Prolonged oral cannabinoid administration prevents neuroinflammation, lowers β-amyloid levels and improves cognitive performance in Tg APP 2576 mice

Abstract

Background: Alzheimer's disease (AD) brain shows an ongoing inflammatory condition and non-steroidal anti-inflammatories diminish the risk of suffering the neurologic disease. Cannabinoids are neuroprotective and anti-inflammatory agents with therapeutic potential.

Methods: We have studied the effects of prolonged oral administration of transgenic amyloid precursor protein (APP) mice with two pharmacologically different cannabinoids (WIN 55,212-2 and JWH-133, 0.2 mg/kg/day in the drinking water during 4 months) on inflammatory and cognitive parameters, and on ¹⁸F-fluoro-deoxyglucose (¹⁸FDG) uptake by positron emission tomography (PET).

Results: Novel object recognition was significantly reduced in 11 month old Tg APP mice and 4 month administration of JWH was able to normalize this cognitive deficit, although WIN was ineffective. Wild type mice cognitive performance was unaltered by cannabinoid administration. Tg APP mice showed decreased ¹⁸FDG uptake in hippocampus and cortical regions, which was counteracted by oral JWH treatment. Hippocampal GFAP immunoreactivity and cortical protein expression was unaffected by genotype or treatment. In contrast, the density of Iba1 positive microglia was increased in Tg APP mice, and normalized following JWH chronic treatment. Both cannabinoids were effective at reducing the enhancement of COX-2 protein levels and TNF-α mRNA expression found in the AD model. Increased cortical β-amyloid (Aβ) levels were significantly reduced in the mouse model by both cannabinoids. Noteworthy both cannabinoids enhanced Aβ transport across choroid plexus cells in vitro.

Conclusions: In summary we have shown that chronically administered cannabinoid showed marked beneficial effects concomitant with inflammation reduction and increased Aβ clearance.

Figure 1

JWH oral administration rescued the cognitive impairment of TgAPP. Cannabinoid agonists did not alter the discrimination of the novel object (NO) compared to the familar one (A) of wt mice. Tg APP failed to distinguish the NO, like those treated with WIN, but JWH treatment restored normal discrimination. Animals were treated with vehicle or cannabinoids (0.2 mg/kg) in the drinking water for 4 months, starting at 7 months of age. Time spent exploring either objects was expressed as percentage of the total exploration time. Results are mean ± SEM (5-7 mice/group). *p < 0.05 vs familiar object (ANOVA followed by Student's t test).

Figure 2

JWH oral administration rescued the decreased ¹⁸F-DG uptake in TgAPP assessed by PET. A: representative images [MR, ¹⁸F-DG uptake and merged] in hippocampus of wild type (Wt) and Tg APP vehicle treated mice. Continuous oral treatment of wild type mice with WIN decreased uptake in all regions studied (B: hippocampus; C: Frontal cortex; D: Temporo-parietal cortex). Tg APP showed decreased uptake which was normalized by JWH oral treatment (A-D). Animals were treated with vehicle or cannabinoids (0.2 mg/kg) in the drinking water for 4 months, starting at 7 months of age. Results are mean ± SEM (n = 4 mice/group) of the ratio of average radiactivity in a given region of interest (ROI) by the radiactivity in cerebellum (Cb) expressed as percentage. *p < 0.05, ** p < 0.01 vs vehicle treated mice; #p < 0.05 vs wild type-vehicle mice (ANOVA followed by Student's t test).

Figure 3

Cannabinoid oral administration did not alter GFAP immunostaining or protein expression. GFAP hippocampal immunostaining (IR) or cerebral cortical protein expression was similar in all groups, irrespective of genotype or treatment. A: representative IR in hippocampus from wild type and Tg APP mice treated with vehicle or cannabinoids. The area assessed is shown (5 hippocampal areas) B: Optical density (OD) was measured by densitometry. C: representative GFAP Western blot (WB) and D: optical density (OD) in cortical samples. Animals were treated with vehicle or cannabinoids (0.2 mg/kg) in the drinking water for 4 months, starting at 7 months of age. Results are mean ± SEM (n = 6-7 mice/group).

Figure 4

Microglial cell density was increased in Tg APP and decreased by continuous JWH oral treatment. A: representative Iba-1 immunostaining in cerebral cortex following the different treatments. B: microglial cortical cell density. Animals were treated with vehicle or cannabinoids (0.2 mg/kg) in the drinking water for 4 months, starting at 7 months of age. Results are mean ± SEM (n = 6-7 mice/group). #p < 0.05 vs wild type vehicle treated mice; * p < 0.05 vs Tg APP vehicle treated mice (ANOVA followed by Student's t test).

Figure 5

Cannabinoid oral treatment decreased inflammatory parameters of Tg APP mice. A, B: Cannabinoids decreased CB₂ protein levels by Western blotting in Tg APP. C, D: Cannabinoids counteracted the increase in COX-2 protein levels by Western blotting. E: IL6 mRNA expression (qRT-PCR) was similar in all groups studied. F: Cannabinoids blocked the increased TNF-α mRNA expression (qRT-PCR) in Tg APP. Animals were treated with vehicle or cannabinoids (0.2 mg/kg) in the drinking water for 4 months, starting at 7 months of age. Results are mean ± SEM (n = 6-7 mice/group) *p < 0.05, ** p < 0.01 vs vehicle treated mice; #p < 0.05 vs wild type mice vehicle treated mice (ANOVA followed by Student's t test).

Figure 6

Cannabinoids decreased Aβ levels of Tg APP mice and increased transport through choroid plexus cell monolayer. A: Both cannabinoids significantly decreased Aβ_1-42 cortical levels. B: JWH decreased Aβ_1-40 cortical levels in Tg APP mice. Animals were treated with vehicle or cannabinoids (0.2 mg/kg) in the drinking water for 4 months, starting at 7 months of age. Results are mean ± SEM (n = 6-7 mice/group) C: rat choroid plexus express both CB₁ and CB₂ receptors. D: time-course of Aβ_1-40 transport through choroid plexus cells. Both cannabinoids (200 nM) reduced the transport time compared to untreated cultures (representative Western blot). E: Cannabinoids enhanced Aβ_1-40 transport through choroid plexus cells at 1 and 3 h after addition vs untreated control cultures (mean ± SEM, n = 4). E: *p < 0.05, *** p < 0.01 vs vehicle treated mice or cultures (Student's t test).

Figure 7

WIN oral treatment counteracted decreased phospho-Ser9 GSK3β in cerebral cortex of Tg APP mice. A: representative Western blot of p-Ser9 GSK3β. B: optical density (OD). C: total-GSK3β. D: optical density (OD). E: representative Western blot of p-Ser21 GSK3α. F: optical density (OD). G: total-GSK3α in cerebral cortex. H: optical density (OD). Wild type (Wt) and Tg APP mice were treated with vehicle or cannabinoids (0.2 mg/kg) in the drinking water for 4 months, starting at 7 months of age. Results are mean ± SEM (n = 6-7 mice/group) *p < 0.05 vs wt-veh mice, # p < 0.05 vs Tg APP-vehicle treated mice; #p < 0.05 vs wild type vehicle treated mice (ANOVA followed by Student's t test).