RESUMEN
Itch is a protective sensation producing a desire to scratch. Pathologic itch can be a chronic symptom of illnesses such as uremia, cholestatic liver disease, neuropathies and dermatitis, however current therapeutic options are limited. Many types of cell surface receptors, including those present on cells in the skin, on sensory neurons and on neurons in the spinal cord, have been implicated in itch signaling. The role of G protein signaling in the regulation of pruriception is poorly understood. We identify here 2 G protein signaling components whose mutation impairs itch sensation. R7bp (a.k.a. Rgs7bp) is a palmitoylated membrane anchoring protein expressed in neurons that facilitates Gαi/o -directed GTPase activating protein activity mediated by the Gß5/R7-RGS complex. Knockout of R7bp diminishes scratching responses to multiple cutaneously applied and intrathecally-administered pruritogens in mice. Knock-in to mice of a GTPase activating protein-insensitive mutant of Gαo (Gnao1 G184S/+) produces a similar pruriceptive phenotype. The pruriceptive defect in R7bp knockout mice was rescued in double knockout mice also lacking Oprk1, encoding the G protein-coupled kappa-opioid receptor whose activation is known to inhibit itch sensation. In a model of atopic dermatitis (eczema), R7bp knockout mice showed diminished scratching behavior and enhanced sensitivity to kappa opioid agonists. Taken together, our results indicate that R7bp is a key regulator of itch sensation and suggest the potential targeting of R7bp-dependent GTPase activating protein activity as a novel therapeutic strategy for pathological itch.
Asunto(s)
Nocicepción/fisiología , Prurito/genética , Prurito/metabolismo , Proteínas RGS/metabolismo , Sensación/genética , Animales , Alcanfor/análogos & derivados , Alcanfor/toxicidad , Células Cultivadas , Cromonas/toxicidad , Endotelina-1/toxicidad , Femenino , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/genética , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Ganglios Espinales/patología , Péptido Liberador de Gastrina/toxicidad , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación/genética , Péptido Natriurético Encefálico/toxicidad , Umbral del Dolor/fisiología , Prurito/inducido químicamente , Desempeño Psicomotor/fisiología , Proteínas RGS/genética , Receptores Opioides kappa/genética , Receptores Opioides kappa/metabolismo , Células Receptoras Sensoriales/efectos de los fármacos , Células Receptoras Sensoriales/fisiologíaRESUMEN
Genomic engineering by the guide RNA (gRNA)-directed CRISPR/CAS9 is rapidly becoming a method of choice for various biological systems. However, pressing concerns remain regarding its off-target activities and wide variations in efficacies. While next generation sequencing (NGS) has been primarily used to evaluate the efficacies and off-target activities of gRNAs, it only detects the imperfectly repaired double strand DNA breaks (DSB) by the error-prone non-homologous end joining (NHEJ) mechanism and may not faithfully represent the DSB activities because the efficiency of NHEJ-mediated repair varies depending on the local chromatin environment. Here we describe a reporter system for unbiased detection and comparison of DSB activities that promises to improve the chance of success in genomic engineering and to facilitate large-scale screening of CAS9 activities and gRNA libraries. Additionally, we demonstrated that the tolerances to mismatches between a gRNA and the corresponding target DNA can occur at any position of the gRNA, and depend on both specific gRNA sequences and CAS9 constructs used.
