PMID: 27128150

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Neurodegenerative disorders including Alzheimer disease (AD) have a devastating impact on the society. AD is clinically characterized by the impaired cognition and memory deficit. The progression of AD results in the depression of the quality of life and a devastating death of the patient. In addition, AD imposes great distress to the families of patients, heavy burdens to caregivers, and enormous cost of care to our society. Due to its expanding threats in developed countries, the measures to prevent, halt, or cure the AD are urgently needed.

To tackle this problem, we started to search a useful substance from natural resources. We adopted an in vitro assay system to test the anti-AD activity of the substance. The assay system tests whether a given compound has the activity to activate the cAMP-response element (CRE)-dependent transcription in PC12 cells. PC 12 cells are neural-crest derived cell line. The CRE-dependent transcription is the central biochemical event in the establishment of learning and memory in the hippocampus (Fig. 1). Thus, the substance that stimulates the CRE-dependent transcription in this assay is expected to become a potential curative medicine for treating AD. After several rounds of screening, we found that nobiletin, a poly-methoxylated flavone from the citrus peel, activated the CRE-dependent transcription in PC12D cells (Nagase et al. 2005). In the following study, nobiletin has been proved to improve the memory deterioration of the various types of neurodegenerative disease model animals including the AD model mice. Importantly, administration of Chinpi, an herb medicine rich with nobiletin, prevented a progression of the cognitive impairment of AD patients treated with donepezil (Seki et al. 2013).

On the other hand, nerve growth factor (NGF) is a neurotrophin that promotes survival, neurite outgrowth, and synthesis of neurotransmitters in neurons. Aging in rats accompanies NGF deficiencies (Larkfors et al. 1987). Chronic deprivation of NGF in mice exhibits the symptoms of AD such as neuronal loss, tau hyperphosphorylation, and deposition of b-amyloid plaques (Ruberti et al. 2000). Hence, NGF is a candidate to combat against AD. Indeed, intracerebral infusion of NGF partly ameliorated the cholinergic neuron atrophy and improved spatial memory impairment in aged rats (Fischer et al. 1987). The weakness of NGF is that it does not cross the blood-brain barrier when administered peripherally. Therefore, a compound that up-regulates the synthesis of NGF or augments the activity of NGF in the central nervous system of patients may be useful. Here, we report the acute effect of NGF on the activity of nobiletin in vitro.

Our screen accidentally identified NGF that potentiated the CRE-dependent transcription induced by nobiletin in PC12 cells. So we started to characterize the effect of NGF on the activity of nobiletin in detail. Nobiletin at 30 mM activated the CRE-dependent transcription by over 20-fold. Combination of NGF at 30 and 50 nM with nobiletin significantly enhanced the activity induced by nobiletin. On the other hand, NGF from 1 to 100 nM had little effect on the activity in the absence of nobiletin. Thus, NGF has the potential to enhance the nobiletin-induced CRE-dependent transcription, although NGF itself does not activate the CRE-dependent transcription.

NGF exerts its action by binding to the high affinity receptor, the tropomyosin-regulating kinase A (TrkA) (Huang and Reichardt 2003). So we tested whether the effect of NGF on the nobiletin-induced CRE-dependent transcription is mediated via TrkA or not. K252a, a TrkA antagonist, dose-dependently inhibited the enhancement of nobiletin-induced activity by NGF to the basal level. In contrast, K252a showed no effect on the nobiletin-induced activation of the CRE-dependent transcription in the absence of NGF. The result suggests that the effect of NGF on the nobiletin-induced activation is mediated by TrkA receptor. Furthermore, nobiletin signaling does not include Trk A signaling pathway. Therefore, nobiletin signaling crosstalks with NGF signaling downstream of TrkA.

Nobiletin signaling for the activation of CRE-dependent transcription involves Erk activation. As expected, an Erk inhibitor, PD98059 dose-dependently inhibited the nobiletin-induced activation of the CRE-dependent transcription. Interestingly, there was no difference in the inhibitory effect of PD98059 with and without K252a. In the presence of NGF, K252a cancelled the NGF-induced enhancement of CRE-dependent transcription activity. K252a combined with PD98085 showed stronger inhibitory effect than that of PD98085 alone. The results accord with a notion that TrkA A signaling crosstalks with nobiletin signaling upstream of Erk signaling. This conclusion was confirmed by Erk phosphorylation experiments.

To further elucidate the nature of interaction between NGF and nobiletin signaling, we examined the effect of delayed addition of NGF. The delayed addition of NGF significantly enhanced the nobiletin-induced CRE-dependent transcription within 60 min of nobiletin addition. But NGF added after 60 min of nobiletin addition showed no activating effect on the nobiletin-induced CRE-dependent transcription. The results suggest that the NGF signal could interact only with the early event(s) of nobiletin signaling. We next examined the effect of NGF preincubation on the nobiletin-induced CRE-dependent transcription. The result suggests that the signal elicited by NGF lasts at least for 2 hours for the crosstalk with nobiletin signaling. These results suggest the narrow time window of NGF signaling to interact with nobiletin signaling.

In this study, we have shown for the first time that NGF signaling via TrkA enhanced the nobiletin-induced CRE-dependent transcription in PC12 cells. The effect of NGF on the CRE-dependent transcription is controversial. Previous study showed that NGF treatment for 4 h had little effect on the CRE-dependent transcription in PC12 cells (Minneman, 2000). On the other hand, Chang et al. reported that the treatment of NGF for 5 h increased the level of CRE-dependent transcription by 2-fold in PC12 cells, but the increase was insensitive to K252a (Chang et al., 2003). NGF treatment for 48 h increased on the CRE-dependent transcription over 20-fold, which was cancelled by K252a. This long time effect of NGF must be the differentiation effect. By our hands, NGF had no effect on the CRE-dependent transcription in PC12 cells. On the other hand, NGF showed an acute effect on the nobiletin-induced CRE-dependent transcription. Our detailed analyses indicated that NGF could interact with the signaling event that occurs within 60 min of nobiletin addition. The signal elicited by NGF lasted at least for 2 hours for the crosstalk with nobiletin signaling. On the basis of these observations, we concluded that NGF has no effect on the CRE-dependent transcription in the undifferentiated PC12 cells in a short time range (several hours). Of course, NGF should exert the differentiating effect on PC12 cells in a long time range (several days). Our present work demonstrated that NGF has the ability to exert the acute effect via interaction with other signaling.

Based on inhibitor experiments, we concluded that the crosstalk of NGF and nobiletin signaling probably occurred between TrkA and Erk phosphorylation. As shown in Fig. 1, nobiletin uses the cAMP/PKA/MEK/Erk/MAPK/CREB pathway (Nagase et al. 2005). Authentic NGF signaling mediates TrkA/Ras/Raf/MEK/Erk/MAPK/CREB pathway (Huang and Reichardt 2003). Thus, the two pathways share the MEK/Erk/MAPK/CREB pathway. The event between TrkA/Ras/Raf signaling in the NGF pathway appears to interact with the early event of nobiletin signaling. This finding was unexpected, because we concluded in our early study that nobiletin and NGF had distinct mechanisms in inducing the dendritic outgrowth (Nagase et al. 2005).

Our results obtained by the in vitro experiments might have relevance to the in vivo effect of nobiletin. Nobiletin not only prevented the progression of memory deficits in various types of model animals, but also showed anti-apoptotic effect on rat neuronal cells (Yasuda et al. 2014), neurotrophic actions on PC12 cells (Nagase et al. 2005), and regenerative action on cholinergic neurons (Nakajima et al. 2007). How could nobiletin exert such variety of effects on the brain activity? Our findings might provide at least one explanation for these actions. Nobiletin might strengthen and/or complement the action of endogenous NGF. This notion is supported by the fact that the concentration of nobiletin in the brain after in vivo administration was similar to that one used in the present study. It further leads to the idea that endogenous NGF reinforces the activity of a given medicine for AD, although NGF itself shows no apparent effect on the diseases.

The efficacies of some anti-AD medicines might depend at some degrees on the level of endogenous NGF in the brain of patients. Thus, our study sheds new light on the role of endogenous NGF in the treatment of AD.

fig1

Fig 1. Model of crosstalk between nobiletin and NGF signaling in PC12 cells. Nobiletin activates the CRE-dependent transcription via the cAMP/PKA/MEK/Erk/MAPK/CREB pathway (Nagase et al. 2005). The target molecule of nobiletin is unknown. Authentic NGF signaling mediates TrkA/Ras/Raf/MEK/Erk/MAPK/CREB pathway (Huang and Reichardt 2003). The NGF signal exerts the long time effect such as differentiation of neuronal cells, leading to the establishment of learning and memory. Our study demonstrated that NGF crosstalks with nobiletin signaling in the early signaling event(s) between TrkA and Erk.

Acknowledgements

We thank Ms. Michi Kawada for her excellent technical assistance.

References

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