King’s Research Portal DOI: 10.1038/s41398-019-0580-9 Document Version Publisher's PDF, also known as Version of record Link to publication record in King's Research Portal Citation for published version (APA): Chong, C. H., Li, Q., Mak, P. H. S., Ng, C. C. P., Leung, E. H. W., Tan, V. H., Chan, A. K. W., McAlonan, G., & Chan, S. Y. (2019). Lrrc7 mutant mice model developmental emotional dysregulation that can be alleviated by mGluR5 allosteric modulation. Translational psychiatry, 9(1), [244]. https://doi.org/10.1038/s41398-019-0580-9 Citing this paper Please note that where the full-text provided on King's Research Portal is the Author Accepted Manuscript or Post-Print version this may differ from the final Published version. If citing, it is advised that you check and use the publisher's definitive version for pagination, volume/issue, and date of publication details. 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Download date: 28. Sep. 2021 Chong et al. Translational Psychiatry (2019) 9:244 https://doi.org/10.1038/s41398-019-0580-9 Translational Psychiatry ARTICLE Open Access Lrrc7 mutant mice model developmental emotional dysregulation that can be alleviated by mGluR5 allosteric modulation Chi Ho Chong1,QiLi2, Priscilla Hoi Shan Mak1, Cypress Chun Pong Ng1,EvaHinWaLeung1, Vicky Huiqi Tan1, Anthony Kin Wang Chan1, Grainne McAlonan3 and Siu Yuen Chan 1 Abstract LRRC7 has been identified as a candidate gene for severe childhood emotional dysregulation. Direct experimental evidence for a role of LRRC7 in the disease is needed, as is a better understanding of its impact on neuronal structure and signaling, and hence potential treatment targets. Here, we generated and analyzed an Lrrc7 mutant mouse line. Consistent with a critical role of LRRC7 in emotional regulation, mutant mice had inappropriate juvenile aggressive behavior and significant anxiety-like behavior and social dysfunction in adulthood. The pivotal role of mGluR5 signaling was demonstrated by rescue of behavioral defects with augmentation of mGluR5 receptor activity by 3-Cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB). Intra-peritoneal injection of CDPPB alleviated abnormal juvenile behavior, as well as anxiety-like behavior and hypersociability at adulthood. Furthermore, mutant primary neurons had impaired neurite outgrowth which was rescued by CDPPB treatment. In conclusion, Lrrc7 mutant mice provide a valuable tool to model childhood emotional dysregulation and persistent mental health comorbidities. Moreover, our data highlight an important role of LRRC7 in mGluR5 signaling, which is a potential new treatment 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; target for anxiety and social dysfunction. Introduction were linked to the highest scores on the CBCL–DP (Child Densin-180, also named Leucine-rich repeat containing Behavior Checklist–Dysregulation Profile)4. High scores protein 7 (LRRC7), is an abundant scaffold protein ori- on CBCL–DP place an individual at an increased risk of ginally isolated from the postsynaptic density1. LRRC7 is developing serious mood disorder, anxiety, personality located at 1p31.1, a region linked to neurodevelopmental difficulties, and aggressive behavior which persist into – conditions including severe language impairment2 and adulthood5 7. Therefore, it is essential to understand the autism spectrum disorder3. Importantly, LRRC7 has function of LRRC7 in brain to elucidate the molecular recently been identified as a candidate gene for serious mechanism of the above neurodevelopmental disorders. childhood emotional dysregulation. Specifically, in a LRRC7 interacts with proteins such as CaV1.2 channel, genome-wide association study of the Child Behavior NMDA (N-methyl-D-aspartate) receptors, CaMKIIα + Checklist in children and adolescents with attention (Ca2 /calmodulin-dependent protein kinase II α), δ-cate- – deficit-hyperactivity disorder (ADHD), SNPs in LRRC7 nin, α-actinin, SHANK, and Maguin-1 (refs. 8 14). These targets have themselves been linked to a broad spectrum of neurodevelopmental disorders including autism and 15–22 Correspondence: Siu Yuen Chan ([email protected]) intellectual disability . mGluR5 dysfunction is also 1Department of Paediatrics and Adolescent Medicine, Li Ka Shing Faculty of associated with neurodevelopmental disorders including Medicine, The University of Hong Kong, Hong Kong, China fi 2 autism spectrum disorder and attention de cit hyper- Department of Psychiatry, Li Ka Shing Faculty of Medicine, The University of 23–27 Hong Kong, Hong Kong, China activity disorder . Emotional dysregulation is a Full list of author information is available at the end of the article. © The Author(s) 2019 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a linktotheCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Chong et al. Translational Psychiatry (2019) 9:244 Page 2 of 12 common characteristic across such neurodevelopmental transcripts (Fig. S1E). Primer sequences are available psychiatric disorders28,29. With increasing numbers of upon request. – patients diagnosed with these lifelong conditions30 32,a We inter-crossed heterozygous mice to maintain the better understanding of the underlying molecular line. Lrrc7 homozygous mutants appeared smaller in size mechanisms is crucial to developing effective since the second week of age and needed to be fed with interventions. wet chow from postnatal day (P)17 to P32, otherwise they Although no experimental evidence suggests molecular died of dehydration. Injuries were found in littermates interaction between LRRC7 and mGluR5, there have been and recorded. A paper house was provided from birth to some valuable first steps. Deletion of Lrrc7 in C57BL/6 reduce fighting which otherwise could be fatal. Mice were mice by gene targeting has been reported to reduce the weaned at around P21 but small mice were allowed to be level of postsynaptic mGluR5, and impaired mGluR- kept with their mother until P28–P32. The weight of lit- induced long term depression. By contrast, recruitment termates was monitored starting from P14 to 36. At P16, and function of NMDA receptors are normal at synap- the hanging wire test was used to assess motor strength ses33. However, to date, there is no direct evidence to link before the genotype was known. The mouse was placed mGluR5 abnormalities to behavioral abnormalities in on the top of the cage lid. After the mouse gripped onto animals with Lrrc7 deletion. the wire, the lid was turned upside down. The time taken It has been well-established that mGluR5 regulates for the mouse to fall off was measured with the cut-off – synaptic strength34 36, and indirect evidence shows that time set at 1 min. mGluR5 also plays a role in early neurite development. In the zebrafish model of Fragile X syndrome, pharmacolo- Behavioral tests gical mGluR5 inhibition corrects neurite branching The estimation of sample size was based on the data defects37; whereas in mice, deletion of mGluR5 in layer 2/ from exploratory studies of behavior. According to PASS 3 pyramidal neurons of somatosensory cortex impairs software (NCSS Statistical Software), eight mice should be dendritic arborization38. The link between mGluR5 and assigned to each group to allow randomized block analysis scaffold proteins in neurite morphogenesis and the of variance power analysis with alpha = 0.05 and power = underlying signaling remains unexplored. 0.80. Mice used in behavioral experiments were litter- Therefore, we analyzed Lrrc7 hypomorphic mutant mates collected from heterozygous pairings. Mice born mice which we had fortuitously generated. We aimed to within the same week were employed as a cohort. Mice confirm a behavioral phenotype relevant to develop- were tested from 8 weeks of age and tests were performed mental emotional dysregulation. We then tested whether during the light cycle. The test was performed and ana- impaired