Mechanism of Neurotrophic Action of Nobiletin in PC12D Cells

Abstract

Nobiletin is a nonpeptide compound with a low molecular weight from a citrus fruit and has the activity to rescue bulbectomy-induced memory impairment. Here we describe that nobiletin itself induces neurite outgrowth in PC12D cells, a rat pheochromocytoma cell line, like NGF, and the molecular mechanism of its neurotrophic action. As cultured in the presence of nobiletin or NGF for 48 h and then assayed using a scanning electron microscope, PC12D cells treated with nobiletin showed morphology with flatter and larger cell bodies than the cells cultured with NGF. Nobiletin-induced neurite outgrowth was inhibited by PD98059 and U0126 but not K252a. Consistently, nobiletin caused a concentration-dependent enhancement of Erk/MAP kinase phosphorylation and a sustained increment of phosphorylation of MEK and Erk/MAP kinase, resulting in a stimulation of CREB phosphorylation and CRE-mediated transcription. This compound also increased intracellular cAMP and CRE-mediated transcription in the presence of forskolin and enhanced PKA activity to stimulate phosphorylation of multiple PKA substrates in PC12D cells. Furthermore, nobiletin preferentially inhibited Ca2+/CaM-dependent phosphodiesterase in vitro. This compound failed to stimulate phosphorylation of Erk5, which is known to be induced by NGF/TrkA signaling. These results suggest that nobiletin induces neurite outgrowth by activating a cAMP/PKA/MEK/Erk/MAP kinase-dependent but not TrkA-dependent signaling pathway coupling with CRE-mediated gene transcription and may thus become a novel type of biochemical probe for elucidation of the molecular mechanism of neuronal differentiation.

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

“…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.…”

“…19) Nobiletin prominently stimulates CRE-dependent transcription in PC12D cells 20) 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.…”

“…The cells were maintained in a humidified atmosphere containing 5% CO 2 at 37°C, as previously described. 15) Cell Treatment and RNA Extraction PC12D cells were plated at a cell density of 1.5×10 6 cells per 60-mm culture dish and grown in high-glucose DMEM at 37°C in 5% CO 2 for 48 h, as previously described. 15) The medium was then replaced with fresh low-serum high-glucose DMEM (2% heatinactivated HS, 1% heat-inactivated FBS) containing 30 µM nobiletin or 0.1% DMSO as a vehicle control.…”

“…15) Cell Treatment and RNA Extraction PC12D cells were plated at a cell density of 1.5×10 6 cells per 60-mm culture dish and grown in high-glucose DMEM at 37°C in 5% CO 2 for 48 h, as previously described. 15) The medium was then replaced with fresh low-serum high-glucose DMEM (2% heatinactivated 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.…”

“…Central nervous systemspecific 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.…”