Alex Rohde

The kisspeptin receptor GPR54 is required for sexual differentiation of the brain and behavior.

GPR54 is a G-protein-coupled receptor, which binds kisspeptins and is widely expressed throughout the brain. Kisspeptin–GPR54 signaling has been implicated in the regulation of pubertal and adulthood gonadotropin-releasing hormone (GnRH) secretion, and mutations or deletions of GPR54 cause hypogonadotropic hypogonadism in humans and mice. Other reproductive roles for kisspeptin–GPR54 signaling, including the regulation of developmental GnRH secretion or sexual behavior in adults, have not yet been explored. Using adult wild-type (WT) and GPR54 knock-out (KO) mice, we first tested whether kisspeptin–GPR54 signaling is necessary for male and female sexual behaviors. We found that hormone-replaced gonadectomized GPR54 KO males and females displayed appropriate gender-specific adult sexual behaviors. Next, we examined whether GPR54 signaling is required for proper display of olfactory-mediated partner preference behavior. Testosterone-treated WT males preferred stimulus females rather than males, whereas similarly treated WT females and GPR54 KO males showed no preference for either sex. Because olfactory preference is sexually dimorphic and organized during development by androgens, we assessed whether GPR54 signaling is essential for sexual differentiation of other sexually dimorphic traits. Interestingly, adult testosterone-treated GPR54 KO males displayed “female-like” numbers of tyrosine hydroxylase-immunoreactive and Kiss1 mRNA-containing neurons in the anteroventral periventricular nucleus and likewise possessed fewer motoneurons in the spino-bulbocavernosus nucleus than did WT males. Our findings indicate that kisspeptin–GPR54 signaling is not required for male or female copulatory behavior, provided there is appropriate adulthood hormone replacement. However, GPR54 is necessary for proper male-like development of several sexually dimorphic traits, likely by regulating GnRH-mediated androgen secretion during “critical windows” in perinatal development.

Developmental specificity of the interaction between the locus control region and embryonic or fetal globin genes in transgenic mice with an HS3 core deletion.

ABSTRACT The human β-globin locus control region (LCR) consists of five erythroid-lineage-specific DNase I-hypersensitive sites (HSs) and is required for activation of the β-globin locus chromatin domain and globin gene expression. Each DNase I-HS of the LCR consists of a highly conserved core element and flanking sequences. To analyze the functional role of the core elements of the HSs, we deleted a 234-bp fragment encompassing the core of HS3 (HS3c) from a β-globin locus residing on a 248-kb β-locus yeast artificial chromosome and analyzed its function in F2 progeny of transgenic mice. Human ɛ-globin gene expression was absent at day 10 and severely reduced in the day 12 embryonic erythropoiesis of mice lacking HS3c. In contrast, γ-globin gene expression was normal in embryonic erythropoiesis but it was absent in definitive erythropoiesis in the fetal liver. These results indicate that the core element of HS3 is necessary for ɛ-globin gene transcription in embryonic cells and for γ-globin gene transcription in definitive cells. Normal γ-globin gene expression in embryonic cells and the absence of γ-globin gene expression in definitive cells show that different HSs interact with γ-globin gene promoters in these two stages of development. Such results provide direct evidence for developmental stage specificity of the interactions between the core elements of HSs and the promoters of the globin genes.

Galanin receptor subtype 2 (GalR2) null mutant mice display an anxiogenic-like phenotype specific to the elevated plus-maze.

The neuropeptide galanin has been implicated in anxiety-related behaviors, cognition, analgesia, and feeding in rodents. Neuromodulatory actions of galanin are mediated by three G-protein coupled receptors, GalR1, GalR2, and GalR3. The present study investigates the role of the GalR2 receptor by evaluating behavioral phenotypes of mice with a targeted mutation in the GalR2 gene. A three-tiered behavioral phenotyping approach first examined control measures of general health, body weight, neurological reflexes, sensory abilities and motor function. Mice were then assessed on several tests for cognitive and anxiety-like behaviors. GalR2 null mutants and heterozygotes were not significantly different from wildtype littermates on two cognitive tests previously shown to be sensitive to galanin manipulation: acquisition of the Morris water maze spatial task, and trace cued and contextual fear conditioning, an emotional learning and memory task. Two independent cohorts of GalR2 null mutant mice demonstrated an anxiogenic-like phenotype in the elevated plus-maze. No genotype differences were detected on several other measures of anxiety-like behavior. The discovery of an anxiogenic phenotype specific to the elevated plus-maze, similar to findings in GalR1 null mutants, highlights the potential therapeutic efficacy of targeting GalR1 and GalR2 receptors in treating anxiety disorders.

Neuromedin U Receptor 2-Deficient Mice Display Differential Responses in Sensory Perception, Stress, and Feeding

ABSTRACT Neuromedin U (NMU) is a highly conserved neuropeptide with a variety of physiological functions mediated by two receptors, peripheral NMUR1 and central nervous system NMUR2. Here we report the generation and phenotypic characterization of mice deficient in the central nervous system receptor NMUR2. We show that behavioral effects, such as suppression of food intake, enhanced pain response, and excessive grooming induced by intracerebroventricular NMU administration were abolished in the NMUR2 knockout (KO) mice, establishing a causal role for NMUR2 in mediating NMUs central effects on these behaviors. In contrast to the NMU peptide-deficient mice, NMUR2 KO mice appeared normal with regard to stress, anxiety, body weight regulation, and food consumption. However, the NMUR2 KO mice showed reduced pain sensitivity in both the hot plate and formalin tests. Furthermore, facilitated excitatory synaptic transmission in spinal dorsal horn neurons, a mechanism by which NMU stimulates pain, did not occur in NMUR2 KO mice. These results provide significant insights into a functional dissection of the differential contribution of peripherally or centrally acting NMU system. They suggest that NMUR2 plays a more significant role in central pain processing than other brain functions including stress/anxiety and regulation of feeding.

An Inducible and Reversible Mouse Genetic Rescue System

Inducible and reversible regulation of gene expression is a powerful approach for uncovering gene function. We have established a general method to efficiently produce reversible and inducible gene knockout and rescue in mice. In this system, which we named iKO, the target gene can be turned on and off at will by treating the mice with doxycycline. This method combines two genetically modified mouse lines: a) a KO line with a tetracycline-dependent transactivator replacing the endogenous target gene, and b) a line with a tetracycline-inducible cDNA of the target gene inserted into a tightly regulated (TIGRE) genomic locus, which provides for low basal expression and high inducibility. Such a locus occurs infrequently in the genome and we have developed a method to easily introduce genes into the TIGRE site of mouse embryonic stem (ES) cells by recombinase-mediated insertion. Both KO and TIGRE lines have been engineered for high-throughput, large-scale and cost-effective production of iKO mice. As a proof of concept, we have created iKO mice in the apolipoprotein E (ApoE) gene, which allows for sensitive and quantitative phenotypic analyses. The results demonstrated reversible switching of ApoE transcription, plasma cholesterol levels, and atherosclerosis progression and regression. The iKO system shows stringent regulation and is a versatile genetic system that can easily incorporate other techniques and adapt to a wide range of applications.

Developmental specificity of recruitment of TBP to the TATA box of the human γ-globin gene

It is unclear whether the core promoter is involved in developmental regulation. To address this question, we mutated the TATA box of the human γ-globin gene, produced transgenic mice, and examined the effect of the mutation during the course of mouse development. In our test system, the γ-globin gene is expressed at similar levels in the embryonic and adult erythroid cells. The TATA box mutation dramatically reduced expression of the γ-globin gene in the adult but not in embryonic erythroid cells. In addition, the disruption of the γ TATA box significantly reduced the recruitment of TATA box-binding protein (TBP) in the adult cells, but not in embryonic cells, suggesting that the recruitment of TBP to the γ gene promoter is developmentally specific. Similarly, the recruitment of transcription factor II B and RNA polymerase II to the γ promoter was affected in the adult but not in embryonic cells. The distinct effects of the TATA mutation in the embryonic and adult developmental stages suggest that the basal transcription apparatus can be recruited to a core promoter in a developmental stage-dependent manner. The TATA mutation resulted in a shift of transcription initiation site 6 bp or longer upstream to the cap site both in the embryonic and adult erythrocytes. We conclude that the TATA box determines the initiation site but not the efficiency of transcription of the γ-globin gene.

N-Acylhydrazones as inhibitors of PDE10A.

Cyclic nucleotide phosphodiesterases (PDEs) are represented by a large superfamily of enzymes. A series of hydrazone-based inhibitors was synthesized and shown to be novel, potent, and selective against PDE10A. Optimized compounds of this class were efficacious in animal models of schizophrenia and may be useful for the treatment of this disease.

Novel 2-methoxyacylhydrazones as potent, selective PDE10A inhibitors with activity in animal models of schizophrenia

A series of 2-methoxyacylhydrazones were optimized to yield compounds with high affinity for PDE10A. Several compounds demonstrated efficacy in animal models of schizophrenia, including conditioned avoidance response and a pro-psychotic phencyclidine hyperactivity model.