Dysregulation of cannabinoid CB1 receptor and associated signaling networks in brains of cocaine addicts and cocaine-treated rodents
Graphical abstract
Introduction
Cocaine addiction is a major medical entity (O’Brien and Anthony, 2005) with complex molecular, cellular and structural brain abnormalities (Nestler, 2005, Thomas et al., 2008) and a lack of effective pharmacologic treatments (Vocci and Elkashef, 2005). Cocaine inhibits dopamine transporter (DAT), which initially increases dopamine neurotransmission (Giros et al., 1996). In the long-term, human cocaine dependence is associated with dysregulation of D2 dopamine receptor signaling networks in the prefrontal cortex (PFC) (Volkow et al., 1993, Álvaro-Bartolomé et al., 2011).
Recent advances in laboratory animals have implicated the endocannabinoid system in the neurobiology of drug addictions (Maldonado et al., 2006). Thus, the reciprocal functional interactions between cannabinoids and cocaine or opiates have been shown to be important in the molecular mechanisms leading to dependence on these drugs (Arnold, 2005, Wiskerke et al., 2008, Parolaro et al., 2010, Trigo et al., 2010). The endocannabinoids (e.g. anandamide) function as retrograde lipid messengers (Vaughan and Christie, 2005) through the activation of inhibitory cannabinoid (CB) CB1 and CB2 receptors (Pertwee, 1997). The CB1 receptor is highly expressed in the cerebral cortex and other limbic areas of the brain reward circuitry (Herkenham et al., 1991). The less abundant (neurons and astrocytes) and less well understood CB2 receptor (Chin et al., 2008) is mainly associated with inflammatory processes mediated by microglia (Onaivi et al., 2012). Chronic cocaine exposure has resulted in inconsistent findings in the regulation of CB1 receptors (mRNA expression and/or radioligand binding sites) in rodent brains (González et al., 2002, Centonze et al., 2004, Adamczyk et al., 2012). Overexpression of CB2 receptors was reported to reduce cocaine self-administration in mice (Aracil-Fernández et al., 2012).
An interesting feature of CB1 receptors is its high level of constitutive activity exerting a tonic control (ligand-independent activity) on its endocytic cycle (Leterrier et al., 2004, Leterrier et al., 2006, McDonald et al., 2007, Rozenfeld, 2011; but also see Howlett et al., 2011). Recently, a small pool of CB1 receptors has been quantified on neuronal mitochondrial membranes revealing its intracellular localization (Bénard et al., 2012). The homologous receptor regulation is induced by G protein-coupled receptor kinases (GRK) that phosphorylate the agonist- or endogenous ligand-activated receptor, which together with β-arrestin leads to receptor desensitization and internalization (Premont and Gainetdinov, 2007). CB1 receptors are mainly regulated by GRK2 and GRK3 (Jin et al., 1999, Rubino et al., 2006, Namkung et al., 2009).
Several intracellular signaling networks mediate the effects of CB1 receptors (Demuth and Molleman, 2006). CB1 receptor stimulation induces the activation of Akt (protein kinase B) and the mammalian target of rapamicin (mTOR) (Ozaita et al., 2007, Puighermanal et al., 2009), mTOR kinase being a downstream target of Akt pathway (Manning and Cantley, 2007). In previous studies, the CB1 receptor agonist WIN55212-2 and cocaine were shown to regulate Akt in rodent brain cortex and in brains of cocaine addicts (Álvaro-Bartolomé et al., 2010, Álvaro-Bartolomé et al., 2011). Given the regulation of Akt, mTOR and its target the 70 kDa ribosomal protein S6 kinase (p70S6K) induced by CB1 receptors, D2 receptors and cocaine (Beaulieu et al., 2007, Ozaita et al., 2007, Perrine et al., 2008, Puighermanal et al., 2009), these signaling molecules are important in the mechanisms of cocaine addiction.
To test the hypothesis that human cocaine addiction results in malfunction of cannabinoid CB1 receptors and associated signaling networks, receptor content and subcellular distribution, the content of regulatory kinases GRK2-6, and the activation of mTOR/p70S6K signaling were measured in postmortem brains (PFC) of well-characterized long-term pure cocaine addicts as well as in mixed cocaine/opiate and pure opiate (heroin, methadone) addicts. For comparison, these molecular targets were also evaluated in the cerebral cortex of cocaine- and cannabinoid-treated mice and rats. The main results reveal that cocaine addiction is associated with the downregulation of CB1, but not CB2, receptors, reduced GRK2/3/5 content and altered receptor subcellular redistribution which leads to dysregulation (dampening) of Akt/mTOR/p70S6K signaling in the brain. The findings provide new insights into the participation of the endocannabinoid system in cocaine addiction in humans.
Section snippets
Subject selection and specimens of postmortem human brain
The subjects and specimens of PFC/Brodmann’s area 9 (PFC/BA9) were selected from larger cohorts of drug abusers and controls whose individual features including history of drug abuse, comorbidity and detailed toxicology have been reported in previous studies on the molecular mechanisms of drug addictions in humans (García-Fuster et al., 2008a, Ramos-Miguel et al., 2009, Álvaro-Bartolomé et al., 2011). The PFC was selected for examination because of its relevant role in cocaine abuse (Bradberry,
Validation of anti-CB1 and anti-CB2 receptor antibodies in brain tissue
Western blot analysis of cerebral cortex of WT mice with the finally selected anti-CB1 receptor antibody (Abcam) readily identified the monomeric species of CB1 receptor (≈60 kDa peptide) (Fig. 1A, left). This immunoreactive cortical protein was absent in CB1 receptor KO mice (Fig. 1A, left), which demonstrated antibody specificity. Identical results were obtained in the corpus striatum and cerebellum of WT and CB1 receptor KO mice (Fig. 1A, right). Other brain proteins (≈35–45 kDa) recognized by
Identification of CB1 and CB2 receptors in brain tissue
The CB1 receptor (≈60 kDa, monomeric species) was readily identified in human, rat and mouse brains using an antibody directed against the C-terminal of the human receptor (Abcam). The antibody specificity was demonstrated in various brain regions of CB1 receptor KO mice (negative control), which is the gold standard method for antibody validation (Bordeaux et al., 2010). Other anti-CB1 receptor antibodies analyzed (Sigma; Santa Cruz) were not validated in brain regions of CB1 receptor KO mice,
Conclusion
This study tested the hypothesis that human cocaine addiction results in malfunction of cannabinoid CB1 receptors, regulatory kinases GRKs and associated signaling networks. The main results in postmortem human brains reveal that cocaine addiction is associated with the downregulation of CB1, but not CB2, receptors, reduced GRK2/3/5 content and altered CB1 receptor subcellular redistribution which leads to dysregulation (dampening) of Akt/mTOR/p70S6K signaling in the PFC/BA9. CB1 receptor
Acknowledgments
The study was supported by grants SAF2008-01311 and SAF2011-29918 from Ministerio de Economía y Competitividad and Fondo Europeo de Desarrollo Regional (MINECO/FEDER), Madrid, Spain. The research was also funded by Redes Temáticas de Investigación Cooperativa en Salud (RETICS, Instituto de Salud Carlos III, MINECO/FEDER, Madrid): Red de Trastornos Adictivos, RTA-RD12/0028/0011. M.A.-B. was supported by a FPI predoctoral fellowship from MINECO (Madrid). The authors thank Dr. Romano La Harpe
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