Research Communication Neuroprotective Effects of a-Melanocytestimulating Hormone Against the Neurotoxicity of 1-Methyl-4-phenylpyridinium

Tao Peng Jingtao Wang Jingjing Lu Hong Lu Junfang Teng Yanjie Jia*

Department of Neurology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan Province, China

Abstract Parkinson’s disease (PD) is the second most common neurodegenerative disease in humans. The hormone a-melanocytestimulating hormone (a-MSH) has been reported to be neuroprotective in previous studies. The aim of this study is to investigate the neuroprotective effects of a-MSH against the neurotoxicity of 1-methyl-4-phenylpyridinium (MPP1). Our results indicated that treatment with a-MSH in M17 cells attenuated MPP1-induced oxidative stress, embodied by exacerbated reactive oxygen species and protein carbonyls. In addition, we found that a-MSH could improve mitochondrial function in M17 cells through increasing the level of adeno-

sine triphosphate and mitochondrial membrane potential. Furthermore, treatment with a-MSH restored the reduction of cell viability and the induction of lactate dehydrogenase release induced by a-MSH. Importantly, Hoechst staining results indicated that a-MSH treatment significantly reduces the number of apoptotic cells after treatment with MPP1. Mechanically, we found that a-MSH prevented apoptosis signals through reducing the level of cleaved caspase-3 and attenuating cytochrome c release. All these data imply that aC 2015 IUBMB Life, MSH produces a protective effect in PD. V 00(00):000–00, 2015

Keywords: Parkinson’s disease; a-melanocyte-stimulating hormone; 1-methyl-4-phenylpyridinium; oxidative stress; apoptosis

Introduction Parkinson’s disease (PD), the second most common neurodegenerative disease, is a movement disorder affecting approximately 3% of the population aged above 65 years (1). 1Methyl-4-phenylpyridinium (MPP1) is a major product of the oxidation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which has been extensively used in a variety of in vivo and in vitro systems to model PD. MPP1 induces neurotoxicity primarily by killing dopamine-producing neurons that reside in the pars compacta of the substantia nigra (2). The underlying mechanisms of neurotoxicity of MPTP/MPP1 is

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Volume 00, Number 00, Month 2015, Pages 000–000 *Address correspondence to: Yanjie Jia, Department of Neurology, The First Affiliated Hospital of Zhengzhou University, 1 Jianshe East Road, Zhengzhou, Henan Province 450052, China. Tel/Fax: 186037166862108. E-mail: [email protected] Received 4 December 2014; Accepted 20 April 2015 DOI 10.1002/iub.1385 Published online 00 Month 2015 in Wiley Online Library (wileyonlinelibrary.com)

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complex. Generally, MPP1 is actively transported into dopaminergic neurons through the plasma membrane in a dopamine transporter (DAT) fashion (3). After taken up by dopaminergic neurons through DAT, MPP1 is stored in vesicles by vesicular monoamine transporter 2 (VMAT2; ref. [4)). It has been found that MPP1 accumulates in mitochondria. On one hand, MPP1 stimulates the generation of superoxide production in mitochondria. One the other hand, MPP1 has been reported to induce mitochondrial dysfunction acting as a potent inhibitor of complex I of the electron transport chain (5). In addition, MPP1 treatment led to reduced adenosine triphosphate (ATP) synthesis (6). Importantly, MPP1 can open the mitochondrial permeability transition pore, release cytochrome c, and activate proapoptotic and/or necrotic pathways (7). Exploring the cellular mechanisms of MPP1induced neurotoxicity will help us to understand the degenerative process of PD and to provide insights into therapeutic targets for PD. The hormone a-melanocyte-stimulating hormone (a-MSH) is a tridecapeptide derived from proopiomelanocortin by posttranslational processing (8). As an endogenous cytokine, it has been reported to suppress the inflammation (9). In addition, aMSH has been reported to act as a prosurvival factor by

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rescuing human melanocytes from DNA damage caused by UV radiation, inhibiting reactive oxygen species (ROS) generation, and activating prosurvival Akt signaling (10–12). The above actions of a-MSH are mediated by binding and activating the melanocortin 1 receptor (MC1R), which is a Gs protein-coupled receptor with seven transmembrane domains (13). Interestingly, recent studies have shown the neuroprotective effects of a-MSH in Alzheimer’s disease by the findings that a-MSH is able to modulate the excitatory–inhibitory balance in the brain by restoring GABAergic inhibition and to improve cognition in a transgenic TgCRND8 mice model (14). However, the neuroprotective effects of a-MSH in PD are still unknown. In this study, we reported that a-MSH induced prosurvival effect against MPP1, suggesting that a-MSH might have therapeutical values for patients with PD.

Materials and Methods Cell Culture and Treatment The M17 neuroblastoma cells were cultured in Opti-MEM medium (Life technologies, Waltham, MA, USA) containing 5% fetal bovine serum and 1% penicillin–streptomycin (P/S) in a humid incubator with 5% CO2 at 378C. The cells were treated with 20 mM MPP1 in the presence or absence of a-MSH at 10 or 20 nM.

Measurement of Intracellular Reactive Oxygen Species The intracellular levels of ROS in M17 cells were investigated using the 20 ,70 -dichlorofluorescein-diacetate (DCFH-DA). After being incubated with the indicated concentrations of MPP1 in the presence or absence of a-MSH, M17 cells were rinsed with Krebs’ ringer solution (100 mM NaCl, 2.6 mM KCl, 25 mM NaHCO3, 1.2 mM MgSO4, 1.2 mM KH2PO4, and 11 mM glucose), and 10 lM DCFH-DA was loaded and incubated at 378C for 2 h. After washing five times with the same buffer, fluorescence signals were examined under a fluorescence microscope.

Protein Carbonyl Assay The cells were lysed and diluted in phosphate-buffered saline (PBS) for 10 times. Protein samples from the cells were adsorbed to wells of an ELISA plate and then reacted with dinitrophenylhydrazine (DNPH). The hydrazone adducts were probed by an anti-DNPH antibody, followed by quantification with horseradish peroxidase (HRP)-conjugated second antibody. Oxidized BSA was used as a standard to calibrate the method as described previously (15,16).

Mitochondrial Membrane Potential Determination The fluorescence dye tetramethylrhodamine methyl ester (TMRM) was used to examine the levels of mitochondrial membrane potential (MMP) according to the manual instructions. Briefly, M17 cells were loaded with 20 nmol/L TMRM (Invitrogen) for 60 min at room temperature. After washing three times, fluorescence signals were captured using a fluorescence microscope. The level of MMP was evaluated by the average fluorescence intensity analyzed by Image-Pro Plus software.

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Cell Viability Determination Cell viability was determined by using a 3-4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) reduction assay. After the completion of indicated experiments, MTT was added to each well with a final concentration of 1.0 mg/ mL and incubated for 2 h at 378C. Then, MTT solution was removed, and the samples were washed for three times with PBS, the formed formazane crystal was dissolved in DMSO. The OD value at 570 nm was measured using a microplate reader to index cell viability (15,16).

Determination of Adenosine Triphosphate Levels via Bioluminescence Assay The level of ATP in M17 cell lines under different conditions was determined by an ATP bioluminescence assay kit following the manual instructions. Briefly, cells were lysed with a lysis buffer in a kit. The samples were centrifuged at 10,000 3 g for 10 min at 48C. Then equal amounts of supernatant and luciferase reagent were mixed, which catalyzed the light production from ATP and luciferin. The signals were measured by a microplate luminometer and used to index ATP concentration.

Western Blot Analysis The cells were lysed in cell lysis buffer (Cell Signaling, Danvers, MA, USA) containing EDTA free protease inhibitor cocktail (CalBiochem, Billerica, Massachusetts, USA). The protein extracts were subject to SDS-PAGE (12% Bis-Tris gel; Invitrogen) and then transferred to hydrophobic polyvinylidene fluoride (PVDF) membranes. The transferred PVDF membranes were blocked in TBST buffer (20 mM Tris-HCl, 150 mM sodium chloride, and 0.1% Tween-20) containing 5% nonfat dry milk (Santa Cruz, Dallas, Texas, USA) for 1 h at room temperature. Then, the membranes were sequentially incubated with primary antibodies and with corresponding HRP-linked secondary antibodies. The intensity of immune-reactive bands was accessed by the NIH ImageJ software. The following antibodies were used in this study: rabbit monoclonal antibody against Bax (#5023; Cell Signaling Technology); rabbit monoclonal antibody against Bcl-2 (#2876; Cell Signaling Technology); rabbit monoclonal antibody against cytochrome c (#4280; Cell Signaling Technology); rabbit monoclonal antibody against COX4 (#4850; Cell Signaling Technology); and rabbit monoclonal antibody against b-actin (#4967; Cell Signaling Technology).

Determination of Lactate Dehydrogenase Release Lactate dehydrogenase (LDH) released from damaged cells was assayed by a commercial kit (Sigma-Aldrich, Saint Louis, MO, USA) according to the manual instructions. Absorbance at 490 nm was used to quantify the amount of LDH. The ratio of LDH activity in the supernatant to total LDH activity was used to index cell death as outlined by the manufacturer’s instructions.

Determination of Cytochrome c Release A cytochrome c detection assay was used to measure the release of cytochrome c from mitochondria. Briefly, on

Neuroprotective Effects of a-MSH in Parkinson’s Disease

FIG 1

Effects of a-MSH on oxidative stress in PD cell models. A: ROS was measured by DCFH-DA. Representative fluorescence photos of intracellular ROS showed increased intracellular ROS accumulation in MPP1-treated cells, which was rescued by a-MSH in a dose-dependent manner (ANOVA: *P < 0.001 vs. nontreatment control; #P < 0.001 vs. MPP1 treatment group; n 5 4–5). Scale bar: 100 mM. B: Protein carbonyl was measured by ELISA. Quantitation showed increased protein carbonyl accumulation in MPP1-treated cells, which was prevented by a-MSH in a dose-dependent manner (ANOVA: *P < 0.001 vs. nontreatment control; #P < 0.001 vs. MPP1 treatment group; n 5 5).

completion of indicated treatment, the cells were washed and treated on ice for 30 min in an ice-cold cytosolic extraction buffer as previously described (17). Then, the cells were gently homogenized using a glass Dounce homogenizer followed by centrifuged at 2,500 rpm at 48C for 15 min. Lysate supernatants were then centrifuged at 15,000 rpm at 48C for 30 min to yield a cytosolic extract. The cytosolic

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extract was used to measure cytochrome c levels by Western blot analysis.

Statistical Analysis Experimental data obtained from different experiments were analyzed using one-way analysis of variance (ANOVA). Data are presented as mean 6 standard error of mean from at least three

Effects of a-MSH against MPP1-induced mitochondrial dysfunction in M17 cells. A: Representative fluorescence photos of mitochondrial membrane potential (MMP) showed reduced intracellular MMP in MPP1-treated M17 cells, which was rescued by aMSH in a dose-dependent manner (ANOVA: *P < 0.001 vs. nontreatment control; #P < 0.001 vs. MPP1 treatment group; n 5 5). Scale bar: 100 mM. B: Quantitative results show reduced intracellular ATP in MPP1-treated M17 cells, which was rescued by aMSH in a dose-dependent manner (ANOVA: *P < 0.001 vs. nontreatment control; #P < 0.001 vs. MPP1 treatment group; n 5 5).

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FIG 3

a-MSH prevents neuronal cell death induced by MPP1. A: Cell viability was measured by MTT assay. B: Cell death was measured by LDH release assay. All experiments were repeated for at least three times (ANOVA: *P < 0.001 vs. nontreatment control; #P < 0.001 vs. MPP1 treatment group; n 5 4–5).

separate experiments. The difference between the two treatments was considered to be statistically significant at P < 0.05.

Results Fluorescence probe DCFH-DA was used to determine the generation of intracellular ROS in M17 cells. The results indicate that the intracellular ROS level in MPP1-treated M17 cells is significantly higher than that in controls, which can be attenuated by a-MSH. Consistent with these results, MPP1 treatment in M17 cells is found to lead to an increased level of protein carbonyl, which can be prevented by treatment with a-MSH (Fig. 1B).

FIG 4

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a-MSH prevents MPP1-induced cell apoptosis. After incubation with MPP1 for 48 h, cells were subjected to Hoechst 33258 staining for morphological assessment of apoptosis. Apoptotic cells were recognized by condensed/fragmented nuclei. Statistical data demonstrated that a-MSH treatment inhibits MPP1induced cell death and apoptosis (ANOVA: *P < 0.001 vs. nontreatment control; #P < 0.001 vs. MPP1 treatment group; n 5 4–5). Scale bar: 100 mM.

Decreased MMP is an essential parameter of mitochondrial dysfunction. The results of our study showed that significantly reduced MMP was found in MPP1-treated M17 cells when compared with nontreatment controls, which was mitigated by treatment with a-MSH (Fig. 2A). In addition, we studied the levels of ATP in various cells in an effort to determine mitochondrial function. Our results indicated that MPP1 treatment leads to a significant decrease in ATP level in M17 cells when compared with nontreatment controls. However, the impaired production of ATP was able to be partially rescued by treatment with a-MSH. These data implicate that a-MSH plays an essential role in maintaining mitochondrial function. We then investigated whether a-MSH has a neuroprotective effect against MPP1-induced cell death. Both MTT (Fig. 3A) and LDH assays (Fig. 3B) displayed that treatment with MPP1 induced significant cell death in M17 cells; however, treatment with a-MSH rescued MPP1-induced neurotoxicity. In the next step, we examined the patterns of apoptosis by using Hoechst 33258 to stain the nuclear condensation characteristics. Our results demonstrated that a-MSH treatment significantly reduces the number of apoptotic cells after treatment with MPP1 (Fig. 4). Disruption of MMP leads to the release of cytochrome c into the cytoplasm, which can trigger activation of caspase-3. Therefore, we then investigated the effects of a-MSH on MPP1-induced cytochrome c release. As shown in Fig. 5A, cytochrome c in the cytosol was significantly increased in cells after MPP1 treatment, and a-MSH treatment markedly attenuated the release of cytochrome c. COX4, another mitochondrial protein, was used to confirm that the purified cytosolic fractions were not contaminated with mitochondrial proteins. The Bcl-2 family members Bax and Bcl-2 play pivotal roles in mitochondrial pathway-mediated apoptosis. Bax is a proapoptotic member, whereas Bcl-2 is an antiapoptotic member. As shown in Fig. 5B, our results indicated that MPP1 treatment led to an increased amount of Bax but a reduced amount of Bcl-2, which were reversed by a-MSH in a dose-dependent manner. Furthermore, it was found that aMSH treatment mitigated the expression level of cleaved caspase-3 induced by MPP1 in M17 cells (Fig. 5B).

Neuroprotective Effects of a-MSH in Parkinson’s Disease

FIG 5

Effects of a-MSH on the levels of cytosol cytochrome c release, Bax, Bcl-2, and cleaved caspase-3 as measured by Western blot analysis. A: a-MSH treatment prevents MPP1-induced release of cytochrome c (ANOVA: *P < 0.001 vs. nontreatment control; #P < 0.001 vs. MPP1 treatment group; n 5 4). B: a-MSH treatment reverses the increase of Bax, the reduction of Bcl-2, and inhibits MPP1-induced levels of cleaved caspase-3 (ANOVA: *P < 0.001 vs. nontreatment control; #P < 0.001 vs. MPP1 treatment group; n 5 4).

Discussion MPP1 has been widely used to damage dopaminergic neurons of the substantia nigra pars compacta and to produce animal and cellular models of PD (2). The action of this toxin in these neurons includes oxidative stress, mitochondrial dysfunction, and apoptosis. In this study, we for the first time showed the antioxidative and antiapoptotic effects of administered a-MSH, a naturally occurring ocular peptide, in MPP1-treated M17 cells. Specifically, a-MSH normalized ROS production and protein carbonyls in MPP1-treated M17 cells. In addition, a-MSH restored the reduction of MMP and ATP production induced by treatment with MPP1. Importantly, a-MSH inhibits MPP1-induced apoptosis. Oxidative stress has been shown to be involved in multiple chronic diseases, including age-related PD (18). The therapeutic regimen and mechanistic study against oxidative stress in one disease may provide clues for another. The antioxidative effects of a-MSH have been studied in several chronic diseases. Typically, it was found that a-MSH has an antioxidative stress ability in skin diseases. For instance, a-MSH counteracts the UVB irradiation-induced oxidative stress by upregulating the expression of the transcription factor Nrf2 and its dependent genes through MC1R-mediated activation of cAMP/PKA pathway in skin keratinocytes and melanocytes (19). In addition, a previous study showed that a-MSH reduces ROS levels and protects cells from DNA damage through activating cAMP/PKA and PI3K/Akt pathway-mediated P53 phosphorylation in UVA-irradiated melanocytes (20). In this study, we hypothesize that a-MSH may exert antioxidative effects in MPP1-treated M17 cells. Indeed, our results in MPP1-treated M17 cells in vitro showed that intracellular levels of ROS and protein carbonyls were significantly suppressed by treatment with a-MSH. In addition, it has been shown that administration of a-MSH elevated total antioxidant capacity in the retina (21). We speculate that the antioxidative stress capacity of a-MSH might be associated with elevated total intracellular antioxidant capacity. Further study will help us to understand the underlying mechanisms. The antiapoptotic effects of a-MSH may partially result from its antioxidative effects against MPP1-induced neurotox-

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icity. Consistent with our current findings, multiple lines of evidence have shown that a-MSH or its analog has a wide spectrum of antiapoptotic function against neurotoxicity. a-MSH has been shown to rescue neurons from apoptosis induced by various different insults in animal disease models, including traumatic brain injury caused by controlled cerebral impact (22), cerebral ischemia generated by common carotid artery occlusion (23), and hippocampal excitotoxicity induced by excitatory neurotransmitter glutamate (24). In summary, administration of a-MSH demonstrated antioxidative, restoring mitochondrial function, and antiapoptotic effects in MPP1-treated M17 cells, supporting the possibility that a-MSH or its chemical derivatives can be developed into an effective and novel intervention approach for PD treatment.

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Neuroprotective Effects of a-MSH in Parkinson’s Disease