Carnosine suppresses neuronal cell death and inflammation induced by 6-hydroxydopamine in an in vitro model of Parkinson's disease

Parkinson's disease is a progressive neurodegenerative disease for which prevention and effective treatments are lacking. The pathogenesis of Parkinson's disease is not clearly understood. It is thought to be caused by oxidative stress-dependent loss of dopamine neurons in the substantia nigra and the promotion of inflammatory responses by microglia at the lesion site. In addition, cell loss occurs in the hypothalamus of Parkinson's disease patients. Carnosine is an endogenous dipeptide that can exert many beneficial effects, including an antioxidant action, metal ion chelation, proton buffering capacity, and inhibition of protein carbonylation and glycolysis. Previously, we found that carnosine inhibits trace metal-induced death of immortalized hypothalamic neuronal GT1-7 cells. In this study, we analyzed the efficacy of carnosine on 6-hydroxydopamine (6-OHDA)-dependent GT1-7 cell death and inflammatory responses. We found that carnosine significantly prevented 6-OHDA-dependent GT1-7 cell death in a dose-dependent manner. Moreover, carnosine significantly suppressed the expression of 6-OHDA-induced integrated stress response (ISR)-related factors and pro-inflammatory cytokines. Carnosine also significantly inhibited 6-OHDA-dependent reactive oxygen species (ROS) production and c-Jun amino-terminal kinase (JNK) pathway activation in GT1-7 cells. These results indicate that carnosine inhibits hypothalamic neuronal cell death and inflammatory responses by inhibiting the ROS-JNK pathway. We therefore suggest that carnosine may be effective in preventing the onset or the exacerbation of Parkinson's disease.


Introduction
Parkinson's disease is a progressive neurodegenerative disease that presents with motor deficits such as resting tremor, muscle rigidity, akinesia, and impaired postural reflexes. As Parkinson's disease progresses, people may have difficulty walking on their own and become wheelchair-bound or bedridden. Age is the main risk factor for Parkinson's disease and it develops in people over the age of 60 at a rate of about 1 in 100 [1,2]. The number of dopamine neurons in the brain progressively decreases in Parkinson's patients; therefore, the dopamine precursor, L-dopa, anticholinergics, and amantadine hydrochloride are used for treatment [3]. However, no methods for prevention or effective long-term treatment of Parkinson's disease have been established. Although the pathogenesis of Parkinson's disease is not clearly understood, it is thought to be caused by loss of dopamine neurons in the substantia nigra and the promotion of inflammatory responses by microglia at the lesion site [4,5]. Dopaminergic neuronal shedding and microglial activation have also been observed in animal models of Parkinson's disease established using 6-hydroxydopamine (6-OHDA) or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) [6]. Dysfunction of the hypothalamo-pituitary-adrenal (HPA) axis is also considered to be involved in Parkinson's disease pathogenesis [7]. The hypothalamus regulates endocrine and autonomic functions. Lower plasma levels of adrenocorticotropic hormone (ACTH) and cortisol have been recorded in Parkinson's disease patients compared to healthy controls [8]. Moreover, cell loss has also been observed in the hypothalamus of Parkinson's disease patients [9]; Thannickal et al. reported 50% hypothalamic cell loss in 10 patients with Parkinson's disease, with cell loss increasing with severity [10]. Therefore, we speculate that inhibiting neuronal cell death and inflammatory responses in the hypothalamus may be important to prevent the development or exacerbation of Parkinson's disease.
Carnosine is an endogenous dipeptide composed of β-alanine and L-histidine that is synthesized by carnosine synthase. Commonly consumed foods such as beef, pork, and fish contain carnosine. Carnosine has many beneficial effects, including antioxidant action, metal ion chelation, proton buffering capacity, and inhibition of protein carbonylation and glycolysis [11][12][13]. Many studies have shown that carnosine has neuroprotective effects against various neurological diseases, such as the neurodegenerative diseases, prion disease and Alzheimer's disease, and autism spectrum disorders and Gulf War Syndrome [13,14]. In addition, we have reported that carnosine inhibits cell death and integrated stress responses (ISR) in hypothalamic neurons caused by excessive amounts of trace metals [15][16][17], indicating that it may be effective in the treatment of neurodegenerative diseases.
Several reports have examined the efficacy of carnosine in Parkinson's disease, especially and the efficacy of carnosine in animal models of Parkinson's disease has been shown. Daily intranasal administration of carnosine improved Parkinson's-like symptoms and reduced the accumulation of alpha-synuclein in Thy1-αSyn transgenic mice, a mouse model of Parkinson's disease [18]. In addition, carnosine pretreatment markedly reduced MPTP-induced oxidative stress and proinflammatory cytokine production in a mouse MPTP-induced model of Parkinson's disease [19]. In a clinical pilot study, carnosine administered as a food additive as part of a basic protocol for the treatment of Parkinson's disease significantly improved neurological symptoms and increased erythrocyte Cu/Zn-SOD levels and decreased plasma protein carbonyl and lipid hydroperoxide levels [20]. These results indicate that carnosine may be useful in the treatment and prevention of Parkinson's disease. However, the effects of carnosine on the inflammatory response and death of hypothalamic neurons in a Parkinson's disease model have not been reported.
In the present study, we analyzed the inhibition of 6-OHDA-dependent neuronal cell death and inflammatory responses by carnosine using GT1-7 cells, which are immortalized hypothalamic neurons. We focused on oxidative stress as the mechanism by which carnosine inhibits 6-OHDA-dependent neuronal death and inflammatory responses.

Chemicals and reagents
Carnosine was kindly provided by Hamri Chemicals, Ltd. (Osaka, Japan).

Cell culture
Mouse GT1-7 cells (provided by Dr. R. Weiner, University of California, San Francisco) were grown in DMEM/Ham's-F12 supplemented with 10% fetal bovine serum. After trypsin digestion, cells were resuspended in serum-free medium, distributed into culture dishes and cultured in a humidified incubator (7% CO 2 ) at 37˚C [21].

Measurement of viable cell number and detection of apoptosis
Viable cell number was measured as previously described [22,23]. Briefly, dissociated GT1-7 cells were distributed into 96-well culture plates at a concentration of 2.0 × 10 4 cells per well in 200 μL culture medium. After incubation for 24 h, cells were treated with carnosine (0-8 mM) and 6-OHDA. After exposure for 24 h, viable cell number was quantified using CellTiter-Glo 1 2.0. To detect apoptotic cell death, cells were prepared as described above. After incubation for 24 h, cells were treated with carnosine (0-8 mM) and 6-OHDA for 8 h. Cells were then harvested and apoptotic cell death was measured using the APOPCYTO Caspase Colorimetric Assay Kit.

Measurement of reactive oxygen species (ROS) levels
GT1-7 cells were pre-cultured in black 96-well microplates (2.0 × 10 4 cells/well) for 24 h. Cells were then treated with carnosine (0-8 mM) and 6-OHDA for 1 h and the levels of ROS were then quantified using ROS-Glo TM (Promega Corporation, Madison, WI).

Real-time RT-PCR
Total RNA was extracted from GT1-7 cells grown in 6-well culture plates (5.0 × 10 5 cells per well in 1.5 mL culture medium) using an RNeasy kit as previously described [24,25]. RNA samples were reverse-transcribed using the PrimeScript 1 1st strand cDNA Synthesis Kit. Real-time RT-PCR was performed using the cDNAs as templates with THUNDERBIRD 1 SYBR qPCR Mix on the Bio-Rad CFX96™ Real-time system with CFX Manager™ software (Hercules, CA). Specificity was confirmed by electrophoretic analysis of reaction products and by the inclusion of template-or reverse transcriptase-free controls. To normalize the amount of total RNA present in each reaction, glyceraldehyde-3-phosphate dehydrogenase (Gapdh) cDNA was used as an internal standard. Primers were designed using Primer-BLAST. Primers sequences will be provided upon request.

Statistical analysis
All data are expressed as the mean ± S.E. Significant differences between groups were examined using one-way of analysis of variance (ANOVA) followed by Tukey's multiple comparison. SPSS24 software was used for all statistical analyses. P< 0.05 was considered to indicate statistical significance. 6-OHDA causes Parkinson's disease-like symptoms in animals and induces neuronal cell death in vitro [6,27]. In this study, we first analyzed 6-OHDA-induced neuronal cell death in GT1-7 cells. We measured intracellular ATP levels using CellTiter-Glo 1 2.0 to determine cell viability. As shown in Fig 1A, 6-OHDA (0-80 μM) dose-dependently decreased intracellular ATP levels of GT1-7 cells. Cell viability after treatment with 40, 50, or 60 μM 6-OHDA was 59.8 ± 1.6, 40.5 ± 0.2, and 31.1 ± 1.2% (mean ± S.E., n = 4) of control, respectively. We then measured caspase-3 activity in GT1-7 cells to monitor apoptotic cell death. As shown in Fig  1B, 6-OHDA (0-60 μM) treatment increased caspase-3 activity in a dose-dependent manner. These results indicate that 6-OHDA induces hypothalamic neuronal cell death.

Mechanisms by which carnosine inhibits cell death and inflammation
6-OHDA induces neuronal death through ROS production [33,34] and ROS production may cause the onset of Parkinson's disease and exacerbate its severity [35]. Carnosine has antioxidant properties [11], therefore, we examined whether 6-OHDA induces ROS production under our experimental conditions using the ROS-Glo TM assay kit. As shown in Fig 4A, 6-OHDA (40 μM) clearly induced ROS production in GT1-7 cells. However, the ROS production induced by 6-OHDA was significantly suppressed by carnosine treatment, indicating that carnosine inhibits 6-OHDA-induced hypothalamic neuronal cell death and inflammatory responses by inhibiting ROS production.
Stress-activated protein kinases/c-Jun amino-terminal kinases (SAPKs/JNKs) are members of the mitogen-activated protein kinase (MAPK) family, and the SAPK/JNK signaling pathway plays an important role in apoptosis, necroptosis, and inflammation [36]. Furthermore, the SAPK/JNK signaling pathway is involved in the development of Parkinson's disease [37]. We therefore examined the effect of carnosine on 6-OHDA-induced activation of SAPK/JNK signaling using western blotting. Treatment of GT1-7 cells with 6-OHDA (40 μM) significantly increased levels of phospho-JNK (p46) and phospho-JNK (p54) at 2 h after treatment (Fig 4B  and 4C). In contrast, carnosine treatment (8 mM) significantly suppressed the levels of these phospho-proteins (Fig 4B and 4C). Finally, we examined the effect of SP600125 (an inhibitor of the SAPK/JNK signaling pathway) on 6-OHDA-induced hypothalamic neuronal cell death and inflammatory responses. As shown in S1A Fig, SP600125 significantly restored 6-OHDA-  decreased intracellular ATP levels in GT1-7 cells in a dose dependent manner. Under these conditions, SP600125 treatment alone did not affect the number of viable GT1-7 cells (S1B Fig). Moreover, SP600125 treatment significantly suppressed the induction of Chop and Il6 mRNAs by 6-OHDA treatment (S1C Fig). These results indicate that carnosine inhibits hypothalamic neuronal cell death and inflammatory responses by inhibiting the ROS-JNK pathway.

Discussion
In the present study, we demonstrated that 6-OHDA-dependent hypothalamic neuronal cell injury and promotion of inflammatory responses are attenuated by carnosine treatment. We also found that carnosine attenuated the 6-OHDA-dependent integrated stress response. Specifically, carnosine markedly inhibited 6-OHDA-induced upregulation of the ISR-related genes, Chop, Gadd34, and Atf4. Cellular stress signals (such as ER stress and viral infection) activate protein kinase R-like ER kinase, RNA-activated protein kinase, heme-regulated inhibitor, and general control non-derepressible 2 that converge on phosphorylation of eukaryotic translation initiation factor 2α (eIF2α), the core of ISR. This initiates preferential translation of ISR-specific mRNAs, such as ATF 4, while global cap-dependent translation is attenuated [38]. ATF4 then activates transcription of CHOP and GADD34, which are involved in cell death induction in various cells [39]. We therefore suggest that carnosine inhibits 6-OHDA-dependent hypothalamic neuronal cell death by inhibiting 6-OHDA-dependent integrated stress response.ROS and the downstream SAPK/JNK signaling pathway are important factors in the development and exacerbation of Parkinson's disease. Alam et al. analyzed the ROS-related DNA damage product, 8-hydroxyguanine (8-OHG), in control and Parkinson's disease brains using gas chromatography/mass spectrometry, and found that levels of 8-OHG tended to be elevated in Parkinson's disease [40]. Another group also showed that cytoplasmic 8-OHG immunoreactivity in substantia nigra neurons in both multiple system atrophy-Parkinsonian type and dementia with Lewy bodies patients was increased compared with controls [41]. Moreover, oxidative stress (thiobarbituric acid reactive substances and advanced oxidation protein products) and inflammatory markers, such as myeloperoxidase, were significantly elevated in the blood of Parkinson's disease patients. In contrast, the ferric reducing ability of plasma and vitamin C were significantly lower in Parkinson's disease patients [42]. In contrast, Wang et al. showed that the levels of phosphorylated JNK in peripheral blood lymphocytes were significantly increased in Parkinson's disease (p<0.001) [43]. Furthermore, in MPTPinduced Parkinson's disease models, both ROS production and JNK activation were observed in vivo (in C57BL/6 mice) and in vitro (in PC12 cells) [44]. These reports suggest that the ROS-JNK pathway is important for the development of Parkinson's disease. Carnosine almost completely suppressed JNK activation by 6-OHDA, while carnosine partially suppressed ROS production. We speculate that JNK activation by 6-OHDA is largely dependent on ROS production under the present experimental conditions, but we speculate that other signals activated by ROS could not be suppressed by carnosine treatment. Therefore, we assume that inhibitors of other ROS-induced pathways, in combination with carnosine, may more strongly inhibit 6-OHDA-induced hypothalamic neuronal cell death. I would like to conduct further experiments in the future.
Numerous studies have tested the preventive and therapeutic effects of antioxidants in Parkinson's disease animal models and in human clinical trials. Randomized, double-blind, placebo-controlled, parallel-group pilot trials showed that the reduced form of CoQ 10 decreased Unified Parkinson's Disease Rating Scale (UPDRS) scores in Parkinson's disease patients compared with a placebo group [45]. Recently, Monti et al. assessed the clinical effects of N-acetylcysteine (NAC), a precursor to the natural antioxidant, glutathione, in Parkinson's disease patients. NAC significantly increased dopamine transporter binding in the caudate and putamen and significantly improved Parkinson's disease symptoms [46]. Moreover, in 6-OHDAinduced Parkinson's disease models, manganese superoxide dismutase reduced striatal lesions and loss of neuronal cell bodies in the substantia nigra [47]. These reports indicate that antioxidant therapies, such as carnosine, may prevent the onset or exacerbation of Parkinson's disease.
In summary, we found that carnosine reduced 6-OHDA-induced neuronal cell death and inflammatory responses of immortalized mouse hypothalamic neuronal cells. Furthermore, we found that carnosine suppressed 6-OHDA-induced activation of the SAPK/JNK signaling pathway by inhibiting ROS production. Carnosine may therefore be effective in preventing the onset and/or exacerbation of Parkinson's disease.