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, 23 (4), 1398-405

Nonpsychotropic Cannabinoid Receptors Regulate Microglial Cell Migration

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Nonpsychotropic Cannabinoid Receptors Regulate Microglial Cell Migration

Lisa Walter et al. J Neurosci.

Abstract

During neuroinflammation, activated microglial cells migrate toward dying neurons, where they exacerbate local cell damage. The signaling molecules that trigger microglial cell migration are poorly understood. In this paper, we show that pathological overstimulation of neurons by glutamate plus carbachol dramatically increases the production of the endocannabinoid 2-arachidonylglycerol (2-AG) but only slightly increases the production of anandamide and does not affect the production of two putative endocannabinoids, homo-gamma-linolenylethanolamide and docosatetraenylethanolamide. We further show that pathological stimulation of microglial cells with ATP also increases the production of 2-AG without affecting the amount of other endocannabinoids. Using a Boyden chamber assay, we provide evidence that 2-AG triggers microglial cell migration. This effect of 2-AG occurs through CB2 and abnormal-cannabidiol-sensitive receptors, with subsequent activation of the extracellular signal-regulated kinase 1/2 signal transduction pathway. It is important to note that cannabinol and cannabidiol, two nonpsychotropic ingredients present in the marijuana plant, prevent the 2-AG-induced cell migration by antagonizing the CB2 and abnormal-cannabidiol-sensitive receptors, respectively. Finally, we show that microglial cells express CB2 receptors at the leading edge of lamellipodia, which is consistent with the involvement of microglial cells in cell migration. Our study identifies a cannabinoid signaling system regulating microglial cell migration. Because this signaling system is likely to be involved in recruiting microglial cells toward dying neurons, we propose that cannabinol and cannabidiol are promising nonpsychotropic therapeutics to prevent the recruitment of these cells at neuroinflammatory lesion sites.

Figures

Fig. 1.
Fig. 1.
Microglial cells express cannabinoid CB1 and CB2 receptors. a, b, RT-PCR was performed with primers that recognize either mouse CB1 (mCB1) or mouse CB2 (mCB2) mRNA. We used reverse-transcribed total RNA from mouse brain and spleen (i.e., positive controls), BV-2 cells, and mouse microglial cells. RT-PCR products were of the appropriate size (502 bp for CB1 and 401 bp for CB2) and sequence. c–h, Immunofluorescent confocal microscopy. Mouse microglial cells (c) and BV-2 cells (d) were stained with an antibody directed against the CB1 C terminus (green) and phalloidin to label actin (red). Scale bars, 50 μm. Insets, Higher magnifications of the CB1 receptors (green) located in the intracellular compartment (MAC1, a plasma membrane macrophage marker, is in red). Scale bars, 10 μm. e, HEK293 cells transiently transfected with rat CB2 receptors tagged with HA11 (red) and stained with antibodies directed against CB2 C terminus (green). Colocalization isyellow. f, Similar immunostaining as ine, but performed in the presence of the immunizing antigen (i.e., the last 42 aa of the mouse CB2 receptor C terminus). Scale bars, 35 μm. Mouse microglial cells (g) and BV-2 cells (h) stained with antibodies directed against the CB2 receptor C terminus (green) and phalloidin (red).Arrowheads indicate CB2 receptors located at the lamellipodia tip. Scale bars, 50 μm. Insets, Higher magnification showing CB2 receptors at the leading edges of lamellipodia (g) and on microspikes (h). Scale bars, 10 μm.
Fig. 2.
Fig. 2.
Cannabinoids increase microglial cell migration.a–d, In the lower chamber of the Boyden chamber assay, we added THC (a), CBD (b), abn-CBD (c), and THC plus abn-CBD (d) (at a 1:1 molar ratio). BV-2 cell migration toward these ligands was quantified, and results are expressed as a percentage of basal BV-2 cell migration (i.e., vehicle = 0.1% DMSO; dashed line) measured in individual experiments. *p < 0.05; **p < 0.01; significantly different from basal BV-2 cell migration (ANOVA followed by Dunnett's post hoc test). Values are mean ± SEM of 9–45 independent quantifications of migration (i.e., 3–15 separate experiments performed in triplicate).
Fig. 3.
Fig. 3.
Endocannabinoids increase microglial cell migration. a–f, In the lower chamber of the Boyden chamber assay, we added AEA (a), meth-AEA (mAEA; b), 2-AG (2AG;c), PEA (d), DEA (e), and HEA (f). BV-2 cell migration toward these ligands was quantified, and results are expressed as a percentage of basal BV-2 cell migration (i.e., vehicle = 0.1% DMSO; dashed line) measured in individual experiments. *p < 0.05; **p < 0.01; significantly different from basal BV-2 cell migration (ANOVA followed by Dunnett's post hoc test). Values are mean ± SEM of 9–45 independent quantifications of migration (i.e., 3–15 separate experiments performed in triplicate).
Fig. 4.
Fig. 4.
2-AG increases microglial cell migration by acting through CB2- and CBD-sensitive receptors and stimulating ERK1/2 activity. a, Effects of various agents on basal migration. Values in a and b are means ± SEM of 9–36 independent quantifications of migration (i.e., 3–12 separate experiments performed in triplicate). Agents that were added to the lower chamber are as follows: 30 nmSR141716A (SR1), 30 nm SR144528 (SR2), 300 nm CBN, 300 nm CBD, 1 μm O-1918, or 10 μm PD98059 (PD). To test for the involvement of Gi/o-proteins, BV-2 cells were pretreated with 1 μg/ml pertussis toxin (PTX) for 18 hr.b, Effects of various agents on 2-AG (2AG)-induced migration. Results are expressed as a percentage of the control 2-AG-induced migration determined in each experiment (i.e., migration induced by 1 μm 2-AG added alone to the lower chamber minus corresponding basal migration obtained with the same agent; dashed line; see a). *p < 0.05; **p < 0.01; significantly different from the control 2-AG response (Student'st test). c, Representative Western blot with phospho-ERK1/2 antibodies. d, Quantification of three separate experiments performed in duplicate (n = 6). **p < 0.01; significantly different from basal (ANOVA followed by Dunnett'spost hoc test).
Fig. 5.
Fig. 5.
Mouse neurons and microglial cells produce four endocannabinoids. a–d, Mouse neurons were incubated with ionomycin (iono; 5 μm) for 2.5 and 5 min or with glutamate (Glut; 100 μm) and carbachol (Carb; 1 mm) for 2.5 min, lipids were extracted from chloroform from cells plus adjacent media and purified by HPLC; AEA (a), 2-AG (2AG; b), HEA (c), and DEA (d) were then quantified by CI-GC/MS.e–h, BV-2 cells were incubated with ionomycin (5 μm) for increasing periods of time, and endocannabinoids were quantified. prot, Protein. The arrowindicates that EGTA (5 mm) prevents the ionomycin response.i–l, Mouse microglial cells were incubated with ionomycin (5 μm) for 2.5 and 5 min or with ATP (1 mm) for 10 min, and endocannabinoids were quantified. Values are means ± SEM of 6–32 independent endocannabinoid quantifications, each performed on one 100 mm dish of cells. *p < 0.05; **p < 0.01; significantly different from basal amount (see Table 1) (ANOVA followed by Dunnett's post hoc test).

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