Differential regulation of behavioral tolerance to WIN55,212-2 by GASP1

Abstract

Cannabinoid agonists have shown some promise clinically as analgesics, in particular for cancer pain, in which they have the additional benefit of decreasing nausea. However, as for most other drugs, the long-term use of cannabinoids is limited by the development of tolerance. Several molecular mechanisms have been proposed to explain drug tolerance, including receptor downregulation. The cannabinoid 1 (CB1) receptors can be downregulated in vitro through an interaction with the G-protein-coupled receptor-associated sorting protein1, GASP1, that targets CB1 receptors for degradation after their agonist-mediated endocytosis. To investigate whether GASP1-mediated postendocytic sorting of the CB1 receptor contributes to tolerance to cannabinoid drugs in vivo, we generated a mouse with a disruption of GASP1. In wild-type mice, repeated administration of the cannabinoid agonist WIN55,212-2 promoted downregulation of CB1 receptor levels and concomitant tolerance to the effects of drug on antinociception, motor incoordination, and locomotor hypoactivity. In contrast, GASP1 knockout mice did not develop tolerance to any of these effects and showed no significant receptor downregulation. Taken together, this study provides evidence that GASP1 regulates CB1 receptor downregulation in vivo, and that postendocytic receptor trafficking has a key role in the development of tolerance to WIN55,212-2.

Figure 1

Generation of GASP1 KO mice. (a) Targeting vector design for generating GASP1 KO mice. A cassette expressing the G418 resistance gene flanked by loxP sites was inserted into the intron upstream of the GASP1 open reading frame (ORF) (intron 4) and a third loxP site was inserted in the intron downstream of the GASP ORF (intron 5). ES cells from C57/Bl6 mice were transfected with this vector. Properly targeted clones (see b) were transfected with Cre-recombinase, and blastocysts from clones in which the GASP1 ORF was disrupted were implanted into C57/BL6 females. (b) Southern blotting analysis identified homologous recombination and single insertion using both (left) 5′ and (right) Neo probes. (c) Homogenates from wild-type (WT) and GASP1 knockout (KO) whole brain, cerebellum, spinal cord, and hypothalamus were analyzed by immunoblot (IB) and shows complete knockout of GASP1. Furthermore, there are no compensatory changes in the expression of the closest homolog, GASP2, in either of these regions.

Figure 2

Acute effect of WIN55,212-2 in antinociception, motor coordination, and body temperature. Wild-type (WT, ▪) and GASP1 knockout (KO, □) mice were injected with increasing doses of WIN55,212-2 (WIN) and analyzed for drug-induced behavioral changes. Data are presented as mean±SEM. (a) Antinociception was assessed by tail flick withdrawal latency after injection with WIN55,212-2 (1–6 mg/kg s.c.; n=38–52). There were no genotype differences in response to drug-induced antinociception. MPE, maximal possible effect. (b) Motor coordination was evaluated by placing mice on an accelerating rotarod after injection with WIN55,212-2 (3.5–7 mg/kg i.p.; n=9–13) and measuring latency to fall off the rod. There were no genotype differences in response to drug-induced motor incoordination. (c) Body temperature was measured after injection with WIN55,212-2 (1–6 mg/kg s.c.; n=25–37). There were no genotype differences in response to drug-induced hypothermia except for the highest dose of WIN55,212-2 tested (6 mg/kg, ^*p<0.05).

Figure 3

Tolerance to the effects of WIN55,212-2 in antinociception, motor incoordination, locomotor hypoactivity, and hypothermia after repetitive administration. Wild-type (WT) and GASP1 KO mice were injected twice daily for 5 days with either vehicle (veh) or 3.5 mg/kg WIN55,212-2 (WIN) and assessed for the development of tolerance to WIN55,212-2. (•) Vehicle-treated and (▪) WIN55,212-2-treated WT mice; (○) vehicle-treated and (□) WIN55,212-2-treated GASP1 KO mice. Data are presented as mean±SEM. (a, b) On days 1 and 7, WT (a) and GASP KO (b) mice were tested for tail flick withdrawal latencies after injection with increasing doses of WIN55,212-2 (1–6 mg/kg s.c.; n=18–24 per group). MPE, maximal possible effect. GASP1 KO mice (b) treated chronically with WIN55,212-2 showed reduced antinociceptive tolerance compared with WT mice (a). ^***p<0.001; ^**p<0.01; ns, not significant (compared with vehicle treatment). (c, d) Mice were tested for motor coordination on an accelerating rotarod after injection with increasing doses of WIN55,212-2 (3.5–14 mg/kg i.p.; n=9–12 per group). GASP1 KO mice (d) treated chronically with WIN55,212-2 showed reduced tolerance compared with WT mice (c). ^***p<0.001; ^**p<0.01; ns: not significant (compared with vehicle treatment). (e, f) Mice chronically treated with either vehicle or drug were placed in activity chambers after injection of vehicle (solid bars) or 3.5 mg/kg WIN55,212-2 (hatched bars), respectively. The distance traveled on day 6 was compared with the distance on day 2 (n=15–16 per group). GASP1 KO mice (f) treated chronically with WIN55,212-2 showed reduced tolerance to the hypolocomotor effects of WIN55,212-2 compared with WT mice (e). ^***p<0.001; ns: not significant (compared with vehicle). (g, h) On days 1 and 7, body temperature was measured after injection with increasing doses of WIN55,212-2 (1–6 mg/kg s.c.; n=8–19 per group). On day 7, both WT (g) and GASP1 KO (h) mice had developed tolerance to the hypothermic effects of WIN55,212-2 compared with vehicle-treated mice. ^***p<0.001; ns: not significant (compared with vehicle treatment).

Figure 4

Colocalization of GASP and CB1 receptors in the mouse central nervous system. Micrographs of coronal sections from drug-naïve WT mice (a) preoptic anterior hypothalamus (POAH, medial part) and (b) thalamus co-stained for GASP (green) and CB1 receptor (red). (a) GASP and CB1 receptor immunoreactivity is present in the majority of POAH neurons. POAH sections were also stained for NeuN (neuronal nuclei, blue). Inset: schematic showing the location of POAH (gray ovals). (b) Thalamic sections were stained for GASP (green), CB1 receptors (red), and also for parvalbumin (blue) to identify GABAergic neurons of the reticular thalamic nucleus (nRT). Both GASP and CB1 receptors were present in GABAergic neurons of nRT and also in nearby glutamatergic cells of the ventrobasal thalamus (VB). Scale bars: a, 50 μm; b,100 μm.

Figure 5

Downregulation of CB1 receptors in WT, but not in GASP1 KO mice, after repetitive administration of WIN55,212-2. Tissue from WT and GASP1 KO mice chronically injected with either vehicle or 3.5 mg/kg WIN55,212-2 was tested in saturation binding assays using the antagonist SR141716a to assess total number of CB1 receptors (B_max). (a–d) WT mice treated with WIN55,212-2 showed significant downregulation of CB1 receptor number in both the spinal cord (a, e) and the cerebellum (c, f), whereas GASP1 KO mice showed reduced receptor downregulation in both tissues (b, d, e, f). (e, f) Data presented as percent B_max±SEM in drug-treated versus vehicle-treated mice. ^***p<0.001; ^**p<0.01; ns: not significant (compared with normalized vehicle-treated mice, n=3–4, performed on independent membrane preparations).