1. Medicine

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

  1. Alba Navarro-Romero
  2. Lorena Galera-López
  3. Paula Ortiz-Romero
  4. Alberto Llorente-Ovejero
  5. Lucía de los Reyes-Ramírez
  6. Iker Bengoetxea de Tena
  7. Anna Garcia-Elias
  8. Aleksandra Mas-Stachurska
  9. Marina Reixachs-Solé
  10. Antoni Pastor
  11. Rafael de la Torre
  12. Rafael Maldonado
  13. Begoña Benito
  14. Eduardo Eyras
  15. Rafael Rodríguez-Puertas
  16. Victoria Campuzano
  17. Andres Ozaita  Is a corresponding author
  1. Laboratory of Neuropharmacology, Department of Medicine and Life Sciences, Universitat Pompeu Fabra, Spain
  2. Department of Biomedical Sciences, School of Medicine and Health Sciences, University of Barcelona, and centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Spain
  3. Department of Pharmacology, Faculty of Medicine and Nursing, University of the Basque Country, Spain
  4. Hospital del Mar Medical Research Institute (IMIM), Autonomous University of Barcelona, Spain
  5. EMBL Australia Partner Laboratory Network at the Australian National University, Australia
  6. The John Curtin School of Medical Research, Australian National University, Australia
  7. Hospital del Mar Medical Research Institute (IMIM), Spain
  8. Group of Cardiovascular Experimental and Translational Research (GET-CV), Vascular Biology and Metabolism, Vall d’Hebron Research Institute (VHIR),, Spain
  9. Department of Medicine, Universitat Autònoma de Barcelona, Spain
  10. Centro de Investigación Biomédica en Red de Enfermedades Cardiovasculares (CIBER-CV), Instituto de Salud Carlos III, Spain
  11. Neurodegenerative Diseases, Biocruces Bizkaia Health Research Institute, Spain
https://doi.org/10.7554/eLife.72560
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Cite this article
  1. Alba Navarro-Romero
  2. Lorena Galera-López
  3. Paula Ortiz-Romero
  4. Alberto Llorente-Ovejero
  5. Lucía de los Reyes-Ramírez
  6. Iker Bengoetxea de Tena
  7. Anna Garcia-Elias
  8. Aleksandra Mas-Stachurska
  9. Marina Reixachs-Solé
  10. Antoni Pastor
  11. Rafael de la Torre
  12. Rafael Maldonado
  13. Begoña Benito
  14. Eduardo Eyras
  15. Rafael Rodríguez-Puertas
  16. Victoria Campuzano
  17. Andres Ozaita
(2022)
Cannabinoid signaling modulation through JZL184 restores key phenotypes of a mouse model for Williams–Beuren syndrome
eLife 11:e72560.
https://doi.org/10.7554/eLife.72560
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7 figures, 3 tables and 5 additional files

Figures

Figure 1 with 2 supplements
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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—source data 1

Complete deletion (CD) mice social and cognitive alterations.

https://cdn.elifesciences.org/articles/72560/elife-72560-fig1-data1-v1.zip
Figure 1—figure supplement 1
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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
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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 1—figure supplement 2—source data 1

Long-term nonemotional, emotional, and spatial memory in complete deletion (CD) mice.

https://cdn.elifesciences.org/articles/72560/elife-72560-fig1-figsupp2-data1-v1.zip
Figure 2 with 1 supplement
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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—source data 1

Cannabinoid type-1 receptor (CB1R) density and coupling to Gi/o proteins in complete deletion (CD) mice.

https://cdn.elifesciences.org/articles/72560/elife-72560-fig2-data1-v1.zip
Figure 2—figure supplement 1
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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 with 2 supplements
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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—source data 1

JZL184 treatment normalizes behavioral traits of complete deletion (CD) mice.

https://cdn.elifesciences.org/articles/72560/elife-72560-fig3-data1-v1.zip
Figure 3—figure supplement 1
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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 1—source data 1

Acute administration of JZL184.

https://cdn.elifesciences.org/articles/72560/elife-72560-fig3-figsupp1-data1-v1.zip
Figure 3—figure supplement 2
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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 3—figure supplement 2—source data 1

Locomotor activity after JZL184 treatment.

https://cdn.elifesciences.org/articles/72560/elife-72560-fig3-figsupp2-data1-v1.zip
Figure 4 with 2 supplements
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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—source data 1

JZL184 treatment restores altered cannabinoid type-1 receptor (CB1R) density and coupling.

https://cdn.elifesciences.org/articles/72560/elife-72560-fig4-data1-v1.zip
Figure 4—figure supplement 1
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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 1—source data 1

JZL184 treatment downregulates cannabinoid type-1 receptor (CB1R) protein levels in the amygdala of complete deletion (CD) mice.

https://cdn.elifesciences.org/articles/72560/elife-72560-fig4-figsupp1-data1-v1.zip
Figure 4—figure supplement 1—source data 2

Original blots of cannabinoid type-1 receptor (CB1R) in the amygdala of complete deletion (CD) mice.

https://cdn.elifesciences.org/articles/72560/elife-72560-fig4-figsupp1-data2-v1.pdf
Figure 4—figure supplement 2
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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 4—figure supplement 2—source data 1

Quantification of total number of cells and neurons in basolateral amygdala.

https://cdn.elifesciences.org/articles/72560/elife-72560-fig4-figsupp2-data1-v1.zip
Figure 5 with 2 supplements
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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—source data 1

JZL184 treatment has an impact on the cardiovascular phenotype of complete deletion (CD) mice.

https://cdn.elifesciences.org/articles/72560/elife-72560-fig5-data1-v1.zip
Figure 5—figure supplement 1
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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 1—source data 1

Additional heart morphology data.

https://cdn.elifesciences.org/articles/72560/elife-72560-fig5-figsupp1-data1-v1.zip
Figure 5—figure supplement 2
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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 5—figure supplement 2—source data 1

Brain weight measurements.

https://cdn.elifesciences.org/articles/72560/elife-72560-fig5-figsupp2-data1-v1.zip
Figure 6 with 1 supplement
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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—source data 1

JZL184 treatment reverses cardiac transcriptional deficits of complete deletion (CD) mice.

https://cdn.elifesciences.org/articles/72560/elife-72560-fig6-data1-v1.zip
Figure 6—figure supplement 1
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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).

Figure 6—figure supplement 1—source data 1

Principal component analysis (PCA).

https://cdn.elifesciences.org/articles/72560/elife-72560-fig6-figsupp1-data1-v1.zip
Author response image 1
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Age distribution of mice used for the characterization of the endocannabinoid system.

Tables

Table 1
Levels of endocannabinoids and related compounds in whole brain homogenates of complete deletion (CD) and WT.

(WT, n = 10; CD, n = 11). Statistical significance was calculated by Student’s t-test. Data are expressed as mean ± standard error of the mean (SEM).

Whole brain
WTCD
2-AG (nmol/g)4.96 ± 0.085.51 ± 0.29
2-LG (nmol/g)0.42 ± 0.030.46 ± 0.03
2-OG (nmol/g)0.96 ± 0.040.96 ± 0.05
AEA (pmol/g)5.57 ± 0.195.65 ± 0.32
DEA (pmol/g)1.98 ± 0.071.93 ± 0.06
DHEA (pmol/g)12.38 ± 0.3511.63 ± 0.60
Table 2
Levels of the two major endocannabinoids (2-AG and AEA) in brain regions relevant for social and cognitive behavior (amygdala and hippocampus) after 10 days treatment with VEH or JZL184.

(WT VEH, n = 6; WT JZL184, n = 6; CD VEH, n = 7; CD JZL184, n = 7). Statistical significance was calculated by Newman–Keuls post hoc test following two-way analysis of variance (ANOVA). ###p < 0.001 (treatment effect). Data are expressed as mean ± standard error of the mean (SEM).

Amygdala
WT VEHWT JZL184CD VEHCD JZL184
2-AG
(nmol/g)		18.72 ± 4.60122.96 ± 21.99###16.99 ± 0.86128.33 ± 18.12###
AEA(pmol/g)6.89 ± 1.045.34 ± 0.536.64 ± 0.414.69 ± 0.41
Hippocampus
WT VEHWT JZL184CD VEHCD JZL184
2-AG
(nmol/g)		9.11 ± 1.4575.55 ± 14.95###9.01 ± 0.4686.46 ± 15.25###
AEA
(pmol/g)		8.30 ± 0.778.26 ± 0.578.80 ± 0.637.85 ± 0.87
Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Strain, strain background (Mus musculus, male)C57BL/6JCharles Rivers, FranceC57Bl/6JMale
Genetic reagent (Mus musculus)CDUniversity Pompeu Fabra(C57BL/6J background, male)
Chemical compound, drugJZL184Abcamab141592
Chemical compound, drugDimethyl sulfoxideScharlau ChemieSU01531000
Chemical compound, drugPolyethylene glycol 400AppliChem142436.1611
Chemical compound, drugTween-80Sigma-AldrichP1754
Chemical compound, drug0.9%, NaCl physiological salineLaboratorios ErnVitulia
Chemical compound, drugKetamidorRichter pharmaK1NAI027
Chemical compound, drugXylazine hydrochlorideSigma-AldrichX1251
Biological sample (Equus asinus)Normal donkey serumSigma-AldrichD9663-10ML3% in PBS with 0.3% Triton X-100
AntibodyAnti-CB1R
(rabbit polyclonal)		Immunogenes1:1000
immunofluorescence
AntibodyAnti-CB1R
(rabbit polyclonal)		Frontier scienceCB1-Rb-Af380-11:500
western blot
AntibodyAnti-NeuN
(mouse monoclonal)		Merck MilliporeMAB3771:1000
AntibodyAnti-rabbit IgG (H+L)-AlexaFluor-555
(donkey polyclonal)		Thermo Fisher ScientificA-315721:1000
AntibodyAnti-mouse IgG (H+L)-AlexaFluor-488
(goat polyclonal)		Jackson ImmunoResearch115-545-0031:1000
Commercial assay, kitImmobilon Forte Western HRP SubstrateMerck MilliporeWBLUF0500
Commercial assay, kitNucleospin RNA isolation kitMacherey-Nagel740955-250
Commercial assay, kitSuperScript III enzymeInvitrogen12574-026
Commercial assay, kitSybrGreen master mixThermo Fisher4309155
Sequence-based reagentPrimers Cnr1 (CB1R) (forward)Sigma-Aldrich5′-CCTGGGAAGTGTCATCTTTGT-3′
Sequence-based reagentPrimers Cnr1 (CB1R) (reverse)Sigma-Aldrich5′-GGTAACCCCACCCAGTTTGA-3′
Sequence-based reagentPrimers Gapdh (GAPDH) (forward)Sigma-Aldrich5′-TGTCGTGGAGTCTACTGGTGTCTT-3′
Sequence-based reagentPrimers Gapdh (GAPDH) (reverse)Sigma-Aldrich5′-TGGCTCCACCCTTCAAGTG-3′
Sequence-based reagentPrimers Hprt1 (HPRT) (forward)Sigma-Aldrich5′-AAGCTTGCTGGTGAAAAGGA-3′
Sequence-based reagentPrimers Hprt1 (HPRT) (reverse)Sigma-Aldrich5′-TTGCGCTCATCTTAGGCTTT-3′
Chemical compound, drugDAPI Fluoromount-G mounting mediaThermo Fisher Scientific00-4959-52
Software, algorithmGraphPad Prism 7GraphPad Software, IncRRID:SCR_002798
Software, algorithmSTATISTICA 6.0StatSoft, USARRID:SCR_014213
Software, algorithmSmart 3.0 videotracking softwarePanlabRRID:SCR_002852
Software, algorithmImageJNational Institutes of Health, Bethesda, Maryland, USARRID:SCR_003070
Software, algorithmThe Quantity One software v4.6.3Bio-RadRRID:SCR_014280
Software, algorithmSalmonPMID:28263959RRID:SCR_017036v0.7.2
Software, algorithmRhttps://www.R-project.org/RRID:SCR_001905v3.6.3
Software, algorithmtximportPMID:26925227RRID:SCR_016752v1.2.0
Software, algorithmDESeq2PMID:25516281RRID:SCR_015687v1.26.0
Software, algorithmclusterProfilerPMID:22455463RRID:SCR_016884v3.14.3
Chemical compound, drugSR141716ATocris158681-13-1
Chemical compound, drugSR144528Tocris192703-06-3
Chemical compound, drugCP55,940Tocris83002-04-4
Chemical compound, drugWIN55,212–2Sigma-Aldrich131543-23-2
Chemical compound, drugTris–HClSigma-Aldrich1185-53-1
Chemical compound, drugBSASigma-Aldrich9048-46-8
Chemical compound, drugHEPESSigma-Aldrich7365-45-9
Chemical compound, drugNaClSigma-Aldrich7647-14-5
Chemical compound, drugMgCl2Sigma-Aldrich7791-18-6
Chemical compound, drugEGTASigma-Aldrich13368-13-3
Chemical compound, drugGDPSigma-Aldrich43139-22-6
Chemical compound, drugDTTSigma-Aldrich3483-12-3
Chemical compound, drugGTPγSSigma-Aldrich10220647001
Chemical compound, drug[3H]CP55,940PerkinElmerNET1051250UC
Chemical compound, drug[35S]GTPγSPerkinElmerNEG030H250UC
Otherβ-Radiation sensitive filmSigma-AldrichF5263-50EASee autoradiography methodology

Additional files

Supplementary file 1

[3H]CP55,940 binding of all analyzed brain regions (WT, n = 11; complete deletion [CD], n = 10) in fmol/mg t.e. of cannabinoid type-1 receptor (CB1R).

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).

https://cdn.elifesciences.org/articles/72560/elife-72560-supp1-v1.docx
Supplementary file 2

[35S]GTPγS binding evoked by WIN55,212–2 (10 µM) of all analyzed brain regions (WT, n = 11; complete deletion [CD], n = 10), expressed as percentage of stimulation over the basal binding.

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).

https://cdn.elifesciences.org/articles/72560/elife-72560-supp2-v1.docx
Supplementary file 3

Additional echocardiogram measurements after JZL184 treatment in complete deletion (CD) mice.

Interventricular septum diastolic (IVSd), left ventricular posterior wall thickness diastolic (LVPWd), LV end-diastolic diameter (LVDd), and left ventricular mass diastolic (Lvmass) measurement relatives to body weight (BW) (WT VEH, n = 8; WT JZL184, n = 7; CD VEH, n = 7; CD JZL184, n = 7). Statistical significance was calculated by Newman–Keuls post hoc test following two-way analysis of variance (ANOVA) ***p < 0.001 (genotype effect). Data are expressed as mean ± standard error of the mean (SEM).

https://cdn.elifesciences.org/articles/72560/elife-72560-supp3-v1.docx
Supplementary file 4

Summary of gene expression reversion after JZL 184 administration in complete deletion (CD) mice of genes implicated in cardiac-related process.

Pathways were obtained from Reactome database and expression changes are expressed as log2 ratios of fold-changes between CD mice treated for 10 days with JZL184 (8 mg/kg) and CD or WT littermates treated with vehicle.

https://cdn.elifesciences.org/articles/72560/elife-72560-supp4-v1.docx
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  1. Alba Navarro-Romero
  2. Lorena Galera-López
  3. Paula Ortiz-Romero
  4. Alberto Llorente-Ovejero
  5. Lucía de los Reyes-Ramírez
  6. Iker Bengoetxea de Tena
  7. Anna Garcia-Elias
  8. Aleksandra Mas-Stachurska
  9. Marina Reixachs-Solé
  10. Antoni Pastor
  11. Rafael de la Torre
  12. Rafael Maldonado
  13. Begoña Benito
  14. Eduardo Eyras
  15. Rafael Rodríguez-Puertas
  16. Victoria Campuzano
  17. Andres Ozaita
(2022)
Cannabinoid signaling modulation through JZL184 restores key phenotypes of a mouse model for Williams–Beuren syndrome
eLife 11:e72560.
https://doi.org/10.7554/eLife.72560