To clarify the mechanism of action of nobiletin, its effects on protein kinase A (PKA)/ERK/CREB signaling, oxidative stress, endoplasmic reticulum (ER) stress, neuroinflammation, and Aβ generation and degradation were examined in cultured cells and slices, as described below.
4.1. Effects of Nobiletin on the cAMP/PKA/ERK/CREB Signaling Pathway
Increases in intracellular levels of cAMP lead to the activation of PKA and subsequent phosphorylation of the transcription factor CREB. cAMP also activates Rap1, a small GTP-binding protein in the Ras family that serves as a selective activator of B-Raf [60
]. The B-Raf-Rap1 complex in turn activates ERK, leading to CREB phosphorylation [60
]. The cAMP/PKA/ERK/CREB signaling pathway plays essential roles in memory formation in vivo and in long-term potentiation (LTP), a synaptic model of memory [62
Aβ was demonstrated to inhibit the formation of hippocampal late LTP through the inactivation of the PKA/CREB signaling pathway [67
]. Aβ oligomers also inhibit ERK and CREB activation in primary hippocampal neurons [68
]. Therefore, agents that enhance the cAMP/PKA/ERK/CREB signaling pathway may be useful for the treatment and prevention of AD.
Nagase et al. previously reported that nobiletin inhibits the phosphodiesterase (PDE) activity catalyzing the hydrolysis of cAMP, thereby increasing the intracellular cAMP concentration to activate PKA in PC12D cells [69
]. Moreover, nobiletin induced CREB phosphorylation in an ERK kinase (MEK)/ERK-dependent manner [19
]. Of note, it also reversed the sublethal Aβ-induced decrease in CREB phosphorylation in cultured hippocampal neurons [23
Matsuzaki et al. demonstrated that nobiletin increases the phosphorylation of multiple PKA substrates and the GluR1 receptor, the subunit of α-amino-3-hydroxy-5-methyl-D-aspartate (AMPA) receptors, at the PKA phosphorylation site Ser 845 in cultured hippocampal neurons and hippocampal slices [72
]. The phosphorylation of GluR1 at Ser 845 is essential for synaptic plasticity, including enhancement of the postsynaptic glutamate response [73
]. Of note, nobiletin was found to potentiate AMPA receptor-mediated synaptic transmission at Schaffer collateral-CA1 pyramidal cell synapses in hippocampal slices [72
]. This potentiation was not accompanied by changes in the paired-pulse ratio, suggesting involvement of a postsynaptic mechanism. Thus, nobiletin may upregulate synaptic transmission via postsynaptic AMPA receptors, at least in part, by stimulating PKA-mediated phosphorylation of the GluR1 receptor in the hippocampus.
In addition to the AMPA receptor, the NMDA receptor plays an important role in synaptic plasticity [75
]. Aβ induces persistent depression of NMDA-evoked currents, suggesting that prolonged depression of NMDA receptor-mediated transmission functions in the initiation of the pathological changes observed in AD [78
]. It has also been reported that the NMDA receptor subunits GluN1 and GluN2B mRNA and protein levels are reduced in AD patients, and the degree of reduction is associated with progression of the neuropathology [79
]. Kimura et al. reported that nobiletin significantly upregulated the mRNA expression of GluN1, GluN2A, and GluN2B in PC12D cells [80
]. It is well known that activation of NMDA receptors leads to an influx of Ca2+
, which in turn stimulates numerous signaling pathways that converge on the ERK signaling cascade [81
]. Collectively, the activation of the cAMP/PKA/ERK/CREB signaling pathway is a plausible mechanism by which nobiletin exerts memory-improving effects in animal models of AD.
4.2. Effects of Nobiletin on Oxidative Stress and ER Stress
The effects of nobiletin on oxidative stress and ER stress have been examined in cultured cells, including PC12 cells, HT22 cells, and SK-N-SH cells. It was reported to have neuroprotective effects against hydrogen peroxide (H2
)-induced apoptosis in PC12 cells, accompanied by inhibition of H2
-induced decrease in GSH levels and SOD activity [82
]. Although nobiletin has negligible direct antioxidant activity, it induces intracellular GSH through the upregulation of glutamate-cysteine ligase, a rate-limiting enzyme for GSH synthesis, and protects against serum withdrawal- and H2
-induced cytotoxicity in PC12 cells [83
]. It was also reported to protect against H2
-induced cell death in HT22 cells, a murine hippocampal cell line, accompanied by the inhibition of c-Jun N-terminal kinase (JNK) and p38 phosphorylation [84
]. In addition, it inhibited the H2
-induced increase in the expression of pro-apoptotic protein Bax, whereas anti-apoptotic protein Bcl-2 was induced by nobiletin in HT22 cells [84
Nobiletin-induced alterations of gene expression have been examined by DNA microarrays in three organ-derived cell lines: 3Y1 rat fibroblasts, HuH-7 human hepatocarcinoma cells, and SK-N-SH human neuroblastoma cells [85
]. Nobiletin administration resulted in greater than 200% increases in the levels of five genes, including the ER stress responsive genes Ddit3, Trib3, and Asns, in all three cell lines. It also induced greater than 50% decreases in the levels of seven genes, including the cell cycle-regulating genes Ccna2, Ccne2, and E2f8, as well as the oxidative stress-promoting gene Txnip. Increases in the levels of DDIT3 and ASNS proteins, and a decrease in the level of the TXNIP protein, were confirmed in each nobiletin-treated cell line. In addition, treatment of SK-N-SH cells with nobiletin suppressed the increase in TXNIP expression and apoptosis induced by tunicamycin, a potent revulsant of ER stress [86
]. These results suggest that nobiletin can suppress oxidative stress and excessive/prolonged ER stress.
4.3. Effects of Nobiletin on Neuroinflammation
Neuroinflammation aids in the progression of neurodegenerative diseases, including AD and PD [87
]. Nobiletin has been demonstrated to have anti-neuroinflammatory capacity by inhibiting the lipopolysaccharide (LPS)-induced production and secretion of proinflammatory mediators, such as NO, tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6, in the mouse microglia BV-2 cell line [10
]. In a recent report, nobiletin administration (100 mg/kg/day, p.o.) to mice for 6 weeks was found to ameliorate LPS-induced memory deficits, accompanied by the suppression of microglial activation and proinflammatory cytokine secretion [91
]. These results suggest that nobiletin is useful to prevent or halt the progression of inflammation-related neurodegenerative diseases, including AD and PD.
4.4. Effects of Nobiletin on Aβ Generation and Degradation
Aβ is continuously generated and degraded in the brain [92
]. Therefore, its steady-state level is determined by the balance between its generation and degradation [93
]. Aβ is generated from the APP through endoproteolysis by two proteases called β- and γ-secretases. As β- and γ -secretases are necessary for Aβ generation; these enzymes are prime drug targets for reducing Aβ levels in AD [94
]. A recent in vitro study demonstrated that nobiletin exhibits β-secretase (BACE1) inhibitory activity in a non-competitive manner with IC50 values of 5.9 × 10−5
M, suggesting it to be a promising BACE1 inhibitory agent to reduce Aβ production in AD [95
Regarding Aβ degradation, neprilysin was identified as the major protease involved in the physiological degradation of Aβ in the brain [93
], and it was found to be down-regulated in the AD brain [96
]. Nobiletin was reported to increase the expression and activity of neprilysin in SK-N-SH cells [97
]. Kimura et al. recently examined whether nobiletin promotes Aβ degradation using human-induced pluripotent stem (iPS) cell-derived AD model neurons, which generate excess Aβ1-42 due to the familial AD presenilin-1 mutation [98
]. The AD-type iPS cell harbors the Polish familial AD mutation of presenilin-1 (i.e., P117L-presenilin-1), and consequently exhibits increased Aβ1-42 generation after differentiation into neurons. In contrast, the wild-type iPS cells have no mutation in this gene and were used as a control. According to real-time quantitative RT-PCR, neprilysin mRNA levels were significantly upregulated by nobiletin in the AD model neurons. Immunostaining with an anti-Aβ antibody demonstrated that nobiletin reduced the intraneuronal Aβ level. Furthermore, it reduced the level of Aβ1-42 released into the culture medium based on ELISA analyses. Therefore, nobiletin reduced the levels of both intra- and extracellular Aβ, likely via the upregulation of neprilysin and promotion of Aβ degradation under in vitro AD pathological conditions. Together, the regulation of APP processing and the promotion of Aβ degradation induced by nobiletin may play a role in the reduced Aβ levels observed in the brains of nobiletin-treated APP-SL 7-5 and 3XTg-AD mice.