The pathogenesis of declining bone mineral density, a universal feature of ageing, is not fully understood. Somatic mitochondrial DNA (mtDNA) mutations accumulate with age in human tissues and mounting evidence suggests that they may be integral to the ageing process. To explore the potential effects of mtDNA mutations on bone biology, we compared bone microarchitecture and turnover in an ageing series of wild type mice with that of the PolgA^mut/mut mitochondrial DNA ‘mutator’ mouse. In vivo analyses showed an age-related loss of bone in both groups of mice; however, it was significantly accelerated in the PolgA^mut/mut mice. This accelerated rate of bone loss is associated with significantly reduced bone formation rate, reduced osteoblast population densities, increased osteoclast population densities, and mitochondrial respiratory chain deficiency in osteoblasts and osteoclasts in PolgA^mut/mut mice compared with wild-type mice. In vitro assays demonstrated severely impaired mineralised matrix formation and increased osteoclast resorption by PolgA^mut/mut cells. Finally, application of an exercise intervention to a subset of PolgA^mut/mut mice showed no effect on bone mass or mineralised matrix formation in vitro. Our data demonstrate that mitochondrial dysfunction, a universal feature of human ageing, impairs osteogenesis and is associated with accelerated bone loss.

For the vast majority of the 6000 known rare disease the pathogenic mechanisms are poorly defined and there is little treatment, leading to poor quality of life and high healthcare costs. Genetic skeletal diseases (skeletal dysplasias) are archetypal examples of rare diseases that are chronically debilitating, often life‐threatening and for which no treatments are currently available. There are more than 450 unique phenotypes that, although individually rare, have an overall prevalence of at least 1 per 4000 children. Multiple epiphyseal dysplasia (MED) is a clinically and genetically heterogeneous disorder characterized by disproportionate short stature, joint pain, and early‐onset osteoarthritis. MED is caused by mutations in the genes encoding important cartilage extracellular matrix proteins, enzymes, and transporter proteins. Recently, through the use of various cell and mouse models, disease mechanisms underlying this diverse phenotypic spectrum are starting to be elucidated. For example, ER stress induced as a consequence of retained misfolded mutant proteins has emerged as a unifying disease mechanisms for several forms of MED in particular and skeletal dysplasia in general. Moreover, targeting ER stress through drug repurposing has become an attractive therapeutic avenue.

Cartilage comprises a single cell type, the chondrocyte, embedded in a highly complex extracellular matrix. Disruption to the cartilage growth plate leads to reduced bone growth and results in a clinically diverse group of conditions known as genetic skeletal diseases (GSDs). Similarly, long-term degradation of articular cartilage can lead to osteoarthritis (OA), a disease characterised by joint pain and stiffness. As professionally secreting cells, chondrocytes are particularly susceptible to endoplasmic reticulum (ER) stress and this has been identified as a core disease mechanism in a group of clinically and pathologically related GSDs. If unresolved, ER stress can lead to chondrocyte cell death. Recent interest has focused on ER stress as a druggable target for GSDs and this has led to the first clinical trial for a GSD by repurposing an antiepileptic drug. Interestingly, ER stress markers have also been associated with OA in multiple cell and animal models and there is increasing interest in it as a possible therapeutic target for treatment. In summary, chondrocyte ER stress has been identified as a core disease mechanism in GSDs and as a contributory factor in OA. Thus, chondrocyte ER stress is a unifying factor for both common and rare cartilage-related diseases and holds promise as a novel therapeutic target.

Cysteine‐rich with epidermal growth factor (EGF)‐like domains 2 (CRELD2) is an endoplasmic reticulum (ER)‐resident chaperone highly activated under ER‐stress in conditions such as chondrodysplasias; however, its role in healthy skeletal development is unknown. We show for the first time that cartilage‐specific deletion of Creld2 results in disrupted endochondral ossification and short limbed dwarfism, whilst deletion of Creld2 in bone results in osteopenia, with a low bone density and altered trabecular architecture. Our study provides the first evidence that CRELD2 promotes the differentiation and maturation of skeletal cells by modulating non‐canonical WNT4 signalling regulated by p38 MAPK. Furthermore, we show that CRELD2 is a novel chaperone for the receptor Low density lipoprotein receptor‐related protein 1 (LRP1), promoting its transport to the cell surface, and that LRP1 directly regulates WNT4 expression in chondrocytes through TGF‐β1 signalling. Therefore, our data provide a novel link between an ER‐resident chaperone and the essential WNT signalling pathways active during skeletal differentiation that could be applicable in other WNT‐responsive tissues.

The unfolded protein response (UPR) is a conserved cellular response to the accumulation of proteinaceous material in endoplasmic reticulum (ER), active both in health and disease to alleviate cellular stress and improve protein folding. Multiple epiphyseal dysplasia (EDM5) is a genetic skeletal condition and a classic example of an intracellular protein aggregation disease, whereby mutant matrilin-3 forms large insoluble aggregates in the ER lumen, resulting in a specific ‘disease signature’ of increased expression of chaperones and foldases, and alternative splicing of the UPR effector XBP1. Matrilin-3 is expressed exclusively by chondrocytes thereby making EDM5 a perfect model system to study the role of protein aggregation in disease. In order to dissect the role of XBP1 signalling in aggregation-related conditions we crossed a p.V194D Matn3 knock-in mouse model of EDM5 with a mouse line carrying a cartilage specific deletion of XBP1 and analysed the resulting phenotype. Interestingly, the growth of mice carrying the Matn3 p.V194D mutation compounded with the cartilage specific deletion of XBP1 was severely retarded. Further phenotyping revealed increased intracellular retention of amyloid-like aggregates of mutant matrilin-3 coupled with dramatically decreased cell proliferation and increased apoptosis, suggesting a role of XBP1 signalling in protein accumulation and/or degradation. Transcriptomic analysis of chondrocytes extracted from wild type, EDM5, Xbp1-null and compound mutant lines revealed that the alternative splicing of Xbp1 is crucial in modulating levels of protein aggregation. Moreover, through detailed transcriptomic comparison with a model of metaphyseal chondrodysplasia type Schmid (MCDS), an UPR-related skeletal condition in which XBP1 was removed without overt consequences, we show for the first time that the differentiation-state of cells within the cartilage growth plate influences the UPR resulting from retention of a misfolded mutant protein and postulate that modulation of XBP1 signalling pathway presents a therapeutic target for aggregation related conditions in cells undergoing proliferation. A significant proportion of genetic skeletal diseases result from misfolding of structural proteins within the endoplasmic reticulum (ER) of cartilage cells (chondrocytes). Interestingly, these diseases often share transcriptomic changes with non-skeletal conditions such as Alzheimer’s disease and diabetes and include changes in the unfolded protein response (UPR). UPR signals are conveyed through three effector molecules, IRE1, ATF6 and PERK, understanding of which is of utmost importance for the development of novel therapeutic approaches. IRE1 is the most conserved UPR pathway, with XBP1 as its main effector. Interestingly, in our study we show that chondrocyte response to ER stress is highly modulated by the differentiation state of the cell. We have deleted the IRE1/XBP1 pathway activity in the chondrocytes of a mouse model of multiple epiphyseal dysplasia (MED), expressing misfolding matrilin-3 predominantly in proliferating chondrocytes, and in metaphyseal chondrodysplasia type Schmid (MCDS), resulting from mutations in type X collagen, expressed by the hypertrophic chondrocytes. Here, using deep phenotyping and extensive transcriptomic analysis we demonstrate that whilst the IRE1/XBP1 pathway is redundant in hypertrophic chondrocyte pathology, it is in fact essential for UPR responses in proliferating cells, making this highly conserved pathway an attractive therapeutic target for a broad range of UPR related conditions.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an endoplasmic reticulum (ER) resident protein that can be secreted due to an imperfect KDEL motif. MANF plays a cytoprotective role in several soft tissues and is upregulated in conditions resulting from intracellular retention of mutant protein, including two skeletal diseases, metaphyseal chondrodysplasia, Schmid type (MCDS) and multiple epiphyseal dysplasia (MED). The role of MANF in skeletal tissue homeostasis is currently unknown. Interestingly, cartilage-specific deletion of Manf in a mouse model of MED resulted in increased disease severity, suggesting its upregulation may be chondroprotective. Treatment of MED chondrocytes with exogenous MANF led to a decrease in the cellular levels of BiP (GRP78), confirming MANF’s potential to modulate ER stress responses. However, it did not alleviate the intracellular retention of mutant matrilin-3, suggesting that it is the intracellular MANF that is of importance in the pathobiology of skeletal dysplasias. The Col2Cre-driven deletion of Manf from mouse cartilage resulted in a chondrodysplasia-like phenotype. Interestingly, ablation of MANF in cartilage did not have extracellular consequences but led to an upregulation of several ER-resident chaperones including BiP. This apparent induction of ER stress in turn led to dysregulated chondrocyte apoptosis and decreased proliferation, resulting in reduced long bone growth. We have previously shown that ER stress is an underlying disease mechanism for several skeletal dysplasias. The cartilage-specific deletion of Manf described in this study phenocopies our previously published chondrodysplasia models, further confirming that ER stress itself is sufficient to disrupt skeletal growth and thus represents a potential therapeutic target.