Progress in FY2017 includes the following: In order to study the specific cells, sites, tissues, and transmitters by which BRS-3 acts, we have generated floxed Brs3 mouse. The floxed mice allow determination of necessity of Brs3 in the particular cells. We are in the process of using this mouse by breeding with germline Cre-expressing mice (Vgat-Cre, Vglut2-Cre). These allow knockout of Brs3 in cells defined by their Cre driver expression pattern. Similarly, we have generated loxTB-Brs3 mice. The loxTB-Brs3 mice allow determination of sufficiency of Brs3 in the particular cells. We are in the process of breeding this mouse with germline Cre-expressing mice (Vgat-Cre, Vglut2-Cre). These allow re-expression of Brs3 in cells defined by their Cre driver expression pattern. Preliminary data using these mice reveal that deletion of Brs3 in glutamatergic neurons expressing Vglut2 reproduced the global null phenotype for regulation of food intake, metabolic rate, body temperature, adiposity, and insulin resistance. These mice also no longer responded to a BRS-3 agonist, MK-5046. In contrast, deletion of Brs3 in GABAergic neurons produced no clear phenotype. Conversely, the wild type phenotype was restored by selective re-expression of Brs3 in glutamatergic neurons, with no normalization achieved by re-expressing Brs3 in GABAergic neurons. We conclude that Brs3 expression in glutamatergic neurons is both necessary and sufficient for full Brs3 function in energy metabolism. In these experiments, no function was identified for Brs3 in GABAergic neurons. The data suggest that the anti-obesity pharmacologic actions of BRS-3 agonists occur via agonism of receptors on glutamatergic neurons. Finally, we have generated Brs3-T2A-CreERT2 mice, in which Cre is expressed as a fusion mRNA with the endogenous Brs3 mRNA. These mice express Cre in a tamoxifen-dependent manner and in the reported distribution pattern of Brs3. This mouse is being used to selectively activate Brs3 neurons using chemogenetics and optogenetics.
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