Citrus Auraptene Induces Glial Cell Line-Derived Neurotrophic Factor in C6 Cells

Abstract

We previously demonstrated that auraptene (AUR), a natural coumarin derived from citrus plants, exerts anti-inflammatory effects in the brain, resulting in neuroprotection in some mouse models of brain disorders. The present study showed that treatment with AUR significantly increased the release of glial cell line-derived neurotrophic factor (GDNF), in a dose- and time-dependent manner, by rat C6 glioma cells, which release was associated with increased expression of GDNF mRNA. These results suggest that AUR acted as a neuroprotective agent in the brain via not only its anti-inflammatory action but also its induction of neurotrophic factor. We also showed that (1) the AUR-induced GDNF production was inhibited by U0126, a specific inhibitor of mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) 1/2, and by H89, a specific inhibitor of protein kinase A (PKA); and (2) AUR induced the phosphorylation of cAMP response element-binding protein (CREB), a transcription factor located within the nucleus. These results suggest that AUR-stimulated gdnf gene expression was up-regulated through the PKA/ERK/CREB pathway in C6 cells.

Citrus Auraptene Induces Expression of Brain-Derived Neurotrophic Factor in Neuro2a Cells

“…A number of recent studies showed that glial cells might be a cellular target for therapeutic approaches to treat several neurodegenerative diseases and neurological disorders, because glial cells, especially astrocytes, serve as key elements in the formation, maintenance, and refinement of synapses [13][14][15]. In our earlier studies regarding the neuroprotective ability of AUR, we revealed that (1) AUR has the ability to suppress inflammatory responses in vivo, namely, hyperactivation of microglia/astrocytes, and over-expression of inflammatory factors by astrocytes, in some mouse models of brain disorders; thus, resulting in the suppression of neuronal death in the hippocampus [3][4][5][6]; and (2) AUR has the ability to induce GDNF expression in C6 cells [9] in vitro. This series of our studies thus suggested that the site of neuroprotective ability of AUR might be astrocytes.…”

“…The present findings suggested that some of the neuroprotective effects exerted by AUR could, in part, have resulted from enhanced synthesis/secretion of BDNF by neurons themselves. The amount of BDNF secreted by neuro2a cells was very low (a few pg/mL; Figure 3), whereas that of GDNF secreted by C6 cells was about 10 times as high (tens of pg/mL) [9], probably depending upon the difference in their mechanism of action; BDNF might be neuroprotective in an autocrine manner, and GDNF, in a paracrine one. As previous reports indicated that astrocytes [16] and C6 cells [12] were able to synthesize BDNF, we investigated whether AUR could induce BDNF expression in C6 cells.…”

“…Cells seeded in a 96-well plate at a density of 1 × 10 4 cells/well were cultured for 24 h in a medium containing 10% FBS, then for a further 24 h in medium containing 2% FBS, after which the cells were treated with samples in a medium containing 2% FBS for 20 or 40 h. Cell viability was determined by the MTT assay, as previously described [9,28].…”

“…It is well known that the ERK-CREB pathway plays an important role in the production of neurotrophic factors, such as glial cell line-derived neurotrophic factor (GDNF) [10] and brain-derived neurotrophic factor (BDNF) [11,12]. In fact, our latest study revealed that AUR has the ability to induce GDNF expression in C6 cells by triggering the protein kinase A (PKA)/ERK/CREB pathway [9]. Using neuro2a cells, an immortalized cell line of murine nerve cells, we thus examined herein whether AUR would have the ability to induce BDNF expression in these cells.…”

“…We previously demonstrated that AUR exerts anti-inflammatory effects in not only peripheral organs but also the brain, as found from studies using a mouse model of global cerebral ischemia [3,4] and lipopolysaccharide (LPS)-induced systemic inflammation [5,6]. In the process of studying about the neuroprotective effects of AUR, we clarified that (1) AUR has the ability to phosphorylate (i.e., activate) extracellular signal-related kinase (ERK), a component of mitogen-activated protein kinase (MAPK), in rat primary cortical neurons [7]; (2) In the rat pheochromocytoma cell line (PC12 cells), AUR can cause phosphorylation of ERK and of cAMP response element-binding protein (CREB), a downstream target of ERK and a crucial transcription factor for neuronal plasticity and long-term potentiation (LTP) in the brain [8]; and (3) AUR phosphorylates ERK and CREB in rat C6 glioma cells [9]. It is well known that the ERK-CREB pathway plays an important role in the production of neurotrophic factors, such as glial cell line-derived neurotrophic factor (GDNF) [10] and brain-derived neurotrophic factor (BDNF) [11,12].…”