Roles of cannabinoids in the control of glial cell function
An ISN Symposium within the VI European Meeting on Glial Cell Function in Health and Disease
Berlin, September 6, 2003
Organized by Manuel Guzmán and Nephi Stella
The study of the role of glial cells in brain function and disease is attracting an increasing number of neuroscientists. The idea of dedicating an international neuroscience meeting to glial cells has turned out to be very successful. Earlier meetings in the series (Heidelberg 1994, Arcachon 1996, Athens 1998, Barcelona 2000, Rome 2002) proved most interesting indeed. As far as we know, these European meetings on glial cells are the only large, international neuroscience meetings devoted to the subject, and have attracted scientists from a large number of countries, both within and outside Europe.
The sixth edition of the meeting (Max-Delbrück-Communications Center, Berlin, 2003) was organized by Prof. Helmut Kettenmann. It gathered almost 500 attendants and has included 6 plenary lectures, 15 symposia (each one with 4-6 speakers) and more than 250 posters. Symposium number 15 was “Roles of cannabinoids in the control of glial cell function” (see http://euroglia2003.glia.mdc-berlin.de).
The actors: cannabinoids
Cannabinoids, the active components of Cannabis sativa L. (marijuana), exert a wide
array of effects on the central nervous system as well as at peripheral sites. It is widely accepted that these effects are mediated by the activation of specific G-protein-coupled receptors (the CB receptors) which are normally engaged by a family of endogenous ligands (the endocannabinoids). In brain it is known that endocannabinoids act as retrograde synaptic messengers inhibiting the release of neurotransmitters such as glutamate, dopamine and GABA, thereby affecting basic neural processes such as memory, learning and motor activity. Furthermore, marijuana and its derivatives have been used in medicine for many centuries, and there is a current renaissance in the study of the therapeutic effects of cannabinoids, which actually constitutes a widely debated issue with ample scientific and social relevance. The discovery of endocannabinoids, their receptors and their specific mechanisms of synthesis and degradation has allowed the definition of a new neuromodulatory system (the endocannabinoid system) and the elucidation of the molecular basis of the effects associated to the recreational and therapeutic uses of marijuana compounds.
The stage: glia
Despite the well-established role of glial cells in the control of neuronal function in health and disease, most of the current knowledge on cannabinoid action applies solely to their direct effects on neurons. However, the direct action of cannabinoids on glial cells with its impact on neuronal function is starting to be unraveled. The major findings in the field were discussed in this symposium, and may be summarized as follows: (i)
Endocannabinoids are not only produced by neurons, but also by glial cells (Stella’s
presentation). (ii) Cannabinoids do not only act on neurons, but also on glial cells, and this results in e.g. inhibition of microglial cell activation (Stella’s presentation), promotion of oligodendrocyte (Molina-Holgado’s presentation) and astrocyte (Velasco’s presentation)
survival, and blockade of astrocyte gap junction communication (Venance’s presentation). (iii) Cannabinoid action on glial cells may have therapeutic consequences. For example, cannabinoids attenuate tremor and spasticity in animal models of multiple sclerosis, and a phase III clinical trial is currently going on to test their efficiency in humans (Pertwee’s presentation). Moreover, cannabinoids inhibit the growth of malignant gliomas in animal models, and a phase I/II clinical trial is trying to determine whether they curb the growth of these tumors in humans (Galve-Roperh’s presentation).
All the aforementioned discoveries in the cannabinoid field have occurred in the last decade and have progressively attracted the attention of many researchers to this topic, which had never been represented at previous Euroglia Meetings. By including data from basic and applied research obtained with molecular, pharmacological and physiological approaches, the symposium presentations interested the multidisciplinary audience.
SUMMARIES OF PRESENTATIONS
Endocannabinoids are produced by glial cells and regulate their function Nephi Stella, Eiron Cudaback, Allyn Franklin, Lisa Walter and Anke Witting Department of Pharmacology, Psychiatry and Behavioural Sciences, University of Washington, Seattle, WA, USA
The cannabinoid signaling system, which includes cannabinoid receptors, endogenous cannabinoid ligands (endocannabinoids) and enzymes that inactivate them, has been identified in astrocytes and microglial cells. We have shown that stimuli involved in neuroinflammation increase the production of endocannabinoids from astrocytes and microglial cells. For example, in astrocytes, endothelin increases the production of two endocannabinoids: anandamide and 2-arachidonylglycerol (2-AG). This response is mediated through ETA receptors. On the other hand, in a response mediated through purinergic P2Y2 receptors in astrocytes, ATP selectively increases 2-AG production. In microglial cells, ATP also selectively increases 2-AG production, but this response is mediated through purinergic P2Y2 and P2X7 receptors. Thus, different stimuli acting through distinct receptors increase different endocannabinoid amounts from astrocytes and microglial cells. When endocannabinoid amounts are expressed in pmol/mg of protein, microglial cells in culture produce about 20 times more endocannabinoids than astrocytes or neurons in culture. Thus, activated microglial cells are likely to produce the majority of the endocannabinoids present under neuroinflammatory conditions. Endocannabinoids modulate microglial cell migration without affecting their ability to phagocyte particles or produce nitric oxide. Cannabinoid CB receptors, as well as the 2
recently identified abnormal-cannabidiol (abn-CBD) receptors mediate the endocannabinoid-induced migration of microglial cells. CB receptors are located at the 2
leading edges of microglial cell lamellipodia, which are cellular protrusions involved in cell migration. Anandamide and 2-AG are taken up and hydrolyzed by astrocytes and microglial cells, although at different rates.
The pharmacological profile of CB receptors, which are abundantly expressed by 1
neurons, is different from CB receptors and abn-CBD receptors. Thus, microglial CB 22
and abn-CBD receptors constitute promising pharmaceutical targets to control microglial cell migration occurring under neuroinflammatory conditions.
OLIGODENDROCYTES AS TARGETS OF CANNABINOIDS: PROTECTIVE
112Eduardo Molina-Holgado, Ángel Arévalo-Martín, José Manuel Vela, Guillermina 3411Almazán, Francisco Molina-Holgado, José Borrell and Carmen Guaza 12Instituto Cajal, CSIC, Madrid, Spain; Universitat Autònoma de Barcelona, Bellaterra, 34Barcelona, Spain; McGill University, Montreal, Canada; University of Cambridge,
Oligodendrocytes are highly vulnerable to hypoxia-ischemia, oxidative stress, and humoral and cellular immune-mediated attack. Synthetic and endogenous cannabinoids exert profound actions in the CNS, modulate inflammatory and immune responses, inhibit pain, and reduce neuronal damage in models of excitotoxicity, ischemia and traumatic brain injury. To gain insights into the underlying physiological function of cannabinoid receptors in conditions where oligodendrocyte survival is compromised, we evaluated the expression and functionality of cannabinoid receptors in oligodendrocytes both in vivo and in culture.
Using mice challenged with the Theiler’s murine encephalomyelitis virus, a model of human multiple sclerosis (MS), we observed that treatment with the synthetic cannabinoids (+)-Win-55,212-2, ACEA and JWH-015 during established disease significantly improved the neurological deficits. Recovery of motor function and diminution of inflammation paralleled extensive remyelination in the spinal cord. On the other hand, expression of CB1 receptors by oligodendrocytes was demonstrated immunocytochemically in postnatal and in adult white matter as well as in oligodendrocyte cultures. The cannabinoid agonists HU210, (+)-Win-55212-2 and ACEA protected cultured oligodendrocyte progenitors from apoptosis induced after deprivation of trophic support, a mechanism dependent on the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway.
The present results may have potential therapeutical implications in demyelinating pathologies such as MS. In addition, our studies identify oligodendrocytes as targets of cannabinoid action in the CNS, and point to a direct role of cannabinoids in promoting survival of oligodendrocyte progenitors.
CANNABINOIDS PROTECT ASTROCYTES FROM APOPTOSIS
Guillermo Velasco, Teresa Gómez del Pulgar, María L. de Ceballos, Arkaitz Carracedo, Tania Aguado, Ismael Galve-Roperh and Manuel Guzmán
Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University, Madrid, Spain
Recent studies have evidenced that cannabinoids could play a major role in the control of neural cell fate. Cannabinoids, have been shown to protect neurons from toxicity as induced by different insults and to inhibit neuronal differentiation. By contrast, much less is known about the role of cannabinoids in the control of the fate of the central nervous system major cell population, namely the astrocytes. Our group has shown that cannabinoids induce apoptosis of astrocytoma cells. Here we firstly tested whether 9cannabinoids could protect astrocytes from death. Our data show that: (i) ;-
tetrahydrocannabinol and other cannabinoids rescue astrocytes from ceramide-induced apoptosis, (ii) this effect is CB receptor- and PI3K/PKB-mediated, and (iii) 1
cannabinoids protect astrocytes from the cytotoxic effects of focal ceramide administration in vivo. In addition, based on the potential role of cannabinoids in the control of neural progenitor cell differentiation, we are currently investigating whether cannabinoids may also modulate astrocyte generation under certain circumstances. In
summary, the actions of cannabinoids on astrocytes differ from those exerted on transformed glial cells, and suggest an “astroprotective” role for these compounds.
ANANDAMIDE CONTROLS GAP JUNCTIONAL PERMEABILITY AND
PROPAGATION OF INTERCELLULAR CALCIUM WAVES IN ASTROCYTIC
Laurent Venance, Thomas Höfer, William Même, Marie Vandecasteele, Jacques Glowinski and Christian Giaume
Neuropharmacologie, INSERM U-114, Collège de France, Paris, France
Two main characteristics of astrocytes are their elaborated intracellular calcium signalling and their high degree of intercellular communication which is underlied by gap junctional channels. The combination of these two properties provides a basis for a long-range signalling system throughout an extended glial network within the brain. Indeed, astrocytes, although non-excitable cells, possess a dynamic way to communicate and propagate information through calcium waves. Various
neurotransmitters (endothelins, glutamate) trigger a short or long range calcium waves in astrocytes. Experimental and mathematical modelling analysis of the initiation and semi-regenerative propagation of intercellular calcium waves revealed at least three critical steps: PLC activity, filing level of internal calcium stores and permeability of intercellular channels to IP3. Anandamide, the prototypical endocannabinoid, appears to regulate at least two of these key steps. Indeed, anandamide is a potent inhibitor of intercellular permeability (electric and metabolic coupling) and leads to the depletion of the internal calcium stores, both through activation of a Gi/Go-protein-coupled receptor distinct from the CB1 and CB2 receptors. The block of intercellular calcium waves and the depletion of intracellular calcium stores may represent an action of anandamide that depresses the activity of surrounding neurones through astrocytic network.
CANNABINOIDS AND MULTIPLE SCLEROSIS
Roger G. Pertwee
Department of Biomedical Sciences, Institute of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, UK
Evidence that cannabis and certain individual cannabinoids can suppress some symptoms of multiple sclerosis and spinal cord injury is sufficiently convincing to have prompted several large clinical trials, the outcomes of which have still to be announced. This evidence is to be found in newspaper reports about multiple sclerosis patients who self-medicate with cannabis, in the responses of such patients to questionnaires, in data obtained in a small number of clinical trials performed with just a few patients, and in results obtained from experiments with animals models of multiple sclerosis. The existing clinical data suggest that cannabis and the cannabinoid receptor agonists, delta-9-tetrahydrocannabinol and nabilone, can produce objective and/or subjective relief from spasticity, pain, tremor and nocturia in patients with multiple sclerosis or spinal cord injury. The animal data have shown that tremor and spasticity can be reduced both by CB- and CB-selective cannabinoid receptor agonists and by the non-psychotropic 12
cannabinoid, HU-211. Some of the animal experiments were performed using mice with chronic relapsing experimental allergic encephalomyelitis (CREAE). These have provided strong evidence that endocannabinoid concentrations are elevated in the brains and spinal cords of CREAE mice with spasticity and that this spasticity can be ameliorated by inhibitors of endocannabinoid membrane transport or enzymic
hydrolysis. This finding raises the possibility that the production of endocannabinoids may increase in multiple sclerosis and that inhibitors of endocannabinoid membrane transport or enzymic hydrolysis have clinical potential. An important area for future research will be to establish whether, in addition to relieving symptoms of multiple sclerosis, cannabinoids can retard the course of this disease, for example through their known neuroprotective and anti-inflammatory actions.
ANTI-TUMORAL ACTION OF CANNABINOIDS IN MALIGNANT GLIOMAS
Ismael Galve-Roperh, Guillermo Velasco, Teresa Gómez del Pulgar,
Cristina Blázquez, Cristina Sánchez, Daniel Rueda and Manuel Guzmán
Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University, Madrid, Spain
Cannabinoids have been shown to exert anti-tumoral actions in vivo against different
types of cancer including gliomas, skin carcinomas and lymphoid tumors. Gliomas constitute the most aggressive brain tumors for which conventional therapies are only palliative. Cannabinoid treatment inhibits the growth of human glioma implants in nude mice, and this process is associated with induction of apoptosis of transformed cells as well as angiogenesis inhibition. Cannabinoid-triggered cell death relies on their ability to stimulate de novo synthesis of the pro-apoptotic lipid messenger ceramide, which in turn results in sustained ERK activation and PKB inhibition. Profound changes in tumor vascularization upon cannabinoid administration are also observed, which correlates with dimished expression of pro-angiogenic factors. Importantly, although the most abundant cannabinoid receptor in brain is the CB receptor, glioma cells also express 1
the CB receptor, a receptor normally expressed by immune cells whose activation is 2
devoid of psychoactivity. We further demonstrated that like CB activation, selective 1
CB agonists are able to exert an anti-tumoral action, therefore opening new therapeutic 2
strategies for the management of gliomas. These and others findings constitute the scientific basis for the first clinical trial aimed at investigating the potential benefits of cannabinoid treatment for the management of human glioblastoma multiforme.
We intended to cover at least the flight and hotel expenses. Unfortunately, the decreased exchange of the dollar with respect to the euro (I estimated an approximately 1:1 exchange when applying for the ISN grant) and the raise in the air ticket fares in the last months has allowed covering not 100% but 87.3% of those expenses. Each speaker was therefore given 87.3% of their expenses (“Amount given” in the Table below). As Velasco, Galve-Roperh and myself work in the same institute, we advanced the money to the rest and will wait for the amount pending (expected 900 ?) to get our whole portions.
Amount given: US$ 4000 (? 3600)
Amount pending: US$ 1000 (estimated ? 900)
Summary of expenses (?)
12Speaker Flight Hotel Amount Amount
to be given given
Stella 937 375 1312 1146
Molina-Holgado 251 375 626 547
Venance 301 375 676 591
Pertwee 479 254 733 641
Velasco 368 233 601 225 (300 pending)
Galve-Roperh 368 233 601 225 (300 pending)
Guzmán 368 233 601 225 (300 pending)
Total 3072 2078 5150 3600 (900 pending)
Based on the following fares (all in tourist class):
Washington-Berlin-Washington Lufhansa via Amsterdam (Stella).
Paris-Berlin-Paris Air France direct flight (Venance).
Aberdeen-Berlin-Aberdeen British Airways via London (Pertwee).
Madrid-Berlin-Madrid Iberia direct flight (Guzmán, Molina-Holgado, Velasco, Galve-Roperh – we three could not get Molina-Holgado’s special fare).
2Based on the following fares (all in the Holiday Inn Humboldt-Park, Hochstrasse, Berlin, in which the organization had reserved a block of rooms at a special rate): 5 nights in double room (150 ? per room per night) (Stella, Venance, Molina-Holgado).
4 nights in triple room (175 ? per room per night) (Guzmán, Velasco, Galve-Roperh).
2 nights in single room (127 ? per room per night) (Pertwee).