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Low bone mass and an increased risk of fracture are predictors of osteoporosis. Individuals who share the same bone-mineral density (BMD) vary in their fracture risk, suggesting that microstructural architecture is an important determinant of skeletal strength. Here, we utilized the rich diversity of the Collaborative Cross mice to identify putative causal genes that contribute to the risk of fractures. Using microcomputed tomography, we examined key structural features that pertain to bone quality in the femoral cortical and trabecular compartments of male and female mice. We estimated the broad-sense heritability to be 50–60% for all examined traits, and we identified five quantitative trait loci (QTL) significantly associated with six traits. We refined each QTL by combining information inferred from the ancestry of the mice, ranging from RNA-Seq data and published literature to shortlist candidate genes. We found strong evidence for new candidate genes, particularly Rhbdf2, whose close association with the trabecular bone volume fraction and number was strongly suggested by our analyses. We confirmed our findings with mRNA expression assays of Rhbdf2 in extreme-phenotype mice, and by phenotyping bones of Rhbdf2 knockout mice. Our results indicate that Rhbdf2 plays a decisive role in bone mass accrual and microarchitecture.

Animals have evolved multiple mechanisms to protect themselves from the cumulative effects of age-related cellular damage. Here we reveal an unexpected link between the TNF (tumour necrosis factor) inflammatory pathway, triggered by the metalloprotease ADAM17/TACE, and a lipid droplet (LD)-mediated mechanism of protecting retinal cells from age related degeneration. Loss of ADAM17, TNF and the TNF receptor Grindelwald in pigmented glial cells of theDrosophilaretina leads to age related degeneration of both glia and neurons, preceded by an abnormal accumulation of glial LDs. We show that the glial LDs initially buffer the cells against damage caused by neuronally generated reactive oxygen species (ROS), but that in later life the LDs dissipate, leading to the release of toxic peroxidated lipids. Finally, we demonstrate the existence of a conserved pathway in human iPS-derived microglia-like cells, which are central players in neurodegeneration. Overall, we have discovered a pathway mediated by TNF signalling acting not as a trigger of inflammation, but as a cytoprotective factor in the retina.

Macrophages are prominent cells in acute and chronic inflammatory diseases. Recent studies highlight a role for macrophage proliferation post-monocyte recruitment under inflammatory conditions. Using an acute peritonitis model, we identify a significant defect in macrophage proliferation in mice lacking the leukocyte transmembrane protease ADAM17. The defect is associated with decreased levels of macrophage colony-stimulating factor 1 (CSF-1) in the peritoneum and is rescued by intraperitoneal injection of CSF-1. Cell surface CSF-1 (csCSF-1) is one of the substrates of ADAM17. We demonstrate that both infiltrated neutrophils and macrophages are major sources of csCSF-1. Furthermore, acute shedding of csCSF-1 following neutrophil extravasation is associated with elevated expression of iRhom2, a member of the rhomboid-like superfamily, which promotes ADAM17 maturation and trafficking to the neutrophil surface. Accordingly, deletion of hematopoietic iRhom2 is sufficient to prevent csCSF-1 release from neutrophils and macrophages and to prevent macrophage proliferation. In acute inflammation, csCSF-1 release and macrophage proliferation are self-limiting due to transient leukocyte recruitment and temporally restricted csCSF-1 expression. In chronic inflammation, such as atherosclerosis, the ADAM17-mediated lesional macrophage proliferative response is prolonged. Our results demonstrate a novel mechanism whereby ADAM17 promotes macrophage proliferation in states of acute and chronic inflammation.

Recent studies highlight a role for macrophage proliferation post monocyte recruitment in inflammatory conditions, such as atherosclerosis. However, the mechanisms regulating macrophage proliferation are not well understood. Using an acute peritonitis model, we identified a 40 ± 8% reduction (mean ± SEM; n=5; p

Osteoporosis, the most common bone disease, is characterized by a low bone mass and increased risk of fractures. Importantly, individuals with the same bone mineral density (BMD), as measured on two dimensional (2D) radiographs, have different risks for fracture, suggesting that microstructural architecture is an important determinant of skeletal strength. Here we took advantage of the rich phenotypic and genetic diversity of the Collaborative Cross (CC) mice. Using microcomputed tomography, we examined key structural parameters in the femoral cortical and trabecular compartments of male and female mice from 34 CC lines. These traits included the trabecular bone volume fraction, number, thickness, connectivity, and spacing, as well as structural morphometric index. In the mid-diaphyseal cortex, we recorded cortical thickness and volumetric BMD.The broad-sense heritability of these traits ranged between 50 to 60%. We conducted a genome-wide association study to unravel 5 quantitative trait loci (QTL) significantly associated with 6 of the traits. We refined each locus by combining information obtained from the known ancestry of the mice and RNA-Seq data from publicly available sources, to shortlist potential candidate genes. We found strong evidence for new candidate genes, includingRhbdf2, which association to trabecular bone volume fraction and number was strongly suggested by our analyses. We then examined knockout mice, and validated the causal action ofRhbdf2on bone mass accrual and microarchitecture.Our approach revealed new genome-wide QTLs and a series of genes that have never been associated with bone microarchitecture. This study demonstrates for the first time the skeletal role ofRhbdf2on the physiological remodeling of both the cortical and trabecular bone. This newly assigned function forRhbdf2can prove useful in deciphering the predisposing factors of osteoporosis and propose new investigative avenues toward targeted therapeutic solutions.Author summaryIn this study, we used the novel mouse reference population, the Collaborative Cross (CC), to identify new causal genes in the regulation of bone microarchitecture, a critical determinant of bone strength. This approach provides a clear advantage in terms of resolution and dimensionality of the morphometric features (versus humans) and rich allelic diversity (versus classical mouse populations), over current practices of bone-related genome-wide association studies.Our genome-wide study revealed 5 loci significantly associated with microstructural traits in the cortical and trabecular bone. We found strong evidence for new candidate genes, in particular,Rhbdf2.We then validated the specific role ofRhbdf2on bone mass accrual and microarchitecture using knockout mice. Importantly, this study is the first demonstration of a physiological role forRhbdf2.

Keratin 16 (K16) is a cytoskeletal scaffolding protein highly expressed at pressure-bearing sites of the mammalian footpad. It can be induced in hyperproliferative states such as wound healing, inflammation and cancer. Here we show that the inactive rhomboid protease RHBDF2 (iRHOM2) regulates thickening of the footpad epidermis through its interaction with K16. K16 expression is absent in the thinned footpads of irhom2−/− mice compared with irhom2+/+mice, due to reduced keratinocyte proliferation. Gain-of-function mutations in iRHOM2 underlie Tylosis with oesophageal cancer (TOC), characterized by palmoplantar thickening, upregulate K16 with robust downregulation of its type II keratin binding partner, K6. By orchestrating the remodelling and turnover of K16, and uncoupling it from K6, iRHOM2 regulates the epithelial response to physical stress. These findings contribute to our understanding of the molecular mechanisms underlying hyperproliferation of the palmoplantar epidermis in both physiological and disease states, and how this ‘stress' keratin is regulated., Keratin 16 is an epithelial protein highly expressed at pressure bearing sites and during wound healing and cancer. Here the authors show that K16 interacts with the inactive protease Rhbdf2, associated with Tylosis with oesophageal cancer, and that this interaction drives increased keratinocyte proliferation.

Irradiated or injured cells enter apoptosis, and in turn, promote proliferation of surrounding unaffected cells. In Drosophila, apoptotic cells have an active role in proliferation, where the caspase Dronc and p53 induce mitogen expression and growth in the surrounding tissues. The Drosophila p53 gene structure is conserved and encodes at least two protein isoforms: a full-length isoform (Dp53) and an N-terminally truncated isoform (DDNp53). Historically, DDNp53 was the first p53 isoform identified and was thought to be responsible for all p53 biological activities. It was shown that DDNp53 induces apoptosis by inducing the expression of IAP antagonists, such as Reaper. Here we investigated the roles of Dp53 and DDNp53 in apoptosis and apoptosis- induced proliferation. We found that both isoforms were capable of activating apoptosis, but that they each induced distinct IAP antagonists. Expression of DDNp53 induced Wingless (Wg) expression and enhanced proliferation in both ‘undead cells’ and in ‘genuine’ apoptotic cells. In contrast to DDNp53, Dp53 did not induce Wg expression in the absence of the endogenous p53 gene. Thus, we propose that DDNp53 is the main isoform that regulates apoptosis-induced proliferation. Understanding the roles of Drosophila p53 isoforms in apoptosis and in apoptosis-induced proliferation may shed new light on the roles of p53 isoforms in humans, with important implications in cancer biology.

The unfolded protein response (UPR) is a specific cellular process that allows the cell to cope with the overload of unfolded/misfolded proteins in the endoplasmic reticulum (ER). ER stress is commonly associated with degenerative pathologies, but its role in disease progression is still a matter for debate. Here, we found that mutations in the ER-resident chaperone, neither inactivation nor afterpotential A (NinaA), lead to mild ER stress, protecting photoreceptor neurons from various death stimuli in adult Drosophila. In addition, Drosophila S2 cultured cells, when pre-exposed to mild ER stress, are protected from H(2)O(2), cycloheximide- or ultraviolet-induced cell death. We show that a specific ER-mediated signal promotes antioxidant defences and inhibits caspase-dependent cell death. We propose that an immediate consequence of the UPR not only limits the accumulation of misfolded proteins but also protects tissues from harmful exogenous stresses.

The Drosophila eye is widely used as a model for studies of development and neuronal degeneration. With the powerful mitotic recombination technique, elegant genetic screens based on clonal analysis have led to the identification of signaling pathways involved in eye development and photoreceptor (PR) differentiation at larval stages. We describe here the Tomato/GFP-FLP/FRT method, which can be used for rapid clonal analysis in the eye of living adult Drosophila. Fluorescent photoreceptor cells are imaged with the cornea neutralization technique, on retinas with mosaic clones generated by flipase-mediated recombination. This method has several major advantages over classical histological sectioning of the retina: it can be used for high-throughput screening and has proved an effective method for identifying the factors regulating PR survival and function. It can be used for kinetic analyses of PR degeneration in the same living animal over several weeks, to demonstrate the requirement for specific genes for PR survival or function in the adult fly. This method is also useful for addressing cell autonomy issues in developmental mutants, such as those in which the establishment of planar cell polarity is affected.

Endoplasmic reticulum (ER) stress has been implicated in neurodegenerative diseases but its relationship and role in disease progression remain unclear. Using genetic and pharmacological approaches, we showed that mild ER stress ("preconditioning") is neuroprotective in Drosophila and mouse models of Parkinson disease. In addition, we found that the combination of mild ER stress and apoptotic signals triggers an autophagic response both in vivo and in vitro. We showed that when autophagy is impaired, ER-mediated protection is lost. We further demonstrated that autophagy inhibits caspase activation and apoptosis. Based on our findings, we conclude that autophagy is required for the neuroprotection mediated by mild ER stress, and therefore ER preconditioning has potential therapeutic value for the treatment of neurodegenerative diseases.