Protective effects of cell permeable Tat-PIM2 protein on oxidative stress induced dopaminergic neuronal cell death

Background Oxidative stress is considered as one of the main causes of Parkinson's disease (PD), however the exact etiology of PD is still unknown. Although it is known that Proviral Integration Moloney-2 (PIM2) promotes cell survival by its ability to inhibit formation of reactive oxygen species (ROS) in the brain, the precise functional role of PIM2 in PD has not been fully studied yet. Objective We investigated the protective effect of PIM2 against apoptosis of dopaminergic neuronal cells caused by oxidative stress-induced ROS damage by using the cell permeable Tat-PIM2 fusion protein in vitro and in vivo. Methods Transduction of Tat-PIM2 into SH-SY5Y cells and apoptotic signaling pathways were determined by Western blot analysis. Intracellular ROS production and DNA damage was confirmed by DCF-DA and TUNEL staining. Cell viability was determined by MTT assay. PD animal model was induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and protective effects were examined using immunohistochemistry. Results Transduced Tat-PIM2 inhibited the apoptotic caspase signaling and reduced the production of ROS induced by 1-methyl-4-phenylpyridinium (MPP+) in SH-SY5Y cells. Furthermore, we confirmed that Tat-PIM2 transduced into the substantia nigra (SN) region through the blood-brain barrier and this protein protected the Tyrosine hydroxylase-positive cells by observation of immunohistostaining. Tat-PIM2 also regulated antioxidant biomolecules such as SOD1, catalase, 4-HNE, and 8-OHdG which reduce the formation of ROS in the MPTP-induced PD mouse model. Conclusion These results indicated that Tat-PIM2 markedly inhibited the loss of dopaminergic neurons by reducing ROS damage, suggesting that Tat-PIM2 might be a suitable therapeutic agent for PD.


Introduction
Parkinson's disease (PD), one of degenerative disorder of the central nervous system, typically occurs in people aged over 60 years old and affects about 4% of the population aged over 80 years old [1] and main symptom has motor dysfunctions by loss of dopaminergic cell in the substantia nigra (SN) [2]. Although the precise mechanism of cell death is poorly understood, it implicates not only by accumulation of Lewy body but also by inflammation, and oxidative stress [3][4][5][6]. In general, treatment of PD mostly involves the use of medications such as levodopa, MAO-B inhibitors or agonists of dopamine receptor, however those drugs lose effectiveness and shows adverse effects like involuntary muscle movements [5,[7][8][9].
To investigate the relationship between PD and oxidative stress, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is usually used. MPTP-induced PD model has pathogenesis similar to human PD symptoms and shows dopaminergic neuronal cell death induced by oxidative stress-induced mitochondria dysfunction [3,10,11]. Several studies have shown increased oxidative stress in both PD patients and MPTP-induced PD animal models induced mitochondrial apoptotic pathway resulting the cell death by direct reactive oxygen species (ROS) action [12][13][14]. Moreover, the expression and regulation of caspase signaling have the critical role of distinguishing the progression of apoptosis caused by mitochondria dysfunction and ROS in PD [15,16].
Proviral Integration Moloney-2 (PIM2) protein, known as a serine/threonine-protein kinase family, is involved in the control of cell survival and apoptosis [17][18][19] and is highly expressed and regulated with external influences such as oxidative stress or inflammation in the brain and lymphoid cells [20][21][22]. Elevation of PIM2 reveals an anti-apoptotic function, which can be blocked by NF-κB inhibitor on multiple myeloma or lymphoma [23][24][25] and PIM2 knockdown mice model of gastric cancer augmented apoptosis and diminished the proliferation of cells by high levels of ROS induced by PIM2 silence [26].
Protein transduction domains (PTDs) facilitate the transduction of proteins into cells as well as various tissues including the brain [27][28][29] and we previously demonstrated that several PTD fusion proteins penetrated into cells and brain tissues passing through the BBB and showed inhibition of apoptosis [30][31][32][33]. Therefore, cell permeable Tat-PIM2 was prepared and investigated the protective effects of this fusion protein against MPP + -and MPTP-induced oxidative damage in dopaminergic neurons using in vitro and in vivo PD models.

Analysis of Tat-PIM2's permeability in SH-SY5Y cells
To assess permeation of Tat-PIM2, it treated in SH-SY5Y cells by concentration-dependent manner (0.5-3 μM) for 1 h or timedependent manner (10-60 min) with a dose of 3 μM, respectively. Then, Tat-PIM2 in intracellular was detected by Western blotting with anti-histidine antibody. To avoid interference of cell membrane surface binding, the cells exposed to Tat-PIM2 were washed with trypsin-EDTA (Gibco Grand Island, NY, USA) and PBS. Furthermore, to determine the stability of permeated Tat-PIM2 in SH-SY5Y, the cells were further incubated (1-48 h) and the levels were determined by Western blotting.

Western blotting
SH-SY5Y cells and mouse brain tissue samples were lysed in PRO-PREP™ Protein Extraction Solution (iNtRON Biotechnology, Gyeonggi-do, Korea), and centrifuged for 30 min at 4 • C in 15,000 rpm. The supernatant was boiled with sample buffer (EBA-1052, Elpis Biotech., Daejeon, Korea) for 5 min. The samples were loaded by 12% SDS-PAGE and were then transferred to a nitrocellulose membrane. The membranes were incubated with 5% skim milk in TBST for 1 h and were incubated with primary antibody overnight at 4 • C. After that these were incubated with HRP-conjugated antibody for 2 h. The protein bands were utilized chemiluminescent reagents (Amersham, Franklin Lakes, NJ, USA), and detected using Chemidac system (BioRad, USA).

Cell viability assay
Cell viability assay for cytotoxicity and effect of Tat-PIM2 was performed using MTT assay [32,38]. In the assay of cytotoxicity, Tat-peptide, PIM2, and Tat-PIM2 were treated with various concentrations (0-3 μM) for 24 h in SH-SY5Y cells. To evaluate the cell viability of Tat-PIM2, the proteins (0-3 μM) were pretreated for 1 h and treated 5 mM MPP + for 12 h in SH-SY5Y cells. Both assays were detected by a Labsystems Multiskan MCC/340 plate reader (Farnborough, UK).

Measurement of ROS production
To measure the ROS production, the cells were pretreated with Tat-PIM2 for 1 h, which were incubated with MPP + (5 mM) for 6 h.

TUNEL assay
Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was used to analyze the DNA damage according to the manufacturer's instructions [32,40]. The cells were pretreated with Tat-PIM2 for 1 h, which were incubated with MPP + (5 mM) for 10 h. Then, TUNEL staining was performed. The stained cells with TUNEL were observed under a fluorescence microscope. The number of TUNEL positive cells did counting from the microscopy imaging data. To identify the distribution of Tat-PIM2 in mice brain, the mice (n = 7/each group) were treated Tat-PIM2 (2 mg/kg) by intraperitoneally (i.p.) injected. The valid concentration of Tat-PIM2 (2 mg/kg) was decided following our previous studies [33,35]. After 12 h, the mice brain was sectioned and stained using an immunohistochemical method as previously described [33,35]. In order to analyze the effects of Tat-PIM2 on PD animal model, Tat-PIM2 (2 mg/kg) was injected into the mice (n = 7/each group) by i.p. method. After one day, MPTP (20 mg/kg) was injected into mice 4 times each at 2 h intervals, respectively [38]. A week later, the mice were sacrificed and performed immunohistochemistry with anti-tyrosine hydroxylase (TH), TH + histidine, TH + cresyl violet (CV). TH-and TH + CV-positive cells were displayed via microscopic counts as described previously [32,36].

Statistical analysis
All statistical data were used Graphpad Prism 8.0 software (Graphpad Software, San Diego, CA, USA). Values are shown as means ± standard error of the mean (S.E.M.) from three or more independent experiments. Statistical comparisons between the different treatments were performed using one-way analysis of variance (ANOVA) with Bonferroni's post-hoc test. A p-value <0.05 was considered to be statistically significant.

Preparation, cellular uptake, and intracellular localization of permeable Tat-PIM2
In a previous study, we constructed recombinant PIM2 protein using Tat-peptide [34]. The purified Tat-PIM2 and control PIM2 were identified using SDS-PAGE and Western blotting (Fig. 1A). To confirm the cellular uptake and intracellular localization of Tat-PIM2, immunofluorescence using anti-histidine antibody and DAPI was used. Cell-permeable Tat-PIM2 was located in cytosol and  Tat-PIM2 for 1 h. Then, transduction of Tat-PIM2 was measured by Western blotting and the intensity of the bands was measured by a densitometer. The non-adjusted full images for immunoblots are shwon in Fig. S1. nucleus of the SH-SY5Y cells half-and-half. On the other hand, PIM2, as a negative control, was not identified in the cells (Fig. 1B). Based on Western blotting, the permeability of Tat-PIM2 was affected by concentration and time (Fig. 1C). We also measured the stability of cell-permeable Tat-PIM2 for 48 h. Tat-PIM2 which had penetrated cells was stable for 36 h (Fig. 1D). analyzed by Western blotting (D). Band intensity was measured by densitometer. *P < 0.05 and **P < 0.01 compared with MPP + treated cells. The non-adjusted full images for immunoblots are shwon in Fig. S2.

Effect of Tat-PIM2 on oxidative stress-induced cell death and ROS production
Prior to evaluating its efficacy, we analyzed the cytotoxicity of Tat-PIM2 on SH-SY5Y cells using an MTT assay, and our results did not show significant cytotoxic properties ( Fig. 2A). MPP + elicits oxidative stress that produces ROS and triggers cell death [35]. As shown in Fig. 2B, Tat-PIM2 exhibited a protective effect against MPP + -induced cell death. In cells treated with Tat-PIM2, viability increased from 62% to 84.3%. By contrast, PIM2 and Tat-peptide did not improve cell viability in MPP + -induced cell death (Fig. 2B).
We also assessed whether Tat-PIM2 could moderate cell damage due to ROS production caused by MPP + . Intracellular ROS production was increased in the MPP + treated group, whereas ROS production was conspicuously reduced in the co-treated Tat-PIM2 group (Fig. 2C). Furthermore, mitochondrial proteins, such as, Bax and Bcl-2, were measured to confirm the protective effects of Tat-PIM2 in mitochondria-mediated cell damage caused by ROS production [26]. Tat-PIM2 showed a protective effect on the cells through decreased Bax level and increased Bcl-2 level as compared with the MPP + treated group (Fig. 2D).

The inhibition effect of Tat-PIM2 on the apoptosis pathway
MPP + generates ROS production which triggers the activation of apoptosis signaling pathways [13]. ROS-induced caspase pathway is particularly important in programmed cell death, including Caspase 8, caused by ASK1 activation, and Caspase 9, which results from mitochondrial damage [15]. Caspase 8 and 9 simultaneously enhance Caspase 3 activity and the expression of PARP, which causes DNA fragmentation in the apoptosis pathway [15,33].
We thus confirmed the effects of Tat-PIM2 in the apoptosis pathway by assessing DNA fragmentation and caspase signaling. As shown in Fig. 3A, MPP + -induced DNA fragmentation by green fluorescence measured by TUNEL assay was not detected in the PIM2 and the Tat-peptide groups. On the contrary, decreasing fluorescent signal in apoptotic cells in the Tat-PIM2 group highlighted that (caption on next page) DNA fragmentation was strongly diminished. To determine the role of Tat-PIM2 in MPP + -induced apoptosis pathway, cells were treated with Tat-PIM2 and 5 mM MPP + for 8 h treated and the expression levels of apoptotic proteins was measured by Western blot analysis. In the 3 μM Tat-PIM2 treated group, expression levels of PARP, Caspases 3, 8, and 9 were restored, whereas in the group treated with PIM2 and Tat-peptide not protective effects were observed (Fig. 3C-G). In SH-SY5Y cells, MPP + can be stimulated by the activation of apoptosis, whereas permeable Tat-PIM2 inhibited the apoptosis pathway thereby inhibiting caspase signaling. Fig. 4. Transduced Tat-PIM2 inhibits dopaminergic neuronal cell death in PD animal model. Transduction of Tat-PIM2 into the SN. Tat-PIM2 (2 mg/kg) was injected i.p. into mice, followed by collecting the brains 12 h later. Brain tissues were immunostained with an anti-histidine antibody (A). Relative density was measured by densitometer (B). Localization of transduced Tat-PIM2 proteins was confirmed by fluorescence microscopy. Mice were treated with single injections of Tat-PIM2 (2 mg/kg) proteins and killed after 12 h. Tat-PIM2 was analyzed by immunohistochemistry using anti-Histidine and tyrosine hydroxylase (TH) (C). Protective effects of transduced Tat-PIM2 on PD animal model. Tat-PIM2 (2 mg/kg) was injected i.p. into mice, followed by collecting the brains for 1 week. Brain sections showing tyrosine hydroxylase (TH) immunoreactivity and double staining with cresyl violet (CV) and TH immunoreactivity (D). Scale bars = 100 μm. The number of TH-positive neurons. Quantification of the number of positive dopaminergic neurons in 250 × 250 μm 2 is shown in the graph (E and F). *P < 0.05 and **P < 0.01, the statistically significant difference between MPTP and other groups. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) Fig. 5. The role of Tat-PIM2 on signaling mechanisms in the MPTP animal model. Tat-PIM2 (2 mg/kg) was injected i.p. into mice, followed by collecting the brains for 1 week. Intracellular antioxidant-related protein expression levels using Western blot analysis by anti-SOD1, Catalase, 4-HNE, and 8-OHdG (A). Band intensity was measured by densitometer (B-E). The apoptotic protein expression levels using Western blotting analysis by anti-Caspase-3, Cleaved Caspase-3, Caspase-8, Cleaved Caspase-8, Caspase-9, Cleaved Caspase-9, PARP, and Cleaved PARP (F). Band intensity was measured by densitometer (G-J). *P < 0.05 and **P < 0.01, the statistically significant difference between MPTP and other groups. The nonadjusted full images for immunoblots are shwon in Fig. S4.

Protective effect of Tat-PIM2 in Parkinson's disease animal model
To confirm whether Tat-PIM2 could reach mice brain by passing through the BBB, Tat-PIM2 (2 mg/kg) was injected intraperitoneally and brain tissues were collected after 12 h. The distribution of Tat-PIM2 was measured by immunohistochemistry using a Histidine antibody. It was found that Tat-PIM2 successfully passed the BBB and was widely spread in the SN region of the mice brain ( Fig. 4A and B). To determine the localization of Tat-PIM2, we performed immunocytochemistry in the SN region using anti-TH, a marker of dopaminergic neuron, and anti-His. As shown in Fig. 4C, Tat-PIM2 was significantly localized on dopaminergic neurons. Furthermore, Tat-PIM2 was situated in cytosol (full arrows) and nuclei (empty arrows) of TH-positive cells.
We also assessed whether Tat-PIM2 could reduce the death of dopaminergic neurons in MPTP-induced PD model using a TH antibody and cresyl violet perchlorate (Fig. 4D). It was found that Tat-PIM2 exhibited a protective activity on dopaminergic neurons by increasing the number of TH-and TH + CV positive cells in the SN, but no protective effects were observed in the presence of PIM2 or Tat-peptide ( Fig. 4E and F).

The role of Tat-PIM2 on signaling mechanisms in the MPTP animal model
To investigate the role of permeable Tat-PIM2 in the MPTP animal model, we identified the alterations of intracellular antioxidant and apoptosis pathways using Western blotting analysis. Using robust antioxidant markers, such as, SOD1 and catalase, we demonstrated that the antioxidant activity in the Tat-PIM2 treated group was higher compared to the control group (Fig. 5A-C). Moreover, we analyzed the levels of 4-HNE and 8-OHdG to confirm the antioxidant effects of permeable Tat-PIM2 in mice brains. As shown in Fig. 5A, D and 5E, 4-HNE and 8-OHdG conspicuously decreased in the Tat-PIM2 treated group as compared to the MPTP, PIM2, and Tat-peptide treated groups. These results indicate that Tat-PIM2 induces the expression of antioxidant proteins, which effectively suppressed oxidative stress-induced lipid peroxidation and DNA damage in MPTP animal model.
In PD patients, apoptosis of dopaminergic neurons in the SN leads to a dopamine deficit [1,2]. Therefore, we also demonstrated the protective effect of Tat-PIM2 via Caspase signaling for the apoptosis pathway in SN (Fig. 5F-J). Tat-PIM2 increased the levels of full-length Caspases and PARP while the activations of Caspases and PARP dropped. In the PD animal model, MPTP stimulated the activation of apoptosis, but, in contrast with PIM2 and Tat-peptide groups, permeable Tat-PIM2 alleviated Caspase signaling. We thus suggest that Tat-PIM2 could exhibit antioxidant and anti-apoptotic effects on the MPTP animal model.

Discussion
PD is one of the neurodegenerative diseases and reveals the death of dopaminergic neurons in SN leading to impair dopamine levels [1,2,5]. The cause of dopaminergic neuronal cell death involves in various factors including oxidative stress induced ROS which is particularly recognized to vitally increase and affect the progression of PD [3,4].
PIM2, known as a serine/threonine kinase, modulates the survival and proliferation of cells [20][21][22] and it has been reported that PIM2 advanced cell survival by controlling growth factor signaling, BAD, NF-κB, and apoptosis signaling [23][24][25]. However, the precise role of PIM2 on oxidative stress-induced cell death in PD has not yet been studied yet. To investigate the protective role of PIM2 protein in PD, PIM2 was fused with PTD like Tat which can transduce the target protein into cells and tissues and several studies demonstrated that PTD fusion proteins can be useful tool for treatment of neuronal diseases [29][30][31][32][33][34]36].
In the present study, we evaluated whether cell permeable Tat-PIM2 fusion protein could prevent ROS induced dopaminergic neuronal cell death in vitro and in vivo. As shown in results, Tat-PIM2 effectively transduced into the nuclei and cytosol of SH-SY5Y cells and dopaminergic neurons in the SN region of mice brain. Although it is generally known that Tat PTD fusion protein is located in the nucleus of cells [41,42], its intracellular location might be slightly different depending on the target proteins [43][44][45].
To prepare ROS induced cell and PD mice model, we used MPTP and its metabolite, MPP + in SH-SY5Y cells and mice. MPP + is well known as an intracellular ROS inducer in dopaminergic cells including SH-SY5Y and elevated ROS promotes pathological processes in various neurodegenerative disorders including PD [35,[46][47][48]. It has been reported that PIM2 act as the anti-apoptotic mediator in various apoptosis signaling-related diseases [23,49]. Microenvironment of multiple myeloma was found to enhance PIM2 expression by activating the JAK2/STAT3 pathway and the NF-κB pathway to advance cell survival and cleavage of caspase-3 was strikingly reduced by the overexpression of PIM2 in IL-3 deprivation cells, which demonstrated that PIM2 prevented apoptosis by inhibiting caspase-3 signaling [22][23][24][25][26]50]. In this study, we performed transduced Tat-PIM2 inhibited the apoptotic caspase signaling and reduced the production of ROS induced by MPP + in SH-SY5Y cells. We confirmed that Tat-PIM2 promoted cell survival by lowering ROS production. It is well known that oxidative stress refers to a condition caused by imbalance in the levels of free radicals and radical scavengers. Oxidative damage leads to neurodegeneration, which is observed mainly in brain disorders and several chronic disorders such as inflammation and diabetes [51][52][53]. Other reports have shown that polyphenols related to oxidative stress and inflammation signaling pathways including MAPK, NF-κB, apoptosis and PI3K/Akt. Since polyphenols can inhibit the cell signaling pathways related oxidative stress and inflammation, they are suggested that polyphenols can be used in the prevention and treatment of depressive disorder [54]. Also, other reports have demonstrated that diabetes is associated with hyperglycemia which mainly due to oxidative stress and inflammation. Terminalia catappa with antioxidant, anti-inflammatory and anti-diabetic activity properties prevents hyperglycemia-induced pathological changes by inhibiting various oxidative stress and inflammatory factors such as apoptosis, oxidative stress and inflammatory factors. Therefore, the authors suggest that Terminalia catappa may provide an effective natural therapy for hyperglycemia and prevention of the progression of associated diabetes and diabetic complications [55].
Moreover we investigated the protective effects of Tat-PIM2 in MPTP-induced PD animal model. Tat-PIM2 was injected through intraperitoneal (i.p) and this fusion protein passed through the BBB and transduced into dopaminergic neurons of SN. Transduced Tat-PIM2 showed protective effect and inhibited the apoptosis signaling against cell death in a concentration-dependent manner. Consistent results were obtained in previous studies in which antioxidant cell permeable PTD fusion proteins transduced into neuronal cells and protected against neuronal cell death [32,33,36]. As well as showing the protective effect, Tat-PIM2 elevated the level of antioxidant enzymes such as SOD1 and catalase in PD animal models and ROS induced lipid and DNA oxidation were reduced in mice brain. In summary, Tat-PIM2 has the ability to transduce into dopaminergic neuronal cells across BBB and exhibited anti-apoptotic effect by reducing caspase signaling related to the reduction of ROS. We thus suggest that cell permeable Tat-PIM2 could be a possible candidate for the treatment of PD based on its ability to inhibit cell death.

Further prospects
Protein transduction technology using PTD is well known for their ability to transduce proteins into cells and tissues including the brain by crossing the BBB. Delivery of therapeutic agents is a key point in the development effective therapeutic agents for the treatment of neuronal diseases because therapeutic agents have difficulty crossing the BBB, which is remains a key obstacle to treatment or the development of effective therapeutic agents. To solve this delivery problem, effective approaches will depend on the development of tools that will allow therapeutic agents to cross the BBB. In this study, we showed the protective effect of Tat-PIM2 against MPP + or MPTP-induced dopaminergic neuronal cell death in vitro and in vivo. Although further study for the exact molecular mechanisms still need to be explained, we expect that Tat-PIM2 may contribute to the development of effective therapeutic agents for neuronal diseases including PD in the further.

Data availability statement
Data included in article/supp. material/referenced in article.

Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.