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Am J Physiol Cell Physiol 293: C1884-C1894, 2007. First published October 3, 2007; doi:10.1152/ajpcell.00305.2007
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NERVOUS SYSTEM CELL BIOLOGY

A dibenzoylmethane derivative protects dopaminergic neurons against both oxidative stress and endoplasmic reticulum stress

Katsura Takano,1,3 Yasuko Kitao,1 Yoshiyuki Tabata,1,2 Hikari Miura,1 Kosuke Sato,4 Kazuhiro Takuma,4 Kiyofumi Yamada,4 Satoshi Hibino,5 Tominari Choshi,5 Munekazu Iinuma,6 Hiroto Suzuki,7 Rika Murakami,7 Masashi Yamada,7 Satoshi Ogawa,1 and Osamu Hori1

1Department of Neuroanatomy and 2Department of Molecular Pharmacology, Kanazawa University Graduate School of Medical Science, Kanazawa City; 3Laboratory of Molecular Pharmacology and 4Laboratory of Neuropsychopharmacology, Kanazawa University Graduate School of Natural Science and Technology, Kanazawa City, Ishikawa; 5Fukuyama University, Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama, Hiroshima; 6Gifu Pharmaceutical University, Gifu City, Gifu; and 7Meiji Dairies Corporation, Tokyo, Japan

Submitted 18 July 2007 ; accepted in final form 1 October 2007


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The enhancement of intracellular stresses such as oxidative stress and endoplasmic reticulum (ER) stress has been implicated in several neurodegenerative disorders including Parkinson's disease (PD). During a search for compounds that regulate ER stress, a dibenzoylmethane (DBM) derivative 14-26 (2,2'-dimethoxydibenzoylmethane) was identified as a novel neuroprotective agent. Analysis in SH-SY5Y cells and in PC12 cells revealed that the regulation of ER stress by 14-26 was associated with its anti-oxidative property. 14-26 prevented the production of reactive oxygen species (ROS) when the cells were exposed to oxidants such as hydrogen peroxide and 6-hydroxydopamine (6-OHDA) or an ER stressor brefeldin A (BFA). 14-26 also prevented ROS-induced damage in both the ER and the mitochondria, including the protein carbonylation in the microsome and the reduction of the mitochondrial membrane potential. Further examination disclosed the presence of the iron-chelating activity in 14-26. In vivo, 14-26 suppressed both oxidative stress and ER stress and prevented neuronal death in the substantia nigra pars compacta (SNpc) after injection of 6-OHDA in mice. These results suggest that 14-26 is an antioxidant that protects dopaminergic neurons against both oxidative stress and ER stress and could be a therapeutic candidate for the treatment of PD.

neuronal cell death; stress response; Parkinson's disease

NEURODEGENERATION is a multifaceted process that leads to progressive neuronal death, and one of the key factors underlying it is intracellular stress. Increased oxidative stress and dysfunction of the mitochondria are implicated in many neurodegenerative diseases (3). Several neurotoxins, including 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), have been used to model Parkinson's disease (PD); most of these agents are associated with mitochondrial damage and enhanced oxidative stress.

Recent studies, however, have raised the possibility that the endoplasmic reticulum (ER) also plays an important role in maintaining neurons in the neuropathological situations. The ER is a target for two types of intracellular stresses: ER stress and oxidative stress. ER stress is characterized by the accumulation of unfolded proteins in the ER, which occurs in conditions such as glucose starvation, oxygen deprivation, inhibition of protein modification, and disturbance of Ca2+ homeostasis. Eukaryotic cells, including neurons, respond to ER stress by activating a set of pathways known as the unfolded protein response (UPR) (36). The UPR is transmitted through the activation of ER resident proteins, such as inositol-required enzyme 1 (Ire1{alpha}/β), protein kinase-like ER kinase (PERK), and activating transcription factor 6 (ATF6), and the UPR-targeted genes include molecular chaperones, folding catalysts, subunits of translocation machinery (Sec61 complex) in the ER, ER-associated degradation (ERAD) molecules, and antioxidant genes (16, 36, 43). However, if the protein load in the ER exceeds its folding capacity, cells tend to die, typically, with apoptotic features (ER stress-induced cell death). Important roles for ER stress and ER stress-induced cell death have been demonstrated in various pathological situations, including brain ischemia and neurodegeneration (12, 20, 30, 34, 40).

The ER is also a place where oxidative stress is generated. In the lumen of the ER, the redox status of glutathione (GSH) is shifted to the oxidized form (GSSG), and disulfide bonds of proteins are effectively formed through the relay of electrons by the ER-resident proteins; protein disulfide isomerase (PDI) and a novel flavoprotein Ero1(5, 29). As a result, reactive oxygen species (ROS), which are believed to be eliminated by GSH, are generated in the ER (6). It has been reported that branches of the UPR such as the PERK-eIF2{alpha}-ATF4 pathway are critical for the regulation of oxidative stress derived from the ER or other sources (36). Cells lacking PERK or ATF4 accumulate ROS (11) and are more susceptible to PD-related neurotoxins such as 6-OHDA (34).

Herp is a UPR-dependent ubiquitin-like protein that is located in the ER in a wide range of cells including neurons (16, 24, 35). We recently reported that targeted disruption of the Herp gene rendered F9 embryonic carcinoma cells vulnerable to ER stress (16). Using this cell line and other neuronal cell lines including SH-SY5Y cells and PC12 cells, we evaluated the protective effect of 300 compounds against ER stress-induced cell death. We report here that a dibenzoylmethane (DBM) derivative, 14-26 (2,2'-dimethoxydibenzoylmethane) protects dopaminergic neurons against both ER stress and oxidative stress. 14-26 prevents ROS production under those conditions and inhibits ROS-induced damage in both the ER and mitochondria. In vivo, 14-26 prevented neuronal death in the substantia nigra pars compacta (SNpc) following injection of 6-OHDA in mice.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell cultures and stress conditions. F9 Herp null cells were developed as previously described (16) and were maintained in DMEM containing 20% fetal bovine serum. Human catecholaminergic neuroblastoma SH-SY5Y cells and rat pheochromocytoma PC12 cells were maintained in DMEM containing 15% FBS and 5% FBS-5% horse serum, respectively. ER stress was induced by treating the cells with tunicamycin (Tm) (0.75–2 µg/ml; Sigma, St Louis, MO), an inhibitor of N-linked glycosylation, or brefeldin A (BFA) (1 µg/ml; Sigma), an inhibitor of protein transport from the ER to the Golgi apparatus, for the indicated times (6–48 h). Oxidative stress was induced by exposing the cells to H2O2 (44 µM; Nacalai Tesque, Kyoto, Japan), 6-OHDA (30 or 75 µM; Sigma), a neurotoxin commonly used in the experimental PD model, or buthionine sulfoximine (BSO), a drug that causes the depletion of intracellular glutathione (1 mM; Wako, Osaka, Japan) for the indicated times. Compounds were added to the cells together with stressors, whereas, in some experiments, cells were also preincubated with each compound for the indicated times.

Compounds and cell viability assays. One hundred and three plant-derived compounds (38, 39) and 200 synthetic compounds, including DBM derivatives, Carbazole derivatives, and pyrimidine derivatives, were analyzed for their protective effects against ER stress in F9 Herp null cells, as previously described (39). DBM derivatives such as 14-17 (4,4'-dimethoxydibenzoylmethane), 14-19 (4,4'-dichlorodibenzoylmethane), 14-26 (2,2'-dimethoxydibenzoylmethane), and 14-28 (3,3'-dimethoxydibenzoylmethane) were synthesized as previously described (7). IN21 (2,2'-dihydroxydibenzoylmethane), another DBM derivative, was synthesized as follows. An ester product of 2-hydroxyacetophenone and 2-benzyloxybenzoic acid was treated with KOH in pyridine to yield 2-benzyloxy-2' -hydroxydibenzoylmethane. The dibenzoylmethane was debenzylated with 10% Pd/C in ethylacetate under a hydrogen atmosphere to give IN21. Dantrolene, N-acetyl cysteine (NAC), {alpha}-tocopherol, and deferoxamine mesylate salt (DFO) were obtained from Sigma; GSH was purchased from Wako. Cell viability under conditions of ER stress or oxidative stress conditions was measured using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay (Nacalai Tesque).

Northern blot analysis. SH-SY5Y cells or PC12 cells were treated with each compound or cultured in the medium alone for 16 h, after which tunicamycin (2 µg/ml) or brefeldin A (1 µg/ml) was added to the cells for 6 h. Total RNA (10 µg/condition) isolated from the cells was subjected to Northern blot analysis using cDNA fragments specific for glucose-regulated protein 78 (GRP78), a molecular chaperone in the ER, C/EBP-homologous protein (CHOP), a mediator of ER stress-induced cell death (36), or β-actin as described previously (16).

Western blot analysis. SH-SY5Y cells were treated with 14-26 or cultured in the medium alone for 16 h, after which tunicamycin (2 µg/ml) or brefeldin A (1 µg/ml) was added to the cells for 12 h. The cells were lysed in ice-cold radioimmunoprecipitation assay (RIPA) buffer containing 10 mM Tris, 1 mM EDTA, 150 mM NaCl, 1% Nonidet-40, 0.1% SDS, 0.2% deoxycholate, 1 mM PMSF, 1 µg/ml aprotinin, and 1 µg/ml luepeptin, and subjected to Western blot analysis with anti-KDEL antibody (Stressgen Bioreagents, Ann Arbor, MI), which recognizes both GRP78 and GRP9 and anti-β-actin antibody (Santa Cruz Biotechnology; Santa Cruz, CA).

Measurement of ROS and mitochondrial membrane potential. Intracellular ROS levels were evaluated by incubating the cells in serum-free medium containing 5 µM 2'7'-dichlorodihydrofluorescein diacetate (H2DCFDA; Invitrogen) for 30 min at 37°, followed by observation under a microscope equipped with a CCD camera (Hamamatsu Photonics, Shizuoka, Japan) or by measurement using a 96-well scanning fluorometer Fluoroskan Ascent FL (Labsystems, Helsinki, Finland). Mitochondrial membrane potential was measured using the Mito-sensor kit (Invitrogen) as described previously (15).

Detection of the oxidative modification of proteins. To assess the levels of protein carbonylation in the ER, SH-SY5Y cells were treated or untreated with 6-OHDA (30 or 75 µM) for 90 min in the absence or presence of 14-26 (60 µM) after pretreatment of the cells with 14-26 for 16 h. The cells were harvested, and whole cell lysates were obtained by incubating the cells in RIPA buffer as described in Western blot analysis. To perform subcellular fractionation, the cells were homogenized using a Dounce homogenizer (50 strokes) in ice-cold buffer, containing (in mM) 250 sucrose, 10 HEPES-KOH, pH 7.4, and 1 EDTA and then subjected to crude cell fractionation by the sequential centrifugation, as described previously (10). Fractionation was confirmed by performing Western blot analysis with anti-mitochondrial complex I (17 kDa subunit) antibody (Invitrogen, Carsbad, CA), anti-calnexin antibody (Stressgen Bioreagents), and anti-β-actin antibody (Santa Cruz Biotechnology). Protein carbonylation in both whole lysates or crude cell fractions was detected using the OxyBlot Protein Oxidation Detection Kit (Chemicon, Temecula, CA), which is based on a two-step reaction, the derivatization of carbonylated proteins with dinitrophenylhydrazine (DNPH), and Western blot analysis with the anti-DNP antibody. The proteins modified by 4-hydroxy-2-nonenal (4-HNE), a cytotoxic endoproduct of lipid peroxidation, were also immunologically detected in vivo as described below.

Measurement of metal chelating activity. The relative binding property of compounds to iron, copper, and zinc was measured using the fluorescence indicator calcein (485 nm/520 nm, Wako) as previously described, with some modification (42). In brief, 100 nM calcein was incubated with Fe(NH4)2SO4 (100 nM), CuSO4 (50 nM), ZnSO4 (200 nM) in HEPES-buffered saline, for 10 min at room temperature, followed by further incubation with the indicated compounds for 50 min at room temperature. The fluorescence intensity was measured using Fluoroskan Ascent FL and shown as percentage of control intensity.

Animal experiments. All animal experiments were performed under the Guidelines for the Care and Use of Laboratory Animals in Kanazawa University. A mouse model of PD was created by a single injection of 6-OHDA (5.25 µg/1.5 µl) into the left medial forebrain bundle (MFB) in ICR mice (male, 8–10 wk of age) as described previously (19). For the administration of 14-26 or vehicle, mice were intraperitoneally injected with 14-26 (10 mg·kg–1·day–1) in saline, including 10% Cremophor EL (Sigma) and 5% DMSO, or with the dissolving solution (vehicle: saline including 10% Cremophor EL and 5% DMSO) daily for 3 days. On the second day, mice were lesioned with 6-OHDA 2 h after the administration of 14-26 or vehicle. Histological analysis was performed as described below, 1 and 3 wk after 6-OHDA injection. Amphetamine (2.5 mg/kg)-induced rotational behavior was tested 3 wk after 6-OHDA injection as described previously (19). The counterclockwise turnings of each animal were counted manually for 3 min every 10-min cycle and shown as turns per minute or turns per 60 minutes.

Histological analysis. Brains were removed from mice after perfusion with 4% of paraformaldehyde, and coronal sections (10 µm) were cut on a cryostat. Sections were processed for Nissl staining or immunostaining with anti-tyrosine hydroxylase (TH) antibody (Chemicon International), anti-KDEL antibody, or anti-4-HNE antibody (Japan Institute for the Control of Aging, Shizuoka, Japan) as described previously (21, 39). FITC- or Cy3-conjugated secondary antibodies were used for the visualization of immunolabeling. TH (+) neuronal cells in the SNpc were counted in two representative sections (Bregma-3.16 and -3.64 mm) as described before (25) after digital images were acquired using a CCD camera (Hamamatsu Photonics, Shizuoka, Japan).

Laser densitometric analysis and statistical analysis. Laser densitometric analysis was performed to standardize the results of Western and Northern blot analyses as described previously (16). For statistical evaluation, Bonferroni/Dunnett test following one-way ANOVA or Student's t-test was employed.


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Prevention of ER stress- and oxidative stress-induced cell death by DBM derivatives. We previously reported that Dantrolene, an antagonist of the ryanodine receptors in the ER, and some antioxidants such as {alpha}-tocopherol, partly prevented Tm-induced cell death in F9 Herp null cells (39). With the use of these agents as positive controls, 103 plant-derived compounds and 200 synthetic compounds were screened in F9 Herp null cells treated with Tm. The DBM derivative 14-26 (2,2'-dimethoxydibenzoylmethane) was identified as a protective agent against ER stress (supplemental figure 1, A and B). 14-26 (20–40 µM) improved cell viability after Tm treatment at a level similar to that achieved by Dantrolene (30 µM) in F9 Herp null cells (supplemental figure 1). To further investigate the cell protection by DBM derivatives under stress, two neuronal cell lines, SH-SY5Y cells and PC12 cells, were exposed to ER stress or oxidative stress, and the protective property of DBM derivatives were compared (Fig. 1A). When SH-SY5Y cells (Fig. 1B) were treated with ER stressors such as Tm (Fig. 1B) and BFA (Fig. 1C) for 48 h, cell viability dropped to 38 (SD 5) % and 28 (SD 6) %, respectively. Among the DBM derivatives, 14-26 restored cell viability to 65 (SD 8) % and 62 (SD 7) % in each condition (Fig. 1, B and C). 14-28 also protected SH-SY5Y cells against ER stress to a slightly lesser degree. Similar results were obtained when PC12 cells were treated with ER stressors (data not shown). The strong cell protection by 14-26 and 14-28 against oxidative stress was also observed when exposing SH-SY5Y cells (Fig. 1, D and E) or PC12 cells (data not shown) to oxidants such as H2O2 (Fig. 1D), 6-OHDA (Fig. 1E), and BSO (data not shown) for 24 h, after pretreatment of the cells with each compound for 24 h (Fig. 1, D and E). The protective activity of 14-26 that is equivalent to NAC (1,000 µM), the precursor of glutathione, was obtained at the range of 40–60 µM (Fig. 1, D and E). Similar levels of cell protection against H2O2 were obtained when SH-SY5Y cells were pretreated with 14-26 for shorter periods (4–16 h), but, in the condition without pretreatment, 14-26 was less effective (Fig. 1F). When SH-SY5Ycells were exposed to higher concentration of H2O2 (440 µM) for a shorter period (6 h), 14-26 also protected cells, but to a lesser degree (supplemental Figure 1C).


Figure 1
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  		Fig. 1. Effect of dibenzoylmethane (DBM) derivatives on endoplasmic reticulum (ER) stress and oxidative stress in neuronal cell lines. A: structure of DBM derivatives. B and C: protection of SH-SY5Y cells against ER stress-induced cell death. SH-SY5Y cells (5 x 105 cells/condition) were treated with tunicamycin (Tm, 1.5 µg/ml; B) or brefeldin A (BFA, 1 µg/ml; C) in the absence or presence of compounds for 48 h. Cell viability was evaluated using the 3-(4,5-dimethyl-2-thiazolyl)2,5-diphenyltetrazolium bromide (MTT) assay. NAC, N-acetyl-cysteine (NAC). Values shown are means (SD) of 4 experiments. *P < 0.01, **P < 0.01. D and E: protection of SH-SY5Y cells against oxidative stress-induced cell death. SH-SY5Y cells (5 x 105 cells/condition) were pretreated with medium alone or with the indicated compounds for 24 h and incubated with H2O2 (44 µM; D) or with 6-hydroxydopamine (6-OHDA, 30 µM; E) for a further 24 h in the presence or absence of compounds. Cell viability was evaluated using the MTT assay as above. Values shown are means (SD) of 4 experiments. *P < 0.01, **P < 0.001. F: enhancement of the cell protection against oxidative stress by pretreatment with 2,2'-dimethoxydibenzoylmethane (14-26). SH-SY5Y cells (5 x 105 cells/condition) were pretreated with 14-26 for the indicated times and incubated with H2O2 (44 µM) for a further 24 h in the presence or absence of 14-26. Cell viability was evaluated using the MTT assay as above. Values shown are means (SD) of 4 experiments. *P < 0.01, **P < 0.001.

 
Effect of 14-26 on the expression of the UPR-targeted genes. To analyze the protective property of 14-26 against ER stress, SH-SY5Y cells (Fig. 2, A and D) and PC12 cells (Fig. 2, B and C) were treated with ER stressors such as Tm (2 µg/ml) or BFA (1 µg/ml) in the presence or absence of the indicated compounds for 6 h (Fig. 2, A–C) or for 12 h (Fig. 2D), after pretreatment of the cells with the compounds or vehicle (culture medium) for 16 h. The inductions of the UPR-targeted genes such as GRP78 and CHOP in response to ER stress were suppressed at the mRNA level by treatment of the cells with 14-26 (Fig. 2, A and B), but not with IN21 (Fig. 2C). Similarly, the enhanced levels of expressions of GRP78 and GRP94, the latter another UPR-targeted gene, under ER stress were suppressed at protein level by treating cells with 14-26. These results suggest that the cell protection by 14-26 against ER stress was not related to the activation of the UPR, which is the case for methoxyflavones (39). Instead, these observations suggest that 14-26 improves the ER luminal environment that reduces the level of ER stress after treating the cells with ER stressors.


Figure 2
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  		Fig. 2. Effect of 14-26 on the unfolded protein response (UPR). A–C: expression of glucose-regulated protein 78 (GRP78) and C/EBR homologous protein (CHOP) mRNA in normal and ER stress conditions. SH-SY5Y cells (A) or PC 12 cells (B and C) were treated with Tm (2 µg/ml) or BFA (1 µg/ml) in the presence or absence of the indicated compounds for 6 h after pretreatment of the cells with each conpound or vehicle (culture medium) for 16 h. Total RNA (10 µg/condition) was isolated and subjected to Northern blot analysis with specific probes against GRP78, CHOP, and β-actin. The intensities of GRP78 mRNA and CHOP mRNA were quantified and standardized with those of β-actin (A). D: expression of GRP78 and GRP94 protein in normal and ER stress conditions. SH-SY5Y cells were treated with Tm (2 µg/ml) or BFA (1 µg/ml) in the presence or absence of the 14-26 for 12 h after pretreatment of the cells with 14-26 or vehicle (culture medium) for 16 h. Cell lysates were subjected to Western blot analysis with anti-KDEL antibody and anti-β-actin antibody. The intensities of GRP78 protein and GRP94 protein were quantified and standardized with those of β-actin.

 
Effect of 14-26 on ROS production in response to oxidative stress and ER stress. We hypothesized that the protective effect of 14-26 and 14-28 against ER stress may be related to their anti-oxidative properties. To analyze the effect of 14-26 on ROS production in response to both oxidative stress and ER stress, SH-SY5Y cells were treated with oxidants such as H2O2 (44 µM) and 6-OHDA (75 µM) for 6 h or an ER stressor BFA (1 µg/ml) for 16 h, and the intracellular production of ROS was estimated using a fluorescent probe DCF. Enhanced levels of intracellular ROS were observed in all stress conditions (Fig. 3, A,II; B,II; C,II). Treatment of the cells with Tm also generated ROS to a lesser degree (data not shown). Treatment of the cells with 14-26 or NAC after pretreatment for 16 h prevented both oxidative stress- and ER stress-derived ROS (Fig. 3, A,III; A,IV; B,III; B,I; C,III; C,IV). Similar tendency was observed when DCF-derived fluorescence was measured by a fluorometer (Fig. 3, D–F).


Figure 3
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  		Fig. 3. Effect of 14-26 and NAC on reactive oxygen species (ROS) production. Detection of ROS in the cells after H2O2 (A and D), 6-OHDA (B and E), or BFA (C and F) treatment. SH-SY5Y cells (5 x 105 cells/condition) were pretreated with 14-26 (60 µM), NAC (1 mM), or medium alone or for 16 h, followed by a further incubation with H2O2 (44 µM; A) or 6-OHDA (75 µM; B) for 6 h, or BFA (1 µg/ml; C) for 16 h in the presence or absence of 14-26 (60 µM) or NAC (1 mM). ROS levels were evaluated by analyzing DCF-derived fluorescence under the microscope (A–C) or by measuring it with a 96-well scanning fluorometer (D–F) as described in the text.

 
Effect of 14-26 on ROS-induced damage in the ER and mitochondria. As the oxidative modification of proteins is an important factor in ROS-induced damage to organelles, especially in the ER (9, 33), the protein carbonylation levels were estimated in both whole cell lysates (Fig. 4A) and crude cell fractions (Fig. 4B) from SH-SY5Y cells after treatment of the cells with 6-OHDA for 90 min as described previously (14). Carbonylated proteins were observed in whole cell lysates after 6-OHDA treatment in a dose-dependent manner (Fig. 4A), and they were predominantly located in the microsome fraction. Treatment of cells with 14-26 diminished carbonylated proteins in both whole cell lysates and in the microsome fraction (Fig. 4, A and B). A similar tendency was observed when the cells were treated with H2O2 (data not shown). As the mitochondria is also a target organelle of the ROS-induced injury, the levels of the mitochondrial membrane potential was assessed after treatment of SH-SY5Y cells with H2O2 (44 µM; Fig. 4C,II), 6-OHDA (75 µM; Fig. 4D,II) or BFA (1 µg/ml; Fig. 4E,II). The reduction of the mitochondrial membrane potential was observed in all stress conditions, although the effect of BFA may be smaller than those of H2O2 or 6-OHDA (Fig. 4, C,II; D,II; E,II). Treatment of the cells with 14-26 restored the membrane potential in all cases (Fig. 4, C,III; D,III; E,III).


Figure 4
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  		Fig. 4. Detection of ROS-induced organelle damage. A and B: detection of carbonylated proteins in whole cell lysates (A) and in subcellular fractions (B). SH-SY5Y cells (5 x 106 cells/condition) were pretreated with 14-26 (60 µM) or with medium alone for 16 h, followed by a further incubation with 6-OHDA (30 or 75 µM) for 90 min in the presence or absence of the compound. Whole cell lysates were derivatized with dinitrophenylhydrazine (DNPH) and subjected to Western blot analysis with anti-DNP antibody as described in the text (A). SH-SY5Y cells (5 x 107 cells/condition) were also pretreated 14-26 (60 µM) or with medium alone for 16 h, followed by a further incubation with 6-OHDA (75 µM) for 90 min in the presence or absence of compounds. Subcellular fractionation was performed as described in the text, and samples were subjected to Western blot analysis with anti-DNP antibody after derivatization with DNPH (OxyBlot) or with the indicated antibodies to confirm the fractionation (bottom). C–E: ROS-induced decrease in the membrane potential of mitochondria. SH-SY5Y cells (5 x 105 cells/condition) were pretreated with 14-26 (60 µM) or with medium alone for 16 h, followed by a further incubation with H2O2 (44 µM; C) or 6-OHDA (75 µM; D) for 6 h, or with BFA (1 µg/ml) for 16 h in the presence or absence of the compound. Mitochondrial membrane potential was measured using the Mito-sensor kit.

 
Iron chelation by DBM derivatives. To dissect in more detail the anti-oxidative properties of 14-26, experiments were performed in a cell-free system. As it has been reported that DBM has the iron-chelating activity and can be used for the analysis of serum iron (44), the binding properties of 14-26 to iron, copper, and zinc were measured using the fluorescence indicator calcein. When calcein (100 nM) was incubated with Fe(II), Cu(II), or Zn(II), the fluorescence intensity went down to 39 (SD 4) % (Fig. 5 A,I), 50 (SD 6) % (Fig. 5A,II), and 58 (SD 7) % (data not shown), respectively, of the intensity seen when calcein was incubated with the control solution without metals. The addition of 14-26 restored the fluorescence intensity of calcein in the calcein-iron solution in a dose-dependent manner (Fig. 5A,I), although this effect was milder than that of a common iron chelator deferoxamine (DFO). NAC did not affect the binding of iron to calcein (Fig. 5A,I). 14-26 also showed chelating activity, to a much lesser degree, for Cu(II) (Fig. 5A,II) but not for Zn(II) (data not shown). Further analysis with different DBM derivatives revealed that 14-26 and 14-28, but not other DBM derivatives, chelated iron effectively (Fig. 5B). These observations suggest that the iron-chelating property of 14-26 may play a role in its protective property.


Figure 5
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  		Fig. 5. Iron chelation by DBM derivatives. The chelating property of the indicated compounds for iron (A,I and B) or copper (A,II) was measured using calcein as described in the text and shown as a percentage of the control calcein fluorescence intensity. Values shown are means (SD) of 4 experiments. *P < 0.01, **P < 0.001.

 
Neuroprotective effect of 14-26 in a mouse model of PD. It has been reported that PD-related neurotoxins such as 6-OHDA and MPTP induce both oxidative stress- and ER stress-signaling pathways (13, 34, 37). As 14-26 prevented 6-OHDA-induced cell death in SH-SY5Y cells (Fig. 2D), the neuroprotective properties of 14-26 were assessed in a unilateral 6-OHDA lesion model of PD in mice (19). When mice were injected with 6-OHDA (5.25 µg/1.5 µl) into the left MFB, the number of TH-positive cells in the left SNpc decreased to 42 (SD 10) % of the number observed in the right SNpc (noninjected side) 1 wk after lesioning (Fig. 6 B,I); by 3 wk after lesioning, this number decreased to 21 (SD 8) % of the control figure (Fig. 6A, a, b, c, and Fig. 6B,II). Nissl staining confirmed neuronal loss in the left SNpc at 3 wk after 6-OHDA lesioning (Fig. 6C, a and b). Consistently, loss of TH immunoreactivity was observed in the left striatum, both 1 wk (data not shown) and 3 wk (Fig. 6D,a) after 6-OHDA lesioning. By contrast, when mice were intraperitoneally injected with 14-26 (10 mg/kg) for 3 consecutive days, beginning the day before 6-OHDA injection, the decrease in the number of TH (+) neurons (Fig. 6A, d, e, f, and Fig. 6B) and neuronal death (Fig. 6C,b) were prevented. The number of TH (+) cells in the left SNpc was recovered to 71 (SD 9) % of the number observed in the right SNpc at 1 wk after lesioning (Fig. 6B,I); by 3 wk after lesioning, the corresponding number was 55 (SD 16) % (Fig. 6B,II). The loss of TH immunoreactivity in the left striatum was also improved by 14-26 administration (Fig. 6D).


Figure 6
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  		Fig. 6. Prevention of neuronal death by 14-26 in a mouse model of Parkinson's disease (PD). A: immunoreactivity of tyrosine hydroxylase (TH) in the substantia nigra pars compacta (SNpc) after 6-OHDA lesioning. Brain sections from mice obtained 3 wk after 6-OHDA injection, with (d, e, f) or without (a, b, c) injection of 14-26 intraperitoneal were subjected to immunostaining with anti-TH antibody. a and d: low magnification, b, c, e, f: high magnification. B: number of TH(+) neuronal cells in the left SNpc after 6-OHDA lesioning. The number of TH (+) cells in the left SNpc was counted as described in the text and shown as a percentage of the number of TH (+) cells in the right SNpc. Values shown are means (SD) of 4 experiments (total 6 mice in each group). *P < 0.01 C, Nissl staining. Nissl staining of the brain sections obtained 3 wk after 6-OHDA lesioning with (c, d) or without (a, b) intraperitoneal injection of 14-26. a, c: low magnification; b, d: high magnification. Arrowheads indicate the location of the left SNpc. D: immunoreactivity of TH in the striatum (caudate-putamen). TH immunostaining was performed as described above using brain sections obtained 3 wk after 6-OHDA lesioning. CPu, caudate-putamen.

 
To assess whether the neuroprotective effect of 14-26 in vivo correlates with the regulation of oxidative stress and ER stress, the levels of immunoreactivities of 4-HNE-modified proteins (oxidative stress markers) and GRP78 and/or GRP94 proteins (ER stress markers) were evaluated 1 wk after 6-OHDA lesioning in mice. Enhanced immunoreactivities for both stress markers were observed in vehicle-treated mice (Fig. 7, A,a and B,a), but not in 14-26-treated mice (Fig. 7A, c and B,c).


Figure 7
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  		Fig. 7. Effect of 14-26 on oxidative stress and ER stress after 6-OHDA injection. Brain sections from mice obtained 1 wk after 6-OHDA injection with (A,c; A,d; B,c; B,d) or without (A,a; A,b; B,a; B,b) intraperitoneal injection of 14-26 were subjected to immunostaining with anti-HNE antibody (A), anti-KDEL antibody (B), and anti-TH antibody (A, B).

 
Further behavioral examination revealed that administration of 14-26 significantly reduced amphetamine (2.5 mg/kg)- induced rotation, which is caused by the asymmetrical loss of TH (+) neurons in SNpc (19) at 3 wk after 6-OHDA lesions (Fig. 8).


Figure 8
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  		Fig. 8. Behavioral examination after 6-OHDA lesioning. Amphetamine (2.5 mg/kg)-induced rotations were counted 3 wk after 6-OHDA lesioning, as described in the text. A: time course of rotational behavior. B: total number of rotations in 60 min. Values shown are means (SD) of 3 experiments (total 5 mice in each group). *P < 0.01.

 

   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
DBM and its derivatives are small β-diketone compounds and are minor constituents of licorice (8). Several beneficial bioactivities of DBM, such as anti-tumor activity (21, 27, 31), anti-inflammatory activity (18), anti-mutagenesis (7), and iron chelation (44), have previously been reported. Some DBM derivatives have also been used as sunscreens because of their UVA-absorbing activities, but an inability of these derivatives to protect keratinocytes against UVA-induced cytotoxicity has also been reported (1).

In this report, we demonstrated that 14-26 (2,2'-dimethoxydibenzoylmethane), a DBM derivative, possesses neuroprotective properties against both ER stress and oxidative stress. Recent studies have revealed that the protection of cells against ER stress/ER stress-induced cell death can be obtained in several ways: enhancement of the chaperone activity in the ER (40), suppression of general protein synthesis (16), maintenance of the Ca2+ homeostasis in the ER (26), and activation of the UPR branches (4, 39). However, the regulation of ER stress by 14-26 was associated with its antioxidative property. Comparative analysis between DBM derivatives revealed that the presence of methoxy groups in the structure may play an important role in the neuroprotective properties of 14-26 (Fig. 1, B and D).

Whereas the ER is where ROS are produced through the formation of disulfide bonds in proteins, it has weaker defense systems against oxidative stress when compared with either the cytosol or mitochondria. As a result, ER proteins become major targets for oxidative modifications such as carbonylation as described previously (9, 33). Protein carbonyls are generated by a direct metal-catalyzed oxidative reaction or by secondary reactions with reactive carbonyl compounds on carbohydrates, lipids, and advanced glycation/lipoxydation end products (32). 14-26 prevented ROS production in response to both oxidative stress and ER stress (Fig. 3) and reduced the levels of ROS-induced protein carbonyls in the microsome (Fig. 4, A and B). 14-26 also prevented the reduction of the mitochondrial membrane potential after exposure of the cells to both oxidative stress and ER stress (Fig. 4, C–E). These results suggest that 14-26 protects cells against stresses by suppressing ROS generation and ROS-derived damage in both the ER and mitochondria. Although dopaminergic neurons are highly susceptible to oxidative stress because of the presence of dopamine and its metabolites, the protective effects of 14-26 may be more general. Our preliminary results revealed that 14-26 protected mouse insulinoma MIN6 cells against both oxidative stress and ER stress, in a similar manner to SH-SY5Y and PC 12 cells.

Interestingly, NAC also prevented the production of ROS in response to both oxidative stress and ER stress (Fig. 3), but, unlike 14-26, failed to protect against Tm-induced cell death in our experiments (Fig. 1B). A possible explanation for this discrepancy is the mechanism underlying the antioxidative property of each compound. 14-24 possessed the iron-chelating activity (Fig. 5AI) that may play a role in its cytoprotective property (Fig. 5B). By contrast, NAC plays an important role in prevention of ROS, but it can shift the redox status of proteins to the reduced forms (6). As a result, secretory proteins in the reduced forms accumulate in the ER and cause ER stress. Another possibility is that, besides the antioxidative property, other mechanisms are involved in the prevention of ER stress-induced cell death by 14-26. In this context, our preliminary results indicated that 14-26 slightly suppresses general protein synthesis (15–20% decrease after 24h treatment), which is an alternative mechanism of regulating ER stress. However, it is not clear at this moment to what extent this slight change of the levels of general protein synthesis contributes to the cell protection by 14-26.

In vivo, 14-26 suppressed neuronal death in the SNpc and improved amphetamine-induced rotational behavior after injection of 6-OHDA into mice. The neuroprotection by 14-26 in vivo correlated with the regulation of both oxidative stress and ER stress (Fig. 7).

Total iron levels in the SNpc are increased in both PD patients and animals after administration of PD-related neurotoxins such as 6-OHDA (2) and MPTP (41). As iron can induce ROS, oxidative stress, and aggregation of {alpha}-synuclein, iron chelation is believed to have the potential as a neuroprotective strategy for PD, which requires long-term treatment (45). Unfortunately, however, some iron chelators, such as DFO, have poor penetration across the blood-brain barrier due to their hydrophilic nature; others, such as clioquinol (CQ), have high toxicity (45). Therefore, it is important to find small, hydrophobic, and nontoxic compounds with iron-chelating activity, which can be used in a long-term treatment. The nature of 14-26 and other DBM derivatives described here makes these good candidates for use in PD. 14-26 is a small hydrophobic compound, and our results suggest that 14-26 crosses the blood-brain barrier. Although it is not clear whether it counteracts 6-OHDA in the cells or predominantly in the extracellular spaces in vivo, our results in cultured cells support its ability to penetrate the cells.

It is noteworthy that curcumin (diferuloylmethane), another β-diketone molecule and a component of turmeric, also has strong antioxidative, radical-scavenging, iron-chelating, and neuroprotective properties in PD models (23, 46). Although curcumin did not display clear cell protection in SH-SY5Y cells or PC12 cells exposed to either tunicamycin or H2O2 (data not shown), probably due to its cytotoxicity in neuroblastoma cells as described previously (28), our results suggest an important role for β-diketone compounds, including 14-26 and curcumin, in promoting the cell survival of dopaminergic neurons.

In summary, a DBM derivative, 14-26, was identified as a novel neuroprotective agent against both ER stress and oxidative stresses. The regulation of ER stress by 14-26 was associated with its antioxidative property. Dopaminergic neuronal death in the SNpc was reduced by 14-26 administration following injection of 6-OHDA in mice. These results suggest that 14-26 could be a therapeutic candidate for the treatment of PD.


   GRANTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by Grant-in Aid for Scientific Research (17500226 and 19500319) from the Ministry of Education, Science, Technology, Sports and Culture of Japan.


   ACKNOWLEDGMENTS
 
We thank Takashi Tamatani, Michiyo Tamatani, and Harumi Nishihama for technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: O. Hori, Dept. of Neuroanatomy, Kanazawa Univ. Graduate School of Medical Science, 13-1 Takara-Machi, Kanazawa City, Ishikawa, 920-8640, Japan (e-mail: osamuh{at}nanat.m.kanazawa-u.ac.jp)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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