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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online July 21, 2005 as doi:10.1096/fj.05-3995fje. |
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,1
,1,1
* Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University, Madrid, Spain;
Max-Planck Institute of Psychiatry, Munich, Germany;
Washington University, Seattle, Washington, USA;
The Skaggs Institute for Chemical Biology and Department of Cell Biology, The Scripps Research Institute, La Jolla, California, USA; and
|| Laboratory of Neural Stem Cell Biology, Lund Strategic Research Center for Stem Cell Biology and Cell Therapy, University Hospital, Lund, Sweden
2 Correspondence: E-mail: igr{at}quim.ucm.es
SPECIFIC AIMS
The endocannabinoid (eCB) system is known to exert an important neuromodulatory role via presynaptic CB1 receptors, and it also takes part in the control of cell fate by controlling the balance between cell death and survival. eCBs are generated on demand as a consequence of brain injury, and through CB1 receptor engagement may exert a neuroprotective action. Whereas the function of the eCB system has been extensively studied in differentiated neurons, its potential role in neural progenitor (NP) cells has not been addressed. The aim of the present work was to study whether progenitor cells possess a functional eCB system, and if so what is its role in the regulation of cell proliferation.
PRINCIPAL FINDINGS
1. Neural progenitors express the endocannabinoid system
To determine whether progenitor cells express a functional eCB system, we employed clonally expanded primary neurospheres. Immunofluorescence studies and Western blot analyses show that NP cells express the CB1 receptor and the FAAH enzyme responsible for the degradation of the eCB anandamide (AEA). CB1 receptors and FAAH were expressed by actively dividing cells and were also coexpressed with nestin, a widely employed neuroepithelial marker. Indeed, NPs produced the eCBs AEA and 2-arachidonoylglycerol (2AG), 2AG being 50–100 times more abundant than AEA. Additional putative CB1 ligands such as N-docosatetraenylethanolamine and N-homo-
-linolenylethanolamine were below detection limits. AEA and 2AG production by NPs showed a robust response to the Ca2+ ionophore A23187.
2. Endocannabinoids promote neural progenitor proliferation and neurosphere generation
To determine whether the eCB system controls NP cell function, cells were incubated with the synthetic cannabinoid agonist WIN-55,212-2 and the selective FAAH inhibitor URB597. Cannabinoid stimulation increased neurosphere generation (control: 100±23%; WIN 55,212-2: 259±64%; URB597: 297±92%; n=4; P<0.01), an effect prevented by the selective CB1 antagonist SR141716, pointing to the involvement of the CB1 receptor. Cannabinoid stimulation also increased the number of neurospheres per well (control: 1.9±0.3; WIN-55,212-2: 13.5±0.2; URB597; 11.3±0.4; n=4; P<0.01) and neurosphere size (data not shown). Cannabinoid challenge stimulated NP proliferation in a CB1-dependent manner as determined by quantification of 5-bromo-2'-deoxyuridine (BrdU)-incorporating cells, [3H]-thymidine incorporation and the expression of Ki-67 (an endogenous marker of mitotic cells), a finding that correlates with higher number of multipotent nestin+ cells (data not shown).
The relevance of the eCB system in NP proliferation was further investigated by evaluating primary clonal neurosphere generation in CB1-deficient mice. Such analysis showed a decreased rate of neurosphere generation (neurospheres per well; CB1+/+: 1.27±0.10, n=4; CB1–/–: 0.96±0.04, n=4; P<0.05). Accordingly, limit-dilution analysis (Fig. 1
A) revealed that the number of cells required to generate at least 1 neurosphere per well was 85 ± 12 for CB1+/+ mice (n=4) and 177±15 for CB1–/– mice (n=4; P<0.01). The involvement of the CB1 receptor was proven by the absence of effect of WIN-55,212-2 and URB597 on neurosphere generation in CB1-deficient NPs (Fig. 1B
). Cannabinoid impact on NP self-renewal was determined by their continuous presence for several neurosphere passages using wild-type and CB1–/– neurospheres. The exponential growth of NPs was significantly enhanced by WIN-55,212-2 and URB597 (Fig. 1C
). This effect was prevented by SR141716 treatment and was impaired in CB1 knockout-derived NPs.
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3. The endocannabinoid system remains functionally active in the regulation of adult brain progenitor cell proliferation
We next tested whether the eCB system was also present and active in adult NPs. Adult brain-derived NPs (nestin+ cells), expressed the CB1 receptor and FAAH as evidenced by immunolabeling and Western blot. Moreover, adult NPs proliferate in response to cannabinoids as shown by increased BrdU+ and nestin+ cells after cannabinoid challenge.
The functional relevance of the eCB system in vivo was determined using FAAH knockout mice that possess increased brain levels of eCBs. As shown in Fig. 2
A, FAAH knockout embryos showed a marked increase in hippocampal BrdU-positive cells. In agreement, BrdU+ cell counting in the adult dentate gyrus showed that hippocampal proliferation is increased in FAAH knockout mice as compared with wild-type littermates (Fig. 2B
).
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CONCLUSIONS AND SIGNIFICANCE
Results presented here evidence the existence of a functional eCB system in NPs (Fig. 3
) constituted by the endogenous ligands AEA and 2AG, the CB1 receptor and the eCB-inactivating FAAH enzyme. The eCB system in NP cells participates actively in the control of NP proliferation. Thus, progenitor-derived eCBs are biologically active and promote cell proliferation and neurosphere generation in a CB1-dependent manner, as demonstrated by the observation that neurosphere generation is impaired in CB1–/– progenitors or by CB1 pharmacological blockade. Cannabinoid action on NP self-renewal ability indicates that eCB regulation of NP proliferation occurs, at least in part, in early progenitor cells, although it does not preclude concomitant actions on intermediate-amplifying progenitors. eCBs may activate different proliferative signal transduction pathways involved in the regulation of neural cell fate including the phosphatidylinositol 3-kinase/Akt pathway and the extracellular signal-regulated kinase pathway. Alternatively, the eCB system may interact with growth factors that are essential for the expansion and regulation of progenitor cells. eCBs can transactivate tyrosine kinase receptors of the EGF receptor family in a CB1-dependent manner, or alternatively, 2AG generation might be boosted by FGF-2 receptor activation resulting in enhanced neural cell growth.
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Our results are in agreement with the observations that the eCBs AEA and 2AG participate in the control of cell fate by modulating the balance between neural cell death and survival. AEA may reduce brain damage in excitotoxicity models, and neuronal degeneration due to traumatic brain injury is attenuated by 2AG. Thus, eCBs generated on demand upon brain injury may result in CB1-induced neuroprotection. The eCB-induced stimulation of NP proliferation reported here, together with their recognized neuroprotective role, may represent a mechanism by which the injured brain primes NPs to minimize damage intensity. In agreement, brain injury results in progenitor proliferation, a process that has been proposed to represent a compensatory mechanism by which the injured brain minimizes damage intensity.
The important implications of neurogenesis in cognitive processes and brain repair have led to intense efforts to identify the endogenous signaling mechanisms responsible for the regulation of NP proliferation. Thus, numerous studies have recently addressed the regulatory mechanisms of progenitor cell proliferation in the adult brain that contribute to the generation of new functional neurons with the ability to integrate properly in hippocampal circuits. This process is considered to be a new form of brain plasticity, in which the eCB-induced NP proliferation reported here may play an important role. Our findings thus constitute an important advance in the understanding of the mechanisms underlying the generation of newly born cells in the brain and in expanding the biological roles assigned to the eCB system, which not only exerts a neuroprotective action, but also controls NP cell fate.
FOOTNOTES
1 Present address: Institute of Physiological Chemistry, Johannes Gutenberg University Mainz, Duesbergweg 6, Mainz 55099, Germany 
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-3995fje; doi: 10.1096/fj.05-3995fje
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) and their wild-type littermates (•). B) Effect of WIN-55,212-2 and URB597 on neurosphere-forming cells derived from wild-type (black bars) and CB1 knockout mice (gray bars). C) Self renewal of NPs derived from wild-type (solid lines) and CB1 knockout mice (dashed lines). The number of neurospheres was quantified after 4 consecutive neurosphere passages in the continuous presence of the indicated stimuli. Inset: Representative logarithmic plot of neurosphere generation. Results correspond to 4 (A, B) or 3 (C) independent experiments. *P<0.05, significantly different from controls.
