Mouse mutant phenotyping at scale reveals novel genes controlling bone mineral density.

The genetic landscape of diseases associated with changes in bone mineral density (BMD), such as osteoporosis, is only partially understood. Here, we explored data from 3,823 mutant mouse strains for BMD, a measure that is frequently altered in a range of bone pathologies, including osteoporosis. A total of 200 genes were found to significantly affect BMD. This pool of BMD genes comprised 141 genes with previously unknown functions in bone biology and was complementary to pools derived from recent human studies. Nineteen of the 141 genes also caused skeletal abnormalities. Examination of the BMD genes in osteoclasts and osteoblasts underscored BMD pathways, including vesicle transport, in these cells and together with in silico bone turnover studies resulted in the prioritization of candidate genes for further investigation. Overall, the results add novel pathophysiological and molecular insight into bone health and disease.

INFRAFRONTIER quality principles in systemic phenotyping.

Improving reproducibility and replicability in preclinical research is a widely discussed and pertinent topic, especially regarding ethical responsibility in animal research. INFRAFRONTIER, the European Research Infrastructure for the generation, phenotyping, archiving, and distribution of model mammalian genomes, is addressing this issue by developing internal quality principles for its different service areas, that provides a quality framework for its operational activities. This article introduces the INFRAFRONTIER Quality Principles in Systemic Phenotyping of genetically altered mouse models. A total of 11 key principles are included, ranging from general requirements for compliance with guidelines on animal testing, to the need for well-trained personnel and more specific standards such as the exchange of reference lines. Recently established requirements such as the provision of FAIR (Findable, Accessible, Interoperable, Reusable) data are also addressed. For each quality principle, we have outlined the specific context, requirements, further recommendations, and key references.

Building for the future: essential infrastructure for rodent ageing studies.

When planning ageing research using rodent models, the logistics of supply, long term housing and infrastructure provision are important factors to take into consideration. These issues need to be prioritised to ensure they meet the requirements of experiments which potentially will not be completed for several years. Although these issues are not unique to this discipline, the longevity of experiments and indeed the animals, requires a high level of consistency and sustainability to be maintained throughout lengthy periods of time. Moreover, the need to access aged stock or material for more immediate experiments poses many issues for the completion of pilot studies and/or short term intervention studies on older models. In this article, we highlight the increasing demand for ageing research, the resources and infrastructure involved, and the need for large-scale collaborative programmes to advance studies in both a timely and a cost-effective way.

Talking welfare: the importance of a common language.

Ontologies describing mouse phenotypes and pathology are well established and becoming more universally used (Smith and Eppig in Mamm Genome 23:653, 2012; Scofield et al. in J Biomed Semant 4:18, 2013). However, the language used to describe and disseminate cage-side observations is less well developed. This article explores the hurdles to unifying a language and terminology, and introduces our initial attempt to do so.

Longitudinal home-cage automated assessment of climbing behavior shows sexual dimorphism and aging-related decrease in C57BL/6J healthy mice and allows early detection of motor impairment in the N171-82Q mouse model of Huntington's disease.

Monitoring the activity of mice within their home cage is proving to be a powerful tool for revealing subtle and early-onset phenotypes in mouse models. Video-tracking, in particular, lends itself to automated machine-learning technologies that have the potential to improve the manual annotations carried out by humans. This type of recording and analysis is particularly powerful in objective phenotyping, monitoring behaviors with no experimenter intervention. Automated home-cage testing allows the recording of non-evoked voluntary behaviors, which do not require any contact with the animal or exposure to specialist equipment. By avoiding stress deriving from handling, this approach, on the one hand, increases the welfare of experimental animals and, on the other hand, increases the reliability of results excluding confounding effects of stress on behavior. In this study, we show that the monitoring of climbing on the wire cage lid of a standard individually ventilated cage (IVC) yields reproducible data reflecting complex phenotypes of individual mouse inbred strains and of a widely used model of neurodegeneration, the N171-82Q mouse model of Huntington's disease (HD). Measurements in the home-cage environment allowed for the collection of comprehensive motor activity data, which revealed sexual dimorphism, daily biphasic changes, and aging-related decrease in healthy C57BL/6J mice. Furthermore, home-cage recording of climbing allowed early detection of motor impairment in the N171-82Q HD mouse model. Integrating cage-floor activity with cage-lid activity (climbing) has the potential to greatly enhance the characterization of mouse strains, detecting early and subtle signs of disease and increasing reproducibility in preclinical studies.

Loss of Frrs1l disrupts synaptic AMPA receptor function, and results in neurodevelopmental, motor, cognitive and electrographical abnormalities.

Loss-of-function mutations in a human AMPA receptor-associated protein, ferric chelate reductase 1-like (FRRS1L), are associated with a devastating neurological condition incorporating choreoathetosis, cognitive deficits and epileptic encephalopathies. Furthermore, evidence from overexpression and ex vivo studies has implicated FRRS1L in AMPA receptor biogenesis, suggesting that changes in glutamatergic signalling might underlie the disorder. Here, we investigated the neurological and neurobehavioural correlates of the disorder using a mouse Frrs1l null mutant. The study revealed several neurological defects that mirrored those seen in human patients. We established that mice lacking Frrs1l suffered from a broad spectrum of early-onset motor deficits with no progressive, age-related deterioration. Moreover, Frrs1l-/- mice were hyperactive, irrespective of test environment, exhibited working memory deficits and displayed significant sleep fragmentation. Longitudinal electroencephalographic (EEG) recordings also revealed abnormal EEG results in Frrs1l-/- mice. Parallel investigations into disease aetiology identified a specific deficiency in AMPA receptor levels in the brain of Frrs1l-/- mice, while the general levels of several other synaptic components remained unchanged, with no obvious alterations in the number of synapses. Furthermore, we established that Frrsl1 deletion results in an increased proportion of immature AMPA receptors, indicated by incomplete glycosylation of GLUA2 (also known as GRIA2) and GLUA4 (also known as GRIA4) AMPA receptor proteins. This incomplete maturation leads to cytoplasmic retention and a reduction of those specific AMPA receptor levels in the postsynaptic membrane. Overall, this study determines, for the first time in vivo, how loss of FRRS1L function can affect glutamatergic signalling, and provides mechanistic insight into the development and progression of a human hyperkinetic disorder.This article has an associated First Person interview with the first author of the paper.

Loss of arylformamidase with reduced thymidine kinase expression leads to impaired glucose tolerance.

Tryptophan metabolites have been linked in observational studies with type 2 diabetes, cognitive disorders, inflammation and immune system regulation. A rate-limiting enzyme in tryptophan conversion is arylformamidase (Afmid), and a double knockout of this gene and thymidine kinase (Tk) has been reported to cause renal failure and abnormal immune system regulation. In order to further investigate possible links between abnormal tryptophan catabolism and diabetes and to examine the effect of single Afmid knockout, we have carried out metabolic phenotyping of an exon 2 Afmid gene knockout. These mice exhibit impaired glucose tolerance, although their insulin sensitivity is unchanged in comparison to wild-type animals. This phenotype results from a defect in glucose stimulated insulin secretion and these mice show reduced islet mass with age. No evidence of a renal phenotype was found, suggesting that this published phenotype resulted from loss of Tk expression in the double knockout. However, despite specifically removing only exon 2 of Afmid in our experiments we also observed some reduction of Tk expression, possibly due to a regulatory element in this region. In summary, our findings support a link between abnormal tryptophan metabolism and diabetes and highlight beta cell function for further mechanistic analysis.

Visceral obesity without insulin resistance in late-onset obesity rats.

We describe a line of transgenic rats in which the males develop a unique autosomal dominant, late-onset obesity (LOB) phenotype. LOB males gradually accumulate fat specifically in visceral, but not peripheral, fat depots despite a normal intake of a low fat diet. LOB females normally develop only mild obesity with advanced age. However, the phenotype can be induced rapidly in young females by ovariectomy and prevented by estrogen replacement. LOB males are highly sensitive to dietary fat. Young, nonobese LOB males gain more weight on a 30% fat diet and lose more weight when treated with the lipase inhibitor, Orlistat, than their nontransgenic littermates. Remarkably, despite severe visceral obesity, LOB rats have normal fasting blood glucose, insulin, and corticosterone; show normal or increased insulin sensitivity in glucose and insulin tolerance tests; have increased plasma adiponectin levels; and display a heightened response to treatment with rosiglitazone. Their visceral adiposity reflects a specific increase in visceral adipocyte number, not size. Analysis of the transgene in LOB rats revealed a deletion in the gene encoding the S26 subunit of the mitochondrial ribosome that results in the production of a truncated protein, which we show to be imported into mitochondria. However, the transgene integrant is complex, so whether this is the sole molecular disruption underlying this phenotype remains to be established. Nevertheless, LOB rats provide a valuable new model of late-onset, male-preponderant, visceral-specific obesity, clearly dissociated from insulin resistance.

Transgenesis and neuroendocrine physiology: a transgenic rat model expressing growth hormone in vasopressin neurones.

Human growth hormone (hGH) and bovine neurophysin (bNP) DNA reporter fragments were inserted into the rat vasopressin (VP) and oxytocin (OT) genes in a 44 kb cosmid construct used to generate two lines of transgenic rats, termed JP17 and JP59. Both lines showed specific hGH expression in magnocellular VP cells in the hypothalamic paraventricular (PVN) and supraoptic nuclei (SON). hGH was also expressed in parvocellular neurones in suprachiasmatic nuclei (SCN), medial amygdala and habenular nuclei in JP17 rats; the rat OT-bNP (rOT-bNP) transgene was not expressed in either line. Immunohistochemistry and radioimmunoassay showed hGH protein in the hypothalamus from where it was transported in varicose fibres via the median eminence to the posterior pituitary gland. Immunogold electron microscopy showed hGH co-stored with VP-NP in the same granules. The VP-hGH transgene did not affect water balance, VP storage or release in vivo. Drinking 2 % saline for 72 h increased hypothalamic transgene hGH mRNA expression, and depleted posterior pituitary hGH and VP stores in parallel. In anaesthetised, water-loaded JP17 rats, hGH was released with VP in response to an acute hypovolumic stimulus (sodium nitrosopentacyano, 400 microg I.V.). JP17 rats had a reduced growth rate, lower anterior pituitary rGH contents, and a reduced amplitude of endogenous pulsatile rGH secretion assessed by automated blood microsampling in conscious rats, consistent with a short-loop feedback of the VP-hGH on the endogenous GH axis. This transgenic rat model enables us to study physiological regulation of hypothalamic transgene protein production, transport and secretion, as well as its effects on other neuroendocrine systems in vivo.