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J. Biol. Chem., Vol. 277, Issue 48, 46645-46650, November 29, 2002

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The Endocannabinoid Anandamide Inhibits Neuronal Progenitor Cell Differentiation through Attenuation of the Rap1/B-Raf/ERK Pathway^*

Daniel Rueda, Beatriz Navarro^§, Alberto Martínez-Serrano^§, Manuel Guzmán, and Ismael Galve-Roperh^¶

From the  Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University, 28040 Madrid, Spain and ^§ Molecular Biology Center "Severo Ochoa," Autónoma University, 28049 Madrid, Spain

Received for publication, July 2, 2002, and in revised form, September 11, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Endocannabinoids are neuromodulators that act as retrograde synaptic messengers inhibiting the release of different neurotransmitters in cerebral areas such as hippocampus, cortex, and striatum. However, little is known about other roles of the endocannabinoid system in brain. In the present work we provide substantial evidence that the endocannabinoid anandamide (AEA) regulates neuronal differentiation both in culture and in vivo. Thus AEA, through the CB₁ receptor, inhibited cortical neuron progenitor differentiation to mature neuronal phenotype. In addition, human neural stem cell differentiation and nerve growth factor-induced PC12 cell differentiation were also inhibited by cannabinoid challenge. AEA decreased PC12 neuronal-like generation via CB₁-mediated inhibition of sustained extracellular signal-regulated kinase (ERK) activation, which is responsible for nerve growth factor action. AEA thus inhibited TrkA-induced Rap1/B-Raf/ERK activation. Finally, immunohistochemical analyses by confocal microscopy revealed that adult neurogenesis in dentate gyrus was significantly decreased by the AEA analogue methanandamide and increased by the CB₁ antagonist SR141716. These data indicate that endocannabinoids inhibit neuronal progenitor cell differentiation through attenuation of the ERK pathway and suggest that they constitute a new physiological system involved in the regulation of neurogenesis.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

During the last decade the endocannabinoid system has been characterized by identification of its endogenous ligands anandamide (AEA)¹ and 2-arachidonoylglycerol (2AG) (1, 2), cloning of their specific seven transmembrane receptors CB₁ and CB₂ (3, 4), and description of their mechanisms of synthesis, uptake, and degradation (5, 6). The CB₁ receptor mediates most cannabinoid responses in brain, where it is highly expressed in the hippocampus, cortex, cerebellum, and basal ganglia (5, 6). Endocannabinoids inhibit the release of neurotransmitters such as GABA, glutamate, and dopamine acting as retrograde synaptic messengers (7, 8). In the hippocampus CB₁ is expressed in GABAergic interneurons, and its activation results in the inhibition of GABA_A synaptic transmission (7-9). In addition, electrical stimulation of Schaffer collaterals in hippocampal slices stimulates 2AG synthesis that in turn activates the CB₁ receptor, resulting in inhibition of long-term potentiation (5). Thus interference with required hippocampal cell firing might explain cannabinoid actions on learning and short-term memory (10). Cannabinoids are also able to control movement by interacting with the dopaminergic system in the striatum (11) and pain perception by interfering with analgesic circuits (5, 7). The signal transduction mechanisms responsible for cannabinoid responses include G_i-mediated inhibition of adenylyl cyclase and modulation of ion channels, including inhibition of voltage-dependent Ca²⁺ channels (N, P/Q type) and activation of inwardly rectifying K⁺ channels (6). In addition, cannabinoids activate different signaling pathways involved in the regulation of cell fate such as the MAP kinase family (ERK, JNK and p38), protein kinase B, and the sphingolipid pathway (6, 12, 13). In fact, cannabinoids may act as modulators of cell fate in both neural and extraneural locations (12, 13), and of special relevance endocannabinoids exert a neuroprotective role in a variety of brain injury models (14, 15). This background prompted us to investigate if the endogenous cannabinoid system could be involved in the control of neurogenesis. Results presented herein show that the endocannabinoid AEA inhibits cortical neuron progenitor differentiation to mature neurons. Moreover, human neural stem cell differentiation and NGF-induced PC12 cell differentiation were also inhibited by cannabinoid challenge. Cannabinoids attenuated in a CB₁-dependent manner Rap1/B-Raf-mediated activation of the ERK signaling pathway. Finally, cannabinoid administration inhibited adult hippocampal neurogenesis in vivo.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- The following materials were kindly donated: plasmids encoding Rap1V12, RasV12, and B-Raf (Dr. P. J. S. Stork, Vollum Institute, Portland, OR), GST-RalGDS fusion protein (Dr. J. L. Bos, Utrecht University, Utrecht, The Netherlands), human CB₁ cDNA (Dr. T. I. Bonner at the National Institute of Health, Bethesda, MD, and Dr. Z. Vogel at The Weizmann Institute, Rehovot, Israel), SR141716 (Sanofi Synthelabo, Montpellier, France), anti-human CB₁ polyclonal antibody (Dr. K. Mackie, University of Washington, Seattle, WA) and HU-210 (Dr. R. Mechoulam, Hebrew University, Jerusalem, Israel). AEA and 2AG were from Cayman Chemicals (Ann Arbor, MI); rabbit polyclonal anti-Rap1 (sc-65), B-Raf (C-19), Trk (C-14), and mouse monoclonal anti-phosphorylated ERK (E-4) and anti-phosphorylated Elk (sc-8406) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA); methanandamide (M-AEA), mouse monoclonal anti--tubulin III and anti--tubulin antibodies from Sigma; anti-phosphotyrosine monoclonal antibody clone PY20 from Transduction Laboratories (Lexington, KY); and [-³²P]ATP from Amersham Biosciences.

Cell Culture and Differentiation-- Cortical neuron progenitors, obtained from 17-day-old rat embryos, were cultured in chemically defined medium supplemented with B27 to differentiate into neurons (16). HNSC.100 cells were cultured as described (17), and differentiation experiments were performed in chemically defined medium in the presence of 1% bovine serum albumin and the mentioned stimuli during 1 week. Cell culture medium was replaced every 2 days. PC12 cells in low serum medium (2% heat inactivated horse serum and 1% calf serum) were transfected with hCB1 cDNA using LipofectAMINE 2000 (Invitrogen) and subsequently stimulated with the indicated agents. Differentiation experiments were carried out in the presence of 100 ng/ml NGF for 48 h. Cell viability was determined by the MTT test, Trypan Blue exclusion, or Hoechst 33258 staining (16). Stock solutions of cellular effectors were prepared in Me₂SO except for NGF and isoproterenol, which were prepared in PBS. No significant influence of Me₂SO on any of the parameters determined was observed at the final concentration used (0.1%, v/v). Control incubations included the corresponding vehicle content.

Western Blot and Immunoprecipitation-- Western blots were performed essentially as previously described (13, 18). After stimulation, cells were washed with ice-cold PBS and scraped in lysis buffer consisting of 50 mM Tris-HCl, pH 7.5, 1% (v/v) Triton X-100, 1% (w/v) sodium deoxycholate, 1 mM EDTA, 1 mM EGTA, 50 mM NaF, 10 mM sodium -glycerophosphate, 5 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 0.1% (v/v) 2-mercaptoethanol, 0.5 µM microcystin-LR, 17.5 µg/ml phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, 2 µg/ml aprotinin, 20 µg/ml soybean trypsin inhibitor, and 5 µg/ml benzamidine. Cell lysates cleared by 15 min of centrifugation at 12,000 × g were subjected to SDS-PAGE and transferred to polyvinylidene difluoride membranes. For Trk A Western blotting high voltage transfer conditions were employed to allow high molecular weight proteins to be efficiently transferred. After incubation with primary antibodies (1:1000), blots were developed with appropriate horseradish peroxidase-coupled secondary antibodies (1:20,000) and an enhanced chemiluminescence detection kit. Loading controls were performed with an anti--tubulin antibody. For immunoprecipitation, 500 µg of protein from cleared cell lysates were incubated with 2 µg of anti-TrkA antibody precoupled to protein G-Sepharose. After washing with lysis buffer TrkA phosphorylation extent was determined in the immunoprecipitate by Western blot using the PY20 antibody.

Rap1 and B-Raf Activation Assays-- Active Rap1 was pulled down from cleared cell lysates (250 µg) with GST-RalGDS fusion protein precoupled to GSH-Sepharose (19) and visualized by Western blot using an anti-Rap1 antibody. Endogenous B-Raf was immunoprecipitated with a rabbit polyclonal antibody, and kinase activity was determined in the presence of myelin basic protein. Substrate phosphorylation was determined after 12% SDS-PAGE by autoradiography and scintillation counting of the excised bands (13).

Animals and Drug Treatments-- Adult Wistar rats were injected intraperitoneal with 5 mg/kg/day M-AEA (n = 6) or 1 mg/kg/day SR141716 (n = 4) dissolved in PBS/Tween 80/ethanol (18:1:1 v/v/v; 1 ml/kg body weight), or equal amounts of vehicle (n = 8) for four consecutive days. At day 2 of cannabinoid treatment, 100 mg/kg 5-bromo-2'-deoxyuridine (BrdUrd) was administered together with the cannabinoid, and a second injection of BrdUrd alone was performed 2 h later. Thereafter, animals received M-AEA, SR141716, or vehicle on alternative days until perfusion on day 16 to allow newly generated cells to acquire appropriate neuronal phenotype and functionality (20). Animal procedures were performed according to the Research Ethical Committee from Complutense University.

Immunohistochemistry and Confocal Microscopy-- Perfusion and immunohistochemistry were performed as described (21) using 30 µm coronal free-floating sections. Sections were incubated with rat anti-BrdUrd (Abcam, Cambridge, UK) and mouse anti-Neu N monoclonal antibodies (Sigma) followed by adequate secondary Alexa Fluor 488 and Alexa Fluor 594 secondary antibodies (Molecular Probes, Leiden, The Netherlands). Five coronal sections per rat, located between 2.8 and 4.3 mm posterior to bregma, were examined using a Microradiance confocal microscope (Bio-Rad, Hercules, CA), three passes with a Kalman filter and a 512 × 512 collection box. BrdUrd-positive cells were counted in the subgranular zone and granule cell layer of the dentate gyrus, and confirmation that BrdUrd staining revealed proliferating neuron progenitors but not DNA-repairing cells was assessed by the following criteria: (i) BrdUrd-labeled cells showed small size and irregular shape characteristic of newly born cells, (ii) cells often appeared in clusters, and in certain cases mitotic figures were observed, (iii) no condensed nuclei characteristic of apoptotic cells were observed at any time. Similar experiments were also performed with a mouse anti-BrdUrd monoclonal antibody (Sigma) followed by incubation with a biotinylated anti-mouse antibody (Vector Laboratories, Burlingame, CA) and subsequent development with nickel 3-3'-diaminobenzidine as chromogen.

Statistical Analysis-- Results shown represent the means ± S.D. of the number of experiments indicated in every case. Statistical analysis was performed by analysis of variance. A post hoc analysis was made by the Student-Neuman-Keuls' test. In vivo data (Fig. 5) were analyzed by a unpaired Student's t test.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Anandamide Inhibits Neuronal Differentiation via CB₁-- We monitored the differentiation of cortical neuron progenitors from 17-day-old rat embryos into mature neurons. As shown in Fig. 1A, AEA treatment decreased the amount of cells with neurites as visualized by immunocytochemistry with an anti--tubulin III antibody. Quantification of AEA action on cortical neurite outgrowth is represented in Fig. 1B. Importantly, AEA-induced inhibition of cortical neuron development was mediated by the CB₁ receptor as shown by SR141716 antagonism (Fig. 1, A and B). AEA-induced reduction of neuronal development was not associated with a decrease in cell viability as determined by the MTT test and Hoechst 33258 staining (data not shown). In parallel with the decrease in neurite outgrowth, AEA delayed the appearance of the early neuronal marker -tubulin III (Fig. 1C, upper panel). Moreover, loss of vimentin expression, a characteristic marker of neuroepithelial progenitors, during the neuronal differentiation process was prevented by AEA (Fig. 1C, lower panel). Finally, AEA decreased the expression of the mature neuronal marker Neu N after 4 days of in vitro differentiation (Fig. 1D). AEA-mediated loss of neuronal markers was prevented by SR141716 (Fig. 1D), thus pointing to the involvement of the CB₁ receptor.

View larger version (52K): [in this window] [in a new window]   Fig. 1.   Anandamide inhibits neuronal differentiation via CB1. E17 cortical neuron progenitor differentiation was performed in the presence of the indicated agents. A, immunofluorescence with anti--tubulin III antibody after 24 h of 5 µM AEA incubation in the absence or presence of 2 µM SR141716. B, percentage of neurons bearing neurites longer than twice (left panel) or five times (right panel) the cell body after quantification of immunofluorescence photographies. C, Western blot of -tubulin III and vimentin expression during the indicated days of cortical neuron development in the absence or presence of AEA. Loading controls were carried out with an anti--tubulin antibody. D, Western blot of -tubulin III (left panel) and Neu N (right panel) in cortical neuron extracts from cultures in the absence or presence of AEA and prevention of the AEA effect by SR141716. Results correspond to four different experiments. Significantly different from controls *, p < 0.01.

Endocannabinoids Inhibit Human Neural Stem Cell and PC12 Cell Differentiation-- To further generalize the AEA effects on neural cell development a human neural stem cell line (HNSC.100) (17) and PC12 cells were also employed. HNSC.100 is a stable multipotent neural stem cell line that can differentiate into the three major cell lineages of the nervous system (neurons, astrocytes, and oligodendrocytes) after serum deprivation. Moreover, this cell line engrafts and differentiates in vivo after transplantation (17, 22). Expression of the CB₁ receptor was first analyzed as its presence would constitute a prerequisite for endocannabinoids to target the HNSC.100 cell endogenous differentiation program. HNSC.100 cells expressed CB₁ at similar levels than cortical neuron progenitors employed as positive control (Fig. 2A). Like in cortical neuron progenitors, the expression of -tubulin during the differentiation process was inhibited by AEA in a CB₁-dependent manner (Fig. 2B). In addition, the AEA-stable analogue M-AEA mimicked AEA action.

View larger version (84K): [in this window] [in a new window]   Fig. 2.   Endocannabinoids inhibit human neural stem cell and PC12 cell differentiation. A, CB1 receptor expression determined by Western blot. Cell extracts from E17 cortical neuron progenitors (lane 1), HNSC.100 (lane 2), and hCB1 cDNA-transfected PC12 cells (lane 3) were employed. B, Western blot of -tubulin III in HNSC.100 cell extracts after 1 week of differentiation in the presence of 25 µM AEA, AEA and 4 µM SR141716 or 15 µM M-AEA. C, phase contrast microscopy of PC12 cells cultured for 48 h in low serum medium containing vehicle (a); 100 ng/ml NGF (b); NGF and 5 µM AEA (c); NGF, AEA and 2 µM SR141716 (d). D, regulation of NGF-induced PC12 cell differentiation by 5 µM AEA, 5 µM M-AEA, 5 µM 2AG, or 50 nM HU-210 alone or in the presence of 2 µM SR141716. Results represent the percentage of differentiated cells bearing neurites longer than twice the cell body size referred to incubations in the presence of NGF alone. Results correspond to six different experiments. Significantly different from controls *, p < 0.01.

We extended our experiments to the widely employed NGF-induced PC12 cell differentiation model (23). Cannabinoids did not modify NGF-induced differentiation of naive PC12 cells in agreement with their lack of CB₁ expression both in the undifferentiated and differentiated state (data not shown). Further experiments were therefore performed in PC12 cells transfected with the human CB₁ cDNA, and confirmation of concomitant CB₁ expression was obtained (Fig. 2A). AEA decreased NGF-induced neurite generation (Fig. 2, C and D), and the same effect was observed with M-AEA, 2AG, and the synthetic cannabinoid HU-210 (Fig. 2C). SR141716 prevented cannabinoid-induced inhibition of PC12 cell differentiation (Fig. 2, C and D), therefore evidencing the involvement of the CB₁ receptor. In contrast, the vanilloid receptor antagonist capsazepin had no effect (data not shown). Together with the observed reduction in the number of neurite-bearing cells, AEA decreased the amount of -tubulin III expression without any cytotoxic action as determined by the MTT test and cell viability counting (data not shown). In summary, endocannabinoids inhibit NGF-induced neuronal-like differentiation of PC12 cells similarly to what occurs with human stem cells and cortical neuron progenitors.

Anandamide Inhibits NGF-induced ERK Activation via CB₁-- As cannabinoids modulate the ERK signaling pathway (6, 12) and ERK activation is a crucial event for PC12 differentiation to a neuronal-like phenotype (23, 24), we hypothesized that endocannabinoid inhibition of PC12 cell differentiation was due to the modulation of this signaling pathway. In agreement with previous reports (25-27), NGF induced a robust and persistent ERK activation in PC12 cells (Fig. 3A), and such effect was greatly attenuated by AEA exposure. Thus AEA decreased the potency of NGF-induced ERK activation and accelerated the decline of such activation (Fig. 3B). Importantly, AEA-mediated ERK inhibition relied on the CB₁ receptor as shown by SR141716 antagonism (Fig. 3C). We subsequently determined the AEA effect on NGF-induced phosphorylation of the transcription factor Elk. ERK-mediated Elk phosphorylation upon NGF stimulation is an essential step for transcriptional regulation required for neuronal differentiation (28). In agreement with its inhibitory action on the ERK signaling pathway, AEA reduced NGF-induced Elk phosphorylation (Fig. 3C). To address the mechanism of AEA action on NGF-induced differentiation, tyrosine phosphorylation of the TrkA receptor was determined following immunoprecipitation. NGF-induced TrkA tyrosine phosphorylation was blocked by AEA (Fig. 3D), and this effect relied on CB₁ activation. Thus, endocannabinoids inhibit NGF-induced PC12 cell differentiation by interfering with NGF signaling events responsible for activation of the differentiation program.

View larger version (53K): [in this window] [in a new window]   Fig. 3.   Anandamide inhibits NGF-induced ERK activation via CB1. Time course of NGF-induced ERK activation in the absence (A) or presence (B) of 5 µM AEA for 15 min determined by Western blot of the phosphorylated (active) form of ERK. C, involvement of the CB1 receptor in AEA-induced ERK inhibition. Activated ERK after stimulation with 100 ng/ml NGF for 30 min in the presence or absence of AEA together or not with 2 µM SR141716. D, phosphorylated Elk after cell stimulation with the indicated stimuli. E, tyrosine phosphorylation of immunoprecipitated TrkA receptor determined with PY20 antibody after cell stimulation with the mentioned agonists. Results correspond to six different experiments.

Anandamide Attenuates NGF-induced Rap1- and B-Raf-mediated ERK Activation-- Comprehensive studies have shown that whereas the classical Ras/Raf-1-mediated short-term ERK activation leads to PC12 cell proliferation, the Rap1/B-Raf-mediated module that results in sustained ERK activation is required for cell differentiation (25, 26, 28). To dissect the specific signaling route affected by cannabinoids, transfection experiments were performed with the cDNA encoding the constitutive forms of the small GTPases Ras and Rap1. Interestingly, while Rap1V12 abolished the inhibitory AEA action on NGF-induced cell differentiation, RasV12 was without significant effect (Fig. 4A), therefore indicating that Rap1- but not Ras-dependent signaling pathway is involved in the inhibition of neuronal-like differentiation by AEA. Moreover, transfection with the cDNA encoding the native form of the MEK kinase B-Raf, a major downstream target of Rap1 activation (24), also abrogated AEA inhibition of neurite outgrowth (Fig. 4A). As increased cyclic AMP levels are known to activate the Rap1/ B-Raf signaling pathway by different mechanisms (28-31), this approach was employed to obtain further evidence that cannabinoids inhibit Rap1/B-Raf signaling. Thus the adenylyl cyclase activator forskolin and the -adrenergic agonist isoproterenol blocked AEA action, both in terms of differentiation (Fig. 4B) and ERK inhibition (data not shown). Finally, AEA action on Rap1 and B-Raf activation was determined. NGF activated both Rap1 and B-Raf, and this effect was blunted by AEA and M-AEA. Importantly, SR141716 was able to antagonize AEA action, indicating the involvement of the CB₁ receptor (Fig. 4, C and D). Moreover, incubation with AEA alone decreased basal Rap1 and B-Raf activity in resting cells (data not shown). In summary, cannabinoid stimulation of PC12 cells decreases NGF-mediated Rap1/B-Raf activation, thereby attenuating ERK activation that ultimately results in reduced neuronal-like PC12 cell differentiation.

View larger version (38K): [in this window] [in a new window]   Fig. 4.   Anandamide attenuates NGF-induced Rap1- and B-Raf-mediated ERK activation. A, percentage of differentiated cells relative to vector-transfected cells in the presence of NGF. Cells were transfected with hBC1 alone or in combination with Rap1V12, RasV12, and B-Raf. B, percentage of differentiated cells in the presence of NGF and when indicated AEA (5 µM), forskolin (2 µM), or isoproterenol (20 µM). C, Western blot of Rap1 after affinity precipitation (AP) of the active Rap1GTP form following stimulation with the indicated agents. D, B-Raf activity of endogenous immunoprecipitated protein after cell stimulation. Results correspond to four different experiments. Significantly different from controls *, p < 0.01.

Cannabinoid Inhibition of Adult Rat Neurogenesis in the Hippocampus-- To analyze if endocannabinoid-mediated reduction of neuronal development occurred in vivo, neurogenesis was determined in the subgranular zone of the dentate gyrus of adult rats injected with vehicle or M-AEA. Neurogenesis in vivo was determined by confocal analysis of newly generated cells labeled with BrdUrd. No significant differences were observed in the total amount of dividing cells (BrdUrd⁺) per rat (Fig. 5A), therefore excluding a toxic effect of M-AEA treatment. However M-AEA increased Neu N-negative cells within the newly generated cells. Thus, in parallel with a significant decrease in the percentage of double positive Neu N/BrdUrd-labeled cells there was an increased percentage of Neu N-negative BrdUrd-positive cells (Fig. 5B). To strengthen the hypothesis that endocannabinoids modulate neurogenesis in vivo we tested the effect of SR141716. Blockade of the endogenous cannabinoid tone with the CB₁ antagonist enhanced neurogenesis as shown by the increase in the percentage of Neu N/BrdUrd-labeled cells. This was accompanied by a decrease of Neu N-negative cells (Fig. 5, A and B). Representative examples of immunohistochemistry images obtained with two different anti-BrdUrd antibodies (Fig. 5, C and D) confirm the existence of newly dividing cells in the dentate gyrus of adult animals. These neural progenitor cells in vivo can develop into neuronal cells with the appropriated phenotype and functionality (32) (Fig. 5D). Thus cannabinoid administration diminishes the ability of neuronal progenitors to reach a mature neuronal phenotype in vivo in line with the observed reduction of neuronal generation in vitro.

View larger version (29K): [in this window] [in a new window]   Fig. 5.   Methanandamide and SR141716 modulate adult hippocampal neurogenesis. A, number of BrdUrd-positive cells colocalizing or not with the neuronal marker Neu N per rat in the dentate gyrus of control (open bars), M-AEA- (close bars), or SR141716-treated rats (hatched bars). B, percentage of BrdUrd-positive cells colocalizing or not with Neu N in the same animals. C, immunohistochemistry of BrdUrd-positive newborn cells in adult dentate gyrus sections stained with a mouse anti-BrdUrd monoclonal antibody followed by biotinylated anti-mouse antibody incubation and immunoperoxidase reaction. Photography corresponds to 200× (upper panel) or 400× (lower panel) magnification. Scale bar: 30 and 15 µm, respectively. D, double immunofluorescence of newborn neurons in adult dentate gyrus sections incubated with a rat anti-BrdUrd monoclonal antibody (red staining) and a mouse anti-Neu N monoclonal antibody (green staining) examined under confocal microscopy with 630× (upper panel) and 2200× (lower panel) magnification. Scale bar: 10 and 3 µm, respectively. Individual confocal images are not meant to represent the relative number of labeled cells. Significantly different from controls *, p < 0.05.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Endocannabinoids and the Regulation of Neurogenesis-- Here we show that endocannabinoids are able to inhibit neuronal differentiation in different cellular models in vitro, and this correlates with their ability to inhibit adult hippocampal neurogenesis in vivo. These findings may have important biological implications as the endocannabinoid system is abundantly expressed in the hippocampus where it plays an active role in normal brain physiology (5, 7, 8). Moreover, endocannabinoid levels and CB₁ receptor expression follow a defined pattern during brain development (33). Our findings indicate for the first time that the endocannabinoid system may be involved in the control of neurogenesis, a notion supported by the observed enhancement of neurogenesis by blockade of the endogenous cannabinoid tone. Several physiological or environmental stimuli are known to modulate adult neurogenesis (20, 34). Thus endocannabinoids may constitute an additional factor to be added to those that negatively influence adult neurogenesis such as stress, age, glucocorticoids, and opiates (34). Adult neurogenesis is proposed to be involved in cognition and brain repair (20, 34). For example, formation and improvement of certain forms of memory are influenced by adult neurogenesis (20, 34) as newly generated neurons are rapidly incorporated into functional hippocampal circuits after their generation (32) where they can act as gatekeepers to memory. Thus, besides cannabinoid-mediated neuromodulation and inhibition of hippocampal cell firing (7, 8, 10), inhibition of neurogenesis in adult hippocampus might help to explain cannabinoid disruption of cognitive processes such as learning and short-term memory.

Cannabinoid Inhibition of Rap1/B-Raf-dependent ERK Signaling-- Results presented herein show that endocannabinoids regulate neuronal development by interfering with the ERK signaling pathway responsible for the differentiation program of neuronal multipotent progenitors. These observations are in agreement with the suggested role of endocannabinoids as modulators of neural cell fate (12-15) and with their ability to modulate signal transduction pathways that are essential for the regulation of cell fate (12, 13, 18, 35). The important role of the ERK signaling pathway in neuronal differentiation has been extensively studied (23, 24) and constitutes a paradigm for the generation of different cellular outcomes depending on the kinetics, intensity, and signaling environment of the cell. Although NGF-induced ERK activation involves both Ras- and Rap-dependent mechanisms (24), Rap1/B-Raf-mediated sustained ERK activation appears to be essential to activate the differentiation program (25, 26, 28). Here we show that endocannabinoids are able to inhibit NGF-induced signaling events that ultimately result in the inhibition of neuronal generation. AEA decreased NGF-induced TrkA tyrosine phosphorylation and Rap1 and B-Raf activation, finally resulting in attenuated ERK activation. Moreover, AEA-mediated inhibition of neuronal differentiation was rescued by enhanced activity of the Rap1/B-Raf signaling module, whereas constitutively active Ras was without effect. These results confirm the crucial role of Rap1/B-Raf-dependent sustained ERK activation in neuronal differentiation and show the existence of an inhibitory coupling between neuronal CB₁ receptors and the Rap1/B-Raf/ERK pathway.

The observed inhibition of NGF-induced ERK activation by cannabinoids via its G-protein-coupled receptor CB₁ constitutes a novel paradigm for the signaling links between heptahelical receptors and tyrosine kinase receptors. For example, it is currently well known that G-protein-coupled receptors may activate tyrosine kinase receptors in a process designated as transactivation (reviewed in Refs. 36, 37). In addition, examples of negative cross-regulation (transinactivation), though less frequent, have also been reported. Thus, bradykinin and ATP can inhibit epidermal growth factor receptor phosphorylation in A431 cells (38, 39). Similarly, estrogen activation of its G-protein-coupled receptor GPR30 mediates the inactivation of the epidermal growth factor receptor (40). Moreover, green tea-derived catechins are able to inhibit platelet-derived growth factor receptor phosphorylation by an as yet unknown mechanism (41). In conclusion, we have deciphered a novel signaling mechanism of the CB₁ receptor that leads to the inhibition of NGF-induced ERK activation through the attenuation of the Rap1/B-Raf signaling pathway with important consequences in neuronal development.

	ACKNOWLEDGEMENTS

The institutional grant from the Ramón Areces Foundation to the Molecular Biology Center "Severo Ochoa" (CBMSO) is gratefully acknowledged. D. L. Altschuller is acknowledged for helpful advice in Rap1 activation assays and I. Ocaña for expert technical assistance.

	FOOTNOTES

^* This work was supported, in part, by Ministerio De Ciencia y Tecnología Grants SAF2002-04687 and PM98/0079, the Ramón Areces Foundation, and Complutense University Grant PR48/01-9846 (to I. G. R. and M. G.) and European Union Grants BIO04-CT98-0530 and QLK3-CT-2001-02120 (to A. M. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed. Tel.: 3491-394-4668; Fax: 3491-394-4672; E-mail: igr@bbm1.ucm.es.

Published, JBC Papers in Press, September 16, 2002, DOI 10.1074/jbc.M206590200

	ABBREVIATIONS

The abbreviations used are: AEA, anandamide (N-arachidonoylethanolamine); MAP, mitogen-activated protein; ERK, extracellular signal-regulated kinase; JNK, c-Jun NH₂-terminal kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; NGF, nerve growth factor; 2AG, 2-arachidonoylglycerol; BrdUrd, 5-bromo-2'-deoxyuridine; M-AEA, methanandamide; NGF, nerve growth factor; PBS, phosphate-buffered saline; GST, glutathione S-transferase; MTT, 3-4-5-dimethylthiazol-2,5-diphenyltetrazolium bromide thiazol blue.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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				M. Rohe, A.-S. Carlo, H. Breyhan, A. Sporbert, D. Militz, V. Schmidt, C. Wozny, A. Harmeier, B. Erdmann, K. R. Bales, et al. Sortilin-related Receptor with A-type Repeats (SORLA) Affects the Amyloid Precursor Protein-dependent Stimulation of ERK Signaling and Adult Neurogenesis J. Biol. Chem., May 23, 2008; 283(21): 14826 - 14834. [Abstract] [Full Text] [PDF]

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				I. Galve-Roperh, T. Aguado, J. Palazuelos, and M. Guzman The Endocannabinoid System and Neurogenesis in Health and Disease Neuroscientist, April 1, 2007; 13(2): 109 - 114. [Abstract] [PDF]

				T. Aguado, A. Carracedo, B. Julien, G. Velasco, G. Milman, R. Mechoulam, L. Alvarez, M. Guzman, and I. Galve-Roperh Cannabinoids Induce Glioma Stem-like Cell Differentiation and Inhibit Gliomagenesis J. Biol. Chem., March 2, 2007; 282(9): 6854 - 6862. [Abstract] [Full Text] [PDF]

				E. Kim, A. L. Clark, A. Kiss, J. W. Hahn, R. Wesselschmidt, C. J. Coscia, and M. M. Belcheva {micro}- and {kappa}-Opioids Induce the Differentiation of Embryonic Stem Cells to Neural Progenitors J. Biol. Chem., November 3, 2006; 281(44): 33749 - 33760. [Abstract] [Full Text] [PDF]

				S. H. Kim, S. J. Won, X. O. Mao, C. Ledent, K. Jin, and D. A. Greenberg Role for Neuronal Nitric-Oxide Synthase in Cannabinoid-Induced Neurogenesis J. Pharmacol. Exp. Ther., October 1, 2006; 319(1): 150 - 154. [Abstract] [Full Text] [PDF]

				W. Jia, V. L. Hegde, N. P. Singh, D. Sisco, S. Grant, M. Nagarkatti, and P. S. Nagarkatti {Delta}9-Tetrahydrocannabinol-Induced Apoptosis in Jurkat Leukemia T Cells Is Regulated by Translocation of Bad to Mitochondria Mol. Cancer Res., August 1, 2006; 4(8): 549 - 562. [Abstract] [Full Text] [PDF]

				P. Berghuis, M. B. Dobszay, X. Wang, S. Spano, F. Ledda, K. M. Sousa, G. Schulte, P. Ernfors, K. Mackie, G. Paratcha, et al. Endocannabinoids regulate interneuron migration and morphogenesis by transactivating the TrkB receptor PNAS, December 27, 2005; 102(52): 19115 - 19120. [Abstract] [Full Text] [PDF]

				D. N. Abrous, M. Koehl, and M. Le Moal Adult Neurogenesis: From Precursors to Network and Physiology Physiol Rev, April 1, 2005; 85(2): 523 - 569. [Abstract] [Full Text] [PDF]

				F. RODRIGUEZ de FONSECA, I. DEL ARCO, F. J. BERMUDEZ-SILVA, A. BILBAO, A. CIPPITELLI, and M. NAVARRO THE ENDOCANNABINOID SYSTEM: PHYSIOLOGY AND PHARMACOLOGY Alcohol Alcohol., January 1, 2005; 40(1): 2 - 14. [Abstract] [Full Text] [PDF]

				K. Jin, L. Xie, S. H. Kim, S. Parmentier-Batteur, Y. Sun, X. O. Mao, J. Childs, and D. A. Greenberg Defective Adult Neurogenesis in CB1 Cannabinoid Receptor Knockout Mice Mol. Pharmacol., August 1, 2004; 66(2): 204 - 208. [Abstract] [Full Text] [PDF]

				M. Maccarrone, M. Di Rienzo, N. Battista, V. Gasperi, P. Guerrieri, A. Rossi, and A. Finazzi-Agro The Endocannabinoid System in Human Keratinocytes: EVIDENCE THAT ANANDAMIDE INHIBITS EPIDERMAL DIFFERENTIATION THROUGH CB1 RECEPTOR-DEPENDENT INHIBITION OF PROTEIN KINASE C, ACTIVATING PROTEIN-1, AND TRANSGLUTAMINASE J. Biol. Chem., September 5, 2003; 278(36): 33896 - 33903. [Abstract] [Full Text] [PDF]

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