Activation of spinal microglia contributes to aberrant pain responses associated with neuropathic pain states. Endocannabinoids (ECs) are present in the spinal cord, and inhibit nociceptive processing; levels of ECs may be altered by microglia which modulate the turnover of endocannabinoids in vitro. Here, we investigate the effect of minocycline, an inhibitor of activated microglia, on levels of the endocannabinoids anandamide and 2-arachidonoylglycerol (2-AG), and the related compound N-palmitoylethanolamine (PEA), in neuropathic spinal cord. Selective spinal nerve ligation (SNL) in rats resulted in mechanical allodynia and the presence of activated microglia in the ipsilateral spinal cord. Chronic daily treatment with minocycline (30 mg/kg, ip for 14 days) significantly reduced the development of mechanical allodynia at days 5, 10 and 14 post-SNL surgery, compared to vehicle-treated SNL rats (P < 0.001). Minocycline treatment also significantly attenuated OX-42 immunoreactivity, a marker of activated microglia, in the ipsilateral (P < 0.001) and contralateral (P < 0.01) spinal cord of SNL rats, compared to vehicle controls. Minocycline treatment significantly (P < 0.01) decreased levels of 2-AG and significantly (P < 0.01) increased levels of PEA in the ipsilateral spinal cord of SNL rats, compared to the contralateral spinal cord. Thus, activation of microglia affects spinal levels of endocannabinoids and related compounds in neuropathic pain states.

BACKGROUND AND PURPOSEMicroglial cells are important mediators of the immune response in the CNS. The phytocannabinoid, cannabidiol (CBD), has been shown to have central anti-inflammatory properties, and the purpose of the present study was to investigate the effects of CBD and other phytocannabinoids on microglial phagocytosis. EXPERIMENTAL APPROACHPhagocytosis was assessed by measuring ingestion of fluorescently labelled latex beads by cultured microglial cells. Drug effects were probed using single-cell Ca 2+ imaging and expression of mediator proteins by immunoblotting and immunocytochemistry. KEY RESULTSCBD (10 μM) enhanced bead phagocytosis to 175 ± 7% control. Other phytocannabinoids, synthetic and endogenous cannabinoids were without effect. The enhancement was dependent upon Ca 2+ influx and was abolished in the presence of EGTA, the Ca 2+ channel inhibitor SKF96365, the transient receptor potential (TRP) channel blocker ruthenium red, and the TRPV1 antagonists capsazepine and AMG9810. CBD produced a sustained increase in intracellular Ca 2+ concentration in BV-2 microglia and this was abolished by ruthenium red. CBD rapidly increased the expression of TRPV2 and TRPV1 proteins and caused a translocation of TRPV2 to the cell membrane. Wortmannin blocked CBD enhancement of BV-2 cell phagocytosis, suggesting that it is mediated by PI3K signalling downstream of the Ca 2+ influx. CONCLUSIONS AND IMPLICATIONSThe TRPV-dependent phagocytosis-enhancing effect of CBD suggests that pharmacological modification of TRPV channel activity could be a rational approach to treating neuroinflammatory disorders involving changes in microglial function and that CBD is a potential starting point for future development of novel therapeutics acting on the TRPV receptor family. AbbreviationsCBD, cannabidiol; LPS, bacterial lipopolysaccharide BJP British Journal of Pharmacology

Cannabinoid receptor interacting protein 1a (CRIP1a) is a CB receptor (CBR) distal C-terminal-associated protein that alters CBR interactions with G-proteins. We tested the hypothesis that CRIP1a is capable of also altering CBR interactions with β-arrestin proteins that interact with the CBR at the C-terminus. Coimmunoprecipitation studies indicated that CBR associates in complexes with either CRIP1a or β-arrestin, but CRIP1a and β-arrestin fail to coimmunoprecipitate with each other. This suggests a competition for CRIP1a and β-arrestin binding to the CBR, which we hypothesized could attenuate the action of β-arrestin to mediate CBR internalization. We determined that agonist-mediated reduction of the density of cell surface endogenously expressed CBRs was clathrin and dynamin dependent and could be modeled as agonist-induced aggregation of transiently expressed GFP-CBR. CRIP1a overexpression attenuated CP55940-mediated GFP-CBR as well as endogenous β-arrestin redistribution to punctae, and conversely, CRIP1a knockdown augmented β-arrestin redistribution to punctae. Peptides mimicking the CBR C-terminus could bind to both CRIP1a in cell extracts as well as purified recombinant CRIP1a. Affinity pull-down studies revealed that phosphorylation at threonine-468 of a CBR distal C-terminus 14-mer peptide reduced CBR-CRIP1a association. Coimmunoprecipitation of CBR protein complexes demonstrated that central or distal C-terminal peptides competed for the CBR association with CRIP1a, but that a phosphorylated central C-terminal peptide competed for association with β-arrestin 1, and phosphorylated central or distal C-terminal peptides competed for association with β-arrestin 2. Thus, CRIP1a can compete with β-arrestins for interaction with C-terminal CBR domains that could affect agonist-driven, β-arrestin-mediated internalization of the CBR.

CB1 cannabinoid receptors (CB1R) are one of the most abundantly expressed G protein coupled receptors (GPCR) in the CNS and regulate diverse neuronal functions. The identification of GPCR interacting proteins has provided additional insight into the fine-tuning and regulation of numerous GPCRs. The Cannabinoid Receptor Interacting Protein 1a (CRIP1a) binds to the distal carboxy terminus of CB1R, and has been shown to alter CB1R-mediated neuronal function [1]. The mechanisms by which CRIP1a regulates CB1R activity have not yet been identified; therefore the focus of this investigation is to examine the cellular effects of CRIP1a on CB1R signaling using neuronal N18TG2 cells stably transfected with CRIP1a over-expressing and CRIP1a knockdown constructs. Modulation of endogenous CRIP1a expression did not alter total levels of CB1R, ERK, or forskolin-activated adenylyl cyclase activity. When compared to WT cells, CRIP1a over-expression reduced basal phosphoERK levels, whereas depletion of CRIP1a augmented basal phosphoERK levels. Stimulation of phosphoERK by the CB1R agonists WIN55212-2, CP55940 or methanandamide was unaltered in CRIP1a over-expressing clones compared with WT. However, CRIP1a knockdown clones exhibited enhanced ERK phosphorylation efficacy in response to CP55940. In addition, CRIP1a knockdown clones displayed a leftward shift in CP55940-mediated inhibition of forskolin-stimulated cAMP accumulation. CB1R-mediated Gi3 and Go activation by CP99540 was attenuated by CRIP1a over-expression, but robustly enhanced in cells depleted of CRIP1a. Conversely, CP55940-mediated Gi1 and Gi2 activation was significant enhanced in cells over-expressing CRIP1a, but not in cells deficient of CRIP1a. These studies suggest a mechanism by which endogenous levels of CRIP1a modulate CB1R-mediated signal transduction by facilitating a Gi/o-protein subtype preference for Gi1 and Gi2, accompanied by an overall suppression of G-protein-mediated signaling in neuronal cells.