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  1. Evaluation Summary:

    This study gives us information about the potential of the endocannabinoid system to become a novel target for the treatment of Williams-Beuren syndrome. The authors found there is an alteration of brain cannabinoid type-1 receptor (CB1R) in a mouse model of Williams-Beuren syndrome (CD mice). Modulation of CB1R by JZL184 treatment improved social and cognitive phenotypes and also cardiac function of CD mice. This study will be of great interest to researchers and clinicians in the field of genetic diseases.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)

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  2. Reviewer #1 (Public Review):

    William-Beuren syndrome (WBS) is a rare genetic disease characterized by manifestation affecting the central nervous system and cardiovascular system. This disease is caused by the hemizygous deletion of several adjacent genes within a specific region of chromosome 7 (7q11.23). Specific treatments for this syndrome are unavailable until now.

    Researchers in this study found an alteration in density and signaling of CB1R in several areas of CD mice brain compared to WT mice by [3H]CP55,940 radioligand binding assay and [35S]GTPγS autoradiography. This result suggested an important role of endocannabinoid signaling in the pathophysiology of WBS. Further experiments showed that administration of monoacylglycerol lipase inhibitor JZL184 ameliorated social behavioral phenotype and cardiac clinical and transcriptional phenotype in CD mice compared to WT mice. Overall, this manuscript provided us new information about a potential target for the treatment of WBS.

    This manuscript showed a comprehensive analysis of WBS in CD mice by several methods including CD mice behavioral phenotype and cardiac phenotype, endocannabinoid signaling alteration, and amelioration of CD mice phenotype after administration of JZL184. Especially for cardiac phenotype, they did cardiac RNA-seq which provides in-depth analysis.

    Change CD mice cardiac phenotype in this study seemed not representative for WBS in humans. CD mice only showed mild cardiac hypertrophy and slight reduction of cardiac ejection fraction, while in humans WBS is characterized by congenital cardiovascular phenotype such as pulmonary artery stenosis, supravalvular aortic stenosis, ventricular septal defects, etc.

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  3. Reviewer #2 (Public Review):

    The authors of this manuscript explored the ECS properties in a mouse model for WS. This is a novel research field in WS, with high interest and relevancy. The authors improved behavioral aspects by modulating the ECS, and demonstrate partial improvements in cardiovascular properties. The advances in our understanding of the ECS in WS is of high interest, and the authors indeed improve our understanding by this work. Given the fact that WS treatment nowadays is relatively limited, this manuscript has high potential to be groundbreaking.

    The use of the MAGL inhibitor, based on the ECS characterization and the alterations found in CD mice compared to control, is elegant and well-designed.

    Nevertheless, it is unclear how exactly the drug affect behavior and cardiovascular properties. Only partial evidence is demonstrated, and therefore the authors do not show enough results to fully present the full picture of this mouse model following drug administration.

    I believe that in its current formation, this manuscript has some major gaps. Some of these issues are:

    - In the abstract, the statement saying "Nowadays, there are no available treatments to ameliorate the main traits of WBS" is not accurate, as there are treatments and procedures to ameliorate the main traits of WBS. The authors indeed mention those in the introduction, but not in the abstract. Authors should moderate this statement, perhaps by changing to "no pharmacological treatment to directly ameliorate main traits of WBS".

    - In general, I have difficulties with the test mice age range, being 8-16 weeks of age. This is relatively a wide range, that can lead to the unclear behavioral and other phenotypes described in the manuscript. Many studies showed dynamic changes in the developmental trajectory of the endocannabinoid system in the mouse brain. It might be that the 8-16 weeks of age range is responsible for the different stages of the endocannabinoid system maturation. This is a major issue in this study in my opinion.

    - In the novel object test, why did the authors decided to exclude from the analysis mice that explored <10s both objects? How many mice were excluded based on this criteria? Raw data should be included to allow the reader better judgement of the raw data (with all excluded mice included) and the presented final graphs.
    - In the novel object test, Fig.1c,d make no sense to me: the CD mice should have higher (!) discrimination index than WT based on Fig.1c, right, while Fig.1d shows the opposite.

    - The behavioral characterization in this study is very limited. Why the authors did not characterize other aspects related to WS, such as anxiety and motor capabilities? Tests such as elevated zero maze, open field exploration, rotarod, etc. will better define mice properties and the effectiveness of the drug to modulate these. Especially given the fact that previous article from this group showed motor deficits in CD mice (Segura-Puimedon et al., 2014) , Figure 5a-b). Also contextual fear conditioning can add more insights on fear and cognition in these mice with and without the drug. Similarly, short (i.e. 1 hour) and long-term memory (i.e. 24 hour) should be studied in the NOR test, to better define the results in Fig.1c,d (see comment below).

    - Can the authors show that the i.p. administration of the drug resulted in increased drug concentration in the brain of treated mice control to placebo? The reported alterations following drug administration might be secondary to other drug-related responses.

    - From Fig.3c in this manuscript it seems that the drug has negative effects on NOR discrimination index in WT animals (together with the positive effects on CD mice). This should be discussed in the manuscript to emphasize the potential negative effects of the drug.

    - Why did the authors not characterize the signaling of the CB1R following drug treatment in Fig.3 as they did in Fig.2? This should be demonstrated, to further study the drug's effect on the ECS.

    - The effect of the drug on the cardiovascular phenotype is not clear. There is no mechanistic explanation for this change. RNAseq is only partial aspect of this surprising effect, and is not sufficient to understand and support the mechanistic explanation of how the drug affected cardiovascular properties in only a few days. Why should it affect heart properties, especially given such a short treatment of only several days? This should be further demonstrated and explained, with more histological and molecular and cellular evidence to support this surprising effect. Moreover, did these changes in weight etc. were specific to the heart only? The different number of mice in each of the tests presented in Fig.4 is unjustified and makes me feel uncomfortable. Why did the author have to use 20 mice in Fig.4b, but used 7 in Fig.4c? Can they show statistical power calculation to support the huge difference in mice number between the tests?

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  4. Reviewer #3 (Public Review):

    This paper describes an interesting research with many meaningful observations to study the etiology and therapeutics for WBS. In particular, the authors report evidence supporting that the endocannabinoid system is impaired in an animal model for WBS, the CD mice. Furthermore, a sub-chronic pharmacological treatment with JZL184 normalized all tested phenotypes of CD mice, that are relevant to human disorder, to the level similar to normal mice. These findings should provide deep insights into developing strategies for WBS therapeutics.

    1. The authors studied behavioral phenotypes of CD mice that are reminiscent of the problems that WBS individuals have. In addition to the CNS phenotype, cardiac hypertrophy was observed in the CD mice model, and the effect of JZL-184 for the cardiac phenotype was evaluated.
    2. Dysfunctional endocannabinoid system in the amygdala was observed by various molecular assessments including CB1 receptor density and its G protein coupling. Brain levels of endocannabinoids and related molecules were also measured.
    3. Results from transcriptomic profiling of heart gene expression supports the idea that subchronic JZL184 treatment may restore the normal cardiovascular function in CD mice. The correlation analysis of differentially expressed genes shown in Fig 5E is appropriate and nicely display the beneficial effects of JZL184 on abnormal cardiac gene expression in CD mice.

    1. It is interesting that the authors tested the effect of subchronic JZL, rather than CB1 inverse agonists, in order to reverse the abnormal elevation of the CB1 receptor in CD mice. The rationale by which they selected prolonged MAGL inhibition-induced CB1 desensitization over more straightforward method of CB1 inhibition is not discussed explicitly.
    2. Endocannabinoid levels were quantified from whole brain homogenate (Table 1), not from separated brain regions such as amygdala. Considering the complex structure of the brain and brain region-selective abnormality of CB1 receptors observed in CD mice (Fig 2a), not much conclusion can be made from current dataset regarding endocannabinoid molecules deficits in WBS.
    3. Although it is shown that subchronic JZL184 induces normalization of CB1 receptors density in basolateral amygdala, it is not clear whether this molecular recovery occurs also in other brain regions where increased CB1 availability had been observed in CD mice. Therefore, it is not clear if JZL184-induced normalization of CB1 receptors mediates a part or all beneficial effects on behaviors of CD mice.

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