Neuroprotective properties of cannabigerol in Huntington's disease: studies in R6/2 mice and 3-nitropropionate-lesioned mice

Abstract

Different plant-derived and synthetic cannabinoids have shown to be neuroprotective in experimental models of Huntington's disease (HD) through cannabinoid receptor-dependent and/or independent mechanisms. Herein, we studied the effects of cannabigerol (CBG), a nonpsychotropic phytocannabinoid, in 2 different in vivo models of HD. CBG was extremely active as neuroprotectant in mice intoxicated with 3-nitropropionate (3NP), improving motor deficits and preserving striatal neurons against 3NP toxicity. In addition, CBG attenuated the reactive microgliosis and the upregulation of proinflammatory markers induced by 3NP, and improved the levels of antioxidant defenses that were also significantly reduced by 3NP. We also investigated the neuroprotective properties of CBG in R6/2 mice. Treatment with this phytocannabinoid produced a much lower, but significant, recovery in the deteriorated rotarod performance typical of R6/2 mice. Using HD array analysis, we were able to identify a series of genes linked to this disease (e.g., symplekin, Sin3a, Rcor1, histone deacetylase 2, huntingtin-associated protein 1, δ subunit of the gamma-aminobutyric acid-A receptor (GABA-A), and hippocalcin), whose expression was altered in R6/2 mice but partially normalized by CBG treatment. We also observed a modest improvement in the gene expression for brain-derived neurotrophic factor (BDNF), insulin-like growth factor-1 (IGF-1), and peroxisome proliferator-activated receptor-γ (PPARγ), which is altered in these mice, as well as a small, but significant, reduction in the aggregation of mutant huntingtin in the striatal parenchyma in CBG-treated animals. In conclusion, our results open new research avenues for the use of CBG, alone or in combination with other phytocannabinoids or therapies, for the treatment of neurodegenerative diseases such as HD.

Fig. 1

Behavioral score after 3-nitropropionic acid (3NP) intoxication. Hindlimb clasping, general locomotor activity, hindlimb dystonia, and truncal dystonia were rated from 0 to 2 based on severity: a score of 0 typically indicates normal function and 2 indicates seriously affected function. Values are expressed as means ± SEM for 6–8 animals per group. Data were subjected to one-way analysis of variance followed by the Student–Newman–Keuls test. *P < 0.05, **P < 0.01, ***P < 0.001 when comparing the control group with the 3NP group. ^# P < 0.05, ^## P < 0.01, ^### P < 0.001 when comparing 3NP group with 3NP + cannabigerol (CBG) group

Fig. 2

Systemic administration of 3-nitropropionic acid (3NP) leads to a progressive and selective degeneration in the striatum. (Left) Cresyl violet staining was performed on brain sections from control, cannabigerol (CBG)-, 3NP-, and 3NP + CBG-treated mice. Low (left column) and high magnification (right column) showing the selective loss of cells in the striatum at day 5 (pale region, outlined). This lesion was not detectable in the group that received CBG. Images were acquired by using light microscopy. (Right) Quantification of Nissl-positive cells in the mouse striatum. Total average number of neurons (100× magnification) is shown. Values are expressed as means ± SEM for 6–8 animals per group. Data were subjected to one-way analysis of variance followed by the Student–Newman–Keuls test. ***P < 0.001 when comparing the control group with the 3NP and CBG group. ^### P < 0.001 when comparing the 3NP group with the 3NP + CBG group

Fig. 3

(Left panels) Photomicrographs of NeuN (10×)-, glial fibrillary acidic protein (GFAP) (10×)-, and Iba1 (20×)-immunostained sections through the coronal section of striatum of control and 3-nitropropionic acid (3NP)-lesioned mice treated with vehicle or cannabigerol (CBG). They show a significant loss of NeuN-positive cells in the striatum of 3NP-treated mice compared with controls. CBG treatment significantly reduced 3NP-induced loss of striatal NeuN-positive cells. (Right panels) Quantification of (A) NeuN-, (B) GFAP-, and (C) Iba1-positive cells in the mouse striatum. Total average number of neurons, astrocytes (100× magnification) and microglia (200× magnification) is shown. Values are expressed as means ± SEM for 6–8 animals per group. Data were subjected to one-way analysis of variance followed by the Student–Newman–Keuls test. *P < 0.05, **P < 0.01, ***P < 0.001 when comparing the control group with the 3NP and CBG group. ^# P < 0.05, ^## P < 0.01, ^### P < 0.001 when comparing the 3NP group with the 3NP + CBG group

Fig. 4

Gene expression of inflammatory markers including (A) cyclooxygenase (COX)-2, (B) tumor necrosis factor (TNF)-α, (C) interleukin (IL)-6, and (D) inducible nitric oxide synthase (iNOS) was significantly downregulated in 3-nitropropionic acid (3NP) + cannabigerol (CBG)-treated mice compared with 3NP mice. Values are expressed as means ± SEM for 6–8 animals per group. Data were subjected to one-way analysis of variance followed by the Student–Newman–Keuls test. *P < 0.05, **P < 0.01, ***P < 0.001 indicates significant changes between controls and 3NP mice. ^# P < 0.05, ^## P < 0.01 indicates significant changes between 3NP- and 3NP + CBG-treated mice

Fig. 5

Effect of cannabigerol (CBG) on antioxidant defenses in the striatum of 3-nitropropionic acid (3NP)-treated and control mice. Estimation of (A) catalase activity, (B) reduced glutathione (GSH) levels, and (C) superoxide dismutase (SOD) activity. Data presented are the percentage of the vehicle-treated control group and are expressed as means ± SEM for 6–8 animals per group. Data were subjected to one-way analysis of variance followed by the Student–Newman–Keuls test. *P < 0.05, **P < 0.01, ***P < 0.001 when comparing the control group with the 3NP and CBG group. ^# P < 0.05, ^## P < 0.01, ^### P < 0.001 when comparing the 3NP group with the 3NP + CBG group

Fig. 6

(A) Weight gain and (B) rotarod performance in R6/2 mice treated from the age of 4 weeks with cannabigerol (CBG) or vehicle (Tween 80-saline). Values are expressed as means ± SEM for 6–8 animals per group. Data were subjected to one-way analysis of variance followed by the Student–Newman–Keuls test (*P < 0.05, **P < 0.01, ***P < 0.005 compared with wild-type animals treated with vehicle)

Fig. 7

Gene expression for (A) cannabinoid receptor type 1 (CB₁) and (B) cannabinoid receptor type 2 (CB₂) receptors, and (C) fatty acid hydrolase (FAAH) and (D) monoacylglycerol lipase (MAGL) measured in the striatum of R6/2 mice (10 weeks after birth) treated from the age of 4 weeks with cannabigerol (CBG) or vehicle (Tween 80-saline). Values correspond to fold of change over wild-type animals and are expressed as means ± SEM for 6–8 animals per group. Data were subjected to one-way analysis of variance followed by the Student–Newman–Keuls test (*P < 0.05, **P < 0.01, ***P < 0.005 compared with wild-type animals treated with vehicle)

Fig. 8

Gene expression for (A) brain-derived neurotrophic factor (BDNF), (B) insulin-like growth factor (IGF)-1, (C) glutamate transporter (GLT)-1, and (D) glutamate aspartate transporter (GLAST) measured in the striatum of R6/2 mice (10 weeks after birth) treated from the age of 4 weeks with cannabigerol (CBG) or vehicle (Tween 80-saline). Values correspond to fold of change over wild-type animals and are expressed as means ± SEM for 6–8 animals per group. Data were subjected to one-way analysis of variance followed by the Student–Newman–Keuls test (*P < 0.05, **P < 0.01, ***P < 0.005 compared with wild-type animals treated with vehicle)

Fig. 9

Gene expression for (A) dopamine- and cyclic adenosine monophosphate-regulated phosphoprotein, Mr 32 kDA (DARPP-32), (C) peroxisome proliferator-activated receptor (PPAR)γ, and (D) tumour necrosis factor (TNF)-α, and (B) DARPP-32 immunostaining measured in the striatum of R6/2 mice (10 weeks after birth) treated from the age of 4 weeks with cannabigerol (CBG) or vehicle (Tween 80-saline). Values correspond to fold of change (gene expression) or percentage (immunostaining) over wild-type animals and are expressed as means ± SEM for 6–8 animals per group. Data were subjected to one-way analysis of variance followed by the Student–Newman–Keuls test (*P < 0.05, **P < 0.01, ***P < 0.005 compared with wild-type animals treated with vehicle)

Fig. 10

EM48 immunostaining (representative of mutant huntingtin aggregates) in the striatum of R6/2 mice (at 10 weeks after birth) treated from the age of 4 weeks with cannabigerol (CBG) or vehicle (Tween 80-saline). The stainings were repeated in 5–6 animals per group. Magnification = 40×. Data were subjected to Student’s t test (***P < 0.005 compared with R6/2 mice treated with vehicle)

Fig. 11

Huntingdon’s disease (HD) array analysis showing the up- or downregulatory responses of some genes specifically affected in the striatum of (A) R6/2 compared with wild-type mice or (B) R6/2 mice treated with CBG compared with wild-type mice. One microgram of RNA was retrotranscribed and the resulting cDNA was analyzed in a mouse HD polymerase chain reaction array. Five housekeeping genes contained on the experimental system were used to standardize the mRNA expression levels in every sample. Values correspond to number of folds that a specific gene is up- or downregulated. Data were assessed by the unpaired two-tailed Student’s t test. The 7 genes presented in (A) were selected because the up- and downregulatory responses found between R6/2 and wild-type mice were more than 2-fold higher, and these differences were statistically significant in all cases, and also because these differences were reversed by the treatment with cannabigerol (CBG) (*P < 0.05, **P < 0.01, ***P < 0.005)