The CB1 receptor interacts with cereblon and drives cereblon deficiency-associated memory shortfalls

Cereblon/CRBN is a substrate-recognition component of the Cullin4A-DDB1-Roc1 E3 ubiquitin ligase complex. Destabilizing mutations in the human CRBN gene cause a form of autosomal recessive non-syndromic intellectual disability (ARNSID) that is modelled by knocking-out the mouse Crbn gene. A reduction in excitatory neurotransmission has been proposed as an underlying mechanism of the disease. However, the precise factors eliciting this impairment remain mostly unknown. Here we report that CRBN molecules selectively located on glutamatergic neurons are necessary for proper memory function. Combining various in vivo approaches, we show that the cannabinoid CB1 receptor (CB1R), a key suppressor of synaptic transmission, is overactivated in CRBN deficiency-linked ARNSID mouse models, and that the memory deficits observed in these animals can be rescued by acute CB1R-selective pharmacological antagonism. Molecular studies demonstrated that CRBN interacts physically with CB1R and impairs the CB1R-Gi/o-cAMP-PKA pathway in a ubiquitin ligase-independent manner. Taken together, these findings unveil that CB1R overactivation is a driving mechanism of CRBN deficiency-linked ARNSID and anticipate that the antagonism of CB1R could constitute a new therapy for this orphan disease.

24th Aug 2023 1st Editorial Decision 24th Aug 2023 Dear Prof. Guzmán, Thank you for the submission of your manuscript to EMBO Molecular Medicine.We have now received feedback from the three reviewers who agreed to evaluate your manuscript.As you will see from the reports below, the referees acknowledge the interest of the study and are overall supporting publication of your work pending appropriate revisions.
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EMBO Molecular Medicine has a "scooping protection" policy, whereby similar findings that are published by others during review or revision are not a criterion for rejection.Should you decide to submit a revised version, I do ask that you get in touch after three months if you have not completed it, to update us on the status.This study demonstrates the involvement of overactive CB1R interacting with CBRN in Glut neurons and their role in memory function using several innovative new mouse lines to study ARNSID.The experimental design is thorough and uses validated pharmacological and genetic tools, as well as established biochemical and mouse behavior paradigms.The technical approaches are sound, the manuscript clearly written, and conclusions and interpretations based on convincing results.I believe that this study will have an important impact on the field of cannabinoid research and neuroscience in general, providing an important foundation for future studies, including novel therapeutic approaches for this Orphan disease.I command the authors for such a strong study that likely will become a landmark reference.Below are key concerns to address followed by suggestions.
Concerns to address •Line 228: here specify how you measure cAMP and if IBMX is added to measure cAMP accumulation.
•Line 233: "PKA inactivation, an effect that was also prevented by CRBN" this statement is unclear.Are the authors referring to "reduced PKA activation"?Please clarify, and here specify the assay that was employed to measure PKA and time point of measure.
•Line 234: Are the authors refereeing to "a response that was absent when expressing CRBN"?Please clarify.
•Line 245: "CB1R function in HEK-293T cells".Were these cells transiently transfected or stable cell line?Please clarify.•Line 247: I do not understand this conclusion and do not see any statistical differences in the results.Please explain / elaborate.•Line 293: Replace "blockade" by "antagonism" •Line 348: After "CRBN", add "that we report here on CB1R-cAMP signaling" •Line 353: Provide background and relevance for discussing AHA1.
•Line 803 Determination of PKA activity.Is this a copy past error as it's already described earlier.
•Figure 2A: Were all mice weighted exactly at P60, especially considering line 620: "Adult mice (2-4-month-old)"?If not, please add the range.Also, specify in the manuscript if both or only one sex was studied.
•Figures 5E and F: "60 s" out of how long total?Suggested experiments, minor comments, and edits.
•Figure 5B: The results show that only a small amount of CB1R is associated with CRBN.Could it be that the lower expression CB1R in Glut subpopulation are binding to CRBN and not the more abundant CB1R in GABA?It would be interesting to extend this result with a similar experiment on the CRBN mouse line and include this new result in the study.•Line 46: replace "the blockade of CB1R" by "CB1R antagonists".•Line 93: replace "constitutes" by "represents".
Referee #2 (Remarks for Author): In this paper, the authors examined the role of cereblon (CRBN) in neural function, using cell type-specific KO mice.They found at the molecular level that CRBN expressed in excitatory neurons was required for maintenance of normal neural function by inhibiting the cannabinoid receptor CB1 (CB1R) pathway.At the whole-animal level, mutant mice lacking CRBN in excitatory neurons showed memory deficit, which was recovered by the CB1R-specific antagonist.Although the elaborated analyses on the role for CRBN in neural functions at the molecular level and the whole-animal level should be highly appreciated, there is a large black box between the two levels.

Major comments:
(1) The authors should at least try to elucidate the mechanism at the synaptic level.For instance, they can analyze the phenotypes of the mutant mice using acute brain slices or cultured neurons.If the authors can show that the CB1R pathway is enhanced in mutant mice electrophysiologically, it would strengthen the conclusions of this paper considerably.Such data would attract many readers of EMBO Molecular Medicine.
(2) Fig. 2D: The result of the motor coordination (the first trial of the rotarod test) should also be shown (or described in the text).The result of motor learning would be meaningless if the motor coordination is impaired.
(3) Fig. 2F, 2I: The authors should analyze the data by using "two"-way ANOVA, because there are two groups (WT and KO) with two different conditions (O and M for Fig. 2F; N and F for Fig. 2I).Since there seem to be some other problems in statistical analyses throughout the paper, all the data should be reexamined by a person who specializes in statistics.(4) Fig. 1B, 1C: Why were the 4 groups (CRBN-WT, Glu-CRBN-KO, GABA-CRBN-KO and CRBN-KO) compared here?In the other experiments, the data of WT and KO mice were compared in each genotype (CRBN-KO, Glu-CRBN-KO and GABA-CRBN-KO).

Minor comments:
(5) Throughout the study, is there any statistical difference between the data of male and female mice?
Referee #3 (Comments on Novelty/Model System for Author): The authors have performed laborious in vitro and in vivo experiments, uncovering the unexplored role of CRBN for CB1Rmediated regulation of brain function.The conceptual novelty of these findings is significant and would be of interest for a broad audience in the field of developmental brain disorders.The manuscript is well-written; however, several weaknesses need be addressed to enhance the rigor and overall quality of the study.

Referee #3 (Remarks for Author):
In this manuscript, Costas-Insua et al. report on the role of CRBN, a genetic risk factor for intellectual disability, in maintaining memory function.Using multiple CRBN deletion mouse models, the authors have identified that CRBN, particularly expressed in glutamatergic neurons, regulates memory function.Mechanistically, the authors reveal that CRBN interacts with CB1R, impeding the CB1R-Gi/o-cAMP-PKA pathway in a manner independent of its ubiquitin ligase function.These mechanisms may underlie the cognitive impact of CRBN, as memory deficits resulting from genetic CRBN deletion were alleviated by the acute pharmacological blockade of CB1R.The authors have performed laborious in vitro and in vivo experiments, uncovering the unexplored role of CRBN for CB1Rmediated regulation of brain function.The conceptual novelty of these findings is significant and would be of interest for a broad audience in the field of developmental brain disorders.The manuscript is well-written; however, several weaknesses need be addressed to enhance the rigor and overall quality of the study.
A major weakness in the present study is the absence of physiological data that reveal that genetic deletion of CRBN accelerates CB1R-mediated suppression of glutamatergic neuronal function, which could be rescued by the CB1R inhibition.Without this data, it remains unclear whether CRBN-CB1R interaction is a critical regulator for glutamatergic neurons.The author should provide this evidence by performing electrophysiological assays, such as brain slice recording experiments.
The forced swim test was used to evaluate depression.Nevertheless, while the forced swim test is still used to characterizing the efficacy of antidepressants, the field recognizes that it is unclear whether the observed phenotypes are valid correlates of depressive symptoms.The authors should rephrase the result section accordingly.
To evaluate CB1R functionality in CRBN-deficient mice, the authors examined the impact of THC treatment on catalepsy.However, cognitive effects of THC have been extensively studied in the literature.Studying cognitive tasks is more appropriate for the current study.
Despite the inclusion of both male and female mice in the study, it is uncertian if any sex differences exist.While sex difference is not the primary scope of the current study, the authors should at least analyze some data separately for male and female mice and discuss potential sex effects.It is worth noting that certain studies have reported sex differences in the effects of THC.Perhaps clinical studies may also reveal sex differences in CRBN mutations genetically associated with intellectual disability?

POINT BY POINT RESPONSE TO THE REFEREES' REPORTS
Referee #1 (Comments on Novelty/Model System for Author): This is an excellent report and my comment are outlined below.
Referee #1 (Remarks for Author): This study demonstrates the involvement of overactive CB1R interacting with CBRN in Glut neurons and their role in memory function using several innovative new mouse lines to study ARNSID.The experimental design is thorough and uses validated pharmacological and genetic tools, as well as established biochemical and mouse behavior paradigms.The technical approaches are sound, the manuscript clearly written, and conclusions and interpretations based on convincing results.I believe that this study will have an important impact on the field of cannabinoid research and neuroscience in general, providing an important foundation for future studies, including novel therapeutic approaches for this Orphan disease.I command the authors for such a strong study that likely will become a landmark reference.Below are key concerns to address followed by suggestions.
We would like to thank the reviewer for his/her positive and constructive comments, which have helped to improve the quality of our study.

Concerns to address
•Line 228: here specify how you measure cAMP and if IBMX is added to measure cAMP accumulation.

Done.
•Line 233: "PKA inactivation, an effect that was also prevented by CRBN" this statement is unclear.Are the authors referring to "reduced PKA activation"?Please clarify, and here specify the assay that was employed to measure PKA and time point of measure.

Done (see also Materials and Methods, "Determination of PKA activity").
•Line 234: Are the authors refereeing to "a response that was absent when expressing CRBN"?Please clarify.

Done.
24th Jan 2024 1st Authors' Response to Reviewers •Line 247: I do not understand this conclusion and do not see any statistical differences in the results.Please explain / elaborate.
•Line 353: Provide background and relevance for discussing AHA1.

Done.
•Line 803 Determination of PKA activity.Is this a copy past error as it's already described earlier.

Done (removed from "Cell culture, transfection and signalling experiments"; sorry for the confusion).
•Figure 2A: Were all mice weighted exactly at P60, especially considering line 620: "Adult mice (2-4-month-old)"?If not, please add the range.Also, specify in the manuscript if both or only one sex was studied.S1).

Corrected in
•Figures 5E and F: "60 s" out of how long total?
The scale of the y-axis was cut at 60 s because this was the maximal duration of the catalepsy test.This has been clarified in the legend to Figs 5EF (now FG; see also Materials and Methods, "Cannabinoid administration", line 666).
Suggested experiments, minor comments, and edits.
•Figure 5B: The results show that only a small amount of CB1R is associated with CRBN.Could it be that the lower expression CB1R in Glut subpopulation are binding to CRBN and not the more abundant CB1R in GABA?It would be interesting to extend this result with a similar experiment on the CRBN mouse line and include this new result in the study.
We agree with the reviewer.This is indeed a very likely possibility.To test it, we used the PLA technique to allow a sensitive CB 1 R-CRBN direct-interaction/closeproximity assessment in brain slices in situ.These new PLA experiments showed abundant fluorescence-positive puncta in WT and GABA-CB 1 R-KO mice, but not in Glu-CB 1 R-KO and CB 1 R-KO animals (Fig 5C ; text, lines 278-281), hence further supporting the CB 1 R-CRBN association in hippocampal glutamatergic but not GABAergic neurons.
We have incorporated all the changes suggested by the reviewer.

Referee #2 (Remarks for Author):
In this paper, the authors examined the role of cereblon (CRBN) in neural function, using cell type-specific KO mice.They found at the molecular level that CRBN expressed in excitatory neurons was required for maintenance of normal neural function by inhibiting the cannabinoid receptor CB1 (CB1R) pathway.At the wholeanimal level, mutant mice lacking CRBN in excitatory neurons showed memory deficit, which was recovered by the CB1R-specific antagonist.Although the elaborated analyses on the role for CRBN in neural functions at the molecular level and the wholeanimal level should be highly appreciated, there is a large black box between the two levels.
We would like to thank the reviewer for his/her positive and constructive comments, which have helped to improve the quality of our study.

Major comments:
(1) The authors should at least try to elucidate the mechanism at the synaptic level.For instance, they can analyze the phenotypes of the mutant mice using acute brain slices or cultured neurons.If the authors can show that the CB1R pathway is enhanced in mutant mice electrophysiologically, it would strengthen the conclusions of this paper considerably.Such data would attract many readers of EMBO Molecular Medicine.(2) Fig. 2D: The result of the motor coordination (the first trial of the rotarod test) should also be shown (or described in the text).The result of motor learning would be meaningless if the motor coordination is impaired.(3) Fig. 2F, 2I: The authors should analyze the data by using "two"-way ANOVA, because there are two groups (WT and KO) with two different conditions (O and M for Fig. 2F; N and F for Fig. 2I).Since there seem to be some other problems in statistical analyses throughout the paper, all the data should be reexamined by a person who specializes in statistics.

We
We agree with the reviewer.Sorry for this error.Those data have now been reanalyzed by two-way ANOVA.We have reviewed all the statistical analyses in the paper.
We agree with this remark.All the behavioral tests were conducted with KO (Cre+) and WT/floxed (Cre-) littermates within each mouse line because, owing to the high inter-individual variability of these tests, they demand high n values for an appropriate statistical analysis to be conducted.From a logistical standpoint, these tests can be readily performed with many animals in our laboratory.Regarding the RNAscope technique, on the one hand, the analysis is very laborious within each animal.On the other hand, we obtained essentially all-ornone, low variability Crbn-transcript expression data in the different brain regions of the three KO lines.Hence, we decided to pool the 6 WT/floxed mice used (2 littermates from the Glu-CRBN-KO colony, 2 littermates from the GABA-CRBN-KO colony, and 2 littermates from the CRBN-KO colony) into a single WT/floxed group.Please note that all these WT/floxed mice share an identical genotype (Crbn fl/fl,Cre-).

Minor comments:
(5) Throughout the study, is there any statistical difference between the data of male and female mice?
Both male and female mice were used in the study, and specific symbols for each of the two sexes are shown where appropriate.Source data were collected and analyzed as disaggregated for sex, but that information was not included in the previous version of the manuscript.Please find it now in the Appendix.We increased the n values of some of the experimental groups to make the statistical analyses more robust.Except -as expected-for body weight, that was slightly higher in males than in females (Fig 2A ), no gross sex-specific differences were found in the numerous parameters measured.We are nonetheless aware that statistical trends appeared in a few cases, and we cannot rule out that sample size was not high enough to enable meaningful post hoc statistical conclusions.
The authors have performed laborious in vitro and in vivo experiments, uncovering the unexplored role of CRBN for CB1R-mediated regulation of brain function.The conceptual novelty of these findings is significant and would be of interest for a broad audience in the field of developmental brain disorders.The manuscript is well-written; however, several weaknesses need be addressed to enhance the rigor and overall quality of the study.
Referee #3 (Remarks for Author): In this manuscript, Costas-Insua et al. report on the role of CRBN, a genetic risk factor for intellectual disability, in maintaining memory function.Using multiple CRBN deletion mouse models, the authors have identified that CRBN, particularly expressed in glutamatergic neurons, regulates memory function.Mechanistically, the authors reveal that CRBN interacts with CB1R, impeding the CB1R-Gi/o-cAMP-PKA pathway in a manner independent of its ubiquitin ligase function.These mechanisms may underlie the cognitive impact of CRBN, as memory deficits resulting from genetic CRBN deletion were alleviated by the acute pharmacological blockade of CB1R.The authors have performed laborious in vitro and in vivo experiments, uncovering the unexplored role of CRBN for CB1R-mediated regulation of brain function.The conceptual novelty of these findings is significant and would be of interest for a broad audience in the field of developmental brain disorders.The manuscript is well-written; however, several weaknesses need be addressed to enhance the rigor and overall quality of the study.
We would like to thank the reviewer for his/her positive and constructive comments, which have helped to improve the quality of our study.
A major weakness in the present study is the absence of physiological data that reveal that genetic deletion of CRBN accelerates CB1R-mediated suppression of glutamatergic neuronal function, which could be rescued by the CB1R inhibition.Without this data, it remains unclear whether CRBN-CB1R interaction is a critical regulator for glutamatergic neurons.The author should provide this evidence by performing electrophysiological assays, such as brain slice recording experiments.
We agree with the reviewer that the standard Y-maze test predominantly assesses short-term habituation to recently experienced stimuli.Nonetheless, here we used a modified version of the test that has been reported to largely measure hippocampal-dependent spatial reference memory (Kraeuter et al.,

2019). We have rephrased in the text the outcome evaluated by this test (lines 167-168).
The forced swim test was used to evaluate depression.Nevertheless, while the forced swim test is still used to characterizing the efficacy of antidepressants, the field recognizes that it is unclear whether the observed phenotypes are valid correlates of depressive symptoms.The authors should rephrase the result section accordingly.

The reviewer is correct. We have rephrased in the text the outcome measured by this test (lines 160-161).
To evaluate CB1R functionality in CRBN-deficient mice, the authors examined the impact of THC treatment on catalepsy.However, cognitive effects of THC have been extensively studied in the literature.Studying cognitive tasks is more appropriate for the current study.
We understand the reviewer's comment.As he/she points out, the aim of these experiments was to assess CB 1 R functionality in CRBN-deficient mice by measuring the behavioral response of the animals to a submaximal dose of a cannabinoid agonist (3 mg/kg THC).However, please note that if we had measured cognitive tasks, it would have been hardly feasible to evaluate CB 1 R functionality as the cognitive shortfalls that are induced by THC administration are already present in CRBN-deficient animals.For example, in the NOR test, neither CRBN-KO nor Glu-CRBN-KO mice discriminated basally between the new and familiar objects (Figs 2H and 6A; discrimination indexes around 0). Hence, conceivably, this bad performance could not be worsened even further by agonizing the CB 1 R with THC.In fact, the memory deficits shown by CRBN-KO and Glu-CRBN-KO mice were rescued by antagonizing the CB 1 R with rimonabant (Fig 6A).We consequently sought to evaluate behavioral traits that i) are unaffected basally by CRBN deficiency, ii) are evoked in a straightforward manner by a CB 1 R agonist, and iii) can be measured rapidly and unambiguously.So, we decided to assess catalepsy and thermal analgesia.
Despite the inclusion of both male and female mice in the study, it is uncertian if any sex differences exist.While sex difference is not the primary scope of the current study, the authors should at least analyze some data separately for male and female mice and discuss potential sex effects.It is worth noting that certain studies have reported sex differences in the effects of THC.Perhaps clinical studies may also reveal sex differences in CRBN mutations genetically associated with intellectual disability?Both male and female mice were used in the study, and specific symbols for each of the two sexes are shown where appropriate.Source data were collected and analyzed as disaggregated for sex, but that information was not included in the previous version of the manuscript.Please find it now in the Appendix.We increased the n values of some of the experimental groups to make the statistical analyses more robust.Except -as expected-for body weight, that was slightly higher in males than in females (Fig 2A ), no gross sex-specific differences were found in the numerous parameters measured.We are nonetheless aware that statistical trends appeared in a few cases, and we cannot rule out that sample size was not high enough to enable meaningful post hoc statistical conclusions.Thank you for the submission of your revised manuscript to EMBO Molecular Medicine.We have now received the enclosed reports from the referees that were asked to re-assess it.As you will see the reviewers are now globally supportive and I am pleased to inform you that we will be able to accept your manuscript pending the following final amendments: -Please move the Acknowledgements to after the Data Availability statement.
2) Please ensure that the sequence for primers used for genotyping in Figure 1A are included in the Materials & Methods.
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Abridged guidelines for figures 1. Data
The data shown in figures should satisfy the following conditions: ➡ ➡ definitions of statistical methods and measures: -are tests one-sided or two-sided?-are there adjustments for multiple comparisons?-exact statistical test results, e.g., P values = x but not P values < x; -definition of 'center values' as median or average; -definition of error bars as s.d. or s.e.m.

Materials
Newly Created Materials Information included in the manuscript?
In which section is the information available?
(Reagents and Tools New materials and reagents need to be available; do any restrictions apply?Yes CRBN-KO, Glu-CRBN-KO and GABA-CRBN-KO mice (Materials and Methods: Animals; Results: Selective genetic inactivation of Crbn in glutamatergic neurons impairs memory) CRBN-expressing plasmids

Antibodies
Information included in the manuscript?
In which section is the information available?
(Reagents and Tools For antibodies provide the following information: -Commercial antibodies: RRID (if possible) or supplier name, catalogue number and or/clone number -Non-commercial: RRID or citation

Yes
The following primary and secondary antibodies (with their dilutions, applications, suppliers, and catalog numbers) were used in this study (Materials and Methods: RNAscope and immunofluorescence, Proximity ligation assay (PLA), Western blot and immunoprecipitation, Antibody-capture [35S]GTPγS scintillation proximity assay).All antibodies were used according

DNA and RNA sequences
Information included in the manuscript?
In which section is the information available?
(Reagents and Tools Short novel DNA or RNA including primers, probes: provide the sequences.

Yes
The sequences of the Crbn primers (Materials and Methods: RNA isolation and quantitative PCR) and the Crbn-targeted siRNA (Materials and Methods: CRBN knock-down) are provided.

Cell materials Information included in the manuscript?
In which section is the information available?
(Reagents and Tools

Yes
The HEK-293T cell line was not recently authenticated and was negative for mycoplasma contamination (Materials and Methods: Cell culture, transfection and signalling experiments).

Experimental animals Information included in the manuscript?
In which section is the information available?
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Not Applicable Please detail housing and husbandry conditions.Yes Throughout the study, animals had unrestricted access to food and water.They were housed (typically, 4-5 mice per cage) under controlled temperature

Plants and microbes
Information included in the manuscript?
In which section is the information available?
(Reagents and Tools If your work benefited from core facilities, was their service mentioned in the acknowledgments section?Yes We thank the personnel of the core microscopy centre, the genomics unit, and the animal facilities of Universidad Complutense de Madrid for their expert technical assistance (Acknowledgements).

Design
Study protocol Information included in the manuscript?
In which section is the information available?
(Reagents and Tools ). a statement of how many times the experiment shown was independently replicated in the laboratory.
-common tests, such as t-test (please specify whether paired vs. unpaired), simple χ2 tests, Wilcoxon and Mann-Whitney tests, can be unambiguously identified by name only, but more complex techniques should be described in the methods section; Please complete ALL of the questions below.Select "Not Applicable" only when the requested information is not relevant for your study.
if n<5, the individual data points from each experiment should be plotted.Any statistical test employed should be justified.Source Data should be included to report the data underlying figures according to the guidelines set out in the authorship guidelines on Data Presentation.
Each figure caption should contain the following information, for each panel where they are relevant: a specification of the experimental system investigated (eg cell line, species name).the assay(s) and method(s) used to carry out the reported observations and measurements.an explicit mention of the biological and chemical entity(ies) that are being measured.an explicit mention of the biological and chemical entity(ies) that are altered/varied/perturbed in a controlled manner.Include a statement about sample size estimate even if no statistical methods were used.

Yes
The sample size for each experiment was estimated based on previous studies conducted by our laboratories using similar in vitro and in vivo models (Materials and Methods: Experimental design and statistical analyses).Were any steps taken to minimize the effects of subjective bias when allocating animals/samples to treatment (e.g.randomization procedure)?If yes, have they been described?

Yes
In all experiments, biological samples (cultured cells, tissue extracts, brain sections) and animals (mice) were allocated randomly into the different groups (Materials and Methods: Experimental design and statistical analyses).
Include a statement about blinding even if no blinding was done.

Yes
In vivo experiments were routinely performed and analysed in a blinded manner for mouse genotype, viral injection, and pharmacological treatment (typically an experimenter prepared the animals and the samples derived from Describe inclusion/exclusion criteria if samples or animals were excluded from the analysis.Were the criteria pre-established?
If sample or data points were omitted from analysis, report if this was due to attrition or intentional exclusion and provide justification.

Yes
No data were excluded for the statistical analyses except when, very rarely, it was obvious that a technical problem had occurred in the measure (Materials and Methods: Experimental design and statistical analyses).
For every figure, are statistical tests justified as appropriate?Do the data meet the assumptions of the tests (e.g., normal distribution)?Describe any methods used to assess it.Is there an estimate of variation within each group of data?Is the variance similar between the groups that are being statistically compared?

Yes
The statistical tests that were applied for each dataset are indicated in each figure legend.All datasets were tested for normality and homoscedasticity prior to analysis (Materials and Methods: Experimental design and statistical analyses).

Sample definition and in-laboratory replication Information included in the manuscript?
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Ethics
Ethics Information included in the manuscript?
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Yes
All the experimental procedures used were performed in accordance with the guidelines and with the approval of the Animal Welfare Committee of Universidad Complutense de Madrid and Comunidad de Madrid (protocol codes PROEX 209/18 and PROEX 032.0/22), and in accordance with the directives of the European Commission (Materials and Methods: Animals) Studies involving specimen and field samples: State if relevant permits obtained, provide details of authority approving study; if none were required, explain why.

Dual Use Research of Concern (DURC)
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Reporting
Adherence to community standards Information included in the manuscript?
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(Reagents and Tools Have primary datasets been deposited according to the journal's guidelines (see 'Data Deposition' section) and the respective accession numbers provided in the Data Availability Section?

Not Applicable
Were human clinical and genomic datasets deposited in a public accesscontrolled repository in accordance to ethical obligations to the patients and to the applicable consent agreement?

Not Applicable
Are computational models that are central and integral to a study available without restrictions in a machine-readable form?Were the relevant accession numbers or links provided?

Not Applicable
If publicly available data were reused, provide the respective data citations in the reference list.

Not Applicable
The MDAR framework recommends adoption of discipline-specific guidelines, established and endorsed through community initiatives.Journals have their own policy about requiring specific guidelines and recommendations to complement MDAR.
10) We replaced Supplementary Information with Expanded View (EV) Figures and Tables that are collapsible/expandable online.A maximum of 5 EV Figures can be typeset.EV Figures should be cited as 'Figure EV1, Figure EV2" etc... in the text and their respective legends should be included in the main text after the legends of regular figures.-For the figures that you do NOT wish to display as Expanded View figures, they should be bundled together with their legends in a single PDF file called *Appendix*, which should start with a short Table of Content.Appendix figures should be referred to in the main text as: "Appendix Figure S1, Appendix Figure S2" etc.
** Reviewer's comments ***** Referee #1 (Comments on Novelty/Model System for Author):This is an excellent report and my comment are outlined below.Referee #1 (Remarks for Author): Fig 2AB and the corresponding figure legends.Animals of both sexes were used and analyzed as disaggregated for sex (see Materials and Methods, "Animals", lines 436-438; and Appendix Table agree with the reviewer.We have therefore conducted electrophysiology experiments in hippocampal slices and measured synaptic plasticity-associated parameters.As previous studies had already investigated the electrophysiological alterations occurring in hippocampal slices from CRBN-KO mice (cf.Bavley et al., 2018, Choi et al., 2018; both cited in the paper), we sought to analyze -as for G protein coupling (Fig 5D)-the effect of viral vector-driven CRBN overexpression in CA1 hippocampal neurons.We measured two archetypical forms of endocannabinoid-mediated short-term synaptic plasticity, namely a depolarization-induced suppression of excitation (DSE) and inhibition (DSI), which rely on the CB 1 R-dependent control of glutamatergic and GABAergic transmission, respectively.As shown in new Fig 5E, CRBN overexpression blunted DSE but had no effect on DSI.Taken together, these electrophysiology data provide further support to the notion that CRBN in vivo blunts CB 1 R activation selectively at glutamatergic terminals.See also Results (lines 286-300) and Materials and Methods (Ex vivo electrophysiological recordings, lines 852-888).
agree with the reviewer.Now we also show the data of the three days tested by separate in Fig EV2A (see Results, line 152; and explanation of the rotarod test in Materials and Methods, "Motor performance tests", lines 452-459).
agree with the reviewer.We have therefore conducted electrophysiology experiments in hippocampal slices and measured synaptic plasticity-associated parameters.As previous studies had already investigated the electrophysiological alterations occurring in hippocampal slices from CRBN-KO mice (cf.Bavley et al., 2018, Choi et al., 2018; both cited in the paper), we sought to analyze -as for G protein coupling (Fig 5D)-the effect of viral vector-driven CRBN overexpression in CA1 hippocampal neurons.We measured two archetypical forms of endocannabinoid-mediated short-term synaptic plasticity, namely a depolarization-induced suppression of excitation (DSE) and inhibition (DSI), which rely on the CB 1 R-dependent control of glutamatergic and GABAergic transmission, respectively.As shown in new Fig 5E, CRBN overexpression blunted DSE but had no effect on DSI.Taken together, these electrophysiology data provide further support to the notion that CRBN in vivo blunts CB 1 R activation selectively at glutamatergic terminals.See also Results (lines 286-300) and Materials and Methods (Ex vivo electrophysiological recordings, lines 852-888).
line 267.Please spell out what "PLA" is.We have spelled out the abbreviation when first used in the text (line 200).

1)
Please place individual sections of the manuscript in the following order: Title page -Abstract & Keywords -Introduction -Results -Discussion -Materials & Methods -Data Availability -Acknowledgements -Disclosure and Competing Interests Statement -The Paper Explained -For More Information -References -Figure Legends -Expanded View Figure Legends.

I
look forward to reading a new revised version of your manuscript as soon as possible.have been addressed.Referee #2 (Remarks for Author): This paper has been improved considerably, and I have no more comments.Referee #3 (Remarks for Author): Table, Materials and Methods, Figures, Data Availability Section) Table, Materials and Methods, Figures, Data Availability Section) Table, Materials and Methods, Figures, Data Availability Section) Table, Materials and Methods, Figures, Data Availability Section) Table, Materials and Methods, Figures, Data Availability Section)

In which section is the information available?
Table, Materials and Methods, Figures, Data Availability Section) Reagents and Tools Table, Materials and Methods, Figures, Data Availability Section) If collected and within the bounds of privacy constraints report on age, sex and gender or ethnicity for all study participants.(Reagents and Tools Table, Materials and Methods, Figures, Data Availability Section) (

Checklist for Life Science Articles (updated January 2022) ideally
Table, Materials and Methods, Figures, Data Availability Section)This checklist is adapted from Materials Design Analysis Reporting (MDAR) Checklist for Authors.MDAR establishes a minimum set of requirements in transparent reporting in the life sciences (see Statement of Task: 10.31222/osf.io/9sm4x).Please follow the journal's guidelines in preparing your manuscript.the data were obtained and processed according to the field's best practice and are presented to reflect the results of the experiments in an accurate and unbiased manner., figure panels should include only measurements that are directly comparable to each other and obtained with the same assay.plots include clearly labeled error bars for independent experiments and sample sizes.Unless justified, error bars should not be shown for technical the exact sample size (n) for each experimental group/condition, given as a number, not a range; a description of the sample collection allowing the reader to understand whether the samples represent technical or biological replicates (including how many animals, litters, cultures, etc.

In which section is the information available?
For clinical trials, provide the trial registration number OR cite DOI.(Reagents and Tools Table, Materials and Methods, Figures, Data Availability Section) If study protocol has been pre-registered, provide DOI in the manuscript.

study design and statistics Information included in the manuscript? In which section is the information available?
(Reagents and Tools Table, Materials and Methods, Figures, Data Availability Section) Table, Materials and Methods, Figures, Data Availability Section) In the figure legends: state number of times the experiment was replicated in laboratory.Yes The number of biological replicates (number of mice, number of mouse hippocampal preparations, number of cellular experiments, number of subcellular experiments) is provided in each figure legend (Materials and Methods: Experimental design and statistical analyses).The number of biological replicates (number of mice, number of mouse hippocampal preparations, number of cellular experiments, number of subcellular experiments) is provided in each figure legend.The number of technical replicates is provided in the corresponding Materials and Methods subsection (Materials and Methods: Experimental design and statistical analyses).
Table, Materials and Methods, Figures, Data Availability Section) Include a statement confirming that informed consent was obtained from all subjects and that the experiments conformed to the principles set out in the WMA Declaration of Helsinki and the Department of Health and Human Services Belmont Report.
Studies involving human participants: State details of authority granting ethics approval (IRB or equivalent committee(s), provide reference number for approval.Not ApplicableStudies involving human participants:Not ApplicableStudies involving human participants: For publication of patient photos, include a statement confirming that consent to publish was obtained.Not Applicable Studies involving experimental animals: State details of authority granting ethics approval (IRB or equivalent committee(s), provide reference number for approval.Include a statement of compliance with ethical regulations.

granting approval and reference number for
Table, Materials and Methods, Figures, Data Availability Section) Could your study fall under dual use research restrictions?Please check biosecurity documents and list of select agents and toxins (CDC): https://www.selectagents.gov/sat/list.htmNot Applicable If you used a select agent, is the security level of the lab appropriate and reported in the manuscript?Not Applicable If a study is subject to dual use research of concern regulations, is the name of the authority the regulatory approval provided in the manuscript?

and III randomized controlled trials
Table, Materials and Methods, Figures, Data Availability Section) State if relevant guidelines or checklists (e.g., ICMJE, MIBBI, ARRIVE, PRISMA) have been followed or provided.Yes The ARRIVE guidelines were followed as closely as possible (Materials and Methods: Animals).For tumor marker prognostic studies, we recommend that you follow the REMARK reporting guidelines (see link list at top right).See author guidelines, under 'Reporting Guidelines'.Please confirm you have followed these guidelines., please refer to the CONSORT flow diagram (see link list at top right) and submit the CONSORT checklist (see link list at top right) with your submission.See author guidelines, under 'Reporting Guidelines'.Please confirm you have submitted this list.Reagents and Tools Table, Materials and Methods, Figures, Data Availability Section) (