The C57BL/6N inbred lines of mice are widely used in genetic research. They are particularly favoured in large scale studies such as the International Mouse Phenotyping Consortium (IMPC), where C57BL/6N mice are genetically altered to generate a collection of null alleles (currently more than 8500 null alleles have been generated). In this project, mice carrying null alleles are subjected to a pipeline of broad-based phenotyping tests to produce wide ranging phenotyping data on each model. We have previously described the development of a Home Cage Analysis system that automatically tracks the activity of group housed mice from a microchip inserted in the groin. This platform allows assessment of multiple biologically relevant phenotypes over long periods of time without experimenter interference, and therefore is particularly suited for high through-put studies. To investigate the impact of microchips on other tests carried out in the IMPC pipeline, we inserted microchips in 12 male and 12 female C57BL/6Ntac mice at seven weeks of age. Starting at nine weeks of age these mice underwent standard phenotyping tests, concurrently with 20 unchipped C57BL/6Ntac mice (10 females, 10 males). Tissues from a subset of the microchipped mice (six males and six females), chosen at random, were also sent for histopathological examination at the end of the phenotyping pipeline. No significant impact of insertion of microchip was observed in any of the phenotyping tests apart from bone mineral density measurement at DEXA due to the nature of the microchip. We therefore recommend that the microchip be inserted during the DEXA procedure, after the measurement is taken but before the mouse has recovered from the anaesthetic. This would avoid multiple anaesthetic exposures and prevent the potential variability in DEXA analysis output.
Male and female mice were anesthetized by intraperitoneal injection with a mixture delivering 0.5 mg/kg medetomidine and 50 mg/kg ketamine to achieve immobilization for whole-body radiographs and bone densitometry, as part of a phenotypic screen for bone and mineral disorders in mice carrying genetic modifications induced through mutagenesis with N'-ethyl-N'-nitrosourea. Morbidity and mortality occurred in 19 of 628 (3%) of male mice 24 to 72 h after a seemingly uneventful recovery from anesthesia. No morbidity or mortality occurred in 1564 female mice that were similar in age to the affected male mice and that underwent the same procedure. Of the 7 male mice that underwent postmortem examinations, 5 had urinary bladders grossly distended with urine and 1 had ascites. In addition, the pelvic or penile urethra in 5 of the examined male mice was obstructed with seminal coagulum associated with varying degrees of erosion of the urothelial lining and inflammation of the urethra. In 2 of these animals, from which plasma samples were recovered, azotemia, hyperphosphatemia, and hyperkalemia were present. The predilection for delayed morbidity and mortality in males after anesthesia suggests that anesthesia with 0.5 mg/kg medetomidine and 50 mg/kg ketamine is a potential risk factor for obstructive uropathy due to release of seminal coagulum. This adverse effect did not recur when we altered our anesthesia protocol to 10 mg/kg xylazine and 100 mg/kg ketamine.
The adaptor protein-2 sigma subunit (AP2σ), encoded by AP2S1, forms a heterotetrameric complex, with AP2α, AP2β, and AP2μ subunits, that is pivotal for clathrin-mediated endocytosis, and AP2σ loss-of-function mutations impair internalization of the calcium-sensing receptor (CaSR), a G-protein-coupled receptor, and cause familial hypocalciuric hypercalcemia type-3 (FHH3). Mice with AP2σ mutations that would facilitate investigations of the in vivo role of AP2σ, are not available, and we therefore embarked on establishing such mice. We screened >10,000 mice treated with the mutagen N-ethyl-N-nitrosourea (ENU) for Ap2s1 mutations and identified 5 Ap2s1 variants, comprising 2 missense (Tyr20Asn and Ile123Asn) and 3 intronic base substitutions, one of which altered the invariant donor splice site dinucleotide gt to gc. Three-dimensional modeling and cellular expression of the missense Ap2s1 variants did not reveal them to alter AP2σ structure or CaSR-mediated signaling, but investigation of the donor splice site variant revealed it to result in an in-frame deletion of 17 evolutionarily conserved amino acids (del17) that formed part of the AP2σ α1-helix, α1-β3 loop, and β3 strand. Heterozygous mutant mice (Ap2s1+/del17 ) were therefore established, and these had AP2σ haplosufficiency but were viable with normal appearance and growth. Ap2s1+/del17 mice, when compared with Ap2s1+/+ mice, also had normal plasma concentrations of calcium, phosphate, magnesium, creatinine, urea, sodium, potassium, and alkaline phosphatase activity; normal urinary fractional excretion of calcium, phosphate, sodium, and potassium; and normal plasma parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D (1,25(OH)2) concentrations. However, homozygous Ap2s1del17/del17 mice were non-viable and died between embryonic days 3.5 and 9.5 (E3.5-9.5), thereby indicating that AP2σ likely has important roles at the embryonic patterning stages and organogenesis of the heart, thyroid, liver, gut, lungs, pancreas, and neural systems. Thus, our studies have established a mutant mouse model that is haplosufficient for AP2σ.
Abstract Mutations of the adaptor protein-2 sigma subunit ( AP2S1 ) gene which encodes AP2σ2, a component of the ubiquitous AP2 heterotetrameric complex involved in endosomal trafficking of the calcium-sensing receptor (CaSR), cause familial hypocalciuric hypercalcemia type 3 (FHH3). FHH3 patients have heterozygous AP2S1 missense Arg15 mutations (p.Arg15Cys, p.Arg15His or p.Arg15Leu) with marked hypercalcemia and occasional hypophosphatemia and osteomalacia. To further characterise the phenotypic spectrum and calcitropic pathophysiology of FHH3, we used CRISPR/Cas9 genome editing to generate mice harboring the AP2S1 p.Arg15Leu mutation, which causes the most severe FHH3 phenotype. Heterozygous ( Ap2s1 +/L15 ) mice were viable, and had marked hypercalcemia, hypermagnesemia, hypophosphatemia, and increased plasma concentrations of parathyroid hormone, fibroblast growth factor 23 and alkaline phosphatase activity, but normal pro-collagen type 1 N-terminal pro-peptide and 1,25 dihydroxyvitamin D. Homozygous ( Ap2s1 L15/L15 ) mice invariably died perinatally. The AP2S1 p.Arg15Leu mutation impaired protein-protein interactions between AP2σ2 and the other AP2 subunits, and the CaSR. Cinacalcet, a CaSR allosteric activator, ameliorated the hypercalcemia and elevated PTH concentrations, but not the diminished AP2σ2-CaSR interaction. Thus, our studies have established a mouse model with a germline loss-of-function AP2S1 mutation that is representative for FHH3 in humans, and demonstrated that cinacalcet corrects the abnormalities of plasma calcium and PTH.
Objective: Tofacitinib is a novel, oral Janus kinase inhibitor being investigated for psoriasis. This study assessed the relationship between pruritus and clinical signs of psoriasis (assessed by Physician’s Global Assessment [PGA]) in patients with moderate-to-severe chronic plaque psoriasis receiving tofacitinib.Methods: In this 16-week (12-week treatment period, 4-week observation period), double-blind, placebo-controlled, phase IIb study (NCT00678210), 197 patients were randomized to tofacitinib 2, 5 or 15 mg BID, or placebo. Pruritus was patient assessed using the Itch Severity Score (ISS), a 0–10 (10 = worst itching) rating scale recorded daily from baseline to week 2 and at study visits. Mediation modeling was used to determine relationships between ISS (average score weeks 2–12), PGA (average score weeks 2–12) and treatment groups.Results: Mediation analysis showed that 70.2–80.5% (p < 0.001 versus placebo) of tofacitinib’s effect on pruritus was direct, and mostly independent of improvements in erythema, induration and scaling. ISS measurements had acceptable test–retest reliability. Correlation analyses with clinical outcomes supported the validity of the ISS as a pruritus measure.Conclusions: Tofacitinib has a direct, beneficial effect on patient-reported pruritus independent from improvements in clinician-reported psoriasis severity signs. The ISS demonstrated favorable psychometric characteristics, supporting its use as a pruritus assessment tool.
The calcium-sensing receptor (CaSR) is a family C G-protein–coupled receptor that plays a pivotal role in extracellular calcium homeostasis. The CaSR is also highly expressed in pancreatic islet α- and β-cells that secrete glucagon and insulin, respectively. To determine whether the CaSR may influence systemic glucose homeostasis, we characterized a mouse model with a germline gain-of-function CaSR mutation, Leu723Gln, referred to as Nuclear flecks (Nuf). Heterozygous- (CasrNuf/+) and homozygous-affected (CasrNuf/Nuf) mice were shown to have hypocalcemia in association with impaired glucose tolerance and insulin secretion. Oral administration of a CaSR antagonist compound, known as a calcilytic, rectified the glucose intolerance and hypoinsulinemia of CasrNuf/+ mice and ameliorated glucose intolerance in CasrNuf/Nuf mice. Ex vivo studies showed CasrNuf/+ and CasrNuf/Nuf mice to have reduced pancreatic islet mass and β-cell proliferation. Electrophysiological analysis of isolated CasrNuf/Nuf islets showed CaSR activation to increase the basal electrical activity of β-cells independently of effects on the activity of the adenosine triphosphate (ATP)–sensitive K+ (KATP) channel. CasrNuf/Nuf mice also had impaired glucose-mediated suppression of glucagon secretion, which was associated with increased numbers of α-cells and a higher α-cell proliferation rate. Moreover, CasrNuf/Nuf islet electrophysiology demonstrated an impairment of α-cell membrane depolarization in association with attenuated α-cell basal KATP channel activity. These studies indicate that the CaSR activation impairs glucose tolerance by a combination of α- and β-cell defects and also influences pancreatic islet mass. Moreover, our findings highlight a potential application of targeted CaSR compounds for modulating glucose metabolism.
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