Human neuroblastoma SH-SY5Y cells were obtained from the American Type Culture Collection (ATCC) and maintained in Dulbecco's modified Eagle's medium (Lonza/BioWhittaker) containing 15% FBS, 4 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (Gibco; Thermo Fisher Scientific, Inc.) at 37°C in a 5% CO₂ incubator.

Dichlorofluorescein diacetate (DCF-DA), methyl-4-phenyl-pyridinium (MPP⁺) and 1-methyl-4-phenyl-1,2,3,6-tetrahydro pyridine (MPTP) were obtained from Sigma-Aldrich; Merck KGaA. Tat peptide was synthesized from Peptron, Inc. Enhanced chemiluminescence agent was purchased from Amersham; Cytiva. Antibodies were obtained from Cell Signaling Technology, Inc. Tyrosine Hydroxylase (TH) was purchased from Santa Cruz Biotechnology, Inc. All other chemicals and reagents, unless otherwise stated, were of the highest analytical grade available.

Tat-AR protein was prepared as described in a previous study (28). To develop a therapeutic protein, a human AR gene fused with a Tat PTD to produce a cell permeable Tat-AR expression vector which contains 6X His, Tat PTD (RKKRRQRRR) and the AR gene. Bovine serum albumin was used as a standard and the purified Tat-AR protein concentration was measured by Bradford assay (29).

To examine the Tat-AR protein transduction efficiency, SH-SY5Y cells were exposed to various concentrations of Tat-AR and AR protein (0.5-3 µM) for 1 h. The SH-SY5Y cells were exposed of Tat-AR and AR protein (3 µM) for various periods of time (15-60 min). The cells were then washed with PBS and treated with trypsin-EDTA (Gibco; Thermo Fisher Scientific, Inc.). The intracellular stability of Tat-AR protein was also determined. To confirm the stability of Tat-AR protein, the cells were further cultured (1-30 h) following transduction. The levels of transduced proteins were measured by western blot analysis using anti-histidine antibody.

Cell lysates were prepared with RIPA lysis buffer containing a cocktail of protease inhibitors (Elpis-Biotech, Inc.). The protein concentration was measured using the Bradford method. Equal amounts of each protein (30 µg) were loaded into 12% SDS-PAGE and electrotransferred to a polyvinylidene difluoride (PVDF) membrane (Thermo Fisher Scientific, Inc.). The membrane was blocked with TBS-T (25 mM Tris-HCl, 140 mM NaCl, 0.1% Tween-20, pH 7.5) buffer containing 5% non-fat dry milk for 1 h at room temperature. After washing with TBST, the membrane was incubated with the indicated primary antibodies overnight at 4°C and followed by incubation with horseradish peroxidase-conjugated secondary antibodies for 1 h at 37°C. His (1:5,000; sc-804; Santa Cruz Biotechnology, Inc.), JNK (1:1,000; #9258), p-JNK (1:1,000; #9251), ERK (1:2,000; #9102), p-ERK (1:2,000; #4376), p38 (1:2,000; #9212), p-p38 (1:2,000; #4631), Bcl-2 (1:1,000; #2876), Bax (1:1,000; #2772), caspase-3 (1:1,000; #9662), cleaved caspase-3 (1:1,000; #9661), β-actin (1:5,000; #4967), and appropriate secondary antibodies (1:10,000; #7074). All of the above-mentioned antibodies were purchased from the Cell Signaling Technology, Inc. The membrane was then washed with TBST buffer 3 times and the protein bands were identified using chemiluminescent reagents as recommended by the manufacturer (Amersham; Cytiva). Bands were quantified using ImageJ software (version 1.48; National Institutes of Health) (25,30).

To determine the intracellular distribution of transduced Tat-AR protein in SH-SY5Y cells, confocal fluorescence microscopy was performed, as previously described (24). SH-SY5Y cells were placed on coverslips and treated with 3 µM Tat-AR protein 1 h. The cells were washed with PBS twice and fixed with 4% paraformaldehyde for 5 min. The cells were treated in PBS containing 3% bovine serum albumin, 0.1% Triton X-100 (PBS-BT) at room temperature for 30 min and washed with PBS-BT. Histidine primary antibody (sc-804; Santa Cruz Biotechnology, Inc.) was diluted 1:1,500 and incubated at room temperature for 3 h. Alexa Fluor 488-conjugated secondary antibody (#32723; Invitrogen; Thermo Fisher Scientific, Inc.) was diluted 1:1,500 and incubated in the dark for 1 h at room temperature. Nuclei were stained with 1 µg/ml DAPI (Roche Applied Science, Mannheim, Germany) for 2 min at room temperature. The stained cells were analyzed by confocal fluorescence microscopy using a confocal laser-scanning system (Bio-Rad MRC-1024ES, Bio-Rad Laboratories, Inc.).

Cell viability was measured by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, as previously described (24). The SH-SY5Y cells were pre-treated with Tat-AR (0.5-3 µM), AR (0.5-3 µM) and Tat peptide (0.5-3 µM) for 1 h and MPP⁺ (5 mM) was then added to the culture medium for 13 h. The absorbance was measured at 570 nm using an ELISA microplate reader (Labsystems Multiskan MCC/340; Thermo Fisher Scientific, Inc.) and the cell viability was defined as the percentage of untreated control cells.

To examine whether transduced Tat-AR proteins protect against MPP⁺-induced DNA damage in cells, the SH-SY5Y cells were pre-treated with Tat-AR (3 µM), AR (3 µM) and Tat peptide (3 µM) for 1 h and MPP⁺ (5 mM) was added to the culture medium for 16 h. TUNEL staining was performed using a Cell Death Detection kit (Roche Applied Science). Nuclei were stained with DAPI (1 µg/ml) for 2 min at room temperature. Fluorescent images were obtained using a fluorescence microscope (Nikon eclipse 80i, Nikon Corporation) and cells exhibiting fluorescence were counted under a phase contrast microscope (×200 magnification; Nikon Corporation), as previously described (24,25).

Intracellular ROS levels were determined by 2′,7′-dichlorofluorescein diacetate (DCF-DA) staining as previously described (28,31). To deter-mine the effects of Tat-AR protein against MPP⁺-induced intracellular ROS production in SH-SY5Y cells, the cells were placed on coverslips in 24-well plates, incubated for 12 h, and washed twice with PBS. After pretreated with Tat-AR, AR, and Tat peptide (3 µM) for 1 h, MPP⁺ (5 mM) was added to the culture medium for 1 h. The cells were washed with PBS and incubated for 30 min with DCF-DA (10 µM). Protein was separately processed as mentioned above to obtain the fluorescent image and fluorescence intensity. One was used to obtain a fluorescent image. To obtain fluorescent images for each well, the cells were washed with PBS, mounted, and the cell images were obtained using a fluorescence microscope (Nikon eclipse 80i, Nikon Corporation).

The other was used to obtain the fluorescence intensity. To detect the fluorescence intensity for each well, the cells were collected and washed with PBS. After added 300 µl of PBS buffer and resuspended the cells, the cells (100 µl) were transferred into 96-well plate reader. The fluorescence intensity of the samples was measured using a Fluoroskan ELISA plate reader (Labsystems Diagnostics Oy) at an excitation wavelength of 485 nm and an emission wavelength of 538 nm.

Male C57BL/6 mice (total, n=35), 8 weeks old (weighing 16-20 g), were acquired from the Hallym University Experimental Animal Center. They were housed at 23°C and a humidity of 60%. They were exposed to regulated 12 h cycles of light and dark and were provided with ad libitum access to food and water. The procedures for the care of animals conformed to the Guide for the Care and Use of Laboratory Animals of the National Veterinary Research and Quarantine Service of Korea and approved by the Institutional Animal Care and Use Committee of Soonchunhyang University (SCH16-0051).

To determine whether transduced Tat-AR protein protects against PD, the mice were divided into 5 groups (n=7/each group) as follows: The normal control, MPTP-treated, Tat-AR-treated, control AR-treated and Tat peptide-treated groups. The mice received 4 injections of MPTP (20 mg/kg) at 2-h intervals. Mice were intraperitoneally (i.p.) injected with Tat-AR (2 mg/kg) 12 h prior to MPTP treatment. At 1 week after the final injection, the animals were deeply anesthetized with a mixture of 3% isoflurane (Baxter Healthcare Corporation) in 33% oxygen and 67% nitrous oxide. The brains of these animals were then harvested for immunohistochemistry.

Immunohistochemistry was performed as described in a previous study (31). The frozen and sectioned midbrains were prepared and fixed with 4% paraformaldehyde for 10 min. For removal of non-specific immunoreactivity, free-floating sections were first incubated with 0.3% Triton X-100 and 10% normal goat serum in PBS for 1 h at room temperature. They were then incubated with a rabbit anti-tyrosine hydroxylase (TH) monoclonal antibody (diluted 1:200; sc-14007; Santa Cruz Biotechnology, Inc.) for 48 h at 4°C and sequentially incubated with a biotinylated goat anti-rabbit IgG (diluted 1:250; BA-1000; Vector Laboratories, Inc.) for 2 h at room temperature. The sections were then visualized with 3,3-diaminobenzidine (DAB) (40 mg DAB, 0.045% H₂O₂ in 100 ml PBS) mounted on gelatin-coated slides. To detect viable cells, cresyl violet (0.1%, Sigma-Aldrich; Merck KGaA) counterstaining for Nissl bodies was conducted for 20 min at room temperature following TH immunostaining. The sections were visualized with 3,3′-diaminobenzidine in 0.1 M Tris buffer and mounted on gelatin-coated slides. Images were captured and analyzed using an Olympus DP72 digital camera and a DP2-BSW microscope digital camera software. Figures were prepared using Adobe Photoshop 7.0. The manipulation of images was restricted to threshold and brightness adjustments applied to the entire image. The images shown are representatives from each group and the sections were processed and analyzed by a blinded observer. To establish the specificity of the immunostaining, a negative control test was carried out with pre-immune serum instead of the primary antibody. The negative control resulted in the absence of immunoreactivity in any structures.

For the quantification of TH immunostaining, a cell count was performed. TH immunostaining images (10 sections/mouse) were captured in the same region. Images were sampled from at least 5 different points within each SN section. Thereafter, the number of TH-positive cells was actu-ally counted within the sampled images. All immunoreactive cells were counted regardless the intensity of labeling. Cell counts were performed by 2 different investigators who were blind to the classification of the tissues.

Data are expressed as the means ± SEM of 3 experiments. Differences between groups were analyzed by ANOVA followed by a Bonferroni's post-hoc test (using GraphPad Prism 8; GraphPad Software, Inc.). Statistical significance was considered at P<0.05.

To produce cell-permeable Tat-AR protein, the Tat-AR protein expression vector was constructed by subcloning the cDNA encoding human AR into a pET-15b plasmid containing a Tat PTD. Tat-AR protein expression vector contained a continuous cDNA sequence encoding human AR, a Tat PTD and 6 histidines. In addition, a control AR expression vector containing no Tat PTD was constructed (Fig. 1A). The purified Tat-AR and AR proteins are presented in Fig. 1B. To investigate the transduction efficiency of Tat-AR protein, SH-SY5Y neuro-blastoma cells were treated with Tat-AR protein (0.5-3 µM) for 1 h or with Tat-AR protein at 3 µM for 15-60 min. Tat-AR protein was transduced into the cells in a concentration- and time-dependent manner (Fig. 2A and B). Tat-AR protein was also transduced into the cytosol and nucleus of the cells (Fig. 2C). Since stability is one of the major factors in protein therapy, the stability of the transduced Tat-AR protein was examined. Transduced Tat-AR protein persisted until 12 h in the cells (Fig. 2D). These results indicate that Tat-AR protein can be efficiently transduced into the SH-SY5Y cells and can exist for at least 12 h in the cells.

It is known that MPP⁺ can induce ROS production in dopaminergic neuronal cells, and can cause DNA damage and cell death (32). Thus, the protective effects of transduced Tat-AR protein against MPP⁺-induced cell death were examined. Cell viability was 53% in the cells exposed only to MPP⁺. However, cell viability was markedly increased up to 84% in the cells treated with Tat-AR protein following exposure to MPP⁺. By contrast, AR protein and Tat peptide failed to prevent cell death under the same experimental conditions (Fig. 3A).

The protective effects of Tat-AR protein against MPP⁺-induced DNA damage and intracellular ROS production were also determined by TUNEL and DCF-DA staining, respectively (Fig. 3B and C). DNA damage and intracellular ROS production levels were increased in the cells exposed only to MPP⁺. However, DNA damage and intracellular ROS production levels were markedly inhibited in the Tat-AR protein-treated cells following MPP⁺ exposure. However, AR protein and Tat peptide failed to inhibit DNA damage and intracellular ROS production. These results indicate that Tat-AR protein can inhibit SH-SY5Y cell death by decreasing DNA damage and intra-cellular ROS production, functioning as an antioxidant in the cells.

MPP⁺ can trigger mitogen-activated protein kinase (MAPK) signaling pathway activation (9,33,34). Thus, the effects of Tat-AR proteins on MPP⁺ induced MAPKs signaling pathways were determined in the present study. In the SH-SY5Y cells exposed to MPP⁺, the expression levels of phosphorylated MAPKs were higher than those in the control cells. By contrast, Tat-AR protein significantly decreased expression the levels of phosphorylated MAPKs in the cells exposed to MPP⁺. However, the expression levels of phosphorylated MAPKs in the cells treated with AR protein or Tat peptide were similar to those in the untreated cells exposed to MPP⁺ (Fig. 4).

It is well known that the neurotoxin, MPP⁺, can cause the overproduction of ROS in cells and activate apoptosis signaling pathways (32). Thus, effects of Tat-AR proteins on the MPP⁺-induced expression levels of Bax, Bcl-2 and caspase 3 were investigated. As shown in Fig. 5A, the Bcl-2 expression levels were decreased in the MPP⁺-exposed SH-SY5Y cells. By contrast, Tat-AR protein significantly increased the Bcl-2 expression levels in cells the exposed to MPP⁺. However, the expression levels of Bax exhibited opposite results from those of Bcl-2. Tat-AR protein also markedly increased the expression of caspase-3 in the MPP⁺-exposed cells. The cleaved caspase-3 expression levels were significantly decreased in the cells exposed only to MPP⁺. In addition, Tat-AR protein reduced the cleaved Caspase-3/Caspase-3 ratio in MPP⁺ treated cells. AR protein and Tat peptide failed to affect the expression levels of caspase-3 and cleaved caspase-3 proteins induced by MPP⁺ (Fig. 5B). These results indicate that transduced Tat-AR protein can prevent SH-SY5Y cell death from MPP⁺ by regulating phosphorylation levels of MAPKs and apoptosis-related protein expression.

After Tat-AR protein (2 mg/kg) was i.p. injected into the mice, immunohistochemistry was performed using histidine antibody to determine whether Tat-AR protein was transduced into the mouse brains (Fig. 6A). Tat-AR protein was highly expressed in the SN region of the midbrain. However, AR protein was not detected in the SN region of the midbrain. These results indicate that Tat-AR protein can be transduced into mouse brains by crossing the blood-brain barrier (BBB).

Dopaminergic neuronal cell death is major indicator of PD. Thus, the protective effects of Tat-AR protein against MPTP-induced dopaminergic neuronal cell death were determined by immunohistochemistry using a TH antibody and cresyl violet staining. As shown in Fig. 6B and C, the TH-positive cell numbers were increased in the SN of the Tat-AR protein-treated group. In addition, neuronal cell survival was markedly increased in the Tat-AR protein-treated group. By contrast, the AR protein- and Tat peptide-treated groups did not exhibit such protective effects. These results indicate that Tat-AR protein can markedly prevent dopaminergic neuronal cell death.