Cannabinoid receptors direct neurogenesis through neuroimmune networks: role of interleukin-1 receptor antagonist (IL-1ra) and interleukin-6 (IL-6)

Eduardo Molina-Holgado1, Uyen Le1, Francisco Molina-Holgado2. 1Laboratory of Neuroinflammation, Unidad de Neurologia Experimental, Hospital Nacional de Parapléjicos 45071, Toledo, Spain, 2Department of Life Sciences, University of Roehampton SW15 4JD, London, UK.

Neuroimmune networks and the brain endocannabinoid system (eCB) contribute to the maintenance of neurogenesis (Molina-Holgado and Molina-Holgado 2010). Activation of cannabinoid receptors suppressed chronic inflammatory responses through the attenuation of pro-inflammatory mediators. Moreover, the eCB directed cell fate specification of neural stem cells (NSC) in the central nervous sytem (CNS), (Arévalo-Martín et al., 2008). The aim of this study was to understand better the relationship between the eCB, the immune system and NSC biology, in order to develop therapeutical strategies on CNS diseases that could facilitate brain repair. NSC derived from the stem cell line COR-1 (embryonic stem cells derived from C57BL6/J mice) expressed functional CB1 and CB2 cannabinoid receptors and the neural stem cell marker SOX-2. First, we detected the presence of functional CB1 and CB2 cannabinoid receptors and the SOX-2 marker by immunoblotting in cell homogenates obtained from adherent NSC cultures. Secondly, we investigated the role of CB1 and CB2 cannabinoid receptors in the control of NSC proliferation and in the release of immunomodulators that lead the NSC fate. Specifically, in the neural stem cell line COR-1, CB1 and CB2 cannabinoid receptors modulated FGF-2 (20ng/ml, t = 24h, n = 6) induced stem cell proliferation by the release of interleukin-1 receptor antagonist (IL-1ra) and interleukin-6 (IL-6). Treatment of NSC with FGF induced the release of IL-1ra (P<0.001 vs Control), which was effectively blocked by the CB1 antagonist SR-141716A (SR-1, 1μM) or by the CB2 antagonist SR-144528 (SR-2, 1μM). FGF-induced release of IL-6 (P<0.001 vs Control) was only blocked when neural stem cells were co-incubated with both antagonists.

The specific antagonists for CB1 (SR-141716A, 1μM, t = 24h, n = 6) or CB2 (SR-144528, 1μM, t = 24h, n = 6) cannabinoid receptors, abolished or decreased (by 50% approximately; P<0.001 vs Control) respectively, indicating a critical role for both CB1 and CB2 receptors in the proliferation of COR-1 stem cells. Next, we studied if endogenous or exogenous cannabinoid agonists interact with the CB1 and CB2 cannabinoid receptors modulating neural stem cell self-renewal or NSC fate specification through diverse downstream neurogenic pathways and immune system modulators. Both cannabinoid agonists ACEA (0.5 μM, t = 24h, n = 6) or JWH-056 (0.5 μM, t = 24h, n = 6) induced a significant increase of IL-1ra in COR-1 cells (by 50% approximately; P<0.001 vs Control). This effect was abolished when the COR-1 stem cells were pre-treated with the above specific antagonist for CB1 or CB2 receptors. As opposed to the above cytokine, only CB2 receptors modulated the release of IL-6, a pleiotropic cytokine, which displayed a degree of functional redundancy due to the presence of a glycoprotein 130 (gp130), in their receptors. Endocannabinoids appeared as new participants connecting the immune system and neurogenesis. Thus, the endocannabinoid system, which has neuroprotective and immunomodulatory actions mediated by different signaling cascades in the brain, could assist the process of proliferation and differentiation of embryonic or adult neural stem cells, and this may be of therapeutic interest in the emerging field of brain repair.

Molina-Holgado E and Molina-Holgado F. (2010) Mending the broken brain: neuroimmune interactions in neurogenesis. J Neurochem. 114 (5):1277-90.

Arévalo-Martín A, García-Ovejero D, Gómez O, Rubio-Araiz A, Navarro-Galve B, Guaza C, Molina-Holgado E, Molina-Holgado F. (2008). CB2 cannabinoid receptors as an emerging target for demyelinating diseases: from neuroimmune interactions to cell replacement strategies. Br J Pharmacol. 153( 2): 216-25.

EM-H and FM-H are funded by Gobierno de Castilla-La Mancha,(Fundación para Investigación Sanitaria en Castilla-La Mancha; FISCAM, G-2009­_C/06), SPAIN. U-Le is supported by Instituto de Salud Carlos III (Ministerio de Ciencia e Innovación, Exp. Nº CA09/00609), Spain.