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Abstract
Glycogen synthase kinase 3β (GSK3β) activity is regulated by dopamine D2 receptor signaling and can be inhibited by psychoactive drugs in a D2 receptor–dependent manner. However, GSK3β is ubiquitously expressed in the brain, and D2 receptor–expressing cells are distributed as a mosaic in multiple cortical regions. This complicates the interrogation of GSK3β functions in cortical D2 cells in a circuit-defined manner using conventional animal models. We used a CRISPR-Cas9-mediated intersectional approach to achieve targeted deletion of GSK3β in D2-expressing neurons of the adult medial prefrontal cortex (mPFC). Isolation and analysis of ribosome-associated RNA specifically from mPFC D2 neurons lacking GSK3β demonstrated large-scale translatome alterations. Deletion of GSK3β in mPFC D2 neurons revealed its contribution to anxiety-related, cognitive, and social behaviors. Our results underscore the viability of an intersectional knockout approach to study functions of a ubiquitous gene in a network-defined fashion while uncovering the contribution of GSK3β expressed in mPFC D2 neurons in the regulation of behavioral dimensions related to mood and emotions. This advances our understanding of GSK3β action at a brain circuit level and can potentially lead to the development of circuit selective therapeutics.
Highlights
Gene products involved in disease processes and drug action are often expressed ubiquitously
Our results underscore the viability of an intersectional knockout approach to study functions of a ubiquitous gene in a network-defined fashion while uncovering the contribution of Glycogen synthase kinase 3b (GSK3b) expressed in medial prefrontal cortex (mPFC) D2 neurons in the regulation of behavioral dimensions related to mood and emotions
Efficient and specific knockout of GSK3b by CRISPR-Cas[9] To investigate the efficacy and specificity of CRISPRCas9-mediated knockout of GSK3b, we tested the ability of previously characterized Gsk3b single-guide RNA (sgRNA) to cut and induce mutations of DNA at on- and off-target sites.[11]
Summary
Gene products involved in disease processes and drug action are often expressed ubiquitously. Their functional implication can be widely different in time (developmental stage) and space (brain region, neuronal circuit, and particular cell type). Traditional approaches have allowed systemic as well as region or cell type-selective gene knockouts. Their resolution remains limited and does not allow for the investigation of gene functions within cellular subpopulations belonging to the same brain region or neuronal circuit at a specific developmental stage. Glycogen synthase kinase 3b (GSK3b) is a serinethreonine kinase that is ubiquitously expressed across the brain during the entire life-span.[1,2] This kinase regulates neurodevelopment,[2,3] neuronal signaling, and plasticity.[4,5,6,7] GSK3b is involved in regulating mood,[8,9,10,11] cognition,[12] social interaction, and depressive-like behaviors.[8,12,13] These functions of GSK3b have been identified by using drugs that can systemically modulate its activity or by knockout or knockdown approaches, including germline heterozygous knockout, knockout only in forebrain CamKII-expressing pyramidal neurons, Cre-mediated knockout in the adult prefrontal cortex (PFC), CRISPRCas9-mediated knockout only in adult medial PFC (mPFC) neurons, shRNA-mediated knockdown in adult nucleus accumbens shell neurons, and germline knockout in serotonergic neurons.[8,9,11,13,14,15,16,17] the ubiquitous expression profile of GSK3b makes it difficult to pinpoint the neuroanatomical correlates of these various functions precisely
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