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Oxidative stress is a common etiological feature of neurological disorders, although the pathways that govern defence against reactive oxygen species (ROS) in neurodegeneration remain unclear. We have identified the role of oxidation resistance 1 (Oxr1) as a vital protein that controls the sensitivity of neuronal cells to oxidative stress; mice lacking Oxr1 display cerebellar neurodegeneration, and neurons are less susceptible to exogenous stress when the gene is over-expressed. A conserved short isoform of Oxr1 is also sufficient to confer this neuroprotective property both in vitro and in vivo. In addition, biochemical assays indicate that Oxr1 itself is susceptible to cysteine-mediated oxidation. Finally we show up-regulation of Oxr1 in both human and pre-symptomatic mouse models of amyotrophic lateral sclerosis, indicating that Oxr1 is potentially a novel neuroprotective factor in neurodegenerative disease.

Inflammation of the middle ear, known clinically as chronic otitis media, presents in different forms, such as chronic otitis media with effusion (COME; glue ear) and chronic suppurative otitis media (CSOM). These are highly prevalent diseases, especially in childhood, and lead to significant morbidity worldwide. However, much remains unclear about this disease, including its aetiology, initiation and perpetuation, and the relative roles of mucosal and leukocyte biology, pathogens, and Eustachian tube function. Chronic otitis media is commonly modelled in mice but most existing models only partially mimic human disease and many are syndromic. Nevertheless, these models have provided insights into potential disease mechanisms, and have implicated altered immune signalling, mucociliary function and Eustachian tube function as potential predisposing mechanisms. Clinical studies of chronic otitis media have yet to implicate a particular molecular pathway or mechanism, and current human genetic studies are underpowered. We also do not fully understand how existing interventions, such as tympanic membrane repair, work, nor how chronic otitis media spontaneously resolves. This Clinical Puzzle article describes our current knowledge of chronic otitis media and the existing research models for this condition. It also identifies unanswered questions about its pathogenesis and treatment, with the goal of advancing our understanding of this disease to aid the development of novel therapeutic interventions.

Otitis media with effusion (OME) is the commonest cause of hearing loss in children, yet the underlying genetic pathways and mechanisms involved are incompletely understood. Ventilation of the middle ear with tympanostomy tubes is the commonest surgical procedure in children and the best treatment for chronic OME, but the mechanism by which they work remains uncertain. As hypoxia is a common feature of inflamed microenvironments, moderation of hypoxia may be a significant contributory mechanism. We have investigated the occurrence of hypoxia and hypoxia-inducible factor (HIF) mediated responses in Junbo and Jeff mouse mutant models, which develop spontaneous chronic otitis media. We found that Jeff and Junbo mice labeled in vivo with pimonidazole showed cellular hypoxia in inflammatory cells in the bulla lumen, and in Junbo the middle ear mucosa was also hypoxic. The bulla fluid inflammatory cell numbers were greater and the upregulation of inflammatory gene networks were more pronounced in Junbo than Jeff. Hif-1α gene expression was elevated in bulla fluid inflammatory cells, and there was upregulation of its target genes including Vegfa in Junbo and Jeff. We therefore investigated the effects in Junbo of small-molecule inhibitors of VEGFR signaling (PTK787, SU-11248, and BAY 43-9006) and destabilizing HIF by inhibiting its chaperone HSP90 with 17-DMAG. We found that both classes of inhibitor significantly reduced hearing loss and the occurrence of bulla fluid and that VEGFR inhibitors moderated angiogenesis and lymphangiogenesis in the inflamed middle ear mucosa. The effectiveness of HSP90 and VEGFR signaling inhibitors in suppressing OM in the Junbo model implicates HIF-mediated VEGF as playing a pivotal role in OM pathogenesis. Our analysis of the Junbo and Jeff mutants highlights the role of hypoxia and HIF-mediated pathways, and we conclude that targeting molecules in HIF-VEGF signaling pathways has therapeutic potential in the treatment of chronic OM.

Acute otitis media, inflammation of the middle ear, is the most common bacterial infection in children and, as a consequence, is the most common reason for antimicrobial prescription to this age group. There is currently no effective vaccine for the principal pathogen involved, non-typeable Haemophilus influenzae (NTHi). The most frequently used and widely accepted experimental animal model of middle ear infection is in chinchillas, but mice and gerbils have also been used. We have established a robust model of middle ear infection by NTHi in the Junbo mouse, a mutant mouse line that spontaneously develops chronic middle ear inflammation in specific pathogen-free conditions. The heterozygote Junbo mouse (Jbo/+) bears a mutation in a gene (Evi1, also known as Mecom) that plays a role in host innate immune regulation; pre-existing middle ear inflammation promotes NTHi middle ear infection. A single intranasal inoculation with NTHi produces high rates (up to 90%) of middle ear infection and bacterial titres (104-105 colony-forming units/µl) in bulla fluids. Bacteria are cleared from the majority of middle ears between day 21 and 35 post-inoculation but remain in approximately 20% of middle ears at least up to day 56 post-infection. The expression of Toll-like receptor-dependent response cytokine genes is elevated in the middle ear of the Jbo/+ mouse following NTHi infection. The translational potential of the Junbo model for studying antimicrobial intervention regimens was shown using a 3 day course of azithromycin to clear NTHi infection, and its potential use in vaccine development studies was shown by demonstrating protection in mice immunized with killed homologous, but not heterologous, NTHi bacteria.

Otitis media (OM) is among the most common illnesses of early childhood, characterised by the presence of inflammation in the middle ear cavity. Acute OM and chronic OM with effusion (COME) affect the majority of children by school age and have heritability estimates of 40-70%. However, the majority of genes underlying this susceptibility are, as yet, unidentified. One method of identifying genes and pathways that may contribute to OM susceptibility is to look at mouse mutants displaying a comparable phenotype. Single-gene mouse mutants with OM have identified a number of genes, namely, Eya4, Tlr4, p73, MyD88, Fas, E2f4, Plg, Fbxo11, and Evi1, as potential and biologically relevant candidates for human disease. Recent studies suggest that this “mouse-to-human” approach is likely to yield relevant data, with significant associations reported between polymorphisms at the FBXO11, TLR4, and PAI1 genes and disease in humans. An association between TP73 and chronic rhinosinusitis has also been reported. In addition, the biobanks of available mouse mutants provide a powerful resource for functional studies of loci identified by future genome-wide association studies of OM in humans. Mouse models of OM therefore are an important component of current approaches attempting to understand the complex genetic susceptibility to OM in humans, and which aim to facilitate the development of preventative and therapeutic interventions for this important and common disease.

The extracellular calcium-sensing receptor (CaSR) plays a pivotal role in the regulation of extracellular calcium such that abnormalities, which result in a loss or gain of function, lead to hypercalcemia or hypocalcemia, respectively, in patients. Mice carrying CaSR knockout alleles develop hypercalcemia that mimics the disorders observed in humans. To date, there is no mouse model for an activating CaSR mutation. Here, we describe such a mouse model, named Nuf, originally identified for having opaque flecks in the nucleus of the lens in a screen for eye mutants. Nuf mice also display ectopic calcification, hypocalcemia, hyperphosphatemia, and inappropriately reduced levels of plasma parathyroid hormone. These features are similar to those observed in patients with autosomal dominant hypocalcemia. Inheritance studies of Nuf mice revealed that the trait was transmitted in an autosomal-dominant manner, and mapping studies located the locus to chromosome 16, in the vicinity of the CaSR gene (Mouse Genome Database symbol Gprc2a). DNA sequence analysis revealed the presence of a Gprc2a missense mutation, Leu723Gln. Transient expression of wild-type and mutant CaSRs in human embryonic kidney 293 cells demonstrated that the mutation resulted in a gain of function of the CaSR, which had a significantly lower EC50. Thus, our results have identified a mouse model for an activating CaSR mutation, and the development of ectopic calcification and cataract formation, which tended to be milder in the heterozygote Nuf mice, indicates that an evaluation for such abnormalities in autosomal dominant hypocalcemia patients who have activating CaSR mutations is required.

GNAS/Gnas encodes G(s)α that is mainly biallelically expressed but shows imprinted expression in some tissues. In Albright Hereditary Osteodystrophy (AHO) heterozygous loss of function mutations of GNAS can result in ectopic ossification that tends to be superficial and attributable to haploinsufficiency of biallelically expressed G(s)α. Oed-Sml is a point missense mutation in exon 6 of the orthologous mouse locus Gnas. We report here both the late onset ossification and occurrence of benign cutaneous fibroepithelial polyps in Oed-Sml. These phenotypes are seen on both maternal and paternal inheritance of the mutant allele and are therefore due to an effect on biallelically expressed G(s)α. The ossification is confined to subcutaneous tissues and so resembles the ossification observed with AHO. Our mouse model is the first with both subcutaneous ossification and fibroepithelial polyps related to G(s)α deficiency. It is also the first mouse model described with a clinically relevant phenotype associated with a point mutation in G(s)α and may be useful in investigations of the mechanisms of heterotopic bone formation. Together with earlier results, our findings indicate that G(s)α signalling pathways play a vital role in repressing ectopic bone formation.

Mutations of UDP-N-acetyl-alpha-D-galactosamine polypeptide N-acetyl galactosaminyl transferase 3 (GALNT3) result in familial tumoural calcinosis (FTC) and the hyperostosis-hyperphosphataemia syndrome (HHS), which are autosomal recessive disorders characterised by soft-tissue calcification and hyperphosphataemia. To facilitate in vivo studies of these heritable disorders of phosphate homeostasis, we embarked on establishing a mouse model by assessing progeny of mice treated with the chemical mutagen N-ethyl-N-nitrosourea (ENU), and identified a mutant mouse, TCAL, with autosomal recessive inheritance of ectopic calcification, which involved multiple tissues, and hyperphosphataemia; the phenotype was designated TCAL and the locus, Tcal. TCAL males were infertile with loss of Sertoli cells and spermatozoa, and increased testicular apoptosis. Genetic mapping localized Tcal to chromosome 2 (62.64-71.11 Mb) which contained the Galnt3. DNA sequence analysis identified a Galnt3 missense mutation (Trp589Arg) in TCAL mice. Transient transfection of wild-type and mutant Galnt3-enhanced green fluorescent protein (EGFP) constructs in COS-7 cells revealed endoplasmic reticulum retention of the Trp589Arg mutant and Western blot analysis of kidney homogenates demonstrated defective glycosylation of Galnt3 in Tcal/Tcal mice. Tcal/Tcal mice had normal plasma calcium and parathyroid hormone concentrations; decreased alkaline phosphatase activity and intact Fgf23 concentrations; and elevation of circulating 1,25-dihydroxyvitamin D. Quantitative reverse transcriptase-PCR (qRT-PCR) revealed that Tcal/Tcal mice had increased expression of Galnt3 and Fgf23 in bone, but that renal expression of Klotho, 25-hydroxyvitamin D-1α-hydroxylase (Cyp27b1), and the sodium-phosphate co-transporters type-IIa and -IIc was similar to that in wild-type mice. Thus, TCAL mice have the phenotypic features of FTC and HHS, and provide a model for these disorders of phosphate metabolism.

Otitis media (OM), inflammation of the middle ear, remains the most common cause of hearing impairment in children. It is also the most common cause of surgery in children in the developed world. There is evidence from studies of the human population and mouse models that there is a significant genetic component predisposing to OM, yet nothing is known about the underlying genetic pathways involved in humans. We identified an N-ethyl-N-nitrosourea-induced dominant mouse mutant Junbo with hearing loss due to chronic suppurative OM and otorrhea. This develops from acute OM that arises spontaneously in the postnatal period, with the age of onset and early severity dependent on the microbiological status of the mice and their air quality. We have identified the causal mutation, a missense change in the C-terminal zinc finger region of the transcription factor Evi1. This protein is expressed in middle ear basal epithelial cells, fibroblasts, and neutrophil leukocytes at postnatal day 13 and 21 when inflammatory changes are underway. The identification and characterization of the Junbo mutant elaborates a novel role for Evi1 in mammalian disease and implicates a new pathway in genetic predisposition to OM.

Chronic otitis media (OM) is common in Down syndrome (DS), but underlying aetiology is unclear. We analysed the entire available mouse resource of partial trisomy models of DS looking for histological evidence of chronic middle-ear inflammation. We found a highly penetrant OM in the Dp(16)1Yey mouse, which carries a complete trisomy of MMU16. No OM was found in the Dp(17)1Yey mouse or the Dp(10)1Yey mouse, suggesting disease loci are located only on MMU16. The Ts1Cje, Ts1RhR, Ts2Yah, and Ts65Dn trisomies and the transchomosomic Tc1 mouse did not develop OM. On the basis of these findings, we propose a two-locus model for chronic middle-ear inflammation in DS, based upon epistasis of the regions of HSA21 not in trisomy in the Tc1 mouse. We also conclude that environmental factors likely play an important role in disease onset.