The intracellular retention of misfolded extracellular matrix proteins is a common disease mechanism in various rare skeletal diseases. This discovery has driven the study of ER stress and the unfolded protein response (UPR) as a promising therapeutic target in several skeletal dysplasias. In the case of COL10A1 mutations, targeting the UPR resulted in a clinical trial of the repurposed drug carbamazepine; however, for other closely related skeletal disorders, treatment with carbamazepine was ineffective, indicating the need for suitable markers for in vitro screenings of potential drug treatments. Mutations in cartilage oligomeric matrix protein (COMP), a cartilage structural protein, cause both multiple epiphyseal dysplasia (MED) and pseudoachondroplasia (PSACH); together referred to as the COMPopathies, which result from the intracellular retention of mutant COMP to varying degrees. In contrast to other closely related skeletal disorders, caused by mutations in cartilage structural proteins, the involvement of the UPR is less clear, and so far, no common COMPopathy marker has been identified. Here, using cell models of COMPopathies, we identified MMP9 upregulation as a common feature of six pathogenic COMP variants that do not induce a prominent UPR. We further show that the archetypal p.V194D matrilin-3 MED variant (which causes MED) does not induce MMP9 expression, suggesting that MMP9 upregulation could serve as a specific marker of COMPopathies in vitro.

The intracellular retention of mutant cartilage matrix proteins and pathological endoplasmic reticulum (ER) stress disrupts ossification and has been identified as a shared disease mechanism in a range of skeletal dysplasias including short limbed-dwarfism, multiple epiphyseal dysplasia type 5 (EDM5). Although targeting ER stress is an attractive avenue for treatment and has proven successful in the treatment of a related skeletal dysplasia, to date no drugs have proven successful in reducing ER stress in EDM5 caused by the retention of mutant matrilin-3. Our exciting findings show that by using our established luciferase ER stress screening assay, we can identify a “natural” chemical, curcumin, which is able to reduce pathological ER stress in a cell model of EDM5 by promoting the proteasomal degradation mutant matrilin-3. Therefore, this is an important in vitro study in which we describe, for the first time, the success of a naturally occurring chemical as a potential treatment for this currently incurable rare skeletal disease. As studies show that curcumin can be used as a potential treatment for range of diseases in vitro, current research is focused on developing novel delivery strategies to enhance its bioavailability. This is an important and exciting area of research that will have significant clinical impact on a range of human diseases including the rare skeletal disease, EDM5.

Cysteine rich with epidermal growth factor (EGF)-like domains 2 (CRELD2) is an endoplasmic reticulum (ER) resident chaperone protein with calcium binding properties. CRELD2 is an ER-stress regulated gene that has been implicated in the pathogenesis of skeletal dysplasias and has been shown to play an important role in the differentiation of chondrocytes and osteoblasts. Despite CRELD2 having an established role in skeletal development and bone formation, its role in osteoclasts is currently unknown. Here we show for the first time that CRELD2 plays a novel role in trafficking transforming growth factor beta 1 (TGF-β1), which is linked to an upregulation in the expression of Nfat2 , the master regulator of osteoclast differentiation in early osteoclastogenesis. Despite this finding, we show that overexpressing CRELD2 impaired osteoclast differentiation due to a reduction in the activity of the calcium-dependant phosphatase, calcineurin. This in turn led to a subsequent block in the dephosphorylation of nuclear factor of activated T cells 1 (NFATc1), preventing its nuclear localisation and activation as a pro-osteoclastogenic transcription factor. Our exciting results show that the overexpression of Creld2 in osteoclasts impaired calcium release from the ER which is essential for activating calcineurin and promoting osteoclastogenesis. Therefore, our data proposes a novel inhibitory role for this calcium-binding ER-resident chaperone in modulating calcium flux during osteoclast differentiation which has important implications in our understanding of bone remodelling and the pathogenesis of skeletal diseases.

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.

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.

Treatments for osteoarthritis (OA) are designed to restore chondrocyte function and inhibit cell apoptosis.Previous studies have shown that activation of the glucagon-like peptide-1 receptor (GLP-1R) leads to anti-inflammatory and anti-apoptotic effects.However, the role of GLP-1R in the pathological process of OA is unclear.In present work, we aimed to demonstrate the potential effect of GLP-1R on chondrocytes and elucidate its underlying mechanisms.We found that activation of GLP-1R with liraglutide could protect chondrocytes against endoplasmic reticulum stress and apoptosis induced by interleukin (IL)-1β or triglycerides (TGs).These effects were partially attenuated by GLP-1R small interfering RNA treatment.Moreover, inhibiting PI3K/Akt signaling abolished the protective effects of GLP-1R by increase the apoptosis activity and ER stress.Activating GLP-1R suppressed the nuclear factor kappa-B pathway, decreased the release of inflammatory mediators (IL-6, tumor necrosis factor α), and reduced matrix catabolism in TGtreated chondrocytes; these effects were abolished by GLP-1R knockdown.In the end, liraglutide attenuated rat cartilage degeneration in an OA model of knee joints in vivo.Our results indicate that GLP-1R is a therapeutic target for the treatment of OA, and that liraglutide could be a therapeutic candidate for this clinical application.

Osteochondritis dissecans (OCD) in equids, especially in sport horses, has become a growing issue as it contributes to the occurrence of lameness.Thus the aim of this study was to investigate the cytophysiological properties of OCD chondrocytes including expression of chondrogenic genes, apoptosis, mitochondria dynamics and autophagy.Horse chondrocytes were isolated from healthy (HE) and OCD cartilages.Properties of cells were evaluated using multiple assays e.g., polymerase chain reaction (PCR), immunofluorescence, Western blot.OCD chondrocytes were characterized by increased apoptosis and senescence.Expression of chondrogenic genes (vimentin, aggrecan) was decreased while mRNA levels of matrix metalloproteinase 13 significantly upregulated in comparison to HE cells.Moreover, OCD cells displayed increased mitochondrial fusion while fission events were diminished.Interestingly, chaperone mediated autophagy was triggered in those cells and it predominated over macroautophagy.Furthermore, co-culture of LPS-treated chondrocytes with adipose-derived stem cells (ASC) decreased p62/sequestosome 1 (SQSTM) and increases MMP-13 expression in OCD cells.Our results suggest that OCD affected horse chondrocytes are characterized by senescent phenotype due to endoplasmic reticulum stress and mitochondria dynamics deterioration.Expression of chondrogenic markers is decreased in those cells while expression of chaperone mediated autophagy (CMA)-related genes increased.Increased malfunctioning of cells leads to loss of their functionality and capacity to maintain tissue homeostasis.