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. 2022 Oct 11:11:e72560.
doi: 10.7554/eLife.72560.

Cannabinoid signaling modulation through JZL184 restores key phenotypes of a mouse model for Williams-Beuren syndrome

Alba Navarro-Romero #  1 , Lorena Galera-López #  1 , Paula Ortiz-Romero  2 , Alberto Llorente-Ovejero  3 , Lucía de Los Reyes-Ramírez  1 , Iker Bengoetxea de Tena  3 , Anna Garcia-Elias  1 , Aleksandra Mas-Stachurska  4 , Marina Reixachs-Solé  5   6 , Antoni Pastor  7 , Rafael de la Torre  7 , Rafael Maldonado  1   7 , Begoña Benito  8   9   10 , Eduardo Eyras  5   6   7 , Rafael Rodríguez-Puertas  3   11 , Victoria Campuzano  2 , Andres Ozaita  1
Affiliations

Cannabinoid signaling modulation through JZL184 restores key phenotypes of a mouse model for Williams-Beuren syndrome

Alba Navarro-Romero et al. Elife. .

Abstract

Williams-Beuren syndrome (WBS) is a rare genetic multisystemic disorder characterized by mild-to-moderate intellectual disability and hypersocial phenotype, while the most life-threatening features are cardiovascular abnormalities. Nowadays, there are no pharmacological treatments to directly ameliorate the main traits of WBS. The endocannabinoid system (ECS), given its relevance for both cognitive and cardiovascular function, could be a potential druggable target in this syndrome. We analyzed the components of the ECS in the complete deletion (CD) mouse model of WBS and assessed the impact of its pharmacological modulation in key phenotypes relevant for WBS. CD mice showed the characteristic hypersociable phenotype with no preference for social novelty and poor short-term object-recognition performance. Brain cannabinoid type-1 receptor (CB1R) in CD male mice showed alterations in density and coupling with no detectable change in main endocannabinoids. Endocannabinoid signaling modulation with subchronic (10 days) JZL184, a selective inhibitor of monoacylglycerol lipase, specifically normalized the social and cognitive phenotype of CD mice. Notably, JZL184 treatment improved cardiovascular function and restored gene expression patterns in cardiac tissue. These results reveal the modulation of the ECS as a promising novel therapeutic approach to improve key phenotypic alterations in WBS.

Keywords: Williams–Beuren syndrome; cannabinoid type-1 receptor; endocannabinoid system; intellectual disability; medicine; mouse.

Plain language summary

Williams-Beuren syndrome (WBS) is a rare disorder that causes hyper-social behavior, intellectual disability, memory problems, and life-threatening overgrowth of the heart. Behavioral therapies can help improve the cognitive and social aspects of the syndrome and surgery is sometimes used to treat the effects on the heart, although often with limited success. However, there are currently no medications available to treat WBS. The endocannabinoid system – which consists of cannabis-like chemical messengers that bind to specific cannabinoid receptor proteins – has been shown to influence cognitive and social behaviors, as well as certain functions of the heart. This has led scientists to suspect that the endocannabinoid system may play a role in WBS, and drugs modifying this network of chemical messengers could help treat the rare condition. To investigate, Navarro-Romero, Galera-López et al. studied mice which had the same genetic deletion found in patients with WBS. Similar to humans, the male mice displayed hyper-social behaviors, had memory deficits and enlarged hearts. Navarro-Romero, Galera-López et al. found that these mutant mice also had differences in the function of the receptor protein cannabinoid type-1 (CB1). The genetically modified mice were then treated with an experimental drug called JZL184 that blocks the breakdown of endocannabinoids which bind to the CB1 receptor. This normalized the number and function of receptors in the brains of the WBS mice, and reduced their social and memory symptoms. The treatment also restored the animals’ heart cells to a more normal size, improved the function of their heart tissue, and led to lower blood pressure. Further experiments revealed that the drug caused the mutant mice to activate many genes in their heart muscle cells to the same level as normal, healthy mice. These findings suggest that JZL184 or other drugs targeting the endocannabinoid system may help ease the symptoms associated with WBS. More studies are needed to test the drug’s effectiveness in humans with this syndrome. Furthermore, the dramatic effect JZL184 has on the heart suggests that it might also help treat high blood pressure or conditions that cause the overgrowth of heart cells.

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Conflict of interest statement

AN, LG, PO, AL, Ld, IB, AG, AM, MR, AP, Rd, RM, BB, RR, VC, AO No competing interests declared, EE Reviewing editor, eLife

Figures

Figure 1.
Figure 1.. Complete deletion (CD) mice show an hypersociable phenotype, no preference for social novelty and cognitive alterations.
(a) Schematic cartoon of the sociability and preference for social novelty procedure. (b) Time spent exploring either empty compartments (E) or stranger mice (S) during the three phases of the Vsocial-maze (WT, n = 11; CD, n = 11–12). (c) Time spent exploring either empty compartments (E) or objects (O) (WT, n = 12; CD, n = 8). Statistical significance was calculated by repeated measures analysis of variance (ANOVA) comparison. $p < 0.05; $$p < 0.01; $$$p < 0.001 (compartment effect); **p < 0.01; ***p < 0.001 (genotype effect). (d) Discrimination index of WT and CD mice (WT, n = 7; CD, n = 6). Statistical significance was calculated by Student’s t-test. *p < 0.05 (genotype effect). Data are expressed as mean ± standard error of the mean (SEM).
Figure 1—figure supplement 1.
Figure 1—figure supplement 1.. Schematic cartoon of behavioral test.
Schematic cartoon of the (a) modification of the sociability and preference for social novelty procedure using unfamiliar objects instead of unfamiliar mice and (b) and short-term object recognition memory procedure.
Figure 1—figure supplement 2.
Figure 1—figure supplement 2.. Long-term nonemotional, emotional, and spatial memory in complete deletion (CD) mice.
(a) Discrimination index in long-term novel object recognition test (WT, n=6; CD, n=6). Statistical significance was calculated by Student’s t-test. (b) Freezing (%) behaviour measured in long-term context fear conditioning paradigm (WT, n=6; CD, n=5). Statistical significance was calculated by Student’s t-test. (c) Primary latency (s) measured during Barnes maze training. Statistical significance was calculated by Newman-Keuls post hoc test following Repeated measures two-way ANOVA ** p< 0.01, *** p< 0.001 (compared to WT Day 1); # p<0.05, ## p<0.01 (compared to CD Day 1). (d) Time spend (%) in each quadrant during Barnes maze test trial (WT, n=14; CD, n=10). Statistical significance was calculated by Newman-Keuls post hoc test following Repeated measures two-way ANOVA *** p< 0.001 (compared to WT Target quadrant); ### p<0.001 (compared to CD Target quadrant). Data are expressed as mean ± S.E.M.
Figure 2.
Figure 2.. Complete deletion (CD) mice show alterations in cannabinoid type-1 receptor (CB1R) density and coupling to Gi/o proteins.
(a) [3H]CP55,940 binding of brain regions with significant changes in CD mice in comparison to WT littermates (WT, n = 11; CD, n = 10). (b) Representative images of [3H]CP55,940-binding autoradiography. (c) Brain regions showing significant changes in [35S]GTPγS binding evoked by WIN55,212–2 (10 µM) in CD mice in comparison to WT littermates (WT, n = 10–11; CD, n = 8–10), expressed as percentage of stimulation over the basal binding. (d) Representative images of WIN55,212–2-evoked [35S]GTPγS binding. [14C]-microscales used as standards in Ci/g t.e. Scale bar = 5 mm. Statistical significance was calculated by Student’s t-test. *p < 0.05; **p < 0.01; (genotype effect). Data are expressed as mean ± standard error of the mean (SEM).
Figure 2—figure supplement 1.
Figure 2—figure supplement 1.. Complete deletion (CD) mice alterations in cannabinoid receptor density and activity are specific for cannabinoid type-1 receptor (CB1R).
(a) Representative image of [3H]CP55,940 radioligand binding autoradiography. [3H]CP55,940 radioligand binding in brain slices was blocked with rimonabant but not with the CB2R antagonist SR144528. (b) Representative images of WIN55,212-2-evoked [35S]GTPγS binding. The increase in WIN55,212-2-stimulated [35S]GTPγS binding in brain slices was blocked in the presence of the CB1R antagonist rimonabant, but not with SR144528.
Figure 3.
Figure 3.. JZL184 treatment normalizes behavioral traits of complete deletion (CD) mice.
(a) Time spent exploring either empty compartments (E) or stranger mice (S) in the Vsocial-maze after 10 days of treatment with vehicle (VEH) or JZL184 (8 mg/kg) (WT VEH, n = 11; WT JZL184, n = 8–9; CD VEH, n = 9–10; CD JZL184, n = 11). Statistical significance was calculated by repeated measures analysis of variance (ANOVA) comparison. $p < 0.05; $$p < 0.01; $$$p < 0.001 (compartment effect); *p < 0.05; ***p < 0.001 (genotype effect); #p < 0.05; ###p < 0.001 (treatment effect). (b) Discrimination index of WT and CD mice treated for 7 days with vehicle (VEH) or JZL184 (8 mg/kg) (WT VEH, n = 11; WT JZL184, n = 12; CD VEH, n = 12; CD JZL184, n = 13). Statistical significance was calculated by Newman–Keuls post hoc test following two-way ANOVA. ***p < 0.001 (genotype effect); ###p < 0.001 (treatment effect). (c) Percentage of time spend in open arms and total entries in the elevated plus maze of WT and CD mice treated for 10 days with vehicle (VEH) or JZL184 (8 mg/kg) (WT VEH, n = 9; WT JZL184, n = 9; CD VEH, n = 6; CD JZL184, n = 5). Statistical significance was calculated by Newman–Keuls post hoc test following two-way ANOVA. *p < 0.05 (genotype effect); #p < 0.05 (treatment effect). Data are expressed as mean ± standard error of the mean (SEM).
Figure 3—figure supplement 1.
Figure 3—figure supplement 1.. Acute administration of JZL184.
Acute administration of JZL184 does not normalise hypersociable phenotype in CD mice. (a) Time spent exploring both empty compartments (E). (b) Time spent exploring either empty compartments (E) or stranger mice (S) after one single of treatment of vehicle (VEH) or JZL184 (8 mg/kg) (WT VEH, n=7-8; WT JZL184, n=7-8; CD VEH, n=5-6; CD JZL184, n=6). Statistical significance was calculated by repeated measures ANOVA comparison. $$$ p<0.001 (compartment effect); ** p<0.01 (genotype effect). Data are expressed as mean ± S.E.M.
Figure 3—figure supplement 2.
Figure 3—figure supplement 2.. Locomotor activity after JZL184 treatment.
JZL184 treatment does not modify locomotor activity in WT and CD mice. Horizontal movements performed in locomotor activity boxes for 30 minutes by mice treated with vehicle (VEH) or JZL184 (8 mg/kg) (WT VEH, n=8; WT JZL184, n=8; CD VEH, n=7; CD JZL184, n=8). Statistical significance was calculated by two-way ANOVA. Data are expressed as mean ± S.E.M.
Figure 4.
Figure 4.. JZL184 treatment restores altered cannabinoid type-1 receptor (CB1R) density and coupling to Gi/o proteins in complete deletion (CD) mice.
(a) Quantification and (b) representative images of CB1R immunodetection in the basolateral amygdala of WT and CD mice after 10 days of treatment with vehicle (VEH) or JZL184 (8 mg/kg) (WT VEH, n = 6; WT JZL184, n = 4; CD VEH, n = 5; CD JZL184, n = 4). Scale bar = 100 µm. Statistical significance was calculated by Newman–Keuls post hoc test following two-way analysis of variance (ANOVA). *p < 0.05 (genotype effect); #p < 0.05, ###p < 0.001 (treatment effect). (c) [35S]GTPγS binding evoked by WIN55,212–2 (10 µM) after 10 days of treatment with vehicle (VEH) or JZL184 (8 mg/kg) (WT VEH, n = 5–8; WT JZL184, n = 6–9; CD VEH, n = 5–6; CD JZL184, n = 5) expressed as percentage of stimulation over the basal binding. Statistical significance was calculated by Newman–Keuls post hoc test following two-way ANOVA. **p < 0.01, ***p < 0.001 (genotype effect); #p < 0.05, ##p < 0.01, ###p < 0.001 (treatment effect). (d) Representative images of WIN55,212–2-evoked [35S]GTPγS binding. [14C]-microscales used as standards in Ci/g t.e. Scale bar = 5 mm. Data are expressed as mean ± standard error of the mean (SEM).
Figure 4—figure supplement 1.
Figure 4—figure supplement 1.. JZL184 treatment downregulates cannabinoid type-1 receptor (CB1R) protein levels in the amygdala of complete deletion (CD) mice (CD VEH, n=7; CD JZL184, n=7).
Statistical significance was calculated by Student’s t-test. # p<0.05 (treatment effect). Data are expressed as mean ± S.E.M.
Figure 4—figure supplement 2.
Figure 4—figure supplement 2.. Quantification of total number of cells and neurons in basolateral amygdala.
The number of cells and neurons is not altered neither in CD mice or JZL184 treatment condition in Basolateral amygdala. (a) Density of NeuN+ and (b) Dapi + cells in basolateral amygdala measured by immunofluorescence. (WT VEH, n=6; WT JZL184, n=4; CD VEH, n=5; CD JZL184, n=4). Statistical significance was calculated by Newman-Keuls post hoc test following two-way ANOVA. Data are expressed as mean ± S.E.M.
Figure 5.
Figure 5.. JZL184 administration regresses cardiac hypertrophy and the expression of cardiac Cnr1 alterations of complete deletion (CD) mice.
(a) Heart/body weight ratios obtained from WT and CD mice treated for 10 days with vehicle (VEH) or JZL184 (8 mg/kg) (WT VEH, n = 14; WT JZL184, n = 12; CD VEH, n = 11; CD JZL184, n = 12). (b) Cardiomyocyte cross-sectional area measured from WT and CD mice after treatment (WT VEH, n = 4; WT JZL184, n = 4; CD VEH, n = 5; CD JZL184, n = 5) and (c) representative images, scale bar = 5 µm. (d) Ejection fraction (%) assessed by echocardiography from measurements performed on bidimensional images (WT VEH, n = 7; WT JZL184, n = 6; CD VEH, n = 7; CD JZL184, n = 7). (e) Systolic blood pressure (mmHg) obtained after 10 days treatment (WT VEH, n = 6; WT JZL184, n = 7; CD VEH, n = 5; CD JZL184, n = 7) (f) Cardiac mRNA levels of Cnr1 gene obtained by qPCR expressed in fold-change after the 10th day administration (WT VEH, n = 9; WT JZL184, n = 8; CD VEH, n = 6; CD JZL184, n = 5). Statistical significance was calculated by Newman–Keuls post hoc test following two-way analysis of variance (ANOVA). *p < 0.05; **p < 0.01; ***p < 0.001 (genotype effect); #p < 0.05; ##p < 0.01; ###p < 0.001 (treatment effect). Data are expressed as mean ± standard error of the mean (SEM).
Figure 5—figure supplement 1.
Figure 5—figure supplement 1.. Additional heart morphology data.
Additional heart morphology data after JZL184 treatment in CD mice. Representative haematoxylin-eosin stained transverse cardiac sections and quantification of muscle proportion from WT and CD mice treated for 10 days with vehicle (VEH) or JZL184 (8 mg/kg) (WT VEH, n=6; WT JZL184, n=4; CD VEH, n=5; CD JZL184, n=5). Statistical significance was calculated by Newman-Keuls post hoc test following two-way ANOVA. Data are expressed as mean ± S.E.M.
Figure 5—figure supplement 2.
Figure 5—figure supplement 2.. Brain weight measurements.
Brain weight (a) and proportion of brain to body weight (b) of WT and CD mice after sub-chronic treatment with vehicle or JZL184. (WT VEH, n=8; WT JZL184, n=9; CD VEH, n=5; CD JZL184, n=4). Statistical significance was calculated by two-way ANOVA. *** p<0.001 (effect of genotype). Data are expressed as mean ± S.E.M.
Figure 6.
Figure 6.. JZL184 treatment reversed alterations of the cardiac transcriptome observed in complete deletion (CD) mice.
(a) Volcano plot of differentially expressed genes (p < 0.05 and |log2FC| > 0) between CD and WT mice. Red indicates relative increased expression and blue indicates relative decreased expression. (b) Gene ontology enrichment analysis for both up- and downregulated genes in CD mice compared with WT. Most significant biological processes terms are represented for each group. (c) Heatmap showing the relative mRNA expression level of genes that reverted their expression in CD mice treated for 10 days with JZL184 (8 mg/kg) and CD or WT littermates treated with vehicle. (d) Venn diagrams and gene ontology enrichment analysis of genes that showed opposite differential expression in CD mice after 10 days treatment with JZL184 (8 mg/kg) compared with CD mice treated with vehicle. (e) Correlation plot of differentially expressed genes in CD mice treated for 10 days with JZL184 (8 mg/kg) and CD or WT littermates treated with vehicle. In green, genes with opposite differential expression between conditions, in orange reverted genes associated with cardiovascular function, in gray genes with no change (WT VEH, n = 2; CD VEH, n = 3; CD JZL184, n = 3).
Figure 6—figure supplement 1.
Figure 6—figure supplement 1.. Principal component analysis (PCA).
Principal component analysis (PCA) of sample-to-sample variation in gene expression (WT VEH, n=2; WT JZL184, n=3; CD VEH, n=3; CD JZL184, n=3).
Author response image 1.
Author response image 1.. Age distribution of mice used for the characterization of the endocannabinoid system.
See this image and copyright information in PMC

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