Positive allosteric modulation of the type 1 cannabinoid receptor reduces the signs and symptoms of Huntington's disease in the R6/2 mouse model

Abstract

Huntington's disease (HD) is an inherited progressive neurodegenerative disease characterized by motor, cognitive, and behavioural changes. One of the earliest changes to occur in HD is a reduction in cannabinoid 1 receptor (CB₁) levels in the striatum, which is strongly correlated with HD pathogenesis. CB₁ positive allosteric modulators (PAM) enhance receptor affinity for, and efficacy of activation by, orthosteric ligands, including the endocannabinoids anandamide and 2-arachidonoylglycerol. The goal of this study was to determine whether the recently characterized CB₁ allosteric modulators GAT211 (racemic), GAT228 (R-enantiomer), and GAT229 (S-enantiomer), affected the signs and symptoms of HD. GAT211, GAT228, and GAT229 were evaluated in normal and HD cell models, and in a transgenic mouse model of HD (7-week-old male R6/2 mice, 10 mg/kg/d, 21 d, i.p.). GAT229 was a CB₁ PAM that improved cell viability in HD cells and improved motor coordination, delayed symptom onset, and normalized gene expression in R6/2 HD mice. GAT228 was an allosteric agonist that did not enhance endocannabinoid signaling or change symptom progression in R6/2 mice. GAT211 displayed intermediate effects between its enantiomers. The compounds used here are not drugs, but probe compounds used to determine the potential utility of CB₁ PAMs in HD. Changes in gene expression, and not protein, were quantified in R6/2 HD mice because HD pathogenesis is associated with dysregulation of mRNA levels. The data presented here provide the first proof of principle for the use of CB₁ PAMs to treat the signs and symptoms of HD.

Keywords: Allosteric modulator; Cannabinoid; G protein-coupled receptor; Huntington's disease; Neurodegeneration; Type 1 cannabinoid receptor.

Figure 1.. Characterization of GAT211-, GAT228-, and GAT229-dependent effects on ERK1/2 phosphorylation and arrestin2 recruitment in STHdh cells.

A-H) STHdh^Q7/Q7 and STHdh^Q111/Q111 cells were treated with 1 nM – 10 μM 2-AG, AEA, CP55,940, GAT211, GAT228, or GAT229 ± 1 μM GAT211, GAT228, or GAT229 for 10 min and ERK1/2 phosphorylation (Y185/204) compared to total ERK1/2 levels was determined via In-cell™ western. I-P) STHdh^Q7/Q7 and STHdh^Q111/Q111 cells were transfected with CB₁-GFP² and arrestin2-Rluc and treated with 1 nM – 10 μM 2-AG, AEA, CP55,940, GAT211, GAT228, or GAT229 ± 1 μM GAT211, GAT228, or GAT229 for 30 min and BRET_Eff was determined. CRCs were fit using non-linear regression analysis with variable slope (4 parameter) using Prism (GraphPad v. 5.0). N = 6.

Figure 2.. GAT211 and GAT229 enhanced the pro-survival effect of AEA in STHdh^Q111/Q111 cells.

STHdh^Q7/Q7 and STHdh^Q111/Q111 cells were treated with vehicle (10% DMSO), 100 nM AEA, 1 μM GAT211, GAT228, or GAT229, or 100 nM AEA + 1 μM GAT211, GAT228, or GAT229 for 18 h and A) cellular esterase activity was quantified as a measure of cellular viability, or B) membrane permeability to the fluorescent dye EthD-1 was quantified as a measure of cell death, using the Live/Dead Cytotoxicity assay. *P < 0.01 compared to STHdh^Q7/Q7 cells within treatment, ^P < 0.01 compared to vehicle treatment within cell type, †P < 0.01 to AEA treatment within cell type, as determined via two-way ANOVA followed by Bonferroni’s post-hoc analysis. N = 6.

Figure 3.. GAT211, GAT228, and GAT229 normalized locomotor activity and delayed behavioural changes in R6/2 mice.

Wild-type (C57BL/6J) and R6/2 mice were treated with vehicle or 10 mg/kg/d i.p. GAT211, GAT228, or GAT229 for 21 d and measurements of total distance travelled (m), number of vertical movements, and time spent immobile (sec) in open field were made. A–C) Total distance travelled (m) in the open field test over 5 min during the 21 d treatment period. D) Total area under the curve (AUC) for total distance travelled (m) in the open field test for the duration of the treatment period. E–G) Number of vertical movements in the open field test over 5 min during the 21 d treatment period. H) Total AUC for the number of vertical movements in the open field test for the duration of the treatment period. I–K) Time spent immobile (sec) in the open field test over 5 min during the 21 d treatment period. L) Total AUC for immobility time in the open field test for the duration of the treatment period. M–O) Behavioural change (Supplementary Table S1) during the 21 d treatment period. P) Total AUC for behavioural change for the duration of the treatment period. Statistical analyses: A–C,E–G,I–K,M–O) *P < 0.01 R6/2 -vehicle versus R6/2 – GAT compound within day. D,H,I,P) ***P < 0.001 wild-type versus R6/2 within treatment; ^P < 0.05, ^^P < 0.01, ^^^P < 0.001 compared to vehicle treatment within genotype. Statistical differences determined via two-way ANOVA followed by Bonferroni’s post-hoc analysis. N = 5 per group.

Figure 4.. GAT211 and GAT229 improved weight gain in R6/2 mice.

A–D) Wild-type (C57BL/6J) and R6/2 mice were treated with vehicle or 10 mg/kg/d i.p. GAT211 (A), GAT228 (B), or GAT229 (C) for 21 d and weight was measured 24 h after GAT treatment every day. D) Summary of total change in weight over 21 d duration of the experiment. *P < 0.01 R6/2 treated with GAT compound versus R6/2 treated with vehicle within day as determined via two-way ANOVA followed by Bonferroni’s post-hoc analysis. N = 5 per group. E,F) DEXA scans of mice were conducted post-mortem to determine the % fat (E) tissue and % lean (F) tissue following the 3 week experiment. *P < 0.01 compared to wild-type within treatment, ^P < 0.01 compared to vehicle within genotype as determined via two-way ANOVA followed by Bonferroni’s post-hoc analysis. N = 5 per group.

Figure 5.. GAT229 normalized gene expression in R6/2 mice.

RNA was collected from the striatum, cortex, visceral adipose tissue, and whole blood of wild-type (C57BL/6J) and R6/2 mice treated with vehicle or 10 mg/kg/d i.p. GAT211, GAT228, or GAT229 for 21 d and converted to cDNA for qRT-PCR measurement of CB1 (A–D), CB2 (E–H), PGC1α (I–K), leptin (L), and BDNF-2 (M,N) relative to β-actin. *P < 0.01 compared to wild-type within treatment, ^P < 0.01 compared to vehicle within genotype as determined via two-way ANOVA followed by Bonferroni’s post-hoc analysis. N = 5 per group.