microRNA-seq of cartilage reveals an overabundance of miR-140-3p which contains functional isomiRs.

miR-140 is selectively expressed in cartilage. Deletion of the entire Mir140 locus in mice results in growth retardation and early-onset osteoarthritis-like pathology; however, the relative contribution of miR-140-5p or miR-140-3p to the phenotype remains to be determined. An unbiased small RNA sequencing approach identified miR-140-3p as significantly more abundant (>10-fold) than miR-140-5p in human cartilage. Analysis of these data identified multiple miR-140-3p isomiRs differing from the miRBase annotation at both the 5' and 3' end, with >99% having one of two seed sequences (5' bases 2-8). Canonical (miR-140-3p.2) and shifted (miR-140-3p.1) seed isomiRs were overexpressed in chondrocytes and transcriptomics performed to identify targets. miR-140-3p.1 and miR-140-3p.2 significantly down-regulated 694 and 238 genes, respectively, of which only 162 genes were commonly down-regulated. IsomiR targets were validated using 3'UTR luciferase assays. miR-140-3p.1 targets were enriched within up-regulated genes in rib chondrocytes of Mir140-null mice and within down-regulated genes during human chondrogenesis. Finally, through imputing the expression of miR-140 from the expression of the host gene WWP2 in 124 previously published data sets, an inverse correlation with miR-140-3p.1 predicted targets was identified. Together these data suggest the novel seed containing isomiR miR-140-3p.1 is more functional than original consensus miR-140-3p seed containing isomiR.

XBP1 signalling is essential for alleviating mutant protein aggregation in ER-stress related skeletal disease.

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.

Loss of matrilin 1 does not exacerbate the skeletal phenotype in a mouse model of multiple epiphyseal dysplasia caused by a Matn3 V194D mutation.

OBJECTIVE: Mutations in matrilin 3 can result in multiple epiphyseal dysplasia (MED), a disease characterized by delayed and irregular bone growth and early-onset osteoarthritis. Although intracellular retention of the majority of mutant matrilin 3 was previously observed in a murine model of MED caused by a Matn3 V194D mutation, some mutant protein was secreted into the extracellular matrix. Thus, it was proposed that secretion of mutant matrilin 3 may be dependent on the formation of hetero-oligomers with matrilin 1. The aim of this study was to investigate the hypothesis that deletion of matrilin 1 would abolish the formation of matrilin 1/matrilin 3 hetero-oligomers, eliminate the secretion of mutant matrilin 3, and influence disease severity. METHODS: Mice with a Matn3 V194D mutation were crossed with Matn1-null mice, generating mice that were homozygous for V194D and null for matrilin 1. This novel mouse was used for in-depth phenotyping, while cartilage and chondrocytes were studied both histochemically and biochemically. RESULTS: Endochondral ossification was not disrupted any further in mice with a double V194D mutation compared with mice with a single mutation. A similar proportion of mutant matrilin 3 was present in the extracellular matrix, and the amount of retained mutant matrilin 3 was not noticeably increased. Retained mutant matrilin 3 formed disulfide-bonded aggregates and caused the co-retention of matrilin 1. CONCLUSION: We showed that secretion of matrilin 3 V194D mutant protein is not dependent on hetero-oligomerization with matrilin 1, and that the total ablation of matrilin 1 expression has no impact on disease severity in mice with MED. Mutant matrilin 3 oligomers form non-native disulfide-bonded aggregates through the misfolded A domain.

A novel form of chondrocyte stress is triggered by a COMP mutation causing pseudoachondroplasia.

Pseudoachondroplasia (PSACH) results from mutations in cartilage oligomeric matrix protein (COMP) and the p.D469del mutation within the type III repeats of COMP accounts for approximately 30% of PSACH. To determine disease mechanisms of PSACH in vivo, we introduced the Comp D469del mutation into the mouse genome. Mutant animals were normal at birth but grew slower than their wild-type littermates and developed short-limb dwarfism. In the growth plates of mutant mice chondrocyte columns were reduced in number and poorly organized, while mutant COMP was retained within the endoplasmic reticulum (ER) of cells. Chondrocyte proliferation was reduced and apoptosis was both increased and spatially dysregulated. Previous studies on COMP mutations have shown mutant COMP is co-localized with chaperone proteins, and we have reported an unfolded protein response (UPR) in mouse models of PSACH-MED (multiple epiphyseal dysplasia) harboring mutations in Comp (T585M) and Matn3, Comp etc (V194D). However, we found no evidence of UPR in this mouse model of PSACH. In contrast, microarray analysis identified expression changes in groups of genes implicated in oxidative stress, cell cycle regulation, and apoptosis, which is consistent with the chondrocyte pathology. Overall, these data suggest that a novel form of chondrocyte stress triggered by the expression of mutant COMP is central to the pathogenesis of PSACH.

A mouse model offers novel insights into the myopathy and tendinopathy often associated with pseudoachondroplasia and multiple epiphyseal dysplasia.

Pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED) are relatively common skeletal dysplasias belonging to the same bone dysplasia family. PSACH is characterized by generalized epi-metaphyseal dysplasia, short-limbed dwarfism, joint laxity and early onset osteoarthritis. MED is a milder disease with radiographic features often restricted to the epiphyses of the long bones. PSACH and some forms of MED result from mutations in cartilage oligomeric matrix protein (COMP), a pentameric glycoprotein found in cartilage, tendon, ligament and muscle. PSACH-MED patients often have a mild myopathy characterized by mildly increased plasma creatine kinase levels, a variation in myofibre size and/or small atrophic fibres. In some instances, patients are referred to neuromuscular clinics prior to the diagnosis of an underlying skeletal dysplasia; however, the myopathy associated with PSACH-MED has not previously been studied. In this study, we present a detailed study of skeletal muscle, tendon and ligament from a mouse model of mild PSACH harbouring a COMP mutation. Mutant mice exhibited a progressive muscle weakness associated with an increased number of muscle fibres with central nuclei at the perimysium and at the myotendinous junction. Furthermore, the distribution of collagen fibril diameters in the mutant tendons and ligaments was altered towards thicker collagen fibrils, and the tendons became more lax in cyclic strain tests. We hypothesize that the myopathy in PSACH-MED originates from an underlying tendon and ligament pathology that is a direct result of structural abnormalities to the collagen fibril architecture. This is the first comprehensive characterization of the musculoskeletal phenotype of PSACH-MED and is directly relevant to the clinical management of these patients.

Increased hippocampal excitability in miR-324-null mice.

MicroRNAs are non-coding RNAs that act to downregulate the expression of target genes by translational repression and degradation of messenger RNA molecules. Individual microRNAs have the ability to specifically target a wide array of gene transcripts, therefore allowing each microRNA to play key roles in multiple biological pathways. miR-324 is a microRNA predicted to target thousands of RNA transcripts and is expressed far more highly in the brain than in any other tissue, suggesting that it may play a role in one or multiple neurological pathways. Here we present data from the first global miR-324-null mice, in which increased excitability and interictal discharges were identified in vitro in the hippocampus. RNA sequencing was used to identify differentially expressed genes in miR-324-null mice which may contribute to this increased hippocampal excitability, and 3'UTR luciferase assays and western blotting revealed that two of these, Suox and Cd300lf, are novel direct targets of miR-324. Characterisation of microRNAs that produce an effect on neurological activity, such as miR-324, and identification of the pathways they regulate will allow a better understanding of the processes involved in normal neurological function and in turn may present novel pharmaceutical targets in treating neurological disease.

Skeletal dysplasias associated with mild myopathy-a clinical and molecular review.

Musculoskeletal system is a complex assembly of tissues which acts as scaffold for the body and enables locomotion. It is often overlooked that different components of this system may biomechanically interact and affect each other. Skeletal dysplasias are diseases predominantly affecting the development of the osseous skeleton. However, in some cases skeletal dysplasia patients are referred to neuromuscular clinics prior to the correct skeletal diagnosis. The muscular complications seen in these cases are usually mild and may stem directly from the muscle defect and/or from the altered interactions between the individual components of the musculoskeletal system. A correct early diagnosis may enable better management of the patients and a better quality of life. This paper attempts to summarise the different components of the musculoskeletal system which are affected in skeletal dysplasias and lists several interesting examples of such diseases in order to enable better understanding of the complexity of human musculoskeletal system.

New developments in chondrocyte ER stress and related diseases.

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.

CRELD2 Is a Novel LRP1 Chaperone That Regulates Noncanonical WNT Signaling in Skeletal Development.

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, whereas 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 noncanonical WNT4 signaling 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-ß1 signaling. Therefore, our data provide a novel link between an ER-resident chaperone and the essential WNT signaling pathways active during skeletal differentiation that could be applicable in other WNT-responsive tissues. © 2020 American Society for Bone and Mineral Research. © 2020 The Authors. Journal of Bone and Mineral Research published by American Society for Bone and Mineral Research..

Calcium activated nucleotidase 1 (CANT1) is critical for glycosaminoglycan biosynthesis in cartilage and endochondral ossification.

Desbuquois dysplasia type 1 (DBQD1) is a chondrodysplasia caused by mutations in CANT1 gene encoding an ER/Golgi calcium activated nucleotidase 1 that hydrolyses UDP. Here, using Cant1 knock-in and knock-out mice recapitulating DBQD1 phenotype, we report that CANT1 plays a crucial role in cartilage proteoglycan synthesis and in endochondral ossification. Specifically, the glycosaminoglycan synthesis was decreased in chondrocytes from Cant1 knock-out mice and their hydrodynamic size was reduced, whilst the sulfation was increased and the overall proteoglycan secretion was delayed. Interestingly, knock-out chondrocytes had dilated ER cisternae suggesting delayed protein secretion and cellular stress; however, no canonical ER stress response was detected using microarray analysis, Xbp1 splicing and protein levels of BiP and ATF4. The observed proteoglycan defects caused deregulated chondrocyte proliferation and maturation in the growth plate resulting in the reduced skeletal growth. In conclusion, the pathogenic mechanism of DBQD1 comprises deregulated chondrocyte performance due to defective intracellular proteoglycan synthesis and altered proteoglycan properties in the extracellular matrix.

Changes in Bcl-2 expression in vaccinia virus-infected human peripheral blood monocytes.

In this study, we show that Bcl-2, one of the most important antiapoptotic agents, is expressed in a phase-dependent manner in the human adherent monocytes after vaccinia virus infection, reflecting the viral infection stages. Early viral infection induced Bcl-2 expression in a level higher than in control cells. At 14 h post-infection (p.i.), the Bcl-2 level measured in the whole cell extracts dramatically decreased, followed by the increase at 24 h p.i. The levels of active dephosphorylated Bcl- 2 protein present in the cells reflected the gene expression character, but were much lower than in case of a heat shock. The dramatic increase of Bcl-2 protein level in the nuclear fraction at 4 h p.i. was observed. Changes in Bcl-2 mRNA content in elutriated human blood monocytes isolated from the same donor showed different kinetics, increasing up to 12 h p.i. and diminishing to undetectable level at 24 h p.i. concomitantly with a severe increase in the number of dead cells. The results indicate that virally infected adherent monocytes remain resistant to apoptosis, while freshly isolated monocytes undergo apoptotic cell death. These results throw new light on the apoptotic mechanism in the monocyte-derived cells after vaccinia virus infection in vitro.

The utility of mouse models to provide information regarding the pathomolecular mechanisms in human genetic skeletal diseases: The emerging role of endoplasmic reticulum stress (Review).

Genetic skeletal diseases (GSDs) are an extremely diverse and complex group of rare genetic diseases that primarily affect the development and homeostasis of the osseous skeleton. There are more than 450 unique and well-characterised phenotypes that range in severity from relatively mild to severe and lethal forms. Although individually rare, as a group of related genetic diseases, GSDs have an overall prevalence of at least 1 per 4,000 children. Qualitative defects in cartilage structural proteins result in a broad spectrum of both recessive and dominant GSDs. This review focused on a disease spectrum resulting from mutations in the non-collagenous glycoproteins, cartilage oligomeric matrix protein (COMP) and matrilin-3, which together cause a continuum of phenotypes that are amongst the most common autosomal dominant GSDs. Pseudoachondroplasia (PSACH) and autosomal dominant multiple epiphyseal dysplasia (MED) comprise a disease spectrum characterised by varying degrees of disproportionate short stature, joint pain and stiffness and early-onset osteoarthritis. Over the past decade, the generation and deep phenotyping of a range of genetic mouse models of the PSACH and MED disease spectrum has allowed the disease mechanisms to be characterised in detail. Moreover, the generation of novel phenocopies to model specific disease mechanisms has confirmed the importance of endoplasmic reticulum (ER) stress and reduced chondrocyte proliferation as key modulators of growth plate dysplasia and reduced bone growth. Finally, new insight into related musculoskeletal complications (such as myopathy and tendinopathy) has also been gained through the in-depth analysis of targeted mouse models of the PSACH-MED disease spectrum.

New therapeutic targets in rare genetic skeletal diseases.

Introduction: Genetic skeletal diseases (GSDs) are a diverse and complex group of rare genetic conditions that affect the development and homeostasis of the skeleton. Although individually rare, as a group of related diseases, GSDs have an overall prevalence of at least 1 per 4,000 children. There are currently very few specific therapeutic interventions to prevent, halt or modify skeletal disease progression and therefore the generation of new and effective treatments requires novel and innovative research that can identify tractable therapeutic targets and biomarkers of these diseases. Areas covered: Remarkable progress has been made in identifying the genetic basis of the majority of GSDs and in developing relevant model systems that have delivered new knowledge on disease mechanisms and are now starting to identify novel therapeutic targets. This review will provide an overview of disease mechanisms that are shared amongst groups of different GSDs and describe potential therapeutic approaches that are under investigation. Expert opinion: The extensive clinical variability and genetic heterogeneity of GSDs renders this broad group of rare diseases a bench to bedside challenge. However, the evolving hypothesis that clinically different diseases might share common disease mechanisms is a powerful concept that will generate critical mass for the identification and validation of novel therapeutic targets and biomarkers.

Abnormal chondrocyte apoptosis in the cartilage growth plate is influenced by genetic background and deletion of CHOP in a targeted mouse model of pseudoachondroplasia.

Pseudoachondroplasia (PSACH) is an autosomal dominant skeletal dysplasia caused by mutations in cartilage oligomeric matrix protein (COMP) and characterised by short limbed dwarfism and early onset osteoarthritis. Mouse models of PSACH show variable retention of mutant COMP in the ER of chondrocytes, however, in each case a different stress pathway is activated and the underlying disease mechanisms remain largely unknown. T585M COMP mutant mice are a model of moderate PSACH and demonstrate a mild ER stress response. Although mutant COMP is not retained in significant quantities within the ER of chondrocytes, both BiP and the pro-apoptotic ER stress-related transcription factor CHOP are mildly elevated, whilst bcl-2 levels are decreased, resulting in increased and spatially dysregulated chondrocyte apoptosis. To determine whether the abnormal chondrocyte apoptosis observed in the growth plate of mutant mice is CHOP-mediated, we bred T585M COMP mutant mice with CHOP-null mice to homozygosity, and analysed the resulting phenotype. Although abnormal apoptosis was alleviated in the resting zone following CHOP deletion, the mutant growth plates were generally more disorganised. Furthermore, the bone lengths of COMP mutant CHOP null mice were significantly shorter at 9 weeks of age when compared to the COMP mutant mice, including a significant difference in the skull length. Overall, these data demonstrate that CHOP-mediated apoptosis is an early event in the pathobiology of PSACH and suggest that the lack of CHOP, in conjunction with a COMP mutation, may lead to aggravation of the skeletal phenotype via a potentially synergistic effect on endochondral ossification.

Mild myopathy is associated with COMP but not MATN3 mutations in mouse models of genetic skeletal diseases.

Pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED) are skeletal disorders resulting from mutations in COMP, matrilin-3 or collagen IX and are characterised by short-limbed dwarfism and premature osteoarthritis. Interestingly, recent reports suggest patients can also manifest with muscle weakness. Here we present a detailed analysis of two mouse models of the PSACH/MED disease spectrum; ΔD469 T3-COMP (PSACH) and V194D matrilin-3 (MED). In grip test experiments T3-COMP mice were weaker than wild-type littermates, whereas V194D mice behaved as controls, confirming that short-limbed dwarfism alone does not contribute to PSACH/MED-related muscle weakness. Muscles from T3-COMP mice showed an increase in centronuclear fibers at the myotendinous junction. T3-COMP tendons became more lax in cyclic testing and showed thicker collagen fibers when compared with wild-type tissue; matrilin-3 mutant tissues were indistinguishable from controls. This comprehensive study of the myopathy associated with PSACH/MED mutations enables a better understanding of the disease progression, confirms that it is genotype specific and that the limb weakness originates from muscle and tendon pathology rather than short-limbed dwarfism itself. Since some patients are primarily diagnosed with neuromuscular symptoms, this study will facilitate better awareness of the differential diagnoses that might be associated with the PSACH/MED spectrum and subsequent care of PSACH/MED patients.