Upregulation of N-Methyl-D-aspartate Receptor Subunits and c-Fos Expressing Genes in PC12D Cells by Nobiletin

Abstract

The N-methyl-D-aspartate (NMDA) receptor plays a key role in learning and memory. Our recent studies have shown that nobiletin from citrus peels activates the cAMP response element-binding protein (CREB) signaling pathway and ameliorates NMDA receptor antagonist-induced learning impairment by activating extracellular signal-regulated kinase. For the first time, we have shown that nobiletin significantly upregulated mRNA expression of the NMDA receptor subunits NR1, NR2A, and NR2B in PC12D cells. Furthermore, c-Fos mRNA expression also increased due to the action of nobiletin. Our results indicate that nobiletin modulates the expression of essential genes for learning and memory by activating the CREB signaling pathway, and suggest that this action mechanism of nobiletin plays a crucial role in improving NMDA receptor antagonist-induced learning impairment in model animals with dementia.

Alzheimer’s disease (AD) is an age-related neurodegenerative disorder with progressive learning and memory impairment. The rise in the number of patients with AD is a severe social problem worldwide. It is now widely recognized that the synaptic dysfunction and neurodegeneration in AD is caused by amyloid β (Aβ).^1,2)

It is well-known that the N-methyl-D-aspartate (NMDA) receptor, a glutamate receptor, plays a very important role in learning and memory functions and is widely distributed in the brain, notably in the CA1 region of the hippocampus.³⁾ This receptor is constructed from NR1 and distinct NR2 (A, B, C, or D) subunits. In particular, binding of glutamate requires both NR1 and NR2 subunits; functional NR1/NR2A and NR1/NR2B receptors have a particularly close relationship with long-term potentiation (LTP) that is a form of synaptic plasticity.⁴⁾ Cognitive impairment is caused by hypofunction of the NMDA receptor.⁵⁾ The NR1 subunit is prerequisite for acquisition of spatial memory, object recognition, olfactory discrimination, and contextual fear memory in the hippocampal CA1,^6,7) whereas the NR2A subunit is required for LTP at the hippocampal CA1 synapses,⁸⁾ and NR2B contributes to the maintenance of spatial performance.⁹⁾ Furthermore, NR1 and NR2B subunit mRNA and protein levels decrease significantly in patients with AD, and the degree of decrease is associated with progression of the neuropathology.¹⁰⁾

Recent studies have demonstrated that learning and memory requires expression of large number of genes that are associated with the cAMP response element (CRE)-binding protein (CREB).^11–13) Elevated Ca²⁺ level then triggers multiple protein kinase signal pathways that activate LTP by activating protein kinase A (PKA)/mitogen-activated protein kinase (MAPK) kinase (MEK)/MAPK/CREB. As a consequence of CREB activation, transcriptional regulators such as c-Fos, c-jun, and fra1 are activated that cause secondary expression of numerous molecules related to learning and memory.¹¹⁾ In particular, c-Fos constructs activator protein-1, a transcription factor, which is assumed to play role in the molecular mechanisms of learning and memory. Central nervous system-specific c-Fos knockout mice exhibit hippocampus-dependent impairment in spatial tasks and reduced LTP magnitude at hippocampal CA3–CA1 synapses.¹⁴⁾

We previously identified nobiletin, a polymethoxylated flavone from Citrus depressa peels, as a natural compound with neurotrophic effects such as neurite outgrowth on PC12D cells,¹⁵⁾ which is a subclone of PC12 cells that is known as a model of neuronal differentiation cell system. Nobiletin improves learning and memory impairment in amyloid precursor protein transgenic mice,¹⁶⁾ chronically Aβ-infused AD model rats,¹⁷⁾ olfactory-bulbectomized mice,¹⁸⁾ and NMDA receptor antagonist MK-801-induced learning impaired mice.¹⁹⁾ Nobiletin prominently stimulates CRE-dependent transcription in PC12D cells²⁰⁾ by activating the PKA/MEK/extracellular signal-regulated kinase (ERK)/CREB signaling pathway.¹⁵⁾ We further revealed that nobiletin restores the CREB phosphorylation suppressed by Aβ peptides in hippocampal neurons.¹⁷⁾ However, it remains unclear whether the genes involved in learning and memory are modulated by nobiletin. This study demonstrates for the first time that nobiletin upregulates mRNA expression of the NR1, NR2A, and NR2B subunits and c-Fos in PC12D cells.

MATERIALS AND METHODS

Preparation of Nobiletin

Nobiletin (Fig. 1) was isolated from the peels of Citrus depressa HAYATA, as described previously.²⁰⁾ The compound was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich Co. LLC, St. Louis, MO, U.S.A.) and stored at −20°C.

Fig. 1. Chemical Structure of Nobiletin, Isolated from the Peels of Citrus depressa

Cell Culture

PC12D cells were cultured in high glucose Dulbecco’s modified Eagle’s medium (DMEM; Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) supplemented with 10% heat-inactivated horse serum (HS; Life Technologies Corp., Carlsbad, CA, U.S.A.), 5% heat-inactivated fetal bovine serum (FBS; MP Biomedicals LLC, Santa Ana, CA, U.S.A.), 4 mM L-glutamine (Sigma-Aldrich), 50 units/mL penicillin, and 50 µg/mL streptomycin (Meiji Seika Pharma Co., Ltd., Tokyo, Japan). The cells were maintained in a humidified atmosphere containing 5% CO₂ at 37°C, as previously described.¹⁵⁾

Cell Treatment and RNA Extraction

PC12D cells were plated at a cell density of 1.5×10⁶ cells per 60-mm culture dish and grown in high-glucose DMEM at 37°C in 5% CO₂ for 48 h, as previously described.¹⁵⁾ The medium was then replaced with fresh low-serum high-glucose DMEM (2% heat-inactivated HS, 1% heat-inactivated FBS) containing 30 µM nobiletin or 0.1% DMSO as a vehicle control. After stimulation, total RNA was extracted using an RNeasy Mini Kit (Qiagen, Venlo, the Netherlands), following the manufacturer’s protocol. The cell lysate was passed through a 25-gauge needle to shear the high molecular weight-cellular components and genomic DNA. Then, total RNA concentration was determined and the preparation was stored at −80°C.

Reverse Transcription and Real-Time Polymerase Chain Reaction (PCR)

Reverse transcription and real-time PCR were performed with a Power SYBR® Green RNA-to-CT™ 1-Step Kit (Applied Biosystems, Foster City, CA, U.S.A.) using 50 ng of total RNA from each sample. A Thermal Cycler Dice® Real-Time System and software were used for detection (TaKaRa Bio Inc., Shiga, Japan). Reverse transcription proceeded at 48°C for 30 min, and then AmpliTaq Gold® DNA polymerase was activated at 95°C for 10 min. cDNA amplification was performed for 40–55 cycles; initial cycle of 95°C for 15 s (denaturation), 60°C for 1 min (annealing and extension), and then subsequent cycles of 95°C for 15 s and 60°C for 30 s, as a dissociation curve. Gene expression was confirmed using the following designed primers (TaKaRa Bio Inc.): NR1 (NCBI Reference Sequences: NM_017010.1, position: 838), NR2A (NCBI Reference Sequences: position: NM_012573.3, Position: 1374), NR2B (NCBI Reference Sequences: NM_012574.1, position: 2734), and β-actin (NCBI Reference Sequences: NM_031144.2, position: 1100) as an internal control. The c-Fos primer sequences were 5′-GAT GTT CTC GGG TTT CAA CG-3′ (forward) and 5′-CTT TCG GAT TCT CCG TTT CT-3′ (reverse). Relative gene expression was calculated by the 2^−ΔΔCt method.²¹⁾

Statistical Analyses

Statistically significant differences between the vehicle-treated group and nobiletin-treated group at each time point were evaluated by unpaired Student’s t-test using GraphPad Prism 5.0 software (GraphPad Software, Inc., La Jolla, CA, U.S.A.). A level of p<0.05 was considered statistically significant.

RESULTS

First, nobiletin was evaluated for its ability to upregulate mRNA levels of the receptor subunits of NMDA, a molecule related to learning and memory, in PC12D cells by real-time PCR. After 24 h of treatment with 30 µM nobiletin, mRNA levels of the NR1 and NR2A NMDA receptor subunits increased significantly due to nobiletin, corresponding to approximately 1.4-fold (p=0.038; Fig. 2A) and 1.7-fold (p=0.011; Fig. 2B) of the vehicle control, respectively. Moreover, NR2A subunit mRNA expression showed approximately 1.3-fold (vs. vehicle control, p=0.048) at 3 h (Fig. 2B). The expression level of the NR2B subunit began to increase higher than the vehicle control at 6 h, and then became approximately 2.5-fold of the vehicle control (p=0.007) at 24 h (Fig. 2C).

Fig. 2. Effects of Nobiletin on Expression of the N-Methyl-D-aspartate (NMDA) Receptor Subunit NR1 (A), NR2A (B), and NR2B (C) mRNA in PC12D Cells

The cells were stimulated with 30 µM nobiletin for the indicated times. Reverse transcription and real-time PCR analyses were performed using a RNA-to-CT™ 1-Step Kit. Each mRNA level was determined by the 2^−ΔΔCt method. Values are presented as mean±S.E.M. (n=3–4). * p<0.05, ** p<0.01 vs. vehicle control.

Then, we examined nobiletin-induced upregulation of c-Fos mRNA levels, an immediate early gene. c-Fos mRNA expression increased markedly by nobiletin, reached a peak 30 min after the start of treatment, and was approximately 3-fold that that of the vehicle control, p=0.024 (Fig. 3). Then, c-Fos gene expression showed the attenuation from 30 min to 180 min, but the expression was significantly high comparison with vehicle control of each time-point.

Fig. 3. Effect of Nobiletin on c-Fos mRNA Expression in PC12D Cells

The cells were stimulated with 30 µM nobiletin for the indicated times. Reverse transcription and real-time PCR analyses were performed using a RNA-to-CT™ 1-Step Kit. Each mRNA level was determined by the 2^−ΔΔCt method. Values are presented as mean±S.E.M. (n=3–4). * p<0.05 vs. vehicle control.

DISCUSSION

The NMDA receptor is crucial for learning and memory, and it plays a key role in the regulation of neuronal synaptic plasticity in the dorsal hippocampus.^4,5) It has been reported that NR1 and NR2B mRNA levels decrease in patients with AD associated with progression of the neuropathology.¹⁰⁾ We reported previously that CRE-dependent transcription was enhanced by nobiletin by activation of the PKA/MEK/ERK/CREB signal pathway in PC12D cells.^15,20) Moreover, our previous report showed that nobiletin ameliorates the NMDA receptor antagonist-induced learning impairment in association with ERK activation in mice.¹⁸⁾ This is the first report demonstrating that nobiletin significantly upregulated NR1, NR2A, and NR2B mRNA levels in PC12D cells (Fig. 2). Particularly, NR2B gene expression was increased in a time-dependent manner by nobiletin. Also, the expression of NR1 and NR2A genes was only modestly upregulated from 3 h after the start of nobiletin treatment. These observations suggest that nobiletin upregulates gene expression of the NMDA receptor subunits by stimulating CRE-dependent transcription due to activation of the PKA/MEK/ERK/CREB signaling pathway. In particular, the expression of NR2B gene was markedly upregulated by nobiletin in comparison to the increase in the NR1 and NR2A mRNA expressions. The reason for this phenomenon is NR2B has CREB-binding site in the promoter region,²²⁾ suggesting this subunit directly controlled through the activation of the PKA/MEK/ERK/CREB signaling pathway by nobiletin. On the other hand, NR1 and NR2A mRNA levels showed modest and delayed increase. For this reason, these subunits are controlled by other transcription factors such as Sp1^23,24) and AP1.²³⁾ It is expected that the expression of these subunit genes is secondarily regulated by other CREB-responsive transcription factors.¹¹⁾ These results suggest that enhanced expression of the NMDA receptor subunits plays an important role in the mechanism of improving action of nobiletin in NMDA receptor antagonist-induced learning impaired mice.

c-Fos almost certainly participates in memory formation by inducing gene expression.¹¹⁾ c-Fos knockout mice display impaired hippocampus-dependent learning correlated with a reduction of LTP in hipocammpus CA3–CA1.¹⁴⁾ We have previously demonstrated that nobiletin-impaired hippocampal LTP is restored by nobiletin in ischemia-induced learning and memory deficient mice in an electrophysiological study.²⁵⁾ Our present results show that c-Fos mRNA expression increases markedly by nobiletin. These results suggest that nobiletin contributes to recovery of reduced LTP by upregulating c-Fos expression in a CREB-dependent gene expression system, resulting in amelioration of learning and memory.

In conclusion, the present results will be useful for developing nobiletin-derived functional foods to prevent dementia including AD.

Acknowledgment

This work was supported by Grants for research projects in “Application in Promoting New Policy of Agriculture, Forestry and Fisheries,” in part, “Development of fundamental technology for analysis and evaluation of functional agricultural products and functional foods” from the Ministry of Agriculture, Forestry and Fisheries, Japan.

REFERENCES

• 1) LaFerla FM, Green KN, Oddo S. Intracellular amyloid-β in Alzheimer’s disease. Nat. Rev. Neurosci., 8, 499–509 (2007).
• 2) Takahashi RH, Milner TA, Li F, Nam EE, Edgar MA, Yamaguchi H, Beal MF, Xu H, Greengard P, Gouras GK. Intraneuronal Alzheimer Aβ42 accumulates in multivesicular bodies and is associated with synaptic pathology. Am. J. Pathol., 161, 1869–1879 (2002).
• 3) Hardingham GE, Bading H. Synaptic versus extrasynaptic NMDA receptor signalling: implications for neurodegenerative disorders. Nat. Rev. Neurosci., 11, 682–696 (2010).
• 4) Riedel G, Platt B, Micheau J. Glutamate receptor function in learning and memory. Behav. Brain Res., 140, 1–47 (2003).
• 5) Melik E, Babar E, Ozen E, Ozgunen T. Hypofunction of the dorsal hippocampal NMDA receptors impairs retrieval of memory to partially presented foreground context in a single-trial fear conditioning in rats. Eur. Neuropsychopharmacol., 16, 241–247 (2006).
• 6) Tsien JZ, Huerta PT, Tonegawa S. The essential role of hippocampal CA1 NMDA receptor-dependent synaptic plasticity in spatial memory. Cell, 87, 1327–1338 (1996).
• 7) Rampon C, Tang YP, Goodhouse J, Shimizu E, Kyin M, Tsien JZ. Enrichment induces structural changes and recovery from nonspatial memory deficits in CA1 NMDAR1-knockout mice. Nat. Neurosci., 3, 238–244 (2000).
• 8) Sakimura K, Kutsuwada T, Ito I, Manabe T, Takayama C, Kushiya E, Yagi T, Aizawa S, Inoue Y, Sugiyama H, Mishina M. Reduced hippocampal LTP and spatial learning in mice lacking NMDA receptor epsilon 1 subunit. Nature, 373, 151–155 (1995).
• 9) von Engelhardt J, Doganci B, Jensen V, Hvalby Ø, Göngrich C, Taylor A, Barkus C, Sanderson DJ, Rawlins JNP, Seeburg PH, Bannerman DM, Monyer H. Contribution of hippocampal and extra-hippocampal NR2B-containing NMDA receptors to performance on spatial learning tasks. Neuron, 60, 846–860 (2008).
• 10) Mishizen-Eberz AJ, Rissman RA, Carter TL, Ikonomovic MD, Wolfe BB, Armstrong DM. Biochemical and molecular studies of NMDA receptor subunits NR1/2A/2B in hippocampal subregions throughout progression of Alzheimer’s disease pathology. Neurobiol. Dis., 15, 80–92 (2004).
• 11) Benito E, Barco A. CREB’s control of intrinsic and synaptic plasticity: implications for CREB-dependent memory models. Trends Neurosci., 33, 230–240 (2010).
• 12) Carlezon WA Jr, Duman RS, Nestler EJ. The many faces of CREB. Trends Neurosci., 28, 436–445 (2005).
• 13) Yin JC, Tully T. CREB and the formation of long-term memory. Curr. Opin. Neurobiol., 6, 264–268 (1996).
• 14) Fleischmann A, Hvalby O, Jensen V, Strekalova T, Zacher C, Layer LE, Kvello A, Reschke M, Spanagel R, Sprengel R, Wagner EF, Gass P. Impaired long-term memory and NR2A-type NMDA receptor-dependent synaptic plasticity in mice lacking c-Fos in the CNS. J. Neurosci., 23, 9116–9122 (2003).
• 15) Nagase H, Yamakuni T, Matsuzaki K, Maruyama Y, Kasahara J, Hinohara Y, Kondo S, Mimaki Y, Sashida Y, Tank AW, Fukunaga K, Ohizumi Y. Mechanism of neurotrophic action of nobiletin in PC12D cells. Biochemistry, 44, 13683–13691 (2005).
• 16) Onozuka H, Nakajima A, Matsuzaki K, Shin RW, Ogino K, Saigusa D, Tetsu N, Yokosuka A, Sashida Y, Mimaki Y, Yamakuni T, Ohizumi Y. Nobiletin, a citrus flavonoid, improves memory impairment and Aβ pathology in a transgenic mouse model of Alzheimer’s disease. J. Pharmacol. Exp. Ther., 326, 739–744 (2008).
• 17) Matsuzaki K, Yamakuni T, Hashimoto M, Haque AM, Shido O, Mimaki Y, Sashida Y, Ohizumi Y. Nobiletin restoring β-amyloid-impaired CREB phosphorylation rescues memory deterioration in Alzheimer’s disease model rats. Neurosci. Lett., 400, 230–234 (2006).
• 18) Nakajima A, Yamakuni T, Haraguchi M, Omae N, Song SY, Kato C, Nakagawasai O, Tadano T, Yokosuka A, Mimaki Y, Sashida Y, Ohizumi Y. Nobiletin, a citrus flavonoid that improves memory impairment, rescues bulbectomy-induced cholinergic neurodegeneration in mice. J. Pharmacol. Sci., 105, 122–126 (2007).
• 19) Nakajima A, Yamakuni T, Matsuzaki K, Nakata N, Onozuka H, Yokosuka A, Sashida Y, Mimaki Y, Ohizumi Y. Nobiletin, a citrus flavonoid, reverses learning impairment associated with N-methyl-D-aspartate receptor antagonism by activation of extracellular signal-regulated kinase signaling. J. Pharmacol. Exp. Ther., 321, 784–790 (2007).
• 20) Nagase H, Omae N, Omori A, Nakagawasai O, Tadano T, Yokosuka A, Sashida Y, Mimaki Y, Yamakuni T, Ohizumi Y. Nobiletin and its related flavonoids with CRE-dependent transcription-stimulating and neuritegenic activities. Biochem. Biophys. Res. Commun., 337, 1330–1336 (2005).
• 21) Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2^−ΔΔCt method. Methods, 25, 402–408 (2001).
• 22) Klein M, Pieri I, Uhlmann F, Pfizenmaier K, Eisel U. Cloning and characterization of promoter and 5′-UTR of the NMDA receptor subunit ϵ2: evidence for alternative splicing of 5′-non-coding exon. Gene, 208, 259–269 (1998).
• 23) Zimmer M, Fink TM, Franke Y, Lichter P, Spiess J. Cloning and structure of the gene encoding the human N-methyl-D-aspartate receptor (NMDAR1). Gene, 159, 219–223 (1995).
• 24) Liu A, Zhuang Z, Hoffman PW, Bai G. Functional analysis of the rat N-methyl-D-aspartate receptor 2A promoter: multiple transcription starts points, positive regulation by Sp factors, and translational regulation. J. Biol. Chem., 278, 26423–26434 (2003).
• 25) Yamamoto Y, Shioda N, Han F, Moriguchi S, Nakajima A, Yokosuka A, Mimaki Y, Sashida Y, Yamakuni T, Ohizumi Y, Fukunaga K. Nobiletin improves brain ischemia-induced learning and memory deficits through stimulation of CaMKII and CREB phosphorylation. Brain Res., 1295, 218–229 (2009).