Neuroimmune networks and the brain endocannabinoid system contribute to the maintenance of neurogenesis. Activation of cannabinoid receptors suppresses chronic inflammatory responses through the attenuation of pro-inflammatory mediators. Moreover, the endocannabinoid system directs cell fate specification of NSCs (neural stem cells) in the CNS (central nervous sytem). The aim of our work is to understand better the relationship between the endocannabinoid and the IL-1β (interleukin-1β) associated signalling pathways and NSC biology, in order to develop therapeutical strategies on CNS diseases that may facilitate brain repair. NSCs express functional CB1 and CB2 cannabinoid receptors, DAGLα (diacylglycerol lipase α) and the NSC markers SOX-2 and nestin. We have investigated the role of CB1 and CB2 cannabinoid receptors in the control of NSC proliferation and in the release of immunomodulators [IL-1β and IL-1Ra (IL-1 receptor antagonist)] that control NSC fate decisions. Pharmacological blockade of CB1 and/or CB2 cannabinoid receptors abolish or decrease NSC proliferation, indicating a critical role for both CB1 and CB2 receptors in the proliferation of NSC via IL-1 signalling pathways. Thus the endocannabinoid system, which has neuroprotective and immunomodulatory actions mediated by IL-1 signalling cascades in the brain, could assist the process of proliferation and differentiation of embryonic or adult NSCs, and this may be of therapeutic interest in the emerging field of brain repair.
Although much has been learnt in recent years about the causes of degeneration within the brain, in terms of therapeutic intervention, research has yet to produce an effective treatment to reverse/repair neuronal damage caused by non-pathological aging or neurodegeneration. Recent reports have suggested that there is a synergy between the immune system and NSCs (neural stem cells). This relationship promotes functional recovery, since immune cells help to maintain neurogenesis in germinal centres of the adult CNS (central nervous system) even under non-pathological conditions . Separate studies have recently established that adult mammalian brains maintain some discrete regions of neurogenesis, with the capacity to generate functional neurons; however, the mechanisms of these actions are still poorly understood. Our research has clearly established that the downstream activation of CB1 and CB2 cannabinoid receptors, as well as the enzymes responsible for the synthesis of the endocannabinoid 2-AG (2-arachidonoylglycerol), DAGLα (diacylglycerol lipase α) and DAGLβ, and degradation, MAGL (monoacylglycerol lipase), can modulate neurogenesis by a number of neuroimmune molecules (cytokines) in glial cells, neurons and NSCs . Neuroimmune networks and the brain endocannabinoid system contribute to the maintenance of neurogenesis . An emerging concept in the field of stem cell biology is that a population of immune mediators directs the cell-fate specification of NSCs in the CNS. Our work supports this concept, as in the present paper we describe work that shows that the lack of the gene encoding IL-1β [interleukin-1β, MGI (Mouse Genome Informatics) accession number 96543] has a key role in regulating the proliferative activity of NSCs. Activation of cannabinoid receptors suppressed chronic inflammatory responses through the attenuation of pro-inflammatory mediators such as IL-1β by increasing the expression of IL-1Ra (IL-1 receptor antagonist), an endogenous antagonist for the actions of IL-1 in the brain . Interestingly, new evidence suggests that IL-1Ra is a potent signal that induces NSC proliferation and migration . Moreover, the endocannabinoid system directs cell fate specification of NSCs in the CNS . The aim of the present study is to understand better the relationship between the endocannabinoid system, the IL-1-associated signalling in regulating NSC fate, in order to develop therapeutical strategies for CNS diseases that could facilitate brain repair. Endocannabinoids appear as new participants connecting the immune system and neurogenesis.
IL-1β signalling and neurogenesis
IL-1β is present at high levels during CNS development , but decreases to low constitutive levels during adulthood . IL-1β is enriched in some cells from neurogenic niches, such as astrocytes at the hippocampus , neuroepithelial cells at the developing spinal cord  or the choroid plexus, near the lateral ventricle SVZ (subventricular zone) . In vitro, NSCs themselves produce IL-1β, although not IL-1α or IL-1Ra . IL-1β may bind IL-1R1 (IL-1 receptor 1) that is expressed by NSCs at the hippocampal SGZ (subgranular zone)  and by Type B cells at the SVZ . IL-1R1 is also expressed by cultured hippocampal and fetal rat mesencephalic precursors and by oligodendroctye precursors [9,11]. Another target for IL-1β could be IL-1R2, a decoy receptor expressed by cultured hippocampal NSCs [9,12]. In general, chronic up-regulation or high doses of IL-1β inhibit proliferation and induce differentiation of NPCs (neural progenitor cells). In vitro, IL-1β inhibits proliferation of neonatal and adult hippocampal NSCs and mesencephalic NSCs. This effect is dose-dependent, mediated by IL-1R1, and involves NF-κB (nuclear factor κB) and GSK-3β (glycogen synthase kinase 3β)[9,12a]. However, in embryonic rat-derived NSCs, part of these effects may be mediated by SAPK (stress-activated protein kinase)/JNK (c-Jun N-terminal kinase) . We observed that genetic deletion of IL-1β increases basal proliferation of NSCs (Figure 1), an effect than can be reversed by exogenous administration of IL-1β, and does not modify the number of nestin-positive precursors.
Genetic deletion of IL-1β increases neural stem cell proliferation
In vivo, IL-1β/NF-κB signalling is involved in stress-induced decreases in proliferation and neurogenesis at the SGZ . In addition, reducing IL-1β increases hippocampal neurogenesis in aging , where IL-1β levels are high, but decreases excitotoxic injury-induced increased neurogenesis . At the SVZ, infusion of IL-1β in the lateral ventricles inhibits proliferation , whereas, in the developing spinal cord, IL-1β modulates proliferation in a region-dependent manner: it decreases proliferation in the ventral quadrants of the neuroepithelium, but increases it in the dorsal quadrants .
Besides inhibiting proliferation, IL-1β is a potent factor for inducing a mature dopaminergic neuronal phenotype from mesencephalic precursors . In addition, IL-1β enhances astrogliogenesis and inhibits neurogenesis via GSK-3β activation and TLX modulation in hippocampal NSCs  or p38 MAPK (mitogen-activated protein kinase) in mesencephalic NSCs . The same effect is found in the SGZ, where a sustained expression of IL-1β produces a shift in cell lineage from neuronal to astroglial that requires the presence of IL-1R . However, in the SVZ, IL-1β prevents lineage progression, maintaining undifferentiated NSCs , whereas in the developing spinal cord, IL-1β induces early neuronal differentiation . In addition, IL-1β induces process outgrowth in human neuroepithelial olfactory cells , and promotes differentiation and maturation of oligodendrocyte precursors . Finally, IL-1β mediates the effects of other molecules on neurogenesis such as interferon and taurine  in the SGZ, or VCAM (vascular cell adhesion molecule) in the SVZ .
Cannabinoid signalling and neurogenesis
Cannabinoid receptor 1 (CB1 receptor) and 2-AG-synthesizing enzymes (DAGLα and DAGLβ) are expressed in neurogenic regions of the developing nervous system [21,21a], where they induce proliferation and, critically, participate in the specification of neuronal cell types . In the adult neurogenic niches, the endocannabinoid system is widely represented. In the SGZ, Galve-Roperh and Guzman for several laboratories reported the existence of a functional endocannabinoid system, constituted by the expression of the CB1 receptor and the anandamide-inactivating FAAH (fatty acid amide hydroxylase) enzyme in nestin+ GFAP+ (glial fibrillary acidic protein) NSCs, that controls NSC proliferation . In this region, neurogenic stimuli such as voluntary exercise running increases anandamide and CB1 receptor levels, but not 2-AG . In the SVZ, DAGLα is expressed by ependymal and proliferating cells, and ablation or inhibition of this enzyme impairs cell proliferation and lowers expression of DCX . CB1 receptors are expressed by ependyma and Type B cells and CB2 receptors are expressed by PSA-NCAM+ (polysialylated neural cell adhesion molecule) precursors . In the adult spinal cord central canal, which harbours neurogenic potential, there is a subpopulation of ependymal cells expressing high levels of CB1 receptors. This subpopulation may proliferate during postnatal development and after a spinal cord injury .
In vivo analysis demonstrated that cannabinoids induce proliferation in the SGZ of adult and aged rats, by acting on CB1 and CB2 receptors . Complementary absence or inhibition of CB1 receptors decreases basal proliferation and the number of DCX+ precursors  and NSC proliferation and survival induced by activity or excitotoxic injuries . However, cannabinoid effects on SGZ proliferation may be more complex, since CB1 receptor deficits induce an acute increase in proliferation followed by long-lasting decreases in proliferation and/or generation of neuronal precursors . Besides, chronic treatment of adult rats with high levels of the CB1/CB2 receptor agonist HU210 increases BrdU (bromodeoxyuridine) labelling; however, an acute delivery or the use of moderate doses of HU210 does not . In vitro, brain-derived NSCs produce 2-AG and anandamide  and respond to endocannabinoids and synthetic cannabinoid agonists increasing their proliferation in a CB1- and CB2-receptor-dependent manner .
Cannabinoids also control differentiation in the hippocampus, although the effects are still in debate: some results suggest that metanandamide, a CB1 receptor agonist, induces neurogenesis in vitro  and CB1 receptor absence impairs maturation of new neurons in vivo . Other studies show that cannabinoids inhibit neuronal differentiation and induce astroglial differentiation in vitro and in vivo . A previous study may shed light on this apparent discrepancy, since it found that anandamide induces an initial glial differentiation followed by neuronal differentiation of cultured NPCs .
In postnatal rat neurogenic SVZ, activation of CB1 receptors augments proliferation and increases the number of Olig2-glial precursors, whereas activation of CB2 receptors induces the expression of PSA-NCAM in migratory precursors, the combination of both resulting in increased myelination at the external capsule . Similarly, methanandamide, through CB1 receptors, promotes proliferation and self-renewal of SVZ-derived NSCs, although long-term treatment stimulates functional neuronal differentiation . In the SVZ, NSCs may secrete the endocannabinoid anandamide . The effects of cannabinoids may be dependent on the region from where NPCs are obtained, since NSCs from the spinal cord show neuronal differentiation after genetic or pharmacological inhibition of CB1 receptors, whereas cannabinoid agonists increase the expression of immature markers such as nestin .
NSCs also express CB2 cannabinoid receptors in vitro. In addition, NSCs contain DAGLα- and CB2-receptor-expressing cells, maintained through several passages . Selective pharmacological activation of CB2 receptors with HU308 or JWH133 promotes proliferation and neurosphere generation by activation of PI3K (phosphoinositide 3-kinase)/Akt and mTORC1 (mammalian target of rapamycin complex 1) that may inhibit p27Kip1 impeding cell cycle progression [34,35]. These effects are not found in the SGZ or in NSCs derived from CB2-receptor-deficient mice .
Interaction between cannabinoids and IL-1β
The first evidence of interaction between cannabinoids and IL-1β arose while studying the mechanisms of how cannabis renders their consumers more susceptible to infections . Cannabinoids antagonize IL-1-induced effects through CB1 and/or CB2 receptors by reducing IL-1β production , increasing IL-1Ra  or interfering with the IL-1β signalling cascade . This cannabinoid-induced suppression of IL-1β-mediated effects is observed in neuroinflammatory pathologies such as experimental models of multiple sclerosis , closed head injury , Alzheimer's disease  and HIV-associated dementia . Although most studies focused on the effect of cannabinoids over IL-1β signalling, the interaction between these two systems is bidirectional since IL-1β suppress CB1-receptor-mediated inhibition of glutamate release in the striatum  and cannabinoids abrogate IL-1β-induced anxiety .
An increasing amount of evidence has established that there is a dynamic interplay between the endocannabinoid system, the immune system and neural stem cells [1,29]. We presented the first results showing that bidirectional cross-talk between the TNFα (tumour necrosis factor α) and endocannabinoid signalling pathways is required to stimulate NSC proliferation . In the present paper we report new evidence for a cross-talk between the IL-1β and the endocannabinoid signalling pathways in the control of NSCs/NPCs: (i) there is co-expression of CB1 and CB2 receptors with IL-1R1 and IL-1R2 in mouse brain neurospheres (Figure 2); (ii) activation of either CB1 or CB2 receptors in embryonic mouse brain derived neurospheres decreases IL-1β levels and increases IL-1Ra (Figure 3). In addition, increases in proliferation of NSCs from IL-1β-knockout mice are blocked by inhibiting 2-AG synthesis using RHC-80267. Other evidence points to the same effect: chronic stress that increases IL-1β also down-regulates CB1 receptor expression and reduces the content of 2-AG within the hippocampus . Indeed, exogenous treatment with cannabinoids can reverse cognitive impairments induced by chronic stress that are also mediated by IL-1β . Moreover, cannabinoid treatment of aged rats restores IL-1β levels and the number of SGZ neuroblasts in an effect dependent on CB1 and CB2 receptors . Interestingly, mice with genetic deletion of CB1 receptor show down-regulation of basal IL-1β levels in the hippocampus, but not in LPS-induced IL-1β concentrations, depicting a complex scenario where doses and context of IL-1β induction are differentially modulated by cannabinoids .
Confocal images showing receptors for cannabinoids (CB1 and CB2 receptors) and interleukin-1 co-expressed in mouse brain NSCs
CB1 and CB2 receptor agonists increase IL-1β and decrease IL-1Ra production by NSCs
In summary, cannabinoids and IL-1β seem to play antagonistic roles in neurogenesis: although cannabinoids increase proliferation and induce formation and maturation of new neurons, IL-1β blocks proliferation and formation of new neurons, inducing a shift towards a glial fate. This may be important in situations such as in aging, neurodegenerative diseases, and lesions of the brain and spinal cord. However, the picture be much more complex, since the effects of cannabinoids are still under debate and it appears that the effects of cannabinoids and IL-1β are dose-, time- and region-dependent, and also depend on the context and entourage molecules that form the niche where these molecules interact.
5th Conference on Advances in Molecular Mechanisms Underlying Neurological Disorders: A joint Biochemical Society/European Society for Neurochemistry Focused Meeting held at the University of Bath, U.K., 23–26 June 2013. Organized and Edited by Marcus Rattray (University of Bradford, U.K.) and Rob Williams (University of Bath, U.K.).
central nervous sytem
glycogen synthase kinase 3β
nuclear factor κB
neural progenitor cell
neural stem cell
polysialylated neural cell adhesion molecule
Supported by the Ministry of Economy and Competitiveness (Instituto de Salud Carlos III) [PI: 11/1729] (Spain), the Fundacion Mutua Madrileña (Spain) and University of Roehampton funds (U.K.).