Polyphenols belong to one of the most abundant phytochemicals in the plant kingdom. They are the result of the secondary metabolism of plants through two fundamental metabolic pathways: the shikimate pathway and the acetate pathway [1
]. There are currently about 8000 different polyphenols, divided into at least 10 different classes based on their chemical structure. They are classified as: (1) flavonoids including flavanols, isoflavones, flavanones, flavonones, and anthocyanidins; and (2) nonflavonoids such as phenolic acids (groups of compounds derived from benzoic and hydroxycinnamic acids), stilbenes, and lignans; tannins are flavonoid polymers [3
Polyphenols, which have nutritional interest as micronutrients, are particularly abundant in several foods (vegetables, fruits), oils (argan and olive oils) and beverages (red wines) associated with in the Mediterranean diet. There is much evidence from in vitro studies, animal models, and clinical studies supporting that polyphenol compounds may have geroprotective activities as well as cytoprotective effects, especially in age-related diseases (cardiovascular diseases, eye diseases (cataracts, age-related macular degeneration) and chronic diseases (bowel diseases)) associated or not with enhanced oxysterol levels [5
], through the control of mitochondrial dysfunctions, oxidative stress, inflammation, angiogenesis, and cell death [7
]. At the brain level, phytosterols could prevent oxytosis, i.e., oxidative stress-induced cell death, which could play major role in neurodegeneration [9
]. There is also lot of evidence that several polyphenols have anti-tumor properties (cell cycle delay, apoptosis induction, metastasis prevention) [11
]. Interestingly, there is now recent evidence that polyphenols (especially resveratrol, a polyphenol of the stilbene family, found in grapes, blackberries, or peanuts for example) have pro-differentiating properties on different cell types: adipocytes, hematopoietic cells, human umbilical cord mesenchymal stem cells, cancer cells (thyroid, glioblastoma, colon), human lung fibroblasts, keratinocytes, embryonic cardiomyoblasts, and myoblasts [13
]. There is also now evidence that many dietary components of the Mediterranean diet (curcumin, resveratrol, and polyunsaturated fatty acids (PUFAs)), and diets enriched with polyphenols and PUFAs, as well as caloric restriction, physical exercise, and learning, are able to induce neurogenesis in the adult brain. It is therefore tempting to speculate that nutritional approaches, functional foods enriched in polyphenols, or functionalized polyphenols (polyphenols coupled with nanoparticles) [19
] could provide promising prospects to stimulate adult neurogenesis and combat neurodegenerative diseases and cognitive decline [21
In addition, several polyphenols, including flavonoids such as baicalein, daidzein, luteolin, and nobiletin as well as non-flavonoid polyphenols such as auraptene, carnosic acid, curcuminoids, and hydroxycinnamic acid derivatives including caffeic acid phentyl ester have neurotrophic effects: they enhance neuronal survival and promote neurite outgrowth in vitro, a hallmark of neuronal differentiation [23
]. Flavonoids are also able to induce neuronal differentiation of mouse embryonic stem cells and human pluripotent stem cells [24
]. Altogether, these data support the neurotrophic effects of polyphenols. They also support the ability of these molecules to mimic the functions and/or to stimulate the production of neurotrophic factors, which are a family of biomolecules (peptides or small proteins such as brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), and octadecaneuropeptide (ODN)) capable of favoring the growth, survival, and/or differentiation of both developing and mature neurons [23
]. This is in contrast to peptidyl compounds such as neurotrophins; since polyphenols are not degraded in the intestinal tract and are able to cross the blood–brain barrier [26
], they could potentially be used as therapeutic agents in neurodegenerative pathologies associated with neuronal loss such as ischemic stroke, Alzheimer’s and Parkinson’s diseases, which require the stimulation of neurogenesis [23
]. At the time, while it is considered that polyphenols can mimic neuroprotective activities, little is still known about the ability of these molecules to favor neuronal differentiation and the associated metabolic pathways. Resveratrol, which is an important component of the Mediterranean diet, has been reported to have antioxidant and antitumor properties, but its effects as a neural plasticity inducer are still debated [30
]. Apigenin (a chemical compound of the family of flavones, a subclass of flavonoids) is a major polyphenol of parsley, which is also much consumed in the Mediterranean diet, mainly in the Middle East where it is also used in traditional and folklore medicines [31
]. Apigenin is found in thyme, rosemary, celery and chamomile; it is also present in honey [32
] as well as in olive oil [33
]. At the moment, apigenin has been shown to modulate GABAergic and glutamatergic transmission in cultured cortical neurons [34
]. Neuroprotective, anti-amyloidogenic, and neurotrophic effects of apigenin have been reported in an Alzheimer’s disease mouse model (APP/PS1) [17
]. These effects were associated with an activation of cyclic adenosine monophosphate response element-binding protein (CREB), characterized by an increased level of phosphorylated CREB [17
In the present study, based on the ability of polyphenols to mimic the action of neurotrophic compounds (cytoprotection and/or differentiation), we asked whether two major polyphenols present in the Mediterranean diet (trans-resveratrol (RSV) and apigenin (API)) were able to induce neuronal differentiation characterized by neurite outgrowth (dendrite and axon formation) on murine neuroblastoma N2a cells, which are cholinergic cells capable of differentiating into either cholinergic or dopaminergic cells depending on the culture conditions [35
]. Interestingly, N2a cells express the PAC1 receptor, which is a member of the G-protein coupled receptor (GPCR) superfamily including the metabotropic receptors which bind octadecaneuropeptide (ODN) [37
]. PAC1 and members of GPCR family activate adenylyl cyclase/cAMP/PKA (via Gs-protein coupling) and phospholipase C (PLC)/DAG/protein kinase C (PKC) (via Gq-protein coupling)-dependent signaling pathways [39
]. The PAC1 receptor also triggers the activation of several other protein kinase cascades such as ERK1/2, JNK1/2, p38 MAPK and PKB [41
]. Consequently, N2a cells have the ability to bind the pituitary adenylate cyclase-activating polypeptide (PACAP), which is widely distributed in the brain and peripheral organs and displays high affinity for the PAC1 receptor [40
]. They can also be used to study other neuropeptides or molecules (natural or synthetic) capable of interacting with receptors of the GPCR superfamily. Thus, N2a cells constitute a suitable model to study neuronal differentiation and to identify the signaling pathways associated with this process. In this study, the effects of RSV and API on the neuronal differentiation of N2a were compared with those of trans-retinoic acid (RA) used as positive control. To this end, N2a cells were cultured without or with 10% FBS (conventional culture condition) since it is known that various factors present in FBS can modulate cell differentiation. Interestingly, RSV and API trigger the neuronal differentiation of N2a cells.
Polyphenols are a broad family of molecules including flavonoids, phenolic acids, lignans and stilbenes (such as resveratrol) [3
]. Flavonoids include flavones (such as apigenin), flavonols, flavanones, flavanols, isoflavones, and anthocyanins. The polyphenols are found in large quantities in the Mediterranean diet which is rich in fruits and vegetables and which can be associated with a consumption of wine [55
]. Polyphenols are for the most part powerful antioxidants, and some of them, such as apigenin and resveratrol, also have anti-proliferative and anti-inflammatory properties and decrease the production of growth factors as insulin growth factor-1 (IGF-1) and vascular endothelial growth factor (VEGF) [15
]. With resveratrol, pro-differentiating activities have also been shown on myoblasts, resulting in the formation of myotubes [57
]. Apigenin has also been shown to promote osteogenic differentiation of human mesenchymal stem cells through JNK and p38 MAPK pathways [58
], to enhance myoblast differentiation by regulating Prmt7 [60
], to induce granulocytic differentiation in human promyelocytic leukemia HL-60 cells [61
], and to activate morphological differentiation and G2-M arrest in rat neuronal cells [62
]. It has also been reported that apigenin from Croton betulaster Mull inhibits proliferation, induces differentiation and regulates the inflammatory profile of C6 glioma cells [63
In response to the increase in age-related diseases, particularly neurodegenerative diseases associated with oxidative stress and inflammation [5
], polyphenols, because of their anti-oxidant and anti-inflammatory activities, could be used for prevention or even as a treatment. Several studies also reveal a benefit of polyphenols to cognitive functions [22
]. Moreover, it is now well established that resveratrol prevents the aggregation of β-amyloid, which is neurotoxic [64
]. In the more common neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease, neuronal loss is associated with the evolution of these diseases. Promoting neurogenesis could be a means of preventing these diseases. In this context, neuropeptides produced by the brain via endothelial cells and astrocytes form a family of molecules called neurotrophins, with cytoprotective and differentiating activities [65
]. However, to exert their cytoprotective effects, these molecules must be injected intracerebrally near the lesions [66
]. As polyphenols have the ability to cross the blood–brain barrier and accumulate in the brain much more efficiently when administered intravenously than orally [27
], these properties reinforce their interest in preventing certain neurodegenerative diseases.
In this context, it was therefore important to clarify whether some polyphenols (resveratrol, apigenin) present in significant amount in the Mediterranean diet could promote neurogenesis, especially the differentiation of neuroblasts into mature neurons characterized by the presence of dendrites and/or axons. Using N2a murine neuroblastoma cells, we demonstrated that these cells cultured in the presence of resveratrol and apigenin in a concentration range from 6.25 to 50 μM differentiate into mature neurons with dendrites and/or axons and that the activation of the PKC signaling pathway plays an important role in this process. These results, which establish that some polyphenols (resveratrol, apigenin) are able to trigger neuronal differentiation, opening new perspectives in terms of treatments for neurodegenerative diseases where only few molecules are effective.
Interestingly, the differentiation observed with resveratrol and apigenin on N2a cells is as effective as that obtained with retinoic acid (used as positive control), which in humans can be associated with various side effects because of its ability to activate or suppress the expression of many genes [69
]. In the present study, in terms of toxicity, the differentiation induced by resveratrol as well as with retinoic acid is associated with an inhibition of cell growth resulting from a decrease of cell viability, as evaluated with the FDA test. On the other hand, with apigenin, neuronal differentiation has only little effect on cell growth and viability, suggesting different mechanisms of differentiation between resveratrol and apigenin. In addition, the differentiation observed in the absence of FBS (0% FBS) demonstrates that exogenous factors present in the serum do not contribute to the neuronal differentiation induced by resveratrol and apigenin as well as with retinoic acid. Moreover, a comparison of the efficiency of resveratrol and apigenin to induce neuronal differentiation in serum-free culture medium (0% FBS) and in culture medium containing 10% FBS clearly shows that FBS attenuates the differentiation induced by the polyphenols and retinoic acid, and that FBS also contributes to the attenuation of the cytotoxicity, mainly with retinoic acid. As the comparison of the differentiation obtained in the presence of delipidized serum (10% delipidized FBS) versus 10% FBS shows a lower differentiation in the presence of 10% delipidized FBS than in the presence of 10% FBS, our results demonstrate that some serum proteins counteract neuronal differentiation. The identification of these molecules could make it possible to optimize the use of polyphenols for neurodifferentiation purposes in humans. The fractionation of the serum associated with a proteomic analysis must make it possible to answer this question. It has been reported that serum factors such as α1
-, and β-globulin fractions can cause the inhibition of neuronal differentiation and neurite growth [70
], whereas serum deprivation increases the phosphorylation of EGFR, ERK1/2, AKt, and other signaling molecules in N2a cells [72
]. On the basis of in vitro tests using patient serum instead of FBS, one can also expect to distinguish between good and bad responders to treatment with polyphenols.
In addition, as the ability of RSV, API, and RA to trigger neuronal differentiation occurs without and with 10% FBS, and is associated with modifications of the cell cycle (RSV and API: marked and slight accumulation of the cells in the S phase of the cell cycle, respectively; RA: accumulation of the cells in the G2 + M phase of the cell cycle), the role taken by cell cycle-associated molecules in the neuronal differentiation of N2a cells will require additional investigation.
Altogether, these data underline that in order to promote neurogenesis with polyphenols, it is necessary to identify the signaling pathways involved in this process to develop effective drugs and to optimize the efficiency of resveratrol and apigenin. Hence, we tried to specify which signaling pathways were activated with resveratrol and apigenin but also with retinoic acid used as a positive control.
Due to the ability of certain neurotrophic factors (characterized by cytoprotective and differentiating properties) such as ODN to exert their activities via metabotropic receptors (G protein-coupled receptors) [73
], some molecules capable of inhibiting the metabotropic receptor-associated response under the effect of ODN were used: H89 (PKA inhibitor), U0126 (MEK inhibitor), U73122 (PLC inhibitor), and chelerythrine (PKC inhibitor) [38
]. Without FBS, when RSV and API were associated with H89, U0126, U73122, and chelerythrine, a total inhibition of neuronal differentiation was observed supporting either that these two polyphenols are able to simultaneously stimulate the PLC/PKC/(MEK/ERK) and PKA/(MEK/ERK) signaling pathways, or that they act via a receptor evocating the metabotropic receptors which simultaneously involves the activation of PKA and PLC/PKC, leading to the activation of MEK/ERK. However, with 10% FBS, important modifications in the signaling pathways activated by RSV and API were observed. With RSV, PLC/PKC as well as PKA were activated, whereas with API, the neuronal differentiation was independent of PKA, PLC/PKC, and MEK/ERK. Thus, with RSV, the PLC/PKC signaling pathway was activated both with 0% and 10% FBS, whereas with API, PKA, PLC/PKC, and MEK/ERK were activated with 0% FBS. None of these pathways were required with 10% FBS. These data bring new evidence supporting that the biological activities of RSV and API (impact on cell growth, viability, and differentiation) involve different mechanisms. With RA (used as positive control), PKA and PLC/PKC were activated both with 0% and 10% FBS. Altogether, these data support that the early metabolic pathways involved in neuronal differentiation depend on the inducer considered, and that the differentiating activities of polyphenols (RSV, API) could involve plasma membrane receptors.
In the absence of serum, as the implication of a receptor evoking the metabotropic receptor and simultaneously activating the PKC and PKA pathways was considered [73
], we attempted to determine whether RSV and API were able to activate the synthesis of DBI, which is an ODN precursor [37
]. In those conditions, the ODN produced could in turn activate the metabotropic receptor in an autocrine or paracrine way. The very low values of Ct obtained for DBI in the control cells as well as in the cells cultured in the absence or presence of RSV and API, without FBS or with 10% FBS, excludes this hypothesis. In addition, no effects of RSV and API were observed on NGF and BDNF mRNA levels.
As RSV and API display more ability to differentiate N2a cells than RA, we suggest the following potential mechanism behind this phenomenon. The growth of dendrites and/or neurons requires much energy and mitochondrial biogenesis [75
]. In addition, dietary micronutrients (including polyphenols) have been shown to favor mitochondrial/nuclear dialogue, which could favor gene expression such as in those involved in neuronal differentiation [76
]. In well-differentiated neuronal cells, a topographical redistribution of mitochondria along the axone is also needed to favor the transmission of the nerve impulse [77
]. As several polyphenols are recognized as molecules capable of modulating pathways involved in mitochondrial biogenesis (induction of sirtuins), mitochondrial activity (modulating complexes I to V activity, ATP production), and control of the intra-mitochondrial oxidative status (inhibition of ROS formation), the different benefits of this family of compounds (including RSV and API) at the mitochondrial level might be predominant [78
]. In addition, the mitochondria also play key roles in lipid metabolism, especially fatty acids, which are required for the biogenesis of lipids (fatty acids, phospholipids) present in the membrane of dendrites and neurons [79
]. In contrast, RA is rather known to favor mitochondrial permeability and transition, leading to apoptosis [80
]. It is therefore suggested that the different impacts of RSV, API, and RA at the mitochondrial level, and also probably in other organelles (lysosome, peroxisome) playing key roles in the control of lipid metabolism and the equilibrium between life and death might contribute, at least in part, to the different neuronal differentiation capacities of these different molecules.