffer (KRP, 1.32 mM NaCl, 4.71 mM KCl2, 47 mM CaCl2, 1.24 mM MgSO4, 2.48 mM Na3PO4, 10 mM HEPES (pH 7.4)) for 10 min at 37 8C, then 0.5 mCi/ml 2-DOG as the final concentration was added to the cells. After 10-min incubation, the medium was aspirated and the cells were washed thrice with ice-cold KRP containing 10 mM glucose to terminate the reaction. The cells were lysed with 0.1 M NaOH, and the radioactivity taken up by the cells was determined using a scintillation counter (Beckman Instruments, Fullerton, CA, USA). The d.p.m. value was corrected by protein content in each well which was measured using a BCA protein assay kit.

L-lactate assay

Intracellular lactate levels were measured using a colorimetric L-lactate assay kit (Abcam, ab65330), according to the manufacturer’s instructions. Cell lysates were deproteinized to eliminate the endogenous lactate dehydrogenases (LDH) and then, incubated in the presence of the lactate probe and enzyme mix at room temperature for 30 min. Absorbance was measured at 570 nm using a microplate reader.

Data and statistical analysis

The data and statistical analysis comply with the recommendations of the British Journal of Pharmacology on experimental design and analysis in pharmacology [21]. The group means were compared using the Mann–Whitney U-test for pairs and the Kruskal–Wallis analysis for multiple groups. P values less than 0.05 were considered statistically significant. Statistical analyses were performed using the commercially available software SPSS (version 12.0).

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