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Non-psychoactive CB₂ cannabinoid agonists stimulate neural progenitor proliferation

Javier Palazuelos^*, Tania Aguado^*, Ainara Egia^*, Raphael Mechoulam, Manuel Guzmán^1,2 and Ismael Galve-Roperh^*,1,2

^* Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University, Madrid, Spain;

Department of Medicinal Chemistry and Natural Products, School of Pharmacy, The Hebrew University, Jerusalem, Israel

²Correspondence: Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University, 28040 Madrid, Spain. E-mail: igr{at}quim.ucm.es; mgp{at}bbm1.ucm.es

SPECIFIC AIMS

Endocannabinoids, the endogenous counterparts of the bioactive components produced by marijuana (Cannabis sativa), are generated on demand as a consequence of brain injury and they exert a neuroprotective action. In addition, endocannabinoids target neural progenitor (NP) cells and regulate cell proliferation and differentiation via the seven-transmembrane CB₁ receptor. However, the pharmacological manipulation of NPs by cannabinoids is hampered by the typical marijuana-like CB₁-mediated psychoactive effects. In contrast to the wide expression of CB₁ in the brain and many other organs, cannabinoid receptor CB₂ is restrictedly expressed in brain cells while abundant in the immune system. The aim of the present work was to examine whether progenitor cells express the non-psychoactive CB₂ receptor and to study its potential involvement in NP cell fate both in normal and injured brain.

PRINCIPAL FINDINGS

1. Neural progenitors express CB₂ receptors in vitro and in vivo
Clonally expanded neurospheres derived from different embryonic stages and adult brain were used to determine whether NP cells express CB₂ receptors. RT-polymerase chain reaction (RT-PCR) and Western blot analyses revealed that NPs express CB₂ receptors during development and that its presence remains evident in adult-derived cells. These findings were corroborated in the human neural stem cell line hNSC1. Immnunofluorescence studies with antibodies directed against the CB₂ receptor and markers for multipotent neuroepithelial (nestin), proliferating (bromodeoxyuridine and phosphorylated vimentin), and radial progenitor (RC2 and vimentin) cells confirmed that NPs, including those actively dividing, express CB₂ receptors. Likewise, CB₂ receptors were present in adult brain progenitors. CB₂ expression during neural differentiation was analyzed by RT-PCR and revealed its disappearance during neural differentiation with the concomitant induction of the neuronal (ßbeta;-tubulin-III) and astroglial markers (GFAP).

Next we determined by confocal microscopy whether CB₂ receptors are expressed in vivo in progenitor cells resident in the subgranular zone of the dentate gyrus of the hippocampus, one of the most prominent neurogenic areas throughout life span, including adulthood. CB₂ receptor expression was found only in nestin-positive cells, while we could not find its presence in differentiated hippocampal neurons (NeuN-positive cells) and astrocytes (GFAP-positive cells).

2. CB₂ receptors control neural progenitor cell proliferation and neurosphere generation in vitro
To determine whether CB₂ receptors control NP cell function, we generated neurospheres from CB₂-deficient mice and their wild-type (WT) littermates. Genetic ablation of the CB₂ receptor impaired primary neurosphere generation (Fig. 1 A, inset). Moreover, NP self-renewal, as determined by neurosphere generation for several consecutive passages, was reduced in CB₂-deficient cells (Fig. 1A ). The observed impairment of NP function in CB₂^–/– cell cultures prompted us to analyze the prominin (cluster of differentiation-133)-positive subpopulation, as these cells are considered the stem cell fraction responsible for neurosphere formation activity. Of interest, CB₂^–/– neurospheres, when compared to WT cultures by flow cytometry analysis, showed a reduction in their cluster of differentiation (CD)-133⁺ subpopulation (cluster of differentiation-133⁺ cells: 5.8±2.0% vs. 7.4±1.5%, respectively).

The functional relevance of the CB₂ receptor was evaluated by incubating neurospheres with the CB₂-selective agonists HU-308 and JWH-133, both of which increased neurosphere generation (Fig. 1B ) and NP self-renewal (Fig. 1C ). These actions were prevented by the CB₂-selective antagonist SR144528. The selectivity of the CB₂ agonists was confirmed in CB₂-deficient NPs, in which HU-308 and JWH-133 were unable to enhance neurosphere generation (Fig. 1B ). Moreover, HU-308 and JWH-133 increased the number of bromodeoxyuridine (BrdU)-incorporating cells in a CB₂-dependent manner (Fig. 1D ), supporting the direct impact of CB₂ receptor activation on cell proliferation. Likewise, increased neurosphere generation was observed on CB₂ activation in postnatal and adult progenitors (data not shown).

To determine the potential signaling mechanism responsible for CB₂-mediated proliferation, neural progenitors were incubated with HU-308 and selective inhibitors of the extracellular signal-regulated kinase (ERK) cascade (PD98059) and the phosphatidylinositol 3-kinase (PI3K)/Akt pathway (LY294,002). HU-308 induction of cell proliferation was prevented by both inhibitors (Fig. 1E , upper panel), a finding that was confirmed in neurosphere generation assays (Fig. 1E , lower panel). These results prompted us to analyze CB₂-mediated regulation of ERK and Akt. Thus, HU-308 stimulated ERK and Akt, and this action was prevented by SR144528 (Fig. 1F ).

3. CB₂ receptors control neural progenitor cell proliferation in vivo
The functional relevance of the CB₂ receptor in controlling neural progenitor cell proliferation in vivo was determined by assessing BrdU incorporation in CB₂-deficient mice and their WT littermates. In both embryonic (Fig. 2 A) and adult (Fig. 2C ) brain, CB₂ knockout animals showed a significant decrease in BrdU-labeled cells in the dentate gyrus of the hippocampus. These results suggest that neural progenitor proliferation in vivo may be suitable for CB₂ pharmacological manipulation. Thus, HU-308 and/or SR144528 were administered for 5 consecutive days and hippocampal proliferation was determined. Importantly, CB₂ activation increased progenitor proliferation, while CB₂ blockade exerted the opposite action (Fig. 2B ). The selectivity of HU-308 in vivo was confirmed by SR144528 antagonism and the lack of HU-308 agonistic effect in CB₂-deficient mice. We further tested whether CB₂ receptors may be implicated in the control of neural progenitor cell proliferation in a situation of brain injury such as kainate-induced excitotoxicity. As shown in Fig. 2C , the remarkable excitotoxic stimulation of neural progenitor cell proliferation was abrogated in CB₂-deficient mice.

CONCLUSIONS AND SIGNIFICANCE

The expression pattern of the CB₁ receptor is tightly regulated during brain development and remains expressed at high levels in differentiated neurons and at lower levels in glial cells of various adult brain areas such as the hippocampus, basal ganglia, and cortex. In contrast, the presence of the "peripheral" CB₂ receptor in differentiated neurons is more restricted. Thus, only recently the expression of CB₂ receptors in normal brain could be demonstrated in the cerebellum and in a subpopulation of neurons of the brainstem that participates in emesis regulation. In addition, CB₂ receptor expression in the brain is also found in microglia and endothelial cells. Here, we show that CB₂ cannabinoid receptors are expressed in NP cells both during development and in the adulthood and become down-regulated during their neural cell differentiation. Of interest, other studies previously suggested an inverse relation between CB₂ receptor expression and stage of cell differentiation. For example, CB₂ receptor expression decreases during B-cell differentiation and increases with dedifferentiation (i.e., with increased malignancy) of glial tumors. Thus, it is tempting to speculate that endocannabinoids may control NP cell function via CB₂ receptors acting as a "cell dedifferentiation signal" by favoring a nondifferentiated, proliferative state.

During mammalian development, the generation of the central nervous system (CNS) relies on a finely regulated balance of NP proliferation, differentiation, and survival controlled by a number of extracellular signaling cues. In addition, adult NPs give rise to newly generated cells that can integrate properly in hippocampal circuits and thus may contribute to synaptic plasticity, cognition, or neuroregeneration on brain damage. Our finding of impaired NP proliferation after neuroexcitotoxic damage in CB₂-deficient mice, together with the protective role of endocannabinoids in a variety of brain damage models, suggest that endocannabinoids generated on demand with brain injury may enhance NP proliferation via CB₂ receptors (Fig. 3 ). The relevance of our results is strengthened by the recent demonstration of the role of the endocannabinoid system in the regulation of adult neurogenesis. Hippocampal progenitors produce endocannabinoids and express CB₁ receptors that regulate cell proliferation and neural differentiation. Moreover, in vivo regulation of cannabinoid signaling during CNS development alters neuronal activity and generation, events that add to the reported impairment of cognitive functions in CB₁ knockout mice.

The therapeutic use of cannabinoids is severely limited by their well-known psychotropic effects that are mediated by CB₁ receptors within the brain. Thus, for the development of cannabinoid-based therapies devoid of side effects, the most conceivable possibility would be to selectively target CB₂ receptors. In this context, the recent synthesis of CB₂-selective agonists opens an attractive clinical possibility. The finding that CB₂ receptor activation is functional in stimulating NP cell proliferation in vitro and in vivo, together with the implication of CB₂ receptors in the control of processes such as pain initiation, emesis, neuroinflammation, and brain-tumor cell death, opens the attractive possibility of cannabinoid-based therapeutic strategies for neural disorders devoid of nondesired psychotropic effects. Specifically, the proliferative effect of cannabinoids reported here may set the basis for the potential pharmacological modulation of NP cell fate by CB₂-selective ligands.

View larger version (28K): [in this window] [in a new window]   Figure 1. CB2 receptors control neurosphere generation and neural progenitor cell proliferation in vitro. A) Self-renewal of E17.5 neural progenitors derived from WT and CB2–/– mice. The number of neurospheres was quantified after 5 consecutive neurosphere passages. Inset) Primary neurosphere generation in the two mouse strains. B) Primary neurosphere generation was determined after 7 d of exposure of neural progenitors (black bars) to vehicle (C), the CB2-selective agonists HU-308 or JWH-133 (30 nM) and/or the CB2-selective antagonist SR144528 (2 µM; SR). CB2–/– progenitors (gray bars) were also employed. C) Self-renewal of WT neural progenitors (solid line) incubated as above for 5 consecutive passages. Self-renewal of CB2-deficient progenitors in the presence of vehicle is also shown (dashed line). D) Quantification of BrdU-positive cells from dissociated neurospheres incubated as above for 16 h. E) Quantification of BrdU-positive cells (upper panel) and neurosphere generation (lower panel) of progenitors treated with vehicle (C), HU-308 (30 nM) and/or PD98059 (10 µM; PD) and/or LY294,002 (5 µM; LY). F) ERK and Akt phosphorylation after progenitor challenge with vehicle (C) or HU-308 (alone or in the presence of SR144528) for 15 min (ERK) or 2 min (Akt). Results correspond to 3 (A, C, E, and F) or 4 (B and D) independent experiments. Significantly different from control WT cells: *P < 0.05, ** P < 0.01.

View larger version (17K): [in this window] [in a new window]   Figure 2. CB2 receptors control neural progenitor cell proliferation in vivo. A) Number of BrdU-positive cells per section in the dentate gyrus of WT (n=5) and CB2–/– (n=7) mouse E17.5 embryos. B) Number of BrdU-positive cells per section in the dentate gyrus of WT (black bars; n=4) and CB2–/– (gray bars; n=3) adult mice injected with the indicated agents. C) Number of BrdU-positive cells per section in the dentate gyrus of WT (black bars; n=4) and CB2–/– (gray bars; n=4) adult mice injected with saline (plain bars) or kainic acid (dashed bars). Significantly different from controls: *P < 0.05, **P < 0.01. Significantly different from WT mice treated with kainic acid: #P < 0.05.

View larger version (29K): [in this window] [in a new window]   Figure 3. Schematic diagram showing cannabinoid action on hippocampal neural progenitor (NP) proliferation and its potential involvement in the regenerative response to brain injury.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-6164fje

¹ These authors contributed equally to this work.

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