RESUMEN
The G protein-coupled receptor melanocortin-4 receptor (MC4R) and its associated protein melanocortin receptor-associated protein 2 (MRAP2) are essential for the regulation of food intake and body weight in humans. MC4R localizes and functions at the neuronal primary cilium, a microtubule-based organelle that senses and relays extracellular signals. Here, we demonstrate that MRAP2 is critical for the weight-regulating function of MC4R neurons and the ciliary localization of MC4R. More generally, our study also reveals that GPCR localization to primary cilia can require specific accessory proteins that may not be present in heterologous cell culture systems. Our findings further demonstrate that targeting of MC4R to neuronal primary cilia is essential for the control of long-term energy homeostasis and suggest that genetic disruption of MC4R ciliary localization may frequently underlie inherited forms of obesity.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Receptor de Melanocortina Tipo 4 , Humanos , Proteínas Adaptadoras Transductoras de Señales/genética , Receptor de Melanocortina Tipo 4/genética , Receptor de Melanocortina Tipo 2/metabolismo , Cilios/metabolismo , HomeostasisRESUMEN
The melanocortin 4 receptor (MC4R) plays a critical role in the long-term regulation of energy homeostasis, and mutations in the MC4R are the most common cause of monogenic obesity. However, the precise molecular and cellular mechanisms underlying the maintenance of energy balance within MC4R-expressing neurons are unknown. We recently reported that the MC4R localizes to the primary cilium, a cellular organelle that allows for partitioning of incoming cellular signals, raising the question of whether the MC4R functions in this organelle. Here, using mouse genetic approaches, we found that cilia were required specifically on MC4R-expressing neurons for the control of energy homeostasis. Moreover, these cilia were critical for pharmacological activators of the MC4R to exert an anorexigenic effect. The MC4R is expressed in multiple brain regions. Using targeted deletion of primary cilia, we found that cilia in the paraventricular nucleus of the hypothalamus (PVN) were essential to restrict food intake. MC4R activation increased adenylyl cyclase (AC) activity. As with the removal of cilia, inhibition of AC activity in the cilia of MC4R-expressing neurons of the PVN caused hyperphagia and obesity. Thus, the MC4R signaled via PVN neuron cilia to control food intake and body weight. We propose that defects in ciliary localization of the MC4R cause obesity in human inherited obesity syndromes and ciliopathies.
Asunto(s)
Peso Corporal , Encéfalo/metabolismo , Cilios/metabolismo , Ingestión de Alimentos , Neuronas/metabolismo , Receptor de Melanocortina Tipo 4/metabolismo , Transducción de Señal , Animales , Cilios/genética , Metabolismo Energético , Ratones , Ratones Transgénicos , Receptor de Melanocortina Tipo 4/genéticaRESUMEN
A wide range of human diseases result from haploinsufficiency, where the function of one of the two gene copies is lost. Here, we targeted the remaining functional copy of a haploinsufficient gene using CRISPR-mediated activation (CRISPRa) in Sim1 and Mc4r heterozygous mouse models to rescue their obesity phenotype. Transgenic-based CRISPRa targeting of the Sim1 promoter or its distant hypothalamic enhancer up-regulated its expression from the endogenous functional allele in a tissue-specific manner, rescuing the obesity phenotype in Sim1 heterozygous mice. To evaluate the therapeutic potential of CRISPRa, we injected CRISPRa-recombinant adeno-associated virus into the hypothalamus, which led to reversal of the obesity phenotype in Sim1 and Mc4r haploinsufficient mice. Our results suggest that endogenous gene up-regulation could be a potential strategy to treat altered gene dosage diseases.
Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Elementos de Facilitación Genéticos , Haploinsuficiencia , Obesidad/genética , Regiones Promotoras Genéticas , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Línea Celular , Dependovirus , Modelos Animales de Enfermedad , Femenino , Regulación de la Expresión Génica , Técnicas de Transferencia de Gen , Heterocigoto , Hipotálamo , Mutación con Pérdida de Función , Masculino , Ratones , Ratones Transgénicos , Obesidad/terapia , Fenotipo , Receptor de Melanocortina Tipo 4/genética , Proteínas Represoras/genética , Regulación hacia Arriba , Aumento de PesoRESUMEN
Most monogenic cases of obesity in humans have been linked to mutations in genes encoding members of the leptin-melanocortin pathway. Specifically, mutations in MC4R, the melanocortin-4 receptor gene, account for 3-5% of all severe obesity cases in humans1-3. Recently, ADCY3 (adenylyl cyclase 3) gene mutations have been implicated in obesity4,5. ADCY3 localizes to the primary cilia of neurons 6 , organelles that function as hubs for select signaling pathways. Mutations that disrupt the functions of primary cilia cause ciliopathies, rare recessive pleiotropic diseases in which obesity is a cardinal manifestation 7 . We demonstrate that MC4R colocalizes with ADCY3 at the primary cilia of a subset of hypothalamic neurons, that obesity-associated MC4R mutations impair ciliary localization and that inhibition of adenylyl cyclase signaling at the primary cilia of these neurons increases body weight. These data suggest that impaired signaling from the primary cilia of MC4R neurons is a common pathway underlying genetic causes of obesity in humans.