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Mechanisms of Neurotrophic Activities via Low-molecular-weight Compounds: Post-transcriptional Regulation in PC12 Cells and Neurons | OMICS International
ISSN: 2167-065X
Clinical Pharmacology & Biopharmaceutics
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Mechanisms of Neurotrophic Activities via Low-molecular-weight Compounds: Post-transcriptional Regulation in PC12 Cells and Neurons

Hiroki Maruoka1,2 and Koji Shimoke1*
1Department of Life Science and Biotechnology, Faculty of Chemistry, Materials, and Bioengineering and Strategic Research Base, Kansai University, Japan
2Technology Research Laboratory, KURABO, Japan
Corresponding Author : Dr. Koji Shimoke
Department of Life Science and Biotechnology
Faculty of Chemistry, Materials
and Bioengineering and Strategic Research Base
Kansai University, 3-3-35, Yamate-cho
Suita, Osaka 564-8680, Japan
Tel: +81-6-6368-0853
Fax: +81-6-6330-3770
Received January 10, 2013; Accepted February 20, 2013; Published February 27, 2013
Citation: Maruoka H, Shimoke K (2013) Mechanisms of Neurotrophic Activities via Low-molecular-weight Compounds: Post-transcriptional Regulation in PC12 Cells and Neurons. Clin Pharmacol Biopharm S1:003. doi:10.4172/2167-065X.S1-003
Copyright: © 2013 Maruoka H, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Recently, it was reported that some low-molecular-weight compounds mimic neurotrophic activities including neurite outgrowth and neuroprotection. Carnosic acid (CA) promotes neurite outgrowth through the activation of Nrf2 in a model of neuron PC12 cells. CA also protects neurons from oxidative stress via the keap/Nrf2 transcriptional pathway. Luteolin induces neurite outgrowth via MAPK, PKC, and cAMP/PKA signaling pathways. In addition, luteolin protects PC12 cells from serum withdrawal-induced oxidative stress. Forskolin-induced neurite outgrowth is mediated by the activation of the PKA signaling pathway, and this PKAmediated neurite outgrowth is achieved by the expression of Nur77 in PC12 cells. In addition, a low concentration of forskolin is closely related to the cAMP-induced protective function against L-DOPA-induced cytotoxicity. The post-transcriptional regulation of gene expression including microRNAs and the acetylation of non-histone protein plays critical roles in neurotrophic activities. Recently, it was revealed that miR-132 modulates luteolin-induced neurite outgrowth via cAMP/PKA- and MAPK-dependent CREB signaling pathways in PC12 cells. Moreover, it has been reported that acetylated Nrf2 binds to the transcriptional activator, CBP/p300 directly, and that Nur77 is acetylated in vivo and in vitro by CBP/p300. The modulation of miR-132 and acetylation of Nrf2 and Nur77 by CBP/p300 may constitute a similar novel regulatory mechanism for low-molecular-weight compounds with neurotrophic activities.

Nerve injury; Carnosic acid; Luteolin; Forskolin; miR-132; PC12 cell
Nerve injury including traumatic brain injury (TBI) is a major public health concern in industrialized countries. It has been estimated that 1.4 million people sustain a TBI annually and 5 million people are disabled in the United States. Young children, adolescents, and the elderly, predominantly male, exhibit the highest rates of TBI [1]. The treatment of nerve injury subjects and improving their outcomes has still not been clarified [2].
Nerve injury results in the formation of contusions, neuronal apoptosis, and axonal tract damage. Promoting neurite outgrowth and protecting neurons from apoptosis are important factors in the treatment of nerve injury [3-6]. It has been shown that the neurotrophic factors NGF, BDNF, NT-3, and NT-4/5 have neuroprotective and neuronal differentiation abilities [7], and are attracting attention as medicines for TBI [8]. Previous studies have demonstrated that NGF promoted electrophysiological, and histomorphological parameters and enhanced axonal regeneration following nerve injury in vivo [9-11]. The specific receptors of the neurotrophic factors NGF, BDNF plus NT-4/5, and NT-3 are TrkA, TrkB, and TrkC, respectively. These Trk family members are membrane-spanning receptors on the cell membrane of neurons [12].
Intracellular signaling pathways contain several protein phosphorylation cascades and at least three signaling pathways have been identified downstream of Trk receptors: the Ras/mitogenactivated protein kinase (MAPK) pathway, phosphatidylinositol 3-kinase (PI3-K)/Akt pathway, and PLC-gamma pathway [13]; however, the delivery of exogenous neurotrophic factors is the greatest obstacle for their therapeutic application since neurotrophic factors are large polypeptide molecules that do not penetrate the bloodbrain barrier (BBB) and are easily metabolized by peptidases when administered peripherally.
In this review, we present recent topics regarding low-molecular weight compounds with neurotrophic activity in PC12 cells and neurons.
Natural Products and Neurotrophic Activities
Natural products may harmonize very well for the treatment of neuronal injury [14-19]. Recently, many compounds from natural sources were demonstrated to possess neurotrophic and neuroprotective abilities [20]. Current research has also confirmed the role of natural products in enhancing the neurite outgrowth activity of NGF in various experimental models [21].
Carnosic acid (CA) is a phenolic diterpene found in the dietary herb rosemary (Rosmarinus officinalis L.) (Figure 1) that exerts antioxidant properties by acting as a radical scavenger [22,23]. Previously, it was demonstrated that CA functions as a peroxisome proliferator-activated receptor g (PPARg) agonist or 5-lipoxygenase inhibitor in mammalian cells [24,25]. However, the role of CA in nerve cells is still unknown.
Recently, it was reported that CA stimulates NGF gene expression through an NF-E2-related factor 2 (Nrf2)-dependent pathway and induces NGF production in astroglial cells [26,27]. Nrf2 is a CNCbZip transcription factor that plays a key role in redox regulation and drug metabolism [28,29]. Previous reports have revealed that Nrf2 is activated by Reactive Oxygen Species (ROS) and exogenous and endogenous electrophiles, such as sulphoraphane and 6-methylsulfinylhexyl isothiocyanate. Moreover, CA can cross the BBB and attenuate middle cerebral artery occlusion (MCAO)-induced neuronal cell death by upregulating the expression of antioxidative Nrf2 target genes, such as HO-1 [30].
In PC12 cells, it has been shown that CA promotes neurite outgrowth and CA-activated Nrf2-induced p62/ZIP expression is essential for the neuronal differentiation of PC12 cells. Furthermore, it has been reported that CA-activated MAPK 1/2 and PI3-K, independent of Nrf2 activation and the activation of these kinases leads to the enhancement of Nrf2 accumulation in CA-mediated neuronal differentiation [26]. In this way, it is thought that Nrf2 contributes to CA-induced neuronal differentiation via the induction of p62/ZIP expression.
In addition to the effect on neurite outgrowth, CA exhibited neuroprotective activity against glutamate/ oxidative stress and cerebral ischemia both in vitro and in vivo. Previous reports revealed that CA activates the keap/Nrf2 transcriptional pathway by binding to specific keap1 cysteine residues, thereby protecting neurons from oxidative stress and excitotoxicity [30-32].
From these findings, it is thought that CA may be a treatment for nerve injury. However, the detailed molecular mechanism by which CA enhances NGF production and the roles of neurotrophic activities in neurons remain unknown. A detailed analysis is expected in the future.
Luteolin (3’,4’,5,7-tetrahydroxyflavone), which is a natural flavonoid that exists in several types of vegetables, fruits, and medicinal herbs also exhibits neurotrophic activity (Figure 2). Luteolin is an ingredient of rosemary, similar to CA. In the mammalian Central Nervous System (CNS), it has been shown that luteolin can permeate through the blood-brain barrier (BBB), show anti-amnesic effects against the toxicity of amyloid in mice, and attenuate scopolamineinduced amnesia in rats [33,34].
In neurite outgrowth, Lin et al. [35] suggested that luteolin promotes neurite outgrowth through the activation of MAPK, PKC and cAMP/PKA signaling pathways in PC12 cells. They also reported that this neurite outgrowth induced TrkA- and EGFR-independent signaling pathways [35,36].
For neuroprotective activity, luteolin has been found to possess anti-inflammatory and neuroprotective activities in microglia [37] and attenuate the neurotoxicity induced by peroxide [38], the neurotoxic agent N-methyl-4-phenyl-pyridinium (MPP+) [39], and amyloid beta protein [40]. Furthermore, luteolin protects PC12 cells from serum withdrawal-induced oxidative stress through Nrf2- mediated transcriptional activation of HO-1 [35]. These findings demonstrate the possibility that luteolin as well as CA may be useful in the treatment of nerve injury.
Similar to the effects of neurotrophic factors, cyclic AMP (cAMP) can also promote neurite outgrowth and neuroprotective activity either on its own or via the activation of MAPK and cyclic AMPdependent protein kinase (PKA) in nerve cells [41]. Previously, it was shown that intracellular cAMP protects against oxidative stress when used alone and in association with the neurotrophic factors, NGF and EGF in PC12 cells [42]. In addition, it has also been reported that the cAMP analogue dbcAMP promotes neurite outgrowth in human neuroblastoma SH-SY5Y cells and PC12 cells [43,44]. Furthermore, we have reported that treatment with dbcAMP leads to the expression of immediate early genes (IEGs), including c-fos and Nur77, as does treatment with NGF in PC12 cells. We also observed that the cAMPPKA- Nur77 pathway is essential for the induction of differentiation by dbcAMP in PC12 cells and their expressions are regulated via the acetylation of histone H3 [44]. It is thought that detailed studies on low-molecular-weight compounds with neurotrophic activity will be necessary for advancing this field. However, the detailed mechanisms of cAMP/PKA have not yet been fully elucidated.
In CNS injury models, several studies have demonstrated that the restoration of cAMP levels improves the outcome. In spinal cord injuries, the application of rolipram to inhibit the degradation of cAMP promotes axon sparing and results in locomotor improvements [45,46]. Similarly, rolipram improves neuronal survival in the hippocampus and hippocampal-dependent learning in transient global ischemia [47-49].
However, it has been reported that rolipram is characterized for its emetic and other problematic effects, and the development of a cAMP activator other than rolipram is expected.
Forskolin, one of the natural products, is a cAMP activator that is used to raise the level of cAMP. Forskolin also plays a useful role in neurite outgrowth and neuroprotective activity. Forskolin is a labdane diterpene that is produced by the Indian Coleus plant (Figure 3). Moreover, it is also known that forskolin is a BBB permeant.
Previously, it was shown that the forskolin induced neurite outgrowth of PC12 cells is mediated by the activation of the PKA signaling pathway and synergistic activation of the ERK signaling pathway [43,50].
On the other hand, Jin et al. [41] suggested that a low concentration of forskolin is closely related with the cAMP-induced protective function against L-DOPA-induced cytotoxicity and that a high concentration of forskolin induces the cAMP-mediated apoptotic process, which enhances L-DOPA-induced cytotoxicity in PC12 cells.
These findings reveal the possibility that forskolin may be a useful tool for the treatment of nerve injury. A detailed investigation is expected in the future.
The Post-Transcriptional Regulation of Gene Expression: MicroRNAs and the Acetylation of Non- Histone Protein
The post-transcriptional regulation of gene expression plays critical roles in neurotrophic activities including neurite outgrowth and neuroprotective activity. MicroRNAs (miRNAs) form part of the post-transcriptional machinery. miRNAs are a class of small, noncoding RNAs of 21-23 nucleotides that regulate gene expression at the posttranscriptional level by binding to the mRNA of protein coding genes [51]. It has been reported that miRNAs are involved in several biological processes, such as development, morphogenesis, cell proliferation, cell differentiation, and apoptosis [52]. In the mammalian CNS, several miRNAs are specifically transcribed and enriched and may play important regulatory roles in neuronal development and brain function [53-55]. A previous study revealed that miR-132, an miRNA that is enriched in mammalian brain tissue, could be induced by neurotrophic factors and that this could represent a mechanism for fine-tuning protein expression following neurotrophic action [56,57].
Recently, it was reported that miR-132 modulates luteolin-induced neurite outgrowth in PC12 cells. Furthermore, it has been revealed that the cAMP/PKA- and MAPK-dependent CRE binding protein (CREB) signaling pathways are involved in the luteolin-mediated miR-132 expression and neuritogenesis of PC12 cells [36]. CREB is a transcription factor that binds to the cAMP-responsive element (CRE), a consensus sequence found in the promoter regions of many target genes. It has been reported that miR-132 is induced by CREB and is involved in the modulation of dendritic morphology, neurite outgrowth, synaptic plasticity and neuroprotection [56,58-60]. Therefore, it is thought that miR-132 also modulates a lot of CREBregulated genes in nerve cells.
The relationship between CA-induced neurite outgrowth and neuroprotective activity, and the regulation of miR-132 has not yet been clarified. Recently, it was revealed that CREB-binding protein (CBP) regulates Nrf2-induced gene transcription [61]. Therefore, it is expected that miR-132 is regulated by Nrf2 via CBP. Moreover, it has been shown that CA activates the MAPK-dependent CREB pathway in addition to Nrf2/ p62/ZIP in PC12 cells [26]. Therefore, it is expected that the MAPK-dependent CREB pathway induced by CA regulates miR-132.
On the other hand, it has been reported that forskolin promotes PKA and CREB phosphorylation and induces miR-132 expression in cultured primary rat neurons [57]. It is known that PKA phosphorylates CREB and that there are more than 100 CREB target genes including IEGs. Furthermore, we reported that dbcAMPinduced neurite outgrowth is regulated by the PKA-CREB-Nur77 pathway in PC12 cells [44]. However, the relationship between PKACREB- dependent neurite outgrowth and the regulation of miR- 132 has not yet been clarified. Recently, it was reported that miR- 132 regulates the differentiation of dopamine neurons by directly targeting Nurr1 expression [62]. It is known that Nur77 and Nurr1 are members of the Nur77 family, which also contains orphan nuclear transcription factors. Therefore, it is expected that miR-132 regulates neurite outgrowth by Nur77 expression, similar to Nurr1. The role of Nur77 on miR-132-mediated PC12 differentiation remains to be investigated. A detailed investigation is expected in the future.
The transcriptional regulation of acetylation is also one of the most important potential mechanisms by which signaling transduction cascades may control their cellular functions [63]. The control of transcription by epigenetic modifications has proven to be important for neurite outgrowth and neuroprotection activity during neuronal development in the nervous system.
Recent studies have shown that many non-histone proteins, particularly transcription factors, are substrates for CBP/p300, greatly expanding the possible mechanisms of CBP/p300 in transcriptional activation [64]. CBP and p300 proteins are common co-activators for a variety of transcription factors [65,66].
Sun et al. revealed that CBP/ p300 directly bound to and acetylated Nrf2 in response to arsenite-induced oxidative stress [67]. Acetylation of Nrf2 by CBP/p300 showed the possibility to constitute a novel regulatory mechanism for Nrf2-dependent neurotrophic activity. Nur77 is acetylated in vivo and in vitro by CBP/p300 and has been detected using acetylation specific antibodies, including anti-Panacetyl and antiacetylated Lys antibodies [68]. We reported that Nur77 was involved in dbcAMP-induced neurite outgrowth in PC12 cells [44]. Acetylation of Nur77 by CBP/ p300 may also constitute a novel regulatory mechanism for Nur77-dependent neurotrophic activity. As shown in figure 4, we propose a similar novel regulatory mechanism by which low-molecular-weight compounds induce neurite outgrowth and neuroprotection.
It has become evident that low-molecular-weight-compounds including natural products may work as therapeutic agents possessing neurotrophic activities and that they may exert many effects on cell function in the central and peripheral nervous systems. However, molecular mechanisms of neurotrophic activities via low-molecularweight- compounds are largely unknown. In this review, we introduced the possibility that three low-molecular-weight compounds CA, luteolin and forskolin showed neurotrophic activities through the same mechanism in the post-translational regulation.
Low-molecular-weight compounds may lead to posttranscriptional regulation including miRNAs and acetylation of histones, and, thus, induce the expression of transcription factors. These transcription factors may be acetylated by CBP/p300. Both acetylated transcription factors and acetylated histones may lead to the increased expression of the genes of proteins involved in neurite outgrowth and neuroprotection (Figure 4).
It is expected that the detailed relationship between the neurotrophic activities of low-molecular-weight compounds and gene expression will be revealed in the future.
This work was supported, in part, by Grants-in-Aid for Scientific Research from MEXT (Ministry of Education, Culture, Sports, Science, and Technology of Japan), HAITEKU (2002-2006) and SENRYAKU (2008-2012) from MEXT, and the Kansai University Special Research Funds, 2007 and 2009.
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