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ObjectiveTo explore whether aberrant transient chondrocyte behaviors occur in the joints of STR/Ort mice (which spontaneously develop osteoarthritis [OA]) and whether they are attributable to an endochondral growth defect.MethodsKnee joints from STR/Ort mice with advanced OA and age‐matched CBA (control) mice were examined by Affymetrix microarray profiling, multiplex polymerase chain reaction (PCR) analysis, and immunohistochemical labeling of endochondral markers, including sclerostin and MEPE. The endochondral phenotype of STR/Ort mice was analyzed by histologic examination, micro–computed tomography, and ex vivo organ culture. A novel protocol for quantifying bony bridges across the murine epiphysis (growth plate fusion) using synchrotron x‐ray computed microtomography was developed and applied.ResultsMeta‐analysis of transcription profiles showed significant elevation in functions linked with endochondral ossification in STR/Ort mice (compared to CBA mice; P < 0.05). Consistent with this, immunolabeling revealed increased matrix metalloproteinase 13 (MMP‐13) and type X collagen expression in STR/Ort mouse joints, and multiplex quantitative reverse transcriptase–PCR showed differential expression of known mineralization regulators, suggesting an inherent chondrocyte defect. Support for the notion of an endochondral defect included accelerated growth, increased zone of growth plate proliferative chondrocytes (P < 0.05), and widespread type X collagen/MMP‐13 labeling beyond the expected hypertrophic zone distribution. OA development involved concomitant focal suppression of sclerostin/MEPE in STR/Ort mice. Our novel synchrotron radiation microtomography method showed increased numbers (P < 0.001) and mean areal growth plate bridge densities (P < 0.01) in young and aged STR/Ort mice compared to age‐matched CBA mice.ConclusionTaken together, our data support the notion of an inherent endochondral defect that is linked to growth dynamics and subject to regulation by the MEPE/sclerostin axis and may represent an underlying mechanism of pathologic ossification in OA.

Background LRRK2 mutations and risk variants increase susceptibility to inherited and idiopathic Parkinson’s disease, while recent studies have identified potential protective variants. This, and the fact that LRRK2 mutation carriers develop symptoms and brain pathology almost indistinguishable from idiopathic Parkinson’s disease, has led to enormous interest in this protein. LRRK2 has been implicated in a range of cellular events, but key among them is canonical Wnt signalling, which results in increased levels of transcriptionally active β-catenin. This pathway is critical for the development and survival of the midbrain dopaminergic neurones typically lost in Parkinson’s disease.MethodsHere we use Lrrk2 knockout mice and fibroblasts to investigate the effect of loss of Lrrk2 on canonical Wnt signalling in vitro and in vivo. Micro-computed tomography was used to study predicted tibial strength, while pulldown assays were employed to measure brain β-catenin levels. A combination of luciferase assays, immunofluorescence and co-immunoprecipitation were performed to measure canonical Wnt activity and investigate the relationship between LRRK2 and β-catenin. TOPflash assays are also used to study the effects of LRRK2 kinase inhibition and pathogenic and protective LRRK2 mutations on Wnt signalling. Data were tested by Analysis of Variance.ResultsLoss of Lrrk2 causes a dose-dependent increase in the levels of transcriptionally active β-catenin in the brain, and alters tibial bone architecture, decreasing the predicted risk of fracture. Lrrk2 knockout cells display increased TOPflash and Axin2 promoter activities, both basally and following Wnt activation. Consistently, over-expressed LRRK2 was found to bind β-catenin and repress TOPflash activation. Some pathogenic LRRK2 mutations and risk variants further suppressed TOPflash, whereas the protective R1398H variant increased Wnt signalling activity. LRRK2 kinase inhibitors affected canonical Wnt signalling differently due to off-targeting; however, specific LRRK2 inhibition reduced canonical Wnt signalling similarly to pathogenic mutations.ConclusionsLoss of LRRK2 causes increased canonical Wnt activity in vitro and in vivo. In agreement, over-expressed LRRK2 binds and represses β-catenin, suggesting LRRK2 may act as part of the β-catenin destruction complex. Since some pathogenic LRRK2 mutations enhance this effect while the protective R1398H variant relieves it, our data strengthen the notion that decreased canonical Wnt activity is central to Parkinson’s disease pathogenesis.Electronic supplementary materialThe online version of this article (doi:10.1186/s13024-017-0153-4) contains supplementary material, which is available to authorized users.

The transmembrane glycoprotein E11/Podoplanin (Pdpn) has been implicated in the initial stages of osteocyte differentiation. However, its precise function and regulatory mechanisms are still unknown. Due to the known embryonic lethality induced by global Pdpn deletion, we have herein explored the effect of bone‐specific Pdpn knockdown on osteocyte form and function in the post‐natal mouse. Extensive skeletal phenotyping of male and female 6‐week‐old Oc‐cre;Pdpn flox/flox (cKO) mice and their Pdpn flox/flox controls (fl/fl) has revealed that Pdpn deletion significantly compromises tibial cortical bone microarchitecture in both sexes, albeit to different extents (p < 0.05). Consistent with this, we observed an increase in stiffness in female cKO mice in comparison to fl/fl mice (p < 0.01). Moreover, analysis of the osteocyte phenotype by phalloidin staining revealed a significant decrease in the dendrite volume (p < 0.001) and length (p < 0.001) in cKO mice in which deletion of Pdpn also modifies the bone anabolic loading response (p < 0.05) in comparison to age‐matched fl/fl mice. Together, these data confirm a regulatory role for Pdpn in osteocyte dendrite formation and as such, in the control of osteocyte function. As the osteocyte dendritic network is known to play vital roles in regulating bone modeling/remodeling, this highlights an essential role for Pdpn in bone homeostasis.

Background One of the principles underpinning our understanding of ageing is that DNA damage induces a stress response that shifts cellular resources from growth towards maintenance. A contrasting and seemingly irreconcilable view is that prompting growth of, for example, skeletal muscle confers systemic benefit. Methods To investigate the robustness of these axioms, we induced muscle growth in a murine progeroid model through the use of activin receptor IIB ligand trap that dampens myostatin/activin signalling. Progeric mice were then investigated for neurological and muscle function as well as cellular profiling of the muscle, kidney, liver, and bone. Results We show that muscle of Ercc1 Δ/− progeroid mice undergoes severe wasting (decreases in hind limb muscle mass of 40–60% compared with normal mass), which is largely protected by attenuating myostatin/activin signalling using soluble activin receptor type IIB (sActRIIB) (increase of 30–62% compared with untreated progeric). sActRIIB‐treated progeroid mice maintained muscle activity (distance travel per hour: 5.6 m in untreated mice vs. 13.7 m in treated) and increased specific force (19.3 mN/mg in untreated vs. 24.0 mN/mg in treated). sActRIIb treatment of progeroid mice also improved satellite cell function especially their ability to proliferate on their native substrate (2.5 cells per fibre in untreated progeroids vs. 5.4 in sActRIIB‐treated progeroids after 72 h in culture). Besides direct protective effects on muscle, we show systemic improvements to other organs including the structure and function of the kidneys; there was a major decrease in the protein content in urine (albumin/creatinine of 4.9 sActRIIB treated vs. 15.7 in untreated), which is likely to be a result in the normalization of podocyte foot processes, which constitute the filtration apparatus (glomerular basement membrane thickness reduced from 224 to 177 nm following sActRIIB treatment). Treatment of the progeric mice with the activin ligand trap protected against the development of liver abnormalities including polyploidy (18.3% untreated vs. 8.1% treated) and osteoporosis (trabecular bone volume; 0.30 mm 3 in treated progeroid mice vs. 0.14 mm 3 in untreated mice, cortical bone volume; 0.30 mm 3 in treated progeroid mice vs. 0.22 mm 3 in untreated mice). The onset of neurological abnormalities was delayed (by ~5 weeks) and their severity reduced, overall sustaining health without affecting lifespan. Conclusions This study questions the notion that tissue growth and maintaining tissue function during ageing are incompatible mechanisms. It highlights the need for future investigations to assess the potential of therapies based on myostatin/activin blockade to compress morbidity and promote healthy ageing.

SummaryObjectivesHuman osteoarthritis (OA) is detected only at late stages. Male STR/Ort mice develop knee OA spontaneously with known longitudinal trajectory, offering scope to identify OA predisposing factors. We exploit the lack of overt OA in female STR/Ort and in both sexes of parental, control CBA mice to explore whether early divergence in tibial bone mass or shape are linked to emergent OA.MethodWe undertook detailed micro-CT comparisons of trabecular and cortical bone, multiple structural/architectural parameters and finite element modelling (FEM) of the tibia from male and female STR/Ort and CBA mice at 8–10 (pre-OA), 18–20 (OA onset) and 40 + weeks (advanced OA) of age.ResultsWe found higher trabecular bone mass in female STR/Ort than in either OA-prone male STR/Ort or non-prone CBA mice. Cortical bone, as expected, showed greater cross-sectional area in male than female CBA, which surprisingly was reversed in STR/Ort mice. STR/Ort also exhibited higher cortical bone mass than CBA mice. Our analyses revealed similar tibial ellipticity, yet greater predicted resistance to torsion in male than female CBA mice. In contrast, male STR/Ort exhibited greater ellipticity than both female STR/Ort and CBA mice at specific cortical sites. Longitudinal analysis revealed greater tibia curvature and shape deviations in male STR/Ort mice that coincided with onset and were more pronounced in late OA.ConclusionGeneralised higher bone mass in STR/Ort mice is more marked in non OA-prone females, but pre-OA divergence in bone shape is restricted to male STR/Ort mice in which OA develops spontaneously.

The secondary dystroglycanopathies are characterized by the hypoglycosylation of alpha dystroglycan, and are associated with mutations in at least 18 genes that act on the glycosylation of this cell surface receptor rather than the Dag1 gene itself. At the severe end of the disease spectrum, there are substantial structural brain defects, the most striking of which is often cobblestone lissencephaly. The aim of this study was to determine the gene-specific aspects of the dystroglycanopathy brain phenotype through a detailed investigation of the structural brain defects present at birth in three mouse models of dystroglycanopathy-the FKRP(KD) , which has an 80% reduction in Fkrp transcript levels; the Pomgnt1null , which carries a deletion of exons 7-16 of the Pomgnt1 gene; and the Large(myd) mouse, which carries a deletion of exons 5-7 of the Large gene. We show a rostrocaudal and mediolateral gradient in the severity of brain lesions in FKRP(KD) , and to a lesser extent Pomgnt1null mice. Furthermore, the mislocalization of Cajal-Retzius cells is correlated with the gradient of these lesions and the severity of the brain phenotype in these models. Overall these observations implicate gene-specific differences in the pathogenesis of brain lesions in this group of disorders.

Tissue inhibitor of metalloproteinases-3 (TIMP-3) regulates extracellular matrix via its inhibition of matrix metalloproteinases and membrane-bound sheddases. Timp-3 is expressed at multiple sites of extensive tissue remodelling. This extends to bone where its role, however, remains largely unresolved. In this study, we have used Micro-CT to assess bone mass and architecture, histological and histochemical evaluation to characterise the skeletal phenotype of Timp-3 KO mice and have complemented this by also examining similar indices in mice harbouring a Timp-3 transgene driven via a Col-2a-driven promoter to specifically target overexpression to chondrocytes. Our data show that Timp-3 deficiency compromises tibial bone mass and structure in both cortical and trabecular compartments, with corresponding increases in osteoclasts. Transgenic overexpression also generates defects in tibial structure predominantly in the cortical bone along the entire shaft without significant increases in osteoclasts. These alterations in cortical mass significantly compromise predicted tibial load-bearing resistance to torsion in both genotypes. Neither Timp-3 KO nor transgenic mouse growth plates are significantly affected. The impact of Timp-3 deficiency and of transgenic overexpression extends to produce modification in craniofacial bones of both endochondral and intramembranous origins. These data indicate that the levels of Timp-3 are crucial in the attainment of functionally-appropriate bone mass and architecture and that this arises from chondrogenic and osteogenic lineages.