Report |
Address correspondence to Patrick Doherty, Molecular Neurobiology Group, Medical Research Council Centre for Developmental Biology, King's College London, New Hunt's House, London Bridge, London SE1 1UL, UK. Tel.: 44-207-848-6813. Fax: 44-207-848-6816. E-mail: patrick.doherty{at}kcl.ac.uk
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Abstract |
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Key Words: CAM; CB1; 2-AG; cannabinoid; N-cadherin
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Introduction |
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The mechanism that couples the hydrolysis of DAG to the calcium response in neurons is not known. The canonical pathway would involve the synthesis of two key second messengers in neurons. The initial hydrolysis of DAG at the sn-1 position (by DAG lipase) will generate 2-arachidonylglycerol (2-AG), with the subsequent hydrolysis of 2-AG generating arachidonic acid. At first sight, arachidonic acid appeared to be the best candidate for the "instructive" signal for axonal growth in the CAM/FGF receptor pathway, as the direct application of arachidonic acid to primary neurons fully mimics the neurite outgrowth response stimulated by FGF2 and the aforementioned CAMs (Williams et al., 1994a, 1994c). However, arachidonic acid can stimulate the accumulation of 2-AG in cells (Ueda et al., 2000), and this raises the possibility that it might be 2-AG that normally couples the FGF receptor signaling cascade to the calcium response. Interestingly, 2-AG is a ligand for the CB1 and CB2 cannabinoid receptors (Di Marzo et al., 1998), and in some instances cannabinoid receptors have been shown to positively couple with calcium channels (Okada et al., 1992; Sugiura et al., 1996; Rubovitch et al., 2002). Based on these observations, we tested for "cross-talk" between the FGF receptor and endocannabinoid signaling systems. Now, we provide compelling evidence that signaling via the CB1 receptor is not only required for, but can also mediate, the neurite outgrowth response stimulated by N-cadherin and FGF2, and that it does so by coupling DAG hydrolysis to a signaling cascade that depends upon calcium influx into neurons via both N- and L-type calcium channels.
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Results and discussion |
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When postnatal cerebellar neurons are cultured over monolayers of transfected 3T3 cells that express physiological levels of N-cadherin, N-cadherin promotes neurite outgrowth via a mechanism that requires activation of a neuronal FGF receptor signal transduction cascade (Williams et al., 2001). Given the requirement of DAG lipase activity for the axonal growth response, and considering that the hydrolysis of DAG will generate the CB1 agonist 2-AG, we decided to test whether CB1 function was required for the N-cadherin response. In this context, there is ample evidence that cultured cerebellar neurons express the CB1 receptor on cell bodies and neurites (for review see Nogueron et al., 2001), and we have extended this observation to cerebellar growth cones (Fig. 1). Our results show that two independent CB1 antagonists (AM 251 and AM 281) completely inhibit the N-cadherin component of the neurite outgrowth response (Fig. 2 A). If the CB1 antagonists are acting at a step downstream from the FGF receptor, they should also inhibit the neurite outgrowth response stimulated by FGF2. Fig. 2 B shows a representative example of an experiment where various doses of AM 251 were tested for their ability to inhibit the response stimulated by 5 ng/ml FGF2. A substantial inhibition (80%) of the response can be seen at an AM 251 concentration of 0.2 µM, with a complete inhibition found at 1 µM. A series of pooled experiments show that at 1 µM, both AM 251 and AM 281 completely inhibit the FGF2 response (Fig. 2 C). In contrast, a specific CB2 receptor antagonist (AM 630) had no effect on the FGF2 response (Fig. 2 C). BDNF stimulates axonal growth by activating the TrkB receptor tyrosine kinase, and as this response does not depend on the hydrolysis of DAG (Lom et al., 1998), there is no obvious basis for postulating a role for the endocannabinoid signaling in the response. In agreement, AM 251 and AM 281 did not inhibit the neurite outgrowth response stimulated by BDNF (Fig. 2 D).
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Biphasic responses to cannabinoids have been noted in biochemical, physiological, and behavioral studies (Okada et al., 1992; Glass and Felder, 1997; Sulcova et al., 1998). The data have been interpreted as evidence for the concomitant activation of two parallel pathways by the activated CB1 receptor, namely a stimulatory pertussis toxininsensitive pathway, and an inhibitory pertussis toxinsensitive pathway. An analogous situation might explain the biphasic nature of the neurite outgrowth response to FGF2 and arachidonic acid, and the fact that in some circumstances activation of the FGF receptor can inhibit axonal growth (Williams et al., 1994a, 1994c, 1995). In this scheme, the more classical CB1-mediated inhibition of calcium channels (Caulfield and Brown, 1992; Mackie and Hille, 1992) might account for the desensitization/inhibitory component of the response.
In summary, to our knowledge, this is the first explicit demonstration of cross-talk between a neuronal receptor tyrosine kinase and the endocannabinoid system. This work also suggests that the emerging roles for adhesion molecules such as N-cadherin in synaptic plasticity in the adult (for review see Goda, 2002; Togashi et al., 2002) might be considered alongside their ability to activate the endocannabinoid signaling system.
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Materials and methods |
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Materials
ACEA (arachidonyl-2¢-chloroethylamide N-[2-chloroethyl]-5Z,8Z,11Z,14Z-eicosatetraenamide), NA (2-arachidonylglycerol ether; 2-[(5Z,8Z,11Z,14Z)-eicosatetraenyloxy]-1,3-propanediol), WIN 55,2122 mesylate ((R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl) pyrrolo-[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate), AM 281 (1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4-morpholinyl-1H-pyrazole-3-carboxamide) and AM 251 (N-[piperidin-1-yl]-5-[4-iodophenyl]-1-[2,4-dichlorophenyl]-4-methyl-1H-pyrazole-3-carboxamide), and AM 630 (6-Iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl](4-methoxyphenyl) methanone) were obtained from Tocris Cookson, Ltd., and were used at concentrations recommended by the supplier. BDNF and FGF2 were obtained from R&D Systems. Arachidonic acid was obtained from Affinity Bioreagents, Inc., and was used as described previously (Williams et al., 1994c). Diltiazem hydrochloride was obtained from Calbiochem, and -conotoxin GVIA was obtained from Alomone Labs; both were used as described previously (Doherty et al., 1991a).
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Footnotes |
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Acknowledgments |
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Submitted: 29 October 2002
Revised: 31 December 2002
Accepted: 2 January 2003
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References |
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