Nobiletin and its related flavonoids with CRE-dependent transcription-stimulating and neuritegenic activities

BBRC Biochemical and Biophysical Research Communications 337 (2005) 1330–1336 www.elsevier.com/locate/ybbrc

Nobiletin and its related flavonoids with CRE-dependent transcription-stimulating and neuritegenic activities Hiroyuki Nagase a, Naoki Omae a, Akiko Omori a, Osamu Nakagawasai b, Takeshi Tadano b, Akihito Yokosuka c, Yutaka Sashida c, Yoshihiro Mimaki c, Tohru Yamakuni a, Yasushi Ohizumi a,* a

c

Department of Pharmaceutical Molecular Biology, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba, Aramaki, Aoba-ku, Sendai 980-8578, Japan b Department of Pharmacology, Tohoku Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, 981-8558 Sendai, Japan Laboratory of Medicinal Plant Science, School of Pharmacy, Tokyo University of Pharmacy and Life Science, Hachioji, Tokyo 192-0392, Japan Received 5 September 2005 Available online 10 October 2005

Abstract cAMP response element (CRE) transcription is dysregulated in neurodegenerative disorders in the central nervous system (CNS), including polyglutamine diseases. As the first step to find natural compounds with protective action against neurodegeneration in the CNS, we here examined whether six citrus flavonoids, namely nobiletin, 5-demethylnobiletin, tangeretin, sinensetin, 6-demethoxytangeretin, and 6-demethoxynobiletin, stimulated CRE-dependent transcription and induced neurite outgrowth in PC12D cells. Among the compounds, nobiletin most potently enhanced CRE-dependent transcription and neurite outgrowth by activating ERK/MAP kinase-dependent signalling to increase CREB phosphorylation. The transcription and neurite outgrowth were stimulated by nobiletin in a concentration-dependent manner, with a strong correlation between them. Furthermore, a 11-day oral administration of nobiletin rescued impaired memory in olfactory-bulbectomized mice documented to be accompanied by a cholinergic neurodegeneration. These results suggest that nobiletin with the activity to improve impaired memory may become a potential leading compound for drug development for neurodegenerative disorders exhibiting the dysregulated CRE-dependent transcription.
2005 Elsevier Inc. All rights reserved. Keywords: Flavonoids; Nobiletin; CRE-dependent transcription; Neurite outgrowth; Neurodegeneration; Bulbectomy; Memory impairment

Increasing evidence indicates that the cAMP-dependent signalling plays crucial roles in a positive regulation of neurite outgrowth via ERK/MAP kinase activation [1], glutamatergic neurotransmission [2,3], and the hippocampal long-term potentiation associated with learning and memory [4,5]. It is also well known that cAMP response element (CRE) transcription is misregulated in neurological disorders, including polyglutamine diseases [6]. In addition, disruption of CREB function in the brain leads to neurodegeneration [7]. In fact, expanded polyglutamine stretches interact with TAFII130, interfering with CREB

*

Corresponding author. E-mail address: [email protected] (Y. Ohizumi).

0006-291X/$ - see front matter
2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.10.001

transcription [8]. Furthermore, since the production and accumulation of b-amyloid (Ab) is thought to be central to the pathogenesis of AlzheimerÕs disease (AD) [9,10], the effects of Ab on the cAMP-dependent signalling have been studied. Recent studies have demonstrated that PKA/CREB pathway and long-term potentiation are inhibited by sublethal Ab1–42 in cultured hippocampal neurons [11], and that sublethal Ab1–42 interferes not only with neuronal activity-dependent signalling to suppress activation of CREB [12], but also with brain-derived neurotrophic factor (BDNF)-induced activation of Ras-mitogen-activated protein kinase/extracellular signal-regulated (ERK) [13]. Moreover, the effect of Ab1–42 on BDNF signalling results in the suppression of the activation of CREB and Elk-1 and CRE-dependent transcription in cultured

H. Nagase et al. / Biochemical and Biophysical Research Communications 337 (2005) 1330–1336 R4 OMe

R3 MeO

O

R2 OR1 O

1, Nobiletin 2, 5-Demethylnobiletin 3, Tangeretin 4, Sinensetin 5, 6-Demethoxytangeretin 6, 6-Demethoxynobiletin

R1 Me H Me Me Me Me

R2 OMe OMe OMe OMe H H

R3 OMe OMe OMe H OMe OMe

R4 OMe OMe H OMe H OMe

Fig. 1. Chemical structures of the tested structurally related flavonoids from a citrus fruit.

cortical neurons [13]. Additionally neuroprotective action of BDNF requires CREB [14,15]. Therefore, it is plausible to consider that like CREB-binding protein (CBP) [6], control of cAMP/CREB and its downstream transcription is a potential target for treatment of neurological diseases, including polyglutamine diseases and AD. Large numbers of compounds from natural resources have provided not only useful pharmacological tools [16– 18] but also novel leading compounds for drug development [19]. Flavonoids form a large family of natural products which are widely distributed in the plant kingdom. In the course of our survey of substances having the activity to potentiate cAMP/CREB signalling to repair dysregulated neuronal functions from natural resources, we examined the effects of six structurally related compounds isolated from Citrus depressa: nobiletin, 5-demethylnobiletin, tangeretin, sinensetin, 6-demethoxytangeretin, and 6-demethoxynobiletin on cAMP/CREB signalling in PC12D cells (Fig. 1). Consequently, nobiletin (Fig. 1A) was successfully found as a flavonoid that potently triggered an activation of cAMP/CREB signalling pathway coupled with CRE-dependent transcription. Here, we first describe the pharmacological properties of nobiletin in comparison with those of the structure-related flavonoids and provide the evidence that nobiletin improves impaired memory in olfactory-bulbectomized mice. Materials and methods Extraction and isolation Citrus depressa fruits were provided through the courtesy of Dr. Masamichi Yano, National Institute of Fruit Tree Science, Shizuoka, Japan. A voucher of the fruits is on file in our laboratory. The peels of C. depressa (dry weight, 500 g) were extracted with hot MeOH (2 L) for 2 h twice. The MeOH extract was passed through a Diaion HP-20 column, eluted successively with H2O–MeOH (7:3; 2:3; 1:4), MeOH, EtOH, and finally with EtOAc (each 2 L). The EtOAc eluate fraction (10 g) was chromatographed over silica gel, eluted with CHCl3–MeOH (19:1), and divided into five fractions (I–V). Fraction II (1.52 g) was subjected to silica gel column chromatography, eluted with hexane–Me2CO (3:2), to give 2 (100 mg). Fraction III (1.63 g) was chromatographed over silica gel, eluted

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with hexane–Me2CO (2:1), to give 3 (389 mg) and 1 (725 mg). Fraction IV (558 mg) was purified by a silica gel column, eluted with hexane–Me2CO (3:2), to give 4 (38.8 mg), 5 (35.8 mg), and 6 (49.0 mg). Nobiletin (1). Pale-yellow needles (CHCl3–MeOH); mp 137–138 C; EIMS m/z 402 [M]+ (C21H22O8); IR (KBr) mmax 2950, 2840, 1640, 1585, 1565, 1510, 1480, 1460, 1415, 1410, 1365, 1335, 1300, 1275, 1255, 1220, 1205, 1170, 1145, 1100, 1075, 1035, 1030, 1015, 965, 950, 905, 860, 835, 810, 800 cm 1; 1H NMR (CDCl3) d 7.55 (1H, dd, J = 8.5, 2.1 Hz), 7.39 (1H, d, J = 2.1 Hz), 6.97 (1H, d, J = 8.5 Hz), 6.59 (1H, s), 4.08 (3H, s), 4.01 (3H, s), 3.96 (3H, s), 3.94 (3H, s), 3.93 (3H · 2, s); 13C NMR (CDCl3) d 177.2 (C@O), 160.9 (C), 151.9 (C), 151.3 (C), 149.2 (C), 148.3 (C), 147.6 (C), 144.0 (C), 137.9 (C), 123.9 (C), 119.5 (CH), 114.8 (C), 111.2 (CH), 108.5 (CH), 106.8 (CH), 62.2 (OMe), 61.9 (OMe), 61.7 (OMe), 61.6 (OMe), 56.0 (OMe), 55.9 (OMe). 5-Demethylnobiletin (2). Yellow powder; EIMS m/z 388 [M]+ (C20H20O8); IR (KBr) mmax 3420, 2945, 2830, 1640, 1610, 1585, 1510, 1480, 1460, 1435, 1430, 1415, 1365, 1340, 1265, 1225, 1190, 1170, 1145, 1115, 1065, 1035, 1030, 1015, 960, 850, 835, 795 cm 1; 1H NMR (CDCl3) d 12.53 (s, OH), 7.58 (1H, dd, J = 8.6, 2.0 Hz), 7.42 (1H, d, J = 2.0 Hz), 6.99 (1H, d, J = 8.6 Hz), 6.60 (1H, s), 4.11 (3 H, s), 3.98 (3H · 2, s), 3.96 (3H, s), 3.95 (3H, s); 13C NMR (CDCl3) d 182.9 (C@O), 163.9 (C), 153.0 (C), 152.5 (C), 149.5 (C), 149.4 (C), 145.7 (C), 136.6 (C), 132.9 (C), 123.7 (C), 120.1 (CH), 111.3 (CH), 108.8 (CH), 107.0 (C), 104.0 (CH), 62.0 (OMe), 61.7 (OMe), 61.1 (OMe), 56.1 (OMe), 56.0 (OMe). Tangeretin (3). Pale-yellow needles (CHCl3–MeOH); mp 150–151 C; EIMS m/z 372 [M]+ (C20H20O7); IR (KBr) mmax 2945, 2835, 1645, 1605, 1580, 1510, 1480, 1460, 1420, 1400, 1365, 1305, 1260, 1215, 1175, 1130, 1105, 1065, 1025, 1015, 1000, 965, 945, 935, 890, 825, 795 cm 1; 1H NMR (CDCl3) d 7.87 (2H, d, J = 8.9 Hz), 7.02 (2H, d, J = 8.9 Hz), 6.59 (1H, s), 4.09 (3H, s), 4.02 (3H, s), 3.94 (3H · 2, s), 3.88 (3H, s); 13C NMR (CDCl3) d 177.3 (C@O), 162.3 (C), 161.2 (C), 151.3 (C), 148.4 (C), 147.7 (C), 144.1 (C), 138.1 (C), 127.7 (CH · 2), 123.8 (C), 114.9 (C), 114.5 (CH · 2), 106.7 (CH), 62.2 (OMe), 62.0 (OMe), 61.8 (OMe), 61.6 (OMe), 55.5 (OMe). Sinensetin (4). Pale-yellow powder; EIMS m/z 372 [M]+ (C20H20O7); IR (KBr) mmax 2990, 2935, 2820, 1635, 1595, 1505, 1485, 1460, 1445, 1425, 1415, 1345, 1320, 1285, 1265, 1255, 1245, 1215, 1205, 1200, 1165, 1145, 1115, 1095, 1060, 1020, 985, 955, 865, 835, 815, 785, 760 cm 1; 1H NMR (CDCl3) d 7.50 (1H, dd, J = 8.5, 2.1 Hz), 7.32 (1H, d, J = 2.1 Hz), 6.96 (1H, d, J = 8.5 Hz), 6.79 (1H, s), 6.58 (1H, s), 3.99 (3H, s), 3.98 (3H, s), 3.97 (3H, s), 3.95 (3H, s), 3.91 (3H, s); 13C NMR (CDCl3) d 177.1 (C@O), 161.1 (C), 157.6 (C), 154.5 (C), 152.6 (C), 151.8 (C), 149.3 (C), 140.3 (C), 124.1 (C), 119.6 (CH), 112.9 (C), 111.2 (CH), 108.7 (CH), 107.4 (CH), 96.2 (CH), 62.2 (OMe), 61.5 (OMe), 56.3 (OMe), 56.1 (OMe), 56.0 (OMe). 6-Demethoxytangeretin (5). Pale-yellow powder; EIMS m/z 342 [M]+ (C19H18O6); IR (KBr) mmax 3000, 2945, 2845, 1635, 1600, 1570, 1505, 1460, 1420, 1405, 1375, 1340, 1305, 1295, 1255, 1245, 1210, 1185, 1175, 1135, 1110, 1045, 1030, 875, 960, 930, 880, 840, 810, 800 cm 1; 1H NMR (CDCl3) d 7.87 (2H, d, J = 9.0 Hz), 7.01 (2H, d, J = 9.0 Hz), 6.58 (1H, s), 6.43 (1H, s), 3.99 (3H, s), 3.97 (3H, s), 3.94 (3H, s), 3.87 (3H, s); 13C NMR (CDCl3) d 177.8 (C@O), 162.1 (C), 160.6 (C), 156.4 (C), 156.3 (C), 151.9 (C), 130.8 (C), 127.6 (CH · 2), 123.9 (C), 114.4 (CH · 2), 109.1 (C), 106.9 (CH), 92.6 (CH), 61.5 (OMe), 56.6 (OMe), 56.3 (OMe), 55.4 (OMe). 6-Demethoxynobiletin (6). Pale-yellow powder; EIMS m/z 372 [M]+ (C20H20O7); IR (KBr) mmax 2930, 2845, 1635, 1595, 1575, 1505, 1455, 1435, 1420, 1400, 1375, 1340, 1320, 1295, 1275, 1255, 1230, 1210, 1205, 1170, 1135, 1120, 1105, 1040, 1035, 1015, 965, 945, 855, 835, 800, 795 cm 1; 1H NMR (CDCl3) d 7.58 (1H, dd, J = 8.5, 2.1 Hz), 7.42 (1H, d, J = 2.1 Hz), 6.98 (1H, d, J = 8.5 Hz), 6.61 (1H, s), 6.44 (1H, s), 4.00 (3H, s), 3.98 (3H, s), 3.97 (3H, s), 3.95 (3H · 2, s); 13C NMR (CDCl3) d 177.8 (C@O), 160.5 (C), 156.5 (C), 156.3 (C), 151.9 (C), 151.8 (C), 149.3 (C), 130.8 (C), 124.1 (C), 119.5 (CH), 111.2 (CH), 109.1 (C), 108.6 (CH), 107.2 (CH), 92.6 (CH), 61.5 (OMe), 56.6 (OMe), 56.3 (OMe), 56.0 (OMe), 55.9 (OMe). PC12D cell culture PC12D cells were cultured in DMEM supplemented with 10% heatinactivated HS, 5% heat-inactivated FCS in a 5% CO2 incubator at 37 C as described previously [20].

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Transient transfection and reporter gene assay Transient transfection and reporter gene assay were conducted as described previously [21]. A firefly luciferase reporter plasmid containing cyclic AMP response (CRE)-element inserted into the upstream of a TATA-like promoter (pTAL) region taken from herpes simplex thymidine kinase promoter was purchased from Clontech. mTH pro0.8-Luc was transfected as described previously [21]. A Renilla luciferase vector, pRLTK (Promega), was used as an internal control to normalize the difference in transfection efficiency. PC12D cells were transfected by using LipofectAMINE 2000 (Invitrogen). Following transfection, cells were treated with vehicle (0.1% DMSO), 100 lM nobiletin, 100 lM of the structurerelated flavonoids from C. depressa fruits or 50 ng/ml NGF for 8 h. Thereafter cells were harvested to assay both the activities of firefly and seapansy luciferase by using a dual luciferase assay kit (Promega).

placed on a stereotaxic instrument. The olfactory bulb (OB) was bilaterally aspirated by suction pump after the scalp was incised, the openings drilled over the bulbs, and the dura cut. All animals were verified histologically. It was confirmed that more than two-thirds of the OB had been removed and some areas of the olfactory nuclei also had lesions. The sham operation was performed in the same way as in the case of OBX without the removal of the OB. Step-through passive-avoidance task Training and retention trials of the passive-avoidance task were conducted as described previously [23]. The latency time of the retention trial of each tested animal was determined on the 14th day after the first administration of nobiletin. Twenty-five to 200 mg/kg/day of nobiletin or vehicle was orally given to olfactory-bulbectomized mice consecutively for 11 days from day 3 after olfactory bulbectomy.

Neurite growth assay Y-maze test Assay of neurite growth from PC12D cells using phase-contrast microscopy was carried out following a 48-h incubation with vehicle (0.1% DMSO), 100 lM nobiletin, 100 lM of its structure-related flavonoids or 50 ng/ml NGF, as described previously [20]. Western blotting Western blotting was performed as described previously [17, 22]. The blotted membrane was blocked in TBST buffer (100 mM NaCl, 0.05% Tween 20, and 10 mM Tris–HCl, pH 7.5) containing 1% BSA for 2 h at room temperature. The membrane was thereafter incubated successively with either anti-phospho-ERK (Thr 202/Tyr 204) or anti-phosphoCREB (Ser 133) antibodies in 1% BSA/TBST buffer overnight at 4 C, and horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Cell Signaling Technology) for 2 h at room temperature. Following stripping the antibodies, the membranes were reprobed with anti-PKA antibody in 1% BSA/TBST buffer for overnight at 4 C and subsequently incubated with HRP-conjugated anti-rabbit IgG. Immunoreactivities were visualized with SuperSignal West Pico Chemiluminescent Substrate (Pierce). For analysis of TH protein expression, following blocking with TBST buffer (100 mM NaCl, 0.05% Tween 20, and 10 mM Tris–HCl, pH 7.5) containing 1% BSA for 2 h at room temperature, the membrane was thereafter incubated successively with mouse monoclonal anti-TH antibody in 1% BSA/TBST buffer overnight at 4 C and horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Cell Signaling Technology) for 2 h at room temperature. Following stripping the antibodies, the membranes were reprobed with mouse monoclonal anti-GAPDH antibody in 1% BSA/TBST buffer for overnight at 4 C and subsequently incubated with HRP-conjugated anti-mouse IgG. Immunoreactivities were visualized as described above. Animals Adult male ddY mice (24.5 ± 1.5 g) were obtained from Nippon SLC (Hamamatsu, Japan). Animals were housed in cages with free access to food and water under the condition of constant temperature (23 ± 1 C) and humidity (55 ± 5%) and adapted to a standard 12-h light/12-h dark cycle (light cycle: 9:00–21:00). All experiments were performed according to the Guide for Care and Use of Laboratory Animals at Tohoku Pharmaceutical University. The procedures used in this study were also approved by the Committee on the Care and Use of Experimental Animals, Tohoku University, in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health. Surgery Surgery was performed as described previously [23]: Mice anesthetized with pentobarbital Na (50 mg/kg, i.p.; Dainippon, Osaka, Japan) were

Short-term memory was examined by monitoring spontaneous alternation behavior in the Y-maze as described previously [24]. Each mouse was placed at the end of one arm and allowed to move freely through the maze during an 8-min session, and the series of arm entries was recorded visually. The alternation behavior was defined by the successive entry into the three arms, on overlapping triplet sets, and such behavior (%) was expressed as the ratio of actual alternations to possible alternations (defined as the total number of arm entries minus two), multiplied by 100. Statistical analyses One-way ANOVA was used to assess the main effect of OBX-lesions and the main effect of drug and interactions between these factors. FisherÕs PLSD posthoc tests were conducted when appropriate. For data from the experiments other than behavioral pharmacology, statistical comparisons were made by using StudentÕs t test. A level of P < 0.05 was considered statistically significant.

Results and discussion Effects of nobiletin and its structurally related compounds on CRE-dependent transcription and neurite outgrowth in PC12D cells Effects of 100 lM of six structurally related flavonoids, that is nobiletin, 5-demethylnobiletin, tangeretin, sinensetin, 6-demethoxytangeretin, and 6-demethoxynobiletin, on CRE-dependent transcription and neurite outgrowth were tested in PC12D cells (Figs. 2 and 3). Nobiletin and its analogues enhanced CRE-dependent transcription with the following rank order: nobiletin  6-demethylnobiletin, sinensetin, tangeretin > 6-demethoxytangeretin > 5-demethylnobiletin. When PC12D cells were treated with 100 lM of each compound, or 50 ng/ml NGF for 48 h, the compounds induced neurite outgrowth with the following rank order: nobiletin  6-demethylnobiletin, sinensetin > tangeretin, 6-demethoxyltangeretin, and 5-demethylnobiletin. Nobiletin also showed the neurite outgrowth-inducing activity which was almost comparable to that of NGF at 50 ng/ ml. These results indicate that nobiletin most potently potentiates CRE-dependent transcription and neurite outgrowth amongst these compounds in PC12D cells (Fig. 3).

A

Relative transcription activity (Fold increase)

H. Nagase et al. / Biochemical and Biophysical Research Communications 337 (2005) 1330–1336

15

**

10

** **

5

0 0

B

**

Neurite-bearing cells (%)

**

100

**

40

** ** 0 10

30

100

Nobiletin(mM) Fig. 4. Concentration-dependent effects of nobiletin on CRE-dependent transcription (A) and neurite outgrowth (B) in PC12D cells. Transfection, reporter gene assay, and morphological assay were carried out as described above. Data are means ± SEM (n = 4). *P < 0.05, **P < 0.01 versus vehicle-treated cells.

40

**

**

0 Control 1

30

80

0 80

10

Nobiletin(mM) Neurite-bearing cells (%)

Fig. 2. Effects of the structurally related flavonoids on CRE-dependent transcription in PC12D cells. PC12D cells were treated with nobiletin (1), 5-demethylnobiletin (2), tangeretin (3), sinensetin (4), 6-demethoxytangeretin (5), and 6-demethoxynobiletin (6). For analysis of CRE-dependent transcription, PC12D cells were plated at a density of 8 · 104 cells/48-well plate and cultured for 24 h. Cells were then transfected with the luciferase reporter construct for CRE for 19 h. Following transfection, cells were treated with the flavonoids for 5 h. Data are means ± SEM (n = 4). *P < 0.05, **P < 0.01 versus vehicle-treated cells.

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2

3

4

5

Nobiletin induces activation of MEK/ERK/MAP kinasedependent signaling accompanied by stimulation of CREB phosphorylation in PC12D cells

6 NGF

Fig. 3. Effects of the structurally related flavonoids on neurite outgrowth in PC12D cells. PC12D cells were treated with the six flavonoids described in Fig. 2. PC12D cells were plated at a density of 2 · 104 cells/24-well plate and cultured for 24 h. Cells were then treated with 100 lM of tested flavonoids or 50 ng/ml NGF for 48 h, and their morphology was then observed using phase-contrast microscopy. Data are means ± SEM of 300 cells obtained from three independent experiments. **P < 0.01 versus vehicle-treated cells.

There is a strong correlation between CRE-dependent transcription and neurite outgrowth in PC12D cells treated with nobiletin Nobiletin also triggered both an activation of CRE-dependent transcription and neurite outgrowth in a concentration-dependent manner in PC12D cells (Figs. 4A and B). There appeared to be a strong correlation between nobiletin-stimulated CRE-dependent transcriptional activation and neurite outgrowth induced with this natural compound, suggesting the involvement of the CRE-dependent transcription in the nobiletin-induced neurite outgrowth in PC12D cells.

Consistent with the morphological observations described above, nobiletin also increased a MEK phosphorylation leading to a sustained rise in phosphorylation of ERK/MAP kinase which has been demonstrated to be required for NGF-induced neurite outgrowth in PC12 cells [25] (Fig. 5). Increased phosphorylation of ERK/MAP kinase by nobiletin appeared at 3 min after treatment and remained elevated for at least 1 h (data not shown). Furthermore, the stimulated phosphorylation of ERK/ MAP kinase was accompanied by a transient augmentation of CREB phosphorylation without any effect on PKAa protein expression in PC12D cells (Fig. 5). Nobiletin induces activation of CRE- but not AP1-dependent transcription accompanied by increases in TH gene transcription and its protein expression in PC12D cells As shown in Fig. 6A, 100 lM nobiletin increased CREbut not AP1-dependent transcription in PC12D cells, whereas NGF enhanced AP1- but not CRE-dependent transcription. TH is the enzyme for the first and rate-limiting step of catecholamine biosynthesis [21]. TH gene promoterdependent transcription was augmented by nobiletin

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Fig. 5. Effects of nobiletin on the phosphorylation of MEK, ERK/MAP kinase, and CREB in PC12D cells. PC12D cells plated at a density of 5 · 105 cells/35-mm dish were cultured for 48 h. Cells were then stimulated with nobiletin (100 lM) for the indicated times or with NGF (50 ng/ml) for 10 min. Western blot analyses were performed using anti-phosphoMEK, anti-phospho-ERK, and anti-phospho-CREB antibodies. Blots were then stripped and reprobed with anti-PKAa antibody, respectively, to verify that equal amount of proteins was present in each sample. Similar results were obtained from at least three independent experiments.

A

B

Fig. 6. Effects of nobiletin and NGF on AP1-, CRE-, and TH gene promoter-dependent transcription (A) and TH protein expression (B) in PC12D cells. (A) PC12D cells were plated at a density of 8 · 104 cells/48-well plate and cultured for 24 h. Cells were then transfected with different luciferase reporters constructs for 16 h. Following transfection, cells were treated with vehicle, 100 lM nobiletin or 50 ng/ml NGF for 8 h. Data are means ± SEM (n = 10). **P < 0.01 versus vehicle-treated cells. (B) PC12D cells were plated at a density of 1 · 106 cells/35-mm dish and cultured for 24 h. Cells were then treated with vehicle (lane 1), 100 lM nobiletin (lane 2) or 1 lM forskolin (lane 3) for 48 h. Western blot analyses were performed using anti-TH and anti-GAPDH antibodies. Blots were first probed with anti-TH antibody, and then stripped and reprobed with anti-GAPDH antibody, to verify that equal amount of proteins was loaded on each lane. Similar results were obtained from at least three independent experiments.

(Fig. 6A). Consistent with this result, nobiletin showed to augment protein expression of TH as well (Fig. 6B). Additionally NGF more potently increased TH gene transcription than nobiletin. Nobiletin improves impaired memory in olfactorybulbectomized mice Olfactory-bulbectomized (OBX) animals have been widely utilized as a useful paradigm that shares some major

clinical features of AD, since the animal model exhibits impaired learning and memory caused by degeneration of the CNS cholinergic system [23,26]. The latency time of the retention trial in OBX mice was shown to be markedly decreased on day 14 after the operation [F5,62 = 17.140; P < 0.01] (Fig. 7A). The successive daily administration of nobiletin raised the latency time in OBX mice in a dose-dependent manner on day 14 after the operation and appreciably increased the latency time at doses of 50 and 100 mg/kg/day [F5,62 = 17.140; P < 0.05] (Fig. 7A). The effects of the successive daily administration of nobiletin on the spontaneous alternation behavior were also tested using the Y-maze test. Nobiletin exerted an increasing action on spontaneous alternation behavior in OBX mice in a dose-dependent manner [F5,58 = 3.482; P < 0.01] (Fig. 7B), without affecting total arm entries [F5,58 = 0.962; P = 0.448] (Fig. 7C). Administration of 50 mg/kg of nobiletin in OBX mice for 11 days significantly increased alternation behavior compared with vehicle-treated OBX mice [P < 0.05]. There was a difference between alternation behavior observed in vehicle-treated OBX and vehicletreated sham mice on the 15th day after the administration [P < 0.01]. These findings suggest that nobiletin improves impaired memory in OBX mice. In the present study, we, for the first time, found that among the six structurally related flavonoids isolated from C. depressa, nobiletin most potently triggered an activation of an ERK/MAP-dependent signalling pathway to elicit CRE transcription and neurite outgrowth, with a strong correlation between them. Also a 11-day oral administration of nobiletin was found to improve the memory impairment in OBX mice documented to undergo a cholinergic neurodegeneration [23]. Several recent studies have revealed that in several neurological disorders, such as polyglutamine diseases [6,8] and AD [11–13], cAMP response element (CRE) transcription is dysregulated. The present structure–activity relationship study suggests that the methyl group at the R1 and the methoxy group at the R2 may be responsible for the activity of nobiletin to elicit CRE-dependent transcription and neurite outgrowth in PC12D cells. Moreover, nobiletin did enhance neuronal function, e.g., transcription of TH gene, a CREB target gene, and its protein expression in PC12D cells, like NGF. It is suggested that new drugs targeted at counteracting CBP loss of function could stand as a valid therapeutic approach in neurodegenerative disorders [6]. In fact, nobiletin exhibited an antagonistic action on suppressing of glutamate-induced activation of CREB by sublethal Ab1–42 in cultured hippocampal neurons (data not shown). It is thus plausible to consider that nobiletin might be a new leading compound for drug development for neurological diseases with the impairment of CRE-dependent transcription. The interesting fact that nobiletin potently elicits CREdependent transcription and neurite outgrowth, with a strong correlation between them, raises the possibility of the involvement of CRE-dependent transcription in

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Fig. 7. Nobiletin improves impaired memory in olfactory-bulbectomized (OBX) mice. (A) Effects of a daily administration (p.o.) of nobiletin for 11 days on the passive-avoidance behavior in OBX mice. Mice were administered nobiletin or vehicle (0.5% Tween 80) for 11 days (from the 3rd day to the 13th day after OBX). The latency time was measured on the 14th day after OBX. Each column represents the mean ± SEM. Numbers in parentheses are the number of animals used. **P < 0.01 versus Sham + vehicle. #P < 0.05 versus OBX + vehicle. (B,C) Effects of a daily administration (p.o.) of nobiletin for 11 days on the spontaneous alternation behavior (B) and the total arm entries (C) in OBX mice. Mice were administered nobiletin or vehicle for 11 days (from the 3rd day to the 13th day after OBX). Y-maze test was carried out on the 15th day after OBX. Each column represents the mean ± SEM. The numbers in parentheses are the number of animals used. **P < 0.01 versus Sham + vehicle. #P < 0.05 versus OBX + vehicle.

nobiletin-induced neurite outgrowth from PC12D cells. In support with this idea, the following recent findings have been documented: (1) cAMP-elicited neurite outgrowth from PC12D cells is blocked by dominant negative ATF1 [27], and (2) axonal growth defects as well as degeneration of peripheral neurons occur in mice lacking CREB [28]. Therefore, it is quite possible to interpret that nobiletin-induced neurite outgrowth from PC12D cells is dependent on CRE-dependent transcription, although the mechanism remains to be defined. Furthermore, the most important finding in this study is that in OBX mice showing memory impairment, nobiletin also rescues this memory deterioration, although the mechanism of improving memory impairment by this compound remains to be identified. The elucidation of the mechanism of action of nobiletin in OBX mice showing memory impairment may give us a new insight into therapeutic drug development for the CNS neurodegeneration. Acknowledgments This work was supported in part by Grant-in-Aid for Scientific Research from the Ministry of Education,

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