ER procollagen storage defect without coupled unfolded protein response drives precocious arthritis.

Collagenopathies are a group of clinically diverse disorders caused by defects in collagen folding and secretion. For example, mutations in the gene encoding collagen type-II, the primary collagen in cartilage, can lead to diverse chondrodysplasias. One example is the Gly1170Ser substitution in procollagen-II, which causes precocious osteoarthritis. Here, we biochemically and mechanistically characterize an induced pluripotent stem cell-based cartilage model of this disease, including both hetero- and homozygous genotypes. We show that Gly1170Ser procollagen-II is notably slow to fold and secrete. Instead, procollagen-II accumulates intracellularly, consistent with an endoplasmic reticulum (ER) storage disorder. Owing to unique features of the collagen triple helix, this accumulation is not recognized by the unfolded protein response. Gly1170Ser procollagen-II interacts to a greater extent than wild-type with specific proteostasis network components, consistent with its slow folding. These findings provide mechanistic elucidation into the etiology of this disease. Moreover, the cartilage model will enable rapid testing of therapeutic strategies to restore proteostasis in the collagenopathies.

Modeling human skeletal development using human pluripotent stem cells.

Chondrocytes and osteoblasts differentiated from induced pluripotent stem cells (iPSCs) will provide insights into skeletal development and genetic skeletal disorders and will generate cells for regenerative medicine applications. Here, we describe a method that directs iPSC-derived sclerotome to chondroprogenitors in 3D pellet culture then to articular chondrocytes or, alternatively, along the growth plate cartilage pathway to become hypertrophic chondrocytes that can transition to osteoblasts. Osteogenic organoids deposit and mineralize a collagen I extracellular matrix (ECM), mirroring in vivo endochondral bone formation. We have identified gene expression signatures at key developmental stages including chondrocyte maturation, hypertrophy, and transition to osteoblasts and show that this system can be used to model genetic cartilage and bone disorders.

Understanding image-text relations and news values for multimodal news analysis.

The analysis of news dissemination is of utmost importance since the credibility of information and the identification of disinformation and misinformation affect society as a whole. Given the large amounts of news data published daily on the Web, the empirical analysis of news with regard to research questions and the detection of problematic news content on the Web require computational methods that work at scale. Today's online news are typically disseminated in a multimodal form, including various presentation modalities such as text, image, audio, and video. Recent developments in multimodal machine learning now make it possible to capture basic "descriptive" relations between modalities-such as correspondences between words and phrases, on the one hand, and corresponding visual depictions of the verbally expressed information on the other. Although such advances have enabled tremendous progress in tasks like image captioning, text-to-image generation and visual question answering, in domains such as news dissemination, there is a need to go further. In this paper, we introduce a novel framework for the computational analysis of multimodal news. We motivate a set of more complex image-text relations as well as multimodal news values based on real examples of news reports and consider their realization by computational approaches. To this end, we provide (a) an overview of existing literature from semiotics where detailed proposals have been made for taxonomies covering diverse image-text relations generalisable to any domain; (b) an overview of computational work that derives models of image-text relations from data; and (c) an overview of a particular class of news-centric attributes developed in journalism studies called news values. The result is a novel framework for multimodal news analysis that closes existing gaps in previous work while maintaining and combining the strengths of those accounts. We assess and discuss the elements of the framework with real-world examples and use cases, setting out research directions at the intersection of multimodal learning, multimodal analytics and computational social sciences that can benefit from our approach.

Proteome profiles of intramuscular connective tissue: influence of aging and physical training.

Both aging and physical activity can influence the amount of intramuscular connective tissue in skeletal muscle, but the impact of these upon specific extracellular matrix (ECM) proteins in skeletal muscle is unknown. We investigated the proteome profile of intramuscular connective tissue by label-free proteomic analysis of cellular protein-depleted extracts from lateral gastrocnemius muscle of old (22-23 mo old) and middle-aged (11 mo old) male mice subjected to three different levels of regular physical activity for 10 wk (high-resistance wheel running, low-resistance wheel running, or sedentary controls). We hypothesized that aging is correlated with an increased amount of connective tissue proteins in skeletal muscle and that regular physical activity can counteract these age-related changes. We found that dominating cellular proteins were diminished in the urea/thiourea extract, which was therefore used for proteomics. Proteomic analysis identified 482 proteins and showed enrichment for ECM proteins. Statistical analysis revealed that the abundances of 86 proteins changed with age. Twenty-three of these differentially abundant proteins were identified as structural ECM proteins (e.g., collagens and laminins) and all of these were significantly more abundant with aging. No significant effect of training or interaction between training and advance in age was found for any proteins. Finally, we found a lower protein concentration in the urea/thiourea extracts from the old mice compared with the middle-aged mice. Our findings indicate that the intramuscular ECM solubility is affected by increased age but is not altered by physical training.NEW & NOTEWORTHY We investigated the impact of aging and exercise on extracellular matrix components of intramuscular connective tissue using proteomics. Middle-aged and old mice were subjected to three different levels of regular physical activity for 10 wk (high-resistance wheel running, low-resistance wheel running, or sedentary controls). We prepared extracts of extracellular matrix proteins depleted of cellular proteins. Our findings indicate that intramuscular connective tissue alters its soluble protein content with age but is unaffected by training.

Using CRISPR/Cas9 to generate a heterozygous COL2A1 p.R719C iPSC line (MCRIi019-A-6) model of human precocious osteoarthritis.

The human iPSC line MCRIi019-A-6 was generated using CRISPR/Cas9-mediated gene editing to introduce a heterozygous COL2A1 exon 33 c.2155C>T (p.R719C) mutation into the control human iPSC line MCRIi019-A. Both the edited and parental lines display typical iPSC characteristics, including the expression of pluripotency markers, the ability to be differentiated into the three germ lines, and a normal karyotype. This cell line, along with the isogenic control line, can be used to study the molecular pathology of precocious osteoarthritis in a human model, more broadly understand type II collagenopathies, and explore novel therapeutic targets for this class of diseases.

The effect of carbamazepine on bone structure and strength in control and osteogenesis imperfecta (Col1a2 +/p.G610C ) mice.

The inherited brittle bone disease osteogenesis imperfecta (OI) is commonly caused by COL1A1 and COL1A2 mutations that disrupt the collagen I triple helix. This causes intracellular endoplasmic reticulum (ER) retention of the misfolded collagen and can result in a pathological ER stress response. A therapeutic approach to reduce this toxic mutant load could be to stimulate mutant collagen degradation by manipulating autophagy and/or ER-associated degradation. Since carbamazepine (CBZ) both stimulates autophagy of misfolded collagen X and improves skeletal pathology in a metaphyseal chondrodysplasia model, we tested the effect of CBZ on bone structure and strength in 3-week-old male OI Col1a2 +/p.G610C and control mice. Treatment for 3 or 6 weeks with CBZ, at the dose effective in metaphyseal chondrodysplasia, provided no therapeutic benefit to Col1a2 +/p.G610C mouse bone structure, strength or composition, measured by micro-computed tomography, three point bending tests and Fourier-transform infrared microspectroscopy. In control mice, however, CBZ treatment for 6 weeks impaired femur growth and led to lower femoral cortical and trabecular bone mass. These data, showing the negative impact of CBZ treatment on the developing mouse bones, raise important issues which must be considered in any human clinical applications of CBZ in growing individuals.

Collagen misfolding mutations: the contribution of the unfolded protein response to the molecular pathology.

Mutations in collagen genes cause a broad range of connective tissue pathologies. Structural mutations that impact procollagen assembly or triple helix formation and stability are a common and important mutation class. How misfolded procollagens engage with the cellular proteostasis machinery and whether they can elicit a cytotoxic unfolded protein response (UPR) is a topic of considerable research interest. Such interest is well justified since modulating the UPR could offer a new approach to treat collagenopathies for which there are no current disease mechanism-targeting therapies. This review scrutinizes the evidence underpinning the view that endoplasmic reticulum stress and chronic UPR activation contributes significantly to the pathophysiology of the collagenopathies. While there is strong evidence that the UPR contributes to the pathology for collagen X misfolding mutations, the evidence that misfolding mutations in other collagen types induce a canonical, cytotoxic UPR is incomplete. To gain a more comprehensive understanding about how the UPR amplifies to pathology, and thus what types of manipulations of the UPR might have therapeutic relevance, much more information is needed about how specific misfolding mutation types engage differentially with the UPR and downstream signaling responses. Most importantly, since the capacity of the proteostasis machinery to respond to collagen misfolding is likely to vary between cell types, reflecting their functional roles in collagen and extracellular matrix biosynthesis, detailed studies on the UPR should focus as much as possible on the actual target cells involved in the collagen pathologies.

Generation of a heterozygous COL2A1 (p.G1113C) hypochondrogenesis mutation iPSC line, MCRIi019-A-7, using CRISPR/Cas9 gene editing.

The human inherited cartilage disease, Hypochondrogenesis, is caused by mutations in the collagen type II gene, COL2A1. To produce an in vitro disease model, we generated a heterozygous patient mutation, COL2A1 p.G1113C, in an established control human induced pluripotent stem cell (iPSC) line, MCRIi019-A, using CRISPR-Cas9 gene editing. The gene-edited mutant line, MCRIi019-A-7, exhibited normal iPSC characteristics, including normal cell morphology, expression of pluripotency markers, the ability to differentiate into three embryonic germ layers, and normal karyotype. Together with its parental isogenic control, this cell line will be useful for Hypochondrogenesis disease modelling and drug testing.

Generation of a miR-26b stem-loop knockout human iPSC line, MCRIi019-A-1, using CRISPR/Cas9 editing.

miR-26b has been implicated in a wide range of human diseases, including cancer, diabetes, heart disease, Alzheimer's disease and osteoarthritis. To provide a tool to explore the importance of miR-26b in this broad context, we have generated and characterized a homozygous miR-26b stem-loop knockout human iPSC line. This gene-edited line exhibited a normal karyotype, expressed pluripotency markers and differentiated into cells representative of the three embryonic germ layers. This iPSC line will be valuable for studies investigating disease mechanisms and testing therapeutic strategies in vitro.

CRISPR/Cas9 editing to generate a heterozygous COL2A1 p.G1170S human chondrodysplasia iPSC line, MCRIi019-A-2, in a control iPSC line, MCRIi019-A.

To develop an in vitro disease model of a human chondrodysplasia, we used CRISPR/Cas9 gene editing to generate a heterozygous COL2A1 exon 50 c.3508 GGT > TCA (p.G1170S) mutation in a control human iPSC line. Both the control and COL2A1 mutant lines displayed typical iPSC characteristics, including normal cell morphology, expression of pluripotency markers, the ability to differentiate into endoderm, ectoderm and mesoderm lineages and normal karyotype. These chondrodysplasia mutant and isogenic control cell lines can be used to explore disease mechanisms underlying type II collagenopathies and aid in the discovery of new therapeutic strategies.

Identification of the skeletal progenitor cells forming osteophytes in osteoarthritis.

OBJECTIVES: Osteophytes are highly prevalent in osteoarthritis (OA) and are associated with pain and functional disability. These pathological outgrowths of cartilage and bone typically form at the junction of articular cartilage, periosteum and synovium. The aim of this study was to identify the cells forming osteophytes in OA. METHODS: Fluorescent genetic cell-labelling and tracing mouse models were induced with tamoxifen to switch on reporter expression, as appropriate, followed by surgery to induce destabilisation of the medial meniscus. Contributions of fluorescently labelled cells to osteophytes after 2 or 8 weeks, and their molecular identity, were analysed by histology, immunofluorescence staining and RNA in situ hybridisation. Pdgfrα-H2BGFP mice and Pdgfrα-CreER mice crossed with multicolour Confetti reporter mice were used for identification and clonal tracing of mesenchymal progenitors. Mice carrying Col2-CreER, Nes-CreER, LepR-Cre, Grem1-CreER, Gdf5-Cre, Sox9-CreER or Prg4-CreER were crossed with tdTomato reporter mice to lineage-trace chondrocytes and stem/progenitor cell subpopulations. RESULTS: Articular chondrocytes, or skeletal stem cells identified by Nes, LepR or Grem1 expression, did not give rise to osteophytes. Instead, osteophytes derived from Pdgfrα-expressing stem/progenitor cells in periosteum and synovium that are descendants from the Gdf5-expressing embryonic joint interzone. Further, we show that Sox9-expressing progenitors in periosteum supplied hybrid skeletal cells to the early osteophyte, while Prg4-expressing progenitors from synovial lining contributed to cartilage capping the osteophyte, but not to bone. CONCLUSION: Our findings reveal distinct periosteal and synovial skeletal progenitors that cooperate to form osteophytes in OA. These cell populations could be targeted in disease modification for treatment of OA.

CRISPR/Cas9 gene editing of a SOX9 reporter human iPSC line to produce two TRPV4 patient heterozygous missense mutant iPSC lines, MCRIi001-A-3 (TRPV4 p.F273L) and MCRIi001-A-4 (TRPV4 p.P799L).

To produce in vitro models of human chondrodysplasias caused by dominant missense mutations in TRPV4, we used CRISPR/Cas9 gene editing to introduce two heterozygous patient mutations (p.F273L and p.P799L) into an established control human iPSC line. This control line expressed a fluorescent reporter (tdTomato) at the SOX9 locus to allow real-time monitoring of cartilage differentiation by SOX9 expression. Both TRPV4 mutant iPSC lines had normal karyotypes, expressed pluripotency markers, and could differentiate into cells representative of the three embryonic germ layers. These iPSC lines, with the parental isogenic control, will be used to study TRPV4 chondrodysplasia mechanisms and explore therapeutic approaches.

Ablation of the miRNA Cluster 24 Has Profound Effects on Extracellular Matrix Protein Abundance in Cartilage.

MicroRNAs (miRNAs) regulate cartilage differentiation and contribute to the onset and progression of joint degeneration. These small RNA molecules may affect extracellular matrix organization (ECM) in cartilage, but for only a few miRNAs has this role been defined in vivo. Previously, we showed that cartilage-specific genetic ablation of the Mirc24 cluster in mice leads to impaired cartilage development due to increased RAF/MEK/ERK pathway activation. Here, we studied the expression of the cluster in cartilage by LacZ reporter gene assays and determined its role for extracellular matrix homeostasis by proteome and immunoblot analysis. The cluster is expressed in prehypertrophic/hypertrophic chondrocytes of the growth plate and we now show that the cluster is also highly expressed in articular cartilage. Cartilage-specific loss of the cluster leads to increased proteoglycan 4 and matrix metallopeptidase 13 levels and decreased aggrecan and collagen X levels in epiphyseal cartilage. Interestingly, these changes are linked to a decrease in SRY-related HMG box-containing (SOX) transcription factors 6 and 9, which regulate ECM production in chondrocytes. Our data suggests that the Mirc24 cluster is important for ECM homoeostasis and the expression of transcriptional regulators of matrix production in cartilage.

Generation of a heterozygous COL2A1 (p.R989C) spondyloepiphyseal dysplasia congenita mutation iPSC line, MCRIi001-B, using CRISPR/Cas9 gene editing.

To produce an in vitro model of the human chondrodysplasia, spondyloepiphyseal dysplasia congenita, we used CRISPR/Cas9 gene editing to generate a heterozygous patient COL2A1 mutation in an established control human iPSC line. The gene-edited heterozygous COL2A1 p.R989C line had a normal karyotype, expressed pluripotency markers, and could differentiate into cells representative of the three embryonic germ layers. When differentiated into cartilage this cell line and the parental isogenic control may be used to explore disease mechanisms and evaluate therapeutic approaches.

Genetic Disorders of the Extracellular Matrix.

Mutations in the genes for extracellular matrix (ECM) components cause a wide range of genetic connective tissues disorders throughout the body. The elucidation of mutations and their correlation with pathology has been instrumental in understanding the roles of many ECM components. The pathological consequences of ECM protein mutations depend on its tissue distribution, tissue function, and on the nature of the mutation. The prevalent paradigm for the molecular pathology has been that there are two global mechanisms. First, mutations that reduce the production of ECM proteins impair matrix integrity largely due to quantitative ECM defects. Second, mutations altering protein structure may reduce protein secretion but also introduce dominant negative effects in ECM formation, structure and/or stability. Recent studies show that endoplasmic reticulum (ER) stress, caused by mutant misfolded ECM proteins, makes a significant contribution to the pathophysiology. This suggests that targeting ER-stress may offer a new therapeutic strategy in a range of ECM disorders caused by protein misfolding mutations. Anat Rec, 2019. © 2019 The Authors. The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.

Generation of a SOX9-tdTomato reporter human iPSC line, MCRIi001-A-2, using CRISPR/Cas9 editing.

To develop an iPSC SOX9 reporter line for monitoring differentiation into SOX9 expressing cells such as chondrocytes, cranial neural crest and Sertoli cells, we used gene editing to introduce sequences encoding the tdTomato fluorescent protein into the SOX9 locus. The gene-edited line had a normal karyotype, expressed pluripotency markers and differentiated into cells representative of the three embryonic germ layers. Endogenous SOX9 expression was undisturbed and the tdTomato fluorescent reporter mirrored SOX9 mRNA expression. This iPSC line will be useful for assessing iPSC differentiation into SOX9-expressing cells and enrichment by cell sorting.

Identification of Two Independent COL5A1 Variants in Dogs with Ehlers-Danlos Syndrome.

The Ehlers-Danlos syndromes (EDS) are a heterogeneous group of heritable disorders affecting connective tissues. The mutations causing the various forms of EDS in humans are well characterized, but the genetic mutations causing EDS-like clinical pathology in dogs are not known, thus hampering accurate clinical diagnosis. Clinical analysis of two independent cases of skin hyperextensibility and fragility, one with pronounced joint hypermobility was suggestive of EDS. Whole-genome sequencing revealed de novo mutations of COL5A1 in both cases, confirming the diagnosis of the classical form of EDS. The heterozygous COL5A1 p.Gly1013ValfsTer260 mutation characterized in case 1 introduced a premature termination codon and would be expected to result in α1(V) mRNA nonsense-mediated mRNA decay and collagen V haploinsufficiency. While mRNA was not available from this dog, ultrastructural analysis of the dermis demonstrated variability in collagen fibril diameter and the presence of collagen aggregates, termed 'collagen cauliflowers', consistent with COL5A1 mutations underlying classical EDS. In the second case, DNA sequencing demonstrated a p.Gly1571Arg missense variant in the COL5A1 gene. While samples were not available for further analysis, such a glycine substitution would be expected to destabilize the strict molecular structure of the collagen V triple helix and thus affect protein stability and/or integration of the mutant collagen into the collagen V/collagen I heterotypic dermal fibrils. This is the first report of genetic variants in the COL5A1 gene causing the clinical presentation of EDS in dogs. These data provided further evidence of the important role of collagen V in dermal collagen fibrillogenesis. Importantly, from the clinical perspective, we showed the utility of DNA sequencing, combined with the established clinical criteria, in the accurate diagnosis of EDS in dogs.

Cartilage endoplasmic reticulum stress may influence the onset but not the progression of experimental osteoarthritis.

BACKGROUND: Osteoarthritis has been associated with a plethora of pathological factors and one which has recently emerged is chondrocyte endoplasmic reticulum (ER) stress. ER stress is sensed by key ER-resident stress sensors, one of which is activating transcription factor 6 (ATF6). The purpose of this study is to determine whether increased ER stress plays a role in OA. METHODS: OA was induced in male wild-type (+/+), ColIITgcog (c/c) and Atf6α-/- mice by destabilisation of the medial meniscus (DMM). c/c mice have increased ER stress in chondrocytes via the collagen II promoter-driven expression of ER stress-inducing Tgcog. Knee joints were scored histologically for OA severity. RNA-seq was performed on laser-micro-dissected RNA from cartilage of +/+ and c/c DMM-operated mice. RESULTS: In situ hybridisation demonstrated a correlation between the upregulation of ER stress marker, BiP, and early signs of proteoglycan loss and cartilage damage in DMM-operated +/+ mice. Histological analysis revealed a significant reduction in OA severity in c/c mice compared with +/+ at 2 weeks post-DMM. This chondroprotective effect in c/c mice was associated with a higher ambient level of BiP protein prior to DMM and a delay in chondrocyte apoptosis. RNA-seq analysis suggested Xbp1-regulated networks to be significantly enriched in c/c mice at 2 weeks post-DMM. Compromising the ER through genetically ablating Atf6α, a key ER stress sensor, had no effect on DMM-induced OA severity. CONCLUSION: Our studies indicate that an increased capacity to effectively manage increases in ER stress in articular cartilage due either to pre-conditioning as a result of prior exposure to ER stress or to genetic pre-disposition may be beneficial in delaying the onset of OA, but once established, ER stress plays no significant role in disease progression.

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.

Extra-skeletal manifestations in mice affected by Clcn7-dependent autosomal dominant osteopetrosis type 2 clinical and therapeutic implications.

Autosomal dominant osteopetrosis type 2 (ADO2) is a high-density brittle bone disease characterized by bone pain, multiple fractures and skeletal-related events, including nerve compression syndrome and hematological failure. We demonstrated that in mice carrying the heterozygous Clcn7 G213R mutation, whose human mutant homolog CLCN7 G215R affects patients, the clinical impacts of ADO2 extend beyond the skeleton, affecting several other organs. The hallmark of the extra-skeletal alterations is a consistent perivascular fibrosis, associated with high numbers of macrophages and lymphoid infiltrates. Fragmented clinical information in a small cohort of patients confirms extra-skeletal alterations consistent with a systemic disease, in line with the observation that the CLCN7 gene is expressed in many organs. ADO2 mice also show anxiety and depression and their brains exhibit not only perivascular fibrosis but also ß-amyloid accumulation and astrogliosis, suggesting the involvement of the nervous system in the pathogenesis of the ADO2 extra-skeletal alterations. Extra-skeletal organs share a similar cellular pathology, confirmed also in vitro in bone marrow mononuclear cells and osteoclasts, characterized by an impairment of the exit pathway of the Clcn7 protein product, ClC7, through the Golgi, with consequent reduced ClC7 expression in late endosomes and lysosomes, associated with high vesicular pH and accumulation of autophagosome markers. Finally, an experimental siRNA therapy, previously proven to counteract the bone phenotype, also improves the extra-skeletal alterations. These results could have important clinical implications, supporting the notion that a systematic evaluation of ADO2 patients for extra-skeletal symptoms could help improve their diagnosis, clinical management, and therapeutic options.