RESUMO
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.
Assuntos
Hidrazonas/química , Hidrazonas/uso terapêutico , Inibidores de Fosfodiesterase/química , Inibidores de Fosfodiesterase/uso terapêutico , Diester Fosfórico Hidrolases/metabolismo , Esquizofrenia/tratamento farmacológico , Esquizofrenia/enzimologia , Animais , Antipsicóticos/química , Antipsicóticos/farmacocinética , Antipsicóticos/uso terapêutico , Hidrazonas/farmacocinética , Camundongos , Inibidores de Fosfodiesterase/farmacocinética , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Hidrazonas/química , Inibidores de Fosfodiesterase/química , Diester Fosfórico Hidrolases/química , Quinolinas/química , Animais , Modelos Animais de Doenças , Humanos , Hidrazonas/farmacocinética , Hidrazonas/uso terapêutico , Inibidores de Fosfodiesterase/farmacocinética , Inibidores de Fosfodiesterase/uso terapêutico , Diester Fosfórico Hidrolases/metabolismo , Quinolinas/farmacocinética , Quinolinas/uso terapêutico , Ratos , Esquizofrenia/tratamento farmacológico , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Expressão Gênica , Marcação de Genes , Transgenes , Animais , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Aterosclerose/tratamento farmacológico , Aterosclerose/patologia , Colesterol/sangue , Doxiciclina/administração & dosagem , Doxiciclina/metabolismo , Células-Tronco Embrionárias/fisiologia , Expressão Gênica/efeitos dos fármacos , Vetores Genéticos/genética , Elementos Isolantes , Camundongos , Camundongos Transgênicos , Mutagênese Insercional , Retroviridae/genética , Transativadores/genética , Transativadores/metabolismoRESUMO
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 NMU's 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.
Assuntos
Comportamento Alimentar/fisiologia , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Dor/genética , Percepção/fisiologia , Receptores de Neurotransmissores/deficiência , Receptores de Neurotransmissores/genética , Estresse Fisiológico/genética , Animais , Ansiedade/genética , Feminino , Masculino , Proteínas de Membrana/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dor/fisiopatologia , Receptores de Neurotransmissores/biossínteseRESUMO
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.
Assuntos
Encéfalo/metabolismo , Receptores Acoplados a Proteínas G/fisiologia , Diferenciação Sexual/fisiologia , Comportamento Sexual Animal/fisiologia , Transdução de Sinais/fisiologia , Análise de Variância , Animais , Comportamento Animal/fisiologia , Encéfalo/citologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Hormônio Liberador de Gonadotropina/metabolismo , Humanos , Kisspeptinas , Hormônio Luteinizante/sangue , Masculino , Camundongos , Camundongos Knockout , Neurônios/classificação , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Proteínas/genética , Proteínas/metabolismo , Receptores Acoplados a Proteínas G/deficiência , Receptores de Kisspeptina-1 , Caracteres Sexuais , Diferenciação Sexual/efeitos dos fármacos , Diferenciação Sexual/genética , Comportamento Sexual Animal/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Testosterona/farmacologia , Proteínas Supressoras de Tumor/farmacologia , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
TRH is a neuropeptide with a variety of hormonal and neurotransmitter/neuromodulator functions. In particular, TRH has pronounced acute antidepressant effects in both humans and animals and has been implicated in the mediation of the effects of other antidepressive therapies. Two G protein-coupled receptors, TRH receptor 1 (TRH-R1) and TRH-R2, have been identified. Here we report the generation and phenotypic characterization of mice deficient in TRH-R1. The TRH-R1 knockout mice have central hypothyroidism and mild hyperglycemia but exhibit normal growth and development and normal body weight and food intake. Behaviorally, the TRH-R1 knockout mice display increased anxiety and depression levels while behaving normally in a number of other aspects examined. These results provide the first direct evidence that the endogenous TRH system is involved in mood regulation, and this function is carried out through TRH-R1-mediated neural pathways.
Assuntos
Ansiedade/metabolismo , Depressão/metabolismo , Receptores do Hormônio Liberador da Tireotropina/genética , Receptores do Hormônio Liberador da Tireotropina/fisiologia , Afeto , Animais , Comportamento Animal , Peso Corporal , Hiperglicemia/genética , Hipotireoidismo , Masculino , Camundongos , Camundongos Knockout , Modelos Genéticos , Movimento , FenótipoRESUMO
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.
Assuntos
Ansiedade/genética , Ansiedade/psicologia , Receptor Tipo 2 de Galanina/genética , Receptor Tipo 2 de Galanina/fisiologia , Animais , Peso Corporal/fisiologia , Condicionamento Psicológico/fisiologia , Comportamento Exploratório/fisiologia , Medo/fisiologia , Medo/psicologia , Feminino , Genótipo , Saúde , Heterozigoto , Masculino , Aprendizagem em Labirinto/fisiologia , Camundongos , Camundongos Knockout , Movimento/fisiologia , Dor/genética , Dor/psicologia , Medição da Dor , Fenótipo , Equilíbrio Postural/fisiologia , Reflexo/fisiologia , Sensação/fisiologia , Estresse Psicológico/genética , Estresse Psicológico/psicologiaRESUMO
We have previously reported that DNase I hypersensitive site 5 (5'HS5) of the human beta-globin locus control region functions as a chromatin insulator in stable transfection assays. In this report we show that a 3.2 kb DNA fragment containing the entire 5'HS5 region can protect a position-sensitive (A)gamma-globin gene against position effects in transgenic mice. Bracketing is required for function of 5'HS5 as an insulator. The 5'HS5 insulator operates in adult as well as in embryonic murine erythroid cells. The insulator has no significant stimulatory effects of its own. These results indicate that 5'HS5 can function as a chromatin insulator in vivo.
Assuntos
Cromatina/genética , Cromatina/metabolismo , Desoxirribonuclease I/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Globinas/genética , Região de Controle de Locus Gênico/genética , Envelhecimento/sangue , Envelhecimento/genética , Animais , Cromatina/química , Citomegalovirus/genética , DNA Recombinante/genética , Desenvolvimento Embrionário e Fetal , Elementos Facilitadores Genéticos/genética , Feminino , Humanos , Integrases/genética , Masculino , Camundongos , Camundongos Transgênicos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transgenes/genética , Proteínas Virais/genética , Saco Vitelino/metabolismoRESUMO
It is unclear whether the core promoter is involved in developmental regulation. To address this question, we mutated the TATA box of the human gamma-globin gene, produced transgenic mice, and examined the effect of the mutation during the course of mouse development. In our test system, the gamma-globin gene is expressed at similar levels in the embryonic and adult erythroid cells. The TATA box mutation dramatically reduced expression of the gamma-globin gene in the adult but not in embryonic erythroid cells. In addition, the disruption of the gamma 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 gamma gene promoter is developmentally specific. Similarly, the recruitment of transcription factor II B and RNA polymerase II to the gamma 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 gamma-globin gene.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Globinas/genética , TATA Box/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular , Núcleo Celular , Cromatina/metabolismo , Eritrócitos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Globinas/metabolismo , Células HeLa , Humanos , Camundongos , Camundongos Transgênicos , Mutação , Reação em Cadeia da Polimerase , Testes de Precipitina , Regiões Promotoras Genéticas , Ligação Proteica , RNA Mensageiro/metabolismo , Proteína de Ligação a TATA-Box , Fatores de Tempo , Transcrição GênicaRESUMO
The beta-globin locus control region (LCR) is a powerful regulatory element required for high-level globin gene expression. We have generated transgenic mouse lines carrying a beta-globin locus yeast artificial chromosome lacking the LCR to determine if the LCR is required for globin gene activation. beta-Globin gene expression was analyzed by RNase protection, but no detectable levels of epsilon-, gamma- and beta-globin gene transcripts were produced at any stage of development. These findings suggest that the presence of the LCR is a minimum requirement for globin gene expression. Next, we tested whether the LCR is necessary to activate globin gene expression in a gamma-globin promoter mutant that causes hereditary persistence of fetal hemoglobin (HPFH). beta-YAC transgenic mice carrying the -117 HPFH mutation and a HS3 core deletion that specifically abolishes gamma-globin gene expression during definitive erythropoiesis were produced to test whether the -117 (A)gamma promoter is activated in the absence of interaction with the LCR. In four transgenic mouse lines, gamma-globin gene expression was absent in adult erythrocytes, suggesting that an interaction between the gamma-globin gene promoter and the LCR is required for gamma gene activation even when the promoter contains an HPFH mutation.